International Review of Cytology, A Survey of Cell Biology, Vol. 193

International Review of

A Survey of

Cytology Cell Biology VOLUME 193

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949–1988 1949–1984 1967– 1984–1992 1993–1995

EDITORIAL ADVISORY BOARD Eve Ida Barak Rosa Beddington Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Laurence Etkin Hiroo Fukuda Elizabeth D. Hay P. Mark Hogarth Anthony P. Mahowald M. Melkonian

Bruce D. McKee Keith E. Mostov Andreas Oksche Vladimir R. Pantic´ Jozef St. Schell Manfred Schliwa Robert A. Smith Wilfred D. Stein Ralph M. Steinman M. Tazawa Donald P. Weeks Robin Wright Alexander L. Yudin

International Review of A Survey of

Cytology Cell Biology

Edited by Kwang W. Jeon Department of Biochemistry University of Tennessee Knoxville, Tennessee

VOLUME 193

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

Front cover photograph: Photomicrograph of the two daughter cells of the desmid, Microsterias. (For more details, see Chapter 3, Figure 1.) This book is printed on acid-free paper. Copyright0 1999 by ACADEMIC PRESS All Riohts Reserved. NOG r t of this publication may be reproduced or transmined in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher The appearance of the code at the bonom of the first page of a chapter in this book indicates the Publisher's consent that copies of the chapter may be made for personal or internal use of specific clients.This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, inc. (222 Rosewood Drive, Danven, Massachusens 01923). for cowing beyond that permined by Sections 107 or 108 of the U S Copyright Law.This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-I999 chapters are as shown on the title pages. if no tee code appears on the title page, the copy fee is the same as far current chapters. 0074-7696199$30.00 Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given. a e. s. s .A.c. -d-e.m . ..i.c.P . r. . A Harcourt Science and Technology Company 525 B Street. Suite 1900. San Diego. California 92101-4495. USA hnp:l/w.apnet.com

Academic Press

24-28 Oval Road, London NWI 7DX, UK hnp:llwww.hbuk.co.uklap/ international Standard Book Number: 0-12-364597-2 PRINTED IN THE UNITED STATES OF AMERICA 99 0 0 0 1 02 03 04 B B 9 8 7 6

5

4

3

2

1

CONTENTS

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Unusual Autonomic Ganglia: Connections, Chemistry, and Plasticity of Pelvic Ganglia Janet R. Keast I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Typical Features of Autonomic Ganglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pelvic Ganglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 11 49 51

Mechanisms and Control of Embryonic Genome Activation in Mammalian Embryos Keith E. Latham I. II. III. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forming the Embryonic Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear Changes during Cleavage Related to EGA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytoplasmic Changes Controlling EGA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Comprehensive Model for Controlling EGA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prospectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

71 73 81 94 109 111 113

vi

CONTENTS

Temporal and Spatial Coordination of Cells with Their Plastid Component Annette W. Coleman and Andrea M. Nerozzi I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basic Background Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Architecture of Plastid DNA Genome Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytology of Plastid Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fundamental ptDNA:Plastome:Cell Size:Nuclear Ploidy Proportionality . . . . . . . . . . . . . Molecular Aspects of Plastid DNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plastid Distribution at Cytokinesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control of Plastid Positioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteins Associated with Plastid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics of Plastid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Choice of Experimental Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

125 128 129 134 136 139 141 143 146 150 153 155 156

Cellular and Molecular Mechanisms of Sexual Incompatibility in Plants and Fungi Simon J. Hiscock and Ursula Ku¨es I. II. III. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Mechanisms of Recognition and Mating in Fungi . . . . . . . . . . . . . . . . . . . . . . Sexual Incompatibility Systems in Nonflowering Plants . . . . . . . . . . . . . . . . . . . . . . . . . Self-Incompatibility Systems in Flowering Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution of Mating-Type and Self-Incompatibility Systems . . . . . . . . . . . . . . . . . . . . . . Prospect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

165 170 213 218 246 258 259

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Annette W. Coleman (125), Division of Biology and Medicine, Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912 Simon J. Hiscock (165), Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom Janet R. Keast (1), Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia Ursula Ku¨es (165), Institute of Microbiology, ETH Zu¨rich, CH-8092 Zu¨rich, Switzerland Keith E. Latham (71), The Fels Institute for Cancer Research and Molecular Biology and the Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Andrea M. Nerozzi (125), Division of Biology and Medicine, Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912

vii

This Page Intentionally Left Blank

Unusual Autonomic Ganglia: Connections, Chemistry, and Plasticity of Pelvic Ganglia Janet R. Keast Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia

The pelvic ganglia provide the majority of the autonomic nerve supply to reproductive organs, urinary bladder, and lower bowel. Of all autonomic ganglia, they are probably the least understood because in many species their anatomy is particularly complex. Furthermore, they are unusual autonomic ganglia in many ways, including their connections, structure, chemistry, and hormone sensitivity. This review will compare and contrast the normal structure and function of pelvic ganglia with other types of autonomic ganglia (sympathetic, parasympathetic, and enteric). Two aspects of plasticity in the pelvic pathways will also be discussed. First, the influence of gonadal steroids on the maturation and maintenance of pelvic reflex circuits will be considered. Second, the consequences of nerve injury will be discussed, particularly in the context of the pelvic ganglia receiving distributed spinal inputs. The review demonstrates that in many ways the pelvic ganglia differ substantially from other autonomic ganglia. Pelvic ganglia may also provide a useful system in which to study many fundamental neurobiological questions of broader relevance. KEY WORDS: Autonomic ganglia, Urinary tract, Sympathetic, Parasympathetic, Pelvic plexus, Androgen, Estrogen. 䊚 1999 Academic Press.

I. Introduction Many neuroscientists working on the central nervous system regard peripheral autonomic neurons as somewhat dull and unchallenging. They are frequently not even appreciated as individual cells, each with integrative and plastic properties, but rather as one of many small, uninteresting reInternational Review of Cytology, Vol. 193 0074-7696/99 $30.00

1

Copyright 䉷 1999 by Academic Press. All rights of reproduction in any form reserved.

2

JANET R. KEAST

peated units of the ‘‘autonomic system.’’ The importance of possessing a fully functional autonomic nervous system, fortunately, is better acknowledged as it influences the many and varied ‘‘involuntary’’ reflexes of the body, from cardiovascular and digestive function to metabolism and the immune system. The focus of this review is the group of neurons which supply the pelvic viscera, providing reflex control of the lower bowel and urinary tract as well as various reproductive functions. Many of the neurons which supply these organs lie in the pelvic ganglia and exhibit numerous features which distinguish them from other autonomic ganglia. Some of these features, such as hormone sensitivity, have provided insights into more fundamental issues in neuroscience (in this case the convergence of endocrine and neural signaling processes). Other aspects, such as the unique mixture of sympathetic and parasympathetic connections, not only add to their complexity and interest but also have contributed significantly to the delay in understanding various clinical conditions such as impotence and urinary incontinence. The aim of this review is to elucidate the unique features of the pelvic ganglia not only for the purpose of allowing a better appreciation of their relationship to other components of the nervous system but also to demonstrate that the pelvic ganglia provide an accessible system for addressing some fundamental questions of neurobiology which are currently more difficult to address elsewhere. First, an overview of autonomic ganglia will be provided in order to place the pelvic innervation in context. Second, pelvic ganglia will be discussed in more detail, highlighting their unusual features and aspects of particular interest. Finally, an argument will be made for exploiting some of these features in order to make progress in some particular problems in basic neurobiology and the clinical arena.

II. Typical Features of Autonomic Ganglia A. An Overview of Sympathetic, Parasympathetic, and Enteric Ganglia The most common definition of the autonomic nervous system is the ‘‘visceral motor system.’’ This places the primary emphasis on the motor component of autonomic reflexes—the groups of neurons which influence smooth and cardiac muscle, exocrine glands, and various other targets (such as adipose tissue and immune cells). Numerous excellent reviews of the autonomic regulation of these tissues have been published (Gibbins, 1991; Ja¨nig and McLachlan, 1987, 1992b; Gabella, 1995; Wang et al., 1995). However, equally important to effective reflex behavior and homeostasis is the visceral

UNUSUAL AUTONOMIC GANGLIA

3

afferent system, which detects changes in the internal and external environments and then triggers the appropriate autonomic motor activity. This sensory system is typically not included in the definition of the autonomic system, but it is difficult to separate this from the definition, either conceptually or experimentally. Therefore, in this review I have included interactions between the visceral sensory and motor pathways where appropriate. The autonomic system can be considered as having three components— the sympathetic, parasympathetic, and enteric nervous systems—although not all texts consider the enteric system separately. The first two exhibit some similarities in their general organization because both consist of preganglionic neurons (lying in the central nervous system) and postganglionic neurons (lying in aggregates, the autonomic ganglia) in the periphery. Although there are numerous distinguishing features of sympathetic and parasympathetic pathways, the most accepted way of classification is by their anatomy, with sympathetic pathways having their preganglionic neurons within thoracic and lumbar spinal segments and parasympathetic pathways originating from cranial nuclei or sacral levels of the spinal cord. In either case, ganglionic activity is primarily determined by central outflow from preganglionic axons, such that if the spinal nerves are damaged, reflex activity ceases. Some exceptions to this are found in the sympathetic system. Sympathetic ganglia are of two types, paravertebral and prevertebral, which differ in their location, connections, integrative properties, channels, receptors, and transmitters ( Ja¨nig and McLachlan, 1987, 1992b; Gabella, 1995). Paravertebral ganglia lie in two linear networks (‘‘chains’’) on each side of the vertebral column. In some places, more commonly in the caudal region, these chains fuse and only one ganglion is found near the midline. The majority of these neurons are noradrenergic and contain the peptide, neuropeptide Y (NPY), although some are cholinergic and contain other peptides, e.g., vasoactive intestinal peptide (VIP). The neurons possess dendrites and typically receive a small number of strong (suprathreshold) preganglionic inputs, each of which activates ganglion cells via nicotinic acetylcholine (ACh) receptors. Most of these neurons supply peripheral vasculature and, as such, their activity is matched closely to other cardiovascular events ( Ja¨nig and McLachlan, 1992a). However, the cholinergic neurons supply other targets, such as piloerector muscles and sweat glands, the central regulation of which is not as well understood. Prevertebral sympathetic ganglia are unpaired and lie near the midline, closer to the peripheral organs that they supply. They are sometimes referred to as the ‘‘abdominal plexus’’ or ‘‘mesenteric ganglia’’ because they lie in the mesentery and their neuronal connectives travel with mesenteric vessels. Most of the neurons are noradrenergic, although a minority are cholinergic and contain peptides such as VIP. Most of the noradrenergic neurons contain NPY, but some contain other peptides or no peptides at

4

JANET R. KEAST

all. The neurons typically possess many dendrites and the complexity of their dendritic arbor may vary between neurons with different targets and firing properties (see Section II,B). Each neuron typically receives numerous synaptic inputs; however, whereas in paravertebral ganglia these arise only from spinal preganglionic axons, in the prevertebral ganglia many neurons also receive inputs from other peripheral neurons (see Section II,D,2). These sympathetic neurons supply the digestive tract and, in turn, some neurons in the gut (‘‘viscerofugal neurons’’) send axons to prevertebral ganglia. This peripheral reflex loop appears to provide the primary excitatory input to such neurons, whereas central inputs are comparatively weak. Both the peripheral and the central inputs to prevertebral neurons are excitatory and dominated by cholinergic nicotinic responses, although other transmitters and receptors may play a minor role. In prevertebral ganglia there is also considerable divergence and convergence of inputs (Szurszewski, 1981; Ja¨nig and McLachlan, 1992a; Miolan and Niel, 1996). Thus, there is integration of central (and in some cases peripheral) signals within the final motor pathway. Prevertebral ganglia control a diverse array of tissues, including cardiac and smooth muscle and glandular and other tissues. Distinguishing features for functionally diverse pathways include the distribution and types of transmitter receptors on the various target tissues and the patterns of activation and strength of their preganglionic innervation. In addition, in some cases the peptide contents is characteristic for neurons with a particular function, a feature known as ‘‘chemical coding’’ (see Section II,C,1). Parasympathetic ganglia are very different from sympathetic ganglia in a number of anatomical and functional ways (Gibbins, 1991; Akasu and Nishimura, 1995; Gabella, 1995; Wang et al., 1995). Apart from their different origin of central outflow, the neurons are located in small ganglia lying very close to their targets. In some cases, these are found embedded within the adventitial tissues or outer musculature of the organs, making experimental or surgical access difficult. However, some investigators have found this problem worth overcoming because the neurons within a parasympathetic ganglion are a relatively homogeneous functional group in comparison with typical sympathetic ganglia that each supply many targets. Parasympathetic neurons are also structurally homogeneous and typically possess few or no dendrites (see Section II,B). Far less convergence and divergence of inputs occurs within parasympathetic ganglia, and many investigators therefore consider them as mere ‘‘relay stations’’ rather than having significant integrative function. This has appeal for detailed studies of neuronal membrane properties and synaptic function due to the ease with which these simple neurons can be voltage clamped with a single microelectrode and their inputs readily manipulated. All parasympathetic neurons are thought to be cholinergic, with various other substances also present,

UNUSUAL AUTONOMIC GANGLIA

5

depending on the target organ. Other substances commonly synthesized by parasympathetic ganglion cells include VIP and nitric oxide synthase (NOS) (see Section II,C,2). Many organs in the body receive both sympathetic and parasympathetic axons, although only in a few cases do the axons supply the same tissues within the organ and have a genuinely antagonistic action (e.g., the sinoatrial node of the heart). More commonly tissues are supplied by either parasympathetic or sympathetic axons (but not both) or have a dual innervation whereby each subserves a different but not opposite function. Thus, the behavior of the visceral organs is determined by the types and numbers of each type of autonomic ganglion cell activated as well as by their temporal pattern of activation. The central integration of sensory information and the subsequent patterning of the appropriate motor outflow through the sympathetic and parasympathetic pathways are obviously critical to adequate autonomic function; however, the events occurring within peripheral ganglia and at their terminals provide the final points at which motor activities can be modified. As discussed in Sections III,G and III,H, it is at these relatively accessible locations where it may be possible to manipulate reflex behaviors in order to maximize their success following various types of injury or chemical defect. The nervous system embedded within the digestive tract, the enteric nervous system, is properly considered as a third component of the autonomic system, although some older texts place it within the parasympathetic category. This has been done primarily because of its close location to its targets, coupled with its sensitivity to vagal or sacral spinal nerve activation. However, it bears only scant resemblance to the other parts of the autonomic system. In particular, it has no dependence on the central nervous system for many of its activities and possesses its own sensory neurons within the gut that respond to chemical and mechanical stimuli (Furness and Costa, 1987; Furness et al., 1995a). These trigger motility and secretomotor reflexes without the aid of spinal preganglionic activation, and the enteric system therefore deserves to be regarded as a unique, independent part of the autonomic system. This independence, however, is not complete in that sympathetic and parasympathetic pathways supply the digestive tract and have particular roles in coordination of distant regions within the gut and integration of gut activity with other body functions (see Section II,D,2). In addition to the obvious need to study autonomic neuronal mechanisms in order to understand regulation of peripheral structures, autonomic ganglia have proved to be appealing to many researchers interested in more fundamental neurobiological issues. For example, autonomic ganglia can be readily removed and studied in vitro while maintaining many or all of their neural connections. Furthermore, in most cases, the input and output pathways lie in different axon bundles which can be monitored and manipu-

6

JANET R. KEAST

lated separately, either in vivo or in vitro. Principles of synaptic activation and integration can therefore be studied in a simpler, more easily accessible system. The enteric system provides additional appeal because it contains sensory neurons, and entire reflex circuits can be studied in vitro. Finally, autonomic ganglia from both young and adult animals are appreciated for being very robust in that the neurons and their synapses can be studied successfully in vitro for many hours.

B. Neuron Morphology The degree of complexity of a neuronal dendritic arbor is commonly thought to reflect the number of synaptic inputs it receives. This also generally holds true in autonomic ganglia. The most complex sympathetic neurons are those referred to as ‘‘tonic’’ neurons because of their firing behavior during a prolonged depolarizing current pulse (Szurszewski, 1981; McLachlan and Meckler, 1989). These exhibit a greater number of dendrites with more extensive branching than other sympathetic neurons (Boyd et al., 1996). They are found most commonly in prevertebral ganglia and many are thought to receive synaptic inputs from enteric neurons (Kreulen and Szurszewski, 1979a,b; Szurszewski, 1981; Kreulen and Peters, 1986; McLachlan and Meckler, 1989). It is therefore believed that these neurons are ‘‘motility regulating,’’ either of the gut or other organs (e.g., the bladder; Ja¨nig and McLachlan, 1987). Other sympathetic neurons in the pre- and paravertebral ganglia also possess dendrites, but these are slightly less complex, in parallel with their smaller number of inputs (McLachlan and Meckler, 1989; Boyd et al., 1996). Parasympathetic neurons contrast strongly with both classes of sympathetic neurons because they have few or no dendrites (Yamakado and Yohro, 1977; Forehand, 1985; Chiang and Gabella, 1986; Snider, 1987; Akasu and Nishimura, 1995; Baluk, 1995; Callister et al., 1997). Where cells have been filled with dye to allow detailed analysis of morphology, many appear to possess very short, fine processes. This morphological simplicity concurs with the proposed relay function of these ganglia, which typically receive only a small number of inputs—many of which are classed as ‘‘strong’’ (i.e., suprathreshold; Akasu and Nishimura, 1995). The function of the vestigial dendrites is not known, although synapses are made with them in some ganglia. Thus, in parasympathetic ganglia there is relative homogeneity of structure and function. A notable exception is some of the intracardiac ganglia, which contain a variety of structural, chemical, and physiological neuron types and may, like the enteric system, contain sensory neurons and interneurons (Edwards et al., 1995; Klemm et al., 1997; Pauza et al., 1997).

UNUSUAL AUTONOMIC GANGLIA

7

In contrast, the enteric nervous system contains a variety of motor neurons, interneurons, and sensory neurons, each with their own morphological features. These are described well in recent reviews (Surprenant, 1994; Furness et al., 1995b) and will not be discussed further here; suffice to say that neurons receiving more synaptic inputs tend to have more complex dentritic arbors. There are also numerous chemical and morphological differences between many of the main functional subclasses of enteric neurons.

C. Neurotransmitters 1. Sympathetic Ganglion Cells The majority of sympathetic ganglion cells synthesize noradrenaline and use it as a transmitter (Elfvin et al., 1993); conversely, the sole supply of noradrenergic axons to visceral tissues is the sympathetic system. Except in a few rare cases [e.g., a small population of neurons in the guinea pig proximal colon (Costa et al., 1971) and some regions of bat intestine (Keast, 1994a)], there are no enteric noradrenergic neurons; therefore, all noradrenergic axons in the gut originate from extrinsic sympathetic nerves. Adrenoceptor blockers attenuate many sympathetic nerve responses in the periphery. They are also abolished when peripheral tissues are pretreated with catecholamine-depleting drugs (e.g., reserpine) or substances which destroy noradrenergic terminals (e.g., 6-hydroxydopamine). However, there have been some errors in describing the location of peripheral noradrenergic neurons when utilizing only immunohistochemical methods. Notably, the presence of tyrosine hydroxylase (TH), the rate-determining enzyme of catecholamine synthesis, is not restricted to catecholamine pathways but can also be expressed by neurons that do not contain the enzyme dopamine-웁-hydroxylase (required for noradrenaline synthesis). Such neurons have been demonstrated in the guinea pig pelvic (paracervical) ganglia (Morris and Gibbins, 1987) and in guinea pig dorsal root ganglia (Kummer et al., 1990) but are not thought to be noradrenergic or dopaminergic because they do not store catecholamines. A small proportion of sympathetic neurons (defined as such by their thoracic or lumbar spinal connections) are not noradrenergic but instead synthesize acetylcholine. The best studied of these are the sympathetic neurons supplying eccrine sweat glands. These neurons are located in the paravertebral ganglia and bear considerable resemblance to parasympathetic neurons, synthesizing ACh and VIP and activating secretion via muscarinic receptors. Interestingly, this adult cholinergic phenotype is not present earlier in development, and until the first postnatal week (i.e., until

8

JANET R. KEAST

they reach their target gland cells) these synapses are noradrenergic and the gland cells express 움-adrenoceptors (Landis, 1996). The developmental ‘‘switch’’ to a cholinergic phenotype can be mimicked in culture by exposure of these neurons to various conditioned media (e.g., from heart cells), and it is thought that a secreted cytokine acts as a differentiation factor in vivo (Habecker et al., 1997). It is not known if the small populations of nonnoradrenergic neurons found in sympathetic ganglia which supply other targets also change their primary transmitter during development. Many sympathetic noradrenergic nerves also contain one or more other substances that may act as cotransmitters (Elfvin et al., 1993). These vary between tissues and species; however, NPY is considered to be a distinguishing feature of sympathetic vasoconstrictor axons in most species, and in some circumstances it may also act as a cotransmitter (Morris and Gibbins, 1992; Gibbins, 1995). Conversely, a peptide which is notably absent from sympathetic noradrenergic neurons is VIP. A substance known to be coreleased from many sympathetic noradrenergic terminals in peripheral organs is ATP, the best known nonnoradrenergic, noncholinergic autonomic transmitter (Burnstock, 1977, 1985; Morris and Gibbins, 1992). Unfortunately, there is no reliable method available for visualizing neurons which use ATP as a transmitter (i.e., purinergic neurons); however, extensive pharmacological studies have demonstrated its role in a variety of peripheral organs. 2. Parasympathetic Ganglion Cells Parasympathetic neurons are thought to synthesize ACh, although until recently it has not been possible to demonstrate this directly due to lack of antisera of appropriate sensitivity for the synthetic enzyme, choline acetyltransferase. More commonly, investigators have relied on blockade of nerve-evoked responses with muscarinic antagonists to demonstrate parasympathetic cholinergic transmission. Many of these studies have also indicated that in some tissues parasympathetic terminals release one or more cotransmitters (Lundberg and Ho¨kfelt, 1986; Morris and Gibbins, 1992; Elfvin et al., 1993). The peptide VIP is frequently colocalized in cholinergic neurons, so it has been the popular choice for cotransmitter status in parasympathetic effectors. However, convincing evidence for its release in response to physiological stimuli has been difficult to obtain, mainly due to lack of pharmacological tools with which to demonstrate its release at synapses. Recently, NOS has been shown to occur in many parasympathetic neurons and NO contributes to various responses in target organs, notably inhibition of smooth muscle (Rand, 1992; Sanders and Ward, 1992; Rand and Li, 1995). Finally, pharmacological studies have demonstrated that ATP can be coreleased from some cholinergic parasympathetic neurons (e.g., in the detrusor muscle of the bladder; Fujii, 1988;

UNUSUAL AUTONOMIC GANGLIA

9

Brading and Mostwin, 1989); therefore, purinergic transmission is not restricted to the sympathetic system. 3. Enteric Ganglion Cells Numerous transmitters and putative transmitters have been demonstrated in the enteric nervous system. These have been the subject of many excellent reviews and will not be considered in detail here (Costa et al., 1986; Furness and Costa, 1987; Surprenant, 1994; Furness et al., 1995b). The enteric system contains many cholinergic motor- and interneurons as well as many other transmitter types within these and noncholinergic pathways. Those most well studied are ATP, substance P, VIP, and the opioid peptides. Recently, glutamate (Liu et al., 1997), aspartate, and glycine ( J. Keast and S. Meusburger, unpublished observations) have been localized in some enteric neurons and there is evidence that glutamate may mediate some excitatory synaptic responses in the myenteric plexus (Liu et al., 1997). With rare exceptions, there are no enteric noradrenergic neurons (see Section II,C,1). The enteric nervous system is perhaps best known because the gut was the first site of isolation, characterization, and neuronal localization for many neuropeptides. Moreover, the coexistence of peptides with each other and with other transmitter types has been particularly well established for the enteric system, giving rise to extensive maps of ‘‘chemical coding,’’ i.e., where the target or projection pattern of a particular functional class of neurons is marked by its unique chemical content (Costa et al., 1986; Furness et al., 1988, 1989).

D. Connections between Different Parts of the Peripheral Nervous System 1. Sensory Ganglia The central processes of primary afferent neurons which supply the visceral tissues are essential for triggering all autonomic reflexes, except some of those activated by sensory neurons within the bowel. Primary afferent neurons activate interneurons which may be restricted to the spinal cord (segmental or intersegmental spinal reflexes, such as many of those which control male internal reproductive organs) or which may be located in various brain stem nuclei (supraspinal reflexes, such as the micturition reflex; de Groat and Steers, 1988; de Groat et al., 1993). Therefore, primary afferent neurons do not communicate directly with preganglionic neurons. In contrast, the peripheral processes of some sensory neurons synapse directly with some postganglionic autonomic neurons. This has been best

10

JANET R. KEAST

demonstrated in prevertebral sympathetic ganglia, in which collateral processes of primary sensory fibers supply some, but not all, of the ganglion cells (Matthews and Cuello, 1982; Le´ranth and Feher, 1983). It is not known which functional class of neurons is supplied by these terminals; however, it is thought that the sensory terminals are responsible for the slow depolarization elicited by bowel distension, mediated at least in part by the release of substance P (Dun and Jiang, 1982; Kreulen and Peters, 1986; Saria et al., 1987). While this would clearly potentiate the pre- to postganglionic nicotinic transmission in the ganglion, it is not clear in which circumstances these collaterals are activated in vivo. The assumption has been commonly made that these sensory connections branch from sensory processes traveling via the ganglion to the peripheral organs. However, it is not known if all peripheral sensory fibers produce collaterals or whether perhaps some sensory neurons project exclusively to ganglion cells. Finally, although it is clear that these sensory projections exist, it has not been possible to demonstrate their complete distribution due to the lack of markers for the entire visceral afferent population. To date, studies have used neuropeptide immunoreactivity [especially substance P (SP)] as an indicator, but this has limitations because only a proportion of visceral afferent neurons contain neuropeptides (Gibbins et al., 1987; Ju et al., 1987; Keast and de Groat, 1992); furthermore, neuropeptides are also present in some preganglionic neurons (Gibbins, 1995). Peripheral processes of sensory neurons also pass through paravertebral sympathetic ganglia, although they are less prevalent than in prevertebral ganglia (Ho¨kfelt et al., 1977). Similarly, some parasympathetic ganglia contain processes of primary afferent neurons, although it is not known if these make functional synapses in the ganglion. Finally, in the enteric system, terminals of primary afferent neurons have been found (as immunolabeled for SP and calcitonin gene-related peptide), where they are thought to excite subpopulations of enteric neurons (Costa et al., 1981; Maggi et al., 1988, 1997; Kojima and Shimo, 1995; Holzer and Holzer-Petsche, 1997a,b). 2. Enteric Ganglia The enteric nervous system is widely regarded as providing a considerable degree of autonomy to various aspects of digestive function because it depends on neither central afferent nor efferent activity for its primary reflexes (e.g., peristalsis). However, many sympathetic terminals synapse in the gut wall throughout the length of the digestive tract to decrease motility and secretion and to cause constriction of the gut vasculature (Furness and Costa, 1987). In the smooth muscle sphincters, sympathetic activation causes contraction; however, most of the sympathetic effects on motility and secretion occur at synapses on enteric neurons rather than

UNUSUAL AUTONOMIC GANGLIA

11

direct actions on smooth muscle or epithelia. Hence, this branch of sympathetic outflow has at least a ‘‘three-neuron chain’’ consisting of pre- and postganglionic sympathetic neurons and one or more enteric neurons. In contrast, the parasympathetic supply to the gut is sparse in most regions, although it is greater at both proximal and distal ends (esophagus through to duodenum and rectum; Berthoud et al., 1990, 1991; Luckensmeyer and Keast, 1998c). Parasympathetic activation increases motility and secretion and in the distal bowel of some species causes vasodilatation (Hulte´n, 1969; Andersson et al., 1983; Hedlund et al., 1985). The enteric neurons on which vagal terminals terminate can be considered parasympathetic postganglionic neurons; however, many gut neurons do not receive these connections and so cannot be considered part of the parasympathetic system. In contrast, sacral preganglionic axons terminate in the pelvic ganglia, which then send axons to the gut; these axons also terminate primarily in the myenteric ganglia and smooth muscle (see Section III,F,2). Hence, many of these neurons are part of a three-neuron chain, such as the sympathetic connection.

III. Pelvic Ganglia A. General Anatomical and Physiological Features Detailed anatomical maps and descriptions of nerve-evoked responses in the pelvic viscera (lower urinary and digestive tracts and internal reproductive organs) were elegantly described many years ago (Langley and Anderson, 1894, 1895a,b, 1896; Trumble, 1933). Various aspects of the anatomy and physiology of pelvic ganglia have since been investigated (Langworthy, 1965; Hulte´n, 1969; Purinton et al., 1973; Gonella et al., 1987; Ja¨nig and McLachlan, 1987; Dail, 1993; de Groat and Booth, 1993b; Traurig and Papka, 1993; Keast, 1995a). However, in many recent diagrammatic representations of the autonomic nervous system, the innervation of the pelvic viscera is difficult to decipher. Frequently, it is illustrated by both sympathetic and parasympathetic pathways converging or more commonly by separate components being present but arising from unspecified sources. When reading about the general physiology of these pathways at the textbook level, there is usually a similar lack of clarity regarding the source of different nerve fiber types, the transmitters, and the central mechanisms for coordinating sympathetic and parasympathetic outflow. Nevertheless, it is clear that both sympathetic and parasympathetic components of the autonomic system have a role to play in all these organs and that disruption

12

JANET R. KEAST

of this nerve supply seriously affects our ability to initiate or complete various digestive, urinary, and reproductive reflexes. The primary difficulty in experimentally investigating the autonomic supply to the pelvic viscera is its complex anatomy. Furthermore, perusing the scientific literature is confused by the array of terminology used to describe these neurons and their connectives. In this review I have chosen to use the term pelvic ganglia to describe all autonomic neurons which supply the pelvic viscera, except neurons of the lumbar paravertebral ganglia, inferior mesenteric ganglia, and enteric neurons of the lower bowel. The major connections between the pelvic ganglia and other parts of the autonomic nervous system are summarized in Fig. 1. Particular features of the ganglion cells are demonstrated in Fig. 2, and their preganglionic connections are shown in Fig. 3. Pelvic ganglia provide axons to all smooth muscle and exocrine glands of the pelvic viscera. In addition, they provide axons to many enteric ganglia of the lower bowel. As such, they provide a vital component of numerous diverse autonomic reflexes. While the parasympathetic supply is best known

FIG. 1 Connections of the major pelvic ganglion (MPG) in the rat. The majority of autonomic neurons supplying the pelvic viscera are located in the MPG, along with the smaller, adjacent accessory ganglia (AG). Only one of the paired MPG and AG is shown. Major nerve bundles are shown as broken lines. The MPG and AG contain both sympathetic and parasympathetic neurons, which receive excitatory synaptic inputs from either the upper lumbar or the sacral spinal cord, respectively. These connections travel via the sympathetic chain and inferior mesenteric ganglion (IMG) to the pelvic ganglia. Sympathetic and parasympathetic preganglionic axons project in the hypogastric and pelvic nerves, respectively. Most IMG neurons supply the intestine, along with neurons in the coeliac/superior mesenteric ganglion complex (CG/SMG). Bundles of postganglionic axons project separately to the various target organs (not shown).

UNUSUAL AUTONOMIC GANGLIA

13

for its role in the micturition, defecation, and erectile reflexes, it also provides a substantial innervation to the glandular epithelia of the internal reproductive organs, particularly in males (Dail, 1993). The sympathetic supply is best known for its excitatory effects on sphincter smooth muscle in the bowel and urinary tract and for similar effects on smooth muscle in the male reproductive organs. It is also thought to play a role in some aspects of penile erection and detumescence, although the precise mechanism is still unclear (Dail, 1993; de Groat and Booth, 1993a). Innervation of the pelvic viscera therefore provides another demonstration of the principle raised earlier (see Section II,A) that sympathetic and parasympathetic axons do not necessarily exert antagonistic effects in the same organ by targeting the same tissues. The relationship between sympathetic and parasympathetic nerves is more difficult to investigate in the pelvic viscera, given that some of the sympathetic terminals in pelvic organs release ACh (see Section III,D,1). Simple pharmacological manipulations are therefore not sufficient to distinguish between lumbar (sympathetic) and sacral (parasympathetic) effects. Irrespective of the level of spinal outflow, each of the pelvic visceral reflex activities depends on intact spinal nerves (to carry both preganglionic and primary afferent fibers) and in many cases intact supraspinal connections (e.g., for the micturition reflex). The effects of disrupting the spinal nerves are particularly interesting because substantial axogenesis occurs within just a few days in the pelvic ganglia, some of which may influence reflex restoration. This will be discussed in Section III,H,1. It is possible that the list of functions of autonomic nerves in the pelvic viscera is incomplete. In particular, various anatomical and histochemical studies have demonstrated populations of axons for which the function is not yet known. An example is the dense plexus of cholinergic axons (also containing NOS and VIP) which underlies the lumenal epithelium of the vas deferens and epididymis (Alm, 1982; McConnell et al., 1982; Dixon and Gosling, 1991; Keast, 1992) and which was originally thought to have a sensory function. However, these axons are now known to be autonomic and, as with many other cholinergic VIP axons, may stimulate secretory water and ion fluxes across the mucosa. They may have an important role in maintaining the optimal ionic environment for sperm transport along the ejaculatory ducts. A second example is the autonomic innervation of the testis (capsule, blood vessels, and interstitium), which has been recently described (Rauchenwald et al., 1995) but for which a function is not yet known. Pelvic ganglia vary considerably between species and genders in their gross anatomical features. In many mammals (including humans) they are numerous and distributed broadly among the pelvic organs (Langley and Anderson, 1895a, 1896; Trumble, 1933; Kuntz and Moseley, 1936; Schnitz-

14

JANET R. KEAST

lein et al., 1960; Owman and Sjo¨strand, 1965; Wozniak and Skowronska, 1967). Of these, many are located close to the wall of the pelvic organs or within their outer layers. Examples include some neurons which supply the bladder (sometimes referred to as ‘‘vesical ganglia’’; Fletcher and Bradley, 1969; Gosling and Thompson, 1977; Klu¨ck, 1980; Dixon et al., 1983) and the lower bowel (‘‘colonic’’ or ‘‘rectal’’ ganglia; de Groat and Krier, 1976; Payette et al., 1987; McRorie et al., 1991; Neuhuber et al., 1993; Luckensmeyer and Keast, 1998b,c). Such ganglia are considered to exclusively supply the organ with which they are closely associated. However, numerous other neurons are located in ganglia which are more distant from target organs and embedded within the connective tissues of the pelvic cavity. It is not known if there is any topography for neurons supplying particular targets or whether they outnumber the pelvic neurons located close to the organs. This large population of neurons supplying a range of targets has been given with various names in the past, including (inferior) hypogastric ganglia, hypogastric plexus, and pelvic plexus. Notably, ‘‘plexus’’ is used in preference to ganglia to indicate the complex meshwork of numerous ganglia and connectives. This arrangement has undoubtedly contributed significantly to the delay in examining their connectivity, neurochemistry, and physiology in laboratory animals and to the lack of progress in understanding clinical disorders such as impotence, urinary incontinence, and various lower bowel complaints. Moreover, there is an alarmingly frequent loss of reproductive and voiding reflexes following various types of surgical intervention, such as prostatectomy and hysterectomy, indicative of damage to neural pathways associated with the pelvic ganglia. This damage could perhaps be minimized or better compensated if the anatomy of pelvic ganglia were better understood. A simpler arrangement of pelvic autonomic neurons exists in guinea pigs (Costa and Furness, 1973a; Yokota and Burnstock, 1983; Morris and Gibbins, 1987), in which the majority of pelvic neurons are clustered in two distinct groups known as the anterior and posterior pelvic plexus. These have different targets, with the anterior plexus projecting to the urinary bladder and reproductive organs and the posterior plexus to the lower bowel (Costa and Furness, 1973a). However, the simplest anatomy described to date belongs to rats and mice, which have become favored for studies of pelvic ganglion structure and function. In particular, rats have been used for a myriad of neuroanatomical, pharmacological, and physiological studies. They are currently the species in which pelvic ganglia are best understood and will therefore be the focus of much of the latter part of this review. In rats (and mice), the pelvic neurons are aggregated into just a few ganglia rather than being scattered broadly throughout the pelvic viscera (Langworthy, 1965; Foroglou and Winckler, 1973; Purinton et al., 1973;

UNUSUAL AUTONOMIC GANGLIA

15

Dail et al., 1975; Baljet and Drukker, 1979, 1980), and the entire population of pelvic neurons can be reliably dissected, manipulated, and quantified. The majority of pelvic neurons are found in the bilateral major pelvic ganglia (MPG), closely associated with the dorsoventral aspect of the prostate gland. In female rodents this ganglion is often referred to as the paracervical ganglion or, in older studies, Frankenha¨user’s ganglion. Close to the MPG and attached to it is a cluster of two to four small ganglia, the accessory ganglia. These lie close to the ureters near the midline. Connections between accessory ganglia and MPGs of both sides consist of both pre- and postganglionic axons projecting to the contralateral ganglion and pelvic organs via the MPG (Kihara and de Groat, 1997). There is also a marked sexual dimorphism in the pelvic ganglia, with many more pelvic neurons in males than in females. For example, in male and female rats, there are 15,000 and 6000 pelvic neurons, respectively (Greenwood et al., 1985). This difference is reflected in the relative density of autonomic nerve terminals within the reproductive organs, with extensive axonal arborizations in the male reproductive organs but a relatively sparse supply to the female organs (apart from to their vasculature). Various other aspects of the chemistry and physiology of male and female pelvic ganglia will be discussed in Section III,G. Application of retrograde tracers to various pelvic organs has demonstrated a distinct topography within the rat MPG, but similar studies have not been carried out extensively in other species (Dail et al., 1983; Keast et al., 1989; Keast, 1992; Kolbeck and Steers, 1993; Kepper and Keast, 1995, 1997; Luckensmeyer and Keast, 1995b). In rats the accessory ganglia contribute mainly to the supply of the reproductive organs. Moreover, bundles of postganglionic axons can be followed from the major pelvic ganglia to each of the major target organs (e.g., the penile or cavernous nerves and rectal nerves). While indicating the major function of each nerve, these names are misleading because they have been derived from gross anatomical dissection studies. For example, retrograde and anterograde tracing studies have shown that a branch of the penile nerve projects to the rectum, internal anal sphincter, and anococcygeus muscle (Dail et al., 1990; Luckensmeyer and Keast, 1998b,c). The penile nerve also contains numerous nerve cell bodies (Dail et al., 1983), giving a falsely high impression of the number of axons it contains. Pelvic ganglia contain the entire parasympathetic nerve supply to the pelvic organs, but provide only a proportion of the sympathetic innervation. Lumbar paravertebral sympathetic neurons supply each of the targets and are thought to be particularly important in controlling their blood supply. The inferior mesenteric (prevertebral) sympathetic ganglion cells also innervate the pelvic viscera, although the range of targets varies considerably between species. In rats, this ganglion mainly supplies the bowel (Luckens-

16

JANET R. KEAST

meyer and Keast, 1994), whereas in other species it also provides substantial innervation to the urinary bladder and internal reproductive organs (Baron et al., 1985a,b; de Groat and Steers, 1988). A comparison of these two sources of sympathetic nerve supply to the pelvic organs has been made in the rat, and substantial differences in chemistry and physiology exist. These differences will be illustrated later. A similarly detailed comparison is not available for other species.

B. Mixed Sympathetic and Parasympathetic Ganglia In addition to their complex anatomy, a compounding feature of pelvic ganglia is that they contain both sympathetic and parasympathetic neurons. Parasympathetic pelvic neurons receive their spinal inputs from the sacral spinal cord via the pelvic nerves, whereas sympathetic pelvic neurons are supplied by preganglionic axons arising from the lumbar spinal cord and traveling in the hypogastric nerves (Figs. 1 and 2a) (Langley and Anderson, 1895a,b, 1896; Kuntz and Moseley, 1936; Baron et al., 1985b; Morgan et al., 1986; Ja¨nig and McLachlan, 1987; Baron and Ja¨nig, 1991). These two groups of nerves are easily identifiable and discrete, entering the complex of pelvic ganglia separately, after which they send off numerous branches to supply individual groups of neurons. Using simple dissection techniques it is not

FIG. 2 Features of pelvic ganglion cells in male rats. (a) There are three main chemical classes of pelvic ganglion cells, which contain acetylcholine (ACh) or noradrenaline (NA) along with one of two peptides, neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). All noradrenergic neurons are sympathetic (i.e., receive lumbar spinal inputs), and all cholinergic NPY neurons are parasympathetic (i.e., receive sacral inputs). Cholinergic VIP neurons form a mixed population, with the majority being parasympathetic. (b) Neurons in whole mounts of pelvic ganglion. All have been impaled with a microelectrode, filled with neurobiotin, and processed with the diaminobenzidine reaction. Rat pelvic neurons are characteristically monopolar and their axons can be traced to the edge of the ganglion. (c) Catecholaminecontaining neurons visualized by the glyoxylic acid reaction. Many but not all neurons display granular reaction product. A cluster of brightly reactive small, intensely fluorescent cells is shown (arrow). (d) Neurons intensely stained with the NADPH diaphorase reaction to demonstrate cells likely to possess nitric oxide synthase activity. (e) Almost all pelvic neurons are either noradrenergic or cholinergic, as demonstrated in this pair of micrographs. Noradrenergic neurons show immunoreactivity for tyrosine hydroxylase (e1), whereas cholinergic neurons are immunoreactive for choline acetyltransferase (e2). The arrows indicate examples of cholinergic neurons; cholinergic preganglionic terminals can also be seen. (f ) Noradrenergic neurons immunostained for tyrosine hydroxylase are often found in clusters and are typically relatively large neurons (cf. cholinergic neurons in g.). (g) Cholinergic VIP neurons are usually much smaller than noradrenergic neurons. Scale bar in b ⫽ 40 애m (b and c), 150 애m (d), 25 애m (e), and 50 애m (f and g).

UNUSUAL AUTONOMIC GANGLIA

17

18

JANET R. KEAST

possible to determine the prevalence or location of each neuron type, and for many years it was not known whether sympathetic or parasympathetic neurons predominated. Nevertheless, it has been more common for pelvic ganglia to be thought of as parasympathetic ganglia primarily because of their relatively close proximity to target organs. Moreover, many pelvic viscera receive sympathetic axons from the lumbar sympathetic chain or inferior mesenteric ganglion (Baron et al., 1985b; Ja¨nig and McLachlan, 1987; Baron and Ja¨nig, 1991; Gabella, 1995); an additional source of sympathetic axons could therefore be considered unnecessary. The first approach used to determine whether both sympathetic and parasympathetic neurons exist in pelvic ganglia was to stimulate either pelvic or hypogastric nerves and monitor responses in target organs. Such studies clearly indicated that pelvic (parasympathetic) nerve stimulation elicits responses such as contraction of the lower bowel and bladder detrusor muscle and penile erection (de Groat et al., 1981; de Groat and Steers, 1988). These responses were all abolished by nicotinic receptor antagonists, indicating that cholinergic synapses in pelvic ganglia were involved. Similar studies of hypogastric nerve activation also demonstrated potent effects on the pelvic viscera, including contraction of smooth muscle in the male internal reproductive organs and contraction of the bladder trigone muscle (de Groat and Steers, 1988; Dail, 1993). Various experiments also demonstrated blockade of these responses by nicotinic antagonists, indicating the presence of cholinergic synapses by sympathetic preganglionic axons (de Groat and Saum, 1972; Saum and de Groat, 1972b; de Groat and Krier, 1976; Gallagher et al., 1982). These observations were particularly important because they distinguished the effects of stimulating pre- and postganglionic sympathetic axons, both of which travel in the hypogastric nerves (the postganglionic sympathetic axons passing through pelvic ganglia originate mainly from the inferior mesenteric ganglion; Baron et al., 1985b; Ja¨nig and McLachlan, 1987). The ratio of pre- to postganglionic axons in the hypogastric nerve varies considerably between species, and in the species in which reflex organization was first investigated (the cat) postganglionic axons are numerous. In contrast, in rats, preganglionic axons are the major population in the hypogastric nerve ( Ja¨nig and McLachlan, 1987). Histochemical studies of catecholamine fluorescence provided further evidence of pelvic sympathetic neurons. Numerous brightly fluorescent ganglion cells exist in the pelvic ganglia of a variety of species (Fig. 2c) (Hamberger and Norberg, 1965a,b; Owman and Sjo¨strand, 1965; Costa and Furness, 1973a; Morris and Gibbins, 1987). However, the unusual resistance of these neurons to reserpine and 6-hydroxydopamine (6-OHDA), as well as their near proximity to their target organs, distinguishes them from other sympathetic noradrenergic neurons; they are therefore sometimes called ‘‘short adrenergic neurons.’’

UNUSUAL AUTONOMIC GANGLIA

19

A particularly interesting observation arising from in vivo studies of pelvic innervation is that some pelvic ganglia (e.g., the vesical ganglia in cats) appear to be supplied by both sympathetic and parasympathetic nerves, and experiments in which both have been stimulated together have provided evidence for close interaction between the two pathways (de Groat and Saum, 1972; Keast and de Groat, 1990; de Groat and Booth, 1993b). Possible explanations are (i) that both types of preganglionic terminals synapse on the same neurons and (ii) that sympathetic postganglionic (noradrenergic) terminals traveling to the organ from more distant pelvic ganglia either synapse on parasympathetic ganglion cells or influence transmitter release from their preganglionic terminals. It has been difficult to determine which occurs, primarily because the cellular neuranatomy is so poorly understood in species in which these potential interactions have been demonstrated in vivo. In particular, more detailed studies on the location of noradrenergic terminals within pelvic ganglia of these species are required. Although there are limited reports of their prevalence in some species (e.g., Hamberger and Norberg, 1965a), no information is available on which particular ganglia were studied or the uniformity of catecholamine terminal distribution. Finally, many sympathetic noradrenergic axons from the caudal sympathetic chain ganglia pass through the pelvic ganglia but apparently do not terminate within it (Kuntz and Moseley, 1936; Hamberger and Norberg, 1965a,b; Costa and Furness, 1973a,b; Hulsebusch and Coggeshall, 1982; Kuo et al., 1984). These may contribute to the difficulty in interpreting experiments in vivo. Intracellular microelectrode recordings from pelvic ganglion cells in vitro have provided further useful insights into the spinal innervation of pelvic neurons (see Section III,E). A limited number of recordings have been made from neurally activated cat, rabbit, and guinea pig vesical ganglia and indicate that both sympathetic and parasympathetic neurons exist (Blackman et al., 1969; Crowcroft and Szurszewski, 1971; de Groat et al., 1979; Griffith et al., 1980, 1981; Gallagher et al., 1982; Akasu et al., 1986). However, the recordings were made only from neurons associated with the bladder wall, and the spinal inputs to bladder-projecting pelvic neurons more distant from the organ remain unknown. Interestingly, similar studies in cat colonic ganglia show no interaction between sympathetic and parasympathetic pathways (de Groat and Krier, 1976, 1978; de Groat et al., 1979). Detailed electrophysiological studies have been made in guinea pig and rat pelvic ganglia (Blackman et al., 1969; Crowcroft and Szurszewski, 1971; de Groat et al., 1979; Tabatabai et al., 1986; Brock and Cunnane, 1992; Zhu et al., 1995; Zhu and Yakel, 1997). In some of these studies it is not clear which ganglion was studied (e.g., in the rat, either the major pelvic or accessory ganglion). Nevertheless, it appears that in these species both sympathetic and parasympathetic neurons are present and, predictably,

20

JANET R. KEAST

synaptic transmission is mediated by nicotinic ACh receptors. Where quantification has been performed, similar numbers of neurons of each type appear to be present. However, because only limited numbers of neurons can be characterized in these experiments, and because different neuron types vary in size (thereby affecting their likelihood of impalement), this methodology cannot give precise information on the prevalence of each population. A particularly interesting observation from intracellular recording studies is that, at least in some species, some neurons receive inputs from both sympathetic and parasympathetic spinal nerves. That is, the pelvic ganglia are not only mixed ganglia but also the neurons are ‘‘mixed neurons.’’ Such neurons have been reported in pelvic ganglia of cats, rabbits, and guinea pigs (Blackman et al., 1969; Crowcroft and Szurszewski, 1971; de Groat et al., 1979) but are rare or absent in rats (Tabatabai et al., 1986). Their role is obscure—it is not known to which organs they project or which transmitters they contain. Furthermore, it is not known whether both inputs to these neurons are ever activated concurrently in physiological circumstances. If they are, then this is an additional peripheral location at which synaptic integration may occur. However, if they are not, then the ‘‘dual input’’ suggests that the neuron is capable of subserving both sympathetic and parasympathetic roles. For example, it is known that there are both sympathetic and parasympathetic cholinergic pelvic neurons (see Section II,C), which may have the same effects on target tissues and which could be achieved by stimulation of either type of spinal input. Neuroanatomical studies have complemented the electrophysiological data and allowed quantification of different neuron types. Immunoreactivity for synaptophysin (a synaptic vesicle protein) has been used to stain all preganglionic nerve terminals in the major pelvic ganglion of male rats (Keast, 1995b). After lesion of the hypogastric or pelvic nerves and allowing sufficient time for terminal degeneration, the neurons which remain innervated can be quantified and classified (e.g., sympathetic neurons will remain innervated after pelvic nerve lesion but will be denervated by hypogastric nerve lesion). These studies have shown that there are approximately equal numbers of sympathetic and parasympathetic neurons in the rat major pelvic ganglion (Figs. 3a and 3b). Similar experiments focused on bowelprojecting neurons have suggested that there is a small population of dually innervated neurons (i.e., receiving both sympathetic and parasympathetic inputs; Luckensmeyer and Keast, 1995b), but it is not known if this is also true for neurons supplying other pelvic viscera. By combining this approach with immunohistochemical identification of cholinergic and noradrenergic neurons, and with retrograde tracing from peripheral target organs, a complete picture of the connectivity and chemis-

UNUSUAL AUTONOMIC GANGLIA

21

try has been obtained (Dail et al., 1975, 1983, 1985; Keast et al., 1984, 1995; Keast and de Groat, 1989; Keast, 1991, 1995b; Ding et al., 1993; Domoto and Tsumori, 1994; Vizzard et al., 1994a). Further details of the chemistry will be discussed later, but in summary most of the sympathetic neurons resemble sympathetic neurons elsewhere and synthesize noradrenaline and NPY, whereas about 25% synthesize ACh and VIP; many of this latter group also contain NOS. All the parasympathetic neurons synthesize ACh (with extremely rare noradrenergic neurons), and of these half contain VIP and half NPY. All this information has been obtained from male rats and has been summarized in Fig. 2a, but less extensive studies in female rats indicate similar patterns (Papka et al., 1987). These experiments have been greatly facilitated by the uniform simplicity of rat pelvic ganglion cells, almost all of which are adendritic or, at most, have a couple of rudimentary processes (Fig. 2b) (Dail et al., 1975; Tabatabai et al., 1986). Because the vast majority of their synapses must therefore be axosomatic, this permits the visual identification of innervated neurons. In many other species, pelvic neurons are structurally more complex and it would not be possible to determine which neurons were innervated (or denervated) solely by the location of stained preganglionic varicosities. Recently, these results have been supplemented by anterograde tracing studies from the spinal cord. Injection of a dextran–Lucifer yellow conjugate into either the upper lumbar or the sacral cord of male rats has allowed visualization of the terminal baskets of a randomly selected subpopulation of preganglionic neurons (Figs. 3c and 3d). The innervated neurons can then be categorized in terms of their target organ (by peripheral application of retrograde tracer) or chemistry (by immunostaining for transmitters or their synthetic enzymes). The results from pilot anterograde tracing experiments concur with the synaptophysin and lesioning experiments in terms of the prevalence, distribution, and chemical features of sympathetic and parasympathetic pelvic neurons in rat major pelvic ganglia. The mixed sympathetic–parasympathetic composition is a very distinctive feature of pelvic ganglia. Although there is chemical heterogeneity in many sympathetic and parasympathetic ganglia, there is no evidence for any other ganglion type having such duality of spinal inputs. Whether this mixed nature of the pelvic ganglia is sufficient reason to abandon the use of the terms sympathetic and parasympathetic in this part of the nervous system is quite a different issue. While this causes a problem in classifying the ganglia, it is easy to classify the individual pelvic neurons because many are exclusively innervated by one or the other spinal level. The only significant confusion arises with neurons that receive both types of inputs; that is, the mixed neurons. These are truly unusual and do not fit into the typical autonomic paradigm.

22

JANET R. KEAST

UNUSUAL AUTONOMIC GANGLIA

23

C. Neuron Morphology Morphological analyses of pelvic neurons have been most thoroughly carried out at the light microscope level following ionophoretic dye injection. While some information has also been obtained from retrograde tracing studies (i.e., with dye applied to peripheral targets or severed postganglionic nerve trunks), this is generally an inferior method for labeling dendritic processes. Caution must also be exercised in interpreting these results because neuronal structure and chemistry may change considerably after injury. Ionophoretically filled rat pelvic neurons have allowed tracing of axons, which typically leave the ganglion (Tabatabai et al., 1986; Rogers et al., 1990). No neurons have been identified which provide varicose terminals to other ganglion cells. Furthermore, all varicose terminals in the ganglion are removed by severing the major nerve bundles associated with it (Keast, 1995b; Luckensmeyer and Keast, 1995b, 1996). Together this indicates that interneurons are likely to be rare or absent in pelvic ganglia. However, this has not been investigated in the more complicated multiganglionated pelvic plexuses of other species. Dye tracing studies have further demonstrated that some neurons project out of the hypogastric or pelvic nerves (Baron et al., 1985b; Dail and Minorsky, 1986; Baron and Ja¨nig, 1991; Nadelhaft and Vera, 1991; Luckensmeyer and Keast, 1995a). This has also been shown electrophysiologically by eliciting antidromic action potentials in neurons after activation of either of these nerves (Tabatabai et al., 1986). While the neurons projecting out of the hypogastric nerve probably supply the bowel (after traversing the inferior mesenteric ganglion), the target of

FIG. 3 Features of preganglionic neurons projecting to pelvic ganglion cells in male rats. (a) Varicose terminals of sympathetic preganglionic neurons, demonstrated by their immunoreactivity for the synaptic vesicle protein, synaptophysin (SYN). Parasympathetic terminals are absent from this field because the pelvic nerves had been lesioned 3 days earlier. (b) Varicose terminals of parasympathetic preganglionic neurons, demonstrated by SYN immunoreactivity in fibers remaining after hypogastric nerve lesion. (c) Sympathetic preganglionic terminals demonstrated directly by anterogradely labeling from the upper lumbar spinal cord using Lucifer yellow–dextran conjugate (Lucifer yellow is then visualized immunohistochemically). Baskets of varicose terminals surround numerous neurons, and often the axon of origin is traceable. (d) Parasympathetic preganglionic terminals similarly localized by injecting the dextran conjugate into the sacral spinal cord. (e) Preganglionic neurons supplying the pelvic ganglion can be identified by injecting retrograde tracer, in this case fast blue, into the ganglion. Brightly labeled neurons in the lumbar cord are shown (e1), all of which are immunoreactive for choline acetyltransferase (e2). (f ) Many but not all preganglionic neurons express one or more neuropeptides. Here a small number of neurons are supplied by galanin-immunoreactive terminals. (g) A delicate basket of terminals immunoreactive for somatostatin supplies some pelvic neurons. Scale bar in a ⫽ 20 애m (a–e) and 30 애m (f and g).

24

JANET R. KEAST

neurons projecting out of the pelvic nerves is not known. It is possible that some axons travel in the pelvic nerves for a short distance but then return to the pelvic ganglion before projecting to the periphery (Dail and Minorsky, 1986). The shape of pelvic neurons varies broadly between species and in some cases also between pelvic neurons of different functions. In cats, dye-filling studies have shown that neurons in ganglia closely associated with the urinary bladder are very complex, possessing on average 6.9 dendrites (Tabatabai et al., 1986), whereas those lying on the adventitial surface of the rectum are much simpler, with many having no dendrites and a mean dendrite number per neuron of 3.6 (de Groat et al., 1985). It is not known whether neurons lying more distant from these target organs also bear distinctive features. Some dye-filling studies have been carried out in the less complex guinea pig pelvic ganglia. However, the literature is unclear on the exact location within the anterior and posterior plexuses of neurons of each morphological type. Nevertheless, dye-filling studies and fluorescence histochemistry have shown that there is a mixture of simple (adendritic) and complex neuron types (Watanabe, 1971; Costa and Furness, 1973a; Yokota and Burnstock, 1983; Morris and Gibbins, 1987). In contrast, in rats and mice virtually all pelvic neurons are very simple, with the majority bearing very few or no dendrites (Kanerva et al., 1972; Foroglou and Winckler, 1973; Tabatabai et al., 1986; Rogers et al., 1990; Papka and McNeill, 1993). Interestingly, many of the sympathetic noradrenergic pelvic neurons possess numerous very short extensions, which under typical magnification of the light microscope give them a ‘‘fluffy’’ appearance (Dail et al., 1975). It is not known if these bear functional similarity to dendrites, although when seen in other parasympathetic ganglia they do possess synapses (see Section II,B). Thus, these neurons resemble typical parasympathetic neurons but are very different from sympathetic neurons found elsewhere in the same species. As seen elsewhere in the nervous system, the complexity of the dendritic arbor of pelvic neurons generally reflects the number of synaptic inputs (Purves and Lichtman, 1985). For example cat bladder neurons receive on average seven cholinergic excitatory synaptic inputs from preganglionic axons, whereas rat pelvic neurons receive just one or two (Tabatabai et al., 1986). Moreover, in cat bladder neurons many inputs are subthreshold, requiring temporal or spatial summation to trigger an action potential (Booth and de Groat, 1979; de Groat and Booth, 1980), whereas the majority of rat pelvic neurons receive a single suprathreshold (‘‘strong’’) input (Tabatabai et al., 1986). Thus, cat bladder ganglia appear more like typical sympathetic ganglia in their structure and synaptology, whereas rat pelvic ganglia resemble typical parasympathetic ‘‘relay stations.’’ In contrast, colonic (adventitial) ganglia in the cat have fewer dendrites and correspond-

UNUSUAL AUTONOMIC GANGLIA

25

ingly fewer synaptic inputs, resembling more closely rat pelvic neurons or typical parasympathetic neurons (de Groat and Krier, 1976; Booth and de Groat, 1979). The level of spinal input to these neurons has not been investigated so they may indeed be parasympathetic. This considerable variation in ganglion morphology and synaptology between species has led some to question whether the mechanism of activation, integration, and output of pelvic reflexes is generally much simpler in rats than in other species. However, while the final motor pathways are quite variable, there is no reason to speculate that sensory and central processing differs significantly between the species. Furthermore, it is possible that there is an additional level of complexity of spinal processing in rats, such that further integration in the ganglion is not required. Apart from ganglion cell complexity, it is also possible to distinguish some neurons on the basis of their topography (Dail et al., 1983, 1985, 1989a, 1990; Keast, 1992; Kolbeck and Steers, 1993; Kepper and Keast, 1995, 1997), chemistry (see Section III,D), electrical properties (see Section III,E), and size. For example, in rat pelvic ganglia, the sympathetic noradrenergic neurons are substantially larger than the cholinergic neurons and some are binucleate (Figs. 2f and 2g) (Dail et al., 1975; Keast and de Groat, 1989). Nonneuronal constituents of pelvic ganglia are similar to other autonomic ganglia. Each neuron is closely associated with a basal lamina and concentric layers of glia (satellite cells); the ganglia are surrounded by a connective tissue capsule (Dail et al., 1975; Kawatani et al., 1989; Wang et al., 1990; Gabella, 1995). Small, intensely fluorescent (SIF) cells, commonly found in many sympathetic ganglia, are also found in clumps, apparently randomly distributed in pelvic ganglia (Fig. 2c) (Becker, 1972; Kanerva and Tera¨va¨inen, 1972; Dail, 1976; Kanerva and Hervonen, 1976; Baker et al., 1977). They are more common in female animals (Kanerva and Hervonen, 1976) but generally are less prevalent in pelvic ganglia than in other sympathetic ganglia (Furness and Costa, 1976). Along with catecholamines, some SIF cells in pelvic ganglia contain neuropeptides, serotonin, or GABA (Dail et al., 1983; Dail and Dziurzynski, 1985; Papka et al., 1987; Karhula et al., 1988). They also stain strongly for the synaptic protein, synaptophysin (Hou and Dahlstrom, 1995; Keast, 1995b), but rarely receive synaptic terminals (Becker, 1972; Dail, 1976). The function of SIF cells is obscure and possibilities include an endocrine or interneuron role (Era¨nko¨, 1978). D. Neurotransmitters 1. Ganglion Cells For many years pharmacological studies have demonstrated the existence of both cholinergic and noradrenergic pelvic neurons by showing the pres-

26

JANET R. KEAST

ence of cholinergic and noradrenergic nerve-evoked responses in the peripheral organs. Histofluorescence reactions for catecholamines have allowed visualization of numerous pelvic neurons in a variety of species, and it is now known that this is mainly due to the presence of noradrenaline (Owman and Sjo¨strand, 1965; Sjo¨berg, 1965; Owman et al., 1971). However, in guinea pigs, many pelvic neurons do not possess the full complement of catecholamine synthesizing enzymes, containing dopamine-웁hydroxylase but not tyrosine hydroxylase or noradrenaline (Morris and Gibbins, 1987). Interestingly, in rats and guinea pigs, noradrenergic pelvic neurons are common in males (comprising about one-third of the whole neuron population in rats) but quite uncommon in females (Costa and Furness, 1973a; Dail et al., 1975; Dail, 1976; Yokota and Burnstock, 1983; Morris and Gibbins, 1987; Papka et al., 1987; Keast and de Groat, 1989; Vera and Nadelhaft, 1992; Houdeau et al., 1995); this is the reverse situation to the distribution of the other ganglionic source of catecholamines, the SIF cells, which are much more common in female rat pelvic ganglia (Papka et al., 1987). While the difference in neurons simply reflects the greater noradrenergic innervation density of male internal reproductive organs, the difference in SIF cells cannot be interpreted until the function of these cells is understood. Cholinergic neurons were not visualized directly for many years due to the unavailability of sufficiently sensitive antisera against the synthetic enzyme, choline acetyltransferase (ChAT). In the meantime, many authors reported instead on the distribution of acetylcholinesterase (Bell and McLean, 1967; Dail et al., 1975; Dunzendorfer et al., 1976; Papka et al., 1985; Kujat et al., 1993; Melo and Machado, 1993; Warburton and Santer, 1993; Persson et al., 1995). Although it is now known that this enzyme is not exclusively located in cholinergic neurons, the patterns of staining were consistent with recent observations using ChAT immunoreactivity (Keast et al., 1995). Recently, various antibodies have also been successfully developed against the vesicular choline transporter (Weihe et al., 1996; Arvidsson et al., 1997), but this method has not been applied to investigations of pelvic autonomic innervation. Although there is no doubt that many pelvic neurons are cholinergic, visualization of cholinergic neurons is essential for addressing two important questions. The first of these was triggered by the observation that many nerve-evoked responses in pelvic viscera are resistant to adreno- and cholinoceptor blockade, implying the existence of additional transmitter types (Dail, 1993; Hoyle and Burnstock, 1993). The question then arose whether cholinergic and noradrenergic pelvic neurons contain other transmitters or whether there is a separate population of nonadrenergic, noncholinergic neurons. A second confounding observation was that when the lumbar sympathetic outflow is activated by stimulating the hypogastric nerve, in

UNUSUAL AUTONOMIC GANGLIA

27

some reproductive organs both noradrenergic and cholinergic responses occur (Dail, 1993). This could mean that there are either separate populations of cholinergic and noradrenergic sympathetic neurons or that both transmitter types are present within one neuron. Although this would be highly unusual in the autonomic system, the pelvic ganglia were already acknowledged as being unusual in many other ways, so the possibility continued to be entertained. These issues have only been resolved using ChAT immunolocalization in the male rat major pelvic ganglion (Keast et al., 1995). Here, virtually all neurons are either noradrenergic or cholinergic, with only 1 or 2% of neurons unstained for either tyrosine hydroxylase or ChAT (Fig. 2e). Noradrenergic neurons immunoreactive for ChAT were also rare (⬍1% pelvic neurons). Therefore, at least in this species, additional transmitters must be released from cholinergic or noradrenergic neurons, i.e., there is cotransmission in the pelvic viscera. It is disappointing that this has not been established in other species or in female rats. In addition to ACh, the majority of pelvic neurons appear to contain one or more neuropeptides (Dail et al., 1983; Gu et al., 1983; Hele´n et al., 1984; Dail and Dziurzynski, 1985; Mattiasson et al., 1985; Papka et al., 1985, 1987; Crowe et al., 1986; Inyama et al., 1987; Morris and Gibbins, 1987; Keast and de Groat, 1989; Papka and Traurig, 1989; Dhami and Mitchell, 1991; Keast, 1991, 1994b; Edyvane et al., 1992; Hedlund et al., 1994; Persson et al., 1995; Jen et al., 1996). The nature of the peptide(s) varies between species and, in species which possess multiple pelvic ganglia, perhaps also between ganglia. Most commonly VIP is present in cholinergic pelvic neurons (Fig. 2g) and, as in other parts of the autonomic system, is thought to potentiate secretory responses and relaxation of smooth muscle (Dockray, 1992). A small number of cholinergic VIP pelvic neurons also contain NPY, the function of which has not been thoroughly examined in pelvic viscera. In addition, many pelvic cholinergic VIP neurons contain NOS (Fig. 2d) (McNeill et al., 1992; Vizzard et al., 1994b; Papka et al., 1995a,b; Persson et al., 1995), and NO may contribute to noncholinergic relaxations in some pelvic viscera (Persson and Andersson, 1992; Andersson, 1993; Leone et al., 1994). Although it has not been possible to demonstrate histochemically, pharmacological studies have shown that many cholinergic pelvic neurons release ATP as a transmitter (Hoyle, 1992). Purinergic transmission from cholinergic axons has been best demonstrated in the detrusor muscle of the urinary bladder (Fujii, 1988; Brading and Mostwin, 1989). Noradrenergic pelvic neurons exhibit similar neurochemical features as other sympathetic neurons (Sjo¨berg, 1965; Owman et al., 1971) and usually contain peptides common to other noradrenergic neurons in the species. For example, in many animals they contain NPY (Papka et al., 1985, 1987; Morris and Gibbins, 1987; Keast and de Groat, 1989; Papka and Traurig,

28

JANET R. KEAST

1989; Keast, 1991), whereas in guinea pigs some also contain somatostatin (Morris and Gibbins, 1987), a peptide found in many prevertebral guinea pig sympathetic neurons (Macrae et al., 1986). It is not known if somatostatin is released at synapses in the pelvic viscera, but there is evidence for NPY being a transmitter in some organs (Morris and Murphy, 1988; Morris, 1990, 1993; Stjernquist and Owman, 1990; Zoubek et al., 1993). Many pelvic noradrenergic neurons are also known to corelease ATP at their peripheral synapses. In particular, the excitatory noradrenergic synapse in vas deferens smooth muscle of rodents was one of the first in which purinergic transmission (and cotransmission) was demonstrated (Hoyle, 1992; Morris and Gibbins, 1992). This has been determined using many different pharmacological strategies and recently was supported by molecular analyses of purine receptors in the smooth muscle (Vulchanova et al., 1996). A particularly unusual feature of pelvic noradrenergic neurons is their relative insensitivity to transmitter-depleting agents such as reserpine or to the destructive catecholamine analog, 6-OHDA (Sjo¨strand, 1965; Owman and Sjo¨berg, 1967; Malmfors and Sachs, 1968; Kanerva and Hervonen, 1976). This has led to the use of 6-OHDA as a tool by which the origin of peripheral noradrenergic neurons can be established—any noradrenergic axons remaining within organs after exposure to 6-OHDA are likely to arise from pelvic ganglia. The biochemical features underlying this difference are not known, but it may reflect a difference in catecholamine uptake or storage mechanisms or a higher rate of noradrenaline synthesis within pelvic neurons. In many other parts of the nervous system, neurons of different functions can be distinguished by the combination of substances present within them—substances which may not necessarily be transmitters. This has been studied most thoroughly in the peripheral nervous system, in which various peptides appear to form a ‘‘chemical code’’ specific to neurons of different targets (Costa et al., 1986; Furness et al., 1989; Kummer, 1992; Gibbins, 1995). This has not been established as well in the pelvic ganglia, although in parasympathetic neurons of male rats NPY predominates in pathways supplying the bladder and bowel, whereas VIP is found mainly in reproductive neurons (Keast et al., 1984; Keast and de Groat, 1989; Keast, 1992; Dail, 1993; Kepper and Keast, 1995, 1997). There is the potential for a much more sophisticated chemical code in guinea pig pelvic ganglia, in which up to 11 combinations of substances have been identified (Morris and Gibbins, 1987). 2. Preganglionic Neurons It is widely accepted that all preganglionic neurons supplying pelvic ganglia are cholinergic. This has been demonstrated by immunohistochemical and

UNUSUAL AUTONOMIC GANGLIA

29

electrophysiological recording studies (see Section III,E). As in all autonomic ganglia, activation of preganglionic axons elicits fast excitatory synaptic potentials mediated by nicotinic acetylcholine receptors (Blackman et al., 1969; Crowcroft and Szurszewski, 1971; Holman et al., 1971; de Groat and Saum, 1972; Griffith et al., 1980; Gallagher et al., 1982; Rogers et al., 1990; Hanani and Maudlej, 1995). While slower inhibitory and excitatory synaptic potentials have also been recorded in cat vesical ganglia (de Groat and Booth, 1980; Gallagher et al., 1982; Akasu et al., 1984, 1986), their importance for normal reflex behavior is not known. Suggested mediators of these slow events include acetylcholine (via muscarinic receptors), adenosine, and various neuropeptides. In addition, responses can be evoked in pelvic neurons after administration of a variety of substances such as serotonin, ATP, and various neuropeptides, indicating the presence of numerous receptor types on pelvic ganglion cells or their preganglionic terminals (de Groat and Booth, 1993b). For many of these, however, a local endogenous neuronal source is unlikely, so their physiological relevance is questionable. Immunohistochemical studies have also demonstrated the presence of various substances in lumbar and sacral preganglionic neurons, retrogradely labeled following injection of tracer dyes into the rat pelvic ganglion. These include ChAT (present in all preganglionic neurons; Fig. 3e), NOS (present in the majority of lumbar and sacral preganglionic neurons), and various neuropeptides (Kawatani et al., 1983; Dail and Dziurzynski, 1985; Morris and Gibbins, 1987; Senba and Tohyama, 1988; Keast and de Groat, 1989; Rogers and Henderson, 1990; Wang et al., 1990; Keast, 1991, 1994b; Papka and McNeill, 1993; Vizzard et al., 1994b; Papka et al., 1995a). Similarly, numerous varicose axon terminals surrounding pelvic neurons can be demonstrated using antisera against various peptides (Figs. 3f and 3g), most commonly the enkephalins (Ho¨kfelt et al., 1978; Dail et al., 1985; Keast and de Groat, 1989; Rogers and Henderson, 1990). These terminals form very closely apposed baskets around particular pelvic neurons, although the types of neurons supplied have not been determined. Selective lesion of either lumbar or sacral inputs to the pelvic ganglion has provided some insight into which peptides are preferentially located in sympathetic and parasympathetic preganglionic terminals, although in many cases they are found in both types (Dail and Dziurzynski, 1985; Dail et al., 1986; Senba and Tohyama, 1988; Pacheco et al., 1989; Papka, 1990; Wang et al., 1990; Papka et al., 1991; Keast, 1994b). No peptide appears to exclusively label sympathetic or parasympathetic inputs or the entire population of preganglionic terminals. The issue is further complicated by the presence of some of these substances in processes of primary afferent neurons which pass through the ganglion (see Section III,F,1) because pelvic or hypogastric nerve lesions will disrupt both these and preganglionic fibers.

30

JANET R. KEAST

Finally, there are also noradrenergic terminals in pelvic ganglia of some species, although these originate from sympathetic ganglia rather than from the spinal cord (Hamberger and Norberg, 1965a; Elbadawi and Schenk, 1973; Klu¨ck, 1980). Nevertheless, their activation could influence normal ganglionic transmission (Saum and de Groat, 1972a,b). They are much less common in rats and guinea pigs than in larger species (Watanabe, 1971; Costa and Furness, 1973b; Morris and Gibbins, 1987). In summary, there are numerous substances present in some preganglionic axons that supply the pelvic ganglion, but their function is largely unknown. In particular, the role of the numerous neuropeptides present in particular groups of preganglionic neurons is obscure. While receptors for some of these substances are expressed by pelvic ganglion cells, their synaptic activation has not been demonstrated. Moreover, the location of these substances has so far provided little clear indication of specific functional groups of neurons that they may regulate.

E. Membrane Properties The electrophysiological properties of sympathetic and parasympathetic ganglion cells have been extensively reviewed (Adams and Harper, 1995; Akasu and Nishimura, 1995). The active and passive membrane properties of pelvic neurons have also been investigated (de Groat and Booth, 1993b). Here, I discuss only those features which distinguish pelvic neurons from other ganglion cells or which identify neurons of particular functions or spinal connections. Given the substantial differences between the properties of sympathetic and parasympathetic neurons elsewhere in the body and the mixed nature of pelvic ganglia, it could be predicted that pelvic neurons are heterogeneous in their membrane properties, underlying differences in mechanisms of synaptic activation and firing behavior. One would expect, for example, that parasympathetic pelvic neurons would be distinguishable by their simple features, having little synaptic integration, a small number of strong synaptic inputs, and uniform firing behavior in response to prolonged depolarization. Conversely, one could predict that the sympathetic neurons resemble other prevertebral sympathetic neurons in possessing numerous synaptic inputs, subthreshold synaptic events, and perhaps a more varied response to depolarization. However, this review has already demonstrated that cat parasympathetic pelvic neurons supplying the bladder are morphologically quite complex, having extensive dendritic arbors (e.g., in cat vesical ganglia; Booth and de Groat, 1979; de Groat and Booth, 1980; Tabatabai et al., 1986); consistent with this, such neurons also receive numerous synaptic inputs and possess a physiological complexity resembling typical sympa-

UNUSUAL AUTONOMIC GANGLIA

31

thetic neurons (e.g., synaptic facilitation; Booth and de Groat, 1979; de Groat and Booth, 1980). Interestingly, in the same species, pelvic ganglion cells supplying a different target, the lower bowel, have much simpler structure and synaptology (such as fewer dendrites, fewer inputs, and no facilitation; de Groat and Krier, 1976; Booth and de Groat, 1979; see Section III,C). In contrast, in other species (e.g., rats and mice) many or all the sympathetic and parasympathetic neurons appear to lack dendrites and receive just one or two strong inputs (Blackman et al., 1969; Crowcroft and Szurszewski, 1971; Holman et al., 1971; Tabatabai et al., 1986; Rogers et al., 1990; Hanani and Maudlej, 1995). Both populations in this species therefore resemble typical parasympathetic neurons. Together this demonstrates that few generalizations can be made about the electrophysiological properties of pelvic neurons as a group; rather, the species, target, and spinal connection of pelvic neurons must be taken into account when interpreting each study. In all pelvic neurons, irrespective of target organ or species, action potentials can be elicited by administration of acetylcholine and synaptically evoked nicotinic fast excitatory synaptic potentials can be readily demonstrated both in vivo (de Groat and Saum, 1972, 1976; Purinton et al., 1976; Steers et al., 1988; Mallory et al., 1989) and in vitro (Blackman et al., 1969; Crowcroft et al., 1971; Holman et al., 1971; Tabatabai et al., 1986; Griffith et al., 1980; Gallagher et al., 1982; Rogers et al., 1990; Warren and Lavidis, 1996; Hanani and Maudlej, 1995). In addition, and particularly in cats (which have more structurally complex pelvic neurons), it is possible to demonstrate slow synaptic events, which may be due to activation of muscarinic receptors or other transmitter systems (de Groat and Booth, 1993b). It is not known if these are also elicited as part of normal reflex behavior in vivo. Some studies identified spontaneous depolarizations and hyperpolarizations in a small population of pelvic neurons in cats and rabbits (Griffith et al., 1980; Nishimura et al., 1988). While numerous types of channels and receptors have been described in pelvic ganglion cells, few studies have investigated their synaptic activation. Of these, many have failed to identify which effects are due to activation of lumbar or sacral preganglionic inputs. In addition, there are numerous peptides present in preganglionic neurons supplying pelvic ganglia which have not been tested in terms of their activity or neuronal release (see Section III,D,2). A possible presynaptic action has not been excluded in all cases and is probably the primary mechanism of action of opioid peptides (Kennedy and Krier, 1987a; Rogers and Henderson, 1990; Warren and Lavidis, 1996). It is unfortunate that the majority of electrophysiological studies conducted at the cellular level on intact ganglia have been performed on pelvic ganglion cells of larger species, in which the pelvic plexus is more complex

32

JANET R. KEAST

and the neurochemistry of individual neuron types not as thoroughly defined. In these cases it is also rarely possible to identify the spinal source of synaptic inputs, particularly because most of these studies are on ganglia located close to the target organs (commonly the vesical ganglia), where anatomical separation and distinction of the two groups of spinal inputs does not occur. Recently, studies have commenced on acutely dissociated rat pelvic neurons (Yoshimura and de Groat, 1996). These have incorporated prior injection of fluorescent retrograde tracer into a peripheral organ, the urinary bladder, to allow characterization of the channel properties of a functionally identified subclass of neurons. These studies have show that rat bladder neurons possess a TTX-sensitive sodium current and three main types of voltage-dependent potassium currents (IA current, calcium-activated current, and a delayed rectifier), all of which are common to other sympathetic neurons in this species (Belluzzi et al., 1985; Schofield and Ikeda, 1989). It is not known whether the characterized pelvic neurons include both noradrenergic and cholinergic groups, although it would be expected that both are present. Their similarity to other sympathetic neurons is surprising given the numerous structural and chemical differences of sympathetic pelvic neurons from other sympathetic neurons in the same species, and that many neurons were probably parasympathetic. Interestingly, studies by another group have identified a unique feature of rat sympathetic noradrenergic neurons, again by patch clamping isolated rat pelvic neurons (Zhu et al., 1995; Zhu and Yakel, 1997). These authors showed that noradrenergic sympathetic neurons uniquely express T-type calcium channels (Zhu et al., 1995), which are not expressed by either parasympathetic pelvic neurons or other sympathetic neurons in this species. Furthermore, Zhu et al. also showed that many noradrenergic neurons could be distinguished by their greater membrane capacitance, as would be predicted by their larger size (Keast and de Groat, 1989). It remains to be determined whether chemical subclasses of parasympathetic neurons in the rat pelvic ganglia can be distinguished or whether these behave differently than the cholinergic sympathetic pelvic neurons. While patch clamp studies can provide valuable insights into membrane and channel properties, there is a great need to determine how various channels and receptors are activated in vivo, with normal patterns of reflex activity. To date, such studies have been limited to extracellular recordings from pre- and postganglionic nerve bundles. Ideally, intracellular recording from single neurons in vivo should be carried out, as has recently occurred in the superior cervical ganglion (McLachlan et al., 1997). This requires accessibility of the neurons for recording, for which the simple major pelvic ganglion or the adventitial vesical ganglia of cats and rabbits would be suitable.

UNUSUAL AUTONOMIC GANGLIA

33

Finally, there is often a temptation to use young animals for electrophysiological recording studies, mainly because they have less and softer connective tissues (making for easier impalement with sharp microelectrodes or dissociation with less harsh reagents for patch clamp studies). However, as discussed in Section III,G, numerous changes occur during puberty to both the pre- and postganglionic neurons, including changes in soma and axon terminal morphology, transmitter levels, and reflex activity. It is also possible that membrane and synaptic properties change during this time and may indeed underlie the changes in reflex activity.

F. Connectivity with Other Parts of the Nervous System 1. Sensory Ganglia Peripheral processes of lumbar and sacral primary afferent neurons travel in the hypogastric and pelvic nerves, respectively, and pass through the pelvic ganglia on their way to the pelvic viscera. Many are known to contain one or more neuropeptides and can therefore be visualized immunohistochemically (Kawatani et al., 1985, 1986; de Groat, 1987; Gibbins et al., 1987; Ju et al., 1987; Keast and de Groat, 1992). However, it is not possible to visualize the entire population of sensory fibers because only a proportion of sensory neurons supplying pelvic viscera contain peptides (Gibbins et al., 1987; Ju et al., 1987; Keast and de Groat, 1992). Nevertheless, some comparisons of peptide distribution can be made between pelvic ganglia and other autonomic ganglia in the context of peptides which may be markers for sensory fibers. In particular, a comparison with sympathetic prevertebral ganglia is of interest because they are supplied by many collaterals of sensory neurons; these are thought to be activated during some digestive reflexes, giving rise to depolarization of sympathetic neurons, to enhance their duration and frequency of firing (Szurszewski, 1981; Kreulen and Peters, 1986). The only available clues come from immunohistochemical localization of peptides, such as substance P and calcitonin gene-related peptide, after selective lesion of either the hypogastric or the pelvic nerves (Dail and Dziurzynski, 1985; Senba and Tohyama, 1988; Papka, 1990). These studies have shown that most peptide-containing fibers have both lumbar and sacral sources, although the sacral (pelvic) nerves usually provide most of the fibers. However, there are two problems in interpreting these studies. First, nerve lesions may lead to substantial and rapid changes in neuronal connectivity within the ganglion (see Section III,H). Second, many lumbar and sacral preganglionic neurons contain peptides common to pelvic visceral afferent neurons and it is currently not possible to visually distinguish

34

JANET R. KEAST

sensory from preganglionic terminals. While it may be possible to speculate that preganglionic terminals will have a particularly dense basket-like organization around neurons (in comparison to a looser, less cell-specific distribution of sensory fibers), this has not been clearly demonstrated. Despite these deficiencies, in some species it is likely that sensory innervation of pelvic ganglion cells is relatively sparse or absent. For example, in rat pelvic ganglia the distribution of SP terminals is sparse in comparison to the very dense innervation of prevertebral ganglia in the same species (Ho¨kfelt et al., 1977; Dail and Dziurzynski, 1985; Keast and Chiam, 1994). Furthermore, there is no evidence for slow synaptic potentials (as one might predict from collateral sensory activation) after activation of either hypogastric or pelvic nerves in this species (Tabatabai et al., 1986). Because all the synaptic excitation is blocked by nicotinic receptor antagonists, this also excludes a role for glutamate release from these sensory fibers. However, it is necessary to investigate other species in which the connectivity, chemistry, and electrophysiological properties of ganglion cells are more complex before ignoring a physiological role for sensory collaterals in the pelvic ganglia. 2. Enteric Ganglia The pelvic ganglia provide axons to much of the lower bowel and many studies have demonstrated that hypogastric and pelvic nerve stimulation cause potent but quite different effects on gut activity (Gonella et al., 1987; Ja¨nig and McLachlan, 1987). Typically, parasympathetic (sacral) nerves stimulate motility, secretion, and, in some regions, blood flow, whereas sympathetic (lumbar) nerves inhibit these activities. Sympathetic nerves also excite smooth muscle of the internal anal sphincter. Pelvic ganglia provide axons to the gut wall via the rectal nerves. In rats there is another substantial projection via the cavernous (penile) nerve, the name of which is misleading (Tanaka et al., 1983; Dail et al., 1990; Luckensmeyer and Keast, 1998c). The rectal and cavernous nerves carry axons of both sympathetic and parasympathetic pelvic neurons to the lower bowel; they also carry a small number of axons from neurons in the inferior mesenteric ganglion which first travel through the hypogastric nerve and pelvic ganglion. Despite the well-known actions of pelvic ganglion cells on the gut, their mechanism has not been determined. In particular, there has been little investigation of whether pelvic ganglion cells projecting to the lower bowel synapse on smooth muscle and glands or whether they primarily exert their effects through synapses on enteric neurons. The latter mechanism is known to occur in more proximal gut regions (see Section II,A). Studies of noradrenergic axon distribution in the bowel following selective nerve lesions

UNUSUAL AUTONOMIC GANGLIA

35

have shown that those originating from pelvic neurons supply the myenteric plexus and blood vessels of the rectum as well as the internal anal sphincter (Costa and Gabella, 1971; Costa and Furness, 1973a,b). In contrast, a combination of retrograde tracing and lesion studies in rats have shown that pelvic neurons supply the myenteric plexus of the distal colon and rectum but not the vasculature or other gut layers (Luckensmeyer and Keast, 1994, 1998c). While the latter group of studies did not distinguish between noradrenergic and cholinergic axons, it is clear that the pelvic sympathetic supply cannot provide a substantial supply to the submucous plexus or vasculature. This differs markedly from the prevertebral sympathetic supply of this and other gut regions which innervates both the vasculature and the submucous plexus. It is not known which enteric neurons receive noradrenergic synapses, although the broad distribution of noradrenergic terminals suggests that the majority are probably influenced by noradrenaline release. A significant contrast can be made regarding the innervation between the small and large intestines, first in the degree of smooth muscle innervation by noradrenergic axons. While sparse in the small intestine (despite the prevalence of adrenoceptors in the muscle; Furness and Costa, 1987), noradrenergic terminals are more common in smooth muscle of the lower bowel (Norberg, 1964; Furness, 1970), particularly the rectum (Furness and Costa, 1973). Concomitantly, the noradrenergic innervation of the myenteric plexus in the lower bowel is less extensive (Schultzberg et al., 1980). Interestingly, nerve-evoked activation of the smooth muscle receptors can be readily demonstrated in the large bowel (Luckensmeyer and Keast, 1998a). In the small bowel most of the nerve-evoked adrenoceptor responses decrease motility by inhibiting myenteric motor neurons, although inhibition can also be evoked by exogenous agonists that activate 웁 receptors in the muscle. In contrast, in smooth muscle of the large bowel there are a mixture of 움-excitatory and 웁-inhibitory responses to nerve activation, with the former evoked at lower stimulation frequencies. Moreover, the pelvic supply to the rectum is purely excitatory, whereas prevertebral innervation of other regions causes a broader range of effects but most commonly inhibition. This excitation challenges the previously held view that sympathetic nerves inhibit motility of nonsphincteric gut regions and raises a number of issues critical to understanding lower bowel physiology. In particular, it indicates that some classes of sympathetic neurons, notably those in the pelvic ganglia, can promote motility, particularly in the rectum. Whereas the parasympathetic nervous system has been given ‘‘sole rights’’ to promoting defecation, it is perhaps time to assign some of this function to pelvic sympathetic neurons; alternatively, the sympathetic nerves may have an important mixing function in the rectum, promoting motility to allow greater absorption of any remaining fluid.

36

JANET R. KEAST

The distribution of terminals arising from cholinergic pelvic neurons has been more difficult to determine because there is no histochemical marker to distinguish them from enteric cholinergic neurons. Most of these cholinergic pelvic neuron terminals originate from parasympathetic neurons, although a few are sympathetic (Luckensmeyer and Keast, 1995b). Lesion studies have also failed to identify which fibers in the gut wall arise from pelvic ganglia because they are greatly outnumbered by fibers from intrinsic sources. Recent tracing studies have used the lipophilic tracer, DiI, in gut explants and connected ganglia maintained in organotypic culture (Luckensmeyer and Keast, 1998c). They have shown that the primary target of pelvic ganglion cells in the gut is the myenteric plexus and, to a lesser degree, the circular muscle. Together with studies on noradrenergic terminal distribution and electrophysiological studies (Fukai and Fukuda, 1985), this suggests that the cholinergic pelvic neurons mainly supply the myenteric plexus (Luckensmeyer and Keast, 1998c) In many species, ganglia are located on the adventitial surface of the lower bowel and have typically been considered part of the pelvic plexus, similar to those lying on the bladder wall. In cats these seem to be simply relay stations, providing parasympathetic input to much of the colon and to the rectum (de Groat and Krier, 1976, 1979; Krier and Hartman, 1984; Kennedy and Krier, 1987b). However, in rats, although there are no other pelvic neurons close to the viscera (e.g., no ganglia on the surface of the urinary bladder), ganglia are present on the adventitial surface of the rectum (Neuhuber et al., 1993; Luckensmeyer and Keast, 1998b). These are congregated over the last few millimeters and innervate the internal anal sphincter and adjacent circular muscle. However, in contrast to cat rectal ganglia, which have most if not all of their projection toward the gut, most rat rectal ganglion cells project away from the gut wall (Neuhuber et al., 1993; Luckensmeyer and Keast, 1998b). They are therefore considered to have an afferent role, perhaps as primary sensory neurons directly transducing changes in the gut environment or perhaps as interneurons projecting to the spinal cord (Neuhuber et al., 1993). They could even be considered as displaced myenteric viscerofugal neurons. Although the function and cellular connections of these neurons are not known, they are clearly very different from other pelvic neurons in this species. Finally, given that the lower bowel has a source of sympathetic axons in addition to the pre- and paravertebral ganglia, it is worth considering if these sympathetic pelvic neurons are also involved in viscerofugal reflexes (see Section II,D,2). This requires knowledge of the areas of bowel supplied by sympathetic neurons of each type and whether any myenteric viscerofugal neurons indeed project to pelvic sympathetic neurons. Tracing studies have shown that whereas the most caudal of the prevertebral ganglia, the inferior mesenteric ganglion, supplies primarily the middle and distal colon,

UNUSUAL AUTONOMIC GANGLIA

37

sympathetic neurons of the pelvic ganglia also supply much of the same area as well as the rectum (Luckensmeyer and Keast, 1994). Surprisingly, the possibility of viscerofugal neuron projections to pelvic sympathetic neurons has not been directly investigated until recently. Using a combination of retrograde tracing, nerve lesion, and immunohistochemical methods, it was shown in rats that very few pelvic sympathetic neurons are innervated by myenteric viscerofugal neurons; however, many of these projections pass through the pelvic ganglion to finally synapse in the inferior mesenteric ganglion (Luckensmeyer and Keast, 1995a, 1996). Therefore, despite innervating similar bowel regions, the prevertebral and pelvic ganglion neurons differ considerably in their degree of involvement with viscerofugal reflexes.

G. Effects of Gonadal Steroids during Development and Adulthood 1. Sexual Dimorphism of Pelvic Autonomic Pathways The pelvic ganglia are highly unusual among autonomic ganglia in regard to their sexual dimorphism and their sensitivity to gonadal steroids during adulthood. While these features have not been as thoroughly examined in most other autonomic ganglia, the involvement of many pelvic neurons in reproductive reflexes makes them particularly important targets for androgens and estrogens. The most obvious difference between male and female pelvic ganglia is their size; the pelvic ganglia in male rats have many more neurons than do females (Greenwood et al., 1985). Similarly, there is a greater number of preganglionic neurons in the lumbar spinal cord of male rats (Baron et al., 1985b; McLachlan, 1985; Nadelhaft and McKenna, 1987) and various gender differences in peptide distribution in ‘‘autonomic’’ areas of the spinal cord (Newton and Hamill, 1988; Newton et al., 1990; Newton, 1992). Underlying these differences is the increased density of innervation of the male internal reproductive organs, in which it plays an important role in their normal regulation (Dail, 1993); in contrast, most of the female internal reproductive organs possess relatively sparse innervation (with the exception of their vasculature) and their activity is largely dependent on fluctuations in circulating steroids (Traurig and Papka, 1993). Although dimorphism in neuroanatomical features of pelvic autonomic pathways has not been documented in species other than rats, the gender difference in reproductive target organ innervation is apparent across species and neuron number is therefore predicted to follow. The sexual dimorphism in pelvic ganglion structure is thought to be initiated prenatally, although the mechanism by which it is established is

38

JANET R. KEAST

not known (Suzuki et al., 1983; Greenwood et al., 1985). It is possible that testosterone ‘‘rescues’’ a portion of pelvic neurons in males, whereas in females these will undergo apoptosis. A consideration of the chemical differences between male and female pelvic ganglion cells may suggest which population of neurons is lost in females. Because the male reproductive organs are supplied by a dense noradrenergic innervation (largely supplying smooth muscle) and a somewhat less prominent cholinergic innervation (supplying secretory epithelia), it could be predicted that male pelvic ganglia contain significantly more noradrenergic neurons than female pelvic ganglia, i.e., mostly noradrenergic neurons will fail to be rescued by androgens in development of female animals and subsequently undergo apoptosis. These predictions are borne out in that noradrenergic pelvic neurons are quite sparse in female rats, whereas in males noradrenergic neurons comprise almost one-third of all pelvic neurons (see Section III,D,1). The mechanisms by which androgens promote survival of these neurons in males are not known but may involve a direct action on the genome of noradrenergic neurons or a less direct action (e.g., by regulating production of target-derived neurotrophic factors) (see Section III,G,3). 2. Effects of Circulating Gonadal Steroids on Pelvic Autonomic Neurons In both males and females there is evidence that one or more components of the pelvic autonomic reflex circuitry are sensitive to the actions of circulating gonadal steroids (Fig. 4). These effects are all thought to be due to actions on the genome of the neurons or their peripheral targets; however, it is possible that some are due to the more rapid nongenomic actions of steroid hormones, which are currently of immense interest in the central nervous system (Nabekura et al., 1986; Murphy and Segal, 1996; Wehling, 1997). Genomic steroid actions are mediated by intracellular steroid receptors, which are transcription factors activating protein synthesis in target cells. The range of effects of steroid exposure on the nervous system is broad, encompassing neuron growth, receptor expression, and transmitter synthesis (Breedlove, 1992; Kawata, 1995). Such effects have been typically demonstrated (i) by comparison of neuronal properties in different reproductive states and (ii) by investigating changes in these properties after steroid deprivation in adulthood, for example, after postpubertal castration. Any differences would indicate that prepubertal increases in circulating steroids initiate maturation of pelvic innervation to its optimal adult form and that steroids are required to maintain certain features of neurons during adulthood. Some of the strongest evidence for steroid effects on pelvic innervation comes from studies of the uterus, in which the noradrenergic nerves change

UNUSUAL AUTONOMIC GANGLIA

39

FIG. 4 Sites of testosterone action on autonomic pathways supplying pelvic viscera. The diagram summarizes the main areas known to be altered when circulating androgen levels are chronically manipulated. Most of the information has been obtained from male rats. The structure and activity of many peripheral organs, particularly those with reproductive functions, are well known to be androgen sensitive. However, many aspects of the ganglion cells supplying these targets also implicate them as important targets of testosterone. In particular, neurons, soma size, axon density, and transmitter levels are dramatically affected by castration. Expression of transmitter receptors on the pre- and postsynaptic membranes is also androgen sensitive, as demonstrated primarily by studies on the vas deferens. There is very little information available on preganglionic neurons; however, the density of their terminals in some pathways is androgen sensitive. Interneurons and primary afferent neurons involved in these circuits have not been examined in the context of androgen sensitivity.

during the estrous cycle, pregnancy, and aging (Kennedy, 1929; Sjo¨berg, 1965; Marshall, 1970; Hervonen et al., 1972, 1973; Kanerva et al., 1972; Owman et al., 1975; Bell and Malcolm, 1978; Alm and Lundberg, 1988; Mitchell and Stauber, 1990; Heaton and Varrin, 1994). Most of the comparisons have been anatomical, demonstrating an increased number and staining intensity of terminals under conditions of high circulating estrogens; interestingly, the increase does not seem to be maintained throughout pregnancy. It is both intriguing and frustrating that the effects of estrogens show considerable species variation. For example, in pregnant rats the myometrial noradrenergic innervation increases transiently and then decreases below normal levels, whereas in guinea pigs it disappears completely and permanently; in rabbits it is unchanged from normal. There has been less work on the functional changes in the sympathetic innervation, in which the most interesting focus is probably the vasculature because this has an extensive nerve supply (Brauer et al., 1992; Sullivan et al., 1994). There have been fewer studies of the cholinergic parasympathetic innervation of female reproductive organs, particularly in the context of hormonal dependence. However, a small number of studies on VIP distribution show

40

JANET R. KEAST

that there is some loss from the uterus during pregnancy but less change in VIP nerves of the cervix (Stjernquist et al., 1985; Alm et al., 1986). While the focus in this area has been the final motor neurons (i.e., the pelvic ganglion cells) that supply the female reproductive organs, other parts of the reflex circuitry should also be considered as possible targets of steroid action. This is supported by recent immunohistochemical studies in which estrogen receptors were demonstrated in many preganglionic and primary afferent neurons which supply the uterus, along with many pelvic ganglion cells (Papka et al., 1997). Estrogen receptor localization has not been carried out in pathways supplying other pelvic viscera in females. However, the receptors may be quite widespread because it is possible that bladder and urethra function can vary with the hormonal environment (Andersson, 1993). In males, various aspects of peripheral pelvic autonomic circuitry change after puberty (Fig. 4). In response to increased circulating androgen levels, the organs grow and their innervation density increases, along with expression and activity of various transmitter receptors; conversely, numerous changes occur following pre- or postpubertal castration (Broberg et al., 1974; Wakade et al., 1975; Sjo¨strand and Swedin, 1976; MacDonald and McGrath, 1980; Hamill and Guernsey, 1983; Hamill et al., 1984; Lara et al., 1985; Anderson and Navarro, 1988; Melvin et al., 1988; Bustamante et al., 1989; Hamill and Schroeder, 1990; Bitran et al., 1991; Andersson et al., 1992; Morita et al., 1992; Heaton and Varrin, 1994; Holmquist et al., 1994). Moreover, in aging humans, and possibly as a consequence of lower androgen levels, impotence becomes more common (Kaiser et al., 1988; Stief et al., 1989). Animal studies on penile erection demonstrate defects in the peripheral autonomic nerves or target cells under conditions of lowered androgen exposure, including aging (Hart, 1967; Anderson and Navarro, 1988; Andersson et al., 1992; Mills et al., 1992; Andersson, 1993; Giuliano et al., 1993; Heaton and Varrin, 1994; Andersson and Wagner, 1995; Garban et al., 1995; Penson et al., 1996). Furthermore, clinical observations in patients administered antiandrogens (e.g., as an adjunct to treatment for prostate cancer) show that many patients experience the side effect of impotence (Schroder, 1991), consistent with autonomic dysfunction. However, despite this possible link, there is not a strong correlation between circulating androgens and impotence in normal adult rat and human populations (Andersson and Wagner, 1995). The precise relationship between androgens and neuronal function in normal adults therefore remains obscure. There has not been a complete study of which parts of the autonomic circuitry are androgen sensitive, although it appears that many components may be involved and not just the peripheral ganglion cells and their targets (Fig. 4). Studies of the pelvic ganglia in male rats have shown that the

UNUSUAL AUTONOMIC GANGLIA

41

soma size of some neurons is dependent on exposure to circulating androgens because their somata fail to grow to normal size after prepubertal castration and fail to maintain their normal volume after postpubertal castration (Hamill and Guernsey, 1983; Hamill et al., 1984; Melvin and Hamill, 1987; Melvin et al., 1988, 1989; Hamill and Schroeder, 1990; Keast and Saunders, 1998). The effects of castration can be prevented by administration of testosterone from the time of operation, indicating that androgens are essential for the maturation and maintenance of pelvic ganglion structure. Recent studies in our laboratory have shown that many preganglionic neurons supplying pelvic ganglion cells not only express androgen receptors but also change markedly after postpubertal castration. In particular, the volume and number of varicose terminals innervating each pelvic neuron decrease; in contrast, the soma volume of these preganglionic neurons does not change, indicating a specific effect of testosterone on maintaining the structure of particular neuronal compartments ( J. Keast and T. Watkins, unpublished observations). The search for androgen-sensitive neurons should be extended to other components of autonomic reflex circuits, including the sensory neurons and interneurons. While it is not known whether morphological changes in neurons translate to altered function, it is commonly assumed from concurrent changes in reflex activity after castration that such a link does exist. Some clues of which neurons contribute to these changes can be obtained by separately analyzing sympathetic and parasympathetic components of the pelvic ganglia. Numerous studies have demonstrated that noradrenergic pelvic neurons decrease significantly in size after castration, along with a decrease in TH and NPY levels (Sjo¨strand and Swedin, 1976; Hamill and Guernsey, 1983; Hamill et al., 1984; Melvin and Hamill, 1987; Melvin et al., 1988, 1989; Hamill and Schroeder, 1990). Recent work has shown that this occurs in all groups of pelvic noradrenergic neurons—not only those supplying reproductive targets but also those supplying the bladder and rectum (Keast and Saunders, 1998). While the function of most noradrenergic neurons has not been tested under different hormonal conditions, it is known that the innervation of the rat and guinea pig vas deferens (supplied by the pelvic ganglia) changes markedly at puberty and in castrated animals (MacDonald and McGrath, 1980, 1984; de Avellar and Markus, 1993; Lamas and Spadori, 1993; Nagao et al., 1994; Ren et al., 1996). In particular, noradrenergic excitation of smooth muscle is decreased, partly because of decreased expression of 움adrenoceptors but also perhaps because of less transmitter release. The responses mediated by the cotransmitter, ATP, are also smaller after castration and it is not clear how much of this difference is due to changes in receptors or transmitter release processes. Moreover, changes in physical structure of the targets or the axon terminals could contribute to altered

42

JANET R. KEAST

responses to nerve activation; for example, in the rat vas deferens there is an increased density of axons containing NOS after puberty (Ventura and Burnstock, 1996). More difficult to interpret are changes in number and catecholamine concentration of SIF cells in pelvic ganglia during aging or after castration (Partanen and Hervonen, 1979; Partanen et al., 1980) because the normal function of these cells is not understood. Interestingly, the cholinergic neurons of the male rat pelvic ganglia are influenced less by castration, at least in the context of structural change. There is no effect of castration on soma size of pelvic cholinergic NPY neurons, although the VIP neurons become slightly smaller (Keast and Saunders, 1998). Similarly, the cholinergic pathway is less sensitive to aging, such that sacral preganglionic innervation is maintained despite a decreased density of lumbar preganglionic terminals associated with pelvic noradrenergic neurons (Warburton and Santer, 1995). The function of the cholinergic neurons under conditions of low or absent circulating androgens has not been thoroughly studied, although it is known that pelvic nerve-evoked penile erection is impaired in rats (Mills et al., 1992; Giuliano et al., 1993; Heaton and Varrin, 1994; Garban et al., 1995; Penson et al., 1996). In other reproductive tissues, the changes have not been investigated because most of the parasympathetic nerves supply glandular epithelia and most pharmacological studies focus on control of smooth muscle. 3. Possible Mechanisms of Gonadal Steroid Action on Autonomic Reflexes Few studies have investigated the mechanism by which the effects of steroids on pelvic ganglia occur, and dissection of direct and indirect actions of steroids on neuron structure and function is notoriously difficult. Indeed, in the central nervous system, in which genomic steroid actions have been investigated much more thoroughly than in the periphery, this issue is largely unresolved. Recently, however, evidence has been provided that at least in some neurons a mixture of direct and indirect effects occurs. For example, in the lumbar motor neurons supplying the sexually dimorphic perineal muscles, the spinal nucleus of the bulbocavernosus, it appears that testosterone determines soma size by a direct action on the neuron but influences dendritic field indirectly by regulating expression of a targetderived trophic factor (Rand and Breedlove, 1995). Because many of the targets of pelvic ganglion cells synthesize neurotrophic factors (MacGrogan et al., 1991; Te et al., 1994; Tuttle et al., 1994b), this should also be considered as a possible mechanism of steroid action in the periphery. Further clues may be obtained by studying the location of androgen receptors in various components of the pelvic circuitry. These receptors were first found in extracts of male pelvic ganglia (Melvin and Hamill,

UNUSUAL AUTONOMIC GANGLIA

43

1989) and recent immunohistochemical studies have shown that nuclear receptors are expressed by a population of the cholinergic VIP- and NOScontaining pelvic neurons (which supply mainly reproductive targets) (Schirar et al., 1997; Keast and Saunders, 1998). These neurons also demonstrate some changes after castration. Interestingly, the noradrenergic neurons change much more markedly after castration but do not contain nuclear androgen receptors and instead display an unusual cytoplasmic staining (Keast and Saunders, 1998); because this cannot be translocated to the nucleus by exposure to high levels of testosterone before tissue removal, it is not known if this is a genuine receptor or an unusual cross-reactivity of the antiserum. Irrespective of the site of androgen action, the question of the nature of the active molecule remains. In many locations (e.g., the male internal reproductive organs), testosterone is metabolized to 5-dihydrotestosterone, which then activates the androgen receptor. It is not known if this occurs in peripheral ganglia. Furthermore, in many central neurons, the presence of an aromatase enzyme converts testosterone to estradiol, which then mediates the major biological effect (Arnold and Gorski, 1984; Breedlove, 1992; Kawata, 1995). It is not known if this enzyme or estrogen receptors are present in male pelvic ganglia. However, there has been one report that the effects of castration on rat pelvic ganglion cell somata are prevented by testosterone but not by estradiol (Hamill et al., 1984), suggesting that testosterone is the primary or sole active steroid mediating the morphological effects. Whether this can also be extrapolated to other actions of androgens in these neurons is not known. While the pelvic ganglia provide much of the innervation to the pelvic viscera, a substantial proportion is also provided by other sympathetic neurons of the inferior mesenteric ganglia and lumbar sympathetic chain ( Ja¨nig and McLachlan, 1987). A recent study showed that after castration the soma size of these neurons did not change at all or only to a small degree (in contrast to sympathetic pelvic neurons); however, possible changes in dendritic field (constituting a significant change in neuron volume) were not investigated (Keast and Saunders, 1998). Some studies of androgen sensitivity of sympathetic ganglia supplying other targets, notably the superior cervical ganglion, have provided mixed results (Dibner and Black, 1976; Hamill et al., 1984; Wright and Smolen, 1985). Although the functional relevance of gonadal hormones to the superior cervical ganglion is not obvious, in rodents this ganglion supplies a sexually dimorphic target, the salivary glands, which produce different pheromone secretions in males than in females; furthermore, testosterone may regulate NGF synthesis by the gland (Levi-Montalcini and Angeletti, 1964; Hendry and Iversen, 1973; Ishii and Shooter, 1975).

44

JANET R. KEAST

In summary, many pelvic neurons are influenced significantly by exposure to gonadal steroids, during both development and adulthood. The mechanism of this influence and its implication for reflex function are not known. Importantly, other components of pelvic reflexes (preganglionic neurons, interneurons, and visceral afferent neurons) must also be considered as potential sites of steroid action. It is appropriate that interest in this area grows. First, for those investigating pelvic autonomic reflexes, the role of steroids may be relevant to understanding various disease processes and changes occurring at different stages in the life cycle; important clues could be obtained with respect to treatment or modification of organ responses. Related to this is the acknowledgment that must occur of the status (age, reproductive activity, and gender) of the tissues studied because this may impact considerably on the innervation profile and activity. In particular, many studies are easier to carry out on tissues taken from young animals (e.g., cellular electrophysiology studies and neuronal culture studies), but these may display considerable differences in hormone exposure and subsequent structure, physiology, and chemistry. Second, many questions on the mechanism of steroid hormone action on synaptic function and plasticity, as well as the possible involvement of neurotrophic factors, have been extremely difficult to answer in the more complicated circuitry of the central nervous system. The well-characterized and relatively simple organization of the pelvic ganglion and its similar sensitivity to hormones provide an excellent opportunity to examine some of the broader issues with respect to hormone effect on neuron function.

H. Responses to Nerve Injury 1. Deafferentation Numerous studies have been conducted on the effects of removing spinal inputs (deafferentation or decentralization) on autonomic ganglion cells and these have consistently demonstrated that regeneration of spinal connections occurs, albeit slowly (Nja˚ and Purves, 1977; Purves et al., 1981; Liestol et al., 1987; Taxi and Euge`ne, 1995). The majority of these studies have been carried out on paravertebral sympathetic ganglia and have demonstrated a considerable degree of specificity in the regeneration process, whereby spinal nerves preferentially reinnervate their original target cells and after which at least some functional reflex circuitry is restored. In the pelvic ganglia, the consequences of spinal nerve injury are predictably more complex, given the distributed nature of the spinal inputs. That is, pelvic ganglia receive both lumbar and sacral inputs (and, in some species, individual neurons receive both; see Section III,B), either some or all of

UNUSUAL AUTONOMIC GANGLIA

45

which may be damaged. This is a markedly different situation from other autonomic ganglia, which are almost all either purely sympathetic or parasympathetic. Interestingly, in cases of partial deafferentation (removal of either lumbar or sacral inputs but not both), there is evidence that one or more components of the intact pathway undergo significant structural changes, perhaps as a compensatory mechanism. This raises the general issue of how adult neurons recognize and respond to loss of innervation from neighboring neurons. Furthermore, the clinical relevance of either type of deafferentation is highlighted by considering the consequences of reflex behavior. Disruption of lumbar pathways leads to lack of neural activation of internal reproductive organs in males or bladder neck closure, whereas intact sacral pathways are required for the micturition, defecation, and erection reflexes. An important indication that remodeling of intact pathways occurs after partial decentralization of pelvic ganglia has been provided by studies of cat bladder ganglia by de Groat and Kawatani (1989). Within 3 months of cutting the pelvic nerves (disrupting micturition reflexes), bladder contractions could instead be evoked by stimulating the hypogastric nerves, indicating that some component of the lumbar pathway was able to activate detrusor muscle activity. This apparently new mechanism was sensitive to adrenoceptor blockade, further supporting the idea that sympathetic nerves were now involved in the response. It is not known whether these new pathways can be activated reflexly; however, it was proposed that this remodeling of intact pathways may not necessarily be a favorable outcome for the animal, unless central pathways are also reorganized (e.g., bladder contraction and relaxation may occur simultaneously, or contraction may occur at an inappropriate time, if these new peripheral circuits are reflexively activated in their original sequence). This remodeling may therefore contribute to clinical phenomena such as autonomous hyperactive bladder which occur after some types of spinal injury. A similar observation was made by Dail et al. (1989b), who investigated the sequelae to sacral spinal nerve damage in male rats in the context of nerve-evoked penile erection. In intact animals erection can be evoked by pelvic (sacral) nerve but not hypogastric (lumbar) nerve stimulation; however, as soon as 3 days after sacral nerve transection, erection can be elicited by hypogastric nerve activation. This is also consistent with a remodeling of intact peripheral pathways to allow activation of denervated ganglion cells. It is not known if the more rapid effects seen in this study were due to species differences or differences between nerves of particular functions. It is not known if and how this remodeling influences regeneration of spinal connections to connect with their appropriate ganglion cells. Finally, a recent study has shown a converse remodeling response, namely,

46

JANET R. KEAST

reinnervation by intact parasympathetic pathways of damaged sympathetic neurons supplying the rat vas deferens (Kihara et al., 1996). The most popular interpretation of these observations has been that intact preganglionic axons grow collateral processes to innervate neurons which have had their spinal inputs removed, as occurs in partially deafferented sympathetic chain ganglia (Maehlen and Nja˚, 1981, 1984; Henningsen et al., 1985). However, this has not been demonstrated in pelvic ganglia, in which the situation differs somewhat. That is, in pelvic ganglia the only preganglionic neurons remaining intact that could contribute to reinnervation would be in very different spinal levels from the damaged pathways. However, there is a second possibility—that ganglion cells with intact spinal inputs are the ones which grow collaterals to supply denervated neurons. Evidence that this occurs comes from anatomical studies which show that many new fibers grow in rat pelvic ganglia just a few days after either lumbar or sacral deafferentation (Dail et al., 1997; Kepper and Keast, 1998). Recent studies have shown that these new fibers specifically target neurons which have been denervated and that at least some originate from neurons which have intact spinal connections (Kepper and Keast, 1998). Whether this mechanism is responsible for all or only some of the restored ganglionic connectivity is not known. However, this is important to establish because the two mechanisms differ considerably in the transmitters released by newly formed terminals and in the neurotrophic factors potentially involved in terminal growth and targeting. Many possible mechanisms may underly this remodeling. For example, the stimulus may be provided by the target organs, many of which change in structure after decentralization. The most obvious example of this is the urinary bladder, which will no longer be reflexly voided, hypertrophies, and secretes high levels of nerve growth factor (NGF) (Steers et al., 1990, 1991b, 1994; Tuttle et al., 1994b). NGF may contribute to the change in structure or chemistry of pelvic ganglion cells supplying this organ because NGF antisera block structural changes occurring simultaneously in sensory neurons (Steers et al., 1996). Alternatively, the stimulus for new fiber growth after partial decentralization may arise within the ganglion from either ganglion cells or glia, which may also be sources of neurotrophic factors. There is no information available on which neurotrophic factors and receptors are present in intact pelvic ganglia and which may therefore contribute to remodeling after injury. However, it is predicted that sympathetic noradrenergic neurons are the ones which would be most responsive to NGF, in line with the selective action of NGF on sympathetic neurons elsewhere in the peripheral autonomic system (Rush et al., 1992). Moreover, studies on cultured pelvic neurons have shown that while many respond to NGF, other factors may also be involved (Tuttle and Steers, 1992; Tuttle et al., 1994a).

UNUSUAL AUTONOMIC GANGLIA

47

A quite different scenario is provided by total deafferentation, in which both lumbar and sacral inputs are removed (Dail et al., 1997; Kepper and Keast, 1998). This provides a stimulus for even more new fiber growth in the pelvic ganglion than after partial lesion. Given that all spinal connections have been removed, it is most likely that these new fibers arise from pelvic ganglion cells (although if unilateral lesions have been performed, some may come from neurons of the contralateral ganglion). This type of axogenesis also occurs in other completely deafferented autonomic ganglia [rabbit ciliary ganglion ( Johnson, 1988) and frog cardiac ganglion (Sargent and Dennis, 1977, 1981)], at least some of which are known to form functional synapses. It is not known if genuine synapses also occur in pelvic ganglia. Moreover, in pelvic ganglia, despite the rapid growth of large numbers of fibers after total deafferentation, these fibers appear not to be maintained at the same high levels for more than a few days (Kepper and Keast, 1998). It is possible that with time they disappear completely, irrespective of whether or not preganglionic nerves reinnervate the ganglion cells. This will depend on whether there is sufficient stimulus to maintain these new connections or whether ingrowing spinal axons displace the intraganglionic connections. While spinal nerve injury has dramatic clinical consequences and is readily mimicked in animal models, the more common type of injury in humans is spinal cord damage. Particularly for supraspinal reflexes, this will lead to a loss of regular activation of pelvic ganglion cells. It is not known if there is remodeling in the pelvic ganglia after this type of injury. Such studies would indicate whether it is the physical absence of the preganglionic terminal which initiates remodeling (e.g., by loss of a secreted substance) or whether it is the lack of ganglion cell depolarization alone which stimulates remodeling. 2. Axotomy Autonomic neurons are known to respond to axotomy in many ways (Taxi and Euge`ne, 1995) and numerous changes in chemistry, membrane properties, and structure have been observed (Morris and Gibbins, 1989; Kummer, 1992; Ho¨kfelt et al., 1994; Lubischer and Arnold, 1994; Yu, 1994; de Castro et al., 1995; Magnusson et al., 1996). While many of these changes may occur in response to loss of target-derived substances (e.g., NGF), some appear to be due directly to nerve trauma. In pelvic ganglia this has not been extensively studied, despite the possibility in some species of identifying and manipulating the entire population of axons supplying some organs (e.g., the penis; Dail et al., 1989a). However, the changes occurring in neurons after axotomy are important to bear in mind because they may occur during a number of experimental procedures.

48

JANET R. KEAST

For example, if axons are severed in order to apply retrograde tracer dye, it should be borne in mind that the labeled neurons may not be the same as unlabeled, intact neurons. In addition, some neurons project in the pelvic and hypogastric nerves, which will be axotomized in deafferentation experiments. In the case of the hypogastric nerves, these axons are thought to traverse the inferior mesenteric ganglion to supply the bowel (Baron and Ja¨nig, 1988; Nadelhaft and Vera, 1991; Luckensmeyer and Keast, 1994). The target of ganglion cells projecting in the pelvic nerves is not known, although some project in this nerve for only a short distance before reentering the pelvic ganglion (Dail and Minorsky, 1986). In rats it has been proposed that axotomy of these neurons may underlie at least some of the new fiber formation after decentralization (Dail and Minorsky, 1986; Dail et al., 1997). This is consistent with the observation that severing the pelvic nerves close to the ganglion induces more axogenesis than cutting further away (Dail et al., 1997). However, the total number of neurons projecting out of these nerves appears to be very small (M. Kepper and J. Keast, unpublished observations) and is unlikely to account entirely for the substantial number of fibers formed in the ganglion after deafferentation.

3. Other Types of Nerve Damage Pelvic ganglia are able to adapt to innervate increasing target fields. This is best demonstrated by experiments in rats in which one pelvic ganglion is removed and reinnervation of the target organs is rapidly achieved by axogenesis from the remaining ganglion (Banns et al., 1980; Gabella and Uvelius, 1993). Where the target is instead increased directly by changing the organ (e.g., bladder size increased by urethral ligation or diabetes), increased peripheral innervation follows; in parallel, the soma size of pelvic neurons supplying the larger targets (but not unmanipulated organs) is increased (Steers et al., 1990, 1994; Gabella et al., 1992; Gabella and Uvelius, 1993; Nadelhaft et al., 1993). Interestingly, while the bladder may determine the features of its nerve supply, the converse is not true. Studies by Gabella and Uvelius (1993) showed that after urethral ligation the bladder hypertrophies irrespective of whether the ganglion cells or their connections are damaged. It has been proposed that bladder inactivity and the subsequent prolonged stretch due to urine accumulation triggers excessive NGF production by the smooth muscle, which in turn increases the size of sensory and motor neurons innervating the bladder (Steers et al., 1990, 1991a, 1996; Tuttle et al., 1994b). The factors mediating concurrent changes in preganglionic neurons are not known, nor is it known whether factors other than NGF are released from the hypertrophic bladder.

49

UNUSUAL AUTONOMIC GANGLIA

IV. Concluding Remarks The pelvic ganglia differ in numerous ways from sympathetic and parasympathetic ganglia, as summarized in Table I. Some of these differences (e.g., their more complex connectivity) have hindered the study of the reflex circuitry in which they are involved and appear to be the primary reason why understanding of urinary and reproductive reflexes lags behind that of peripheral cardiovascular control. Furthermore, the extensive and ramifying nature of the pelvic plexus in humans may lead to additional clinical problems because the location and connections of peripheral nerve bundles

TABLE I Unusual Features of Pelvic Ganglia Feature

Unusual because

Mixture of sympathetic and parasympathetic neurons (various species)

Sympathetic and parasympathetic neurons typically in separate ganglia

Strong synaptic inputs (rats and mice)

Most sympathetic neurons receive smaller, graded subthreshold inputs

Convergence of sympathetic and parasympathetic inputs (guinea pigs and cats)

Other autonomic neurons receive either sympathetic or parasympathetic inputs, not both

T-type calcium channels (rats)

Not commonly expressed by sympathetic neurons of other types

Neuron morphology (rats)

Pelvic sympathetic neurons unusually simple, mostly adendritic

Neuron morphology (cats)

Pelvic parasympathetic neurons unusually complex, many with dendrites

Acetylcholine synthesis by some sympathetic neurons (rats and guinea pigs)

Most sympathetic neurons noradrenergic

Catecholamine storage (various species)

Most catecholamine neurons easily influenced by reserpine, 6hydroxydopamine, but pelvic neurons relatively resistant

Innervation of lower bowel (rats)

Pelvic ganglia target different layers than do other sympathetic ganglia; minimal involvement in viscerofugal reflexes; sympathetic innervation to lower bowel causes excitation rather than inhibition

Steroid sensitivity (various species)

Structure, chemistry, and function of pelvic neurons particularly sensitive to androgens and estrogens

50

JANET R. KEAST

are not well documented and therefore may be readily damaged during procedures such as prostatectomy and hysterectomy. Surprisingly, even the most fundamental issues of autonomic control, such as the nature of the transmitter in each pelvic neuroeffector, have not been clearly elucidated, and roles for ATP, nitric oxide, and various neuropeptides may outweigh those of the ‘‘classical’’ transmitters, noradrenaline and acetylcholine, in some instances. This prompts the need for clinically useful methods for manipulating these other transmitter systems for treatment of conditions such as urinary incontinence and impotence. In contrast, in some species, such as mice and rats, the anatomy and cellular structure of pelvic neurons are notably simpler than their counterparts in larger species, promoting their use as popular models of pelvic circuitry for both neurochemical and physiological study. The adendritic structure of many pelvic neurons in these species is particularly valuable for studies of neuronal membrane properties and synaptic function, although surprisingly they have not been used extensively for this purpose. Such dramatic differences between species in the connectivity and complexity of the pelvic ganglia beg the question of how pelvic reflexes function in each case, and whether the entire circuit is simpler in rodents or whether the increased peripheral simplicity is counterbalanced by more complex central processing. The steroid hormone sensitivity of some neuron types within pelvic autonomic circuits has only just begun to be systematically investigated. While the logical starting point is the motor neurons that connect directly with steroid-sensitive organs, there may be equally important sites of steroid hormone action at other points in the circuit, such as interneurons and sensory neurons. Furthermore, steroid sensitivity is not restricted to neurons with reproductive function. The emphasis of most studies has been the morphological changes that occur with hormone manipulation because this is a relatively fast way to ‘‘screen’’ hormone-sensitive neuron populations. However, it may well ignore other neurons which respond in their receptor expression, chemistry, or excitability but which give no morphological indicators of this change. Given the relative ease of investigating membrane and synaptic properties in peripheral neurons, the pelvic ganglia seem to be ideally suited to investigate how chronic and acute exposure to steroids influences the excitability of neurons, an issue which is much more difficult to study in central steroid-sensitive neurons. The pelvic ganglia also provide an interesting model in which to investigate the peripheral consequences of spinal injury, whether it be of the cord itself or the spinal nerves that connect with the pelvic ganglia. Whereas other autonomic ganglia are typically innervated by a limited range of spinal levels, the mixed nature of the pelvic ganglia gives rise to situations in which only a proportion of neurons may be incapacitated after spinal

UNUSUAL AUTONOMIC GANGLIA

51

damage. The rapid and pronounced remodeling of peripheral autonomic pathways that ensues suggests that localized and powerful signaling processes exist in order to detect and respond to this damage. Because many of the pelvic neurons express neurotrophic factor receptors and are predicted to access a variety of these factors, the ganglionic remodeling may well prove valuable to investigating the regulation of neurotrophic factor signaling processes and their impact on plasticity of adult neurons.

References Adams, D. J., and Harper, A. A. (1995). Electrophysiological properties of autonomic ganglion neurons. In ‘‘Autonomic Ganglia’’ (E. M. McLachlan, Ed.), pp. 153–212. Harwood Academic, Luxembourg. Akasu, T., and Nishimura, T. (1995). Synaptic transmission and function of parasympathetic ganglia. Prog. Neurobiol. 45, 459–522. Akasu, T., Shinnick-Gallagher, P., and Gallagher, J. P. (1984). Adenosine mediates a slow hyperpolarizing synaptic potential in autonomic neurones. Nature 311, 62–65. Akasu, T., Shinnick-Gallagher, P., and Gallagher, J. P. (1986). Evidence for a catecholaminemediated slow hyperpolarizing synaptic response in parasympathetic ganglia. Brain Res. 365, 365–368. Alm, P. (1982). On the autonomic innervation of the human vas deferens. Brain Res. Bull. 9, 673–677. Alm, P., and Lundberg, L.-M. (1988). Co-existence and origin of peptidergic and adrenergic nerves in the guinea pig uterus. Retrograde tracing and immunocytochemistry, effects of chemical sympathectomy, capsaicin treatment and pregnancy. Cell Tissue Res. 254, 517–530. Alm, P., Owman, C., Sjo¨berg, N.-O., Stjernquist, M., and Sundler, F. (1986). Histochemical demonstration of a concomitant reduction in neural vasoactive intestinal polypeptide, acetylcholinesterase, and noradrenaline of cat uterus during pregnancy. Neuroscience 18, 713–726. Andersson, G. F., and Navarro, S. P. (1988). The response of autonomic receptors to castration and testosterone in the urinary bladder of the rabbit. J. Urol. 140, 885–889. Andersson, K.-E. (1993). Pharmacology of lower urinary tract smooth muscles and penile erectile tissues. Pharmacol. Rev. 45, 253–308. Andersson, K.-E., and Wagner, G. (1995). Physiology of penile erection. Physiol. Rev. 75, 191–236. Andersson, K. E., Holmquist, F., and Bødker, A. (1992). Castration enhances NANC nervemediated relaxation in rabbit isolated corpus cavernosum. Acta Physiol. Scand. 146, 405–406. Andersson, P.-O., Bloom, S. R., and Ja¨rhult, J. (1983). Colonic motor and vascular responses to pelvic nerve stimulation and their relation to local peptide release in the cat. J. Physiol. 334, 293–307. Arnold, A. P., and Gorski, R. A. (1984). Gonadal steroid induction of structural sex differences in the central nervous system. Annu. Rev. Neurosci. 7, 413–442. Arvidsson, U., Riedl, M., Elde, R., and Meister, B. (1997). Vesicular acetylcholine transporter (VAChT) protein: A novel and unique marker for cholinergic neurons in the central and peripheral nervous systems. J. Comp. Neurol. 378, 454–467. Baker, H. A., Burke, J. P., Bhafnagar, R. K., Van Orden, D. E., Van Orden, L. S., and Hartman, B. K. (1977). Histochemical and biochemical characterization of the rat paracervical ganglion. Brain Res. 132, 393–405.

52

JANET R. KEAST

Baljet, B., and Drukker, J. (1979). The extrinsic innervation of the abdominal organs of the female rat. Acta Anat. 104, 243–267. Baljet, B., and Drukker, J. (1980). The extrinsic innervation of the pelvic organs in the female rat. Anat. Rec. 107, 241–267. Baluk, P. (1995). Structure of autonomic ganglia. In ‘‘Autonomic Ganglia’’ (E. M. McLachlan, Ed.), pp. 13–71. Harwood Academic, Luxembourg. Banns, H., Ekstro¨m, J., and Mann, S. P. (1980). Recovery of choline acetyltransferase activity in the rat urinary bladder deprived of half of its innervation. Acta Physiol. Scand. 109, 85–88. Baron, R., and Ja¨nig, W. (1988). Neurons projecting rostrally in the hypogastric nerve of the cat. J. Auton. Nervous System 24, 81–86. Baron, R., and Ja¨nig, W. (1991). Afferent and sympathetic neurons projecting into the lumbar visceral nerves of the male rat. J. Comp. Neurol. 314, 429–436. Baron, R., Ja¨nig, W., and McLachlan, E. M. (1985a). The afferent and sympathetic components of the lumbar spinal outflow to the colon and pelvic organs in the cat. III. The colonic nerves, incorporating an analysis of all components of the lumbar prevertebral outflow. J. Comp. Neurol. 238, 158–168. Baron, R., Ja¨nig, W., and McLachlan, E. M. (1985b). The afferent and sympathetic components of the lumbar spinal outflow to the colon and pelvic organs in the cat. I. The hypogastric nerve. J. Comp. Neurol. 238, 135–146. Becker, K. (1972). Paraganglienzellen in Ganglion cervicale uteri der Maus. Z. Zeitschrift. 130, 249–261. Bell, C., and Malcolm, S. J. (1978). Observations on the loss of catecholamine fluorescence from intrauterine adrenergic nerves during pregnancy in the guinea-pig. J. Reprod. Fertil. 53, 51–58. Bell, C., and McLean, J. R. (1967). Localisation of norepinephrine and acetylcholinesterase in separate neurons supplying the guinea-pig vas deferens. J. Pharmacol. Exp. Ther. 69, 69. Belluzzi, O., Sacchi, O., and Wanke, E. (1985). Identification of delayed potassium and calcium current in the rat sympathetic neuron under voltage-clamp conditions. J. Physiol. 358, 109–129. Berthoud, H.-R., Jedrzejewsk, A., and Powley, T. (1990). Simultaneous labeling of vagal innervation of the gut and afferent projections from the visceral forebrain with DiI injected into the dorsal vagal complex in the rat. J. Comp. Neurol. 301, 65–79. Berthoud, H.-R., Carlson, N. R., and Powley, T. (1991). Topography of efferent vagal innervation of the rat gastrointestinal tract. Am. J. Physiol. 260, R200–R207. Bitran, M., Torres, G., Fournier, A., St. Pierre, S., and Huidobro-Toro, J.P. (1991). Age and castration modulate the inhibitory action of neuropeptide Y on neurotransmission in the rat vas deferens. Eur. J. Pharmacol. 203, 267–274. Blackman, J. G., Crowcroft, P. J., Devine, C. E., Holman, M. E., and Yonemura, K. (1969). Transmission from preganglionic fibres in the hypogastric nerve to peripheral ganglia of male guinea-pigs. J. Physiol. 201, 723–743. Booth, A. M., and de Groat, W. C. (1979). A study of facilitation in vesical parasympathetic ganglia of the cat using intracellular recording techniques. Brain Res. 169, 388–392. Boyd, H. D., McLachlan, E. M., Keast, J. R., and Inokuchi, H. (1996). Three electrophysiological classes of guinea pig sympathetic neurone have distinct morphologies. J. Comp. Neurol. 369, 372–387. Brading, A. F., and Mostwin, J. L. (1989). Electrical and mechanical responses of guinea-pig bladder muscle to nerve stimulation. Br. J. Pharmacol. 98, 1083–1090. Brauer, M. M., Lincoln, J., Blundell, D., and Corbacho, A. (1992). Postnatal development of noradrenaline-containing nerves of the rat uterus. J. Auton. Nervous System 39, 37–50. Breedlove, S. M. (1992). Sexual dimorphism in the vertebrate nervous system. J. Neurosci. 12, 4133–4142.

UNUSUAL AUTONOMIC GANGLIA

53

Broberg, A., Nybell, G., Owman, C., Rosengren, E., and Sjo¨berg, N. O. (1974). Consequence of neonatal androgenization and castration for future levels of norepinephrine transmitter in uterus and vas deferens of the rat. Neuroendocrinology 15, 308–312. Brock, J. A., and Cunnane, T. C. (1992). Impulse conduction in sympathetic nerve terminals in the guinea-pig vas deferens and the role of the pelvic ganglia. Neuroscience 47, 185–196. Burnstock, G. (1977). The purinergic nerve hypothesis. Ciba Foundation Symp. 48, 295–314. Burnstock, G. (1985). Nervous control of smooth muscle by transmitters, cotransmitters and modulators. Experientia 41, 869–874. Bustamante, D., Lara, H., and Belmar, J. (1989). Changes of norepinephrine levels, tyrosine hydroxylase and dopamine-beta-hydroxylase activities after castration and testosterone treatment in vas deferens of adult rats. Biol. Reprod. 40, 541–548. Callister, R. J., Keast, J. R., and Sah, P. (1997). Ca2⫹ activated K⫹ channels in rat otic ganglion cells: Role of calcium entry via Ca2⫹-channels and nicotinic receptors. J. Physiol. 500, 571–582. Chiang, C. H., and Gabella, G. (1986). Quantitative study of the ganglion neurons of the mouse trachea. Cell Tissue Res. 246, 243–252. Costa, M., and Furness, J. B. (1973a). Observations on the anatomy and amine histochemistry of the nerves and ganglia which supply the pelvic viscera and on the associated chromaffin tissue in the guinea-pig. Z. Anat. Entwickl. 140, 88–108. Costa, M., and Furness, J. B. (1973b). The origins of the adrenergic fibres which innervate the internal anal sphincter, the rectum, and other tissues of the pelvic region in the guineapig. Z. Anat. Entwickl. 140, 129–142. Costa, M., and Gabella, G. (1971). Adrenergic innervation of the alimentary canal. Z. Zellforsch. 122, 357–377. Costa, M., Furness, J. B., and Gabella, G. (1971). Catecholamine containing nerve cells in the mammalian myenteric plexus. Histochemie 25, 103–106. Costa, M., Furness, J. B., Llewellyn-Smith, I. J. and Cuello, A. C. (1981). Projections of substance P-containing neurons within the guinea-pig small intestine. Neuroscience 6, 411–424. Costa, M., Furness, J. B., and Gibbins, I. L. (1986). Chemical coding of enteric neurons. Prog. Brain Res. 68, 217–239. Crowcroft, P. J., and Szurszewski, J. H. (1971). A study of the inferior mesenteric and pelvic ganglia of guinea-pigs with intracellular electrodes. J. Physiol. 219, 421–441. Crowe, R., Haven, A. J., and Burnstock, G. (1986). Intramural neurones of the guinea-pig urinary bladder: Histochemical localization of putative neurotransmitters in cultures and newborn animals. J. Auton. Nervous System 15, 319–339. Dail, W. G. (1976). Histochemical and fine structural studies of SIF cells in the major pelvic ganglion of the rat. In ‘‘SIF Cells. Structure and Function of the Small, Intensely Fluorescent Sympathetic Cells’’ (O. Era¨nko¨, Ed.), Fogarty Int. Cr. Proc., Vol. 30, pp. 8–18. Natl. Inst. Health, Bethesda, MD. Dail, W. G. (1993). Autonomic innervation of male genitalia. In ‘‘Nervous Control of the Urogenital System’’ (C. A. Maggi, Ed.), pp. 69–102. Harwood Academic, Chur, Switzerland. Dail, W. G., and Dziurzynski, R. (1985). Substance P immunoreactivity in the major pelvic ganglion of the rat. Anat. Rec. 212, 103–109. Dail, W. G., and Minorsky, N. (1986). Composition of the pelvic nerve. Exp. Neurol. 92, 278–283. Dail, W. G., Evan, A. P., and Eason, H. R. (1975). The major ganglion in the pelvic plexus of the male rat: A histochemical and ultrastructural study. Cell Tissue Res. 159, 49–62. Dail, W. G., Moll, M. A., and Weber, K. (1983). Localization of vasoactive intestinal polypeptide in penile erectile tissue and in the major pelvic ganglion of the rat. Neuroscience 10, 1379–1386. Dail, W. G., Manzanares, K., Moll, M. A., and Minorsky, N. (1985). The hypogastric nerve innervates a population of penile neurons in the pelvic plexus. Neuroscience 16, 1041–1046.

54

JANET R. KEAST

Dail, W. G., Minorsky, N., Moll, M. A., and Manzanares, K. (1986). The hypogastric nerve pathway to penile erectile tissue: Histochemical evidence supporting a vasodilator role. J. Auton. Nervous System 15, 341–349. Dail, W. G., Trujillo, D., de la Rosa, D., and Walton, G. (1989a). Autonomic innervation of reproductive organs: Analysis of the neurons whose axons project in the main penile nerve in the pelvic plexus of the rat. Anat. Rec. 224, 94–101. Dail, W. G., Walton, G., and Olmsted, M. P. (1989b). Penile erection in the rat: Stimulation of the hypogastric nerve after chronic interruption of the sacral parasympathetic outflow. J. Auton. Nervous System 28, 251–258. Dail, W. G., Carrillo, Y., and Walton, G. (1990). Innervation of the anococcygeus muscle of the rat. Cell Tissue Res. 259, 139–146. Dail, W. G., Galindo, R., Leyba, L., and Barba, V. (1997). Denervation-induced changes in perineuronal plexuses in the major pelvic ganglion of the rat: Immunohistochemistry for vasoactive intestinal polypeptide and tyrosine hydroxylase and histochemistry for NADPHdiaphorase. Cell Tissue Res. 287, 315–324. de Avellar, M. C. W., and Markus, R. P. (1993). Age-related changes in norepinephrine release and its modulation by presynaptic alpha-2 adrenoceptors in the rat vas deferens. J. Pharmacol. Exp. Ther. 267, 38–44. de Castro, F., Sa´nchez-Vives, M. V., Munoz-Martı´nez, E. J., and Gallego, R. (1995). Effects of postganglionic nerve section on synaptic transmission in the superior cervical ganglion of the guinea-pig. Neuroscience 67, 689–695. de Groat, W. C. (1987). Neuropeptides in pelvic afferent pathways. Experientia 43, 801–813. de Groat, W. C., and Booth, A. M. (1980). Inhibition and facilitation in parasympathetic ganglia of the urinary bladder. Fed. Proc. 39, 2990–2996. de Groat, W. C., and Booth, A. M. (1993a). Neural control of penile erection. In ‘‘Nervous Control of the Urogenital System’’ (C. A. Maggi, Ed.), pp. 467–524. Harwood Academic, Chur, Switzerland. de Groat, W. C., and Booth, A. M. (1993b). Synaptic transmission in pelvic ganglia. In ‘‘Nervous Control of the Urogenital System’’ (C. A. Maggi, Ed.), pp. 291–348. Harwood Academic, Chur, Switzerland. de Groat, W. C., and Kawatani, M. (1989). Reorganization of sympathetic preganglionic connections in cat bladder ganglia following parasympathetic denervation. J. Physiol. 409, 431–449. de Groat, W. C., and Krier, J. (1976). An electrophysiological study of the sacral parasympathetic pathway to the colon of the cat. J. Physiol. 260, 425–445. de Groat, W. C., and Krier, J. (1978). The sacral parasympathetic reflex pathway regulating colonic motility and defecation in the cat. J. Physiol. 276, 481–500. de Groat, W. C., and Krier, J. (1979). The central control of the lumbar sympathetic pathway to the large intestine of the cat. J. Physiol. 289, 449–468. de Groat, W. C., and Saum, W. R. (1972). Sympathetic inhibition of the urinary bladder and of pelvic ganglionic transmission in the cat. J. Physiol. 220, 297–314. de Groat, W. C., and Saum, W. R. (1976). Synaptic transmission in parasympathetic ganglia in the urinary bladder of the cat. J. Physiol. 256, 137–158. de Groat, W. C., and Steers, W. D. (1988). Neural control of the urinary bladder and sexual organs: Experimental studies in animals. In ‘‘Autonomic Failure: A Textbook of Clinical Disorders of the Autonomic Nervous System’’ (R. Bannister, Ed.), pp. 196–222. Oxford Univ. Press, Oxford. de Groat, W. C., Booth, A. M., and Krier, J. (1979). Interaction between sacral parasympathetic and lumbar sympathetic inputs to pelvic ganglia. In ‘‘Integrative Functions of the Autonomic Nervous System’’ (C. M. Brooks, K. Koizumi, and A. Sato, Eds.), pp. 234–247. Univ. of Tokyo Press, Tokyo.

UNUSUAL AUTONOMIC GANGLIA

55

de Groat, W. C., Nadelhaft, I., Milne, R., Booth, A. M., Morgan, C., and Thor, K. (1981). Organization of the sacral parasympathetic reflex pathways to the urinary bladder and large intestine. J. Auton. Nervous System 3, 135–160. de Groat, W. C., Booth, A. M., Kawatani, M., and Lowe, I. P. (1985). A study of synaptic transmission and neuropeptide function in extramural colonic ganglia of the cat. Jpn. J. Smooth Muscle Res. 21(Suppl.), 95. de Groat, W. C., Booth, A. M., and Yoshimura, N. (1993). Neurophysiology of micturition and its modification in animal models of human disease. In ‘‘Nervous Control of the Urogenital System’’ (C. A. Maggi, Ed.), pp. 227–290. Harwood Academic, Chur, Switzerland. Dhami, D., and Mitchell, B. S. (1991). Specific patterns of immunoreactivity in neuronal elements of the anterior major pelvic ganglia of the male guinea-pig. J. Anat. 176, 197–210. Dibner, M. D., and Black, I. B. (1976). Elevation of sympathetic ganglion tyrosine hydroxylase activity in neonatal and adult rats by testosterone treatment. J. Neurochem. 27, 323–324. Ding, Y.-Q., Wang, Y.-Q., Qin, B.-Z., and Li, J.-S. (1993). The major pelvic ganglion is the main source of nitric oxide synthase-containing nerve fibers in penile erectile tissue of the rat. Neurosci. Lett. 164, 187–189. Dixon, J. S., and Gosling, J. A. (1991). Neuropeptide studies on the human vas deferens. J. Auton. Nervous System 33, 112. Dixon, J. S., Gilpin, S. A., Gilpin, C. J., and Gosling, J. A. (1983). Intramural ganglia of the human urinary bladder. Br. J. Urol. 55, 195–198. Dockray, G. J. (1992). Transmission: Peptides. In ‘‘Autonomic Neuroeffector Mechanisms’’ (G. Burnstock and C. H. V. Hoyle, Eds.), pp. 409–464. Harwood Academic, Chur, Switzerland. Domoto, T., and Tsumori, T. (1994). Co-localization of nitric oxide synthase and vasoactive intestinal peptide immunoreactivity in neurons of the major pelvic ganglion projecting to the rat rectum and penis. Cell Tissue Res. 278, 273–278. Dun, N., and Jiang, Z. G. (1982). Non-cholinergic excitatory transmission in inferior mesenteric ganglia of the guinea-pig: Possible mediation by substance P. J. Physiol. 325, 145–159. Dunzendorfer, U., Jonas, D., and Weber, W. (1976). The autonomic innervation of the human prostate. Histochemistry of acetylcholinesterase in the normal and pathologic states. Urol. Res. 4, 29–31. Edwards, F. R., Hirst, G. D., Klemm, M. F., and Steele, P. A. (1995). Different types of ganglion cell in the cardiac plexus of guinea-pigs. J. Physiol. 486, 453–471. Edyvane, K. A., Trussell, D. C., Jonavicius, J., Henwood, A., and Marshall, V. R. (1992). Presence and regional variation in peptide-containing nerves in the human ureter. J. Auton. Nervous System 39, 127–138. Elbadawi, A., and Schenk, E. A. (1973). Parasympathetic and sympathetic postganglionic synapses in ureterovesical autonomic pathways. Z. Zellforsch. 146, 147–154. Elfvin, L.-G., Lindh, B., and Ho¨kfelt, T. (1993). The chemical neuroanatomy of sympathetic ganglia. Annu. Rev. Neurosci. 16, 471–507. Era¨nko¨, O. (1978). Small intensely fluorescent (SIF) cells and nervous transmission in sympathetic ganglia. Annu. Rev. Pharmacol. Toxicol. 18, 417–430. Fletcher, T. F., and Bradley, W. F. (1969). Comparative morphologic features of urinary bladder innervation. Am. J. Vet. Res. 30, 1655–1662. Forehand, C. J. (1985). Density of somatic innervation on mammalian autonomic ganglion cells is inversely related to dendritic complexity and preganglionic convergence. J. Neurosci 5, 3403–3408. Foroglou, C., and Winckler, G. (1973). Characteristiques du plexus hypogastrique inferieur (pelvien) chez le rat. Bull. Assoc. Anat. Paris 57, 853–857. Fujii, K. (1988). Evidence for adenosine triphosphate as an excitatory transmitter in guineapig, rabbit and pig urinary bladder. J. Physiol. 404, 39–52.

56

JANET R. KEAST

Fukai, K., and Fukuda, H. (1985). Three serial neurones in the innervation of the colon by the sacral parasympathetic nerve of the dog. J. Physiol. 362, 69–79. Furness, J. B. (1970). The origin and distribution of adrenergic nerve fibres in the guinea-pig colon. Histochemie 21, 295–306. Furness, J. B., and Costa, M. (1973). The ramifications of adrenergic nerve terminals in the rectum, anal sphincter and anal accessory muscles of the guinea-pig. Z. Anat. Entwickl. Gesch. 140, 109–128. Furness, J. B., and Costa, M. (1976). Some observations on extra-adrenal chromaffin cells of the lower abdomen and pelvis. In ‘‘Chromaffin, Enterochromaffin and Related Cells’’ (R. E. Coupland and T. Fujita, Eds.), pp. 25–34. Elsevier, Amsterdam. Furnes, J. B., and Costa, M. (1987). ‘‘The Enteric Nervous System.’’ Churchill Livingstone, Edinburgh UK. Furness, J. B., Llewellyn-Smith, I. J., Bornstein, J. C., and Costa, M. (1988). Chemical neuroanatomy and the analysis of neuronal circuitry in the enteric nervous system. Hbk. Chem. Neuroanat. 6, 161–218. Furness, J. B., Morris, J. L., Gibbins, I. L., and Costa, M. (1989). Chemical coding of neurons and plurichemical transmission. Annu. Rev. Pharmacol. Toxicol. 29, 289–306. Furness, J. B., Johnson, P. J., Pompolo, S., and Bornstein, J. C. (1995a). Evidence that enteric motility reflexes can be initiated through entirely intrinsic mechanisms in the guinea-pig small intestine. Neurogastroenterol. Mot. 7, 89–96. Furness, J. B., Young, H. M., Pompolo, S., Bornstein, J. C., Kunze, W. A. A., and McConalogue, K. (1995b). Plurichemical transmission and chemical coding of neurons in the digestive tract. Gastroenterology 108, 554–563. Gabella, G. (1995). Autonomic nervous system. In ‘‘The Rat Nervous System’’ (G. Paxinos, Ed.), pp. 81–103. Academic Press, New York. Gabella, G., and Uvelius, B. (1993). Effect of decentralization or contralateral ganglionectomy on obstruction-induced hypertrophy of rat urinary bladder muscle and pelvic ganglion. J. Neurocytol. 22, 827–834. Gabella, G., Berggren, T., and Uvelius, B. (1992). Hypertrophy and reversal of hypertrophy in rat pelvic ganglion neurons. J. Neurocytol. 21, 649–662. Gallagher, J. P., Griffith, W. H., and Shinnick-Gallagher, P. (1982). Cholinergic transmission in cat parasympathetic neurones. J. Physiol. 332, 96–109. Garban, H., Marquez, D., Cai, L., Rajfer, J., and Gonzalez-Cadavid, N. F. (1995). Restoration of normal adult penile erectile response in aged rats by long-term treatment with androgens. Biol. Reprod. 53, 1365–1372. Gibbins, I. (1991). Autonomic nervous system. In ‘‘Encyclopedia of Human Biology,’’ pp. 535–550. Academic Press, New York. Gibbins, I. L. (1995). Chemical neuroanatomy of sympathetic ganglia. In ‘‘Autonomic Ganglia’’ (E. M. McLachlan, Ed.), pp. 73–122. Harwood Academic, Luxembourg. Gibbins, I. L., Furness, J. B., and Costa, M. (1987). Pathway-specific patterns of the coexistence of substance P, calcitonin gene-related peptide, cholecystokinin and dynorphin in neurons of the dorsal root ganglion of the guinea-pig. Cell Tissue Res. 248, 417–437. Giuliano, F., Rampin, O., Schirar, A., Jardin, A., and Rousseau, J.-P. (1993). Autonomic control of penile erection: Modulation by testosterone in the rat. J. Neuroendocrinol. 5, 677–683. Gonella, J., Bouvier, M., and Blanquet, F. (1987). Extrinsic nervous control of motility of small and large intestines and related sphincters. Physiol. Rev. 67, 902–961. Gosling, J. A., and Thompson, S. A. (1977). A neurohistochemical and histological study of peripheral autonomic neurons of the human bladder neck and prostate. Urol. Int. 32, 269–276. Greenwood, D., Coggeshall, R. E., and Hulsebosch, C. E. (1985). Sexual dimorphism in the numbers of neurons in the pelvic ganglia of adult rats. Brain Res. 340, 160–162.

UNUSUAL AUTONOMIC GANGLIA

57

Griffith, W. H., Gallagher, J. P., and Shinnick-Gallagher, P. (1980). An intracellular investigation of cat vesical pelvic ganglia. J. Neurophysiol. 43, 343–354. Griffith, W. H., Gallagher, J. P., and Shinnick-Gallagher, P. (1981). Sucrose-gap recordings of nerve-evoked potentials in mammalian parasympathetic ganglia. Brain Res. 209, 446–451. Gu, J., Polak, J. M., Islam, K. N., Marangos, P. J., Mina, S., Adrian, T., McGregor, G. P., O’Shaughnessy, D. J., and Bloom, S. R. (1983). Peptidergic innervation of the human male genital tract. J. Urol. 130, 386–391. Habecker, B. A., Symes, A. J., Stahl, N., Francis, N. J., Economides, A., Fink, J. S., Yancopoulos, G. D., and Landis, S. C. (1997). A sweat gland-derived differentiation activity acts through known cytokine signalling pathways. J. Biol. Chem. 272, 30421–30428. Hamberger, B., and Norberg, K.-A. (1965a). Adrenergic synaptic terminals and nerve cells in bladder ganglia of the cat. Int. J. Neuropharmacol. 4, 41–45. Hamberger, B., and Norberg, K. A. (1965b). Studies on some systems of adrenergic synaptic terminals in the abdominal ganglia of the cat. Acta Physiol. Scand. 65, 235–242. Hamill, R. W., and Guernsey, L. A. (1983). Hormonal regulation of sympathetic neuron development: The effects of neonatal castration. Dev. Brain Res. 11, 303–307. Hamill, R. W., and Schroeder, B. (1990). Hormonal regulation of adult sympathetic neurons: The effects of castration on neuropeptide Y, norepinephrine, and tyrosine hydroxylase activity. J. Neurobiol. 21, 731–742. Hamill, R. W., Earley, C. J., and Guernsey, L. A. (1984). Hormonal regulation of adult sympathetic neurons: The effects of castration on tyrosine hydroxylase activity. Brain Res. 299, 331–337. Hanani, M., and Maudlej, N. (1995). Intracellular recordings from intramural neurons in the guinea pig urinary bladder. J. Neurophysiol. 74, 2358–2365. Hart, B. J. (1967). Testosterone regulation of sexual reflexes in spinal male rats. Science 155, 1283–1284. Heaton, J. P. W., and Varrin, S. J. (1994). Effects of castration and exogenous testosterone supplementation in an animal model of penile erection. J. Urol. 151, 797–800. Hedlund, H., Fa¨ndriks, L., Delbro, D., and Fasth, S. (1985). On the transmission of sacral parasympathetic nervous influence on distal colonic and rectal motility in the cat. Acta Physiol. Scand. 125, 225–234. Hedlund, P., Alm, P., Hedlund, H., Larsson, B., and Andersson, K.-E. (1994). Localization and effects of pituitary adenylate cyclase-activating polypeptide (PACAP) in human penile erectile tissue. Acta Physiol. Scand. 150, 103–104. Hele´n, P., Panula, P., Yang, H.-Y. T., Hervonen, A., and Rapoport, S. I. (1984). Location of substance P-, bombesin-gastrin-releasing peptide-, [Met5] enkephalin- and [Met5] enkephalin-Arg6-Phe7-like immunoreactivities in adult human sympathetic ganglia. Neuroscience 12, 907–916. Hendry, I. A., and Iversen, L. L. (1973). Reduction in the concentration of nerve growth factor in mice after sialectomy and castration. Nature 243, 500–504. Henningsen, I., Liestol, K., Maehlen, J., and Nja˚, A. (1985). The selective innervation of guinea-pig superior cervical ganglion cells by sprouts from intact preganglionic neurons. J. Physiol. 358, 239–253. Hervonen, A., Kanerva, L., Lietzen, R., and Partanen, S. (1972). Effects of steroid hormones on the differentiation of the catecholamine storing cells of the paracervical ganglion of the rat uterus. Z. Zellforsch. 134, 519–527. Hervonen, A., Kanerva, L., and Lietzen, R. (1973). Histochemically demonstrable catecholamines and cholinesterases in the rat uterus during the oestrus cycle and pregnancy and after estrogen treatment. Acta Physiol. Scand. 87, 283–288. Ho¨kfelt, T., Elfvin, L.-G., Schultzberg, M., Goldstein, M., and Nilsson, G. (1977). On the occurrence of substance P-containing fibers in sympathetic ganglia: Immunohistochemical evidence. Brain Res. 132, 29–41.

58

JANET R. KEAST

Ho¨kfelt, T., Schultzberg, M., Elde, R., Nilsson, G., Terenius, L., Said, S., and Goldstein, M. (1978). Peptide neurons in peripheral tissues including the urinary tract: Immunohistochemical studies. Acta Pharmacol. Toxicol. 43, 79–89. Ho¨kfelt, T., Zhang, X., and Wiesenfeld-Hallin, Z. (1994). Messenger plasticity in primary sensory neurons following axotomy and its functional implications. TINS 17, 22–30. Holman, M. E., Muir, T. C., Szurszewski, J. H., and Yonemura, K. (1971). Effect of iontophoretic application of cholinergic agonists and their antagonists to guinea-pig pelvic ganglia. Br. J. Pharmacol. 41, 26–40. Holmquist, F., Persson, K., Bødker, A., and Andersson, K.-E. (1994). Some pre- and postjunctional effects of castration in rabbit isolated corpus cavernosum and urethra. J. Urol. 152, 1011–1016. Holzer, P., and Holzer-Petsche, U. (1997a). Tachykinins in the gut. Part I. Expression, release and motor function. Pharmacol. Ther. 73, 173–217. Holzer, P., and Holzer-Petsche, U. (1997b). Tachykinins in the gut. Part II. Roles in neural excitation, secretion and inflammation. Pharmacol. Ther. 73, 219–263. Hou, X. E., and Dahlstrom, A. (1995). Effects of decentralization on the levels of GAP-43 and p38 (synaptophysin) in sympathetic adrenergic neurons: A semiquantitative study using immunofluorescence and confocal laser scanning microscopy. Brain Res. 679, 49–63. Houdeau, E., Prud’homme, M.-J., Rousseau, A., and Rousseau, J.-P. (1995). Distribution of noradrenergic neurons in the female rat pelvic plexus and involvement in the genital tract innervation. J. Auton. Nervous System 54, 113–125. Hoyle, C. H. V. (1992). Transmission: Purines. In ‘‘Autonomic Neuroeffector Mechanisms’’ (G. Burnstock and C. H. V. Hoyle, Eds.), pp. 367–408. Harwood Academic, Chur, Switzerland. Hoyle, C. H. V., and Burnstock, G. (1993). Postganglionic efferent transmission in the bladder and urethra. In ‘‘Nervous Control of the Urogenital System’’ (C. A. Maggi, Ed.), pp. 349–382. Harwood Academic, Chur, Switzerland. Hulsebusch, C. E., and Coggeshall, R. E. (1982). An analysis of the axon populations in the nerves to the pelvic viscera in the rat. J. Comp. Neurol. 211, 1–10. Hulte´n, L. (1969). Extrinsic nervous control of colonic motility and blood flow. An experimental study in the cat. Acta Physiol. Scand. (Suppl.) 335, 1–116. Inyama, C. O., Hacker, G. W., Gu, J., Dahl, D., Bloom, S. R., and Polak, J. M. (1987). Cytochemical relationships in the paracervical ganglion (Frankenha¨user) of rat studied by immunocytochemistry. Neurosci. Lett. 55, 311–316. Ishii, D. N., and Shooter, E. M. (1975). Regulation of nerve growth factor synthesis in mouse submaxillary glands by testosterone. J. Neurochem. 25, 843–851. Ja¨nig, W., and McLachlan, E. M. (1987). Organization of the lumbar spinal outflow to distal colon and pelvic organs. Physiol. Rev. 67, 1332–1404. Ja¨nig, W., and McLachlan, E. M. (1992a). Characteristics of function-specific pathways in the sympathetic nervous system. TINS 15, 475–481. Ja¨nig, W., and McLachlan, E. M. (1992b). Specialized functional pathways are the building blocks of the autonomic nervous system. J. Auton. Nervous System 41, 3–14. Jen, P. Y. P., Dixon, J. S., Gearhart, J. P., and Gosling, J. A. (1996). Nitric oxide synthase and tyrosine hydroxylase are colocalized in nerves supplying the male genitourinary organs. J. Urol. 155, 1117–1121. Johnson, D. A. (1988). Regulation of intraganglionic synapses among rabbit parasympathetic neurones. J. Physiol. 397, 51–62. Ju, G., Ho¨kfelt, T., Brodin, E., Fahrenkrug, J., Fischer, J. A., Frey, P., Elde, R. P., and Brown, J. C. (1987). Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells. Cell Tissue Res. 247, 417–431.

UNUSUAL AUTONOMIC GANGLIA

59

Kaiser, F. E., Viosca, S. P., Morley, J. E., Mooradian, A. D., Davids, S. S., and Korenman, S. C. (1988). Impotence and aging: Clinical and hormonal factors. J. Am. Geriatric Soc. 36, 511–519. Kanerva, L., and Hervonen, A. (1976). SIF cells, short adrenergic neurons and vacuolated nerve cells of the paracervical (Frankenha¨user) ganglion. In ‘‘SIF Cells. Structure and Function of the Small, Intensely Fluorescent Sympathetic Cells’’ (O. Era¨nko¨, Ed.), Fogarty Int. Cr. Proc., Vol. 30, pp. 19–34. Natl. Inst. Health, Bethesda, MD. Kanerva, L., and Tera¨va¨inen, H. (1972). Electron microscopy of the paracervical (Frankenha¨user) ganglion of the adult rat. Z. Zellforsch. 129, 161–177. Kanerva, L., Lietzen, R., and Tera¨va¨inen, H. (1972). Catecholamines and cholinesterases in the paracervical (Frankenha¨user) ganglion of normal and pregnant rats. Acta Physiol. Scand. 86, 271–277. Karhula, T., Ha¨ppo¨la¨, O., Joh, T., and Wu, J.-Y. (1988). Localization of l-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac–superior mesenteric ganglion complex of the rat. Histochemistry 90, 255–260. Kawata, M. (1995). Roles of steroid hormones and their receptors in structural organization in the nervous system. Neurosci. Res. 24, 1–46. Kawatani, M., Lowe, I., Booth, A. M., and de Groat, W. C. (1983). The presence of leucineenkephalin in the sacral preganglionic pathways to the urinary bladder of the cat. Neurosci. Lett. 39, 143–148. Kawatani, M., Erdman, S. L., and de Groat, W. C. (1985). Vasoactive intestinal polypeptide and substance P in primary afferent pathways to the sacral spinal cord of the cat. J. Comp. Neurol. 241, 327–347. Kawatani, M., Nagel, J., and de Groat, W. C. (1986). Identification of neuropeptides in pelvic and pudendal nerve afferent pathways to the sacral spinal cord of the cat. J. Comp. Neurol. 249, 117–132. Kawatani, M., Shioda, S., Nakai, Y., Takashige, C., and de Groat, W. C. (1989). Ultrastructural analysis of enkephalinergic terminals in parasympathetic ganglia innervating the urinary bladder of the cat. J. Comp. Neurol. 288, 81–91. Keast, J. R. (1991). Patterns of co-existence of peptides and differences of nerve fibre types associated with noradrenergic and non-noradrenergic (putative cholinergic) neurons in the major pelvic ganglion of the male rat. Cell Tissue Res. 266, 405–415. Keast, J. R. (1992). Location and peptide content of pelvic neurons supplying the muscle and lamina propria of the rat vas deferens. J. Auton. Nervous System 40, 1–12. Keast, J. R. (1994a). Catecholamine innervation of the intestine of flying foxes (Pteropus spp.)—A substantial supply from enteric neurons. Cell Tissue Res. 276, 403–410. Keast, J. R. (1994b). Neuropeptide-containing axon terminals in the male rat major pelvic ganglion are primarily of sacral origin. J. Auton. Nervous System 47, 151–158. Keast, J. R. (1995a). Pelvic ganglia. In ‘‘Autonomic Ganglia’’ (E. M. McLachlan, Ed.), pp. 445–480. Harwood Academic, Luxembourg. Keast, J. R. (1995b). Visualization and immunohistochemical characterization of sympathetic and parasympathetic neurons in the male rat major pelvic ganglion. Neuroscience 66, 655–662. Keast, J. R., and Chiam, H.-C. (1994). Selective association of nerve fibres immunoreactive for substance P or bombesin with putative cholinergic neurons of the male rat major pelvic ganglion. Cell Tissue Res. 47, 151–158. Keast, J. R., and de Groat, W. C. (1989). Immunohistochemical characterization of pelvic neurons which project to the bladder, colon, or penis in rats. J. Comp. Neurol. 288, 387–400. Keast, J. R., and de Groat, W. C. (1990). Sympathetic modulation of cholinergic transmission in cat vesical ganglia is mediated by 움1- and 움2-adrenoceptors. Am. J. Physiol. 27, R44–R50. Keast, J. R., and de Groat, W. C. (1992). Segmental distribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats. J. Comp. Neurol. 319, 1–9.

60

JANET R. KEAST

Keast, J. R., and Saunders, R. J. (1998). Testosterone has potent, selective effects on the morphology of pelvic autonomic neurons which control the bladder, lower bowel and internal reproductive organs. Neuroscience 85, 543–546. Keast, J. R., Furness, J. B., and Costa, M. (1984). Origins of peptide and norepinephrine nerves in the mucosa of the guinea-pig small intestine. Gastroenterology 86, 637–644. Keast, J. R., Booth, A. M., and de Groat, W. C. (1989). Distribution of neurons in the major pelvic ganglion of the rat which supply the bladder, colon or penis. Cell Tissue Res. 256, 105–112. Keast, J. R., Luckensmeyer, G. B., and Schemann, M. (1995). All pelvic neurons in male rats contain synthetic enzymes for either noradrenaline or acetylcholine. Neurosci. Lett. 196, 209–212. Kennedy, C., and Krier, J. (1987a). ␦-Opioid receptors mediate inhibition of fast excitatory post-synaptic potentials in cat parasympathetic colonic ganglia. Br. J. Pharmacol. 92, 437–443. Kennedy, C., and Krier, J. (1987b). [Met5]enkephalin acts via ␦-opioid receptors to inhibit pelvic nerve-evoked contractions of cat distal colon. Br. J. Pharmacol. 92, 291–298. Kennedy, W. P. (1929). The ganglion cervicalia uteri and The oestrus hormone. Edin. Med. J. 36, 75–88. Kepper, M., and Keast, J. R. (1995). Location, immunohistochemical properties and spinal connections of pelvic autonomic neurons which innervate the rat prostate gland. Cell Tissue Res. 281, 533–542. Kepper, M., and Keast, J. R. (1997). Location, immunohistochemical features and spinal connections of autonomic neurons innervating the rat seminal vesicles. Biol. Reprod. 157, 1164–1174. Kepper, M. E., and Keast, J. R. (1998). Specific targeting of ganglion cell sprouts provides an additional mechanism for restoring peripheral motor circuits in pelvic ganglia after spinal nerve damage. J. Neurosci. 18, 7987–7995. Kihara, K., and de Groat, W. C. (1997). Sympathetic efferent pathways projecting bilaterally to the vas deferens in the rat. Anat. Rec. 248, 291–299. Kihara, H., Kakizaki, H., and de Groat, W. C. (1996). Reorganization of the innervation of the vas deferens after sympathetic decentralization. Am. J. Physiol. 271, R1481–R1488. Klemm, M., Wallace, D. J., and Hirst, G. D. (1997). Distribution of synaptic boutons around identified neurones lying in the cardiac plexus of the guinea-pig. J. Auton. Nervous System 66, 201–207. Klu¨ck, P. (1980). The autonomic innervation of the human urinary bladder, bladder neck and urethra: A histochemical study. Anat. Rec. 198, 439–447. Kojima, S., and Shimo, Y. (1995). Calcitonin gene-related peptide (CGRP)-enhanced nonadrenergic non-cholinergic contraction of guinea-pig proximal colon. Br. J. Pharmacol. 115, 1290–1294. Kolbeck, S. C., and Steers, W. D. (1993). Origin of neurons supplying the vas deferens of the rat. J. Urol. 149, 918–921. Kreulen, D. L., and Peters, S. (1986). Non-cholinergic transmission in a sympathetic ganglion of the guinea-pig elicited by colon distension. J. Physiol. 374, 315–334. Kreulen, D. L., and Szurszewski, J. H. (1979a). Electrophysiologic and morphologic basis for organization of neurons in prevertebral ganglia. In ‘‘Frontiers of Knowledge in the Diarrhoeal Diseases’’ (H. D. Janowitz and D. B. Sachar, Eds.), pp. 211–226. Projects in Health, New York. Kreulen, D. L., and Szurszewski, J. H. (1979b). Nerve pathways in celiac plexus of the guinea pig. Am. J. Physiol. 237, E90–E97. Krier, J., and Hartman, D. A. (1984). Electrical properties and synaptic connections to neurons in parasympathetic colonic ganglia of the cat. Am. J. Physiol. 247, G52–G61.

UNUSUAL AUTONOMIC GANGLIA

61

Kujat, R., Rose, C., and Wrobel, K.-H. (1993). The innervation of the bovine ductus deferens: Comparison of a modified acetylcholinesterase-reaction with immunoreactivities of cholinacetyltransferase and panneuronal markers. Histochemistry 99, 231–239. Kummer, W. (1992). Neuronal specificity and plasticity in the autonomic nervous system. Ann. Anat. 174, 409–417. Kummer, W., Gibbins, I. L., Stefan, P., and Kapoor, V. (1990). Catecholamines and catecholamine-synthesizing enzymes in guinea pig sensory ganglia. Cell Tissue Res. 261, 595–606. Kuntz, A., and Moseley, R. L. (1936). An experimental analysis of the pelvic autonomic ganglia in the cat. J. Comp. Neurol. 64, 63–75. Kuo, D. C., Hisamitsu, T., and de Groat, W. C. (1984). A sympathetic projection from sacral paravertebral ganglia to the pelvic nerve and to postganglionic nerves on the surface of the urinary bladder and large intestine of the cat. J. Comp. Neurol. 226, 76–78. Lamas, J. L. T., and Spadori, R. C. (1993). Effect of testosterone on the response of young rat vas deferens to norepinephrine. Gen. Pharmacol. 24, 185–189. Landis, S. C. (1996). The development of cholinergic sympathetic neurons: A role for neuropoietic cytokines? Perspect. Dev. Neurobiol. 4, 53–63. Langley, J. N., and Anderson, H. K. (1894). The constituents of the hypogastric nerves. J. Physiol. 17, 177–191. Langley, J. N., and Anderson, H. K. (1895a). The innervation of the pelvic and adjoining viscera. II. The bladder. J. Physiol. 19, 71–84. Langley, J. N., and Anderson, H. K. (1895b). On the innervation of the pelvic and adjoining viscera. Part I. The lower portion of the intestine. J. Physiol. 18, 67–105. Langley, J. N., and Anderson, H. K. (1896). The innervation of the pelvic and adjoining viscera. VII. Anatomical observations. J. Physiol. 20, 372–406. Langworthy, O. R. (1965). Innervation of the pelvic organs of the rat. Invest. Urol. 2, 491–511. Lara, H., Galleguillos, X., Arrau, J., and Belmar, J. (1985). Effect of castration and testosterone on norepinephrine storage and on the release of [3H]norepinephrine from rat vas deferens. Neurochem. Int. 7, 667–674. Leone, A. M., Wiklund, N. P., Ho¨kfelt, T., Brundin, L., and Moncada, S. (1994). Release of nitric oxide by nerve stimulation in the human urogenital tract. NeuroReport 5, 733–736. Le´ranth, C., and Feher, E. (1983). Synaptology and source of vasoactive intestinal polypeptide and substance P containing axons of the cat coeliac ganglion. An experimental electron microscopic immunohistochemical study. Neuroscience 10, 947–958. Levi-Montalcini, R., and Angeletti, P. U. (1964). Hormonal control of NGF in the submaxillary glands of mice. In ‘‘Salivary Glands and Their Secretion’’ (L. M. Sreebny and J. Meyer, Eds.), pp. 129–141. Pergamon, New York. Liestol, K., Maehlen, J., and Nja˚, A. (1987). Two types of synaptic selectivity and their interrelation during sprouting in the guinea-pig superior cervical ganglion. J. Physiol. 384, 233–245. Liu, M. T., Rothstein, J. D., Gershon, M. D., and Kirchgessner, A. L. (1997). Glutamatergic enteric neurons. J. Neurosci. 17, 4764–4784. Lubischer, J. L., and Arnold, A. P. (1994). Axotomy of developing rat spinal motoneurons: Cell survival, soma size, muscle recovery, and the influence of testosterone. J. Neurobiol. 26, 225–240. Luckensmeyer, G. B., and Keast, J. R. (1994). Projections from the prevertebral and major pelvic ganglia to the ileum and large intestine of the rat. J. Auton. Nervous System 49, 247–259. Luckensmeyer, G. B., and Keast, J. R. (1995a). Distribution and morphological characterisation of viscerofugal projections from the large intestine to the inferior mesenteric and pelvic ganglia in the male rat. Neuroscience 66, 663–671. Luckensmeyer, G. B., and Keast, J. R. (1995b). Immunohistochemical characterisation of sympathetic and parasympathetic pelvic neurons which project to the large intestine in the male rat. Cell Tissue Res. 281, 551–559.

62

JANET R. KEAST

Luckensmeyer, G. B., and Keast, J. R. (1996). Immunohistochemical characterisation of viscerofugal neurons projecting to the inferior mesenteric and major pelvic ganglia in the male rat. J. Auton. Nervous System 61, 6–16. Luckensmeyer, G. B., and Keast, J. R. (1998a). Activation of 움- and 웁-adrenoceptors by sympathetic nerve stimulation in the large intestine of the rat. J. Physiol. 510, 549–561. Luckensmeyer, G. B., and Keast, J. R. (1998b). Characterisation of the adventitial rectal ganglia in the male rat by their immunohistochemical features and projections. J. Comp. Neurol, 396, 429–441. Luckensmeyer, G. B., and Keast, J. R. (1998c). Projections of pelvic autonomic neurons within the lower bowel of the male rat: An anterograde labelling study. Neuroscience 84, 263–280. Lundberg, J. M., and Ho¨kfelt, T. (1986). Multiple coexistence of peptides and classical transmitters in peripheral autonomic and sensory neurons—Functions and pharmacological implications. Prog. Brain Res. 68, 241–262. MacDonald, A., and McGrath, J. C. (1980). The effects of castration on neurotransmission in the rat vas deferens. Br. J. Pharmacol. 69, 49–58. MacDonald, A., and McGrath, J. C. (1984). Post-natal development of functional neurotransmission in rat vas deferens. Br. J. Pharmacol. 82, 23–34. MacGrogan, D., Despre`s, G., Romand, R., and Dicou, E. (1991). Expression of the 웁-nerve growth factor gene in male sex organs of the mouse, rat, and guinea pig. J. Neurosci. Res. 28, 567–573. Macrae, I. M., Furness, J. B., and Costa, M. (1986). Distribution of subgroups of noradrenaline neurons in the coeliac ganglion of the guinea-pig. Cell Tissue Res. 244, 173–180. Maehlen, J., and Nja˚, A. (1981). Selective synapse formation during sprouting after partial denervation of the guinea-pig superior cervical ganglion. J. Physiol. 319, 555–567. Maehlen, J., and Nja˚, A. (1984). Rearrangement of synapses on guinea-pig sympathetic ganglion cells after partial interruption of the preganglionic nerve. J. Physiol. 348, 43–56. Maggi, C. A., Giuliani, S., Santicioli, P., Patacchini, R., and Meli, A. (1988). Neural pathways and pharmacological modulation of defecation reflex in rats. Gen. Pharmacol. 19, 517–524. Maggi, C. A., Giuliani, S., and Santicioli, P. (1997). CGRP potentiates excitatory transmission to the circular muscle of guinea-pig colon. Regul. Pept. 69, 127–136. Magnusson, S., Alm, P., and Kanje, M. (1996). Inducible nitric oxide synthase increases in regenerating rat ganglia. NeuroReport 96, 2046–2050. Mallory, B., Steers, W. D., and de Groat, W. C. (1989). Electrophysiological study of micturition reflexes in rats. Am. J. Physiol. 257, R410–R421. Malmfors, T., and Sachs, C. (1968). Degeneration of adrenergic nerves produced by 6-hydroxydopamine. Eur. J. Pharmacol. 3, 89–92. Marshall, J. M. (1970). Adrenergic innervation of the female reproductive tract: Anatomy, physiology and pharmacology. Ergeb. Physiol. 62, 6–67. Matthews, M. R., and Cuello, A. C. (1982). Substance P-immunoreactive peripheral branches of sensory neurones innervate guinea-pig sympathetic neurons. Proc. Natl. Acad. Sci. USA 79, 1668–1672. Mattiasson, A., Ekblad, E., Sundler, F., and Uvelius, B. (1985). Origin and distribution of neuropeptide Y-, vasoactive intestinal polypeptide- and substance P-containing nerve fibres in the urinary bladder of the rat. Cell Tissue Res. 239, 141–146. McConnell, J., Benson, G. S., and Wood, J. G. (1982). Autonomic innervation of the urogenital system: Adrenergic and cholinergic elements. Brain Res. Bull. 9, 679–694. McLachlan, E. M. (1985). The components of the hypogastric nerve in male and female guinea pigs. J. Auton. Nervous System 13, 327–342. McLachlan, E. M., and Meckler, R. L. (1989). Characteristics of synaptic input to three classes of sympathetic neurone in the coeliac ganglion of the guinea-pig. J. Physiol. 415, 109–129. McLachlan, E. M., Davies, P. J., Ha¨bler, H.-J., and Jamieson, J. (1997). On-going and reflex synaptic events in rat superior cervical ganglion cells. J. Physiol. 501, 165–182.

UNUSUAL AUTONOMIC GANGLIA

63

McNeill, D. L., Traugh, N. E., Vaidya, A. M., Hua, H. T., and Papka, R. E. (1992). Origin and distribution of NADPH-diaphorase-positive neurons and fibers innervating the urinary bladder of the rat. Neurosci. Lett. 147, 33–36. McRorie, J., Krier, J., and Adams, T. (1991). Morphology and projections of myenteric neurons to colonic fiber bundles of the cat. J. Auton. Nervous System. 32, 205–216. Melo, R. C. N., and Machado, C. R. S. (1993). Noradrenergic and acetylcholinesterase-positive nerve fibres of the uterus in sexually immature and cycling rats. Histochem. J. 25, 213–218. Melvin, J. E., and Hamill, R. W. (1987). The major pelvic ganglion: Androgen control of postnatal development. J. Neurosci. 7, 1607–1612. Melvin, J. E., and Hamill, R. W. (1989). Hypogastric ganglion perinatal development: Evidence for androgen specificity via androgen receptors. Brain Res. 485, 11–19. Melvin, J. E., McNeill, T. H., and Hamill, R. W. (1988). Biochemical and morphological effects of castration on the postorganizational development of the hypogastric ganglion. Dev. Brain Res. 38, 131–139. Melvin, J. E., McNeill, T. H., Hervonen, A., and Hamill, R. W. (1989). Organizational role of testosterone on the biochemical and morphological development of the hypogastric ganglion. Brain Res. 485, 1–10. Mills, T. M., Wiedmeier, V. T., and Stopper, V. S. (1992). Androgen maintenance of erectile function in the rat penis. Biol. Reprod. 46, 342–348. Miolan, J. P., and Niel, J. P. (1996). The mammalian sympathetic prevertebral ganglia: Integrative properties and role in the nervous control of digestive tract motility. J. Auton. Nervous System 58, 125–138. Mitchell, B. S., and Stauber, V. V. (1990). Morphological, histochemical and immunohistological studies of the paracervical ganglion in prepubertal, pregnant and adult non-pregnant guinea-pigs. J. Anat. 172, 177–189. Morgan, C., de Groat, W. C., and Nadelhaft, I. (1986). The spinal distribution of sympathetic preganglionic and visceral primary afferent neurons that send axons into the hypogastric nerves of the cat. J. Comp. Neurol. 243, 23–40. Morita, T., Tsuchiya, N., Tsujii, T., and Kondo, S. (1992). Changes of autonomic receptors following castration and estrogen administration in the male rabbit urethral smooth muscle. Tohoku J. Exp. Med. 166, 403–405. Morris, J. L. (1990). Neuropeptide Y inhibits relaxations of the guinea pig uterine artery produced by VIP. Peptides 11, 381–386. Morris, J. L. (1993). Co-transmission from autonomic vasodilator neurons supplying the guinea pig uterine artery. J. Auton. Nervous System 42, 11–22. Morris, J. L., and Gibbins, I. L. (1987). Neuronal colocalization of peptides, catecholamines, and catecholamine-synthesizing enzymes in guinea pig paracervical ganglia. J. Neurosci. 7, 3117–3130. Morris, J. L., and Gibbins, I. L. (1989). Co-localization and plasticity of transmitters in peripheral autonomic and sensory neurons. Int. J. Dev. Neurosci. 7, 521–531. Morris, J. L., and Gibbins, I. L. (1992). Co-transmission and neuromodulation. In ‘‘Autonomic Neuroeffector Mechanisms’’ (G. Burnstock and C. H. V. Hoyle, Eds.), pp. 33–120. Harwood Academic, Chur, Switzerland. Morris, J. L., and Murphy, R. (1988). Evidence that neuropeptide Y released from noradrenergic axons causes prolonged contraction of the guinea-pig uterine artery. J. Auton. Nervous System 24, 241–249. Murphy, D. D., and Segal, M. (1996). Regulation of dendritic spine density in cultured rat hippocampal neurons by steroid hormones. J. Neurosci. 16, 4059–4068. Nabekura, J., Oomura, Y., Minami, T., Mizuno, Y., and Fukuda, A. (1986). Mechanism of the rapid effect of 17웁-estradiol on medial amygdala neurons. Science 233, 226–228. Nadelhaft, I., and McKenna, K. E. (1987). Sexual dimorphism in sympathetic preganglionic neurons of the rat hypogastric nerve. J. Comp. Neurol. 256, 308–315.

64

JANET R. KEAST

Nadelhaft, I., and Vera, P. L. (1991). Neurons labelled after the application of tracer to the distal stump of the transected hypogastric nerve in the rat. J. Auton. Nervous System 36, 87–96. Nadelhaft, I., Vera, P. L., and Steinbacher, B. (1993). Hypertrophic neurons innervating the urinary bladder and colon of the streptozotocin-diabetic rat. Brain Res. 609, 277–283. Nagao, T., Fujito, A., Takeuchi, T., and Hata, F. (1994). Changes in neuronal contribution to contractile responses of vas deferens of young and adult guinea pigs. J. Auton. Nervous System 50, 87–92. Neuhuber, W. L., Appelt, M., Polak, J. M., Baier-Kustermann, W., Abelli, L., and Ferri, G.-L. (1993). Rectospinal neurons: Cell bodies, pathways, immunocytochemistry and ultrastructure. Neuroscience 56, 367–378. Newton, B. W. (1992). A sexually dimorphic population of galanin-like neurons in the rat lumbar spinal cord: Functional implications. Neurosci. Lett. 137, 119–122. Newton, B. W., and Hamill, R. W. (1988). Neuropeptide Y immunoreactivity is preferentially located in rat lumbar sexually dimorphic nuclei. Neurosci. Lett. 94, 10–16. Newton, B. W., Unger, J., and Hamill, R. W. (1990). Calcitonin gene-related peptide and somatostatin immunoreactivities in the rat lumbar spinal cord: Sexually dimorphic aspects. Neuroscience 37, 471–489. Nishimura, T., Tokimasa, T., and Akasu, T. (1988). Calcium-dependent potassium conductance in neurons of rabbit vesical pelvic ganglia. J. Auton. Nervous System 24, 133–145. Nja˚, A., and Purves, D. (1977). Specific innervation of the guinea-pig superior cervical ganglion cells by different preganglionic fibres arising from different levels of the spinal cord. J. Physiol. 264, 565–583. Norberg, K.-A. (1964). Adrenergic innervation of the intestinal wall studied by fluorescence microscopy. Int. J. Neuropharmacol. 3, 379–382. Owman, C., and Sjo¨berg, N. O. (1967). Difference in rates of depletion and recovery of noradrenaline in ‘‘short’’ and ‘‘long’’ sympathetic nerves after reserpine treatment. Life Sci. 6, 2549–2556. Owman, C., and Sjo¨strand, C. (1965). Short adrenergic neurons and catecholamine-containing cells in vas deferens and accessory male genital glands of different mammals. Z. Zellforsch. 66, 300–320. Owman, C. L., Sjo¨berg, N.-O., and Swedin, G. (1971). Histochemical and chemical studies on pre- and postnatal development of the different systems of ‘‘short’’ and ‘‘long’’ adrenergic neurons in peripheral organs of the rat. Z. Zellforsch. 116, 319–341. Owman, C., Alm, P., Rosengren, E., Sjo¨berg, N.-O., and Thorbert, G. (1975). Variations in the level of uterine norepinephrine during pregnancy in the guinea pig. Am. J. Obstet. Gynecol. 127, 811–817. Pacheco, P., Martinez-Gomez, M., Whipple, B., Beyer, C., and Komisaruk, B. R. (1989). Somato-motor components of the pelvic and pudendal nerves of the female rat. Brain Res. 490, 85–94. Papka, R. E. (1990). Some nerve endings in the rat pelvic paracervical autonomic ganglia and varicosities contain calcitonin gene-related peptide and originate from dorsal root ganglia. Neuroscience 39, 459–470. Papka, R. E., and McNeill, D. L. (1993). Light- and electron-microscopic study of synaptic connections in the paracervical ganglion of the female rat: Special reference to calcitonin gene-related peptide-, galanin- and tachykinin (substance P and neurokinin A)immunoreactive nerve fibers and terminals. Cell Tissue Res. 271, 417–428. Papka, R. E., and Traurig, H. H. (1989). Galanin-immunoreactive nerves in the female rat paracervical ganglion and uterine cervix: Distribution and reaction to capsaicin. Cell Tissue Res. 257, 41–51. Papka, R. E., Cotton, J. P., and Traurig, H. H. (1985). Comparative distribution of neuropeptide tyrosine, vasoactive intestinal polypeptide-, substance P-IR, acetylcholinesterase-positive

UNUSUAL AUTONOMIC GANGLIA

65

and noradrenergic nerves in the reproductive tract of the female rat. Cell Tissue Res. 242, 475–490. Papka, R. E., Traurig, H. H., and Klenn, P. (1987). Paracervical ganglia of the female rat: Histochemistry and immunohistochemistry of neurons, SIF cells, and nerve terminals. Am. J. Anat. 179, 243–252. Papka, R. E., Newton, B. W., and McNeil, D. L. (1991). Origin of galanin-immunoreactive nerve fibres in the rat paracervical autonomic ganglia and uterine cervix. J. Auton. Nervous System 33, 25–34. Papka, R. E., McCurdy, J. R., Williams, S. J., Mayer, B., Marson, L., and Platt, K. B. (1995a). Parasympathetic preganglionic neurons in the spinal cord involved in uterine innervation are cholinergic and nitric oxide-containing. Anat. Rec. 241, 554–562. Papka, R. E., McNeill, D. L., Thompson, D., and Schmidt, H. H. H. W. (1995b). Nitric oxide nerves in the uterus are parasympathetic, sensory, and contain neuropeptides. Cell Tissue Res. 279, 339–349. Papka, R. E., Srinivasan, B., Miller, K. E., and Hayashi, S. (1997). Localization of estrogen receptor protein and estrogen receptor messenger RNA in peripheral autonomic and sensory neurons. Neuroscience 79, 1153–1163. Partanen, M., and Hervonen, A. (1979). The effect of long-term castration on the histochemically demonstrable catecholamines in the hypogastric ganglion of the rat. J. Auton. Nervous System 1, 139–147. Partanen, M., Hervonen, A., and Santer, R. M. (1980). The effect of ageing on the SIF-cells in the hypogastric (main pelvic) ganglion of the rat. Histochemistry 66, 99–112. Pauza, D. H., Skripkiene, G., Skripka, V., Pauziene, N., and Stropus, R. (1997). Morphological study of neurons in the nerve plexus on heart base of rats and guinea pigs. J. Auton. Nervous System 62, 1–12. Payette, R. F., Tennyson, V. M., Pham, T. D., Mawe, G. M., Pomeranz, H. D., Rothman, T. P., and Gershon, M. D. (1987). Origin and morphology of nerve fibers in the aganglionic colon of the lethal spotted (ls/ls) mutant mouse. J. Comp. Neurol. 257, 237–252. Penson, D. F., Ng, C., Cai, L., Rajfer, J., and Gonza´lez-Cadavid, N. F. (1996). Androgen and pituitary control of penile nitric oxide synthase and erectile function in the rat. Biol. Reprod. 55, 567–574. Persson, K., and Andersson, K. E. (1992). Nitric oxide and relaxation of pig lower urinary tract. Br. J. Pharmacol. 106, 416–422. Persson, K., Alm, P., Johansson, K., Larsson, B., and Andersson, K.-E. (1995). Coexistence of nitrergic, peptidergic and acetylcholinesterase-positive nerves in the pig lower urinary tract. J. Auton. Nervous System 52, 225–236. Purinton, P. T., Fletcher, T. F., and Bradley, W. E. (1973). Gross and light microscopic features of the pelvic plexus in the rat. Anat. Rec. 175, 697–706. Purinton, P. T., Fletcher, T. F., and Bradley, W. E. (1976). Innervation of pelvic viscera in the rat: Evoked potentials in nerves to bladder and penis (clitoris). Invest. Urol. 14, 28–32. Purves, D., and Lichtman, J. W. (1985). Geometrical differences among homologous neurons in mammals. Science 228, 298–302. Purves, D., Thompson, W., and Yip, J. W. (1981). Re-innervation of ganglia transplanted to the neck from different levels of the guinea-pig sympathetic chain. J. Physiol. 313, 49–63. Rand, M. J. (1992). Nitrergic transmission: Nitric oxide as a mediator of non-adrenergic, noncholinergic neuroeffector transmission. Clin. Exp. Pharmacol. Physiol. 19, 147–169. Rand, M. J., and Li, C. G. (1995). Nitric oxide as a neurotransmitter in peripheral nerves: Nature of transmitter and mechanism of transmission. Annu. Rev. Physiol. 57, 659–682. Rand, M. N., and Breedlove, S. M. (1995). Androgen alters the dendritic arbors of SNB motoneurons by acting upon their target muscles. J. Neurosci. 15, 4408–4416. Rauchenwald, M., Steers, W. D., and Desjardins, C. (1995). Efferent innervation of the rat testis. Biol. Reprod. 52, 1136–1143.

66

JANET R. KEAST

Ren, L.-M., Hoyle, C. H. V., and Burnstock, G. (1996). Developmental changes in sympathetic contraction of the circular muscle layer in the guinea-pig vas deferens. Eur. J. Pharmacol. 318, 411–417. Rogers, H., and Henderson, G. (1990). Activation of 애- and ␦-opioid receptors present on the same nerve terminals depress transmitter release in the mouse hypogastric ganglion. Br. J. Pharmacol. 101, 505–512. Rogers, H., Kennedy, C., and Henderson, G. (1990). Characterization of the neurons of the mouse hypogastric ganglion: Morphology and electrophysiology. J. Auton. Nervous System 29, 255–270. Rush, R. A., Saltis, J., Smet, P. J., and Williams, R. (1992). Survival factors for autonomic neurones. In ‘‘The Autonomic Nervous System: Development, Regeneration and Plasticity of the Autonomic Nervous System’’ (I. A. Hendry and C. E. Hill, Eds.), pp. 305–364. Harwood Academic, Chur, Switzerland. Sanders, K. M., and Ward, S. M. (1992). Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission. Am. J. Physiol. 262, G379–G392. Sargent, P. B., and Dennis, M. J. (1977). Formation of synapses between parasympathetic neurones deprived of preganglionic innervation. Nature 268, 456–458. Sargent, P. B., and Dennis, M. J. (1981). The influence of normal innervation upon abnormal synaptic connections between frog parasympathetic neurons. Dev. Biol. 81, 65–73. Saria, A., Ma, R. C., Dun, N. J., Theodorsson-Norheim, E., and Lundberg, J. M. (1987). Neurokinin A in capsaicin-sensitive neurons of the guinea-pig inferior mesenteric ganglia: An additional putative mediator for non-cholinergic excitatory postsynaptic potentials. Neuroscience 21, 951–958. Saum, W. R., and de Groat, W. C. (1972a). Antagonism by bulbocapnine of adrenergic inhibition in parasympathetic ganglia in the urinary bladder. Brain Res. 37, 340–344. Saum, W. R., and de Groat, W. C. (1972b). Parasympathetic ganglia: Activation of an adrenergic inhibitory mechanism by cholinomimetic agents. Science 175, 659–661. Schirar, A., Chang, C., and Rousseau, J. P. (1997). Localization of androgen receptor in nitric oxide synthase- and vasoactive intestinal peptide-containing neurons of the major pelvic ganglion innervating the rat penis. J. Neuroendocrinol. 9, 141–150. Schnitzlein, H. N., Hoffman, H. H., Tucker, C. C., and Quigley, M. B. (1960). The pelvic splanchnic nerves of the male rhesus monkey. J. Comp. Neurol. 114, 51–65. Schofield, G. G., and Ikeda, S. R. (1989). Potassium currents of acutely isolated adult rat superior cervical ganglion neurons. Brain Res. 485, 205–214. Schroder, F. H. (1991). Endocrine therapy: Where do we stand and where are we going? Cancer Surv. 11, 177–194. Schultzberg, M., Ho¨kfelt, T., Nilsson, G., Terenius, L., Rehfeld, J. F., Brown, M., Elde, R., Goldstein, M., and Said, S. (1980). Distribution of peptide- and catecholamine-containing neurons in the gastrointestinal tract of rat and guinea-pig: Immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/cholecystokinin, neurotensin and dopamine 웁-hydroxylase. Neuroscience 5, 689–744. Senba, E., and Tohyama, M. (1988). Calcitonin gene-related peptide containing autonomic efferent pathways to the pelvic ganglia of the rat. Brain Res. 449, 386–390. Sjo¨berg, N.-O. (1965). The adrenergic transmitter of the female reproductive tract. Acta Physiol. Scand. (Suppl.) 305, 5–26. Sjo¨strand, N. O. (1965). The adrenergic innervation of the vas deferens and the accessory male genital glands. Acta Physiol. Scand. (Suppl.) 65, 1–82. Sjo¨strand, N. O., and Swedin, G. (1976). Influence of age, growth, castration and testosterone treatment on the noradrenaline levels of the ductus deferens and the auxiliary male reproductive glands in the rat. Acta Physiol. Scand. 98, 323–338. Snider, W. D. (1987). The dendritic complexity and innervation of submandibular neurons in five species of mammals. J. Neurosci. 7, 1760–1768.

UNUSUAL AUTONOMIC GANGLIA

67

Steers, W. D., Mallory, B., and de Groat, W. C. (1988). Electrophysiological study of neural activity in penile nerve of the rat. Am. J. Physiol. 254, R989–R1000. Steers, W. D., Ciambotti, J., Erdman, S., and de Groat, W. C. (1990). Morphological plasticity in efferent pathways to the urinary bladder of the rat following urethral obstruction. J. Neurosci. 10, 1943–1951. Steers, W. D., Ciambotti, J., Etzel, B., Erdman, S., and de Groat, W. C. (1991a). Alterations in afferent pathways from the urinary bladder of the rat in response to partial urethral obstruction. J. Comp. Neurol. 310, 401–410. Steers, W. D., Kolbeck, S., Creedon, D. J., and Tuttle, J.B. (1991b). Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J. Clin. Invest. 88, 1709–1715. Steers, W. D., Mackway-Gerardi, A. M., Ciambotti, J., and de Groat, W. C. (1994). Alterations in neural pathways to the urinary bladder of the rat in response to streptozotocin-induced diabetes. J. Auton. Nervous System 47, 83–94. Steers, W. D., Creedon, D. J., and Tuttle, J. B. (1996). Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy. J. Urol. 155, 379–385. Stief, C. G., Bahren, W., Scherb, W., and Gall, H. (1989). Primary erectile dysfunction. J. Urol. 141, 315–319. Stjernquist, M., and Owman, C. (1990). Further evidence for a prejunctional action of neuropeptide Y on cholinergic motor neurons in the rat uterine cervix. Acta Physiol. Scand. 138, 95–96. Stjernquist, M., Alm, P., Ekman, R.,Owman, C., Sjo¨berg, N.-O., and Sundler, F. (1985). Levels of neural vasoactive intestinal polypeptide in rat uterus are markedly changed in association with pregnancy as shown by immunocytochemistry and radioimmunoassay. Biol. Reprod. 33, 157–163. Sullivan, K. A., Traurig, H. H., and Papka, R. E. (1994). Ontogeny of neurotransmitter systems in the paracervical ganglion and uterine cervix of the rat. Anat. Rec. 240, 377–386. Surprenant, A. (1994). Control of the gastrointestinal tract by enteric neurons. Annu. Rev. Physiol. 56, 117–140. Suzuki, Y., Ishi, H., and Arai, Y. (1983). Prenatal exposure of male mice to androgen increases neuron number in the hypogastric ganglion. Dev. Brain Res. 10, 151–154. Szurszewski, J. H. (1981). Physiology of mammalian prevertebral ganglia. Annu. Rev. Physiol. 43, 53–68. Tabatabai, M., Booth, A. M., and de Groat, W. C. (1986). Morphological and electrophysiological properties of pelvic ganglion cells in the rat. Brain Res. 382, 61–70. Tanaka, S., Zukeran, C., and Nakagawa, S. (1983). A macroscopical study of the somatic and visceral nerves innervating the male rat lowest digestive tract. Jpn. J. Anat. 58, 1–13. Taxi, J., and Euge`ne, D. (1995). Effects of axotomy, deafferentation, and reinnervation on sympathetic ganglionic synapses: A comparative study. Int. Rev. Cytol. 159, 195–263. Te, A. E., Santarosa, R. P., Koo, H. P., Buttyan, R., Greene, L. A., Kaplan, S. A., Olsson, C. A., and Shabsigh, R. (1994). Neurotrophic factors in the rat penis. J. Urol. 152, 2167–2172. Traurig, H. H., and Papka, R. E. (1993). Autonomic efferent and visceral sensory innervation of the female reproductive system: Special reference to the functional roles of nerves in reproductive organs. In ‘‘Nervous Control of the Urogenital System’’ (C. A. Maggi, Ed.), pp. 103–142. Harwood Academic, Chur, Switzerland. Trumble, H. C. (1933). The plan of the visceral nerves in the lumbar and sacral outflows of the autonomic nervous system. Br. J. Surg. 21, 664–676. Tuttle, J. B., and Steers, W. D. (1992). Nerve growth factor responsiveness of major pelvic ganglion neurons from the adult rat. Brain Res. 588, 29–40. Tuttle, J. B., Mackey, T., and Steers, W. D. (1994a). NGF, bFGF and CNTF increase survival of major pelvic ganglion neurons cultured from the adult rat. Neurosci. Lett. 173, 94–98.

68

JANET R. KEAST

Tuttle, J. B., Steers, W. D., Albo, M., and Nataluk, E. (1994b). Neural input regulates tissue NGF and growth of the adult rat urinary bladder. J. Auton. Nervous System 49, 147–158. Ventura, S., and Burnstock, G. (1996). Variation in nitric oxide synthase-immunoreactive nerve fibres with age and along the length of the vas deferens in the rat. Cell Tissue Res. 285, 427–434. Vera, P. L., and Nadelhaft, I. (1992). Afferent and sympathetic innervation of the dome and the base of the urinary bladder of the female rat. Brain Res. 29, 651–658. Vizzard, M. A., Erdman, S. L., Fo¨rstermann, U., and de Groat, W. C. (1994a). Differential distribution of nitric oxide synthase in neural pathways to the urogenital organs (urethra, penis, urinary bladder) of the rat. Brain Res. 646, 279–291. Vizzard, M. A., Erdman, S. L., Roppolo, J. R., Fo¨rstermann, U., and de Groat, W. C. (1994b). Differential localization of neuronal nitric oxide synthase immunoreactivity and NADPHdiaphorase activity in the cat spinal cord. Cell Tissue Res. 278, 299–309. Vulchanova, L., Arvidsson, U., Riedl, M., Wang, J., Buell, G., Surprenant, A., North, R. A., and Elde, R. (1996). Differential distribution of two ATP-gated channels (P2x receptors) determined by immunocytochemistry. Proc. Natl. Acad. Sci. USA 93, 8063–8067. Wakade, A. R., Garcia, A. G., and Kirkepar, S. M. (1975). Effect of castration on the smooth muscle cells of the internal sex organs of the rat: Influence of the smooth muscle on the sympathetic neurons innervating the vas deferens, seminal vesicle and coagulating gland. J. Pharmacol. Exp. Ther. 193, 424–434. Wang, B. R., Senba, E., and Tohyama, M. (1990). Met5-enkephalin-Arg6-Gly7-Leu8- like immunoreactivity in the pelvic ganglion of the male rat: A light and electron microscopic study. J. Comp. Neurol. 293, 26–38. Wang, F. B., Holst, M.-C., and Powley, T. L. (1995). The ratio of pre- to postganglionic neurons and related issues in the autonomic nervous system. Brain Res. Rev. 21, 93–115. Warburton, A. L., and Santer, R.M. (1993). Localisation of NADPH-diaphorase and acetylcholinesterase activities and of tyrosine hydroxylase and neuropeptide-Y immunoreactivity in neurons of the hypogastric ganglion of young adult and aged rats. J. Auton. Nervous System 45, 155–163. Warburton, A. L., and Santer, R. M. (1995). Decrease in synapsin I staining in the hypogastric ganglion of aged rats. Neurosci. Lett. 194, 157–160. Warren, D. A., and Lavidis, N. A. (1996). Effect of opiates on transmitter release from visualized hypogastric boutons innervating the rat pelvic ganglia. Br. J. Pharmacol. 118, 1913–1918. Watanabe, H. (1971). Adrenergic nerve elements in the hypogastric ganglion of the guinea pig. Am. J. Anat. 130, 305–330. Wehling, M. (1997). Specific, nongenomic actions of steroid hormones. Annu. Rev. Physiol. 59, 365–393. Weihe, E., Tao Cheng, J. H., Schafer, M. K., Erickson, J. D., and Eiden, L. E. (1996). Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles. Proc. Natl. Acad. Sci. USA 93, 3547–3552. Wozniak, W., and Skowronska, U. (1967). Comparative anatomy of pelvic plexus in cat, dog, rabbit, macaque and man. Anat. Anz. 120, 457–473. Wright, L. L., and Smolen, A. J. (1985). Synaptogenic effects of neonatal estradiol treatment in rat superior cervical ganglia. Dev. Brain Res. 21, 161–165. Yamakado, M., and Yohro, T. (1977). Population and structure of nerve cells in mouse submandibular ganglion. Anat. Embryol. (Berlin) 150, 301–312. Yokota, R., and Burnstock, G. (1983). Synaptic organisation of the pelvic ganglion in the guinea-pig. Cell Tissue Res. 232, 379–397. Yoshimura, N., and de Groat, W. C. (1996). Characterization of voltage-sensitive Na⫹ and K⫹ currents recorded from acutely dissociated pelvic ganglion neurons of the adult rat. J. Neurophysiol. 76, 2508–2521.

UNUSUAL AUTONOMIC GANGLIA

69

Yu, W. A. (1994). Nitric oxide synthase in motor neurons after axotomy. J. Histochem. Cytochem. 42, 451–457. Zhu, Y., and Yakel, J. L. (1997). Modulation of Ca2⫹ currents by various G protein-coupled receptors in sympathetic neurons of male rat pelvic ganglia. J. Neurophysiol. 78, 780–780. Zhu, Y., Zboran, E. L., and Ikeda, S. R. (1995). Phenotype-specific expression of T-type calcium channels in neurons of the major pelvic ganglion of the adult male rat. J. Physiol. 489, 363–375. Zoubek, J., Somogyi, G. T., and de Groat, W. C. (1993). A comparison of inhibitory effects of neuropeptide Y on rat urinary bladder, urethra and vas deferens. Am. J. Physiol. 265, R537–R543.

This Page Intentionally Left Blank

Mechanisms and Control of Embryonic Genome Activation in Mammalian Embryos Keith E. Latham The Fels Institute for Cancer Research and Molecular Biology and the Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Activation of transcription within the embryonic genome (EGA) after fertilization is a complex process requiring a carefully coordinated series of nuclear and cytoplasmic events, which collectively ensure that the two parental genomes can be faithfully reprogrammed and restructured before transcription occurs. Available data indicate that inappropriate transcription of some genes during the period of nuclear reprogramming can have long-term detrimental effects on the embryo. Therefore, precise control over the time of EGA is essential for normal embryogenesis. In most mammals, genome activation occurs in a stepwise manner. In the mouse, for example, some transcription occurs during the second half of the one-cell stage, and then a much greater phase of genome activation occurs in two waves during the two-cell stage, with the second wave producing the largest onset of de novo gene expression. Changes in nuclear structure, chromatin structure, and cytoplasmic macromolecular content appear to regulate these periods of transcriptional activation. A model is presented in which a combination of cell cycledependent events and both translational and posttranslational regulatory mechanisms within the cytoplasm play key roles in mediating and regulating EGA. KEY WORDS: Embryonic genome activation, Zygotic genome activation, Transcriptional control, Embryo, Chromatin structure, Nuclear reprogramming, Fertility, Fertilization, Oocyte. 䊚 1999 Academic Press.

I. Introduction The union of sperm and egg results in the union of two parental genomes within a common egg cytoplasm. The egg cytoplasm then executes what International Review of Cytology, Vol. 193 0074-7696/99 $30.00

71

Copyright 䉷 1999 by Academic Press. All rights of reproduction in any form reserved.

72

KEITH E. LATHAM

may be considered its primary function—the conversion of the two parental genomes into a single embryonic genome that is both developmentally totipotent and endowed with the necessary epigenetic programming to permit it to initiate the developmental pathway leading to the formation of a new organism. To accomplish this task, the oocyte cytoplasm must terminate the nuclear programs that led to the formation of sperm and egg and then restructure both genomes to the embryonic state. In addition, recent studies indicate that in mammals the oocyte cytoplasm may have the ability to modulate genomic imprinting information in the paternal genome to make it compatible with its own maternal genome (Latham, 1994; Latham and Sapienza, 1998; Latham and Solter, 1991). The ability of the oocyte cytoplasm to reprogram nuclear function is extensive, and many nuclear transplantation studies have documented the ability of the oocyte cytoplasm to terminate gene expression programs of transplanted nuclei and then reactivate expression of appropriate stage-specific genes (Barnes et al., 1987; Borsuk et al., 1996; DiBerardino, 1997; Howlett et al., 1987; Latham et al., 1991b, 1994; Prather and Rickords, 1992; van Stekelenburg-Hamers et al., 1994). Recently, the remarkable ability of the oocyte cytoplasm to reprogram nuclear function was demonstrated by the ability of fetal and adult somatic cell nuclei to support development to adulthood when transplanted to enucleated oocytes (Cibelli et al., 1998a,b; Wakayama et al., 1998; Wilmut et al., 1997). As remarkable as is the ability of the oocyte cytoplasm to reprogram nuclear function, there must exist an equally remarkable molecular mechanism for controlling the time at which gene transcription begins. Although the oocyte is endowed with an abundant supply of RNA and protein to support embryonic metabolism, these resources are limited, thus creating a need to initiate embryonic gene expression before this maternal endowment becomes depleted through degradation. This need is made all the more urgent as the bulk of maternal mRNA becomes polyadenylated, recruited for translation, and then degraded within a defined period after fertilization, before embryonic genome activation. This need to initiate embryonic gene transcription before maternal resources are exhausted, however, must be balanced by the need to delay gene transcription until nuclear remodeling and reprogramming are completed. If gene transcription occurs before reprogramming is completed, there is a chance that an inappropriate array of new embryonic transcripts may be produced, and this could result in embryo lethality or abnormal development. Moreover, data from nuclear transplantation studies indicate that exposure of transcriptionally active genes to oocyte cytoplasm can result in irreversible repression of some genes (Latham et al., 1994; Smith et al., 1996); the results of other nuclear transplantation studies exploring the effect of recipient cell cycle stage on development may be at least partly explicable on this basis (Robl et al.,

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

73

1986; Smith et al., 1988, 1990). These observations support the idea that delaying gene transcription until after nuclear reprogramming is completed may serve an essential protective function that prevents inappropriate epigenetic modifications and gene repression. Thus, it appears that the time of onset of embryonic gene transcription must be controlled precisely in order for development to succeed. Controlling embryonic genome activation (EGA) poses a significant regulatory problem for the embryo. The transcription of literally thousands of genes must at first be repressed and then initiated coordinately. Along with activation of thousands of structural genes (i.e., pol II transcription units), nucleolar formation and onset of synthesis of ribosomal RNA must also occur (Brinsko et al., 1995; Geuskens and Alexandre, 1984; Baran et al., 1996, 1997; Chartrain et al., 1987; Grondahl and Hyttel, 1996; Kopecny et al., 1989). The available data indicate that EGA is a multistep process consisting of profound changes in the oocyte cytoplasm and nuclear structure. EGA must occur within the context of a dramatically changing chromatin structure that does not appear to support initially the same regulatory controls as the genomes of somatic cells. EGA apparently requires several incremental activation events that cause certain groups of genes to become transcriptionally activated at different times. In addition, EGA must initially be mediated and regulated entirely by posttranscriptional mechanisms (although the possibility exists that the expression of some of the genes transcribed in one of the early bursts of transcription promotes later activation events). In order to explore the mechanisms that mediate and regulate EGA, this review will summarize the available information related to the formation of the embryonic genome, changes in genome structure, and the role of DNA replication, protein synthesis, and posttranslational processes in controlling the time of EGA. In addition, the relevance of early embryonic genome modifications to understanding nuclear reprogramming after nuclear transplantation will be discussed.

II. Forming the Embryonic Genome A critical step in the overall process leading to EGA is the formation of the embryonic genome from the two gametic genomes. This is a complex process involving changes in the nuclear envelope and extensive changes in chromatin proteins as the pronuclei are formed and eventually united into a single embryonic genome. The available data, obtained from a variety of species and from many different approaches, including in vivo observations, in vitro systems based on oocyte lysates, and nuclear transplantation and intracytoplasmic sperm injection (ICSI) studies, indicate that both

74

KEITH E. LATHAM

kinds of change are required to establish the ability for the embryo to regulate its genome appropriately. Immediately after fertilization, many essential events must occur before pronuclei can form. The sperm nucleus must be broken down and then reformed with a new, permeable nuclear envelope. Subsequently, the paternal (sperm) pronucleus undergoes chromatin decondensation and repackaging and swelling, followed by the formation of nucleoli and additional modifications, culminating in the early transcriptional activation of some genes during the one-cell stage. The egg must also complete meiosis and form a maternal pronucleus. The formation of both pronuclei appears to be under tight maternal control so that the pronuclei form at specific times. The events that contribute to pronucleus formation and the timing of paternal and maternal pronuclear formation may be important determinants of the transcriptional activity that will be observed within the two pronuclei and possibly later in the embryonic genome after the pronuclei are united. Therefore, it is worthwhile to review the molecular and structural aspects of pronucleus formation as well as differences in the timing of formation and transcriptional properties of the paternal and maternal pronuclei. A. Pronucleus Formation Pronucleus (PN) formation has been most extensively studied with respect to the formation of the paternal PN for the obvious reason that the transition from a highly condensed, inert sperm nucleus to a large and in some cases transcriptionally active paternal PN is a very dramatic event. The two major components of paternal PN formation that need to be considered are how a nonpermeable sperm nuclear envelope is replaced with a permeable nuclear envelope and how the chromatin packaging proteins of the sperm are replaced with oocyte-specific or cleavage stage-specific chromatin proteins. Many studies of paternal PN formation have been conducted in invertebrate species, such as the sea urchin, and these studies have provided useful insight into the molecular mechanisms that may be exploited during the process. Although PN formation obviously differs in significant ways between mammals and other organisms, it is nevertheless useful to review what has been learned from the studies of mammalian embryos and nonmammalian embryos, and to point out aspects of PN formation that differ. 1. Replacement of Sperm Nuclear Envelope with a Permeable PN Envelope The first step in paternal PN formation occurs during the fertilization process as the sperm perinuclear theca binds to the oocyte microvilli and

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

75

becomes disrupted (Sutovsky et al., 1997). Failure of this step inhibits paternal PN formation. The next step in paternal PN formation is the breakdown of the sperm nuclear envelope. In fish, and probably most organisms, sperm nuclear envelope breakdown is essential before PN formation can occur (Yamashita et al., 1990). In the sea urchin, sperm nuclear envelope breakdown requires phosphorylation of lamin B by protein kinase C (Poccia and Collas, 1997; Collas et al., 1997). The ability of mammalian oocyte cytoplasm to break down the sperm nuclear envelope is developmentally regulated, being present in maturing oocytes but absent in activated eggs (Borsuk, 1991; Borsuk and Tarkowski, 1989; Tesarik and Kopecny, 1989); however, there are reports that sperm can form PN in activated eggs when the embryos are examined at the two-cell stage (Maleszewski, 1992). The ability to remodel introduced somatic nuclei and injected sperm nuclei is also developmentally regulated (Cothren and Poccia, 1993; Czolowska et al., 1984; Szollosi et al., 1986, 1988; Tarkowski and Balakier, 1980). The factor(s) responsible for sperm nuclear envelope breakdown appears to be present in limiting quantities because penetration of cytoplasts by multiple sperm appears to inhibit complete transformation to a PN (Borsuk, 1991; Clarke and Masui, 1986, 1987; Tesarik and Kopecny, 1989). One interesting feature of some mammalian oocytes is the ability to break down the membranes of intact sperm following ICSI. This resembles the ability of Drosophila eggs to break down sperm plasma membranes as a normal part of fertilization (Fitch and Wakimoto, 1998), suggesting that some mammals may express factors that are analogous in their activities to insect ooplasmic factors that break down sperm cell membranes. In mammalian eggs treated by ICSI, it appears that non-acrosome-reacted sperm form pronuclei slower than acrosome-reacted sperm (Lee et al., 1997) and that the presence of these membranes may lead to alterations in the normal progression of chromatin decondensation (Sutovsky et al., 1996). The delay in PN formation and chromatin decondensation following ICSI with intact sperm may create significant asynchrony between the paternal PN and both cytoplasm and maternal PN, and this might render reprogramming of the paternal genome less complete than with normal fertilization. This asynchrony may be overcome during later rounds of DNA replication in some species, provided that the factors responsible for reprogramming are not specific to the oocyte cytoplasm and do not become degraded soon after fertilization. It will be useful to ascertain whether any significant deficiencies in epigenetic paternal genome modification are associated with ICSI using intact sperm. Once the sperm nuclear envelope is broken down, the next phase of PN formation is the formation of the PN membrane. After the PN envelope is initially formed, PN swelling occurs, and this appears to be essential for normal chromatin decondensation. Like the factors that break down the

76

KEITH E. LATHAM

sperm nuclear envelope, factors that regulate PN formation also appear to be developmentally regulated. The GV-stage oocyte contains a repressor of PN formation (Fulka et al., 1996). Mitogen-activated protein kinase appears to have a role in controlling PN formation (Moos et al., 1995), and paternal PN formation appears to be coupled to processes that promote maternal PN formation (Wright and Longo, 1988). Some of the molecular steps in PN envelope formation and growth have been characterized for the sea urchin. The PN envelope is formed by the accumulation of membrane vesicles around the sperm chromatin. Binding of the vesicles requires lipophilic structures that are derived from the sperm nuclear envelope (Collas and Poccia, 1995). Three distinct membrane vesicle populations contribute to the PN envelope and are required for its formation, and these membrane vesicle populations differ in their compositions and sites of binding (Collas and Poccia, 1996). One of these membrane vesicle populations contains a lamin B receptor-like, 56-kDa protein, which appears to mediate chromatin binding, recruitment of lamin B and other vesicles during PN formation, and later binding of the membrane to the lamina (Collas et al., 1996). In mammals, but not sea urchins, microtubules appear to be required for PN formation and lamin acquisition, possibly due to a role for microtubules in the transition from meiosis to interphase (Schatten et al., 1989). Interestingly, the effect of colcemid on paternal PN formation is influenced by the presence of maternal chromosomes (Borsuk and Manka, 1988). Microtubules also appear to be required for paternal and maternal PN apposition and the eventual union of the two PN (Palermo et al., 1997; Reinsch and Karsenti, 1997; Williams et al., 1997). As with the factors that promote nuclear envelope breakdown, the factors that support PN formation may also exist in limiting quantities, as polyspermic and polygynic hamster eggs show PN of reduced size (Witkowska, 1981; Wright and Longo, 1988). In sea urchin, mouse, and Drosophila, PN formation is associated with changes in lamin composition (Liu et al., 1997; Schatten et al., 1985). Embryonic or somatic cell nuclei transplanted into oocytes undergo changes in their lamin compositions to resemble authentic pronuclei (Kubiak et al., 1991; Leno and Munshi, 1997), indicating that the ooplasm has the ability to modify existing nuclear envelopes as well as the newly formed paternal PN envelope. These changes in lamin composition appear to be associated with changes in nuclear envelope permeability. The formation of porous PN membranes appears to be essential for subsequent changes in the pronuclei. This most likely reflects a need for exchange of proteins and other macromolecules between the nuclear and cytoplasmic compartments (Kunkle et al., 1978; Kopecny et al., 1986; DiBerardino, 1997). Permeabilized nuclei obtained from somatic cells are able to undergo DNA replication in Xenopus oocytes, whereas nonpermeabilized nuclei are not (Leno and Munshi, 1997). Changes in PN lamina composition also appear to be re-

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

77

quired for PN DNA replication in Drosophila (Liu et al., 1997). In addition, access of cytoplasmic factors to the sperm chromatin is essential for normal chromatin decondensation. Sperm chromatin decondensation is critically dependent on replacement of sperm chromatin proteins with the appropriate array of cleavage-stage histone and nonhistone chromatin proteins. Thus, formation of a permeable PN envelope that permits access of proteins to the genome is a prerequisite for successful genome reprogramming and activation. 2. Repackaging Chromatin during PN Formation The replacement of chromatin-packaging proteins has also been studied extensively in the sea urchin and other invertebrate species. These studies are instructive from the standpoint of understanding how such a transition can be achieved and how maternal control over the process can be maintained. The sea urchin sperm chromatin is packaged with five sperm-specific (Sp) histone variants, which are removed under the influence of the ooplasm and replaced with cleavage stage (CS) histone variants (Imschenetzky et al., 1991; Mandl et al., 1997; Poccia et al., 1981; Poccia, 1986). There is a maternal store of CS histones in the unfertilized egg, as well as maternal CS transcripts in the nucleus of the unfertilized sea urchin egg, which are released and translated after fertilization (De Leon et al., 1983; Herlands et al., 1982; Poccia et al., 1981, 1984; Venezky et al., 1981). The egg also contains the factors needed to direct histone replacement (Imschenetzky et al., 1991). The replacement of Sp histones by CS histones appears to be mediated by a combination of protein phosphorylation of Sp H1 and Sp H2B by a specific Sp histone kinase, and specific proteolysis of Sp histones by a cysteine protease, which is present in the unfertilized egg and becomes activated after fertilization (Poccia et al., 1990; Imschenetzky et al., 1997). A result of the histone replacement is a dramatic decrease in nucleosome repeat length to a value that is similar to that of somatic cells (Poccia et al., 1984). This transition can occur in the absence of substantial DNA replication or protein synthesis, but it is correlated with the accumulation of CS H2A and H2B within the PN (Poccia et al., 1984). The replacement of Sp H1 by CS H1 precedes the transition in nucleosome repeat length (Savic et al., 1981). Cleavage-stage histones are also observed in vertebrates, and in Xenopus CS histones appear to be able to displace somatic histones (which become phosphorylated) in transplanted nuclei, just as they can displace Sp histones (Dimitrov and Wolffe, 1996). The Xenopus CS H1 variant (H1M or B4) is homologous to the sea urchin CS H1, indicating evolutionary conservation of both structure and function (Mandl et al., 1997). The B4 linker histone is less basic than the somatic H1 histone,

78

KEITH E. LATHAM

should bind to DNA less tightly, and thus may facilitate rapid cycles of DNA replication (Dimitrou et al., 1996). A variety of factors support the replacement of sperm basic proteins with histones H2A/H2B following fertilization, including nucleoplasmin, and these factors exist in the ooplasm as maternally inherited molecules (Dimitrov et al., 1994). Along with these changes in histone proteins, changes in nonhistone chromatin proteins also occur during PN formation (Imschenetzky et al., 1988, 1996; Kunkle et al., 1978). For mammals, sperm chromatin decondensation requires the removal of protamines and their replacement with histones. This occurs before DNA replication, indicating independence from replication (Nonchev and Tsanev, 1990). Removal of protamines apparently requires the reduction of disulfide bonds, which is promoted by oocyte-derived glutathione (Naish et al., 1987, Sutovsky and Schatten, 1997). The activity of glutathione can be mimicked by treatment of sperm with reducing agents (Zakhidov et al., 1985; Perreault et al., 1987) and facilitated in cultured embryos by the inclusion of cystein in the culture medium (Yoshida et al., 1992, 1993). Proteolysis mediated by a sperm-derived protease has been suggested to contribute to protamine removal and sperm nucleus decondensation, although this has been reported to be true only for decondensation in vitro and not in vivo (Marushige and Marushige, 1978; Perreault and Zirkin, 1982). Other in vitro studies indicate that changes in ion concentration may be involved in protamine removal (Rodman et al., 1982). Mammals do not appear to exhibit as extensive a repertoire of oocyte-specific or cleavage stage-specific histones as the sea urchin. The mouse oocyte contains histones H2A, H2B, H3, and H4 and synthesizes H3 and H4 from maternal transcripts during the one-cell stage (Wiekowski et al., 1997). Like Xenopus and many invertebrate species, however, early mammalian embryos do not exhibit somatic-type histone H1, and instead possess an oocyte-specific variant, denoted H1o which is replaced by somatic histone H1 during cleavage (Clarke et al., 1992, 1998; Smith et al., 1995). The formation of the mammalian PN appears to be controlled entirely by the ooplasmic components that exist at the time of fertilization (Chian and Sirard, 1996). Core histones replace protamines during paternal PN formation within 8 h postinsemination (McClay and Clarke, 1997). This ability to replace protamines with histones is developmentally regulated within the oocyte, being acquired during meiotic maturation, and requires protein synthesis during maturation but does not require egg activation (McClay and Clarke, 1997). Interestingly, microinjected somatic histone H1 can incorporate into developing PN without disrupting development (Lin and Clarke, 1996). As in Xenopus, somatic histone H1 can be removed from nuclei transplanted to oocytes, although the ability of the cytoplasm to direct this transition is lost over time, and the factors responsible may be present in limiting quantities (V. Bordignon, H. Clarke, and L. Smith, personal communication).

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

79

B. Differences between Pronuclei The foregoing description of PN formation focused primarily on the formation of the paternal PN. In addition to paternal PN formation, the maternally derived genome must also form a PN. As a result, there exists a period of time in which the two parental genomes are physically separated from one another, despite their residence within a common cytoplasm. This initial period of separation provides an interesting opportunity for differences between the two parental genomes to be established. This may have immediate consequences for what genes are expressed from the two genomes in the early embryo and also long-lasting consequences on the expression of the parental genomes and epigenetic effects on genes that are regulated by genomic imprinting (Latham, 1994; Latham and Sapienza, 1998). Given these interesting possibilities, it is worth reviewing what is known about the differences that exist between paternal and maternal PN in mammalian embryos. In mice, the paternal and maternal PN are typically distinguishable on the basis of their locations within the egg and their sizes. The maternal PN is typically closest to the polar body and smaller than the paternal PN. The paternal PN may be larger because it may begin to form first, or its larger size may reflect other differences in chromatin structure within the two pronuclei, accompanied by functional differences. Abundant evidence for different chromatin structures and functional properties between the two PNs has been obtained. One of the earliest indications that the two PNs differ functionally resulted from experiments in which reporter gene constructs were injected into the two PNs of one-cell embryos. DePamphilis and coworkers showed that when reporter constructs are injected into the paternal PN of embryos that are arrested during the first S phase with aphidicolin, they are more highly transcribed than when they are injected into the maternal PN (Henery et al., 1995; Wiekowski et al., 1993). Similar results have been obtained for endogenous genes (Aoki et al., 1997). Transcription of reporter genes injected into the paternal PN becomes repressed only after mitosis (Henery et al., 1995). The repression of transcription in maternal PN and in paternal PN upon mitosis cannot be overcome by a strong enhancer, even though a strong enhancer can promote transcription when the constructs are injected into two-cell stage nuclei (Henery et al., 1995). The immediate repression of transcription within maternal but not paternal PN may be the result of (i) a difference in chromatin structure, (ii) the presence of a repressor in maternal PN, or (iii) the absence or insufficiency of essential transcription factors within the maternal PN. Available data have provided support for differences in chromatin structure and limiting availability of transcription factors, with preferential access to those factors by the paternal PN.

80

KEITH E. LATHAM

That maternal and paternal PNs possess different chromatin structures has been evidenced in several ways. In one series of experiments, fusion of metaphase II oocytes with half-zygotes containing just one maternal or one paternal PN resulted in faster chromatin condensation within the maternal PN than the paternal PN (Ciemerych and Czolowska, 1993). This indicates that the maternal PN chromatin can respond more quickly to the condensing factors than can the paternal PN chromatin. It has also been reported that maternal and paternal chromosomes assemble onto the first mitotic spindle in a nonrandom manner and remain segregated from each other within two-cell stage nuclei, providing another indication of a difference in chromatin structure and function (Brandriff et al., 1991). Immediately after fertilization, the paternal PN stains positively for hyperacetylated histone H4, but the maternal PN does not (Adenot et al., 1997). This initial difference persists through G1 phase but disappears by the S/G2 stage, when differential transcription becomes apparent. DNA replication is not required for this transition (Adenot et al., 1997). These observations indicate that paternal PN chromatin initially outcompetes the maternal PN for the maternal pool of hyperacetylated histone H4. This further supports the idea that a variety of chromatin proteins and other nuclear proteins are present in limiting quantities in the early mammalian embryo, just as factors that promote sperm nuclear envelope break down and PN envelope formation are limiting. Because the maternal pool of H4 is predominantly diacetylated initially (Wiekowski et al., 1997), the preferential uptake of hyperacetylated H4 by the paternal PN may contribute to its initially greater transcriptional capacity. Ongoing synthesis of histones H3 and H4, and initiation of synthesis of histones H2A, H2B, and H1 near the end of the one-cell stage (Wiekowski et al., 1997), may permit the acquisition of transcriptional ability within the maternal PN. In addition to differences in histone protein content, maternal PNs possess lower abundances of certain transcription factors, such as Sp1 (Worrad et al., 1994), again indicating a limiting quantity of such factors and preferential access to them by the paternal PN. Functional support for this explanation has come from experiments with parthenogenetic embryos. For example, whereas the paternal PN incorporates four to five times more BrUTP than the maternal PN, parthenogenetic embryos show an intermediate rate of incorporation of BrUTP, equal between the two maternal PNs and the total rate of BrUTP incorporation into the two parthenogenetic PNs is equal to the total incorporation into maternal plus paternal PNs of fertilized embryos (Aoki et al., 1997). In addition, in parthenogenetic embryos the PNs accumulate hyperacetylated histone H4, in contrast to maternal PNs of fertilized embryos (Adenot et al., 1997). These observations indicate that, in the absence of a paternal PN, maternal PN can acquire many of the same properties of paternal PN. The limiting quantities of chromatin

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

81

proteins and transcription factors revealed by these differences between maternal and paternal PNs is an important aspect of the early embryo that must be taken into account when considering the mechanism of EGA. In addition, the initially greater rate of transcription within the paternal PN raises the interesting possibility that, if gene products encoded by the limited array of genes that are transcribed during the one-cell stage help to control genome remodeling and EGA, then these essential early events may be predominantly under paternal control in fertilized embryos so that significant paternal effects may be realized. It should also be noted that the initial differences in chromatin structure and transcription factor content between the two PN may contribute to early epigenetic modifications of the embryonic genome, possibly including genomic imprinting modifications. The egg contains maternal factors that modify the two PN, possibly in a reciprocal manner (Latham and Solter, 1991, Latham, 1994, Latham and Sapienza, 1998, Reik et al., 1993). While some of these factors may act directly upon imprinted genes (i.e., genes bearing gametic imprinting marks), differences in chromatin structure between the two PN may also lead to differential access of these factors to non-imprinted target genes, with the end result being essentially indistinguishable from gametic imprinting effects. In addition, initial differences in transcriptional activity of some genes and initial differences in binding site occupancy by factors like Sp1 may affect the course of early epigenetic modifications, like DNA methylation, again leading to an outcome essentially indistinguishable from gametic imprinting modifications. It will be interesting to identify the factors in the egg cytoplasm that modify the pronuclei, and to discover how they mediate their effects and how their functions might be affected by the initial differences in chromatin structure and transcription factor content of the two PN.

III. Nuclear Changes during Cleavage Related to EGA The foregoing section emphasized the many complex events that must occur in order to create the maternal and paternal PNs which are eventually united to form the embryonic genome. Subsequent events must prepare the newly formed embryonic genome to function as a template for transcription. An important part of this transition is the continued modification of the embryonic nuclear chromatin structure to create a genome that can be regulated appropriately. That the factors required for chromatin remodeling are initially present in limiting quantities raises the important question of how the embryo increases its supplies of these essential nuclear components in a timely manner, thus allowing continued DNA replication and cell

82

KEITH E. LATHAM

divisions but at the same time controlling the activities of these proteins so that it can manifest the necessary specificity of gene expression during the early phase of transcription, the major genome activation event, and subsequent developmental stages as the embryo begins to execute its first differentiative programs. The apparent differences in chromatin structure that accompany the differences in transcriptional activities between the maternal and paternal PNs argue strongly for a central role of chromatin structure in controlling EGA. Data from many studies support this view and reveal a complex series of changes involving the establishment of an initially repressive chromatin state followed by changes in chromatin structure that permit transcriptional activation by certain trans-acting factors, leading to the transcription of specific genes in a regulated manner. The changes in chromatin structure and function and how these parameters relate to DNA replication and cell cycle will be reviewed in the following sections.

A. Changes in Histones during Cleavage Two major changes in embryonic histones occur during the cleavage divisions. First, changes in the array of histone subtypes that are expressed and assembled onto chromatin occur as maternally derived histones and histone mRNAs are replaced with embryonically encoded histone variants and their mRNAs. Second, significant posttranslational changes, most notably changes in histone acetylation status, occur during cleavage. Not surprisingly, the kinetics of the changes in histone proteins differs among species, and these differences correlate somewhat with differences in the timing of EGA and subsequent major changes in gene expression activity. In some reports, it appears that these changes in histone content and modification do not occur all at once but rather occur in a progressive manner as the genome is made ready for EGA. It is likely that these changes and other changes in nonhistone chromatin proteins play a major role in controlling both the timing of EGA and the array of genes that are activated.

1. Changes in the Array of Histones After pronuclei have formed and sperm proteins have been replaced by maternally encoded histone variants, histone composition of the embryonic chromatin continues to change. The most extensively studied replacement is the replacement of oocyte-specific histone H1 variants with somatic variants. In the sea urchin, the 움 form of histone H1 first replaces CS H1, and then the 웁 and 웂 forms of H1 appear. In the frog, the two major somatic

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

83

variants, histones H1A and H1B, first appear around the late blastula stage (Poccia, 1986; Dimitrov et al., 1993). In Xenopus, assembly of somatic-type linker histones onto DNA may serve to repress certain oocyte-specific genes, such as the 5S rRNA genes and some pol II genes, thus providing for a passive mechanism of gene repression that is related to chromatin structure and consequent acquisition of the ability to regulate transcription more precisely (Bouvet et al., 1994). Thus, assembly of somatic-type linker histones may be an important early step in establishing the ability to regulate embryonic gene activity. The changes in histone composition are perhaps not as well characterized for mammals as for other species, but some notable changes have been documented. In the mouse, somatic-type histone H1 is first detected cytochemically in a portion of embryos at the 4-cell stage and by the 8-cell stage in all the nuclei in all embryos (Clarke et al., 1992). In one study (Wiekowski et al., 1997), histone H1 synthesis was detectable in 1-cell embryos and 2-cell embryos but was transcription independent during these stages, indicating that this was due to maternally encoded mRNA and, given the results of Clarke et al. (1992), was probably not of the somatic type. The rate of synthesis of histone H1 apparently decreases substantially during the 2-cell stage and then increases again at the 4-cell stage (Wiekowski et al., 1997), consistent with the degradation of maternal histone H1 mRNAs during the 2-cell stage followed by expression of embryonic (i.e., somatic variant) transcripts at the 4-cell stage. In the cow, somatic histone H1 first appears between the 8-cell and 32-cell stages (Smith et al., 1995). The appearance of somatic histone H1 in mouse embryos apparently requires transcription and synthesis during the late 2-cell and/or 4-cell stages (Clarke et al., 1992), and somatic-type histone H1 appearance in the bovine requires transcription during the 8-cell stage (Smith et al., 1995). Thus, somatic-type histone H1 may be one of the most important genes that is transcriptionally activated during EGA at the 2-cell stage in the mouse and the 8-cell stage in the cow, and this activation may be an essential prerequisite for further development and chromatin remodeling. The absence of somatic-type histone H1 during earlier cleavage stages distinguishes the chromatin that exists at the time of EGA from that of later stage embryos. Interestingly, the appearance of somatic-type histone H1 anticipates temporally the upregulation in transcription of many housekeeping genes, such as actin, and the shift in two-dimensional protein gel patterns to a more somatic-like pattern, which occurs at the eight-cell stage, for example, in mice (Latham et al., 1991a, 1992a). Because the binding of linker histone (i.e., histone H1) to chromatin can have a transcription inhibitory effect even when acetylated core histones are present (Ura et al., 1997), the switch to somatic histone H1 may contribute to the embryo’s ability to regulate gene transcription appropriately and may precede the

84

KEITH E. LATHAM

expression of certain transcription factors that are responsible for fully activating housekeeping genes such as actin. The appearance of somatictype histone H1 during the four-cell stage may thus be regarded as an important part of completing the transition from comparatively unique chromatin state of the oocyte and newly fertilized embryo to a more typical somatic-like chromatin state that supports a more somatic-like pattern of gene expression and represses an oocyte-type gene expression pattern. Because microinjecting somatic-type histone H1 into early mouse embryos does not interfere with subsequent preimplantation development (Lin and Clarke, 1996), it does not appear that precocious assembly of somatic histones into chromatin is problematic. The functional significance of the absence of the somatic variant of histone H1 from mature oocytes and early embryos has not been established, but this absence may reflect a need for a specialized chromatin to allow expression of genes during oocyte maturation that might be sensitive to somatic variant histone H1 assembly on chromatin (Lin and Clarke, 1996). In this case, the appearance of somatic-type histone H1 would be merely a prerequisite for later gene activation events, and the early addition of somatic-type histone H1 would not necessarily be expected to have a detrimental effect on the preimplantation embryo because it would only accelerate the eventual acquisition of gene regulatory capacity. The transition in histone H1 isotype expression, however, indicates that although the major EGA event in the mouse occurs at the two-cell stage, some regulatory mechanism probably exists to ensure that the genome is not fully activated until after these later changes in histone composition occur. In the mouse, the synthesis of histones H2A and H3 appears to be slightly upregulated between the one-cell and two-cell stages (Wiekowski et al., 1997). A slight effect of 움-amanitin on H2A and H3 synthesis has been reported (see Fig. 4C in Wiekowski et al., 1997), indicating that the synthesis of these histones in both one-cell and two-cell embryos may be directed by a combination of maternal and embryonic transcripts. Early transcription of these histone genes by the embryonic genome may facilitate early development and changes in chromatin that lead to EGA. Histone H4 synthesis varies little during the one-cell and two-cell stages, and like histone H1 synthesis is transcription independent during these stages, indicating a strong initial maternal contribution of histone H4 mRNA. 2. Changes in Histone Acetylation during Cleavage From the above discussion, it is clear that changes in the array of histone variants expressed in the early mammalian embryo generally occur after the time of the major EGA event and are more likely to contribute to further maturation of the embryonic genome, which allows the elaboration

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

85

of a more somatic-type pattern of gene expression during the later cleavage stages. Thus, if changes in histones are to contribute to the regulation of EGA, they are most likely to involve posttranslational modifications of the maternally inherited histone proteins and newly synthesized proteins produced from the maternal mRNAs. The available data have indeed revealed some striking changes in histone modification, predominantly acetylation state, and even some unique spatial patterns of acetylated isoform distribution within embryonic nuclei that are seen during no other stage of development. Because histone acetylation is highly correlated with increased rates of gene transcription (Wade et al., 1997), these observations are consistent with a major role for such modifications in controlling genome activity during EGA. Acetylation of core histones facilitates gene transcription and is widely correlated with transcriptionally active chromatin (Ura et al., 1997; Wade et al., 1997). An exciting discovery during recent years is that a number of transcriptional coactivators possess core histone acetyltransferase activity, and that recruitment of these coactivators to the template by other transcription factors may promote a local destabilization of chromatin structure in order to facilitate transcription (Wade et al., 1997). Other transcriptional regulators (i.e., repressors) possess histone deacetylase activity, thus providing the potential requirement for continuous histone acetylation to balance an ongoing tendency toward deacetylation in order to maintain transcriptional activity. The initial transcriptional silence in mammalian embryos could thus be supported either by an abundance of deacetylase activity or by a paucity of active coactivators with acetyltransferase activity or the other factors that recruit them to the template. With such a model, one would expect there to be a significant shift toward a greater degree of histone acetylation accompanying EGA. In both Xenopus and mouse, histone H4 is stored in the diacetylated form, a form to which it typically is modified immediately after synthesis (Dimitrov et al., 1993; Wiekowski et al., 1997). Interestingly, it appears that in many cases histone H4 becomes deacetylated as it is assembled into chromatin, and indeed this occurs in the early embryos of both Xenopus and the mouse (Dimitrov et al., 1993; Wiekowski et al., 1997). Therefore, assembly of the stored histone onto chromatin as the early rounds of DNA replication take place would tend to reduce the degree of histone H4 acetylation and thus tend to repress transcription. Further repression would result from the assembly of linker histone following deposition of the deacetylated H4 into chromatin (Dimitrov et al., 1993). The available data thus indicate that the chromatin state of the newly fertilized embryo initially shifts toward a repressive, deacetylated state as histone H4 is assembled into chromatin during DNA replication. This may account for why DNA replication in the early mouse embryo can lead to repression of some

86

KEITH E. LATHAM

injected reporter constructs and for how this repression can be prevented by arresting the embryos in S phase or by treatment with sodium butyrate to inhibit histone deacetylases (Wiekowski et al., 1997). After the initial deacetylation of H4 as new chromatin is assembled, a shift in the acetylation state back to the hyperacetylated state occurs in order for gene transcription to occur. Many studies have documented striking changes in core histone acetylation during the one-cell and two-cell stages in mice and positive effects of histone deacetylase inhibitors on the transcription of endogenous genes and injected reporter genes (Aoki et al., 1997; Henery et al., 1995; Worrad et al., 1995; Stein et al., 1997; Thompson et al., 1995; Wiekowski et al., 1993). One of the most intriguing aspects of core histone acetylation in the early embryo is a striking difference in spatial localization of specific acetylated H4 isoforms within the early embryonic nuclei (Worrad et al., 1995; Thompson et al., 1995; Stein et al., 1997). Histone H4 acetylated on lysine 16 is uniformly distributed throughout the nucleus, but histone H4 isoforms acetylated on lysines 5, 8, or 12 are specifically enriched at the nuclear periphery in the late one-cell and twocell stage mouse embryo. This enrichment for these histone H4 isoforms at the nuclear periphery diminishes after the two-cell stage (Worrad et al., 1995; Thompson et al., 1995) but appears to be enhanced at all preimplantation stages by treatment with trichostatin A, an inhibitor of histone deacetylases (Thompson et al., 1995). More recent studies have revealed enhanced staining at the nuclear periphery for histone H3 acetylated on lysines 9 and/or 18 and histone H2A acetylated on residue 5 (Stein et al., 1997). Histone H3 acetylated on residues 14 or 23 and histone H2B are not enriched at the nuclear periphery (Stein et al., 1997). Taken together, these observations indicate that a specialized chromatin structure containing hyperacetylated histones is established at the nuclear periphery and may be uniquely permissive for transcription in the two-cell mouse embryo. Neither 움-amanitin nor cytochalasin D inhibit the formation of this nuclear domain, but inhibition of DNA replication with aphidicolin does inhibit formation of the domain (Stein et al., 1997). It has been proposed that as DNA is replicated during S phase of the two-cell stage, the chromatin is passed to the nuclear periphery where histone H4 (having been initially diacetylated on residues 5 and 12 before assembly) becomes further acetylated on residue 8 and histone H3 becomes acetylated on residues 9 or 18 (Stein et al., 1997). This would explain the replication dependence of the domain. While at the periphery, genes become exposed to essential transcription factors and then are passaged back to the nuclear interior, where they may subsequently be regulated by more conventional mechanisms, including coactivators and repressors that provide for site-specific histone deacetylation and acetylation. By this model, passage of newly replicated chromatin through this domain would constitute an essential step toward transcriptional activation

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

87

and could account for the uniqueness of this domain to the two-cell stage. Stein et al. (1997) also point out that this would explain why the major EGA event, wherein the majority of housekeeping genes become activated (Latham et al., 1991a), occurs after the second S phase. The question then arises whether those genes that are transcriptionally activated in the late one-cell embryo (Bouniol et al., 1995; Aoki et al., 1997) or transiently activated in the early two-cell stage (Conover et al., 1991; Christians et al., 1995; Davis et al., 1996; Latham et al., 1991a, 1995) must also pass thorugh this nuclear domain or instead can undergo histone hyperacetylation independently, possibly through the binding of specific transcription factors that may recruit coactivators with acetyltransferase activity.

B. Changes in Nonhistone Chromatin Proteins during Cleavage Other chromatin proteins undergo stage-specific changes in the early embryo, and these transitions may contribute to the control of EGA either directly by controlling access of the template to transcription factors or indirectly by supporting DNA replication, which itself appears to be important for controlling EGA (see below). High-mobility group (HMG) proteins are especially interesting in this context. HMG I/Y proteins have been examined in the early mouse embryo because of their role in supporting transcription from scaffold attachment regions (SARs), which can enhance transgene expression in the early embryo (Thompson et al., 1995). These proteins are readily detectable in the nuclei of early embryos, disappear transiently in eight-cell embryos, and reappear in blastocyst-stage embryos. Treatment of eight-cell embryos with trichostatin A increases the nuclear staining for HMG-I/Y but is not able to stimulate transcription of a SARcontaining heat shock protein transgene, possibly because of an excess amount of linker histone relative to HMG-I/Y; transcriptional stimulation is recovered in blastocysts (when the HMG-I/Y normally reappears) under conditions of heat shock. Other HMGs, such as HMG 14 and 17, may also undergo stage-specific changes.

C. Role of the Cell Cycle in Controlling Chromatin Changes and EGA The timing of EGA appears to be coordinated with the cell cycle and in fact may be dependent on successful completion of certain cell cycleassociated events, such as DNA replication. These links between the cell cycle and EGA provide a potentially useful means of coordinating nuclear

88

KEITH E. LATHAM

and cytoplasmic events that mediate and control EGA with each other and with completion of nuclear remodeling. 1. Role of DNA Replication in Permitting and Controlling EGA The foregoing discussion alluded to a link between DNA replication and EGA. The existence of this link has come to light slowly during the past several years. Some early studies concluded that DNA replication during the one-cell stage was not required for EGA (Howlett, 1986; Poueymirou and Schultz, 1987). In those studies, however, DNA replication may have been inhibited too late to affect EGA (Davis et al., 1996). Several studies have revealed that some injected reporter genes and some endogenous genes can exhibit at least a basal rate of transcription beginning during late S or G2 phase of the one-cell stage (Bouniol et al., 1995; Ram and Schultz, 1993; Aoki et al., 1997; Davis et al., 1996; Christians et al., 1995; Vernet et al., 1992; Matsumoto et al., 1994). Inhibiting DNA replication with aphidicolin during the one-cell stage reduces, but does not prevent, the normal expression of endogenous genes that occurs during the one-cell stage, as assayed by BrUTP incorporation, and the degree of inhibition is affected by the time of addition of the drug (Aoki et al., 1997; Davis et al., 1996). This indicates that passage through the first S phase may be required for certain nuclear events that permit early transcription of some genes—most likely events involved in chromatin remodeling. Along with this facilitating effect on early gene transcription, the first round of DNA replication also appears to initiate the establishment of a chromatin state that permits transcription to be regulated efficiently. As pointed out above, this may be due in part to histone deacetylation after maternal histone incorporation into replicated chromsomes during DNA replication. Earlier results with some injected reporter constructs indicated that a repressive state must arise during S or G2 of the one-cell stage, and that this repressive state can effectively silence many promoters. A common observation has been that in mouse embryos completion of the first cell cycle following microinjection at the one-cell stage is often accompanied by partial or complete repression of the injected reporter gene (Henery et al., 1995; Martinez-Salas et al., 1989; Wiekowski et al., 1991). The repression occurs before cleavage in one-cell embryos when the reporter construct is injected into the maternal pronucleus, but it does not occur until the two-cell stage when the paternal pronucleus is injected (Wiekowski et al., 1993). In other studies, passage through the first S phase has been reported to silence injected reporter genes (Ram and Schultz, 1993). Nuclear transfer experiments, in which the injected paternal pronucleus is transplanted to two-cell stage cytoplasm, reveal that the injected

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

89

reporter gene can become repressed within the paternal pronucleus once it is within the two-cell cytoplasm (Henery et al., 1995). This indicates that the paternal pronucleus does not simply exclude repressive factors but instead that the relevant repressive factors are not in the one-cell cytoplasm, and that the maternal pronucleus probably inherits some repressive quality (possibly associated with its chromatin composition) from the oocyte (Nothias et al., 1995). When the injected mouse embryos are cultured in the presence of aphidicolin to inhibit DNA replication, the repression is largely alleviated (Henery et al., 1995; Wiekowski et al., 1991). In addition, repression can be relieved by treatment with sodium butyrate to increase histone acetylation (Henery et al., 1995; Nothias et al., 1995). These results indicate that the repressive effects observed during the one-cell stage are chromatin structure dependent and replication dependent, consistent with a possible role for histone deacetylation after DNA replication. In the rabbit, injected reporter gene repression also occurs, but additional repression arises as a consequence of events that occur in association with the normal activation of the embryonic genome at about the eight-cell stage (Christians et al., 1994). Other studies indicate that the second round of DNA replication in the mouse embryo also has a repressive effect on transcription, and that this repressive effect is more pronounced than that attributable to the first S phase. Aphidicolin treatment of two-cell mouse embryos prevents the normal repression of the eIF-4C gene (now called eIF-1A), which is transiently transcribed in the early embryo (Davis et al., 1996). Microinjection of reporter genes into two-cell embryos reveals that enhancers become necessary for high rates of transcription at this stage (Martinez-Salas et al., 1989). Assay of expression of injected reporter genes in embryos that are aphidicolin treated but chronologically at the two-cell stage reveals that inhibiting both of the first two rounds of DNA replication greatly increases expression of the reporter gene and thus that the normal repression of the reporter gene that occurs during the two-cell stage is prevented by inhibiting the second S phase (Wiekowski et al., 1991; Henery et al., 1995; Nothias et al., 1995). Interestingly, aphidicolin treatment of one-cell embryos followed by assay of endogenous gene transcription by BrUTP incorporation at the two-cell stage (rather than the one-cell stage as discussed above) reveals a nearly two-fold increase in the total amount of incorporation relative to untreated two-cell embryos (Aoki et al., 1997). This indicates that inhibiting the second round of replication leads to a very pronounced increase in total transcription, and that the second S phase must normally be associated with a strongly repressive event. The establishment of this repressive state within two-cell stage nuclei thus creates the condition in which strong transcription-activating mechanisms are required in order for transcription to occur, thus providing the ability to regulate transcription appropriately.

90

KEITH E. LATHAM

Interestingly, it has been shown recently that enhancer elements in some reporter gene constructs are able to stimulate transcription in one-cell embryos when located proximal to the promoter but cannot provide longrange stimulation of transcription until after the late one-cell stage (Forlani et al., 1998). This long-range activation requires completion of the first round of DNA replication. Interestingly, these enhancers are able to promote expression in 100% of transgenic lines and a copy number-dependent increase in expression in two-cell and later embryos, indicating that they function much like a locus control region and thus probably alter chromatin structure (Forlani et al., 1998). Because the effects of both butyrate and enhancers overcome the effects of a repressive chromatin structure, it appears that the repressive environment that is established during the two-cell stage involves an alteration in chromatin structure (Majumder et al., 1993; Nothias et al., 1995). This altered chromatin structure appears to have a global effect on endogenous genes as well as reporter genes. The rate of transcription in two-cell embryos can be markedly increased with trapoxin treatment to induce histone hyperacetylation (Aoki et al., 1997). These observations thus indicate that, as the embryo develops from a newly fertilized zygote to the stage at which the major EGA event typically occurs (two-cell stage in mouse, and four- to eight-cell stage in most other mammals), the embryo acquires the ability to transcribe both endogenous genes and injected reporter genes, but it concurrently acquires what appears to be a repressive chromatin state. This repressive chromatin state most likely represents the earliest establishment of a chromatin state that is conducive to tightly regulated gene regulation because it is at this stage when enhancers (or other cis regulatory elements that facilitate transcription) first become necessary for transcription from weak promoters. The establishment of this chromatin state may be essential for minimizing precocious expression of genes that are associated with later differentiative events. Both transitions are dependent to some degree on DNA replication. DNA replication during the first cell cycle in the mouse embryo is associated with the dual effect of promoting the ability to undergo transcriptional activation and exerting the first repressive change in chromatin structure, and later rounds of replication appear to exert further repressive effects to provide the embryo with the ability to regulate transcription appropriately. The dependence of these transitions on DNA replication indicates that DNA replication during the first few cell cycles may provide an important window of opportunity for restructuring the chromatin in order to permit precise gene regulation. Incorporation and deacetylation of histones during S phase may be an important part of the repressive mechanism. Subsequent histone acetylation and chromatin restructuring probably then play an essential role in transcriptional activation of specific genes, in con-

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

91

junction with enhancers or specific promoter elements, and the factors that bind to them. This restructuring may also involve the unique chromatin domain (described above) which is revealed by the spatial distributions of acetylated forms of core histones. It should be noted that the genes that are transcribed transiently during the first burst of transcription (i.e., before S of the two-cell stage) may serve important functions in the early embryo, for example, by facilitating the second round of activation, or they may be activated merely as a consequence of incomplete restructuring of the genome. If the latter were the case, then completion of the genome remodeling would be expected to result in repression of many of these genes, which in fact occurs. If the earliest phases of transcription are indeed ‘‘accidental’’ then minimizing this early transcription may be advantageous. The existence of other mechanisms outside of chromatin structure for delaying EGA would thus be expected. 2. Cleavage Arrest in Cultured Embryos May Reflect an Inability to Undergo EGA A common observation related to mammalian embryo culture is that suboptimal culture conditions frequently result in cleavage arrest in embryos at about the time of EGA ( Johnson and Nasr-Esfahani, 1994). In the mouse embryo, this cleavage arrest most commonly occurs during the two-cell stage, but under certain conditions arrest can occur during the one-cell stage. It was recently demonstrated that the posttranslational modifications and expression of cell cycle regulators is altered in some embryos arrested by culture conditions, indicating that suboptimal culture conditions may prevent normal cell cycle-dependent events (Haraguchi et al., 1996). An interesting by-product of this failure to undergo EGA may be the activation of an apoptotic pathway or possibly the failure to activate the expression of genes that suppress a default pathway leading to apoptosis; it has been observed that apoptosis, while a normal part of mammalian preimplantation development, appears to occur more frequently in cultured embryos and probably contributes to reduced rates of success in such endeavors as in vitro fertilization ( Jurisicova et al., 1995). It has also been suggested that the presence of a default apoptotic pathway that requires EGA in order to be circumvented may provide a useful mechanism for the early elimination of unfit embryos that are derived from normal conception involving inferior oocytes ( Jurisicova et al., 1998). Moreover, other cell cycledependent events that affect EGA through other mechanisms may fail to occur after the one-cell stage, leading to a failure in EGA and arrest at the time when EGA should occur (e.g., during the two-cell stage in the mouse), despite completion of earlier cell cycle(s).

92

KEITH E. LATHAM

D. Effects of Extrinsic Factors on Chromatin Remodeling The importance of chromatin remodeling to EGA and subsequent development is highlighted by the effects of a number of agents that are believed to alter chromatin structure. When applied to early embryos, these agents disrupt normal development either immediately during the preimplantation period or much later during fetal life. For example, transient exposure of one-cell stage mouse embryos to certain DNA-modifying compounds leads to long-term developmental defects through an epigenetic mechanism that does not appear to involve mutation but rather an uncharacterized change in chromatin structure (Katoh et al., 1989; Generoso et al., 1991). Longterm embryonic and fetal development in humans may also be affected by chemical exposure (Siffel and Czeizel, 1995; Rutledge et al., 1992). Developmental and embryological defects have been observed in one mouse derived by in vitro fertilization of an oocyte that had been grown and matured in vitro after removal from a newborn female (Eppig and O’Brien, 1997). This may have resulted from an inability of the oocyte cytoplasm to modify chromatin correctly or to support normal EGA. Exposure of two-cell stage mouse embryos to lithium chloride has been found to disrupt embryonic axis development, and again it was proposed that this resulted from an effect on chromatin structure (Rogers and Varmuza, 1996). Although the mechanism by which such treatments might lead to chromatin alterations and the nature of those modifications remain largely uncharacterized, these examples nevertheless indicate that extrinsic factors that may interfere with the normal progression of chromatin maturation during the one-cell and two-cell stages may have significant long-term effects on the developmental program.

E. Changes in DNA Methylation Another change that may contribute to EGA through an effect on chromatin structure is the global change in DNA methylation that begins in the early embryo and continues through later cleavage stages (Monk et al., 1987; Monk, 1995). It was proposed recently that the principal function of DNA methylation is to suppress the transcription of genomic parasites such as retrotransposons (Yoder et al., 1997). The wave of global demethylation between the two-cell and blastocyst stages may permit the transcription of such elements, and indeed some intracisternal type A particle genes, which are endogenous retrotransposons, appear to be transcriptionally activated in the two-cell embryo (Szollosi and Yotsuyanagi, 1985; Piko´ et al., 1984). Global demethylation may permit the activation of these elements, and other regulatory changes in chromatin structure may be required to silence

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

93

these genes as development progresses. Whether global DNA methylation has any other effect on endogenous gene transcription, such as remodeling the chromatin structure or reprogramming the embryonic genome, remains to be determined. Studies of Xenopus embryos indicate that DNA methylation may direct an important repressive effect on gene expression. This repressive effect requires a chromatin environment and probably involves the formation of a complex that includes the methylated DNA-binding protein MeCP2 plus Sin3, histone deacetylase, and components of the replication origin recognition complex ( Jones et al., 1998). Further evidence that DNA methylation may serve an important transcription-repressing function in the early embryo has come from experiments in which male rats or mice were treated with 5-azacytidine pulses. Mating these males to untreated females produces many embryos that arrest development during the one-cell stage. An apparent dependence of this effect on the time of treatment and spermatogenic stage indicated that the embryo lethality was not the result of chemical carryover but was instead most likely the result of deregulated gene expression during the one-cell stage that resulted from hypomethylation of the paternal genome (Doerksen and Trasler, 1996). Support for this interpretation has been obtained by treating the one-cell embryos with 움-amanitin, which reduces developmental arrest during the one-cell stage (T. Doerksen and J. Trasler, personal communication). These observations indicate that DNA methylation during gametogenesis may help to prevent the early transcription of genes (the products of which may be deleterious to the embryos) that, if hypomethylated, can be transcribed during the early vulnerable stage before chromatin remodeling has been completed. The greater tendency for transcription within the paternal PN as compared with the maternal PN may make the greater degree of DNA methylation of the paternal genome advantageous for controlling (i.e., limiting) early transcription.

F. Summary of Chromatin Regulation of EGA The results described here indicate that DNA replication leads to significant changes in chromatin structure that are associated with an initial establishment of a transcriptionally repressive state followed by changes that permit transcriptional activation of specific genes in an appropriately regulated manner by cis regulatory elements, such as enhancers and multiple promoter elements (e.g., Sp1 binding sites) found in many housekeeping genes. DNA replication may support these changes by providing an opportunity for core histone replacement and access of certain essential transcription factors to the DNA template. The result of these changes is a progressive maturation of the chromatin as reflected by a change in the ability of certain cis

94

KEITH E. LATHAM

regulatory elements to stimulate transcription and in the dependence of transcription upon such elements. Another link between DNA replication and gene expression is that certain enhancer binding sites apparently can function as replication origins (Nothias et al., 1995). In addition to the facilitating role of passage through S phase, a unique nuclear subdomain, as reflected in peripheral staining for hyperacetylated histones, may be responsible for posttranslational modifications of core histones, which in turn promote transcription. Finally, in addition to these two periods of change that appear to be directly related to EGA, additional changes occur during later cleavage stages to permit the embryo to shift to a more somatictype pattern of gene expression.

IV. Cytoplasmic Changes Controlling EGA The changes described above for chromatin structure serve the purpose of preparing the DNA template to be transcriptionally activated and to be activated with the necessary control over the array of genes that are transcribed. These changes, while clearly essential to the task of EGA, comprise only one part of the overall regulatory mechanism that controls EGA. Changes in the cytoplasm apparently play an equally important role by controlling the activities and abundances of essential transcription factors as well as factors that regulate mRNA translation. These cytoplasmic changes are likely to involve a combination of translational and posttranslational mechanisms acting on maternally derived mRNAs and proteins and possibly on some of the newly transcribed zygotic mRNAs produced during the earliest period of gene transcription. These cytoplasmic changes are likely to be rate limiting for EGA and are likely to be coordinated with nuclear events to the extent that they are delayed until the nuclear events that are required for correct EGA have been completed. As such, these cytoplasmic events are likely to be controlled at least partially by the cell cycle. The nature of these cytoplasmic changes and how they may be controlled are considered in the following sections.

A. Conversion from a Transcriptionally Nonpermissive to a Transcriptionally Permissive State Many studies, including many of those cited above and others by Piko´ and coworkers, revealed that gene transcription can occur during the one-cell stage (Bouniol et al., 1995; Ram and Schultz, 1993; Aoki et al., 1997; Davis et al., 1996; Christians et al., 1995; Vernet et al., 1992; Matsumoto et al.,

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

95

1994; Clegg and Piko´, 1982, 1983a,b). That the maturing one-cell stage cytoplasm changes in a way that might contribute to the control of EGA was revealed by nuclear transplantation studies. Transcriptionally competent but silent (i.e., 움-amanitin inhibited) tester nuclei from chronologically two-cell stage mouse embryos were transplanted to enucleated one-cell stage cytoplasm of different ages (Latham et al., 1992b). The expression of a transcription-dependent, two-cell stage-specific group of proteins, the 70-kDa TRC, was used as an indicator of whether the recipient one-cell cytoplasm was transcriptionally permissive. If the one-cell recipient cytoplasm could provide a source of functional RNA polymerase II and was not otherwise repressive for transcription, the 70-kDa TRC was expected to be expressed. Those studies revealed that the early one-cell cytoplasm (20–26 h post-hCG) was not permissive for transcription, whereas the late one-cell cytoplasm (26–32 h post-hCG) was permissive (Latham et al., 1992b). The shift from nonpermissive to permissive state could be accelerated by treatment with dibutyryl cyclic AMP, indicating that protein kinase A (PKA) may normally be involved in this transition and that the posttranslational modification of maternal proteins is involved in the process (Latham et al., 1992b). The molecular basis for this shift is unknown. Because the tester nuclei were 움-amanitin inhibited, the transition to a permissive state may have involved the posttranslational activation of RNA polymerase II. Other possibilities include a PKA-dependent translational recruitment of maternal mRNAs encoding RNA polymerase II or other essential transcription factors or a PKA-dependent inactivation or degradation of inhibitory transcription factors (e.g., Maid; Hwang et al., 1997).

B. Changes in Transcription Factor Content As explained above, the mouse embryo and apparently other mammalian embryos become competent for transcription of injected reporter genes and some endogenous genes well before the major genome activation event; in the mouse, this occurs during late S/G2 of the one-cell stage, whereas the major EGA event occurs during the second half of the two-cell stage. How, then, is the major EGA event delayed for so long after the embryo actually becomes transcriptionally permissive? The most likely explanation is that, although the embryo becomes permissive for transcription of some genes in advance of the major EGA event, its overall capacity for transcription remains limited until a later time, thus preventing the activation of the majority of housekeeping genes and other genes until its transcriptional capacity has been augmented. This increase in transcriptional capacity can be achieved by either of two means: posttranslational activation of existing maternally inherited transcription factors (TFs) or the synthesis of addi-

96

KEITH E. LATHAM

tional TFs using maternal mRNAs as templates. The available data indicate that both levels of control are used and that both probably play important roles in controlling the timing of the major EGA event. Previous studies indicated that cAMP-dependent PKA is required for EGA during the twocell stage of the mouse (Poueymirou and Schultz, 1987), consistent with a role for this enzyme in the posttranslational activation of one or more essential TFs. An apparent need for protein synthesis to achieve activation of housekeeping genes also indicates that a combination of constitutive synthesis of some TFs and temporally regulated recruitment of maternal mRNAs encoding other TFs is probably also critical for EGA (Wang and Latham 1997). 1. Posttranslational Activation of Transcription Factors Because the cAMP-dependent PKA is required for EGA, posttranslational activation of maternally inherited proteins, very likely including transcription factors, must contribute to EGA. PKA may directly activate TFs or may act indirectly through a protein kinase cascade. In order for such a control mechanism to elicit a global onset gene transcription, it is reasonable to suspect that among the TFs that become activated are components of the core transcription complex, such as RNA polymerase II subunits and associated proteins. a. Regulation of RNA Polymerase II Activity One facile way in which transcriptional activity might be regulated globally would be by regulating the activity of maternally inherited RNA polymerases, for example, by modifying RNA pol II subunits. The RNA polymerase II transcription complex, of course, contains many subunits, some of which mediate RNA synthesis, some that interact with splicing or 5⬘ capping factors, and some that regulate these activities. The carboxy-terminal domain (CTD) of the large pol II subunit (RPB1) contains a group of tandem repeats of a consensus heptapeptide that becomes phosphorylated predominantly on serine residues but also on tyrosine and threonine (Patturajan et al., 1998). The hypophosphorylated form of pol II (form IIA) apparently associates with preinitiation complexes, and then phosphorylation of the CTD, which generates form IIO, appears to be essential for transcriptional elongation for most genes. Many viral proteins promote CTD hyperphosphorylation (Shilatifard, 1998; Cujec et al., 1997; Herrmann et al., 1996). The CTD also appears to be involved in recruiting enzymes responsible for mRNA capping and splicing (McCracken et al., 1997; Cho et al., 1997; Nekhai et al., 1997; Okamoto et al., 1996; Chun and Jeang, 1996; Yang et al., 1996a,b; Shuman, 1997; Bourquin et al., 1997; Du and Warren, 1997; Kim et al., 1997; Mortillaro et al., 1996)

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

97

so that its phosphorylation in the early embryo could also contribute to mRNA processing, transport, and translation. The hyperphosphorylated CTD also appears to be required for recruitment of certain TFs to the transcription complex (Grondin et al., 1997). Many different kinases have been implicated in phosphorylation of the CTD, including cdc2, cyclins, TFIIH, Abl, MAP kinases, CTD kinases, ERK2, and other kinases that regulate TFIIH (Kuchin and Carlson, 1998; Lee and Greenleaf, 1997; Spencer et al., 1997; Baskaran et al., 1997; Marshall et al., 1996; Valay et al., 1996; Markowitz et al., 1995; Shiekhattar et al., 1995; Serizawa et al., 1995; Serizawa, 1998; Venetianer et al., 1995; Okhuma et al., 1995). Recycling of pol II to the hypophosphorylated state appears to be mediated by specific phosphatases (Chambers and Kane, 1996). Pol II activity and CTD phosphorylation are regulated with the cell cycle (Patturajan et al., 1998; Long et al., 1998; Leresche et al., 1996) and also by heat shock (Patturajan et al., 1998; Dubois et al., 1997). Comparatively little work has been reported for analyses of RNA polymerase activities and modifications in embryos in general, and especially in mammalian embryos. Some suggestive results, however, have been obtained. In Xenopus, the CTD is hypophosphorylated in growing oocytes and becomes hyperphosphorylated upon oocyte maturation as a result of MAP kinase activity. Dephosphorylation of the CTD then occurs quickly after fertilization (Bellier et al., 1997b). A maternal store of a phosphatase may be responsible for the dephosphorylation after fertilization (Leresche et al., 1996). Mouse oocytes and embryos also undergo changes in CTD phosphorylation. As in Xenopus, the CTD is hyperphosphorylated in the unfertilized mouse and rabbit eggs and becomes dephosphorylated after fertilization (Bellier et al., 1997a). In the rabbit, nuclear staining for the RPB1 subunit of pol II is very faint in 1-cell embryos and increases gradually during subsequent cleavage divisions. In the mouse, nuclear staining for RPB1 first becomes evident 3 to 4 h after PN formation and then increases abruptly with development to the 2-cell stage. Phosphorylation sites associated with TFIIH kinase activity in somatic cells are unphosphorylated until the time of the major EGA event (i.e., 2-cell stage in mouse and 8- to 16-cell stage in rabbit) (Bellier et al., 1997a). Thus, nuclear localization is correlated with establishment of a CTD phosphorylation state similar to that seen in somatic cells (Bellier et al., 1997a). The low level of nuclear staining in pronuclei of 1-cell mouse embryos is consistent with a limited capacity for transcription during the late 1-cell stage, and the increase during the 2-cell stage is consistent with a possible role for the regulated phosphorylation of pol II CTD in controlling the time of the major EGA event. The nuclear translocation of RPB1 is insensitive to inhibitors of protein synthesis, DNA replication, and transcription (Bellier et al., 1997a), indicating that it is regulated by posttranslational modification of maternally inherited protein.

98

KEITH E. LATHAM

Other studies reveal not only that pol II translocates to the nucleus at the time of EGA but also that in two-cell mouse embryos it is preferentially localized to the special nuclear domain detected by staining for certain acetylated histone isoforms (Worrad et al., 1995). Such a localization of pol II may be another indication of a specific property of this domain that relates to nuclear reprogramming, as discussed above. Alternatively, this distribution may only reflect the necessary consequence of pol II phosphorylation associated with transcriptional elongation and its subsequent interaction with other nuclear proteins involved in mRNA processing. In somatic cells, phosphorylated RPB1 localizes to ‘‘speckles’’ within cell nuclei, where it is associated with proteins involved in mRNA processing (Bregman et al., 1995). In transcriptionally inhibited cells treated with 움-amanitin, the RPB1 redistributes and takes on a different subnuclear localization (Bregman et al., 1995). This latter domain would presumably not exist in the embryo because during the time leading up to EGA, pol II CTD is predominantly hypophosphorylated. The relationship between the peripheral domain of two-cell embryos and the speckles of somatic cells is not known, but it would be interesting if the two are related with regard to structure, composition, or function. It would also be interesting to learn whether some special property of the nuclear envelope or nuclear matrix is responsible for specifically localizing pol II, histone acetyltransferases, splicing factors, and other TFs, and whether passage through this domain is indeed a prerequisite for reprogramming chromatin. b. Posttranslational Regulation of Other Transcription Factors Posttranslational modifications may also regulate the activities of other maternally inherited TFs, such as Sp1 and TATA binding protein (TBP). Sp1 and TBP are both phosphorylated by a DNA-dependent protein kinase (DNA-PK). This phosphorylation does not prevent the formation of promoter complexes containing these factors but may affect further interaction of the promoter complex with pol II and may modulate gene activity (Chibazakura et al., 1997; Jackson et al., 1990; Chun et al., 1998). This phosphorylation by DNA-PK can also be influenced by other nuclear factors (Chun et al., 1998). The Sp1 phosphorylation state is regulated by a variety of extrinsic factors, and dephosphorylation is often associated with enhanced DNA binding and gene activity (Sanceau et al., 1995; Schafer et al., 1997; Vlach et al., 1995; Zutter et al., 1997). In terminally differentiating cells, Sp1 is phosphorylated by casein kinase II in its carboxy-terminal domain, and this decreases Sp1 DNA binding activity (Armstrong et al., 1997). Sp1 is also phosphorylated and its DNA binding activity downregulated in differentiating liver cells (Leggett et al., 1995). Interestingly, Sp1 is also phosphorylated by PKA, and this phosphorylation increases DNA binding and reporter gene activity (Rohlff et al., 1997). Sp1 is also regulated by

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

99

glycosylation ( Jackson and Tjian, 1988). TBP is phosphorylated by a mitotic kinase, and a specific phosphatase that reverses this phosphorylation may reactivate TBP and relieve transcriptional repression following mitosis (Leresche et al., 1996; Segil et al., 1996; White et al., 1995). These observations indicate that both Sp1 and TBP activities might be regulated posttranslationally in the egg and newly fertilized embryo. Several interesting possibilities can be envisioned by which this might contribute to EGA. First, TBP and/or Sp1 activities might be reduced by phosphorylation by the mitotic kinase so that dephosphorylation after fertilization may reactivate these factors. This could contribute to the acquisition of transcriptional ability during the late one-cell stage in mice and possibly other species. Final maturation of the oocyte (i.e., terminal differentiation) may also inactivate Sp1 by casein kinase II or other kinases in a manner similar to differentiating somatic cells, and again dephosphorylation may be required to reactivate maternally derived Spl. Finally, the possible regulation by phosphorylation of Sp1 by PKA raises the possibility that part of the demonstrated role for PKA in EGA may involve Sp1 activation. Evidence supporting a possible connection between Sp1 phosphorylation status and EGA was obtained recently for the mouse embryo. An increase in the total amount of Sp1 occurs between G1 and G2 of the one-cell stage, and this is largely attributable to an increase in the amount of the hypophosphorylated form of Sp1 (Worrad and Schultz, 1997). It is not clear, however, whether maternally inherited Sp1 or TBP protein can be reactivated after fertilization, whether the glycosylation status of maternally inherited forms prevents their efficient participation in transcription complexes, or whether newly synthesized molecules provide the only source of hypophosphorylated forms. To understand fully the role of posttranslational control of Sp1 and TBP activities in EGA will require a greater understanding of which protein kinases and phosphatases modulate Sp1 and TBP activities, which of these are expressed in the egg and early embryo, and how the phosphorylation status of specific sites on these TFs changes. An investigation of the expression in the embryo of other TFs that interact with Sp1 and TBP, and are themselves regulated by phosphorylation, will also be valuable. 2. Protein Synthesis Contributes to EGA Protein synthesis during the late one-cell and/or early two-cell stage is required for the second major burst of transcription during the two-cell stage, during which housekeeping genes are activated. Treatment of embryos from the late one-cell stage (i.e., G2 phase) onward with the protein synthesis inhibitor cycloheximide resulted in an essentially complete inhibition in the normal transcription-dependent increase in expression of all housekeeping genes tested (Wang and Latham, 1997). These results indicate

100

KEITH E. LATHAM

that, although the late one-cell cytoplasm may be permissive for transcription of some endogenous genes and injected reporter genes, it does not contain all the transcription factor activities necessary for the normal widescale activation of numerous housekeeping genes, many of which possess comparatively weak promoters that may require a high nuclear concentration of certain transcription factors (e.g., Sp1 and TBP) in order to complete effectively and to be activated. The simplest explanation for this result is that the ability to transcribe housekeeping genes as a part of the major EGA event during the two-cell stage requires the synthesis of one or more essential transcription factors during the late one-cell and/or early two-cell stages. This requirement may reflect a need for continuous synthesis of some TFs or stage-specific synthesis of other TFs following maternal mRNA recruitment. Stability of Maternally Derived Transcription Factors In addition to posttranslational modifications of TFs such as Sp1 and TBP, the relative stability of these factors may also have an important impact on whether the embryo can undergo EGA. A protease has been described that specifically degrades certain TFs, including Sp1 (Nishinaka et al., 1997). EGF treatment of rat pituitary cells can reduce Spl-mediated transcription by promoting proteolysis of Spl, and okadaic acid can enhance this effect. This proteolysis occurs after the de novo production of a protease (Mortensen et al., 1997). The expression of such proteases, or their activation either during oocyte maturation or following fertilization as a means of inhibiting transcription, might reduce the relative stability of these TFs. Ongoing synthesis of these TFs, therefore, might be required to maintain an adequate supply of these factors to permit EGA. This might partially account for the pronounced inhibition of housekeeping gene transcription during EGA in cycloheximide-treated embryos (Wang and Latham, 1997) because housekeeping genes require primarily Spl, TBP, and other general transcription factors (i.e., components of the core pol II transcription complex) in order to be transcribed. Evidence that Sp1 and TBP stability may indeed be a factor in the early embryo has come from studies of Sp1 and TBP nuclear content in cycloheximide-treated embryos. Cycloheximide treatment of one-cell embroys during culture to the two-cell stage resulted in a significant reduction of nuclear staining for both TBP (four-fold reduction) and Sp1 (30% reduction) (Worrad et al., 1994). This decrease is largely attributable to inhibition of synthesis of these factors. Synthesis normally promotes an increase in nuclear staining that occurs progressively during most of the one-cell stage. Interestingly, the normal increase in nuclear concentration is greater for Sp1 than for TBP even though the effect of cycloheximide is greater for TBP, suggesting that the relative stability of TBP is less than that of Spl, the increase in Sp1 nuclear staining is partially attributable to

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

101

posttranslational modifications as discussed above, or Sp1 synthesis normally increases more than TBP synthesis. As mentioned above, the degradation of inhibitory TFs after fertilization may also contribute to EGA. One potentially inhibitory TF identified in the mouse embryo, Maid, is expressed as a maternally derived protein and mRNA (Hwang et al., 1997). This factor contains a conserved helix–loop– helix motif without a DNA-binding domain and thus may serve as a negative regulator of bHLH transcription factors. This inhibitory activity has been demonstrated in cultured cells but has not been established for the embryo. If maternally derived factors such as Maid indeed inhibit transcription in the embryo, the kinetics of their degradation may provide one level of control to limit when the activation of some genes can occur. 3. Role of Maternal mRNA Recruitment and Synthesis of Transcription Factors during EGA The apparent effect of cycloheximide on the nuclear abundance of Sp1 and TBP, the increase in nuclear Sp1 and TBP nuclear staining during the onecell stage (Worrad et al., 1994), and the increase in Sp1 activity between the one-cell and two-cell stages (Majumder et al., 1993) indicate that ongoing synthesis of these factors is probably necessary to attain a sufficiently high nuclear concentration in order to accomplish EGA. The ability of cycloheximide to prevent the normal activation of housekeeping genes (Wang and Latham, 1997) supports this interpretation. The increase in Sp1 nuclear staining during the one-cell stage is 움-amanitin insensitive (Worrad and Schultz, 1997). This indicates that the increase in Sp1 and TBP content during the period leading up to EGA is supported by maternal mRNA and may involve increased translation of maternal mRNAs encoding these TFs. Enhanced stability of these proteins might also contribute to their increase in abundance. Just as ongoing synthesis of some TFs, such as Sp1 and TBP, may contribute to the embryo’s ability to undergo EGA, time-dependent translational recruitment of maternal mRNAs encoding other TFs may also be essential for complete EGA. Many reports have documented the appearance of a variety of TF activities during the period leading up to EGA and also during the remainder of preimplantation development. Two factors are most notable in this regard, namely, an unidentified coactivator that is required for enhancer function and the enhancer binding factor mTEAD2. Majumder et al., (1997) found that the polyoma virus F101 enhancer cannot stimulate transcription of injected reporter genes in one-cell embryos, even when provided with a source of enhancer binding factor (exogenously supplied Ga14:VP16). In addition, competition between enhancers of different microinjected constructs was observed in injected two-cell stage

102

KEITH E. LATHAM

embryos but not in injected one-cell stage embryos. These results indicate that enhancers, which earlier studies indicated serve to alleviate chromatinmediated repression (Majumder et al., 1993; Nothias et al., 1995), can promote increased gene transcription in two-cell stage or later embryos but not in one-cell embryos. That the promoters of the injected constructs are active in the one-cell embryo, but not stimulated by the enhancer, indicates that the lack of enhancer effect in one-cell embryos is not attributable to a simple generalized inability to perform transcription. Rather, the data have been interpreted within the context of a stage-dependent appearance of a transcriptional coactivator that allows the enhancer to stimulate promoter usage beginning at the two-cell stage if enhancer binding factors are present (Majumder et al., 1997). This coactivator is unknown, but it is tempting to speculate that it may be related to one of several proteins that have a role in forming multiprotein complexes that bring together many different transcription factors and histone acetyltransferase activities, such as p300/CBP or associated factors such as P/CAF or components of the mammalian equivalents of yeast SWI/SNF complexes (Wang et al., 1996; Yang et al., 1996b; Eckner et al., 1996). The other TF activity that appears in the early embryo is the mTEAD2 factor. The previously observed activation of the polyoma virus F101 enhancer has been attributed to a ‘‘TEF-1-like’’ activity in the early embryo. The assay of expression of TEF-1-like activity in the one-cell embryo is complicated by the inability of enhancers to stimulate transcription before the two-cell stage. To circumvent this problem, a construct was prepared by placing the TEF-1 binding site proximal to the transcription start site to create a TEF-1-dependent promoter. Microinjection of either the TEF1-dependent promoter or two different Spl-dependent promoters into onecell embryos revealed that the Spl-dependent promoter supported transcription but the TEF-1-dependent promoter did not (Kaneko et al., 1997). This indicated that the TEF-1-like activity was not present until the twocell stage, when the major EGA event occurs. The TEF family of transcription factors encompasses at least four distinct proteins: mTEAD-1 (also referred to as mTEF-1), mTEAD-2, mTEAD-3, and mTEAD-4. The expression of the mRNAs encoding these factors was recently examined in early mouse embryos and oocytes (Kaneko et al., 1997). These studies revealed that mTEAD-1, -2, and -3 mRNAs were detectable in oocytes by reverse transcriptase-polymerase chain reaction followed by Southern blot hybridization, and that only mTEAD-2 mRNA remained detectable by this highly sensitive method in two-cell embryos. The mTEAD-1 and mTEAD-3 mRNAs became degraded during the one-cell stage. Quantitative analysis of expression of these mRNAs revealed that the mTEAD-2 mRNA also declined in abundance during the one-cell and two-cell stages but was much more abundant than the other mRNAs (⬎100-fold more abundant

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

103

in oocytes), and it increased dramatically in abundance during preimplantation development. These data indicate that the mTEAD-2 mRNA is by far the most abundantly expressed member of the mTEAD family in the egg and one-cell embryo, that mTEAD-2 most likely constitutes the TEF1-like activity that appears during development to the two-cell stage, and that mTEAD-2 is encoded by maternally inherited mRNAs that may become translationally recruited and then degraded during the first two cell cycles. The appearance of these factors during early development is consistent with a model in which limited availability of certain TFs such as mTEAD2, the enhancer coactivator, and possibly general TFs such as Sp1 and TBP plays an important role in delaying EGA until the appropriate time. Either increases in abundance of some TFs or time-dependent de novo synthesis of other TFs is an essential prerequisite for EGA, and this limitation is likely to provide an important mechanism for controlling the timing of EGA (Wang and Latham, 1997). Similar conclusions have been drawn for the Xenopus embryo (Almouzni and Wolffe, 1995). As already alluded to with respect to mTEAD-2, the time-dependent appearance of these factors may be the result of developmentally controlled translational recruitment of maternal mRNAs. Both 2-D protein gel analyses and studies of specific mRNA polyadenylation patterns have revealed that multiple patterns of maternal mRNA polyadenylation and utilization exist in the mouse oocyte and one-cell embryo (Bachvarova et al., 1985; Bachvarova and DeLeon, 1980; Latham et al., 1991a, 1992a; Vassalli et al., 1989; Huarte et al., 1987; West et al., 1996; Hwang et al., 1997; Oh et al., 1997). Similar results have been obtained for Xenopus oocytes and embryos, and in fact it appears that there exists an evolutionarily conserved mechanism for recruiting maternal mRNAs at different stages (Verotti et al., 1996; Paris et al., 1991; Sheets et al., 1994; Simon et al., 1992, 1996; Varnum and Wormington, 1990). In addition, it appears not only that cytoplasmic polyadenylation control elements (CPEs) may differ among different mRNAs but also that the spacing of the CPE relative to other cis regulatory elements within the mRNA may affect the timing of recruitment (de Moor and Richter, 1997). Thus, fertilized embryos can recruit for translation at specific stages mRNAs encoding different proteins so that recruitment of mRNAs encoding essential TFs may be an important part of the mechanism that regulates EGA. Translational mRNA recruitment is under the control of diverse CPEs, typically in the 3⬘ untranslated region of the mRNAs. The CPE directs polyadenylation of the mRNA at the time of recruitment. Different CPEs can probably promote recruitment at different stages. Polyadenylation is often associated with mRNA degradation as the mRNA is translated. One interesting aspect of the changes in mRNA polyadenylation is that mRNAs lacking the CPE become deadenylated during oocyte maturation through

104

KEITH E. LATHAM

a default mechanism, and many of these can become readenylated following fertilization, in addition to stage-specific polyadenylation and recruitment of other mRNAs. Both mechanisms may apply to the synthesis of TFs required for EGA. Readenylation of previously silenced housekeeping mRNAs might account for the recruitment of mRNAs encoding general TFs such as Sp1 and TBP, whereas stage-specific recruitment under the control of specific CPEs might account for the timed appearance of other specific TFs (e.g., mTEAD-2) that are required for specific enhancer function and gene activation. In either case, mRNA degradation would be likely to follow recruitment, and indeed Spl, TBP, and mTEAD-2 mRNAs are degraded over the course of the one-cell and two-cell stages. An important link may exist between the regulated recruitment of mRNAs encoding TFs and the cell cycle. Because maternal mRNA sequestration is mediated by a variety of masking proteins that bind to the mRNA and prevent its translation, posttranslational modification of these proteins would provide a means for permitting the release of the mRNA for translation as the culmination of a protein phosphorylation cascade. In Xenopus, a c-mos-dependent pathway regulates the polyadenylation of certain mRNAs, and this regulation is mediated by cdc2-dependent phosphorylation of the CPE binding protein (de Moor and Richter, 1997; Paris et al., 1991). This provides a means by which mRNA recruitment can be controlled by either extrinsic (e.g., hormonal or growth factor stimulation or cell–cell interactions such as fertilization) or intrinsic cues (e.g., cell cycledependent events). The possible effect of cell cycle on maternal mRNA recruitment is intriguing because it provides a useful mechanism for delaying EGA until other essential events are completed, most notably nuclear events such as DNA replication and chromatin remodeling. In addition, cell cycle dependence of TF mRNA recruitment until a repressive chromatin state (e.g., in the two-cell mouse embryo) is established would minimize the impact of ‘‘accidental’’ transcription of genes before the major EGA event. The ability of aphidicolin treatment during the one-cell stage to inhibit transcription may result in part from an inhibition of synthesis of certain TFs in addition to a block in the chromatin remodeling that may favor transcription. Thus, cell cycle dependence of TF mRNA recruitment would provide a useful regulatory link between TF mRNA recruitment, DNA replication, nuclear remodeling, and EGA. An additional factor in maternal mRNA recruitment may be that the products encoded by mRNAs that become recruited early may in turn promote the recruitment of other mRNAs later. Thus, a single stimulus (e.g., fertilization or hormonal stimulation) may have the ability to activate one or more protein phosphorylation cascades, which then culminate in a complex temporal pattern of maternal mRNA recruitment directed by diversity in CPE sequence, cis element spacing, and masking proteins and

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

105

the proteins that regulate them. Moreover, this complex temporal pattern of maternal mRNA recruitment may be modulated in a cell cycle-dependent manner in order to achieve a more refined coordination between maternal mRNA recruitment and other events, such as DNA replication and chromatin remodeling. In this way, the controlled use of maternal mRNAs during the period leading up to the major EGA event may play a vital role in controlling the timing of EGA and maintaining long-term embryo viability.

C. Effect of Translational Control on Early Embryonic Gene Expression One final aspect of embryonic genome activation that remains to be considered with respect to its ability to affect embryo phenotype is whether the earliest embryonic transcripts are actually translated into protein product. In most cases, it appears that there is no barrier to the translation of newly synthesized embryonic transcripts, even in the one-cell embryo. Reporter genes encoding such proteins as luciferase are transcribed during the onecell stage and this is followed quickly by the appearance of the protein (Christians et al., 1995). EGA is readily apparent in 2-D protein gels of two-cell mouse embryos by the appearance of many novel proteins (Davis et al., 1996; Latham et al., 1991a and references therein). In a recent study, however, it was found that transcripts synthesized from one microinjected luciferase reporter gene construct during the one-cell stage were not translated, whereas transcripts produced during the late two-cell stage were translated (Nothias et al., 1996). This result might have been due to instability and rapid degradation of the mRNA produced from the reporter gene. Alternatively, the structure of the transcript might have been lacking certain elements that promote processing or polyadenylation or that make the mRNA resistant to cytoplasmic deadenylation. Whatever the reason for translational failure, the results indicate that a mechanism may exist in the early embryo for discriminating among different zygotic mRNAs (as well as maternal mRNAs) and selecting only certain of these transcripts for translation or targeting certain early transcripts for translational silencing. Thus, in addition to many different controlling mechanisms that delay embryonic gene transcription until the appropriate time after fertilization, translational control mechanisms may provide an additional level of control whereby only certain transcripts produced during the earliest period of gene transcription (i.e., before the major EGA event) are permitted to be translated. This additional level of control may be required to prevent the expression of certain proteins that might impede development if produced early but for which mRNAs are either transcribed nonspecifically as a result of incomplete transcriptional repression or unavoidably transcribed

106

KEITH E. LATHAM

in advance of the major EGA event simply because they share regulatory elements with other early transcribed genes that must be transcribed for normal development to occur. Testing this possibility will require the identification of additional mRNAs that are transcribed before the major EGA event and characterization of their translational capacities and the potential effects of their proteins on the early embryo.

D. Reprogramming of Transplanted Nuclei Further clues to the nature of cytoplasmic control of transcription in the early embryo can be gained from observing the effects of early cytoplasm on transcriptional activity in transplanted nuclei taken from later stage donor cells. In general, it appears that transplanted nuclei are made to recapitulate many of the nuclear events that precede and accompany EGA in normal embryos. The first step toward this recapitulation is the conversion of the transplanted nucleus to a pronucleus-like state. In addition to changes in such properties as nuclear envelope composition mentioned previously, transplanted nuclei typically undergo widespread transcriptional silencing. In mouse embryos, proteins characteristic of 8-cell stage embryos are not observed in the 2-D protein gel pattern of 2-cell stage nuclear transplant embryos derived by transplanting 8-cell stage nuclei into 1-cell enucleated recipients (Latham et al., 1994). In bovine embryos, 16- to 32cell stage nuclei may continue to be transcribed for a period of time after transplantation. Heterogenous nuclear RNA synthesis occurs in nuclear transplant bovine embryos through at least the 4-cell stage, and nucleolar structures typical of transcriptionally active nuclei remain visible during the first cell cycle (Lavoir et al., 1997). Eventually, however, some degree of transcriptional silencing is achieved so that granular structures within the nucleoli disappear during the 2-cell through 8-cell stages (Lavoir et al., 1997). No effect of transcription in the nuclear transplant embryos is evident in 1-dimensional gel patterns (Lavoir et al., 1997). The TEC-3 antigen present on bovine morulae and blastocysts disappears after fusion of morula-stage nuclei to unfertilized metaphase II eggs, indicating a silencing of this gene (van Stekelenburg-Hamers et al., 1994). Other studies in rabbit embryos revealed that [3H]uridine incorporation is quickly decreased and totally inhibited within 15 h after fusion of 32-cell stage blastomeres to enucleated oocytes (Kanka et al., 1996). Extensive nucleolar remodeling occurs, and the granular components of the nucleoli disappear by 6 h after fusion (Kanka et al., 1996). The protein synthesis pattern in rabbit nuclear transplant embryos, however, is unlike the pattern of either donor cells or recipient oocytes, indicating that although a generalized transcriptional

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

107

silencing may occur in the nuclear transplant embryos, some genes may continue to be transcribed for some period of time (Liu et al., 1995). The transcriptional silencing observed in transplanted nuclei in some embryos may be associated with premature chromatin condensation (PCC). PCC does not occur in a portion of other embryos. In rabbit embryos, when PCC is avoided embryos typically develop with greater success (Adenot et al., 1997). It appears likely that mechanisms other than PCC probably contribute to the silencing of transcription within transplanted nuclei, possibly including the phosphorylation and deactivation of transcription factors within the nucleus and histone deacetylation. The replacement of somatic histone variants with oocyte-specific variants may also contribute to silencing. Understanding the molecular basis of how transplanted nuclei become transcriptionally silenced should provide valuable insight into what molecular mechanisms normally delay the major EGA event until the appropriate time. In addition, the identification of genes that do not become repressed in nuclear transplant embryos may provide insight into what specific gene regulatory elements may permit some genes to be transcribed in advance of the major EGA event and also provide clues as to how cloning technologies might be improved if the repression of these genes can be elicited. Reactivation of transcription within nuclear transplant embryos resembles in many respects EGA in normal embryos. A variety of nuclei (e.g., eight-cell stage nuclei, inner cell mass nuclei, oocyte germinal vesicles, and embryonic stem cell nuclei) not expressing the 70-kDa TRC protein complex characteristic of mouse two-cell stage embryos will direct the synthesis of this protein after transplantation to enucleated mouse one-cell stage embryos (Latham et al., 1994). In fact, the overall pattern of protein synthesis observed in these embryos is very similar to that of normal two-cell stage embryos. Many other proteins that are characteristic of two-cell embryos are also synthesized. Some proteins are underexpressed, however, indicating that reactivation of the genome is incomplete (Latham et al., 1994). As discussed above, this has been interpreted within the context of inappropriate, irreversible inactivation of some genes by nuclear remodeling factors of the one-cell embryo, which may act differently upon these genes due to their transcriptionally open chromatin configuration. The TEC-3 antigen, the gene for which is silenced after transplantation of bovine morula-stage nuclei to unfertilized eggs, reappears on schedule when the nuclear transplant embryos reach morula stage (van Stekelenburg-Hamers et al., 1994). In rabbit embryos, ribonuclear RNA synthesis is restored at the eight-cell stage in nuclear transplant embryos and resembles that of normal embryos, but there appears to be excessive deposition of ribosomes into the cytoplasm (Kanka et al., 1996). Fibrillarin, a component of nucleoli that is indicative of ribosomal RNA synthesis, reappears in nuclear transplant rabbit embryos at the appropriate stage but does not reappear in every blastomere, indicat-

108

KEITH E. LATHAM

ing that reactivation of the genome is incomplete and variable between blastomeres (Pinto-Correia et al., 1995). The degree to which such phenotypic mosaicism in EGA occurs in normal embryos has not been widely tested. Other aspects of nuclear reprogramming include the restoration of cell proliferative potential in adult or fetal, senescent or near-senescent somatic cell genomes, which indicates that the developmentally programmed onset of senescence can be reversed under the influence of ooplasmic factors (Cibelli et al., 1998a). Presumably, such changes reflect epigenetic reprogramming of the transplanted nucleus that likely resembles the epigenetic programming that occurs during normal development. This reactivation of previously silent genes (i.e., genes not expressed in the donor cell) or silent genes that are repressed following transplantation but then reactivated, most likely reflects the culmination of a whole array of events that normally promote EGA, both global and gene specific in nature. If transcriptional repression, either after transplantation or during normal development, involves alterations in chromatin structure, then this chromatin structure must be acted upon by the cytoplasm. This effect may involve transitions in DNA methylation, histone acetylation, and changes in histone and nonhistone chromatin protein content. If transcription factors within the donor nucleus are inactivated or degraded by cytoplasmic factors, then these same factors must either be reactivated or replaced with newly synthesized active molecules. Changes in nuclear envelope composition (e.g., expression of lamin A/C epitopes) also occur in transplanted blastomere nuclei and are correlated with successful reprogramming of the nuclei (Kubiak et al., 1991; Prather et al., 1991). This change in nuclear envelope composition may be required for access to the nuclear compartment by cytoplasmic factors that first repress gene transcription and then reactivate gene expression or may simply reflect the effects of the oocyte cytoplasm that are normally associated with PN formation. One other feature of nuclear reprogramming following nuclear transplantation that has been observed is a striking genetic effect on the ability of the egg cytoplasm to activate expression of some genes in two-cell embryos. For example, an Hsp70.1 transgene is expressed more highly when ooplasm is of C3H strain origin than when the ooplasm is of BALB/ c origin. After nuclear transfers, the same relationship prevails regardless of the genetic origin of the donor nucleus, indicating that the epigenetic modification responsible for the difference in gene expression is reversible and essentially under the total control of the ooplasm to which the nuclei are exposed immediately before the two-cell stage (Chastant et al., 1996). These observations provide another example of how nuclear transplantation can be used to investigate normal nuclear–cytoplasmic interactions related to EGA and, more importantly, illustrate that genetic variability can exist in the level to which certain genes are activated during EGA.

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

109

These genetic differences may play an important role in determining overall vigor of preimplantation-stage embryos and their differential sensitivity to extrinsic factors such as culture conditions.

V. A Comprehensive Model for Controlling EGA From the previous review it appears likely that a complex series of events organized into multiple, interacting pathways and involving bidirectional interactions between cytoplasm and nucleus are required in order to establish within the mammalian embryo the ability to transcribe its genes, the ability to regulate appropriately the array of genes transcribed, and the ability to undergo the major genome activation event (EGA) at the correct time, thereby avoiding disruptive epigenetic events that might compromise later development. A summary of these events, based on a time scale appropriate for mouse embryos as an example, is provided in Fig. 1. A similar set of events likely holds for other species, but relevant adjustments are required in the timing of these events. Perhaps the earliest events required to accomplish EGA are those involved in sperm nuclear decondensation and formation of maternal and paternal pronuclei, including the formation of a permeable nuclear envelope that permits access of cytoplasmic factors to the nuclear compartment. Of course, some interactions of cytoplasmic factors with chromosomes and DNA likely occur even before PN envelope formation. Changes in nuclear lamins, replacement of protamines with histones, changes in the array and acetylation status of histones, and changes in the phosphorylation state of histones and other nuclear proteins would be included among the events that establish a suitable PN structure that is amenable to subsequent programming and activation. The factors responsible for these nuclear changes appear to be present in limiting quantities. This, combined with the differences in the ability of the maternal and paternal pronuclei to acquire certain nuclear factors, can lead to initial differences in parental genome expression. These nuclear changes initially impose a repressive character on the early genome. Events related to DNA replication during the first cell cycle and subsequent cell cycles probably alter chromatin structure to permit efficient regulation of gene transcription, as evidence by the acquisition of enhancer dependence of transcription and acquisition of the ability of enhancers to mediate long-range promoter activation. In this way, nuclear remodeling and changes in chromatin structure not only establish the ability to undergo EGA but also establish a chromatin structure within which gene transcription can be adequately regulated in a gene-specific manner. Furthermore, changes in histone acetylation accompanying increased expression of certain coactivators, which

110

KEITH E. LATHAM

FIG. 1 Schematic summary of interactions between nucleus and cytoplasm that are involved in mediating and controlling embryonic genome activation. Time scale and stages of events are based on the mouse embryo.

recruit acetyltransferases to the genes, or decreased expression of histone deacetylases probably play an essential role in EGA. While nuclear changes are occurring, the cytoplasm undergoes a transition from a transcriptionally nonpermissive state to a permissive state. This transition may involve both

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

111

the inactivation of repressive factors and the synthesis or activation of positive acting factors. Progress through S phase and completion of the first round of DNA replication (and possibly later rounds in other species) may promote EGA by triggering cell cycle-dependent events within the cytoplasm which eventually drive EGA. These cytoplasmic events likely include translational recruitment of maternal mRNAs, posttranslational activation of certain proteins including maternally derived transcription factors, and posttranslational inactivation or degradation of inhibitory or repressor proteins. Synthesis of essential transcription factors is likely to be especially important given the pronounced inhibitory effect of cycloheximide on EGA. At least some of the posttranslational modifications are likely to be directly or indirectly under the control of PKA, which is essential for EGA. Cytoplasmic events are timed in such a way as to prevent widespread gene transcription until nuclear programming is completed. Cytoplasmic events and nuclear events may well engage in a reciprocal regulation of timing of events within the two compartments. Disrupting the coordination between the cytoplasmic and nuclear compartments, for example, by nuclear transplantation or treatment with metabolic inhibitors, can lead to either gain of function (e.g., expression of injected reporter genes in aphidicolin-treated embryos) or loss of function events (e.g., irreversible repression of some genes following nuclear transplantation). The impact of these events may depend on the degree of disruption, which may in turn depend on elements of timing that are species-specific. This may contribute to differences in the ability of nuclear transplant embryos to develop in different species.

VI. Prospectives The summary provided in Fig. 1, although complex, highlights some of the areas of investigation in which biochemical, molecular, and embryological studies are likely to provide important new insight during the next few years regarding how EGA is mediated and regulated. Perhaps the most critical area of study that will be addressed is the characterization of which specific transcription factors exist in the fertilized egg and how the expression and activities of these factors are regulated. In contrast to studies of gene regulation in well-defined cell culture models, in which terminal differentiation can be elicited and in which the emphasis is typically on learning how certain cell type-specific marker genes are regulated, the study of how EGA is controlled in the embryo is likely to lead eventually to the discovery of novel mechanisms for regulating global transcriptional capacity through modification of ‘‘basal’’ or ‘‘core’’ transcription complexes, such

112

KEITH E. LATHAM

as RNA polymerase II complexes. This regulation may well involve key alterations in factors involved in forming initiation complexes as well as factors that promote transcriptional elongation and termination. The regulation of factors involved in RNA processing, export, and translation is also likely to be of great importance in the overall control of EGA. As more is learned about the molecular components of transcription complexes, mRNA processing factors, mRNA binding factors, and translation regulatory factors, it should become increasingly feasible to obtain specific information about how these molecules are expressed and regulated in the early embryo. In addition, our continually increasing knowledge of how cell cycle-dependent events are regulated and coordinated should provide much needed information with which to investigate how the nuclear and cytoplasmic events associated with nuclear programming and EGA are coordinated. A combination of approaches employing analyses of endogenous genes and analyses of stably integrated gene constructs with wellcharacterized regulatory elements introduced into transgenic animals should provide the necessary functional tests of how specific transcription factors may be involved in these processes. The study of EGA will likely reveal novel mechanisms for achieving global effects on gene expression and improve our understanding of how EGA is controlled, how embryonic nuclei are programmed, and how these processes affect embryo viability and reproductive fertility. Because nuclear programming and extensive changes in gene transcription constitute important parts of the process of cellular differentiation, the mechanisms of gene regulation discovered through the study of EGA should be relevant to understanding differentiation in a variety of cell types and how some pathologies may arise. Indeed, in some cases of cellular differentiation or other physiological responses to specific stimuli, the modulation of activities of general transcription factors such as Sp1 can lead to apparent specific changes in gene expression (Leggett et al., 1995; Schafer et al., 1997; Zutter et al., 1997). Although further elucidation of the molecular mechanisms that mediate and control EGA in mammalian embryos constitutes a significant challenge, this area of investigation is both important and well worth the effort that will be required to meet the challenge because it should not only clarify how mammalian embryos regulate gene expression at the start of life but also provide new information that will be relevant to understanding gene regulation during later developmental stages as cells differentiate. Acknowledgments I thank Hugh Clarke, Jacquetta Trasler, and Rich Chaillet for communicating results of experiments prior to publication. I also thank Hugh Clarke for comments on the manuscript. This work was supported in part by National Institutes of Health Grant GM56682.

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

113

References Adenot, P. G., Mercier, Y., Renard, J. P., and Thompson, E. M. (1997). Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos. Development 124, 4615–4625. Almouzni, G., and Wolffe, A. P. (1995). Constraints on transcriptional activator function contribute to transcriptional quiescence during early Xenopus embryogenesis. EMBO J. 14, 1752–1765. Armstrong, S. A., Barry, D. A., Leggett, R. W., and Mueller, C. R. (1997). Casein kinase IImediated phosphorylation of the C terminus of Sp1 decreases its DNA binding activity. J. Biol. Chem. 272, 13489–13495. Aoki, F., Worrad, D. M., and Schultz, R. M. (1997). Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev. Biol. 181, 296–307. Bachvarova, R., and De Leon, V. (1980). Polyadenylated RNA of mouse ova and loss of maternal RNA in early development. Dev. Biol. 74, 1–8. Bachvarova, R., De Leon, V., Johnson, A., Kaplan, G., and Paynton, B. V. (1985). Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes. Dev. Biol. 108, 325–331. Baran, V., Flechon, J. E., and Pivko, J. (1996). Nucleogenesis in the cleaving bovine embryo: Immunocytochemical aspects. Mol. Reprod. Dev. 44, 63–70. Baran, V., Mercier, Y., Renard, J. P., and Flechon, J. E. (1997). Nucleolar structure of rabbit cleaving embryos: An immunocytochemical study. Mol. Reprod. Dev. 48, 34–44. Barnes, F. L., Robl, J. M., and First, N. L. (1987). Nuclear transplantation in mouse embryos: Assessment of nuclear function. Biol. Reprod. 36, 1267–1274. Baskaran, R., Chiang, G. G., Mysliwiec, T., Kruh, G. D., and Wang, J. Y. (1997). Tyrosine phosphorylation of RNA polymerase II carboxyl-teminal domain by the Abl-related gene product. J. Biol. Chem. 272, 18905–18909. Bellier, S., Chastant, S., Adenot, P., Vincent, M., Renard, J.-P., and Bensaude, O. (1997a). Nuclear translocation and carboxyl-terminal domain phosphorylation of RNA polymerase II delineate the two phases of zygotic gene activation in mammalian embryos. EMBO J. 16, 6250–6262. Bellier, S., Dubois, M. F., Nishida, E., Almouzni, G., and Bensaude, O. (1997b). Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation. Mol. Cell. Biol. 17, 1434–1440. Borsuk, E. (1991). Anucleate fragments of parthenogenetic eggs and of maturing oocytes contain complementary factors required for development of a male pronucleus. Mol. Reprod. Dev. 29, 150–156. Borsuk, E., and Manka, R. (1988). Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid. Gamete Res. 20, 365–376. Borsuk, E., and Tarkowski, A. K. (1989). Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs. Gamete Res. 24, 471–481. Borsuk, E., Szollosi, M. S., Besomebes, D., and Debey, P. (1996). Fusion with activated mouse oocytes modulates transcriptional activity of introduced somatic cell nuclei. Exp. Cell Res. 225, 93–101. Bouniol, C., Nguyen, E., and Debey, P. (1995). Endogenous transcription occurs at the late 1-cell stage in the mouse embryo. Exp. Cell Res. 218, 57–62. Bourquin, J. P., Stagljar, I., Meier, P., Moosmann, P., Silke J., Baechi, T., Georgiev, O., and Schaffner, W. (1997). A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II. Nucleic Acids Res. 25, 2055–2061.

114

KEITH E. LATHAM

Bouvet, P., Dimitrov, S., and Wolffe, A. P. (1994). Specific regulation of Xenopus chromosomal 5S rRNA gene transcription in vivo by histone H1. Genes Dev. 8, 1147–1159. Brandriff, B. F., Gordon, L. A., Segraves, R., and Pinkel, D. (1991). The male-derived genome after sperm–egg fusion: Spatial distribution of chromosomal DNA and paternal–maternal genomic association. Chromosoma 100, 262–266. Bregman, D. B., Du, L., van der Zee, S., and Warren, S. L. (1995). Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains. J. Cell Biol. 129, 287–298. Brinsko, S. P., Ball, B. A., Ignotz, G. G. Thomas, P. G., Currie, W. B., and Ellington, J. E. (1995). Initiation of transcription and nucleogenesis in equine embryos. Mol. Reprod. Dev. 42, 298–302. Chambers, R. S., and Kane, C. M. (1996). Purification and characterization of an RNA polymerase II phosphatase from yeast. J. Biol. Chem. 271, 24498–24504. Chartrain, I., Niar, W. A., Picard, L., and St.-Pierre, H. (1987). Development of the nucleolus in early goat embryos. Gamete Res. 18, 201–213. Chian, R. C., and Sirard, M. A. (1996). Protein synthesis is not required for male pronuclear formation in bovine zygotes. Zygote 4, 41–48. Chibazakura, T., Watanabe, F., Kitajima, S., Tsukada, K., Yasukochi, Y., and Teraoka, H. (1997). Phosphorylation of human general transcription factors TATA-binding protein and transcription factor IIB by DNA-dependent protein kinase—Synergistic stimulation of RNA polymerase II basal transcription in vitro. Eur. J. Biochem. 247, 1166–1173. Christians, E., Rao, V. H., and Renard, J.-P. (1994). Sequential acquisition of trancriptional control during early embryonic development in the rabbit. Dev. Biol. 164, 160–172. Christians, E., Campion, E., Thompson, E. M., and Renard, J.-P. (1995). Expression of the HSP 70.1 gene, a landmark of early zygotic gene activity in the mouse embryo, is restricted to the first burst of transcription. Development 112, 113–122. Chun, R. F., and Jeang, K. T. (1996). Requirements for RNA polymerase II carboxyl-terminal domain for activated transcription of human retroviruses human T-cell lymphotropic virus I and HIV-1. J. Biol. Chem. 271, 27888–27894. Chun, R. F., Semmes, O. J., Neuveut, C., and Jeang, K. T. (1998). Modulation of Sp1 phosphorylation by immunodeficiency virus type 1 Tat. J. Virol. 72, 2615–2629. Cibelli, J. B., Stice, S. L., Golueke, P. J., Kane, J. J., Jerry, J., Blackwell, C., Ponce de Leon, F. A, and Robl, J. M. (1998a). Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280, 1256–1258. Cibelli, J. B., Stice, S. L., Golueke, P. J., Kane, J. J., Jerry, J., Blackwell, C., de Leon, F. A., and Robl, J. M. (1998b). Transgenic bovine chimeric offspring produced from somatic cellderived stem-like cells. Nat. Biotech. 16, 642–646. Ciemerych, M. A., and Czolowska, R. (1993). Differential chromatin condensation of female and male pronuclei in mouse zygotes. Mol. Reprod. Dev. 34, 73–80. Clarke, H. J., and Masui, Y. (1986). Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse. J. Cell Biol. 102, 1039–1046. Clarke, H. J., and Masui, Y. (1987). Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes. J. Cell Biol. 104, 831–840. Clarke, H. J., Oblin, C., and Bustin, M. (1992). Development regulation of chromatin composition during mouse embryogenesis: Somatic histone H1 is first detectable at the 4-cell stage. Development 115, 791–799. Clarke, H. J., Bustin, M., and Oblin, C. (1997). Chromatin modifications during oogenesis in the mouse: Removal of somatic subtypes of histone H1 from oocyte chromatin occurs postnatally through a post-transcriptional mechanism. J. Cell Sci. 110, 477–487. Clarke, H. J., McClay, D. W., and Mohamed, O. A. (1998). Linker histone transitions during mammalian oogenesis and embryogenesis. Dev. Genet. 22, 17–30.

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

115

Clegg, K. B., and Piko´, L. (1982). RNA synthesis and cytoplasmic polyadenylation in the onecell mouse embryo. Nature 295, 342–345. Clegg, K. B., and Piko´, L. (1983a). Quantitative aspects of RNA synthesis and polyadenylation in 1-cell and 2-cell mouse embryos. J. Embryol. Exp. Morphol. 74, 169–182. Clegg, K. B., and Piko´, L. (1983b). Poly(A) length, cytoplasmic adenylation and synthesis of poly(A)⫹RNA in early mouse embryos. Dev. Biol. 95, 331–341. Collas, P., and Poccia, D. (1995). Lipophilic organizing structures of sperm nuclei target membrane vesicle binding and are incorporated into the nuclear envelope. Dev. Biol. 169, 123–135. Collas, P., and Poccia, D. (1996). Distinct egg membrane vesicles differing in binding and fusion properties contribute to sea urchin male pronuclear formation in vitro. J. Cell Sci, 109, 1275–1283. Collas, P., Courvalin, J. C., and Poccia, D. (1996). Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein. J. Cell Biol. 135, 1715–1725. Collas, P., Thompson, L., Fields, A. P., Poccia, D. L., and Courvallin, J. C. (1997). Protein kinase C-mediated interphase lamin B phosphorylation and solubilization. J. Biol. Chem. 272, 21274–21280. Conover, J. C., Temeles, G. L., Zimmerman, J. W., Burke, B., and Schultz, R. M. (1991). Stage-specific expression of a family of proteins that are major products of zygotic genome activation in the mouse embryo. Dev. Biol. 144, 392–404. Cothren, C. C., and Poccia, D. (1993). Two steps are required for male pronucleus formation in the sea urchin egg. Exp. Cell Res. 205, 126–133. Cujec, T. P., Okamoto, H., Fujinaga, K., Meyer, J., Chamberlin, H., Morgan, D. O., and Peterlin, B. M. (1997). The HIV transactivator TAT binds to the CDK-activating kinase and activates the phosphorylation of the carboxy-terminal domain of RNA polymerase II. Genes Dev. 11, 2645–2657. Czolowska, R., Modlinski, J. A., and Tarkowski, A. K. (1984). Behaviour of thymocyte nuclei in non-activated and activated mouse oocytes. J. Cell Sci. 69, 19–34. Davis, W., De Sousa, P. A., and Schultz, R. M. (1996). Transient expression of translation initiation factor eIF-4C during the 2-cell stage of the preimplantation mouse embryo: Identification by mRNA differential display and the role of DNA replication in zygotic genome activation. Dev. Biol. 174, 190–201. De Leon, D. V., Cox, K. H., Angerer, L. M., and Angerer, R. C. (1983). Most early variant histone mRNA is contained in the pronucleus of sea urchin eggs. Dev. Biol. 100, 197–206. de Moor, C. H., and Richter, J. D. (1997). The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes. Mol. Cell. Biol. 17, 6419–6426. DiBerardino, M. A. (1997). ‘‘Genomic Potential of Differentiated Cells,’’ pp. 180–213. Columbia Univ. Press, New York. Dimitrov, S., and Wolffe, A. P. (1996). Remodeling of somatic nuclei in Xenopus laevis egg extracts: Molecular mechanisms for the selective release of histones H1 and H1(0) from chromatin and the acquisition of transcriptional competence. EMBO J. 15, 5897–5906. Dimitrov, S., Almouzni, G., Dasso, M., and Wolffe, A. P. (1993). Chromatin transitions during early Xenopus embryogenesis: Changes in histone H4 acetylation and in linker histone type. Dev. Biol. 160, 214–227. Dimitrov, S., Dasso, M. C., and Wolffe, A. P. (1994). Remodeling of sperm chromatin in Xenopus egg extracts: The role of core histone hyposphorylation and linker histone B4 in chromatin assembly. J. Cell Biol. 126, 591–601. Du, L., and Warren, S. L. (1997). A functional interaction between the carboxy-terminal domain of RNA polymerase II and pre-mRNA splicing. J. Cell Biol. 136, 5–18. Dubois, M. F., Vincent, M., Vigneron, M., Adamczewski, J., Egly, J. M., and Bensaude, O. (1997). Heat-shock inactivation of the TFIIH-associated kinase and change in the phosphorylation sites on the C-terminal domain of RNA polymerase II. Nucleic Acids Res. 25, 694–700.

116

KEITH E. LATHAM

Eckner, R., Yao, T.-P., Oldread, E., and Livingston, D. M. (1996). Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation. Genes Dev. 10, 2478–2490. Eppig, J. J., and O’Brien, M. J. (1997). Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo. Theriogenology 49, 415–422. Fitch, K. R., and Wakimoto, B. T. (1998). The paternal effect gene Ms(3)sneaky is required for sperm activation and the initiation of embryogenesis in Drosophila melanogaster. Dev. Biol. 197, 270–282. Forlani, S., Bonnerot, C., Capgras, S., and Nicolas, J.-F., (1998). Relief of a repressed gene expression state in the mouse 1-cell embryo requires DNA replication. Development 125, 3153–3166. Fulka, J., Jr., Horska, M., Moor, R. M., Fulka, J., and Kanka, J. (1996). Oocyte-specific modulation of female pronuclear development in mice. Dev. Biol. 178, 1–12. Generoso, W. M., Shourbaji, A. G., Piegorsch, W. W., and Bishop, J. B. (1991). Developmental response of zygotes exposed to similar mutagens. Mutat. Res. 250, 439–446. Geuskens, M., and Alexandre, H. (1984). Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos. Cell Differ. 14, 125–134. Grondahl, C., and Hyttel, P. (1996). Nucleologenesis and ribonucleic acid synthesis in preimplantation equine embryos. Biol. Reprod. 55, 769–774. Grondin, B., Cote, F., Bazinet, M., Vincent, M., and Aubry, M. (1997). Direct interaction of the KRAB/Cys2-His2 zinc finger protein ZNF74 with a hyperphosphorylated form of the RNA polymerase II largest subunit. J. Biol. Chem. 272, 27877–27885. Haraguchi, S., Naito, K., Azuma, S., Sato, E., Nagahama, Y., Yamashita, M., and Toyoda, Y. (1996). Effects of phosphate on in vitro 2-cell block of AKR/N mouse embryos based on changes in cdc2 kinase activity and phosphorylation states. Biol. Reprod. 55, 598–603. Henery, C. C., Miranda, M., Wiekowski, M., Wilmut, I., and DePamphilis, M. L. (1995). Repression of gene expression at the beginning of mouse development. Dev. Biol. 169, 448–460. Herlands, L., Allfrey, V. G., and Poccia, D. (1982). Translational regulation of histone synthesis in the sea urchin Strongylocentrotus purpuratus. J. Cell Biol. 94, 219–213. Herrmann, C. H., Gold, M. O., and Rice, A. P. (1996). Viral transactivators specifically target distinct cellular protein kinases that phosphorylate the RNA polymerase II C-terminal domain. Nucleic Acids Res. 24, 501–508. Howlett, S. K. (1986). The effect of inhibiting DNA replication in the one-cell mouse embryo. Roux’s Arch. Dev. Biol. 195, 499–505. Howlett, S. K., Barton, S. C., and Surani, M. A. (1987). Nuclear cytoplasmic interactions following nuclear transplantation in mouse embryos. Development 101, 915–923. Huarte, J., Belin, D., Vassalli, A., Strickland, S., and Vassalli, J.-D. (1987). Meiotic maturation of mouse oocytes triggers the translation and polyadenylation of dormant tissue-type plasminogen activator mRNA. Genes Dev. 1, 1201–1211. Hwang, S. Y., Oh, B., Fuchtbauer, A., Fuchtbauer, E. M., Johnson, K. R., Solter, D., and Knowles, B. B. (1997). Maid: A maternally transcribed novel gene encoding a potential negative regulator of bHLH proteins in the mouse egg and zygote. Dev. Dyn. 209, 217–226. Imschenetzky, M., Puchi, M., Massone, R., Oyarce, A. M., and Rocco, M. (1988). Remodeling of chromatin during male pronucleus formation in the sea urchin Tetrapygus niger. Arch. Biol. Med. Exp. 21, 409–416. Imschenetzky, M., Puchi, M., Pimental, C., Bustos, A., and Gonzales, M. (1991).Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins. J. Cell. Biochem. 47, 1–10.

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

117

Imschenetzky, M., Oliver, M. I., Gutierrez, S., Garrido, C., Bustos, A., and Puchi, M. (1996). Hybrid nucleoprotein particles containing a subset of male and female histone variants form during male pronucleus formation in sea urchins. J. Cell. Biochem. 63, 385–394. Imschenetzky, M., Diaz, F., Montecino, M., Sierra, F., and Puchi, M. (1997). Identification of a cysteine protease responsible for degradation of sperm histones during male pronucleus remodeling in sea urchins. J. Cell. Biochem. 67, 304–315. Jackson, S. P., and Tjian, R. (1988). O-glycosylation of eukaryotic transcription factors: Implications for mechanisms of transcriptional regulation. Cell 55, 125–133. Jackson, S. P., MacDonald, J. J., Lees-Miller, S., and Tjian, R. (1990). GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell 73, 155–165. Johnson, M. H., and Nasr-Esfahani, M. H. (1994). Radical solutions and cultural problems: Could free oxygen radicals be responsible for the impaired development of mammalian embryos in vitro? BioEssays 16, 31–38. Jones, P. L., Veenstra, G. J. C., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., Strouboulis, J., and Wolffe, A. P. (1998). Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nature Genet. 19, 187–190. Jurisicova, A., Varmuza, S., and Casper, R. F. (1995). Involvement of programmed cell death in preimplantation embryo demise. Hum. Reprod. Update 1, 558–566. Jurisicova, A., Latham, K. E., Casper, R. F., and Varmuza, S. (1998). Expression and regulation of genes associated with cell death during preimplantation embryo development. Mol. Reprod. Dev., 51, 243–253. Katoh, M., Cacheiro, N. L., Cornett, C. V., Cain, K. T., Rutledge, J. C., and Generoso, W. M. (1989). Fetal anomalies produced subsequent to treatment of zygotes with ethylene oxide or ethylmethanesulfonate are not likely due to the usual genetic causes. Mutat Res. 210, 337–344. Kim, E., Du, L., Bregman, D. B., and Warren, S. L. (1997). Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-mRNA. J. Cell Biol. 136, 19–28. Kopecny, V., Babusik, P., Tesarik, J., and Pavlok, A. (1986). Selective association of some hamster-egg-synthesized proteins with decondensing human sperm chromatin. Histochemistry 84, 197–200. Kopecny, V., Flechon, J. E., Camous, S., and Fulka, J., Jr. (1989). Nucleogenesis and the onset of transcription in the eight-cell bovine embryo: Fine-structural autoradiographic study. Mol. Reprod. Dev. 1, 79–90. Kubiak, J. Z., Prather, R. S., Maul, G. G., and Schatten, G. (1991). Cytoplasmic modification of the nuclear lamina during pronuclear-like transformation of mouse blastomere nuclei. Mech. Dev. 35, 103–111. Kuchin, S., and Carlson, M. (1998). Functional relationships of Srb10-Srb11 kinase, carboxyterminal domain kinase CTDK-I, and transcriptional corepressor Ssn6-Tup1. Mol. Cell. Biol. 18, 1163–1171. Kunkle, M., Longo, F., and Magun, B. E. (1978). Nuclear protein changes in the maternally and paternally derived chromatin at fertilization. J. Exp. Zool. 203, 371–378. Latham, K. E. (1994). Strain-specific differences in mouse oocytes and their contributions to epigenetic inheritance. Development 120, 3419–3426. Latham, K. E., and Sapienza, C. (1998). Localization of genes encoding egg modifiers of paternal genome function to mouse chromosomes one and two. Development, 125, 929–935. Latham, K. E., and Solter, D. (1991). Effect of egg composition on the developmental capacity of androgenetic mouse embryos. Development 113, 561–568. Latham, K. E., Garrels, J. I., Chang, C., and Solter, D. (1991a). Quantitative analysis of protein synthesis in mouse embryos I. Extensive re-programming at the one- and two-cell stages. Development 112, 921–932.

118

KEITH E. LATHAM

Latham, K. E., Solter, D., and Schultz, R. M. (1991b). Activation of a two-cell stage-specific gene following transfer of heterologous nuclei into enucleated mouse embryos. Mol. Reprod. Dev. 30, 182–186. Latham, K. E., Garrels, J. I., Chang, C., and Solter, D. (1992a). Analysis of protein synthesis in mouse embryos: Construction of a high-resolution, two-dimensional gel protein database. Appl. Theor. Electrophoresis 2, 163–170. Latham, K. E., Solter, D., and Schultz, R. M. (1992b). Acquisition of a transcriptionally permissive state during the 1-cell stage of mouse embryogenesis. Dev. Biol. 149, 457–462. Latham, K. E., Garrels, J. I., and Solter, D. (1994). Alterations in protein synthesis following transplantation of mouse 8-cell stage nuclei to enucleated 1-cell embryos. Dev. Biol. 163, 341–350. Latham, K. E., Rambhatla, L., Hayashizaki, Y., and Chapman, V. M. (1995). Stage-specific induction and regulation by genetic imprinting of the imprinted mouse U2afbp-rs gene in the preimplantation mouse embryo. Dev. Biol. 168, 670–676. Lee, J. M., and Greenleaf, A. L. (1997). Modulation of RNA polymerase II elongation efficiency by C-terminal heptapeptide repeat domain kinease I. J. Biol. Chem. 272, 10990– 10993. Lee, D. R., Lee, J. E., Yoon, H. S., and Roh, S. I. (1997). Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection. Fertil. Steril. 67, 315–320. Leggett, R. W., Armstrong, S. A., Barry, D., and Mueller, C. R. (1995). Sp1 is phosphorylated and its DNA binding activity down-regulated upon terminal differentiation of the liver. J. Biol. Chem. 270, 25879–25884. Leno, G. H., and Munshi, R. (1997). Reactivation of DNA replication in nuclei from terminally differentiated cells: Nuclear membrane permeabilization is required for initiation in Xenopus egg extract. Exp. Cell Res. 232, 412–419. Leresche, A., Wolf, V. J., and Gottesfeld, J. M. (1996). Repression of RNA polymerase II and III transcription during M phase of the cell cycle. Exp. Cell Res. 229, 282–288. Lin, P., and Clarke, H. J. (1996). Somatic histone H1 microinjected into fertilized mouse eggs is transported into pronuclei but does not disrupt subsequent development. Mol. Reprod. Dev. 44, 185–192. Liu, J., Lin, H., Lopez, J. M., and Wolfner, M. F. (1997). Formation of the male pronuclear lamina in Drosophila melanogaster. Dev. Biol. 184, 187–196. Long, J. J., Leresche, A., Kriwacki, R. W., and Gottesfeld, J. M. (1998). Repression of TFIIH transcriptional activity and TFIIH-associated cdk7 kinase activity at mitosis. Mol. Cell. Biol. 18, 1467–1476. Majumder, S., Miranda, M., and DePamphilis, M. L. (1993). Analysis of gene expression in mouse preimplantation embryos demonstrates that the primary role of enhancers is to relieve repression of promoters. EMBO J. 12, 1131–1140. Majumder, S., Zhao, Z., Kaneko, K., and DePamphilis, M. L. (1997). Developmental acquisition of enhancer function requires a unique coactivator activity. EMBO J. 16, 1721–1731. Maleszewski, M. (1992). Sperm nuclei entering parthenogenetically activated mouse oocytes before the first mitosis transform into pronuclei. An ultrastructural study. Anat. Rec. 243, 516–518. Mandl, B., Brandt, W. F., Superti-Furga, G., Graninger, P. G., Birnstiel, M. L., and Busslinger, M. (1997). The five cleavage-stage (CS) histones of the sea urchin are encoded by a maternally expressed family of replacement histone genes: Functional equivalence of the CS H1 and frog H1M (B4) proteins. Mol. Cell. Biol. 17, 1189–1200. Markowitz, R. B., Hermann, A. S., Taylor, D. F., He, L., Anthony-Cahill, S., Ahn, N. G., and Dynan, W. S. (1995). Phosphorylation of the C-terminal domain of RNA polymerase II by the extracellular-signal-regulated protein kinase ERK2. Biochem. Biophys. Res. Commun. 207, 1051–1057.

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

119

Marshall, N. F., Peng, J., Xie, Z., and Price, D. H. (1996). Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase. J. Biol. Chem. 271, 27176– 27183. Martinez-Salas, E., Linney, E., Hassell, J., and DePamphilis, M. L. (1989). The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus. Genes Dev. 3, 1493–1506. Marushige, Y., and Marushige, K. (1978). Dispersion of mammalian sperm chromatin during fertilization: An in vitro study. Biochem. Biophys. Acta 519, 1–22. Matsumoto, K., Anzai, M., Nakagata, N., Takahashi, A., Takahashi, Y., and Miyata, K. (1994). Onset of paternal gene activation in early mouse embryos fertilized with transgenic mouse sperm. Mol. Reprod. Dev. 39, 136–140. McClay, D. W., and Clarke, H. J. (1997). The ability to organize sperm DNA into functional chromatin is acquired during meiotic maturation in murine oocytes. Dev. Biol. 186, 73–84. McCracken, S., Fong, N., Rosonina, E., Yankulov, K., Brothers, G., Siderovski, D., Hessel, A., Foster, S., Amgen EST Program, Shuman, S., and Bentley, D. L. (1997). 5⬘-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 11, 3306–3318. Monk, M. (1995). Epigenetic programming of differential gene expression in development and evolution. Dev. Genet. 17, 188–197. Monk, M., Boubelik, M., and Lehnert, S. (1987). Temporal and regional changes in DNA methylation in the embryonic, extraembryonic, and germ cell lineages during mouse embryo development. Development 99, 371–382. Moos, J., Visconti, P. E., Moore, G. D., Schultz, R. M., and Kopf, G. S. (1995). Potential role of mitogen-activated protein kinase in pronuclear envelope assembly and disassembly following fertilization of mouse eggs. Biol. Reprod. 53, 692–699. Mortensen, E. R., Marks, P. A., Shiotani, A., and Merchant, J. L. (1997). Epidermal growth factor and okadaic acid stimulate Sp1 proteolysis. J. Biol. Chem. 272, 16540–16547. Mortillaro, M. J., Blencowe, B., Wei, X., Nakayasu, H., Du, L., Warren, S. L., Sharp, P. A., and Berezney, R. (1996). A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix. Proc. Natl. Acad. Sci. USA 93, 8253–8257. Naish, S. J., Perreault, S. D., Foehner, A. L., and Zirkin, B. R. (1987). DNA synthesis in the fertilizing hamster sperm nucleus: Sperm template availability and egg cytoplasmic control. Biol. Reprod. 36, 245–253. Nekhai, S., Shukla, R. R., and Kumar, A. (1997). A human primary T-lymphocyte-derived human immunodeficiency virus type 1 Tat-associated kinase phosphorylates the C-terminal domain of RNA polymerase II and induces CAK activity. J. Virol. 71, 7436–7441. Nishinaka, T., Fu, Y. H., Chen, L. I., Yokoyama, K., and Chiu, R. (1997). A unique cathepsinlike protease isolated from CV-1 cells is involved in rapid degradation of retinoblastoma susceptibility gene product, RB, and transcription factor Sp1. Biochem. Biophys. Acta 351, 274–286. Nonchev, S., and Tsanev, R. (1990). Protamine-histone replacement and DNA replication in the male mouse pronucleus. Mol. Reprod. Dev. 25, 72–76. Nothias, J.-Y., Majumder, S., Kaneko, K., and DePamphilis, M. L. (1995). Regulation of gene expression at the beginning of mammalian development. J. Biol. Chem. 270, 22077–22080. Nothias, J.-Y., Miranda, M., and DePamphilis, M. L. (1996). Uncoupling of transcription and translation during zygotic gene activation in the mouse. EMBO J. 15, 5715–5725. Oh, B., Hwang, S. Y., Solter, D., and Knowles, B. B. (1997). Spindlin, a major maternal transcript expressed in the mouse during the transition from oocyte to embryo. Development 124, 493–503. Ohkuma, Y., Hashimoto, S., Wang, C. K., Horikoshi, M., and Roeder, R. G. (1995). Analysis of the role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain

120

KEITH E. LATHAM

phosphorylation through structure–function studies of TFIIE-alpha. Mol. Cell. Biol. 15, 4856–4866. Okamoto, H., Sheline, C. T., Corden, J. L., Jones, K. A., and Peterlin, B. M. (1996). Transactivation by human immunodeficiency virus Tat protein requires the C-terminal domain of RNA polymerase II. Proc. Natl. Acad. Sci. USA 93, 11575–11579. Palermo, G. D., Colombero, L. T., and Rosenwaks, Z. (1997). The human sperm centrosome is responsible for normal syngamy and early embryonic development. Rev. Reprod. 2, 19–27. Paris, J., Swenson, K., Piwnica-Worms, H., and Richter, J. D. (1991). Maturation-specific polyadenylation: In vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE binding protein. Genes Dev. 5, 1697–1708. Patturajan, M., Schulte, R. J., Sefton, B. M., Berezney, R., Vincent, M., Bensaude, O., Warren, S. L., and Corden, J. L. (1998). Growth-related changes in phosphorylation of yeast RNA polymerase II. J. Biol. Chem. 273, 4689–4694. Perreault, S. D., and Zirkin, B. R. (1982). Sperm nuclear decondensation in mammals: Role of sperm-associated proteinase in vivo. J. Exp. Zool. 224, 253–257. Perreault, S. D., Naish, S. J., and Zirkin, B. R. (1987). The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content. Biol. Reprod. 36, 239–244. Piko´, L., Hammons, M. D., and Taylor, K. D. (1984). Amounts, synthesis, and some properties of intracisternal A particle-related RNA in early mouse embryos. Proc. Natl. Acad. Sci. USA 81, 488–492. Poccia, D., (1986). Remodelling of nucleoproteins during gametogenesis, fertilization, and early development. Int. Rev. Cytol. 105, 1–65. Poccia, D., and Collas, P. (1997). Nuclear envelope dynamics during male pronuclear development. Dev. Growth Differ. 39, 541–550. Poccia, D., Salik, J., and Krystal, G. (1981). Transitions in histone variants of the male pronucleus following fertilization and evidence for a maternal store of cleavage-stage histones in the sea urchin egg. Dev. Biol. 82, 287–296. Poccia, D., Greenough, T., Green, G. R., Nash, E., Erickson, J., and Gibbs, M. (1984). Remodeling of sperm chromatin following fertilization: Nucleosome repeat length and histone variant transitions in the absence of DNA synthesis. Dev. Biol. 104, 274–286. Poccia, D., Pavan, W., and Green, G. R. (1990). 6DMAP inhibits chromatin decondensation but not sperm histone kinase in sea urchin male pronuclei. Exp. Cell Res. 188, 226–234. Poueymirou, W. T., and Schultz, R. M. (1987). Differential effects of activators of cAMPdependent protein kinase and protein kinase C on cleavage of one-cell mouse embryos and protein synthesis and phosphorylation in one- and two-cell embryos. Dev. Biol. 121, 489–498. Prather, R. S., and Rickords, L. F. (1992). Developmental regulation of an snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning. Mol. Reprod. Dev. 33, 119–123. Prather, R. S., Kubiak, J., Maul, G. G., First, N. L., and Schatten, G. (1991). The expression of nuclear lamin A and C epitopes is regulated by the developmental stage of the cytoplasm in mouse oocytes or embryos. J. Exp. Zool. 257, 110–114. Ram, P. T., and Schultz, R. M. (1993). Reporter gene expression in G2 of the 1-cell mouse embryo. Dev. Biol. 156, 552–556. Reik, W., Ro¨mer, I., Barton, S. C., Surani, M. A., Howlett, S. K., and Klose, J. (1993). Adult phenotype in the mouse can be affected by epigenetic events in the early embryo. Development 119, 933–942. Reinsch, S., and Karsenti, E. (1997). Movement of nuclei along microtubules in Xenopus egg extracts. Curr. Biol. 7, 211–214. Robl, J. M., Gilligan, B., Critser, E. S., and First, N. L. (1986). Nuclear transplantation in mouse embryos: Assessment of recipient cell stage. Biol. Reprod. 34, 733–739.

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

121

Rodman, T. C., Pruslin, F. H., and Allfrey, V. G. (1982). Mechanism of displacement of sperm basic nucler proteins in mammals. An in vitro simulation of post-fertilization results. J. Cell Sci. 53, 227–244. Rogers, I., and Varmuza, S. (1996). Epigenetic alterations brought about by lithium treatment disrupt embryo development. Mol. Reprod. Dev. 45, 163–170. Rohlff, C., Ahmad, S., Borellini, F., Lei, J., and Glazer, R. I. (1997). Modulation of transcription factor Sp1 by cAMP-dependent protein. J. Biol. Chem. 272, 21137–21141. Sanceau, J., Kaisho, T., Hirano, T., and Wietzerbin, J. (1995). Triggering of the human interleukin-6 gene by interferon-gamma and tumor necrosis factor-alpha in monocytic cells involves cooperation between interferon regulatory factor-1, NF kappa B, and transcription J. Biol. Chem. 270, 27920–27931. Savic, A., Richman, P., Williamson, P., and Poccia, D. (1981). Alterations in chromatin structure during early sea urchin embryogenesis. Proc. Natl. Acad. Sci. USA 78, 3706–3710. Schafer, D., Hamm-Kunzelmann, B., and Brand, K. (1997). Glucose regulates the promoter activity of aldolase A and pyruvate kinase M2 via dephosphorylation of Sp1. FEBS Lett. 417, 325–328. Schatten, G., Maul, G. G., Schatten, H., Chaly, N., Simerly, C., Balczon, R., and Brown, D. L. (1985). Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins. Proc. Natl. Acad. Sci. USA 82, 4727–4731. Schatten, H., Simerly, C., Maul, G., and Schatten, G. (1989). Microtubule assembly is required for the formation of pronuclei, nuclear lamin acquisition, and DNA synthesis during mouse, but not sea urchin, fertilization. Gamete Res. 23, 309–322. Segil, N., Guermah, M., Hoffmann, A., Roeder, R. G., and Heintz, N. (1996). Mitotic regulation of TFIID: Inhibition of activator-dependent transcription and changes in subcellular localization. Genes Dev. 10, 2389–2400. Serizawa, H. (1998). Cyclin-dependent kinase inhibitor p16INK4A inhibits phosphorylation of RNA polymerase II by general transcription factor TFIIH. J. Biol. Chem. 273, 5427–5430. Serizawa, H., Makela, T. P., Conaway, J. W., Conaway, R., Weinberg, R. A., and Young, R. A. (1995). Association of Cdk-activating kinase subunits with transcription factor TFIIH. Nature 374, 280–282. Sheets, M. D., Fox, C. A., Hunt, T., Vande Woude, G., and Wickens, M. (1994). The 3⬘ untranslated regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic polyadenylation. Genes Dev. 8, 926–938. Shiekhattar, R., Mermelstein, F., Fisher, R. P., Drapkin, R., Dynlacht, B., Wessling, H. C., Morgan, D. O., and Reinberg, D. (1995). Cdk-activating kinase complex is a component of human transcription factor TFIIH. Nature 374, 283–287. Shilatifard, A. (1998). The RNA polymerase II general elongation complex. Biol. Chem. 379, 27–31. Shuman, S. (1997). Origins of mRNA identity: Capping enzymes bind to the phosphorylated C-terminal domain of RNA polymerase II. Proc. Natl. Acad. Sci. USA 94, 12758–12760. Simon, R., Tassan, J.-P., and Richter, J. D. (1992). Translational control by poly(A) elongation during Xenopus development: Differential repression and enhancement by a novel cytoplasmic polyadenylation element. Genes Dev. 6, 2580–2591. Simon, R., Wu, L., and Richter, K. D. (1996). Cytoplasmic polyadenylation of activin receptor mRNA and the control of pattern formation in Xenopus development. Dev. Biol. 179, 239–250. Smith, L. C., Wilmut, I., and Hunter, R. H. (1988). Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos. J. Reprod. Fertil. 84, 619–624. Smith, L. C., Wilmut, I., and West, J. D. (1990). Control of first cleavage in single-cell reconstituted mouse embryos. J. Reprod. Fertil. 88, 655–663. Smith, L. C., Meirelles, F. V., Bustin, M., and Clarke, H. J. (1995). Assembly of somatic histone H1 onto chromatin during bovine early embryogenesis. J. Exp. Zool. 273, 317–326.

122

KEITH E. LATHAM

Smith, S. D., Soloy, E., Kanka, J., Holm, P., and Callesen, H. (1996). Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos. Mol. Reprod. Dev. 45, 444–450. Spencer, C. A., Dahmus, M. E., and Rice, S. A. (1997). Repression of host RNA polymerase II transcription by herpes simplex virus type 1. J. Virol. 71, 2031–2040. Stein, P., Worrad, D. M., Belyaev, N., Turner, B. M., and Schultz, R. M. (1997). Stagedependent redistributions of acetylated histones in nuclei of the early preimplantation mouse embryo. Mol. Reprod. Dev. 47, 421–429. Sutovsky, P., and Schatten, G. (1997). Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization. Biol. Reprod. 56, 1503–1512. Sutovsky, P., Hewitson, L., Simerly, C. R., Tengowski, M. W., Navara, C. S., Haavisto, A., and Schatten, G. (1996). Intracytoplasmic sperm injection for rhesus monkey results in unusual chromatin, cytoskeletal, and membrane events, but eventually leads to pronuclear development and sperm aster assembly. Hum. Reprod. 11, 1703–1712. Sutovsky, P., Oko, R., Hewitson, L., and Schatten, G. (1997). The removal of the sperm perinuclear theca and its association with bovine surface during fertilization. Dev. Biol. 188, 75–84. Szollosi, D., and Yotsuyanagi, Y. (1985). Activation of paternally derived regulatory mechanism in early mouse embryo. Dev. Biol. 111, 256–259. Szollosi, D., Czolowska, R., Soltynska, M. S., and Tarkowski, A. K. (1986). Remodeling of thymocyte nuclei in activated mouse oocytes: An ultrastructural study. Eur. J. Cell Biol. 42, 140–151. Szollosi, D., Czolowska, R., Szollosi, M. S., and Tarkowski, A. K. (1988). Remodeling of mouse thymocyte nuclei depends on the time of their transfer into activated, homologous oocytes. J. Cell Sci. 91, 603–613. Tarkowski, A. K., and Balakier, H. (1980). Nucleo-cytoplasmic interactions in cell hybrids between mouse oocytes, blastomeres, and somatic cells. J. Embryol. Exp. Morphol. 55, 319–330. Tesarik, J., and Kopecny, V. (1989). Developmental control of the human male pronucleus by ooplasmic factors. Hum. Reprod. 4, 962–968. Thompson, E. M., Legouy, E. M., Christians, E., and Renard, J.-P. (1995). Progressive maturation of chromatin structure regulates HSP70.1 gene expression in the preimplantation mouse embryo. Development 121, 3425–3437. Ura, K., Kurumizaka, H., Dimitrov, S., Almouzni, G., and Wolffe, A. P. (1997). Histone acetylation: Influence on transcription, nucleosome mobility and positioning, and linker histone-dependent transcriptional repression. EMBO J. 16, 2096–2107. Valay, J. G., Dubois, M. F., Bensaude, O., and Faye, G. (1996). Ccl1, a cyclin associated with protein kinase Kin28, controls the phosphorylation of RNA polymerase II largest subunit and mRNA transcription. Comptes Rendus Acad. Sci. Ser. I ii Sci. Vie 319, 183–189. van Stekelenburg-Hamers, A. E., Rebel, H. G., van Inzen, W. G., de Loos, F. A., Drost, M., Mummery, C. L., Weima, S. M., and Trounson, A. O. (1994). Stage-specific appearance of the mouse antigen TEC-3 in normal and nuclear transfer bovine embryos: Re-expression after nuclear transfer. Mol. Reprod. Dev. 37, 27–33. Varnum, S. M., and Wormington, W. M. (1990). Deadenylation of maternal mRNAs during Xenopus oocyte maturation does not require specific cis-sequences: A default mechanism for translational control. Genes Dev. 4, 2278–2286. Vassalli, J.-D., Huarte, J., Belin, D., Gubler, P., Vassalli, A., O’Connell, M. L., Parton, L. A., Rickles, R. J., and Strickland, S. (1989). Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes. Genes Dev. 3, 2163–2171. Venetianer, A., Dubois, M. F., Nguyen, V. T., Bellier, S., Seo, S. J., and Bensaude, O. (1995). Phosphorylation state of the RNA polymerase II C-terminal domain (CTD) in heat-shocked

MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION

123

cells. Possible involvement of the stress-activated mitogen-activated protein (MAP) kinases. Eur. J. Biochem. 233, 83–92. Venezky, D. L., Angerer, L. M., and Angerer, R. C. (1981). Accumulation of histone repeat transcripts in the sea urchin egg pronucleus. Cell 24, 385–391. Vernet, M., Bonnerot, C., Briand, P., and Nicolas, J. F. (1992). Changes in permissiveness for the expression of microinjected DNA during the first cleavages of mouse embryos. Mech. Dev. 36, 129–139. Verotti, A. C., Thompson, S. R., Wreden, C., Strickland, S., and Wickens, M. (1996). Evolutionary conservation of sequence elements controlling cytoplasmic localization. Proc. Natl. Acad. Sci. USA 93, 9027–9032. Vlach, J., Garcia, A., Jacque, J. M., Rodriguez, M. S., Michelson, S., and Virelizier, J. L. (1995). Induction of Sp1 phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid. Virology 208, 753–761. Wade, P. A., Pruss, D., and Wolffe, A. P. (1997). Histone acetylation: Chromatin in action. Trends Biochem. Sci. 22, 128–132. Wakayama, T., Perry, A. C. F., Zuccotti, M., Johnson, K. R., and Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374. Wang, Q., and Latham, K. E. (1997). A role for protein synthesis during embryonic genome activation in mice. Mol. Reprod. Dev. 47, 265–270. Wang, W., Xue, Y., Zhou, S., Kuo, A., Cairns, B. R., and Crabtree, G. R. (1996). Diversity and specialization of mammalian SWI/SNF complexes. Genes Dev. 10, 2117–2130. West, M. F., Verrotti, A. C., Salles, F. J., Tsirka, S. E., and Strickland, S. (1996). Isolation and characterization of two novel cytoplasmically polyadenylated oocyte-specific mouse maternal mRNAs. Dev. Biol. 175, 132–141. White, R. J., Gottlieb, T. M., Downes, C. S., and Jackson, S. P. (1995). Mitotic regulation of a TATA-binding-protein-containing complex. Mol. Cell. Biol. 15, 1983–1992. Weikowski, M., Mirands, M., and DePamphilis, M. L. (1991). Regulation of gene expression in preimplantation mouse embryos: Effects of the zygotic clock and the first mitosis on promoter and enhancer activities. Dev. Biol. 147, 403–414. Wiekowski, M., Miranda, M., and DePamphilis, M. L. (1993). Requirements for promoter activity in mouse oocytes and embryos distinguish paternal pronuclei from maternal and zygotic nuclei. Dev. Biol. 159, 366–378. Wiekowski, M., Miranda, M., Nothias, J.-Y., and DePamphilis, M. L. (1997). Changes in histone synthesis and modification at the beginning of mouse development correlate with the establishment of chromatin mediated repression of transcription. J. Cell Sci. 110, 1147–1158. Williams, B. C., Dernburg, A. F., Puro, J., Nokkala, S., and Goldberg, M. L. (1997). The Drosophila kinesin-like protein KLP3A is required for proper behavior of male and female pronuclei at fertilization. Development 124, 2365–2376. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., and Campbell, K. H. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813. Witkowska, A. (1981). Pronuclear development and the first cleavage division in polyspermic mouse eggs. J. Reprod. Fertil. 62, 493–498. Worrad, D. M., and Schultz, R. M. (1997). Regulation of gene expression in the preimplantation mouse embryo: Temporal and spatial patterns of expression of the transcription factor Sp1. Mol. Reprod. Dev. 46, 268–277. Worrad, D. M., Ram, P. T., and Schultz, R. M. (1994). Regulation of gene expression in the mouse oocyte and early preimplantation embryo: Developmental changes in Sp1 and TATA box binding protein, TBP. Development 120, 2347–2357. Worrad, D. M., Turner, B. M., and Schultz, R. M. (1995). Temporally restricted spatial localization of acetylated isoforms of histone H4 and RNA polymerase II in the 2-cell mouse embryo. Development 121, 2949–2959.

124

KEITH E. LATHAM

Wright, S. J., and Longo, F. J. (1988). Sperm nuclear enlargement in fertilized hamster eggs is related to meiotic maturation of the maternal chromatin. J. Exp. Zool. 247, 155–165. Yamashita, M., Onozato, H., Nakanishi, T., and Nagahama, Y. (1990). Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: Direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii. Dev. Biol. 137, 155–160. Yang, X., Herrmann, C. H., and Rice, A. P. (1996a). The human immunodeficiency virus Tat proteins specifically associate with TAK in vivo and require the carboxyl-terminal domain of RNA polymerase II for function. J. Virol. 70, 4576–4584. Yang, X.-J., Ogryzko, V. V., Nishikawa, J.-I., Howard, B. H., and Nakatani, Y. (1996b). A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382, 319–324. Yoder, J. A., Walsh, C. P., and Bestor, T. H. (1997). Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 13, 335–340. Yoshida, M., Ishigaki, K., and Pursel, V. G. (1992). Effect of maturation media on male pronucleus formation. Mol. Reprod. Dev. 31, 68–71. Yoshida, M., Ishigaki, K., Nagai, T., Chikyu, M., and Pursel, V. G. (1993). Glutathione concentration during maturation and after fertilization in pig oocytes: Relevance to the ability of oocytes to form male pronucleus. Biol. Reprod. 49, 89–94. Zutter, M. M., Ryan, E. E., and Painter, A. D. (1997). Binding of phosphorylated Sp1 protein to tandem Sp1 binding sites regulates alpha-2 integrin gene core promoter activity. Blood 90, 678–689.

Temporal and Spatial Coordination of Cells with Their Plastid Component Annette W. Coleman and Andrea M. Nerozzi Division of Biology and Medicine, Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912

Careful coordination of cell multiplication with plastid multiplication and partition at cytokinesis is required to maintain the universal presence of plastids in the major photosynthetic lines of evolution. However, no cell cycle control points are known that might underlie this coordination. We review common properties, and their variants, of plastids and plastid DNA in germline, multiplying, and mature cells of phyla capable of photosynthesis. These suggest a basic level of control dictated perhaps by the same mechanisms that coordinate cell size with the nuclear ploidy level. No protein synthesis within the plastid appears to be necessary for this system to operate successfully at the level that maintains the presence of plastids in cells. A second, and superimposed, level of controls dictates expansion of the plastid in both size and number in response to signals associated with differentiation and with the environment. We also compare the germane properties of plastids with those of mitochondria. With the advent of genomics and new cell and molecular techniques, the players in these control mechanisms should now be identifiable. KEY WORDS: Cell cycle, Mitochondrion, Plastid, Plastid division, Plastid DNA, Plastid mutations. 䊚 1999 Academic Press.

I. Introduction The endosymbiont origin of plastids from a free-living photosynthetic prokaryote precursor is now widely accepted. This momentous evolutionary development required a remarkable mutual accommodation, akin to that of any nonlethal parasite vis-a`-vis its host but with the added requirement of germline transmission. Intracellular accommodation of a foreign organInternational Review of Cytology, Vol. 193 0074-7696/99 $30.00

125

Copyright 䉷 1999 by Academic Press. All rights of reproduction in any form reserved.

126

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

ism, and its permanent possession and transmission through the germline, appears to be a rarity in evolution, although the mitochondrion is the other well-known example. Cases in which intracellular bacteria are found within germ cells of insects (Moran, 1996), affecting their fertility, are known, and numerous protistan examples exist in which intracellular prokaryotes or eukaryotes are maintained, perhaps because they confer a nutritional advantage on their hosts. However, none of these latter examples match the sophistication of parasite control found with plastids and mitochondria, in which germline inheritance is accompanied by somatic cell-controlled expansion or restriction of endosymbiont numbers and development. The ability of plastids to proliferate to a large population within a cell implies that the cell must be able to control the population lest it be overgrown. The biochemical mechanism that provides this control is unknown, although the potential for such control is obvious. The change from free-living prokaryote to tamed, intracellular symbiont was accompanied by a loss of 90% of the symbiont DNA, as determined from a comparison of prokaryote and plastid genome sizes (Gray, 1991). Analysis of total plastid genome sequences also suggests why no plastid has ever been successfully cultivated in vitro. In many cases, even tRNAs for protein synthesis must be provided by the host cell because most of the necessary genes are absent from the plastid genome. The opposite problem is the total loss of plastids from members of a photosynthetic clade of organisms. Culture lines totally lacking mitochondria and mitochondrial DNA have been isolated from yeasts, chicken fibroblasts, and human cell lines (Desjardins et al., 1985; King and Attardi, 1989; Haffter and Fox, 1992). By contrast, no photosynthetic organism has been ‘‘cured’’ of its plastids, even in tissue culture, with one exception— strain B of Euglena gracilis, a unicell (Rawson and Boerma, 1976; Conkling et al., 1993). This observation implies that the eukaryote hosts, including unicell species and parasitic plants lacking photosynthesis, have developed a metabolic requirement for one or more plastid-located activities [for examples of multiple metabolic pathways with plastid-located steps, see Sears (1980), Bogorad and Vasil (1991), Doremus and Jagendorf (1995), Taylor (1996), Hrazdina and Jensen (1992), Salisbury and Ross (1992), and Weeden (1981)]. Apparently, only photosynthetic eukaryote lines that developed highly effective selection for retention of the endosymbiont have survived, i.e, loss of the plastid results in death of the host cell. The exception that supports the rule is provided by the Euglena example. Euglena is recognized as being related to the Kinetoplastida lineage, which comprises very animal-like protozoans, that obtained a plastid by secondary endosymbiosis, uptake of a photosynthetic eukaryote, followed by permanent retention of only its plastid. In the Euglena, the endosymbiont–host combination

PLASTID–CELL COORDINATION

127

may be of relatively recent evolutionary vintage because it lacks an absolute metabolic requirement for a plastid-located activity. Euglena is known to have two pathways for porphyrin synthesis, one in the plastid and one in the mitochondrion; the latter is typical of animal cells generally but not plants (Hoober, 1987). Analysis of plastid DNA deletions characterizing different pollen-derived rice callus cell cultures (Harada et al., 1991, 1992) suggests that the DNA region common to all the survivors contains the plastid gene for glutamyl tRNA, a cofactor necessary for porphyrin synthesis in the plant pathway (Hoober, 1987). Plastidless Euglena survives by using its animal-like pathway. Thus, in the major photosynthetic eukaryote lines, the host is metabolically dependent on its plastid component, not just for photosynthesis, whereas the plastid is totally dependent on its host cell for many of its basic nucleic acid and protein synthesis components. How do plastid and host cell coordinate? There are many excellent reviews of plastids and the history of their cytological and molecular research (Herrmann and Possingham, 1980; Boffey and Lloyd, 1988; Boyer et al., 1989; Clegg et al., 1991; Bogorad and Vasil, 1991; Kuroiwa, 1991; Gillham, 1994). However, the fundamental interrelationships of plastids and plastid DNA genomes with events of the cell and life cycle have largely been ignored; thus, this topic will be examined here. Despite the wealth of papers delineating factors associated with checkpoints in the cell cycle, essentially all discussions of the eukaryote cell cycle omit mention of a requirement for plastid or mitochondrial replication. No gene has been implicated in directly controlling the coordination of the organelle cycle with that of its nucleus. A recent review on cells and their DNA content did not discuss plastid DNA. This is not solely because of a lack of progress in research; however, the data are widely scattered in the literature. Newly developed techniques can be used to answer fundamental questions concerning the organization and behavior of plastid genomes and plastids. Prominent among these techniques as applied to single cells and their plastids are fluorescence microscopy methods, including marker fluorescent dyes, DNA-binding fluorochromes applicable to both fixed and living cells (Fig. 1A; see color plate), and nucleotide analog incorporation detectable with fluorescently labeled antibody. For quantification, microspectrofluorimetry and computer-assisted image analysis quantification are invaluable, replacing biochemical averages with actual single cell and single organelle data. After summarizing the basic background information on the cytological variations of plastid DNA locale among photosynthetic eukaryotes, we will review the available information on (i) structural features of the plastid DNA multigenome aggregate of a plastid; (ii) plastid division and its rela-

128

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

tionship to cell division; (iii) proportionality of plastid DNA to nuclear DNA and to cell size; (iv) plastid DNA replication sites and timing of synthesis in cell multiplication and differentiation; and (v) mechanisms associated with delivery of plastids to daughter cells, an apparent challenge in the absence of any recognizable physical device akin to the mitotic spindle. We will try to present as concisely as possible the basic observations, both cytological and molecular, concerning plastids of all types and clarify their implications. Rather than an exhaustive bibliography, we will cite review articles or at least recent references suitable to serve as entries into the literature. Where appropriate and useful, parallel information on mitochondrial DNA will be presented. The aim is to suggest ways in which the wedding of molecular and cytological methods, particularly fluorescence microscopy, might be most useful for decoding the basis of plastid–cell coordination.

II. Basic Background Information Plastid (pt) is a useful generic term. Plastids can become differentiated for functions other than the photosynthesis associated with chloroplasts (Herrmann and Possingham, 1980). Highly pigmented plastids of fruits and flowers are termed chromoplasts, starch reservoirs in potato tuber are amyloplasts, and various oil-accumulating plastid types are known. In terrestrial plants, all are derived ultimately from the plastid type found in meristematic cells, the proplastid. Even root cells contain plastids, though these are not obviously differentiated. Another useful term is plastome, which we use here to include the composite of all the plastids in a single cell and their DNA. A single cell may have from one (e.g., Chlamydomonas or Ochromonas growing rapidly in the dark) to more than 1000 plastids (chromoplasts of tomato pericarp). Plastid division (Whatley, 1988) is typically observed as a simple pinching in two of the plastid, although larger plastids may sometimes bud off a smaller lobe. Among the green algae, plastids may assume very large and elaborate shapes of diagnostic constancy, whereas most other organisms are content with small discoid or flattened ovals as their plastid form. All plastid forms contain plastid DNA (Scott and Possingham, 1980; Scott et al., 1984) with two general exceptions (discussed later). Both biochemical analyses and microspectrofluorometry of DNA of individual plastids have shown that multiple copies of the basic pt genome exist within a single plastid. Rarely if ever do plastid genome numbers per cell, or per plastid, decrease to below a multiplicity of 5 (Maguire et al., 1995), and in larger

PLASTID–CELL COORDINATION

129

plastids they can exceed 100. Thus, plastids are fundamentally multiploid organelles. Molecular studies have shown that the plastid genome is a circular doublestranded DNA, in most cases ranging in size from 80 to 160 kbp in various photosynthetic eukaryotes. The entire plastid genome has been sequenced and almost all the genes have been identified for several terrestrial plants, the diatom Odontella (Kowallik et al., 1995), the red alga Porphyra (Reith and Munholland, 1995), Euglena (Hallick et al., 1993), and the endosymbiont of Cyanophora (Stirewalt et al., 1995). From studies of plant plastids, it is generally assumed that plastid DNA replication is initiated at a site forming a D loop, with DNA replication of the Cairns and perhaps also of the rolling circle type (Kolodner and Tewari, 1975a,b). Bendich and Smith (1990) demonstrated that ptDNA molecules are not all present as monomeric circles. Deng et al. (1989) reported that a significant proportion are dimers, trimers, and even tetramers of the basic genome, present in exponentially decreasing amounts. This was true in both young and mature spinach leaves, tomato, pea and maize, and the green unicellular alga Mesotaenium caldarioram; all are organisms with plastids containing scattered nucleoids. To this list can now be added a chrysophyte, Ochromonas, in which nucleoids are arranged in a continuous ring (Nerozzi, 1995).

III. Architecture of Plastid DNA Genome Complexes The multiple copies of the plastid DNA in the stroma of a plastid are not dispersed singly throughout the plastid stroma but are aggregated to form macrostructures easily visible with a fluorescence microscope after staining with a DNA fluorochrome such as DAPI (4⬘, 6-diamidino-2-phenylindole). These aggregates are called nucleoids. Comparisons among organisms of the more than 10 different photosynthetic eukaryote clades (variously called algae, protozoa, protistans, and plants) reveal some commonalities and apparent limitations to the characteristics of the arrangement of these nucleoids into the organized plastid genome complex of a plastid.

A. Mature Plastids There are basically two different conformations of the nucleoids of a plastid, each characterizing a subset of the various phyla of photosynthetic eukaryotes (Coleman 1985a). The first typifies green algae and plant plastids as

130

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

well as several other algal phyla (Fig. 2). One to several genomes form each of the multiple small DNA aggregates scattered among the thyllakoids of the plastid. Although these scattered nucleoids often appear quite separate from each other, several authors have emphasized that further study may reveal interconnections among them. The second major plastid DNA genome arrangement is the ring nucleoid arrangement (Figs. 3 and 4), a necklace of the multiple genome copies that lies just within the girdle lamellae (the encircling lamellae beneath the plastid envelope membranes). In face view it is an unbroken ring, often beaded in appearance like a necklace; in side view a section reveals only a short line (grazing section) or two densities at plastid extremes, the cross section of the necklace. The ring genome is clearly continuous in the physical sense since it can be isolated intact and stretched to more than three times its length without breaking the apparent continuity of the DNA (Fig. 5). Kuroiwa and Suzuki (1981) isolated ring genomes from Ectocarpus, fixed them briefly with glutaraldehyde, and then found that DNase but not RNase caused breaks in the ring and gradual dissolution. Application of trypsin caused an apparent swelling of the structure. More than 99.9% of photosynthetic eukaryote species display one of these two major conformations in mature cells—the scattered aggregates and the ring of aggregates. The only significant difference between the two conformations is that the former is strikingly three-dimensional whereas the latter is planar. Rare additional plastid genome architectures are found, but only in a very few organisms, all classified as Xanthophyceae, a diverse class in other respects as well. These additional forms, with net-like webbing near the periphery of the plastid, are described by Coleman (1985a). Three simple observations can be made. First, all the arrangements of the multiple plastid genomes in a plastid, since they extend throughout the plastid, would seem to guarantee ptDNA to each daughter plastid no matter how unequal the division. Second, there is no sign of innate polarity to the plastid. Finally, none of the arrangements resemble that of the DNA in blue-green algae, the presumed prokaryote ancestors of plastids (Coleman, 1985b). A point of interest, particularly regarding the ultimate resolution of whether plastid DNA nucleoid architecture is dictated by the plastid type or the cell type, is the presence of exceptional plastid genome DNA arrangements among organisms assumed to be uniform in heritage. For example, Vaucheria plastids, alone among examined xanthophyte algae, have scattered nucleoids rather than the expected ring genome. However, molecular biology studies fail to suggest why this should be. Plastid genome analyses relate Vaucheria plastid DNA closely to that of other xanthophytes, as does nuclear DNA analysis (rDNA) (Daugbjerg and Andersen, 1997). One additional set of exceptions concerns the organisms originally lumped as Cyanidium, a genus of unicellular red algae often found associ-

PLASTID–CELL COORDINATION

131

FIGS. 2–5 (2) Photomicrograph of an isolated, slightly flattened young mesophyll cell of Arabidopsis fixed and stained with DAPI. Visible are one nucleus and about 35 plastids, each containing multiple scattered nucleoids of DNA. Scale bar ⫽ 15 애m. (3) Region of an Ectocarpus cell fixed and stained with DAPI to reveal the ring nucleoids of the plastids. Scale bar ⫽ 15 애m. (4) Cell of Ochromonas danica prepared to preserve and display all the DNA. Cell contains a slightly broken nucleus, four plastids, and approximately 119 mitochondrial DNA nucleoids. Microspectrofluorometry reveals that there are a total of about 220 plastid genomes and 246 mitochondrial genomes in this cell. Scale bar ⫽ 15 애m. (Reproduced with permission from Maguire, M. J., Goff, L. J., and Coleman, A. W. (1995). In situ plastid and mitochondrial DNA determination; Implication of the observed minimal plastid genome number. Am. J. Bot. 82, 1496–1506. (5) Fluorescence photomicrograph of stretched ring nucleoids from isolated Gonyostomum plastids fixed and stained with DAPI. Longest ring visible is 55 애m in circumference. Scale bar ⫽ 15 애m. (Reproduced with permission from Coleman, A. W., and Heywood, P. (1981). Structure of the chloroplast and its DNA in Chloromonadophycean algae. J. Cell Sci. 49, 401–409, Company of Biologists Ltd.

132

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

ated with hot springs. Although all these organisms are considered red algae, they have been split on various grounds into Cyanidium caldarium (formerly Cyanidium caldarium RK-1 and A), Cyanidioschyzon merolae (formerly CD), and Galdieria sulphuraria (formerly Cyanidium caldarium M-8⫽B). Of these, the first, two have scattered nucleoids typical of red algae (Coleman, 1985a), whereas the latter has a ring nucleoid in its plastid (Kuroiwa, 1981; Kuroiwa et al., 1994). In summary, plastid DNA nucleoid macroarchitecture does not correlate with primary versus secondary origin of the plastid (Table I) nor with the presence or absence of the classic girdle lamellae (Gibbs, 1990). However, it is consistently uniform within each major clade of algae, with three exceptions: the xanthophytes, a subgroup of dinoflagellates containing plastids harvested from various secondary symbioses, and some unicells considered red algae.

TABLE I Plastid Characteristics of Major Photosynthetic Groups ptDNA type

Organism

Membranea

GLb

Scattered nucleoids Scattered nucleoids Scattered nucleoids Scattered nucleoids Scattered nucleoids Scattered nucleoids (except secondary symbioses) Scattered nucleoids Scattered nucleoids Scattered nucleoids Scattered nucleoids Ring nucleoid Ring nucleoid Ring nucleoid Ring nucleoid Ring nucleoid (except Vaucheria and rare additional ptDNA arrangements)

Dictyocha Chloroarachnion Rhodophyta Chlorophyta Euglenophyta Pyrrhophyta

3/4 4 2 2 3 3

⫹ ⫺ ⫺ ⫺ ⫺ ⫺

Synurophyceae Cryptophyta Prymnesiophyta Eustigmatophyta Chrysophyta Bacillariophyta Phaeophyta Raphidiophyta Xanthophyta

4 4 4 4 4 4 4 4 4

⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

Note. From Gibbs (1962, 1990). a Total number of membranes surrounding plastid; the two plastid membranes ⫾ additional ones external to the plastid membranes. b GL, girdle lamellae; a triplet of thylakoid membranes just within the two plastid bounding membranes.

PLASTID–CELL COORDINATION

133

B. Developmental Variation Two aspects of development clearly affect ptDNA disposition. The first relates to the difference between the undeveloped or proplastid state compared with its more developed state in the same organism, described thoroughly by Kuroiwa et al. (1981, 1994). The proplastid occurs in meristematic cells of plants, in tip cells of some red algae, and in plastids of small unicells and such algae as Ochromonas when grown in the dark. In these cases, ptDNA typically forms a single nucleoid aggregate, even in Ochromonas in which the more developed plastid has a plastid ring nucleoid. The simplest explanation is that in the absence of internal thylakoids, there is no subcompartmentation of the plastid interior, which is then occupied by its DNA genomes alone as a single aggregate or nucleoid. This is, in fact, the stage which most resembles photosynthetic prokaryotes. Hashimoto (1985) described the transition from proplastid to full-blown chloroplast in the leaf of Avena sativa. In the basal meristem of the 2-mm leaf primordium, the proplastid central nucleoid expands into an arrangement of small nucleoids in the periphery of the small plastid. Cell division ceases by the time the primordium reaches 20 mm, and nucleoids in the enlarging and dividing plastids take on their mature arrangement, scattered through the plastid stroma. One final situation involves green organisms which make pyrenoids in their plastids. Nakamura et al. (1986) found a progressively greater restriction of plastid DNA to the region of the pyrenoid in starving Chlamydomonas cells, a situation rapidly reversed upon introduction of fresh nutrients. In the siphonaceous marine chlorophytes of the genus Caulerpa and its relatives, Miyamura and Hori (1991) found ptDNA predominantly restricted to the pyrenoid matrix of Caulerpa species in vegetative cells, but this DNA disposition changed to the more typical dispersed nucleoids at gametogenesis.

C. Variation with the Cell Cycle For organisms with ring nucloid genomes, no variation in ptDNA disposition has been observed during the cell cycle nor has variation been reported for terrestrial plants, representing the scattered nucleoid type. However, for the green unicell Chlamydomonas reinhardtii, several authors have described the alteration of dispersed, discrete nucleoids in newly formed G1 cells into larger nucleoids and, finally, associated with mitosis and cytokinesis, the apparent stretching out of these condensed nucleoids into a network or skein of fine thin threads of DNA. Postcytokinesis, the plastid

134

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

nucleoids are once again small, scattered, and discrete. Although the initial studies were done on cells fixed in alcohol or acid alcohol fixative, recent studies used glutaraldehyde. Ehara et al. (1990b) evaluated the effect of different glutaraldehyde concentrations, from 0.1 to 1% on the appearance of the DNA nucleoids stained with DAPI. They report that levels of glutaraldehyde ⬎0.5% produce images of multiple scattered nucleoids at all stages of the cell cycle. This suggests that the variations seen by others, after using lower concentrations of the fixative, reflect properties of proteins involved in the organization of multiple genomes into nucleoids. Some of the proteins responsible for maintaining the conformation of the nucleoids may be differentially sensitive to the fixative during the cell cycle. Even more detailed positional alterations of pt DNA during the cell cycle have been reported (Ehara et al., 1996).

IV. Cytology of Plastid Division The typical plastid approaching division takes on an hourglass morphology as a result of a median constriction. Although such views have often been labeled as dividing plastids in the literature, the morphology can be remarkably stable, lasting hours in a cell and being maintained in plastids isolated from cells (Whatley, 1980; Kuroiwa, 1991). Thus, such a shape is not necessarily indicative of immediately incipient cleavage of the plastid into two.

A. Division Apparatus Electron microscopy has revealed a ring of material external to the plastid at its isthmus, and sometimes between the inner and outer plastid membranes, just prior to plastid cleavage (Hashimoto and Possingham, 1989; Kuroiwa, 1991; Kuroiwa et al., 1998). Although nothing as organized has been found in angiosperms, the structure can be observed in the green algae Trebouxia and Pyramimonas and in the moss Funaria, as well as in C. caldarium RK1 (Mita et al., 1986). In this latter cell, cytochalasin B, but not colchicine, delays plastid cleavage, suggesting that actin filaments may play some role. The recent discovery (Osteryoung and Vierling, 1995) in plants of homologs of a family of prokaryote genes ( ftsZ, ftsH, and ftsW ) that control the formation of the septum in dividing bacteria (Erickson, 1995) offers promise for understanding events of plastid division. In plants, these homologs are nuclear genes, with products targeted to the plastid, whereas in red, green, and chromophyte algae they are part of the plastid genome. The gene

PLASTID–CELL COORDINATION

135

family shares a conserved region with clear affinity to tubulin genes. At least one puzzle to be answered is how a bacterial product that forms a structure just inside the (prokaryote) cell membrane is related to a structure that forms just outside the plastid. The plastid dividing ring may be more complex than originally described and may include material just inside the plastid (Miyagishima et al., 1998). Events at the final separation of two daughter plastids are very poorly understood, but one characteristic is noted in several disparate studies in the literature. In Arabidopsis, Leech et al. (1981) noted that the daughter plastids twist about the isthmus, and Slankis and Gibbs (1972) noted that daughter plastids rotate with respect to each other in Ochromonas. The breakage and restoration of the ring nucleoid, an inevitable consequence of separation of two daughter plastids in ring nucleoid types, must be rapid since it is not customary to observe any breaks in ring nucleoids and a search for such breaks in dividing plastids was fruitless (A. Coleman, unpublished observations).

B. Division Timing versus the Cell Cycle Multiplying cells must obviously increase their plastid component as well as their cytoplasm and nucleus lest they be lost. Rapidly dividing cells tend to have a minimal plastid content, whereas in some developing tissues the plastome becomes greatly expanded in each cell. 1. Rapidly Dividing Cells To determine the relative timing of plastid cleavage with respect to the cell cycle, extremely small uniplastidic cell types are perhaps the optimal material. Kuroiwa (1998) compared the timing among small unicellular red algae and found alternative patterns which are species specific. In Galdaria sulphuraria, karyokinesis occurs first, followed by plastid and then mitochondrial fission (Kuroiwa et al., 1989). In C caldarium, plastid division precedes nuclear division, with mitochondrial fission occurring last, and in Cyanidioschizon merolae the plastid cleaves, the mitochondrion cleaves, and finally the nucleus undergoes cytokinesis (Kuroiwa et al., 1998). In the latter organism, it has been shown that plastids can continue to divide at least twice after nuclear DNA synthesis is blocked (Itoh et al., 1996). In very large cells with only one plastid, coordination with nuclear division is quite clear. The desmid Micrasterias undergoes nuclear division and each parental semicell begins forming a new semicell that is invaded by the growing plastid of the parental semicell. Daughter plastid separation is completed long after mitosis (Fig. 1B).

136

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

In the case of Chlamydomonas, the single plastid encompasses a major proportion of the cell, and cleavage of the plastid is a continuation of the furrow cleaving the cell from base to apex (Harris, 1989). This process approximately accompanies nuclear division. In Scenedesmus, plastid and cell cleavage lag behind nuclear division, but plastid division continues for several rounds even if nuclear DNA synthesis is blocked (Zachleder et al., 1996). In the chrysophyte unicell Ochromonas growing exponentially, in which cells average one or two plastids per cell, it has been assumed that plastid division customarily occurs just prior to nuclear division. Nerozzi and Coleman (1997) found no significant consistency in plastid division time, and many plastids were seen to divide just subsequent to nuclear division. 2. Differentiating Tissue In the special case of cell differentiation in mesophyll cells of the developing leaf, numerous studies have contributed to our knowledge of the general pattern, which varies only slightly in the relative timing of events among different plants (Possingham, 1980; Lawrence and Possingham, 1986; Leech and Pyke, 1988; Kuroiwa, 1991). Briefly, proplastids in leaf meristem cells both synthesize DNA rapidly and grow and divide, not just keeping pace with the multiplying leaf progenitor cells but exponentially increasing the plastome component per cell throughout the period of cell division. During early stages of cell expansion, ptDNA synthesis continues as plastids enlarge and divide. Then plastid DNA synthesis ceases and plastids continue to divide for a short period while continuing to enlarge and differentiate. Thus, in expanding leaf cells of angiosperm species, plastid division continues after nuclear division has ceased. No single pattern of linkage to the nuclear events seems to apply to all plastids, and nuclear DNA inhibitor studies suggest no tight linkage of plastid division with a stage of the cell cycle.

V. Fundamental ptDNA:Plastome:Cell Size:Nuclear Ploidy Proportionality Eukaryote cells clearly have mechanisms that assess relative amounts of nuclear DNA, cytoplasm, plastome, and plastid DNA per cell, a property that is most obvious in mature cells. In mature cells that contain multiple plastids, plastid size is typically extremely uniform. This is particularly obvious in multiplastidic cells of green, red, brown, and raphidophyte algae and diatoms and in plant mesophyll cells (Figs. 2 and 3). There is an

PLASTID–CELL COORDINATION

137

apparent upper limit to the plastid size characteristic of particular cell types at maturity, e.g., about 50 애m2 in Arabidopsis mesophyll cells (Pyke and Leech, 1991) and with a ring nucleoid contour size of about 26 애m in Ectocarpus filaments (Fig. 3) (Kuroiwa et al., 1981). This phenomenon seems to be an extension of the basic proportional relationship between cell volume and the nucleus, although the appropriate cell or plastome volume is not always easy to measure, nor can individual plastid DNA quantity always be assessed directly in the same material. The history of the concept is summarized by Herrmann and Possingham (1980).

A. Mature Cells An approximately constant ratio of cell size (cytoplasmic volume) to nuclear ploidy level has long been recognized in eukaryote cells. This is obvious both in comparisons of plants of different ploidy levels and within the same plant in which endopolyploidy occurs in a tissue (Melaragno et al., 1993). Likewise, in mature cells of a particular cell type, plastome amount is directly proportional to ploidy level. For plant cells, Butterfass (1973, 1988) cited the amounts of plastids in guard cells of euploids versus polyploids in many plant species. Even for multinucleate cells of haploid versus diploid generations of the red alga Griffthsia, the cytoplasmic area attributable to each nucleus is proportionately larger and contains twice as many plastids in a diploid cell as in its haploid counterpart (Goff and Coleman, 1987). An additional component of the proportionality is the constancy of plastid DNA quantity per plastid, at least in mature/differentiated cells. In the Griffithsia, plastid size and plastid DNA quantity per plastid are nearly identical in the two generations so that the diploid nucleus, surrounded by twice as many plastids, is consequently associated with twice as much ptDNA. In uniplastidic cells, plastid number does not change, but the plastid of the diploid vegetative cells of C. reinhardtii has twice the DNA of its counterpart in the haploid cell (Whiteway and Lee, 1977). Where ploidy is unchanged, larger cells of the same differentiated cell type (influenced by growth conditions) often have increased numbers of plastids. Since the amount of ptDNA is proportional to plastid size in such cells, the total amount of plastid DNA is also proportional to cell volume in any one cell type.

B. Multiplying and Differentiating Cells In the transition from one resting stage to another, as in exponential growth of a unicell or production of mature leaf cells from the apical meristem, it

138

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

appears that both cell cycle length and differentiation signals influence plastid DNA per cell. Environmental conditions (e.g., light, temperature, and nutrients) can affect ptDNA per plastid as well as total plastome per cell (Herrmann and Possingham, 1980). For example, Rawson and Boerma (1976) found darkgrown stationary phase cells of Euglena to contain a mean of 200 plastid genome equivalents, whereas their light-grown counterparts averaged 600 and even as much as 1000 equivalents in totally autotrophic conditions. Maguire et al. (1995) assessed plastid size and ptDNA content in individual plastids of Ochromonas danica cells growing exponentially in two conditions—the presence or absence of light. Because of the heterotrophic properties of Ochromonas, the generation times were very similar. The ranges of plastid size (2.5–5.5 애m ring nucleoid contour length in the dark versus 9–18 애m in light) did not even overlap; however, the measured ranges of ptDNA content per ring nucleoid (앑20–60 genome equivalents) were the same for both conditions. The simplest conclusion is that this ptDNA amount characterizes the DNA synthetic capacity of the plastids during this minimal cell generation time, but that light-grown cells can manufacture larger plastids for the same amount of plastid DNA synthesis, ones that contain more thylakoids. In this same organism, postexponential cells in the light had a plastid size range of 13–24 애m, which was larger than but overlapping that of the light-grown exponential cells. However, these plastids had three times the amount of ptDNA (80–190 genome equivalents). On average, there were more than six genome equivalents per micrometer of plastid ring nucleoid in the postexponential cells, whereas most exponentially growing cells had two to four genomes per micrometer. The difference in genome density per micrometer of the ring genome is cytologically correlated to the observed thickening of the ring and the increase of knobs and larger localized DNA aggregates along the periphery of the ring in stationary phase cells. Here, there appear to be constraints on the total plastid materials versus the plastid DNA content in comparison with exponentially growing cells. An additional observation from the Ochromonas studies is germane to the proportionality question. The cell size decreases moderately during the first several cell divisions after transfer to fresh medium and then increases again. Total plastid area per cell, and also plastid DNA per cell, follow the same pattern (Maguire et al., 1995; Nerozzi, 1995; Nerozzi and Coleman, 1997). This contrasts with the earliest stages of study reported for leaf primordia, in which plastid DNA accumulation per cell increases steadily. However, no one has quantitated the stages of leaf formation prior to the 2-mm stage; this might yield results more similar to those of a unicell resuming growth.

PLASTID–CELL COORDINATION

139

C. Effects of Mutation Studies of leaf plastid mutants have contributed significant data to the recognition of this basic characteristic of the plastome versus the cell. Plastome volume and effective cell volume per se are not always easy to measure, and alternative estimations can serve as proxies. For example, in leaf mesodermal cells, in which there is a pavement of plastids in the cortical cytoplasm surrounding the central vacuole, the total surface area of plastids is proportional to the surface area of the cell they subtend. A study of Arabidopsis thaliana mutants displays the constancy of plastome to cell cytoplasm ratio most clearly. A series of papers (Pyke and Leech, 1992, 1994; Pyke et al., 1994; Robertson et al., 1995) analyzed several ‘‘arc’’ mutants, all at different nuclear loci, that altered plastid numbers but not cell size in mature leaf mesophyll cells. arc-1, 3, and -5 in cultivar explanation Landsberg all have similar plastome area to cell area ratios, but arc-3 and arc-5 plastids numbers per cell are lower than 20, whereas the wild-type has a mean of 50. Each plastid of arc-3 and arc-5 is compensatingly larger. Since the number of proplastids in meristematic cells at the cessation of cell division is 13 or 14 per cell, this suggests that subsequent to this point in development the plastids grow but do not divide. In the arc-6 mutant obtained in variety Wassilewskiya, mature mesophyll cells never contain more than three plastids, and these are very large (up to 1000 애m2 in area compared to 50 애m2 in wild type) and of complex shapes. In this mutant there are apparently only 2 or 3 proplastids in the postmitotic cells of the developing leaf, implying that plastid division ceased several cell divisions prior if the apical meristem cells still contain the number of proplastids typical in plants (10–16) (see Section VII). Thus, the constraint on plastid division in arc-6 is of a different type and timing, perhaps expressed in the organization of the plastome of the apical meristem and then subsequently maintained. The set of mutants also shows that when plastid division is severely curtailed, resulting plastids can be very nonuniform in size and shape, in contrast to wild-type behavior. Nevertheless, the constancy of plastome to cell size remained, and presumably so too did the total plastid DNA ratio to nucleus and cell size. The biochemical nature of these mutants is not known.

VI. Molecular Aspects of Plastid DNA Synthesis A. Cell Cycle Numerous studies on plants, green algae, and Ochromonas all indicate that ptDNA synthesis is essentially continuous throughout the cell cycle,

140

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

a situation also typical of both prokaryote DNA and mitochondrial DNA (with one exception, trypanosomal mitochondrial DNA; Ferguson et al., 1992). For light:dark synchronized unicells, there may be a diminution of plastid DNA synthetic rate in the latter part of the light period (corresponding to late G1 in these organisms), but there is no indication that plastid DNA synthesis is limited to the nuclear DNA synthetic stage (the S phase of the cell cycle) (Harris, 1989; Gillham, 1994). The second general observation is that ptDNA synthesis in not stringent (Birky, 1983). Nuclear DNA synthesis is stringent; that is, all nuclear DNA is replicated only once in each cell cycle. By contrast, biochemical analyses indicate that ptDNA molecules (and mitochondrial DNA; Bogenhagen and Clayton, 1977) appear to be selected at random for replication, and some are replicated more than once during a cell cycle whereas others may not be replicated at all in cells growing exponentially. The result, nevertheless, is an approximate doubling of ptDNA per cell cycle. From cytological studies of nucleotide analog incorporation in Ochromonas (Nerozzi and Coleman, 1997) it is clear that all plastids in a cell carry out ptDNA replication during a cell cycle, and that all plastids exhibit similar accumulation of analog incorporation (there is no indication of a master plastid). Furthermore, at any one instant there is an average of one synthesis site per 2 애m of ring nucleoid within a single plastid ring nucleoid (in these rapidly growing cells, 2 애m ⫽ four to eight copies of the plastid genome). The sites of nucleotide incorporation are not grouped at one or both ends of the plastid but distributed along the pt ring nucleoid. Pulsechase-pulse double-labeling studies suggested that new sites of synthesis appear steadily, and that some prior sites of synthesis may continue for several hours at least (total cell cycle ca 8.5 h). Only the largest plastids had significantly lower numbers of DNA synthesis sites, and plastids in stationary phase cells had only one or no sites of detectable DNA synthesis. All these cytological observations are in agreement with biochemical analyses indicating nonstringent DNA synthesis and further suggest the functional equivalence of all plastids in a cell and perhaps all genomes in a plastid.

B. Evidence That Cells Measure Plastid DNA (or Some Equivalent) in Cycling Cells The most striking example of a situation in which a cell must measure its DNA content (or some quantitative characteristic of its plastome) is found among unicells which exhibit a relatively prolonged period of growth in size and then undergo a rapid series of alternating S and M stages in the nucleus, like the fertilized eggs of animals, producing 2n daughter cells as

PLASTID–CELL COORDINATION

141

all cell cleavages are finally completed. Here, accumulation of sufficient materials necessary for the number of rapid nuclear doublings and divisions must occur in a prolonged G1 phase. Zachleder et al. (1996) recorded the accumulation of plastid DNA during the initial growth period in Scenedesmus quadricauda prior to any nuclear DNA synthesis. Plastid DNA accumulation is similar in C. reinhardtii prior to the onset of the successive mitoses and cell cleavages (Harris, 1989). The same cell cycle characterizes the related organism, Volvox carteri, but here the plastid DNA accumulation and ultimate distribution problem is exacerbated because ⬎29 daughter cells are formed rapidly from the material of just one large parental cell containing one large plastid. By the end of the prolonged G1 phase, the mother cell contains the necessary ingredients for ⬎1000 daughter cells; at this point, these cells contain more plastid DNA than nuclear DNA (Coleman and Maguire, 1982). Next, a series of alternations between nuclear DNA synthesis and karyokinesis accompanied by cytokinesis occurs that produces the many daughter cells within a period of a few hours. Exactly how does this system recognize when to stop the series of nuclear DNA syntheses and mitoses to avoid forming cells lacking plastid? In fact, occasional plastid maldistributions do occur (Coleman and Maguire, 1982). About 1 in 1000 daughter cells fails to receive any plastid material, as determined by fluorescence of chlorophyll or staining of plastid DNA. This contrasts with the situation for nuclei; in the same sample, only about 1 in 10,000 cells failed to receive a nucleus. These rare mistakes presumably have no impact on the organism because in Volvox the overwhelming majority of daughter cells are somatic rather than germline, destined to die without progeny. A few other examples of presumed errors are known in which plastids lack DNA or cells lack plastids, including a high proportion of genomeless plastids in very large cells of Acetabularia at one stage in the life cycle (Luttke and Bonotto, 1981). None of these examples are involved in the germline. We do not know whether their lack of ptDNA is genetically programmed or not.

VII. Plastid Distribution at Cytokinesis Since it has already been shown that ptDNA is spread throughout the plastid as a result of the architectural arrangements of its multiple genomes, the problem of delivering ptDNA to each daughter becomes a problem of delivering a plastid or significant plastid remnant to each daughter. There are multiple evolutionary solutions to both the number of plastids in the cell and their arrangement.

142

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

One might well ask why plastids divide into smaller bodies if arc-6 mutants survive so well. Multiple versus single plastids per cell seems to be functionally trivial since among the many species of Chlaydomonas, all fundamentally similar in cell size and habitat, both uniplastidic and multiplastidic types are found. Many species of green algae have but one very large plastid per cell (e.g., Spirogyra,) and its size and position are such that cell cleavage would always allocate plastid material to each daughter. In addition to the familiar bean-shaped plastids of leaf tissue, plastids may be pancake shaped, cup shaped with external fluting, or even star shaped. Regarding surface area to volume, essentially all plastids have conformations where no portion of the plastid interior is more than about 2 애m from the plastid surface. In the case of small numbers of plastids with respect to cell size, the problem of distribution at cell partitioning is particularly obvious because it is easy to imagine accidents of misdivision resulting in an unfortunate cell lacking any plastid. For plants especially, this reverts to the problem of proplastid numbers in meristem cells. The plant germline consists of small apical meristem cells that have only a small number of proplastids, but these must be replicated and consistently dealt to both daughters. This can be treated as a population problem, subject to random drift (Birky, 1982). A theoretical treatment of the problem by Birky and Skavaril (1984) showed that as plastome volume approaches 50% of the available cell volume, even cells with only 4 or 6 plastids produce ⬍10% plastid-null daughters. For cells with ⬎10 plastids, daughter cells may receive unequal numbers but will receive at least 1 plastid ⬎95% of the time. The number of proplastids per cell in shoot apical meristems of plants is estimated to be 10–14 from three different methods of estimate: serial section reconstruction of apical meristem cells, extrapolation back from plastid numbers per cell size to a size typical of apical meristem, and the rate of sorting out (observed in the phenotype) of the mutant versus wild-type plastids in heteroplasmic apical meristem cells (Herrmann and Possingham, 1980; Pyke and Leech, 1992). Twenty to 40 proplastids have been reported in root meristem cells by Whatley (1983). Each proplastid is multiploid (Miyamura et al., 1990). Therefore, plastid segregation at cell division in apical meristems should be successful ⬎95% of the time by random chance. Is this only a theoretical problem? Do cells actually distribute plastids stochastically? What happens to cells that receive excessive plastids, and to those that receive very few, at subsequent mitoses? The theory predicts that cells without plastids would be generated at a low rate by random chance, independent of the average number of plastids per cell in the population. Sublineages of cells might continue to receive decreasing numbers of plastids until nulls were generated. Also, nulls have indeed been observed but rarely (Maguire et al., 1995).

PLASTID–CELL COORDINATION

143

An initial effort to obtain data on this problem used a unicellular organism (Olisthodiscus) in which the numbers of plastids could be easily tallied in sister daughter cells after each division (Hennis and Birky, 1984). Plastid distribution to daughters appeared to fit the stochastic prediction, given the mean number of plastids per cell in the culture, except that the extremes of predicted high and low numbers seemed to be missing. Similar data characterize Ochromonas growing exponentially in both light and dark conditions. The dividing parental cells average only 2.5 plastids per cell, but there is a paucity of plastid-null daughters (A. Coleman, unpublished observations). As suggested by Hennis and Birky, this implies a compensation mechanism that operates on extremes of distribution, perhaps by altering the timing of the next mitosis allowing low-plastid cells to accumulate more plastids and causing high-plastid cells to divide before they have fully doubled their plastome.

VIII. Control of Plastid Positioning If one were to design a plastid system for guaranteed delivery of ptDNA to each daughter, given the typical pinching into two of plastids, one might concentrate plastid DNA in the two polar ends of a plastid or at least target these genomes for preferential replication. Such a general conformation actually characterizes mitochondrial DNA arrays in one group of organisms, the trypanosomes (Perez-Morga and Englund, 1993). One might also assign at least one plastid to a site in the cell certified to segregate faithfully at cytokinesis. Since the nucleus is not available, a sensible choice might be the centriole (or centrosome). Are these ideas merely teleologically appealing or do they have any counterpart in nature?

A. Control of Plastid Position in the Cell We have seen that there is no support for a bipolar plastid model. In fact, almost every aspect of plastid internal structure is without discernible polarization. Plastid genomes are positioned so that no region of the plastid is distant from a genome, and DNA replication shows no distributional bias. Although plastids may be elongated just prior to plastid cleavage, there is no feature which allows one to predict where the next cleavage plane will fall, given that plastids are dynamic and not static as in electron micrographs. Plastids can be redistributed in a cell, and cylic changes in form and number of plastids have been described for Euglena (Ehara et al., 1990a).

144

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

Plastid positionings attributable to microtubule arrays have been demonstrated for multinucleate cells of green algae (McNaughton and Goff, 1990; Shihira-Ishikawa, 1987). The plastids might be as dynamic in form as the mitochondria of yeast (Nunnari et al., 1997), fusing and separating at rates detectable within an hour’s observation. This is suggested by the observations of Koehler et al. (1997) on protein exchange among intact plastids in vivo. It is known that actin bundles serve as highways for the plastid streaming seen in plant cells, and myosin has been detected on plastid surfaces (Williamson, 1993). However, rapid plastid streaming is a phenomenon limited to green organisms; it is not observed in multiplastidial cells of red or brown organisms. Instead, red algal plastids and those of a few multinucleate green algae undergo a slower migration related to the diurnal cycle and cell division (Lloyd, 1991). Here, there is evidence for actin involvement in plastid migration (Russel et al., 1996). On the other hand, in the same organisms, plastids are initially oriented in the cortical cytoplasm by microtubule arrays emanating from the spindle poles or from the surface of the nucleus in interphase (Sylvester, 1987). The cytoskeletal elements controlling position and movement of plastids are a major focus of study. One marker of plastid substructure is available in flagellated green unicells; the eyespot is located just beneath the plastid bounding membranes. The eyespot (stigma) in a cell assumes a characteristic position that is often species specific. Painstaking reconstruction from electron micrographs of the cytoskeleton of the cell show that the plastid eyespot region is always adjacent to a particular bundle of microtubules radiating from the basal body region (Holmes and Dutcher, 1989; Lechtreck et al., 1997). This clearly affects plastid positioning and might contribute to plastid separation via indirect linkage to basal bodies, although any continuity of connection is not yet clear. There remains the possibility that some other aspect of the cell cytoskeleton plays a definitive role in ensuring that each daughter cell obtains a plastid, although this possibility has never been recognized. Two areas of research are germane to this question and both employ fluorescence microscopy of whole cells as well as electron microscopy of thin sections. The first concerns the remarkable microtubule arrays found in sporocytes of lower plants at meiosis (Brown and Lemmon, 1997); these sporocytes initially have only one plastid. Plastid division, eventually into four plastids, is intricately coordinated and the four plastids are arranged at the four potential poles of the initial cell by fasces of microtubules, anticipating production of four meiospores. The radiations of microtubules from the tips of the four plastids position them and surround the area of the meiotic nuclei, appearing to meld with the spindle. Examples of this solution to the initially uniplastidial condition are known among liverworts, mosses,

PLASTID–CELL COORDINATION

145

hornworts, Selaginella, and some ferns, and a similar behavior is seen in the meiotic cells of the alga Coleochaete belonging to the green clade presumed to have been ancestral to terrestrial plants. In all these cases, the solution to plastome distribution appears to lie in microtubule arrays controlling positioning long before nuclear division.

B. Possible Alternatives Although plastids of all eukaryotes seem remarkably similar in their properties, those plastids recognized to be products of secondary symbiosis may retain vestiges of this heritage which make their plastid positioning different. Their plastids are located fundamentally in an extracytoplasmic compartment, as indicated by their extra membranes (Table I), rather than directly in the cytoplasm (Gibbs, 1962). This may help to explain some unusual cell positioning phenomena observed in chromophytes. In a very thorough electron microscopic study of Ochromonas cell structure, Slankis and Gibbs (1972) observed the association of the two basal bodies of Ochromonas with each other and with the nucleus via the rhizoplast, a ribbed structure found in many types of flagellates that extends down from the basal bodies to the nucleus and then splays out over its surface. In many rapidly growing Ochromonas cells there is only one plastid, closely associated with the nuclear membrane, that divides prior to mitosis. Slankis and Gibbs interpreted their electron microscopic studies of dividing cells as indicating that a region of the nuclear membrane adjacent to each plastid failed to disperse at mitosis, which was perhaps stabilized by the duplicated rhizoplast. Since the two rhizoplasts migrated apart prior to nuclear division because of their attachment to the separating pairs of basal bodies, each plastid with its nuclear membrane fragment would also be carried along by its rhizoplast. Although physical evidence of this continuity of connections is lacking, it seems very likely that there exists some underlying cytoskeletal scaffolding. Additional observations made on whole cells add to the curiosity. In observations of living Ochromonas, even when several plastids are present in a cell, one plastid always has an apparent attachment to the shorter of the two flagella, an association made strikingly visible as a result of the innate fluorescence of the short flagellum (Coleman, 1988) and the brilliant fluorescence of chlorophyll, even when the extension from the plastid body is extremely fine (Maguire et al., 1995). Such structures would not be obvious in a study of thin sections (Figs. 1D and 1F). At cell division, as the new flagellar basal pairs continue to separate after nuclear division (Figs. 1C–1E), each short flagellum is observed to have an associated plastid—an association that is formed perhaps as late as prophase. Even more striking,

146

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

rare misdividing cells have been observed (Figs. 1F and 1G) that have a pair of flagella with attached plastid toward one side but a single long flagellum with no plastid in the bilaterally symmetrical position. The missing short flagellum, with a plastid attached, lies elsewhere in the cell. An examination of published electron micrographs failed to reveal any recognizable structure which could explain this constant association between plastid and basal body (A. Coleman, manuscript in preparation). Perhaps other kinds of fixation, or a more direct approach, could identify the underlying structure. A similar phenomenon has been reported for cells of brown algae forming multiple spores (Motomura et al., 1997). Here, a centrosome body, the residence of the centrioles, recognizable by staining with fluorescent anticentrin antibody, associates with one end of each of the eight plastids in the sporangium, just as the four nuclei enter anaphase to become eight. Each telophase nucleus is then adjacent to its spindle pole, the site of the centrosome, and thus also adjacent to a plastid, guaranteeing that each of the eight daughter cells will have one nucleus and one plastid.

IX. Proteins Associated with Plastid DNA A. The Plastid Multigenome Complex The plastid genome complex of a single plastid contains from 앑5 to ⬎100 copies of the plastid genome, apparently arranged approximately sequentially around the ring genome complex or in some kind of network of genome aggregates in the scattered nucleoid complex. Electron micrographs of ptDNA–protein complexes from plant and other plastid sources, spread on electron microscope grids (Woodcock and Fernandez-Moran, 1968; Herrmann and Kowallik, 1970; Hansmann et al., 1985), display remarkably organized structures. Numerous loops, both supercoiled and relaxed, emanate from a granular source to form a bouquet of ptDNA (Fig. 6). In size, each loop is considerably smaller than a single genome (about 1/20), suggesting that proteins associated with the granule bind each genome at 앑20 different sites. Strings of such bouquets, some obviously encompassing more than one genome’s worth of DNA, are observed. Although plastids are known to lack histones, they do contain DNA binding proteins (Briat et al., 1984; Nemoto et al., 1991; Wu et al., 1989) and it is possible to isolate ptDNA–protein complexes representing nucloids after treatment with detergent that retain much of their original appearance (Yoshida et al., 1978; Herrmann and Kowallik, 1970). Chemical analyses vary greatly with different techniques and materials, such as daffodil leaf and flower, Ectocarpus, and tobacco, but on a weight basis, protein content

PLASTID–CELL COORDINATION

147

FIG. 6 (a) Visualization of putative membrane-associated DNA released by Triton X-100 lysis from Spinacia oleracea chloroplasts. The DNA protein complexes were isolated by centrifugation in an isopycnic CsCl equilibrium gradient and spread by cytochrome monolayer techniques. Note the coexistence of supertwisted and relaxed (thick arrow) loops in the same complex. Free ends are clearly discernible (small arrow). (b) Detail micrograph from a complete section series through a proplastid of Beta vulgaris showing a DNA–membrane association (arrow). The matrix was partially removed by proteolytic digestion in order to expose DNA. The DNA is associated with protein remnants. The diameter of the plastid is 1.1 애m. (Reproduced with permission from Herrmann, R. G., and Possingham, J. V. (1980). In ‘‘Results and Problems in Cell Differentiation’’ (J. Reinert, Ed.), Vol. 10. SpringerVerlag, Berlin.

148

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

exceeds DNA content, with a more variable level of associated RNA. The set of proteins found associated with these ptDNA aggregates also varies, even between developmental stages (proplastid vs chloroplast of tobacco) (Nemoto et al., 1991). The subset of more tightly bound proteins (more resistant to high salt extraction) has been investigated with the goal of finding one or more proteins that bind one or more specific sequences of the ptDNA, but results remain unclear. From occasional transmission electron microscope images (Fig. 6), and by analogy with prokaryote genomes ( Jacob et al., 1963), such bouquets of ptDNA were proposed to be attached to thylakoid membranes. Again by analogy with prokaryotes, this would provide an explanation for orderly segregation of DNA. The dynamics of membrane attachment could also possibly influence DNA replication, although the possible existence and importance of DNA membrane attachment in prokaryotes has recently been questioned. Evidence that ptDNA genomes are bound to membranes is still not clear-cut, although methods which select for such subregions have been attempted (Kuroiwa, 1991; Wu et al., 1989). A promising development is the discovery in developing pea leaves of a protein, PEND, that binds to several plastid DNA sites and is also a protein of the plastid envelope (Sato et al., 1998).

B. DNA Replication Information on this topic is frustratingly sparse [Tewari (1988) and Gillham (1994); for a review of DNA polymerases, see Wang (1996); for a review of topoisomerases, see Drlica and Franco, (1988) and Andersen et al., (1996) for a discussion of inhibitors, see Ye and Sayre (1990)]. A plastid DNA primase has been identified in pea (Nielsen et al., 1991). Origins of DNA replication require association with a multienzyme complex in order for replication to proceed. The enzymes include at least one nuclear DNA polymerase and several topoisomerases. McKown and Tewari (1984), using a DNA polymerase preparation isolated from pea plastids, reported plastid DNA synthesis to be resistant to aphidocolin, a specific inhibitor of DNA polymerase alpha. However, aphidocolin blocks both nuclear and plastid DNA increase in Cyanidioschyzon, and the plastids divide at least twice more in the absence of DNA synthesis (Itoh et al., 1996). A gamma-like DNA polymerase has been reported in Chlamydomonas (Wang et al., 1991). Topoisomerase molecules have been localized to ptDNA in dividing wheat plastids (Marrison and Leech, 1992), and topoisomerase I and II activities were detected in pea plastids (Lam and Chua, 1987). A purified pea plastid topoisomerase I was characterized by Nielsen and Tewari (1988). Thompson and Mosig (1985) reported an ATP-dependent topoisomerase

PLASTID–CELL COORDINATION

149

activity in Chlamydomonas that is inhibited by nalidixic acid, as is bacterial gyrase, but the role in DNA synthesis is not clear. In Ochromonas, growth in nalidixic acid leads to distorted nucleoid rings with large aggregates of DNA molecules (Nerozzi, 1995). Characterization of the molecules responsible for plastid DNA synthesis is very much needed.

C. Origins of ptDNA Replication In general, neither the DNA sequence nor the position of the D loops in plastid genomes are highly conserved among examined species (Koller and Delius, 1982; Waddell et al., 1984; Meeker et al., 1988; Takeda et al., 1992; Chiu and Sears, 1992; Nielsen et al., 1993), but they are often found close to the rRNA genes, indicating a position within or near the inverted repeats. Although sequences of the plastid Ori regions have been determined, other ptDNA regions that might be implicated in affecting ptDNA replication have yet to be uncovered; one recent exception, however, resolves a genetic puzzle of 80 years. Oenothera is one of the minority of angiosperms with biparental inheritance of ptDNA. In crosses, certain parental plastid types are predictably dominant among the progeny. Sears et al. (1996) found that a region of short and variable repeats in the plastid genome near OriB may confer this property, playing some role in plastid genome competition in the zygote and subsequent growth. In Chlamydomonas the presumed standard origins of plastid DNA are OriA and OriB, which are separated by 6.5 kb. Woelfe et al. (1993) reported that in the presence of novobiocin, ptDNA synthesis at OriA and OriB ceased almost immediately. After 8 h in the antibiotic, ptDNA synthesis resumed despite the presence of the antibiotic by using different origins near a known recombination hot spot in the two inverted repeats. This lag of 8 h might reflect accumulated transcription events, some of which might open the plastid DNA to synthesis starting at an unusual position.

D. ‘‘Mutant’’ Plastids There is strong evidence in the literature that ptDNA replication is primarily or solely controlled and carried out by nuclear-generated materials. Sequenced plastid genomes lack DNA polymerase genes. Moreover, ptDNA and plastids continue to be maintained in certain albino mutants in which plastids are reported to lack ribosomes and protein synthesis (Walbot and Coe, 1979; Scott et al., 1982; Day and Ellis, 1984, 1985). Not only does the nucleus provide all the necessary molecules, but also perhaps almost any region of the ptDNA genome is sufficient to serve as origin. Callus cell

150

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

lines in culture, subcloned from albino plantlets derived from rice pollen regenerates, that lack major portions of the plastid genome including all plastid ribosomal RNA genes continue to multiply and to maintain their chacteristic mutant plastid DNA genome (Harada et al., 1991, 1992). Mitochondrial DNA replication in yeast also continues, no matter what small remnant of the mitochondrial genome remains in rho-mutants (Azpiroz and Butow, 1993). If these observations are correct, the implication is that cytoplasmic protein synthesis provides the necessities for ptDNA replication and organelle maintenance, and that almost any region of the ptDNA is adequate as a template. Furthermore, this implies that control of plastid genome numbers does not result from cell sensing of any plastid-synthesized protein. Alas, the regulatory mechanisms of mitochondrial numbers are also not understood (Clayton, 1996).

E. Possible Metabolic Pathways for Coordination Whether or not the previous perplexing conclusion is correct, there is an additional level of plastid genome multiplication control. In normal cells some aspects of the environment can lead to modification of genome numbers (e.g., light vs dark conditions). Among the possible signals, one should include such aspects as redox state, which is obviously of great importance to both photosynthetic and respiratory organelles (Allen, 1993). Zhang and Wu (1993) described a protein, frxb, that is closely associated with OriA of ptDNA in Chlamydomonas. frxb is an Fe-S protein related to subunits of NADH dehydrogenase and is presumably involved in plastid chlororespiration (Bennoun, 1994). In 1993, Zhang and Wu reported that this protein is concentrated in vesicles or tubules near the plastid pyrenoids that maintain a heighted membrane potential, as indicated by their concentrating DiOC6 (3-3⬘-dihexyloxacarbocyanine iodide). They suggest that the state of membrane potential could modulate the association of frxb with OriA and consequently the rate of plastid DNA replication (Wu et al., 1993).

X. Genetics of Plastid DNA A. Uniparental Inheritance Although all cells of a photosynthetic organism contain plastids, and all plastids contain copies of the ptDNA, there are frequently observed exceptions. These are basically of two kinds. The first exception, as discussed previously, is a consequence of misdivision or somatic cell death, and it is

PLASTID–CELL COORDINATION

151

rare and trivial. The second is extremely important biologically. In the vast majority of photosynthetic organisms examined, ptDNA inheritance is uniparental, usually through the female gamete (Kuroiwa, 1991). This is not solely a consequence of small male gamete size but instead due to an active differentiation act in the life cycle. Plastid DNA and/or plastids may be lost from male gametes during their differentiation (the majority of angiosperms and some algae) (Gillham, 1994), they may be excluded at fertilization (as happens to mitochondria in many animals) (Mogensen, 1996), or they may be selectively degraded subsequent to fertilization (species of Chlamydomonas) (Kuroiwa et al., 1982; Coleman and Maguire, 1983), the remaining genomes being multiplied at meiosis of the zygote (Coleman, 1984). Consequently, most organisms have uniform plastid DNA genomes rather than mixtures and/or recombinants of two parental types. B. Plastid DNA Sorting A second aspect of plastid DNA genetics is that even in cases of biparental inheritance most organisms are genetically uniform for ptDNA. In cells containing two types of ptDNA, the types sort out to homogeneity in subsequent mitotic cell lineages (so-called somatic sorting). Where one of the two ptDNA genomes carries a mutation causing albinism, such mitotic sorting becomes obvious as patches or stripes of white versus green leaf or even as white branches (e.g., Pelargonium) where the plastids of a cell and its subsequent lineage have become homoplasmic for the mutant ptDNA. The sectoring pattern of plants of biparental inheritance can be analyzed to determine the approximate number of plastids that must have been present in the zygote, subject to considerations of unknown partitioning ratios in early development. In heteroplasmic Chlamydomonas zygotes that proceed to grow vegetatively instead of undergoing meiosis, the two genetic types of ptDNA sort out after 10–20 mitoses, yielding homoplasmic cells (VanWinkle-Swift, 1980). This is much too rapidly to fit predictions based on random distribution of the number of plastid genomes present in the initial cell. However, the packaging of plastid genomes into nucleoids in plastids (VanWinkleSwift, 1980), together with the DNA replication pattern which keeps plastid genomes that are products of a common parental molecule contiguous (Nerozzi and Coleman 1997), provides a reasonable explanation. Rapid sorting out is a consequence of grouping of ptDNA genomes, which is in turn a consequence of the architecture of DNA synthesis localization. C. Plastid DNA Exchange Methods The previously discussed phonomena make manipulations of plastid genomes by genetic methods standard to nuclear genes very difficult. Opportu-

152

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

nities for crossover between recognizably different plastid genomes are confined to plants species in which biparental inheritance occurs with some frequency or to such organisms as Chlamydomonas, in which recognizably mutant plastid genomes can be engendered and crosses can be made under selective conditions that increase zygotes biparental for the ptDNA. In both cases, crossover genomes have been found among the progeny, implying that plastids fuse, that ptDNA genomes mix, and that some kind of machinery exists that can produce genome exchanges. These requirements appear to be satisfied. Plastids can be observed to fuse in zygotes of Chlamydomonas and many other green algae. Plastid fusion in plants has been questioned in the past, but some exchange of materials occurs continually, as demonstrated by observations of green fluorescent protein transfer among plastids of tobacco and petunia leaves (Koehler et al., 1997). This indicates both the rapid spread of a protein throughout a plastid and the capacity for plastid fusion (and scission), as reported for mitochondria (Okamoto et al., 1998). An intramolecular breakage, crossover, and repair mechanism is known to operate in plastid genomes with two inverted repeat regions, typical of most plastid genomes (Palmer, 1983). This mechanism could also operate between genomes. A recA-mediated homologous recombination system has been observed in Chlamydomonas plastids that is associated with repair of radiation damage to plastid DNA (Cerutti et al., 1995). Direct evidence for crossover between parental plastid genomes in analyses of hybrid progeny has been presented by Boynton et al. (1976) for Chlamydomonas and by Thanh and Medgyesy (1989) and Medgyesy et al. (1985) for plants.

D. Why Plastid DNA? The compartmentalization of high-energy reactions that produce dangerous oxygen radicals, such as by-products of the light reactions of photosynthesis, seems to be a reasonable justification for the plastid (Allen, 1993), but it is also clear that the chemiosmotic aspects of plastids require small compartments to work. Earlier suggestions that plastid genomes were retained because certain very hydrophobic gene products could not enter the plastid have become less compelling as information accumulates on which gene products are made where and how the importation mechanisms function. An additional suggestion, that some fundamental plastid gene product must act as the core to guide and retain the imported proteins of the multimeric functional enzyme complexes typifying plastids, is still being explored. The real reason may be that plastid DNA itself is the material that the nuclear mechanisms sense and use to control plastid growth.

PLASTID–CELL COORDINATION

153

E. Why Genome Multiplicity? The multiplicity of ptDNA per cell and per plastid has generally been justified in terms of allowing for the relatively uncertain distribution at cell division. The additional multiplicity exemplified by the multitudes of ptDNA molecules in a typical leaf mesoderm cell may reflect a general metabolic demand for pt genome products and/or an intensive demand for one set of ptDNA products, the plastid ribosomal RNAs, as suggested by Bendich (1987). Nuclei have hundreds of copies of their rRNA genes, but the plastid genome has only two (sometimes one). Multiplicity of the ptDNA would provide the correct number of rDNA genes needed for making sufficient ribosomes in the developing chloroplast. A genetic argument for multiple DNA copies can also be made. The nature of the plastid activities, engendering mutagenic materials, may subject ptDNA to high levels of mutational events. There may consequently be a tremendous genetic advantage to ptDNA redundancy. In the absence of standard genetic recombination to create new plastid genotypes and/or correct accumulated mutations, exchange among the many genomes in the plastome may serve to combat Muller’s (1964) ratchet, the inevitable accumulation of deleterious mutations in a lineage lacking recombination.

XI. Choice of Experimental Materials Although ‘‘model organisms’’ have reigned for several decades, one consequence of genome sequencing has been the realization that organisms are much more similar genetically than once thought, and it should be possible to chose an experimental material most appropriate for the research question. Many of the sophisticated gene manipulations will be possible on organisms not well studied genetically. With this possibility, other aspects of cells important to a research project should be considered.

A. Considerations Unicells and small organisms are much easier to handle and to view than large multicellular organisms. Cells with little or no wall offer better access and better viewing. For many kinds of experiments, cells capable of heterotrophy as well as autotrophy offer more experimental possibilities. Also, cells or subcellular materials that can be reduced to two dimensions on a slide without destruction (e.g., Ochromonas, with its lack of wall and characteristic plastid genome necklace) are easier to analyze than three-

154

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

dimensional objects. In practical terms, use of confocal microscopes requires more time. Far more remains to be learned from observations of living cells, particularly using fluorescence microscopy. Naturally occuring autofluorescent molecules, of which chlorophyll is the prime example, can be supplemented by fluorescent compounds concentrated in membrane-bound organelles or engineered fluorescent gene products. Fluorescently labeled compounds or fluorescent antibodies can be injected into single cells. In fixed cells, fluorescent antibodies to cell proteins and fluorescently labeled nucleic acid probes specific for regions of the genome or RNAs of interest can be applied (Webb et al., 1997). When studying DNA synthesis, one should be aware that not all cells take up and incorporate thymidine (and its analogs) into nuclear and plastid DNA, as do plant and metazoan cells. Some large tropical green algae can utilize exogenous thymidine in the plastid (Motomura, 1996). Some green unicells will incorporate it into plastid DNA but not nuclear DNA (e.g., C. reinhardtii; Swinton and Hanawalt, 1972; Munaut et al., 1990), whereas others incorporate thymidine analogs preferentially into nuclear DNA and not ptDNA (Zachleder et al., 1996). Euglena fails to incorporate thymidine into either nuclear or plastid DNA (Sagan, 1965), as does Oedogonium (A. Coleman, unpublished observations). Among brown algae, Laminaria and Fucus do not utilize exogenous thymidine (T. Motomura, personal communication). The salvage pathway necessary for exogenous thymidine utilization also converts fluorodeoxyuridine to the phosphorylated form, a compound that is a noncompetitive inhibitor of endogenous thymidine synthesis. This aspect should be useful to screen organisms not previously studied since growth in fluorodeoxyuridine suggests that the thymidine salvage pathway is lacking. Finally, the limits of culture and molecular manipulations possible with plastids and plastid genomes are unknown. However, homologous recombination with an introduced gene construct has been obtained in Chlamydomonas (Kindle et al., 1991) and with plants, and techniques improve continuously (Lumbreras and Purton, 1998). Chlamydomonas plastids are capable of stably maintaining an introduced episome (Kindle et al., 1994). A source for the many methodologies applicable to unicellular organisms and their genetic manipulation is Harris (1989).

B. Albino Callus Lines Comparisons of albino callus cultures (see Section IX,D) with normal callus cultures kept in the dark to encourage the proplastid form of the plastid might suggest whether the deletion mutant plastids are similar to wild type

PLASTID–CELL COORDINATION

155

in protein content, lipid, and other materials. Likewise, whether their size and DNA content are truly similar to normal proplastids is unknown, but a comparative study could yield leads to molecules potentially involved in coordinating expansion of the plastome in response to environmental or differentiation signals. The pollen-derived callus lines, harboring major plastid DNA deletions, might also be prime material for determining which specific components of the medium are required for their continued growth and how these relate to the number of activities attributed to the plastid (see Section I).

XII. Conclusions It is instructive to compare the major characteristics of the two DNAcontaining eukaryote cell organelles—the plastid and the mitochondrion: Both contain multiple copies of the organelle genome; whether these are interconnected within an organelle is not known (except for plastids with ring nucleoid arrangements). Both are inherited predominantly uniparentally. Both are compartments in which redox reactions capable of generating free radicals are housed. Both are known to be capable of forming a single compartment at some stages and/or to fuse and separate. Both can fail at their primary function (respiration and photosynthesis) and still be maintained in the cell (petites and albinos). Both, when cells are maintained in complex media, can omit protein synthesis and reduce their genetic information to a fragmentary content. Both DNA synthesis and organelle division proceed independent of any one stage of the nuclear cell cycle. Furthermore, DNA replication is relaxed and not stringent. Neither ptDNA synthesis nor plastid division is limited to any cell cycle stage. Mitochondria can be lost altogether, at least if cells are in suitably enriched media. Plastid loss has only been induced experimentally in Euglena (which has an additional pathway of porphyrin synthesis). Among all photosynthetic eukaryotes, however, evolutionarily diverse, the fundamental characteristics of ptDNA and plastids are the same, with one possible exception: the plastid locating mechanism(s) within the cell, which is yet to be fully explained. At least three levels of coordination between plastome and cell exist; those concerned with (1) matching cell multiplication rate (basic mainte-

156

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

nance and control), (2) response to cell- or tissue-type signals (internal), and (3) response to exogenous conditions (external). The first level of coordination monitors some aspect of ptDNA and/or plastid number, preventing the formation of plastid-free cells at cytokinesis and likewise setting an upper limit on ptDNA and/or pt numbers per cell, thus preventing excessive expansion of the plastome. Cells with plastid content below the lower threshold level may respond by increasing plastid DNA synthetic rate or slowing progress through the cell cycle. Cells exceeding the upper threshold may slow ptDNA synthesis or, if dividing, divide precociously. The result is most striking in mature cells of multicellular algae and plants where pavements of almost identical plastids characterize each cell type. The DNA-deletion plastids of albino callus cells, lacking many genes and all protein synthesis, apparently no longer respond to signals of levels 2 or 3 but must continue to coordinate at level 1 to exist. Communication at level 1 must, on the plastid side, occur in undistinguished DNA sequences or in some possible transcription product of them. Levels 2 and 3 can reset the upper limit on ptDNA and plastid number per cell as well as the nature and amount of plastid development. Levels 2 and 3 may share the same controls, responding to cell size. For mature cells, data are lacking but could be obtained. For growing cells, the exponential increase in plastome seen in the differentiating leaf of angiosperms provides support for this proportionality. With the rapid advances in genomics, there should soon be a catalog of all the potential players in plastid–cell coordination, both in the plastid genome and in the nuclear genome. The deciphering of the tools for conversation between the plastome and its host cell is just beginning. At the most basic level, maintenance of the plastid, the only molecules left to represent the plastid side are its DNA and/or some transcription product. No plastid proteins are involved. Imaginative application of the new knowledge concerning gene content, advances in understanding of DNA replication, and use of both molecular biology and microscopic techniques will produce the next steps. Acknowledgment Figure 1B is the gracious gift of Dr. Jeremy Pickett-Heaps, University of Melbourne.

References Allen, J. F. (1993). Control of gene expression by redox potential and the requirement for chloroplast and mitochondrial genomes. J. Theor. Biol. 165, 609–631.

PLASTID–CELL COORDINATION

157

Andersen, A. H., Bendixen, C., and Westergaard, O. (1996). DNA topoisomerases. In ‘‘DNA Replication in Eukaryote Cells’’ (M. L. DePamphilis, Ed.), pp. 587–617. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Azpiroz, R., and Butow, R. A. (1993). Patterns of mitochondrial sorting in yeast zygotes. Mol. Cell Biol. 4, 21–36. Bendich, A. J. (1987). Why do chloroplasts and mitochondria contain so many copies of their genome? BioEssays 6, 279–282. Bendich, A. J., and Smith, S. B. (1990). Moving pictures and pulsed-field gel electrophoresis show linear DNA molecules from chloroplasts and mitochondria. Curr. Genet. 17, 421–425. Bennoun, P. (1994). Chlororespiration revisited: Mitochondrial–plastid interaction in Chlamydomonas. Biochem. Biophys. Acta 1186, 59–66. Birky, C. W., Jr. (1982). The partitioning of cytoplasmic organelles at cell division. Int. Rev. Cytol. Suppl. 15, 49–89. Birky, C. W., Jr. (1983). Relaxed cellular controls and organelle heredity. Science 222, 468–475. Birky, C. W., and Skavaril, R. V. (1984). Random partitioning of cytoplasmic organelles at cell division: The effect of organelle and cell volume. J. Theor. Biol. 106, 441–447. Boerner, T., Herrmann, F., and Hagemann, R. (1973). Plastid ribosome deficient mutants of Pelargonium zonale. FEBS Lett. 37, 117–119. Boffey, S. A., and Lloyd, D. (Eds.) (1988). ‘‘The Division and Segregation of Organelles.’’ Cambridge Univ. Press, Cambridge, UK. Bogenhagen, D., and Clayton, D. A. (1977). Mouse L cells mitochondrial DNA molecules are selected randomly for replication throughout the cell cycle. Cell 11, 719–728. Bogorad, L., and Vasil, I. K. (1991). ‘‘Molecular Biology of Plastids’’ Academic Press, San Diego. Boyer, C. T., Shannon, J. C., and Hardison, R. C. (1989). ‘‘Physiology, Biochemistry, and Genetics of Nongreen Plastids.’’ American Society of Plant Physiologists, Rockville, MD. Boynton, J. E., Gillham, N. W., Harris, E. H., Tingle, C. L., Van Winkle-Swift, K., and Adams, G. M. W. (1976). Transmission, segregation and recombination of chloroplast genes in Chlamydomonas. In ‘‘Genetics and Biogenesis of Chloroplasts and Mitochondria’’ (T. Bucher, Ed.), pp. 313–322. Elsevier/North Holland, Amsterdam. Briat, J. F., Letoffe, S., Mache, R., and Rouviere- Yaniv, J. (1984). Similarity between the bacterial histone-like protein HU and a protein from spinach chloroplasts. FEBS Lett. 172, 75–79. Brown, R. C., and Lemmon, B. E. (1997). The quadripolar microtubule system in lower land plants. J. Plant Res. 110, 93–106. Butterfass, T. (1973). Control of plastid division by means of nuclear DNA amount. Protoplasma 76, 167–195. Butterfass, T. (1988). Nuclear control of plastid division. In ‘‘The Division and Segregation of Organelles’’ (S. A. Boffey and D. Lloyd, Eds.), pp. 21–38. Cambridge Univ. Press, Cambridge, UK. Cerutti, H. J., Johnson, A. M., Boynton, J. E., and Gillham, N. W. (1995). Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli recA. Mol. Cell Biol. 15, 3003–3011. Chiu, W.-L., and Sears, B. B. (1992). Electron microscopic localization of replication origins in Oenothera chloroplast DNA. Mol. Gen. Genet. 232, 33–39. Clayton, D. A. (1996). Mitochondrial DNA replication. In ‘‘DNA Replication in Eukaryotic Cells’’ (M. L. DePamphilis, Ed.), pp. 1015–1028. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Clegg, M. T., Learn, G. H., and Golenberg, E. M. (1991). Molecular evolution of chloroplast DNA. In ‘‘Evolution at the Molecular Level’’ (R. K. Selander, Ed.), pp. 135–331. Sinauer, Sunderland, MA.

158

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

Coleman, A. W. (1984). The fate of chloroplast DNA during cell fusion, zygote maturation and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining. Exp. Cell Res. 152, 528–540. Coleman, A. W. (1985a). Diversity of plastid DNA configuration among classes of eukaryote algae. J. Phycol. 21, 1–16. Coleman, A. W. (1985b). Cyanophyte and cyanelle DNA; A search for the origins of plastids. J. Phycol. 21, 483–508. Coleman, A. W. (1988). The autofluorescent flagellum: A new phylogenetic enigma. J. Phycol. 24, 118–120. Coleman, A. W., and Heywood, P. (1981). Structure of the chloroplast and its DNA in chloromonadophycean algae. J. Cell Sci. 49, 401–409. Coleman, A. W., and Maguire, M. (1982). Microspectrofluorometric analysis of nuclear and chloroplast DNA in Volvox. Dev. Biol. 94, 441–450. Coleman, A. W., and Maguire, M. J. (1983). Cytological detection of the basis of uniparental inheritance of plastid DNA in Chlamydomonas moewusii. Curr. Genet. 7, 211–218. Conkling, B. A., Thomas, E. J., and Ortiz, W. (1993). Delayed but complete loss of chloroplast DNA in heat-bleaching cultures of Euglena gracilis. Plant Physiol. 142, 307–311. Daugbjerg, N., and Andersen, R. A. (1997). A molecular phylogeny of the heterkont algae based on analyses of chloroplast-encoded rbcL sequence data. J. Phycol. 33, 1031–1041. Day, A., and Ellis, T. H. N. (1984). Chloroplast DNA deletions associated with wheat plants regenerated from pollen: Possible basis for maternal inheritance of chloroplasts. Cell 39, 359–368. Day, A., and Ellis, T. H. N. (1985). Deleted forms of plastid DNA in albino plants from cereal anther culture. Curr. Genet. 9, 671–678. Deng, X.-W., Wing, R. A., and Gruissem, W. (1989). The chloroplast genome exists in multimeric forms. Proc. Natl. Acid. Sci. USA 86, 4156–4160. Desjardins, P., Frost, E., and Morais, R. (1985). Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts. Mol. Cell. Biol. 5, 1163–1169. Doremus, H. D., and Jagendorf, T. (1995). Subcellular localization of the pathway of de novo pyrimidine nucleotide biosynthesis in pea leaves. Plant Physiol. 79, 856–861. Drlica, K., and Franco, R. J. (1988). Inhibitors of DNA topoisomerases. Biochemistry 27, 2253– 2259. Ehara, T., Osafuna, T., and Hase, E. (1990a). Interactions between the nucleus and cytoplasmic organelles during the cell cycle of Euglena gracilis in synchronized cultures IV. An aggregate form of chloroplasts in association with the nucleus appearing prior to chloroplast division. Exp. Cell Res. 196, 104–112. Ehara, T., Ogasawara, Y., Osafune, T., and Hase, E. (1990b). Behaviour of chloroplast nucleoids during the cell cycle of Chlamydomonas reinhardtii (Chlorophyta) in synchronized culture. J. Phycol. 26, 317–323. Ehara, T., Osafune, T., and Hase, E. (1996). Association of nuclear and chloroplast DNA molecules in synchronized cells of Chlamydomonas reinhardtii. J. Electron Microsc. 45, 159–162. Ellis, J. R., and Leech, R. M. (1985). Cell size and chloroplast size in relation to chloroplast replication in light grown wheat leaves. Planta 165, 120–125. Erickson, H. P. (1995). FtsZ, a prokaryotic homolog of tubulin? Cell 80, 367–370. Ferguson, M., Torri, A. F., Ward, D. C., and Englund, P. T. (1992). In situ hybridization to the Crithidia fasciculata kinetoplast reveals two antipodal sites involved in kinetoplast DNA replication. Cell 70, 621–629. Gibbs, S. P. (1962). Nuclear envelope–chloroplast relationships in algae. J. Cell Biol. 14, 433–444. Gibbs, S. P. (1990). The evolution of algal chloroplasts. In ‘‘Experimental Phycology; Cell Walls and Surfaces, Reproduction, Photosynthesis’’ (W. Wiessner, D. G. Robinson, and R. C. Starr, Eds.), pp. 145–157. Springer-Verlag, Berlin.

PLASTID–CELL COORDINATION

159

Gillham, N. W. (1994). ‘‘Organelle Genes and Genomes.’’ Oxford Univ. Press, New York. Goff, L. J., and Coleman, A. W. (1987). The solution to the cytological paradox of isomorphy. J. Cell Biol. 104, 739–748. Gray, M. W. (1991). Origin and evolution of plastids. In ‘‘Molecular Biology of Plastids’’ (L. Bogorad and I. K. Vasil, Eds.), pp. 303–332. Academic Press, San Diego. Haffter, P., and Fox, T. D. (1992). Nuclear mutations in the petite-negative yeast Schizosaccharomyces pombe allow growth of cells lacking mitochondrial DNA. Genetics 131, 255–260. Hallick, R. B., Hong, L., Drager, R. G., Favreau, M. R., Monfort, A., Ursat, B., Spielmann, A., and Stutz, E. (1993). Complete sequence of Euglena gracilis chloroplast DNA. Nucleic Acids Res. 21, 3537–3544. Hansmann, P., Falk, H., Ronai, K., and Sitte, P. (1985). Structure, composition and distribution of plastid nucleoids in Narcissus pseudonarcissus. Planta 164, 459–472. Harada, T., Sato, T., Asaka, D., and Matsukawa, I. (1991). Large scale deletions of rice plastid DNA in anther culture. Theor. Appl. Genet. 81, 157–162. Harada, T., Ishikawa, R., Niizeki, M., and Saito, K.-I. (1992). Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth. Mol. Gen. Genet. 233, 145–150. Harris, E. H. (1989). ‘‘The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use.’’ Academic Press, New York. Harris, E. H., Boynton, J. E., and Gillham, N. W. (1994). Chloroplast ribosomes and protein synthesis. Microbiol. Rev. 58, 700–754. Hashimoto, H. (1985). Changes in distribution of nucleoids in developing and dividing chloroplasts and etioplasts of Avena sativa. Protoplasma 127, 119–127. Hashimoto, H., and Possingham, J. V. (1989). Effect of light on the chloroplast division cycle and DNA synthesis in cultured leaf discs of spinach. Plant Physiol. 89, 1178–1183. Hennis, A. S., and Birky, C. W. J. (1984). Stochastic partitioning of chloroplasts at cell division in the alga Olisthodiscus, and compensating control of chloroplast replication. J. Cell Sci. 70, 1–15. Herrmann, R. G., and Kowallik, K. V. (1970). Multiple amounts of DNA related to the size of chloroplasts II. Comparison of electron microscope and autoradiographic data. Protoplasma 69, 365–372. Herrmann, R. G., and Possingham, J. V. (1980). Plastid DNA—The plastome. In ‘‘Results and Problems in Cell Differentiation’’ ( J. Reinert, Ed.), Vol. 10, pp. 45–96. SpringerVerlag, Berlin. Herrmann, R. G. K., Kowallik, V., and Bohnert, H. J. (1974). Structural and functional aspects of the plastome. I. The organization of the plastome. Portugaliae Acta Biol. Ser. A 14, 91–110. Holmes, J. A., and Dutcher, S. K. (1989). Cellular asymmetry in Chlamydomonas reinhardtii. J. Cell Sci. 94, 273–285. Hoober, J. K. (1987). The molecular basis of chloroplast development. In ‘‘The Biochemistry of Plants’’ (M. D. Hatch and N. K. Boardman, Eds.), Vol. 10, pp. 1–74. Academic Press, Orlando, FL. Hrazdina, B., and Jensen, R. A. (1992). Spatial organization of enzymes in plant metabolic pathways. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 241–267. Itoh, R., Takahashi, H., Toda, K., Kuroiwa, H., and Kuroiwa, T. (1996). Aphidicolin uncouples the chloroplast division cycle from the mitotic cycle in the unicellular red alga Cyanidioschyzon merolae. Eur. J. Cell Biol. 71, 303–310. Jacob, F., Brenner, S., and Cuzin, F. (1963). On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28, 329–348. Kindle, K. L., Richards, K. L., and Stern, D. B. (1991). Engineering the chloroplast genome: Techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 88, 1721–1725.

160

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

Kindle, K. L., Suzuki, H., and Stern, D. B. (1994). Gene amplification can correct a photosynthetic growth defect caused by mRNA instability in Chlamydomonas chloroplasts. Plant Cell 6, 187–200. King, M. P., and Attardi, G. (1989). Human cells lacking mtDNA: Repopulation with exogenous mitochondria by complementation. Science 246, 500–503. Koehler, R. H., Cao, J., Zipfel, W. R., Webb, W. W., and Hanson, M. R. (1997). Exchange of protein molecules through connections between higher plant plastids. Science 276, 2039– 2042. Koller, B., and Delius, H. (1982). Origin of replication in chloroplast DNA of Euglena gracilis is located close to the region of variable size. EMBO J. 1, 995–998. Kolodner, R. D., and Tewari, K. K. (1975a). Chloroplast DNA from higher plants replicates by both the Cairns and rolling circle mechanism. Nature 256, 708–711. Kolodner, R. D., and Tewari, K. K. (1975b). Presence of displacement loops in the covalently closed circular chloroplast deoxyribonucleic acid from higher plants. J. Biol. Chem. 250, 8840–8847. Kostrzewa, M., and Zetsche, K. (1992). Large ATP synthase operon of the red alga Antithamnion sp. resembles the corresponding operon in cyanobacteria. J. Mol. Biol. 227, 961–970. Kowallik, K. V. (1993). Origin and evolution of plastids from chlorophyll a ⫹ c containing algae: Suggested ancestral relationships to red and green algal plastids. In ‘‘Origins of Plastids: Symbiogenesis, Prochlorophytes and the Origins of Chloroplasts’’ (R. A. Lewin, Ed.), pp. 223–264. Chapman & Hall, New York. Kowallik, K. V., and Herrmann, R. G. (1972). Variable amounts of DNA related to the size of chloroplasts IV. Three dimensional arrangement of DNA in fully differentiated chloroplasts of Beta vulgaris L. J. Cell Sci. 11, 357–377. Kowallik, K. V., Stoebe, B., Schaffran, I., Kroth-Pancic, P., and Freier, U. (1995). The chloroplast genome of a chlorophyll a⫹c-containing alga, Odontella sinensis. Plant Mol. Biol. Rep. 13, 336–342. Kuroiwa, T. (1991). The replication, differentiation, and inheritance of plastids with emphasis on the concept of organelle nuclei. Int. Rev. Cytol. 128, 1–61. Kuroiwa, T. (1998). The primitive red algae Cyanidium caldarium and Cyanidioschyzon merolae as model system for investigating the dividing apparatus of mitochondria and plastids. BioEssays 20, 344–354. Kuroiwa, T., and Suzuki, T. (1981). Circular nucleoids isolated from chloroplasts in a brown alga Ectocarpus indicus. Exp. Cell Res. 134, 457–461. Kuroiwa, T., Suzuki, T., Ogawa, K., and Kawano, S. (1981). The chloroplast nucleus: Distribution, number, size and shape, and a model for the multiplication of the chloroplast genome during chloroplast development. Plant Cell Physiol. 22, 381–396. Kuroiwa, T., Kawano, S., and Nishibayashi, S. (1982). Epifluorescent microscopic evidence for maternal inheritance of chloroplast DNA. Nature 298, 481–483. Kuroiwa, T., Nagashima, H., and Fukuda, I. (1989). Chloroplast division without DNA synthesis during the life cycle of the unicellular alga Cyanidium caldarium M-8 as revealed by quantitative fluorescence microscope. Protoplasma 149, 120–129. Kuroiwa, T., Kuroiwa, H., Mita, T., and Ohta, N. (1994). Cyanidium caldarium as a model cell for studying division of chloroplasts. In ‘‘Evolutionary Pathways and Enigmatic Algae’’ ( J. Seckbach, Ed.), pp. 239–253. Kluwer, Dordrecht. Kuroiwa, T., Kuroiwa, H., Saki, A., Takahashi, H., Toda, K., and Itoh, R. (1998). The division apparatus of plastids and mitochondria. Int. Rev. Cytol. 181, 1–41. Lam, E., and Chua, N. H. (1987). Chloroplast DNA gyrase and in vitro regulation of transcription by template topology and novobiocin. Plant Mol. Biol. 8, 415–424. Lawrence, M. E., and Possingham, J. V. (1986). Microspectrofluorometric measurement of chloroplast DNA in dividing and expanding leaf cells of Spinacia oleracea. Plant Physiol. 81, 708–710.

PLASTID–CELL COORDINATION

161

Lechtreck, K.-F., Reize, I. B., and Melkonian, M. (1997). The cytoskeleton of the naked green flagellate Spermatozopsis similis: Flagellar and basal body developmental cycle. J. Phycol. 33, 254–265. Leech, R. M., and Pyke, K. A. (1988). Chloroplast division in higher plants. In ‘‘Division and Segregation of Organelles’’ (S. A. Boffey and D. Lloyd, Eds.), pp. 39–62. Cambridge Univ. Press, Cambridge, UK. Leech, R. M., Thomson, W. W., and Platt-Aloia, K. A. (1981). Observations of the mechanism of chloroplast division in higher plants. New Phytol. 87, 1–9. Lloyd, C. W. (1991). ‘‘The Cytoskeletal Basis of Plant Growth and Form.’’ Academic Press, New York. Lumbreras, V., and Purton, S. (1998). Recent advances in Chlamydomonas transgenics. Protist 149, 23–27. Luttke, A., and Bonotto, S. (1981). Chloroplast DNA of Acetabularia mediterranea: Cell cycle related changes in distribution. Planta 153, 536–542. Maguire, M. J., Goff, L. J., and Coleman, A. W. (1995). In situ plastid and mitochondrial DNA determination; Implication of the observed minimal plastid genome number. Am. J. Bot. 82, 1496–1506. Marrison, J. L., and Leech, R. M. (1992). Co-immunolocalization of topoisomerase II and chloroplast DNA in developing, dividing and mature wheat chloroplasts. Plant J. 2, 783–790. McKown, R. L., and Tewari, K. K. (1984). Purification and properties of a pea chloroplast DNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2354–2358. McNaughton, E. E., and Goff, L. J. (1990). The role of microtubules in establishing nuclear spatial patterns in multinucleate green algae. Protoplasma 157, 19–37. Medgyesy, P., Fejes, E., and Maliga, P. (1985). Interspecific chloroplast DNA recombination in a Nicotiana somatic hybrid. Proc. Natl. Acad. Sci. USA 82, 6960–6964. Meeker, R., Nielsen, B., and Tewari, K. K. (1988). Localization of replication origins in pea chloroplast DNA. Mol. Cell. Biol. 8, 1216–1223. Melaragno, J. E., Mehrotra, B., and Coleman, A. W. (1993). The relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis thaliana. Plant Cell 5, 1661–1668. Mita, T., Kanbe, T., Tanaka, K., and Kuroiwa, T. (1986). A ring structure around the dividing plane of the Cyanidium caldarium chloroplast. Protoplasma 130, 211–213. Miyagishima, S., Itoh, R., Toda, K., Takahashi, H., Kuroiwa, H., and Kuroiwa, T. (1998). Identification of a triple ring structure involved in plastid division in the primitive red alga Cyanidioschyzon merolae. J. Electron Microsc. 47, 269–272. Miyamura, S., and Hori, T. (1991). DNA is present in the pyrenoid core of the siphonous green algae of the genus Caulerpa and yellow-green algae of the genus Pseudodichotomosiphon. Protoplasma 161, 192–196. Miyamura, S., Kuroiwa, T., and Nagata, T. (1990). Multiplication and differentiation of plastid nucleoids during development of chloroplasts and etioplasts from proplastids in Triticum aestivum. Plant Cell Physiol. 31, 597–602. Mogensen, H. L. (1996). The hows and whys of cytoplasmic inheritance in seed plants. Am. J. Bot. 83, 383–404. Moran, N. A. (1996). Accelerated evolution and Muller’s rachet in endosymbiotic bacteria. Proc. Natl. Acad. Sci. USA 93, 2873–2878. Motomura, T. (1996). Cell cycle analysis in a multinucleate green alga, Boergesenia forbesii (Siphonocladales, Chlorophyta). Phycol. Res. 44, 11–17. Motomura, T., Ichimura, T., and Melkonian, M. (1997). Coordinative nuclear and chloroplast division in unilocular sporangia of Laminaria angustata (Laminariales, Phaeophyceae). J. Phycol. 33, 266–271. Muller, H. J. (1964). The relation of recombination to mutational advance. Mutat. Res. 1, 2–9. Munaut, C., Dombrowicz, D., and Matagne, R. F. (1990). Detection of chloroplast DNA by using fluorescent monoclonal antibromodeoxyuridine antibody and analysis of its fate during zygote formation in Chlamydomonas. Curr. Genet. 18, 259–263.

162

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

Nakamura, S., Itoh, S., and Kuroiwa, T. (1986). Behavior of chloroplast nucleus during chloroplast development and degeneration in Chlamydomonas reinhardtii. Plant Cell Physiol. 27, 775–784. Nemoto, Y., Kawano, S., Nagata, I., and Kuroiwa, T. (1991). Studies on plastid–nuclei (nucleoids) in Nicotiana tabacum L. IV. Association of chloroplast–DNA with proteins at several specific sites in isolated chloroplast–nuclei. Plant Cell Physiol. 32, 131–141. Nerozzi, A. M. (1995). The structure, replication and maintenance of the polyploid plastid DNA ring nucleoid in Ochromonas danica. PhD dissertation, Brown University, Providence, RI. Nerozzi, A. M., and Coleman, A. W. (1997). Localization of plastid DNA replication on a nucleoid structure. Am. J. Bot. 84, 1028–1041. Nielsen, B. L., and Tewari, K. K. (1988). Pea chloroplast topoisomerase I: Purification, characterization, and role in replication. Plant Mol. Biol. 11, 3–14. Nielsen, B. L., Rajasekhar, V. K., and Tewari, K. K. (1991). Pea chloroplast DNA primase: Characterization and role in initiation of replication. Plant Mol. Biol. 16, 1019–1034. Nielsen, B. L., Lu, Z., and Tewari, K. K. (1993). Characterization of the pea chloroplast DNA ori A region. Plasmid 30, 197–211. Nunnari, J., Marshall, W. F., Straight, A., Murray, A., Sedat, J. W., and Walter, P. (1997). Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA. Mol. Biol. Cell 8, 1233–1242. Okamoto, K., Perlman, P. S., and Butow, R. A. (1998). The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: Preferential transmission of mitochondrial DNA to the medial bud. J. Cell Biol. 142, 613–623. Osteryoung, K. W., and Vierling, E. (1995). Conserved cell and organelle division. Nature 376, 473–474. Palmer, J. D. (1983). Chloroplast DNA exists in two orientations. Nature 301, 92–93. Perez-Morga, D. L., and Englund, P. T. (1993). The attachment of minicircles to kinetoplast DNA networks during replication. Cell 74, 703–711. Possingham, J. V. (1980). Plastid replication and development in the life cycle of higher plants. Annu. Rev. Plant Physiol. 31, 113–129. Pyke, K. A., and Leech, R. M. (1992). Chloroplast division and expansion is radically altered by nuclear mutations in Arabidopsis thaliana. Plant Physiol. 99, 1005–1008. Pyke, K. A., and Leech, R. M. (1994). A genetic analysis of chloroplast division and expansion in Arabidopsis thaliana. Plant Physiol. 104, 201–207. Pyke, K. A., Rutherford, S. M., Robertson, E. J., and Leech, R. M. (1994). arc6, a fertile Arabidopsis mutant with only two mesophyll cell chloroplasts. Plant Physiol. 106, 1169–1177. Rawson, J. R. Y., and Boerma, C. (1976). Influence of growth conditions upon the number of chloroplast DNA molecules in Euglena gracilis. Proc. Natl. Acad. Sce. USA 73, 2401–2404. Reith, M., and Munholland, J. (1995). Complete nucleotide sequence of the Porphyra purpurea chloroplast genome. Plant Mol. Biol. Rep. 13, 333–335. Robertson, E. J., Pyke, K. A., and Leech, R. M. (1995). arc6, an extreme chloroplast division mutant of Arabidopsis also alters proplastid proliferation and morphology in shoot and root apices. J. Cell Sci. 108, 2937–2944. Russel, C. A., Guiry, M. D., McDonald, A. R., and Garbary, D. J. (1996). Actin mediated chloroplast movement in Griffithsia pacifica (Ceramiales, Rhodophyta). Phycol. Res. 44, 57–61. Sagan, L. (1965). An unusual pattern of tritiated thymidine incorporation in Euglena. J. Protozool. 12, 105. Salisbury, F. B., and Ross, C. W. (1992). ‘‘Plant Physiology.’’ Wadsworth, Belmont, CA. Sato, N., Ohshima, K., Watanabe, A., Ohta, N., Nishiyama, Y., Joyard, J., and Douce, R. (1998). Molecular characterization of the PEND protein, a novel bZip protein present in

PLASTID–CELL COORDINATION

163

the envelope membrane that is the site of nucleoid replication in developing plastids. Plant Cell 10, 859–872. Scott, N. S., and Possingham, J. V. (1980). Chloroplast DNA in expanding spinach leaves. J. Exp. Bot. 31, 1081–1092. Scott, N. S., Tymms, M. J., and Possingham, J. V. (1984). Plastid-DNA levels in different tissues of potato. Planta 161, 12–19. Scott, S., Cain, P., and Possingham, J. V. (1982). Plastid DNA levels in albino and green leaves of the albostrians mutant of Hordeum vulgarea. Z. Pflanzenphysiol. 108, 187–191. Sears, B. B. (1980). Elimination of plastids during spermatogenesis and fertilization in the plant kindom. Plasmid 4, 233–255. Sears, B. B, Stoike, L. L., and Chiu, W.-L. (1996). Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication. Mol. Biol. Evol. 13, 850–863. Shihira-Ishikawa, I. (1987). Cytoskeleton in cell morphogenesis of the coenocytic green alga Valonia ventricosa. 1. Two microtubule systems and their roles in positioning of chloroplasts and nuclei. Jpn. J. Phycol. 35, 251–258. Slankis, T., and Gibbs, S. P. (1972). The fine structure of mitosis and cell division in the chrysophycean alga Ochromonas danica. J. Phycol. 8, 243–256. Stirewalt, V. L., Michalowski, C. B., Loffelhardt, W., Bohnert, H. J., and Bryant, D. A. (1995). Nucleotide sequence of the cyanelle genome from Cyanophora paradoxa. Plant Mol. Biol. Rep. 13, 327–332. Swinton, D., and Hanawalt, P. C. (1972). In vivo specific labelling of Chlamydomonas chloroplast DNA. J. Cell Biol. 54, 592–597. Sylvester, A. W. (1987). Organization of the cytoplasm during cell growth of the red alga Anotrichium tenue. PhD dissertation, University of Washington, Seattle. Takeda, Y., Hirokawa, H., and Nagata, T. (1992). The replication origin of proplastid DNA in cultured cells of tobacco. Mol. Gen. Genet. 232, 191–198. Taylor, C. B. (1996). Control of cyclic carotenoid biosynthesis: No lutein, no problem! Plant Cell 8, 1447–1450. Tewari, K. K. (1988). Replication of plant organelle DNA. In ‘‘DNA Replication in Plants’’ ( J. A. Bryant and V. L. Dunham, Eds), pp. 69–116. CRC Press, Boca Raton, FL. Thanh, N. D., and Medgyesy, P. (1989). Limited chloroplast gene transfer via recombination overcomes plastome–genome incompatibility between Nicotiana tabacum and Solanum tuberosum. Plant Mol. Biol. 12, 87–93. Thompson, R. J., and Mosig, G. (1985). An ATP-dependent supercoiling topoisomerase of Chlamydomonas reinhardtii affects accumulation of specific chloroplast transcripts. Nucleic Acids Res. 13, 873–891. VanWinkle-Swift, K. P. (1980). A model for the rapid vegetative segregation of multiple chloroplast genomes in Chlamydomonas: Assumptions and predictions of the model. Curr. Genet. 1, 113–125. Waddell, J., Wang, X.-M., and Wu, M. (1984). Electron microscopic localization of the chloroplast DNA replicative origin in Chlamydomonas reinhardtii. Nucleic Acids Res. 12, 3843– 3856. Walbot, V., and Coe, E. H., Jr. (1979). Nuclear gene iojap conditions a programmed change to ribosome-less plastids in Zea mays. Proc. Natl. Acad. Sci. USA 76, 2760–2764. Wang, T. S.-F. (1996). Cellular DNA polymerases. In ‘‘DNA Replication in Eukaryote Cells’’ (M. L. DePamphilis, Ed.), pp. 461–494. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Wang, Z. F., Yang, J., Nie, Z. Q., and Wu, M. (1991). Purification and characterization of a gamma-like polymerase from Chlamydomonas reinhardtii. Biochemistry 30, 1127–1131. Webb, C. D., Teleman, A., Gordon, S., Straight, A., Belmont, A., Lin, D. C.-H., Grossman, A. D., Wright, A., and Losick, R. (1997). Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B. subtilis. Cell 88, 667–674.

164

ANNETTE W. COLEMAN AND ANDREA M. NEROZZI

Weeden, N. F. (1981). Genetic and biochemical implications of the endosymbiotic origin of the chloroplast. J. Mol. Evol. 17, 133–139. Whatley, J. M. (1980). Plastid growth and division in Phaseolus vulgaris. New Phytol. 86, 1–16. Whatley, J. M. (1983). The ultrastructure of plastids in roots. Int. Rev. Cytol. 85, 175–220. Whatley, J. M. (1988). Mechanisms and morphology of plastid division. In ‘‘Division and Segregation of Organelles’’ (S. A. Boffey and D. Lloyd, Eds.), pp. 63–83. Cambridge Univ. Press, Cambridge, UK. Whiteway, M. S., and Lee, R. W. (1977). Chloroplast DNA content increases with nuclear ploidy in Chlamydomonas. Mol. Gen. Genet. 157, 11–15. Williamson, R. E. (1993). Organelle movements. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 181–202. Woelfe, M. A., Thompson, R. J., and Mosig, G. (1993). Roles of novobiocin-sensitive topoisomerases in chloroplast DNA replication in Chlamydomonas reinhardtii. Nucleic Acids Res. 21, 4231–4238. Woodcock, C. L. F., and Fernandez-Moran, H. (1968). Electron microscopy of DNA conformations in spinach chloroplasts. J. Mol. Biol. 31, 627–631. Wu, M., Nie, Z. Q., and Yang, J. (1989). The 18 kd protein that binds to the chloroplast DNA replicative origin is an iron-sulfur protein related to a subunit of NADH dehydrogenase. Plant Cell 1, 551–557. Wu, M., Chang, C. H., Yang, J., Zhang, Y., Nie, Z. Q., and Hsieh, C.-H. (1993). Regulation of chloroplast DNA replication in Chlamydomonas reinhardtii. Bot. Bull. Acad. Sin. 34, 115–131. Ye, J., and Sayre, R. T. (1990). Reduction of chloroplast DNA content in Solanum nigrum suspension cells by treatment with chloroplast DNA synthesis inhibitors. Plant Physiol. 94, 1477–1483. Yoshida, Y., Laulhere, J.-P., Rozier, C., and Mache, R. (1978). Visualization of folded chloroplast DNA from spinach. Biol. Cell. 32, 187–190. Zachleder, V., Kawano, S., and Kuroiwa, T. (1996). Uncoupling of chloroplast reproductive events from cell cycle division processes by 5-fluorodeoxyuridine in the alga Scenedesmus quadricauda. Protoplasma 192, 228–234. Zhang, Y., and Wu, M. (1993). Fluorescence microscopy on dynamic changes of frx B distribution in Chlamydomonas reinhardtii. Protoplasma 172, 57–63.

Cellular and Molecular Mechanisms of Sexual Incompatibility in Plants and Fungi Simon J. Hiscock* and Ursula Ku¨es† *Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom; and †Institute for Microbiology, ETH Zurich, CH-8092 Zurich, Switzerland

Plants and fungi show an astonishing diversity of mechanisms to promote outbreeding, the most widespread of which is sexual incompatibility. Sexual incompatibility involves molecular recognition between mating partners. In fungi and algae, highly polymorphic mating-type loci mediate mating through complementary interactions between molecules encoded or regulated by different mating-type haplotypes, whereas in flowering plants polymorphic self-incompatibility loci regulate mate recognition through oppositional interactions between molecules encoded by the same self-incompatibility haplotypes. This subtle mechanistic difference is a consequence of the different life cycles of fungi, algae, and flowering plants. Recent molecular and biochemical studies have provided fascinating insights into the mechanisms of mate recognition and are beginning to shed light on evolution and population genetics of these extraordinarily polymorphic genetic systems of incompatibility. KEY WORDS: Self-incompatibility, Mating type, Molecular recognition, Fungi, Algae, Tracheophytes, Angiosperms, Genetic polymorphism, Evolution. 䊚 1999 Academic Press.

I. Introduction A. Sex and Outbreeding The evolution of sex, cell fusion followed by nuclear fusion and meiosis, is thought to have been a key event in the ‘‘Cambrian explosion’’ of eukaryotic lineages. The evolutionary advantage of sexual reproduction appears obviInternational Review of Cytology, Vol. 193 0074-7696/99 $30.00

165

Copyright 䉷 1999 by Academic Press. All rights of reproduction in any form reserved.

166

SIMON J. HISCOCK AND URSULA KU¨ES

ous—increased variation between individuals as a consequence of recombination—but the immediate advantage to an individual, or more precisely its genes (the real units of natural selection), is less obvious. Indeed, asexual reproduction appears to offer more direct benefits to an individual than does sexual reproduction because it avoids the vagaries of finding a mate, preserves coadapted gene complexes, and does not incur inherent ‘‘costs’’ of sex (Maynard Smith, 1978). This apparent paradox has prompted intense speculation as to how and why sexual reproduction evolved and why diploidy is advantageous (Crow and Kimura, 1965; Williams, 1975; Maynard Smith, 1978; Michod and Levin, 1988; Goldstein, 1992; Otto and Goldstein, 1992; Wilmsen Thornhill, 1993; Barton and Charlesworth, 1998). Goodenough et al. (1995) stress that in most simple haploid eukaryotes sex is closely associated with the production of spores and suggest that this association may have been a crucial factor in the evolution of sex and diploidy: The ability to make spores is a highly adaptive trait which allows an organism to survive adverse environmental conditions; therefore, sporemaking genes will not be subject to selection during environmentally stable conditions because they are not expressed. This means that mutations in sporulation genes will accumulate during periods of environmental stability only to be exposed when the environment becomes inhospitable and when the sporulation genes are needed to secure the survival of the individual. Under such stressful conditions there would be strong selection for individuals that could gain diploid status by fusing with other ‘‘like’’ individuals so as to complement any mutated sporulation genes. This primitive ‘‘sex’’ would thus provide an immediate survival advantage to those individuals that aquired it. Meiosis would then be expected to evolve as a means to control runaway ploidy levels by reinstating the haploid condition, while mating types and anisogamy (leading to the evolution of sperm and eggs) probably evolved as mechanisms to control cellular and nuclear fusion and to coordinate the uniparental inheritance of ‘‘selfish’’ cytoplasmic organelles (Goodenough et al., 1995; Hurst and Hamilton, 1992; Hurst, 1996; Partridge and Hurst, 1998). The ability to generate variation that is provided by meiosis has a major selective advantage if the genotypes of some of the offspring produced by sexual reproduction are significantly better adapted to the ambient environment (e.g., in a changing environment or because of dispersal) than the genotypes of their parents. This benefit from genetic recombination can only occur if the individuals (or gametes) which fuse (originally to complement one another) are not derived from the same vegetative clone. This will produce strong selection for mechanisms that can reduce or preclude ‘‘self-fusions’’ and promote ‘‘cross-fusions’’—hence the elaboration of genetic mechanisms that reduce inbreeding and promote outbreeding, most notably sexual incompatibility systems. The binary mating-type sys-

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

167

tems which probably evolved early during the diversification of simple isogamous eukaryotes allow some control over selfing, provided dispersal is efficient and the costs of finding a mate are low, and they are still widespread among extant protists, algae, and fungi. Higher costs of finding a mate are thought to have favored the evolution of the multiple matingtype systems found in slime molds and basidiomycetes (Hurst, 1996). Many eukaryote lineages followed the evolutionary path of anisogamy to oogamy and subsequently to dimorphic sex (analogous to binary mating types) in which selfing is impossible because individuals exist as males or females. In sessile oogamous organisms, however, dimorphic sex (dioecy in plants) imposes too great a cost on finding a mate, so these organisms tend to be hermaphrodite which presents the possibility of selfing. To avoid selfing hermaphrodite flowering plants have evolved systems of genetically determined self-incompatibility which parallel the multiple mating-type systems of slime molds and basidiomycetes, except that they are superimposed on an oogamous sexual system of sperm and egg.

B. Life Cycles The complexities of the cellular and molecular mechanisms which promote outcrossing in plants and fungi are in part a reflection of the relative complexities of their life cycles. Thus, in algae and fungi there is a haploid– haploid recognition reaction which involves ‘‘complementary’’ interactions between molecules derived from different mating-type loci leading to compatibility, whereas in flowering plants there is a haploid–diploid recognition reaction involving an ‘‘oppositional’’ interaction between molecules derived from the same self-incompatibility locus which leads to incompatibility. These subtle but fundamental differences in sexual recognition are a consequence of the different life cycles of these organisms (Lewis, 1979a,b). A brief survey of eukaryotic life cycles is therefore appropriate. The simplest sexual life cycle would be a continuous succession of cell and nuclear fusion followed by meiosis followed by cell and nuclear fusion and so on, but such life cycles do not exist because of the occurrence of mitosis in the haploid phase, the diploid phase, or both (Fig. 1). The position of mitosis in the sexual cycle determines the type of life cycle, and the extent of mitosis determines the level of complexity obtained by the organism. In a haplontic life cycle only the haploid cells can divide by mitosis so that the diploid phase is represented solely by the zygote. This life cycle is characteristically found in protists, certain fungi (e.g., Schizosaccharomyces pombe), and many unicellular algae (e.g., Chlamydomonas), where mitosis in the haploid phase generates new individuals by binary fission until environmental stress cues induce these asexual mitotic products to behave as

FIG. 1 Schematic representation of the life cycles of plants and fungi. (A) Haplontic, typical of many algae and fungi; (B) haplo-diplontic, usually referred to as ‘‘alternation of generations,’’ is typical of all land plants and many algae and certain fungi; (C) diplontic, generally found in animals but also typical of diatoms and certain brown algae and fungi; (D and E) haplodikaryotic and dikaryotic life cycles, respectively, are unique to higher fungi. Cells with haploid nuclei are shown with simple lines, cells with diploid nuclei are shown with bold lines, and dikaryotic cells having two types of haploid nuclei are shown with two lines (for further details see Meinhardt and Esser, 1990).

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

169

gametes and fuse to form diploid ‘‘survival spores’’ in which meiosis takes place. In a diplontic life cycle mitosis occurs only in diploid cells. Diplontic life cycles are usually found in multicellular organisms, particularly animals and certain brown algae (e.g., Fucus), but they also occur in unicellular diatoms and certain fungi (e.g., Saccharomyces cerevisiae). The haploid phase of this life cycle is therefore restricted to the gametes, the immediate products of meiosis. In the haplo-diplontic life cycle, usually referred to as alternation of generations, mitosis takes place in both haploid and diploid phases. This life cycle is typically found in land plants and in many algae. The two phases, gametophyte (haploid) and sporophyte (diploid), may be equally developed and morphologically identical as in algae such as Ectocarpus and Ulva (isomorphic alternation of generations) or, more commonly, the gametophyte and sporophyte may be unequally developed (heteromorphic alternation of generations) with either dominance of the gametophyte (many green and red algae and the Bryophytes) or, more commonly, dominance of the sporophyte, as in the large brown algae (Laminarians) and all nonbryophyte land plants. An important modification of alternation of generations typifies seed plants (gymnosperms and angiosperms): Instead of producing a single type of spore (homospory) they produce two types of spore (heterospory). The large megaspore which is not released from the sporophyte develops into a female gametophyte in situ on the sporophyte within the protective tissue of the ovule, whereas the smaller microspore (pollen grain) which contains the reduced male gametophyte is dispersed. Thus, for passage of sperm cells from the male gametophyte to the egg(s) of the female gametophyte the pollen grain must be dispersed to the vicinity of the entrance to the ovule. In gymnosperms the ovules are born ‘‘naked’’ in cones, and once a pollen grain is delivered successfully to the opening of the ovule (usually by wind) there is no major structural barrier to fertilization. However, in angiosperms the ovule is protected by further diploid (sporophytic) tissue—the carpel/pistil, which consists of a stigma, style, and ovary. The pollen grain must therefore produce a tube that can grow through the sporophytic tissue of the pistil (syphonogamy) and locate an ovule before releasing its sperm cells—the so-called pollen–pistil interaction (Heslop-Harrison, 1975). Most eukaryotes exhibit a life cycle that corresponds to one of the three basic life cycle patterns. Fungi, however, are the notable exception. Haplontic, diplontic, and haplo-diplontic life cycles are certainly found in Protozoans, Oomycetes, and Zygomycetes, but the majority of higher fungi (Ascomycetes and Basidiomycetes) have life cycles that are characterized by the fact that fusion of gametes (plasmogamy) is not followed immediately by nuclear fusion (karyogamy). A new phase is therefore introduced into the life cycle between the haploid and diploid phases—the dikaryotic (heterokaryotic) phase. During this dikaryotic phase, which is frequently the domi-

170

SIMON J. HISCOCK AND URSULA KU¨ES

nant phase of the life cycle, both types of haploid nuclei can multiply by mitosis. Because these different nuclei usually divide synchronously, the zygote that results from nuclear fusion at the end of the dikaryotic phase is generally derived from descendents of the two original gametic nuclei. Meinhardt and Esser (1990) distinguish two uniquely fungal life cycles (Fig. 1): In the haplo-dikaryotic life cycle the haploid phase consists of both the vegetative body (asexual) and the fruit body (sexual); the dikaryotic phase is restricted to early stages in fruit body development, and the short-lived diploid phase is confined to the zygote which undergoes meiosis to produce the spores that reinstate the haploid vegetative phase. This life cycle is typical of filamentous ascomycetes such as Neurospora and Sordaria. The true dikaryotic life cycle exists when both the vegetative body and the fruit body exist as dikaryons. This life cycle is made possible by immediate fusion of (germinated) haploid meiospores (gametes). The diploid phase is therefore confined to the zygote and the haploid phase to the meiospores (and sometimes cells generated by mitoses before fusion). The dikaryotic life cycle is typical of the Basidiomycetes such as Schizophyllum and Coprinus.

II. Molecular Mechanisms of Recognition and Mating in Fungi A. Genetic Control of Mating Type Within the fungi four distinct phyla are recognized: Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota. Other groups formerly classified as fungi are usually separated from the true fungi and placed into the Protozoa (e.g., slime molds) or Chromista (e.g., the Oomycota) (Hawksworth et al., 1995). The majority of fungi are not differentiated into separate ‘‘male’’ and ‘‘female’’ sexes, and morphologically identical individuals are distinguished only by their mating types (Blakeslee, 1904; Kniep, 1928). Consistent with theoretical predictions (Hurst, 1996), mating type in ascomycetes, zygomycetes, and oomycetes is governed by a single locus with two distinctive forms. Notable exceptions are certain slime molds, the unusual ascomycete Glomerella cingulata, and many basidiomycetes which have multiple mating types determined by one, two, or three different genetic loci, each of which can have two or more different forms (Whitehouse, 1949a, b; Ku¨es and Casselton, 1992a; Bailey, 1995; Coppin et al., 1997; Cisar and TeBeest, 1999). Usually, mating-type systems are referred to as ‘‘bipolar’’ if mating type is controlled by a single locus and ‘‘tetrapolar’’ if mating type is controlled by two loci; bipolar and tetrapolar refer to the

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

171

number of different mating types that result from meiosis following a compatible mating. In rare cases of mating type being controlled by three loci, fungi are referred to as octopolar ( Jurand and Kemp, 1973). Many recent reviews have emphasized the rapid progress toward elucidation of the mechanisms of mating-type determination in fungi made through molecular biological studies (Glass and Kuldau, 1992; Ku¨es and Casselton, 1992a, 1993a; Ku¨es and Stahl, 1992; Bo¨lker and Kahmann, 1993; Casselton and Ku¨es, 1994; Glass and Nelson, 1994; Kahmann et al., 1995; Metzenberg and Randall, 1995; Kahmann and Bo¨lker, 1996; Kothe, 1996; Nelson, 1996; Staben, 1996; Vaillancourt and Raper, 1996; Coppin et al., 1997; Kronstad and Staben, 1997; Casselton and Olesnicky, 1998; Turgeon, 1998). All mating-type loci characterized to date in the ascomycetes have been found to encode sets of various transcription factors (Table I). In the yeasts S. cerevisiae and S. pombe, mating-type-encoded transcription factors control the expression of mating-type-specific pheromones and pheromone receptors which mediate recognition of putative mates (Herskowitz, 1989; Dolan and Fields, 1991; Nielsen, 1993; Johnson, 1995; Nielsen and Davey, 1995; Banuett, 1998). In basidiomycetes, such pheromones and their receptors have been shown to be encoded by one of the two possible matingtype loci (Bo¨lker and Kahmann, 1993; Casselton and Olesnicki, 1998; Table I)—the other mating-type locus encodes transcription factors, as in the ascomycetes (Casselton and Ku¨es, 1994; Kahmann and Bo¨lker, 1996; Table I). The relationship between pheromone–pheromone receptor systems and transcription factors is thus a recurring but variable theme in the determination of mating type in fungi. Consistent with the occurrence of secreted pheromones, mating-type recognition in most fungi is extracellular and is accompanied by a phase of mate ‘‘courtship’’ which precedes cellular fusion ( Jackson and Hartwell, 1990; Snetselaar et al., 1996). In the predominantly dikaryotic life cycle of basidiomycetes (see Section I, B), however, in which there is little or no restriction on hyphal fusions even within and between identical individuals, there has been a progression from extracellular to intracellular mate recognition such that self/non-self-recognition appears to be a function of the individual nuclei. Although mechanisms of matingtype determination in fungi all appear to be similar at a superficial level, detailed analyses of the various systems reveal an underlying complexity and variability that is not immediately apparent from classical genetic studies. B. Cellular and Molecular Mechanisms Determining Mating Type 1. Mating-Type Determination in Protozoa Heterothallic slime molds may have up to three different mating-type loci, each of which can have two or more alleles (Collins, 1980; Youngman

TABLE I Fungal Mating-Type Loci, Genes, and Gene Products

Species

Mating type locus

Haplotypes

Ascomycetes Alternaria alternata (asexual)

MAT-2 Others? MAT-2

Bipolaris saccheri (asexual)

172

Cochliobolus bicolor, C. chloridis, C. miyabeanus, C. peregianensis, C. sativum (all bipolar, heterothallic) C. carbonum (bipolar, heterothallic)

C. heterostrophus (bipolar, heterothallic)

C. homomorphus (homothallic) C. victoriae (bipolar, heterothallic?) Cryphonectria parasitica (bipolar, heterothallic)

Genea

MAT

MAT-1 MAT-2

? MAT-2

A

MAT-1

a

MAT-2

MAT-1

MAT-1

MAT-2

MAT-2

MAT-1/2

MAT-1/2

MAT

MAT-2

MAT-2 Others? MAT-2

MAT

MAT-1 MAT-2

MAT-2 ?

MAT

MAT

Protein function

HMG box transcription factor HMG box transcription factor

Reference

Arie et al. (1997) Sharon et al. (1996) Arie et al. (1997)

HMG box transcription factor 움1 domain transcription factor HMG box transcription factor 움1 domain transcription factor HMG box transcription factor HMG box transcription factor HMG box transcription factor HMG box transcription factor

Arie et al. (1997), Christiansen et al. (1998) Turgeon et al. (1993a)

Arie et al. (1997) Arie et al. (1997), Christiansen et al. (1998) Arie et al., (1997)

Gaeumannomyces graminis (homothallic) Mycosphaerella zeaemaydis (homothallic) Nectria haematococca (homothallic or bipolar, heterothallic) Neurospora africana (homothallic) N. crassa (bipolar, heterothallic)

MAT

⫹/⫺ ⫹ ⫺

mat

MAT-2 Others? MAT-2 Others? MAT-2

mat a

MAT-2 ? mt A-1 Others? mat a-1

mat A

mat A-1 mat A-2

173

mat A-3 Podospora anserina (bipolar, heterothallic)

mat

mat⫹

FPR1

mat-

FMR1 SMR1 SMR2

Pyrenophora teres (bipolar, heterothallic) P. tritici-repentis (homothallic)

MAT

⫹ ⫺

MAT-2 ? MAT-2 Others?

HMG box transcription factor HMG box transcription factor HMG box transcription factor HMG box transcription factor 움1 domain transcription factor HMG box transcription factor 움 1 domain transcription factor Unclassified putative transcription factor HMB box transcription factor HMG box transcription factor 움1 domain transcription factor Unclassified putative transcription factor HMG box transcription factor HMG box transcription factor HMG box transcription factor

Arie et al. (1997) Arie et al. (1997) Arie et al. (1997)

Glass and Smith (1994) Glass et al. (1990a), Staben and Yanofsky (1990), Debuchy et al. (1993)

Debuchy and Coppin (1992), Debuchy et al. (1993)

Arie et al. (1997) Arie et al. (1997)

(continued)

TABLE I (continued )

Species Saccharomyces cerevisiae (bipolar, homothallic due to mating-type switching)

Mating type locus MAT

Haplotypes MATa

Genea a1 a2

MAT움

움1

174

움2 Schizosaccharomyces pombe (bipolar, homothallic due to mating-type switching)

mat1

mat-1P

mat1-Pc mat1-Pm

mat1-M

mat1-Mc mat1-Mm

Setosphaeria rostrata (bipolar, heterothallic) S. turica (bipolar, heterothallic) Sordaria macrospora (homothallic)

MAT MAT

MAT

A a MAT-1 MAT-2

MAT-2 ? ? MAT-2

A/a

Smt-a1

Protein function Homeodomain transcription factor (HD2 type) Homeodomain protein (HD1 type), unfunctional 움1 domain transcription factor Homeodomain transcription factor (HD1 type) 움1 domain transcription factor Homeodomain transcription factor (HD1 type) HMG box transcription factor Unclassified putative transcription factor HMG box transcription factor

Reference Astell et al. (1981), Dolan and Fields (1991), Johnson (1995)

Kelly et al. (1988), Dooijes et al. (1993), Nielsen et al. (1996)

Arie et al. (1997) Arie et al. (1997)

HMG box transcription factor HMG box transcription factor

Po¨ggeler et al. (1997)

SmtA-1 SmtA-2 SmtA-3

Basidiomycetes Coprinus bilanatus (tetrapolar, secondary homothallic)

175

C. cinereus (tetrapolar, heterothallic)

A (matT)

B (matP) A (matT)

A1⭈⭈⭈A7

B1⭈⭈⭈B7 A1⭈⭈⭈A160 (postulated)

a1-x a2-x Others? ? a1-x a2-x b1-x b2-x c1-x d1-x d2-x e2-x

움1 domain transcription factor Unclassified putative transcription factor Protein related to mat A-3 of N. crassa and SMR2 of P.anserina but without HMG box Homeodomain transcription factor (HD1 type) Homeodomain transcription factor (HD2 type) Homeodomain transcription factor (HD1 type) Homeodomain transcription factor (HD2 type) Homeodomain transcription factor (HD1 type) Homeodomain transcription factor (HD2 type) Homeodomain transcription factor (HD1 type) Homeodomain transcription factor (HD1 type) Homeodomain transcription factor (HD2 type) Homeodomain transcription factor (HD2 type)

Kemp (1974), U. Ku¨es and M. P. Challen (unpublished data) Kemp (1974) Raper (1966), Casselton and Ku¨es (1994), Gieser and May (1994), Ku¨es et al. (1992, 1994a,b,c.), Pardo et al. (1996)

(continued)

TABLE I (continued )

Species

176

Cryptococcus neoformans (bipolar, heterothallic) Rhodotorula toruloides (bipolar, heterothallic)

Schizophyllum commune (tetrapolar, heterothallic)

Mating type locus

Haplotypes

Genea

B (matP)

B1⭈⭈⭈B80 (postulated)

MAT (matT/ P?) (matT/P?)

MATa Mat움

rcb1.x phb1-1.x phb1-2.x rcb2.x phb2-1.x phb2-2.x rcb3.x phb3-1.x phb3-2.x ? MF움

A (matT)

A

a A1⭈⭈⭈A288 (postulated)

RHA1 RHA2 RHA3 ? Yx Zx Vx

B (matP)

B1⭈⭈⭈B81 (postulated)

barx bapx(1) bapx(2) bapx(3)

Protein function Pheromone receptor Pheromone Pheromone Pheromone receptor Pheromone Pheromone Pheromone receptor Pheromone Pheromone Pheromone Pheromone Pheromone Pheromone Homeodomain transcripton factor (HD1 type) Homeodomain transcription factor (HD2 type) Homeodomain transcription factor (HD2 type) Pheromone receptor Pheromone Pheromone Pheromone

Reference Raper (1966), Casselton and Olesnicky (1998), O’Shea et al. (1998)

Kwon-Chung et al. (1992), Moore and Edman (1993) Akada et al. (1989)

Raper (1966), Stankis et al. (1992). Specht et al. (1992, 1994), Magae et al. (1995), Shen et al. (1996) Raper (1966), Wendland et al. (1995), Vaillancourt et al. (1997)

Ustilago hordei (bipolar, heterothallic)

MAT (matT/P)

MAT-1 -a1 genes -b1 genes

bbrx bbpx(1) bbpx(2) bbpx(3)

Pheromone receptor Pheromone Pheromone Pheromone

pra1 mfa1 bE1

Pheromone receptor Pheromone Homeodomain trancription factor (HD1 type) Homeodomain trancription factor (HD2 type)

bW1 MAT-2 -a2 genes -b2 genes

? bE2

177

bW2 U. maydis (tetrapolar, heterothallic)

b (matT)

b1⭈⭈⭈b33 (postulated)

bEx bWx

a (matP)

a1 a2

U. scitaminea (bipolar, heterothallic)

(matT/P?)

bE1⫹ bE1-

a

x refers to different altermorphs.

mfa1 pra1 (mfa) pra2 lga2 rga2 mfa2 E1 Others? ?

Homeodomain transcription factor (HD1 type) Homeodomain transcription factor (HD2 type) Homeodomain transcription factor (HD1 type) Homeodomain transcription factor (HD2 type) Pheromone Pheromone receptor Pheromone, nonexpressed Pheromone receptor Mitochondrial protein? Mitochondrial protein? Pheromone Homeodomain transcription factor (HD1 type)

Bakkeren and Kronstad (1993, 1994, 1996)

Wong and Wells (1985), Kronstad and Leong (1990), Schulz et al. (1990), Gillissen et al. (1992) Bo¨lker et al. (1992), Urban et al. (1996a)

Albert and Schenck (1996)

178

SIMON J. HISCOCK AND URSULA KU¨ES

et al., 1981; Chang and Raper, 1981; Kirouac-Brunet et al., 1981; Betterly and Collins, 1983; Kawano et al., 1987; Urushihara, 1996; Clark and Haskins, 1998). Strains of Physarum and Dictyostelium species that have multiple mating types can be arranged in a mating-type hierarchy (O’Day et al., 1987; Meland et al., 1991; Kawano et al., 1995). In Physarum the mode of action of the mating-type products is not known. The matB and matC products appear to have an extracellular function because both loci influence fusion of amebas (gametes). The third locus, matA, has no influence on fusion but controls subsequent development of the fusion cell; matA is therefore thought to act intracellularly (Youngman et al., 1981; Kawano et al., 1987; Bailey, 1995). In Dictyostelium there is strong evidence suggesting that the mating-type hierarchy operates via excreted pheromones (O’Day et al., 1987). Gametes—tiny, highly motile ameboid cells with a condensed haploid nucleus—appear to be the source of these pheromones in Dictyostelium. Following contact between two compatible gametes of opposite mating type, binucleate cells are formed that will develop into giant zygotes by swelling and ingesting surrounding amebae. This process of fertilization is mediated by a signal transduction pathway involving calcium and protein kinase C. Fertilization is terminated by an autoinhibitor secreted by the giant zygote which blocks the signal transduction pathway and induces maturation of the zygotes into macrocysts (O’Day et al., 1995). Selfincompatible cells can be rendered self-compatible by mild protease treatment which allows cells to fuse independent of mating type, although subsequent sexual development will be arrested before macrocyst development, indicating a two-checkpoint system in determining selfincompatibility. Self-incompatibility in mating is therefore tightly linked to self-recognition (Urushihara, 1996; Urushihara and Aiba, 1996). Selfrecognition also has consequences for vegetative growth of the organism because breakdown of self/non-self-recognition can lead to cannibalism even in the absence of a sexual cycle (Waddell and Duffy, 1986). Pheromones have yet to be isolated from slime molds, but polypeptide pheromones (‘‘gamones’’) of the ciliates Euplotes raikovi and E. octocarinatus have been well characterized and their involvement in mating-type determination (attraction of cells of compatible mating types and induction of conjugation) demonstrated (Luporini et al., 1996; Kuhlmann et al., 1997). Indeed, the Euplotes system may be considered a model for the molecular determination of mating type in the mutliallelic mating-type systems of the protozoa: Pheromones of E. raikovi are 37–40 amino acids long and are derived, by at least two proteolytic cleavages during exocytosis, from precursors of about 75 amino acids. The putative pheromone receptors are produced by alternative splicing of the same genes that encode the prepropheromones. These receptors are type II membrane-bound proteins of 130 amino acids which have an extracellular C-terminal domain that is identical

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

179

to the soluble pheromone. The C-terminal prepro-signal sequence of the pheromone forms the anchoring transmembrane domain of the receptor, while the cytoplasmic N-terminal sequence is composed of 55 new amino acids (Raffioni et al., 1992; Miceli et al., 1992; Stewart et al., 1992; Luporini et al., 1995). Pheromones have very little overall sequence identity—only 7 amino acids are conserved between all nine of the molecules analyzed. However, the pheromones do share a well-preserved three-dimensional architecture consisting of a tightly packed bundle of three regular or nearly regular 움-helices and a similar disulfide bond pattern (Stewart et al., 1992; Luginbu¨hl et al., 1994, 1996; Luporini et al., 1995, 1996; Anderson et al., 1997). Pheromones have been found to dimerize by two alternative modes—with their N-terminal ends pointing in either the same direction or in opposite directions. This observation led to a proposed model for pheromone–pheromone receptor binding whereby antiparallel-orientated soluble pheromones interact with the parallel-oriented N-terminal ends of the membrane-bound pheromone receptors to mediate cell–cell aggregation (Weiss et al., 1995; Anderson et al., 1997; Fig. 2). In addition to functioning in a paracrine manner to mediate mating between different cells, ciliate pheromones also play an autocrine role in mediating the mitogenic activity of the cells that produce them, thereby controlling the organism’s growth. It is therefore suggested that this pheromone–pheromone receptor mechanism evolved primarily as a mechanism to regulate asexual growth but was later recruited to a role in mating type determination (Vallesi et al., 1995).

FIG. 2 Model of pheromone–pheromone receptor interaction in Euplotes. Pheromones are similar or identical in sequence to the extracellular C-terminal region of their respective receptors. Crystalization studies revealed that these common sequences interact in two different ways, either parallel (N-terminal/N-terminal interaction) or antiparallel (N-terminal/Cterminal interaction) (Weiss et al., 1995). In the model it is proposed that interaction between pheromones and receptors occurs via an interaction between the N-terminal regions of the pheromones and the C-terminal regions of the receptors; interaction between the dimerizing receptors is via the N-proximal regions of their extracellular domains (circled N’s).

180

SIMON J. HISCOCK AND URSULA KU¨ES

2. Mating-Type Determination in Oomycetes A variety of molecules have been implicated in the control of mating in heterothallic oomycetes. For example, in Achlya species (which are unusual in that they show dimorphic sexual differentiation, ‘‘dioecy’’), male and female hyphae produce the sterol hormones antheridiol and oogoniol, respectively. In the phytophatogenic genera Phytophthora and Pythium, however, sterols can also stimulate mating but the fungi are unable to synthesize them—mating is instead controlled by small mating-type-specific lipophillic molecules (움1 and 움2) of unknown structure. Interestingly, the heterothallic A1 and A2 mating types can be induced into self-fertility by 움2 and 움1, respectively (Elliott, 1994; Mullins, 1994; Judelson, 1997). Unlike other fungi, mating type in Phytophthora operates in a heterozygous diploid condition. Heterozygosity (M/m) at the single mating-type locus determines A1 mating type and homozygosity (m/m) determines the A2 type ( Judelson, 1997). The mating-type locus displays non-Mendelian inheritance in most but not all isolates ( Judelson et al., 1995; Fabritius and Judelson, 1997) and is unusually prone to rearrangements, such as duplications and deletions, which could account for the non-Mendelian genetics ( Judelson, 1996a,b, 1997). The nature of the mating-type-specific genes is unknown.

3. Mating-Type Determination in Zygomycetes Within the Zygomycetes only the Mucorales have been studied in terms of sexual differentiation. In these fungi, formation of sexual structures (zygophores with protogametangia) is induced by the carotene derivative trisporic acid. Trisporic acid is synthesized by a cooperative biosynthetic pathway which is shared between the complementary mating types of a given species (Elliott, 1994; Gooday, 1994; Fig. 3). Although this fascinating biochemical interplay between organisms of different mating types is long established, little information is available on the genetics of mating-type determination and the mating-type genes have yet to be identified and characterized (Wo¨stemeyer et al., 1995). One gene from the sexual hormone pathway has been isolated from the (⫺) mating type of Mucor mucedo. This gene, which encodes 4-dihydromethytrisporate dehydrogenase, is expressed specifically in the (⫺) mating type but a (nonexpressed) copy of the gene is also present in the (⫹) mating type (Czempinski et al., 1996). Matingtype recognition molecules in the zygomycetes are not species specific because many Mucor-like fungi can induce formation of zygophores in other Mucor-like species as long as they belong to complementary mating types. Following such ‘‘interspecific’’ mating-type interactions, fusion of the gametangia formed from the zygophores rarely takes place. An interesting exception, however, is the interaction between complementary mating types of Absidia glauca and Parasitella parasitica: Under the mediation of trisporic

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

181

FIG. 3 Coordinated synthesis of trisporic acid between the two mating types in Mucorales. ⫹, reactions performed by the ⫹ mating type;⫺,reactions performed by the ⫺ mating type (after Wo¨stemeyer et al., 1995).

acid, protogametangia from both species fuse, and nuclei from Parasitella invade the Absidia partner (Kellner et al., 1993; Wo¨stemeyer et al., 1995). Mating-type-specific surface components have been proposed to be involved in the process of recognition and fusion of gametangia of opposite mating type. To date, however, only one cell wall protein specific to the (⫹) mating type of A. glauca has been identified. PSSP15 is a 15-kDa plasmid-encoded protein that localizes specifically to the cell wall of the (⫹) mating type; the role of this protein in mating is undetermined (Ha¨nfler et al., 1992). 4. Mating-Type Determination in Ascomycetes a. The Diplontic Yeast Saccharomyces cerevisiae In the simplest and best studied ‘‘model’’ ascomycete, the yeast S. cerevisiae, haploid cells

182

SIMON J. HISCOCK AND URSULA KU¨ES

produce and secrete specific peptide pheromones—either the a pheromone (a-factor) or the 움 pheromone (움-factor), depending on mating type. Recognition and response to pheromones of the opposite mating type is essential for the mating of Mata and Mat움 cells leading to the production of an a/움 diploid (Bender and Sprague, 1989; Jackson and Hartwell, 1990; Herskowitz, 1989; Kurjan, 1992, 1993; Marsh and Rose, 1997). Response to pheromones results in arrest of the cell cycle, transcriptional activation of genes required for mating, cytoskeletal reorganization, and polarized growth toward a mating partner which eventually culminates in mating between the cells (Wittenberg and Reed, 1996; Leberer et al., 1997; Madden and Snyder, 1998). Polarized mating projections form at the site of highest pheromone concentration, indicating that the morphological response is initiated at the point at which a pheromone interacts with its cell surface receptor (Segall, 1993; Schrick et al., 1997). The pheromone response is transmitted through pheromone receptors coupled to a heterotrimeric G protein composed of Gpal (움), STE4 (웁), and STE18 (웂). Interaction of the receptor with its pheromone activates bound G움 via guanine nucleotide exchange and the G움-GTP then dissociates from the G웁웂 subunits. The pheromone response signal is thought to be transmitted from the released G웁웂 dimer via the STE20 kinase to the MAP kinase cascade, consisting of MAP KKK (STE11), MAP KK (STE7), and MAP kinase (FUS3 or KSS1), that ultimately regulates the action of the transcription factor STE12, which coordinates expression of genes relevant to mating, and FAR1, which controls cell cycle arrest. Many other proteins have been shown to interact with components of the MAP cascade; for example the ‘‘scaffolding’’ protein STE5 associates with all four kinases and G웁 in the G웁웂 dimer, and STE50 appears to provide another link between the kinases and the G protein [for further details and extensive review see Kurjan (1992, 1993), Sprague and Thorner (1992), Bardwell et al. (1994), Elion (1995), Herskowitz (1995), Davis and Davey (1997), and Banuett (1998); Fig. 4]. In addition STE20 contributes to a less well-understood complex of proteins that transmits a signal from an activated pheromone receptor to proteins involved in cytoskeletal reorganization, oriented cell growth, and localization of the mating projection tip. STE20 associates with the Rho-like GTPase CDC42 and also with BEM1, which in turn interacts with CDC24 (the guanine nucleotide exchange factor for CDC42 that interacts with the G웁 subunit), STE5, FAR1 (the regulator of cell cycle arrest), and actin (Leeuw et al., 1995; Wittenberg and Reed, 1996; Leberer et al., 1997; Cabib et al., 1998; Madden and Snyder, 1998; Nern and Arkowitz, 1998; Fig. 4). BEM1 is therefore a good candidate for the link between the signaling molecules and the cytoskeleton (Leeuw et al., 1995). Another candidate for this link could be the formin BNI1, which interacts with CDC42, actin, and actin-

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

183

FIG. 4 Interactions between components of the pheromone response pathway in S. cerevisiae. Seven-transmembrane receptors, either STE2 in a cells, or STE3 in 움 cells, are coupled to a heterotrimeric G protein. Activation of the G protein by binding of a pheromone to a receptor (either the a factor to STE2 or the 움 factor to STE3) releases the G웁웂 subunits to activate the MAP kinase signaling pathway (STE11 씮 STE7 씮 FUS3 and KSS1) via protein kinase STE20. Activated FUS3 and KSS1 ultimately regulate the action of the transcription factor STE12 and the cell cycle control protein FAR1. Other proteins have been shown to interact with proteins in the signaling cascade but their specific function is not known (e.g., SYG1 and AKR1). In a way not fully understood, the pheromone response pathway is also linked to the actin cytoskeleton via CDC42/CDC24 and BEM1 and probably BNI1. BEM1 appears to play a central role in this interaction because it is in contact with the protein kinase STE20 and STE5, the scaffolding protein of the MAP kinase complex (for further information see text and Leberer et al., 1997).

associated proteins (Evangelista et al., 1997; Cabib et al., 1998; Madden and Snyder, 1998; Fig. 4). Despite their apparent functional equivalence, the pheromones of S. cerevisiae are basically different and belong to two distinct classes of molecules. The a-factor is a prenylated, carboxyl-methylated dodecamer that is generated from a precursor encoded by the two functionally redundant genes MFA1 and MFA2. The a-factor precursors consist of a 21-

184

SIMON J. HISCOCK AND URSULA KU¨ES

or 23-amino acid (aa) N-terminal sequence, the mature a-factor peptide sequence, and a C-terminal CAAX motif (C, cys; A, aliphatic residue; X, any residue) (Caldwell et al., 1995; Quinby and Deschenes, 1997). Pheromone maturation begins with the farnelysation of the cysteine in the CAAX motif by the farnesyl transferase complex RAM1/RAM2, followed by cleavage of the last three amino acids by the CAAX prenyl proteases RCE1 or STE24 and carboxymethylation by the carboxyl methyltransferase STE14. The consecutive N-terminal processing involves two proteolytic steps, the first mediated by STE24 and the second alternatively by AXL1 or STE23 (Chen et al., 1997; Davis and Davey, 1997; Tam et al., 1998). Following the successive stages of maturation, the a-factor is secreted by a nonclassical secretory pathway mediated by the ATP-binding cassette (ABC) transporter STE6 (Berkower and Michaelis, 1991; Caldwell et al., 1995). The 움factor is also encoded by two functionally redundant genes, MF움1 and MF움2, encoding polypeptide precursors of 165 and 120 aa, respectively, that contain four and two tandem repeats (respectively) of the 움-factor peptide behind a hydrophobic N-terminal peptide leader sequence that is removed from the precursors while translocated to the endoplasmic reticulum. The pro-pheromone is processed consecutively into subunits by the endopeptidase KEX2, which cleaves on the C-terminal side of a pair of basic residues. These subunits are trimmed by the carboxypeptidase KEX1, which removes the two basic residues from the C terminus, and by the aminopeptidase STE13, which trims the N terminus to give the 13-aa-long mature 움-factor that is transported out of the cell by the classical yeast secretory pathway (Caldwell et al., 1995; Davis and Davey, 1997). Once secreted, the a-factor binds to the pheromone receptor STE3 expressed in Mat움 cells, whereas the 움-factor binds the pheromone receptor STE2 expressed in Mata cells. STE3 and STE2 belong to the rhodopsinlike receptor family which has seven hydrophobic transmembrane-spanning helices (7TM-type receptors), an extracellular N-terminal tail, three outer and three cytoplasmic loops, and an inner C-terminal tail. Although STE3 and STE2 are structurally very similar, they show no sequence homology to each other (Nakayama et al., 1985; Hagen et al., 1986). Analysis of chimeric 움-factor receptors of the related yeasts S. cerevisiae and S. kluyveri suggests that pheromones interact in more than one way with their receptors. The 움-factor appears to make specific contacts in the N-terminal tail and in the first and the third outer loops. Contacts in the N-terminal tail and the first loop affect specificity of pheromone binding, while contacts in the first and the third loops affect pheromone specificity for activation of cellular responses (Sen et al., 1997). Binding of the pheromone leads to phosphorylation, ubiquination, and conformational changes of the receptor which are believed to regulate the coupling of the receptor to the G protein and to signal its removal from the cell surface by endocytosis (Chen and

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

185

Konopka, 1996; Bu¨ku¨s¸og˘lu and Jenness, 1996; Hicke and Riezman, 1996; Roth and Davis, 1996). Mutations within the third cytoplasmic loop or in the sixth transmembrane domain can constitutively activate the G proteincoupled pheromone receptors implicating the third intracellular loop in G protein activation and/or desensitization (Boone et al., 1993; Stefan and Blumer, 1994; Konopka et al., 1996). Cell-type-specific expression of pheromone and pheromone receptor genes underlies control of the genes at the mating-type locus ( Johnson, 1995). Two genes are encoded in Mat움: 움1, the product of which defines a specific class of fungal transcription factor (움1 domain transcription factors), and 움2, which encodes a homeodomain transcription factor. The Mata locus contains one functional gene, a1 which encodes a homeodomain transcription factor unrelated to 움2, and a second gene, a2 that is a 5⬘ truncated nonfunctional version of 움2 (Astell et al., 1981; Dolan and Fields, 1991; Johnson, 1995). Because the sequences that reside at the Mat locus within the two different mating types are totally unrelated in character and origin, Metzenberg and Glass (1990) introduced the term ‘‘idiomorph’’ to distinguish this situation from the familiar case of alleles, in which different versions of a gene at a given locus do have a common origin. Because the mating-type loci are complex and contain more than one gene, the different forms of mating-type information may also be called haplotypes (May et al., 1991; Coppin et al., 1997). A remarkable feature of the sexual incompatibility system of S. cerevisiae is the ability of mother cells to switch the genetic information located at the mating-type locus in approximately 86% of all cell divisions. Adjacent to the active mating-type information present at the MAT locus, there are two loci with unexpressed, silenced matingtype information, (HML and HMR) located about 200 and 100 kb away from the MAT locus, respectively. These loci serve as ‘‘storage loci’’—HML in most cases stores 움 mating-type information, (HML움) and HMR usually stores a mating-type information (HMRa). During switching, the a or 움 information prevailing momentarily at MAT is replaced by the opposite mating-type sequences copied from one of the two donor loci by a programmed genetic rearrangement [for further information on the mechanism of mating-type switching see Herskowitz et al. (1992), Klar (1993), Schmidt and Gutz (1994), Haber (1998), and Wu et al. (1998)]. Interestingly, gene products specific to the mating-type locus are not needed for establishing the a cell type of S. cerevisiae. a-Specific functions, such as MAF1, MFA2, STE2, and STE6, are produced constitutively under the guidance of the MADS box transcription factor MCM1 and STE12, a transcription factor containing a divergent homeodomain. MCM1 and STE12 activate transcription of a-specific genes by cooperatively binding the promotor sequence of the regulated genes: MCM1 binds as a homodimer to the a-specific gene operator asg, and STE12 binds a sequence termed PRE,

186

SIMON J. HISCOCK AND URSULA KU¨ES

denoting pheromone-response element (Dolan and Fields, 1991; Johnson, 1995; Fig. 5). Conversely, the exclusive expression of 움-specific genes in 움 cells of S. cerevisiae, including MF움1, MF움2, and STE2, is ensured by binding of a protein complex, consisting of MCM1, 움1, and STE12, to the QP’ operator sequences in promotors of 움-specific genes and by the repression of a-specific functions through cooperative binding of MCM1 and 움2 homodimers whose homeodomains recognize the asg operator. Following binding of the 움2–MCM1 complex, the 움2 dimer recruits the

FIG. 5 Cell type-specific gene regulation in S. cerevisiae. In a cells, the general transcription factors MCM1 and STE12 bind coordinately to their operator sites and activate transcription of a-specific genes. In 움 cells and in diploid a/움 cells, protein complexes of homodimers of 움2 and of MCM1 bind to the asg operator sites of a-specific genes. The SSN6/TUP1 repressor complex is then recruited by 움2 to the promotor and as a consequence a-specific genes are turned off. In contrast, 움-specific genes are activated in 움-cells by binding of an 움1/MCM1/ STE12 protein complex to promotors of 움-specific genes (Q, operator for 움1; P⬘, operator for MCM1). Haploid-specific genes are constitutively expressed in a and 움2 cells but are switched off in a/움 cells by binding of the heterotrimeric a1/움2 homeodomain transcription factor complex to hsg operator sites and recruitment of TUP1/SSN6 to the promotor sites. In a/움 cells, the a1/움2/TUP1/SSN6 protein complex represses also gene 움1 of the Mat움 idiomorph (compiled from Dolan and Fields, 1991; Ku¨es and Casselton, 1992a; Johnson, 1995).

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

187

general transcription factors TUP1 and SSN6 to the protein complex via interaction with the WD40 repeats of TUP1 and the TPR repeats of SSN6 (Dolan and Fields, 1991; Johnson, 1995; Fig. 5). Following fusion of a and 움 cells, the expression of genes required for pheromone production and perception is repressed (Herskowitz, 1989; Herskowitz et al., 1992; Kurjan, 1992, 1993; Bardwell et al., 1994). Expression of a-specific genes in the diploid is similarly repressed by the 움2–MCM1–TUP1–SSN6 protein complex (Dolan and Fields, 1991; Johnson, 1995; Fig. 5), whereas 움-specific genes are not expressed because the 움1 protein is not present in the a/움 cell. Cell fusion brought together a1 and 움2 and these two distinct homeodomain proteins interact to give a new regulatory complex with a changed DNA binding preference. Therefore, a1/움2 heterodimers, together with TUP1 and SSN6, repress expression of 움1 and all haploid-specific genes including rme1, which encodes a transcriptional repressor of meiosis-specific and sporulation-specific genes (Fig. 5, Dolan and Fields, 1991; Johnson, 1995). Due to its central role in the regulation of sexual development in S. cerevisiae, 움2, not surprisingly, is one of the best characterized of all proteins, both structurally and functionally ( Johnson, 1995; Wolberger, 1996; see Section II,B,5,a). Following NMR analysis of the three-helical homeodomain which binds DNA, the crystal structure of the DNA-bound 움2–MCM1 complex and the DNA-bound a1/움2 heterodimer was determined and amino acids and protein domains that interact with DNA and protein partners were established (T. Li et al., 1995; Tan and Richmond, 1998). A special role in a1/움2 activity has been attributed to the 20-aa tail of 움2 which forms a short amphipathic helix that packs against the a1 homeodomain between helix 1 and helix 2. This interaction induces a pronounced 60⬚ bend in the DNA which in turn enables new protein–protein and protein–DNA contacts to be established (Phillips et al., 1991; Wolberger et al., 1991; T. Li et al., 1995; Smith et al., 1995a). The short 움2 tail is the major dimerization interface with a1 (Mak and Johnson, 1993; Phillips et al., 1994; Stark and Johnson, 1994), while a secondary, weaker, dimerization domain is localized to the N termini of 움2 and a1 (Ho et al., 1994) which have recently been implicated in protein stabilization by masking proteolytic signals for the ubiquitin–proteasome pathway ( Johnson et al., 1998). Other defined dimerization interfaces in 움2 are 110–128 aa N terminal to the homeodomain for interaction with MCM1 (Smith and Johnson, 1992; Vershon and Johnson, 1993; Tan and Richmond, 1998) and the homeodomain for interaction with the TPR repeats of SSN6 (Smith et al., 1995b) and the extreme N terminus for interaction with the WD40 repeats in TUP1 (Komachi and Johnson, 1997). b. The Haplontic Yeast Schizosaccharomyces pombe Pheromone communication in the fission yeast S. pombe has been found to be very important

188

SIMON J. HISCOCK AND URSULA KU¨ES

for both fusion of haploid cells of opposite mating type (conjugation) and sequential meiosis (Egel et al., 1990; Nielsen, 1993; Nielsen and Davey, 1995; Yamamoto, 1996a,b; Yamamoto et al., 1997). Responses to pheromone exposure include cell elongation, pheromone-dependent transcription, arrest of cell cycle, and induction of meiosis in diploid cells (Davey and Nielsen, 1994; Imai and Yamamoto, 1994; Wang et al., 1994; Willer et al., 1995). All mating activities in S. pombe are repressed during mitotic growth and only when nitrogen sources are depleted do any sexual activities commence. Nitrogen starvation reduces the intracellular cAMP level and this appears to be the signal to derepress sexual development (Maeda et al., 1994; Kawamukai et al., 1991; Mochizuki and Yamamoto, 1992). Nutritional depletion activates the key regulator of mating and meiosis, the HMG box transcription factor Ste11 (Sugimoto et al., 1991; Okazaki et al., 1998), followed by genes responsible for cellular communication, including the mating-type genes, pheromone and pheromone receptor genes, and genes involved in pheromone activity (Kelly et al., 1988; Kitamura and Shimoda, 1991; Tanaka et al., 1993; Imai and Yamamoto, 1992, 1994; Kjaerulff et al., 1994; Christensen et al., 1997b). The mating-type locus in S. pombe has two idiomorphs designated mat1P and mat1-M. The P idiomorph is related to the Mat움 idiomorph of S. cerevisiae and contains two genes; mat1-Pc encodes an 움1 domain protein and mat1-Pm encodes a homeodomain protein. The M idiomorph contains genes which are totally unrelated to any S. cerevisiae mating-type genes: mat1-Mc encodes an HMG-box transcription factor and mat1-Mm encodes an unknown class of protein (Kelly et al., 1988; Dooijes et al., 1993; Nielsen et al., 1996; Coppin et al., 1997). As with S. cerevisiae, mating-type interconversion is commonly observed in S. pombe, although it functions in a slightly different way. For example, in S. pombe switching is asymmetrical and only one of four grand-daughter cells will switch. Changing cell type in S. pombe also involves two silenced loci (mat2 and mat3) from which mating-type information can transpose to the active locus mat1 and replace the prevailing information of the opposite mating type (Herskowitz et al., 1992; Klar, 1992, 1993; Arcangioli, 1998; Schmidt and Gutz, 1994; Klar et al., 1998). As mentioned previously, expression of mating-type genes in S. pombe occurs under conditions of nitrogen starvation and is activated by Ste11. mat1-Pc and mat1-Mc are required to induce production of the mating pheromones and receptors which generate the pheromone signal that mediates conjugation, whereas all four mating-type genes are required for meiosis in diploid P/M cells. Establishment of the pheromone signal induces expression of mat1-Pm and mat1-Mm, the major pheromone-dependent step in the control of meiosis. The presence of all four mating-type gene products allows expression of the mei3 gene which encodes a direct inducer of meiosis (McLeod et al., 1987; Kelly et al., 1988; Nielsen et al., 1992; Aono et al., 1994;

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

189

Willer et al., 1995). At least two of the mating-type gene products, the HMG box transcription factor Mc and the homeodomain transcription factor Pm appear to bind to the mei3 promotor in vivo to activate mei3 transcription. This is interesting because the Pm homologous homeodomain protein 움2 from S. cerevisiae always functions as a repressor (van Heeckeren et al., 1998). Recent molecular analysis of expression of P-specific and M-specific pheromone and pheromone receptor genes suggests that the 움1 domain factor Mat1-Pc forms a complex with the MCM1-like MADS-box transcription factor, Map1, and that the HMG box protein Mat1-Mc binds of the HMG-box transcription factor Ste11, which then bind to adjacent operator sites in a concerted manner (Nielsen et al., 1996; Kjaerulff et al., 1997). Pheromones produced by the opposite mating types of S. pombe [Minus (M ) and Plus (P ) or h⫺ and h⫹, respectively] structurally resemble those of S. cerevisiae. The M-factor is encoded by three functionally redundant genes (mfm1, mfm2, and mfm3) which contain the same mature pheromone sequence as part of a 29- to 32-aa precusor ending with the CAAX motif, Cys-Val-Ile-Ala. The mature protein, which consists of 9-aa with a Cterminal carboxyl-methylated and S-farnesylated cysteine residue, is secreted by cells of the M mating type through an ABC transporter encoded by mam1 (Davey, 1992; Kjaerulff et al., 1994; Wang et al., 1994; Christensen et al., 1997a,b). The P-factor (specific to the P cells) is an unmodified 23aa peptide encoded by map2. Its precurser has a signal sequence and four repeats of the mature P-factor sequence separated by spacer regions which are cleaved at pairs of basic residues by the KEX2-related endopeptidase Krp (Davey et al., 1994; Imai and Yamamoto, 1994). The receptors for the M-factor and P-factor are seven-transmembrane proteins coupled to heterotrimeric G protein and are found in the opposite cell types (Kitamura and Shimoda, 1991; Tanaka et al., 1993). As in S. cerevisiae, pheromonereceptor binding leads to activation of a MAP kinase signaling pathway, the main components of which in S. pombe are MAP KKK Byr2, MAP KK Byr1, and MAP K Spk1 (Nadin-Davis and Nasim, 1990; Toda et al., 1991; Wang et al., 1991). However, there are numerous differences between the respective signaling pathways of these two yeasts (Neiman et al., 1993; Nielsen, 1993; Nielsen and Davey, 1995; Yamamoto, 1996a,b Yamamoto et al., 1997; Banuett, 1998). For instance, in S. pombe the G움 subunit plays the postive role in signal transduction (Obara et al. 1991) in contrast to S. cerevisiae, in which G웁웂 plays this role (Whiteway et al., 1989; Blinder et al., 1989); also, in S. pombe the mating response requires ras (Nielsen et al., 1992), which is not required during mating in S. cerevisiae (Wigler et al., 1988). Finally and most important, activation of the pheromone response pathway in S. pombe is entirely dependent on nitrogen starvation. The mechanism of this nutritional control is not entirely clear but both cyr1 and pka mutants, defective in adenylate cyclase and cAMP-dependent

190

SIMON J. HISCOCK AND URSULA KU¨ES

protein kinase respectively (Imai and Yamamoto, 1994; Maeda et al., 1994) and mutations in pat1, encoding the Pat1 (Ran1) kinase (Davey and Nielsen, 1994; Mach et al., 1998), interrupt the link between nutritional control and the pheromone response and lead to unscheduled sexual differentiation, probably by a failure to control the activity of the transcription factor Ste11 and the meiosis initiation factor Mei2 by phosphorylation (Sugimoto et al., 1991; Li and McLeod, 1996; Stettler et al., 1996; Watanabe et al., 1997). To release repression of sexual development the Pat1 kinase has to be inactivated in a stepwise manner involving three broad cellular events: (i) the derepression of mating activities by nitrogen starvation; (ii) the induction of particular genes by the pheromone signaling pathway, and (iii) following successful zygote formation total inactivation of Pat1 is brought about when the pseudosubstrate Mei3 binds Pat1, thus preventing Pat1 binding to Ste11 (Nielsen and Egel, 1990; Li and McLeod, 1996). This final inactivation releases repression of Mei2, an RNA-binding protein that forms a complex with a specific RNA species (meiRNA) to promote meiosis I (Yamamoto, 1996a,b; Watanabe et al., 1997; Yamamoto et al., 1997). c. Filamentous Ascomycetes Fertile filamentous ascomycetes form male and female organs (spermatia and ascogonia, respectively) which usually develop on the same mycelium. From the ascogonium an apical receptive hyphal element, the trichogyne, develops. Although sexual differentiation occurs, sexual incompatibility, ensuring outcrossing, is controlled by mating. Usually, heterothallic (self-sterile) filamentous ascomycetes have two mating-type loci, and spermatia of one mating type can fuse only with the trichogyne of the other mating type (Raju, 1992; Nelson, 1996; Coppin et al., 1997). Indirect evidence for the existence of mating-type-specific pheromone/receptor mechanisms in these fungi came from observations in Ascobolus stercorarius, Bombardia lunata, Neurospora crassa, and Podospora anserina in which trichogynes were seen to grow in a targeted direction toward spermatia of the opposite mating type, and spermatial filtrates from one mating type were found to induce the directed growth of trichogynes from the other mating type (Zickler, 1952; Bistis, 1956, 1981, 1983, 1998; Esser, 1959). Moreover, the 움-factor of S. cerevisiae was shown specifically to inhibit development of plant infection structures (appressoria) in MAT1-2 strains of Magnaporthe grisea but not in MAT1-1 strains, indicating the presence of a mating-type-specific pheromone receptor and of a pheromone response pathway in this filamentous rice blast fungus (Beckerman et al., 1997). Recently, the first pheromone genes were cloned from the filamentous ascomycetes Cryphonectria parasitica (Zhang et al., 1993, 1998) and N. crassa (D. Bell-Pedersen, personal communication). All the pheromone genes are present in both mating types but they are expressed in a matingtype-dependent manner. mf1/1 from C. parasitica encodes a pheromone

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

191

precursor, Mf1/1 of the Mat-1 mating type, that has all the structural features of the prepro-움-factor of S. cerevisiae and the prepro-P-factor of S. pombe. Mf1/1 is a 530-aa polypeptide with a typical N-terminal signal peptide (directing secretion) and seven identical decapeptides surrounded by protease signal motifs known to be required for processing of the 움-factor in S. cerevisiae. The other two genes, mf2/1 and mf2/2, encode Mat-2-specific identical 23-aa peptides with a CAAX prenylation/farnesylation motif at their C-terminal ends. Proteolytic cleavage is thought to occur at an asparagine 11aa from the CAAX motif. The overall structure of the mf2/1 and mf2/2 products resembles those of the a-factor of S. cerevisiae, the M-factor of S. pombe, and the lipopeptide pheromones of the basidiomycetes (Zhang et al., 1993, 1998; Caldwell et al., 1995; see Section II,B,5,b). By analogy with ascomycete yeasts, pheromone and pheromone receptor genes are expected to be targets of mating-type gene control in the filamentous ascomycetes (Coppin et al., 1997). Despite the identification of G움 subunits in C. heterostrophus (Horwitz et al., 1999), C. parasitica (Gao and Nuss, 1996), N. crassa (Ivey et al., 1996; Baasiri et al., 1997), and M. grisea (Liu and Dean, 1997), a G웁 subunit in C. parasitica (Kasahara and Nuss, 1997), a MAP kinase and an adenylate cyclase in M. grisea (Xu and Hamer, 1996; Choi and Dean, 1997), and a MAP kinase kinase and a Ras-like protein in N. crassa (Ito et al., 1997; Kothe and Free, 1998), insights into the nature of the pheromone response pathway and signaling cascades relating to mating and sexual development are lacking. However, the N. crassa expressed sequence tag sequencing project (Nelson et al., 1997a) promises to identify many more genes whose products function upstream and downstream of the pheromone–receptor interactions and will also identify target genes for mating-type control. In heterothallic ascomycetes mating-type loci have been cloned and shown to be idiomorphic (Metzenberg and Glass, 1990). Idiomorphs have been analyzed in detail from Cochliobolus heterostrophus, N. crassa, and P. anserina (Glass et al., 1988, 1990a; Staben and Yanofsky, 1990; Debuchy and Coppin, 1992; Debuchy et al., 1993; Turgeon et al., 1993a; LeubnerMetzger et al., 1997; Randall and Metzenberg, 1998; Wirsel et al., 1998; Coppin et al., 1997; Kronstad and Staben, 1997; Table I). Mating-type loci have also been isolated from other fertile ascomycetes of different classes and orders (including Cochliobolus sp., M. grisea, and Tapesia yallundae) and, where possible, classified with respect to other ascomycete matingtype genes by their homology with heterologous mating-type gene probes and by PCR using primer sequences deduced from the mating-type genes of the three well-characterized species (Dyer et al., 1993; Turgeon et al., 1993b; Glass and Smith, 1994; Kang et al., 1994; Arie et al., 1997; Singh and Ashby, 1998; Table I). Potentially functional mating-type genes have also been identified in asexual ascomycetes, such as Bipolaris saccheri,

192

SIMON J. HISCOCK AND URSULA KU¨ES

indicating an absence of other genes required for the sexual process in this species (Sharon et al., 1996). Functional conservation of MAT genes in different ascomycetes was demonstrated by transformation studies: Opposite heterologous MAT genes transformed C. heterostrophus and P. anserina strains into homothallic (self-fertile) dual maters, which were capable of mating with both mating types (Arnaise et al., 1993; Turgeon et al., 1993a, 1995; Glass and Smith, 1994; Sharon et al., 1996; Wirsel et al., 1996; Po¨ggeler et al., 1997; Christiansen et al., 1998). Similar effects were seen in C. heterostrophus and P. anserina transformed with opposite homologous MAT genes (Picard et al., 1991; Turgeon et al., 1993a; Wirsel et al., 1996) but not in N. crassa (Glass et al., 1988). Consistent with these observations, homothallic filamentous ascomycetes often contain mating-type information from the two idiomorphs of heterothallic species within the same nucleus, but various elements are regularly missing from these sets of genes and some homothallic species appear to carry sequences from only one of the idiomorphs (Glass et al., 1988, 1990b; Beatty et al., 1994; Cisar et al., 1994; Glass and Smith, 1994; Po¨ggeler et al., 1997). Mating-type switching has been observed occasionally in some heterothallic ascomycetes (notably, Botryotinia fuckeliana, Ceratocystis coerulescens, Chromocrea spinulosa, Glomerella cingulata, and Sclerotinia trifolium) but, unlike yeasts, switching in filamentous ascomycetes is always unidirectional, i.e., one specific mating type might be converted into the second but the second never converts into the first (Wheeler, 1950; Mathieson, 1952; Uhm and Fujii, 1983a,b; Perkins, 1987; Faretra et al., 1988; Faretra and Pollastro, 1996; Harrington and McNew, 1997). Switching typically involves a self-fertile mating type converting into a self-sterile mating type and it is expected that the selffertile strains of these species contain genetic information for both mating types, whereas the self-sterile strains contain genetic information for only one mating type (Coppin et al., 1997; Harrington and McNew, 1997). Idiomorphs from the three best studied filamentous ascomycetes have basic similarities but they do differ from each other in gene numbers and some of the phenotypes that they control. The Mat locus of C. heterosporus has the simplest organization with one gene per idiomorph and these genes (MAT-1 and MAT-2) confer mating-type specificity. MAT-1 encodes a 383aa 움1 domain protein and MAT-2 a putative 343-aa HMG box transcription factor (Turgeon et al., 1993a; Leubner-Metzger et al., 1997). The mat a locus of N. crassa and the mat⫹ locus of P. anserina both contain a single gene which, like MAT-2 of C. heterostrophus, encodes an HMG box protein: Mat a-1 (283 aa) and FPR1 (402 aa), respectively (Staben and Yanofsky, 1990; Debuchy and Coppin, 1992). Within the second mating-type locus, mat A in N. crassa and mat⫺ in P. anserina, three genes reside: In N. crassa, mat A-1 encodes a 293-aa 움1 domain protein, mat A-2 encodes a 373-aa protein with no known motif, and mat A-3 encodes a 324-aa HMG box

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

193

protein (Glass et al., 1990a; Ferreira et al., 1996). The analogous genes of the mat⫺ locus in P. anserina encode the 305-aa 움1 domain protein FMR1, the 356-aa protein SMR1 (which shares a short sequence of 14 identical amino acids and 5 synonymous amino acids with MAT A-2 of N. crassa), and the 288-aa HMG box protein SMR2 (Debuchy et al., 1993). Based on the motifs present in these mating-type proteins, it has been proposed that they all act as transcription factors, with Mat A-2 and SMR1 defining a new class of transcription factor (Debuchy et al., 1993; Coppin et al., 1997). To date, however, only MAT a-1 and Mat A-3 of N. crassa have been shown to bind to DNA. The DNA target sites encompass the 5⬘-CAAG3⬘ sequence common to the DNA binding sites of HMG box transcription factors (Philley and Staben, 1994; Kronstad and Staben, 1997). Direct targets of mating-type proteins are not known in any of the filamentous ascomycetes but a number of putative target genes have been isolated (Nelson and Metzenberg, 1992; Nelson et al., 1997; Ferreira et al., 1998). Deletion of mating-type loci has no effect on vegetative characteristics of the filamentous ascomycetes but it abolishes mating ability. Reintroduction of mating-type information at ectopic sites restores mating competence in an idiomorph-dependent manner. However, ectopic mating-type transformants of ⌬mat strain of N. crassa are infertile and fail to produce ascospores after mating, in contrast to ectopic mat transformants of the analogous ⌬mat strains of C. heterostrophus and P. anserina (Coppin et al., 1993; Wirsel et al., 1996; Ferreira et al., 1998). Similarly, when sterile strains of N. crassa carrying mutated mating-type sequences (mat Am or mat am) were transformed with functional mating-type idiomorphs the introduced mating-type information was only fully functional in directing postfertilization events (such as ascospore formation) when it replaced information at the resident mat locus, suggesting that sequences surrounding the mat locus are required for correct expression (Glass et al., 1988, 1990a; Staben and Yanofsky, 1990; Chang and Staben, 1994). Transformation studies also revealed that MAT a-1 and MAT A-1 of N. crassa and FMR1 and FPR1 of P. anserina are responsible for mating specificity, as are the related proteins MAT-1 and MAT-2 in C. heterostrophus. Moreover, all these proteins are essential for postfertilization events (Glass et al., 1990; Debuchy and Coppin, 1992; Coppin et al., 1993; Turgeon et al., 1993a; Chang and Staben, 1994; Saupe et al., 1996; Ferreira et al., 1998; Wirsel et al., 1998). A unique feature of the MAT a-1 and MAT A-1 proteins of N. crassa is their additional role in vegetative incompatibility which manifests itself in the death of heterokaryotic cells generated by vegetative fusions of A and a hyphae (Beadle and Coonradt, 1944; Griffiths and Delange, 1978; Griffiths, 1982; Glass et al., 1990a; Glass and Kuldau, 1992; Chang and Staben, 1994; Saupe et al., 1996; Ferrera et al., 1998). The C-terminal region of the MAT a-1 protein controls this vegetative incompatibility and is dispensable for mating and postfertilization events (Staben and Yanofsky, 1990; Philley

194

SIMON J. HISCOCK AND URSULA KU¨ES

and Staben, 1994). In contrast, overlapping domains of the MAT A-1 protein direct vegetative incompatibility and mating (Glass et al., 1990a; Saupe et al., 1996). Although the presence of mat A-2 and mat A-3 in N. crassa increases the efficiency of mating, neither is essential for mating but both are necessary for postfertilization events (Ferreira et al., 1998). Likewise, the additional genes SMR1 and SMR2 at the mat⫺ idiomorph of P. anserina are only required after fertilization when, together with FMR1, they promote fruit body (perithecia) maturation and ascospore formation (Debuchy and Coppin, 1992; Debuchy et al., 1993). Following cell–cell (spermatium–trichogyne) mating interactions, fruit body maturation requires nucleus–nucleus interactions to direct the precise migration of nuclei into a specialized dikaryotic cell (the crozier cell) where assorted fusion takes place, followed by meiosis and the formation of ascospores (Raju, 1992; Nelson, 1996; Coppin et al., 1997). Under normal conditions mating types segregate in a 1 : 1 ratio at meiosis, except in rare cases in which mating-type switching occurs (as discussed previously). At the time of crozier formation it is essential that the two types of nuclei present are of different mating type, which raises the fascinating question of how nuclear recognition is achieved. Gene replacement studies of FMR1 and SMR2 in the resident mat⫺ idiomorph lead (in addition to reduction of ascus production) to uniparental progeny, indicating that FMR1 and SMR2 are involved in nuclear recognition. An SMR2 gene present in a mat⫹ nucleus cannot complement a defective SMR2 mat⫺ nucleus; in contrast, partial internuclear complementation was achieved between a mat⫺ nucleus with a disrupted FMR1 gene and a mat⫹ nucleus containing an intact FMR1 copy (Arnaise et al., 1997). Because FMR1 and SMR2 were shown to interact in the yeast two-hybrid system (Coppin et al., 1997), the current model for nuclear recognition postulates SMR2 to act as a carrier protein to return FMR1 to the mat⫺ nucleus (Arnaise et al., 1997). FPR1, on the other hand forms homodimers and returns specifically to the mat⫹ nucleus (R. Debuchy, personal communication). Gene disruption of SMR1 leads to complete sterility in crosses with mat⫹ strains, barren perithecia being formed which lack the ascogenous hyphae from which the dikaryotic croziers develop. SMR1 is therefore proposed to function in development of the ascogenous hyphae and not in establishing nuclear identity (Arnaise et al., 1997), even though an earlier study in which ectopic insertion of mutated SMR1 genes in ⌬mat nuclei led to uniparental progenies in crosses with mat⫹ wild-type strains suggested otherwise (Zickler et al., 1995). Interestingly, in crosses with such backgrounds the biparental progeny can be restored when a C-terminal domain of the mat⫹ protein FPR1 is deleted (Zickler et al., 1995). This domain was previously shown to be essential for postfertilization events but not for mating (Debuchy and Coppin, 1992).

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

195

In contrast to certain crosses involving strains of P. anserina carrying mutant copies of either SMR1 or SMR2 (Zickler et al., 1995; Arnaise et al., 1997), uniparental progenies were not observed in matings involving strains of N. crassa carrying mutations in the analogous genes mat A-1 or mat A-3 genes (Glass and Lee, 1992; Ferreira et al., 1998), indicating no apparent role for these genes in the control of nuclear identity. However, MAT A-1, MAT A-2, and MAT A-3 together were found to control the expression of at least some genes specifically expressed at the sexual development stage. This observation led Ferreira et al. (1998) to propose that the three proteins may interact and that such an interaction may be important in maintaining nuclear identify before karyogamy. If such protein–protein interactions are essential for nuclear recognition, the question remains as to how nuclear recognition is achieved in species such as C. heterostrophus and other ascomycetes which lack MAT A-2/SMR1 and MAT A-3/SMR2like proteins (Wirsel et al., 1998). Moreover, genetic data from sporecolor mutants in homothallic species (such as Sordaria macrospora), which contain both mating-type idiomorphs linked together within the same nucleus, indicate that nuclei are still differentially recognized, probably due to expression of either of the two mating-type idiomorphs, perhaps as a consequence of imprinting (Esser and Straub, 1958; Heslot, 1958; Glass et al., 1990b; Coppin et al., 1997). To add to the contradictions, sequencing of the S. macrospora mating-type locus revealed that the mat A-3/SMR2 analog is present only in an incomplete form (Po¨ggeler et al., 1997). It is thus possible that within filamentous ascomycetes there are variations in the molecular mechanisms through which nuclear recognition takes place. Evidence for such differences is derived from cytological and biochemical observations of heterothallic and homothallic species which implicate the cytoskeleton, spindle-pole bodies, and microtubules in the control of nuclear recognition of mating-type and nuclear migration (Thompson-Coffe and Zickler, 1994). Neurospora crassa and P. anserina mutants with random spindle body positioning show deviation from the normal dikaryotic pattern and crozier cells become multinucleate (Raju, 1992; Berteaux-Lecellier et al., 1998). 5. Mating-Type Determination in Basidiomycetes The occurrence and role of pheromones in courtship and mating in basidiomycetes has long been a matter of controversy. Observations of selective attraction between cells of opposite mating type in the Sporidiales (Abe et al., 1975), Ustilaginales (Dickinson, 1927; Bauch, 1932; Thren, 1940; Day, 1976; Snetselaar et al., 1996), and Tremellales (Bandoni, 1965; Flegel, 1981) indicated that pheromones must be involved in mating. Pheromones have been isolated from various hemibasidiomycetous yeasts, such as Rho-

196

SIMON J. HISCOCK AND URSULA KU¨ES

dosporidium toruloides, Tremella spp., and Ustilago maydis, and shown to induce conjugation tube formation that precedes cell fusion (Kamiya et al., 1978, 1979; Sakagami et al., 1979, 1981a,b Fujino et al., 1980; Flegel, 1981; Ishibashi et al., 1983, 1984; Wong and Wells, 1985; Martinez-Espinosa et al., 1993; Spellig et al., 1994). In accordance with production of distinct pheromones, cells of bipolar R. toruloides and Ustilago sp. will only fuse if they are of different mating type, and similarly in the tetrapolar U. maydis and Tremella sp. cell fusion will only occur if cells differ at their a and A mating-type loci, respectively (Bandoni, 1963; Puhalla, 1969; Day, 1972; Abe et al., 1975; Truehart and Herskowitz, 1992; Martinez-Espinosa et al., 1993; Snetselaar, 1993; Fig. 6). In contrast to the findings for hemibasidiomycetes, attempts to purify pheromones from homobasidiomycetes have proved unsuccessful. In these filamentous fungi all mycelia can fuse regardless of mating type (Sicari and Ellingboe, 1967; Leary and Ellingboe, 1970), so it is usually assumed that there is no extracellular pheromone reaction before cellular fusion (Kothe, 1996; Vaillancourt and Raper, 1996; Casselton and Olesnicky, 1998). Nevertheless, there are several observations that could relate to pheromone reactions within homobasidiomycetes, although from these observations it is not clear which particular mating-type locus (A or B ) is involved. For instance, in Schizophyllum commune there is mutual attraction between hyphae, and hyphal fusion between different mating types is stimulated by a secreted substance; furthermore, high fusion frequencies have been correlated with heterozygosity at the A mating-type locus (Ahmad and Miles, 1970). In contrast to these findings, in Lenzites betulinus sexual barriers are formed between mycelia having different A but the same B mating types, and this phenomenon is correlated with the presence of a secreted substance (pheromone) (Hennebert et al., 1994). Similarly, observations for Coprinus cinereus reveal that reduction of hyphal fusion is associated with homozygosity at the B mating-type locus (Smythe, 1973). Also in Coprinus species, uninucleate haploid mitotic spores (oidia) are known to attract hyphae; however, this ‘‘homing’’ reaction can occur between hyphae and oidia of the same mating type and even sometimes between oidia and hyphae from different species (Kemp, 1975, 1977). Comparable observations have also been made for sexual basidiospores and hyphae in Leccinum (Fries, 1981). A possible reason for this apparent variability in hyphal reactions and fusions in homobasidiomycetes may be because hyphae fuse for different reasons. For instance, vegetative fusions occur independently of mating type to create a hyphal complex that eases nutritional translocation within mycelium (Rayner, 1991; Rayner et al., 1994; Olsson and Gray, 1998), whereas fusions between hyphae of different mating types not only facilitate nutritional translocation but also lead to sexual development. Thus, the effects of mating-type-encoded pheromones

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

197

FIG. 6 Cell–cell communication in Ustilago maydis. a1 and a2 cells produce and secrete mating-type-specific pheromones that bind to their specific seven-transmembrane receptors which are localized in the cell walls of the opposite mating type. Pheromone binding activates an incompletely understood signaling cascade (involving elements of a MAP kinase pathway and elements linked to nitrogen and cAMP metabolism) that ultimately acts via the transcription factor Prf1 to regulate expression of the two classes of homeodomain transcription factors encoded in the b mating-type locus. Following fusion, bE (HD1) and bW (HD2) proteins from different b altermorophs recognize each other and dimerize to regulate gene expression in the dikaryotic cells (for further information see text).

on these two types of hyphal fusion will need careful evaluation once the respective pheromones have been isolated. There is little doubt, however, that pheromones do play a role in the fusion of clamp connections with hyphal cells in the growing dikaryon

198

SIMON J. HISCOCK AND URSULA KU¨ES

(Kothe, 1996). Division of the two different nuclei (each derived from a separate mating partner) in the apical hyphal cells is synchronized and formation of clamp cells and their fusion with the subapical cells ensures that every hyphal cell of the dikaryon contains two nuclei of different mating type (Tanabe and Kamada, 1994; Iwasa et al., 1998). Classical genetic studies in C. cinereus and S. commune demonstrated that the A locus controls the synchronized nuclear division and formation of clamp cells and the B locus controls septal resolution and reciprocal migration of nuclei into homokaryotic hyphae of compatible mating types together with clamp cell fusion in the established dikaryon (Papazian, 1951; Swiezynski and Day, 1960a,b; Giesy and Day, 1965; Casselton, 1978; Raper, 1966; Raper, 1983; Raudaskoski, 1998). Given that hyphal fusion between monokaryons is not restricted by mating type, recognition of nuclei derived from different mating partners must take place intracellularly. Thus, mating types must somehow mark the identity of the respective nuclei and these nuclei must be able to communicate with each other. Recent studies in S. commune indicate that nuclear distances within dikaryotic cells can differentially affect transcription of genes within the aerial mycelium: Dikaryon-specific genes are expressed when nuclei reside close to each other, whereas genes also expressed in the monokaryotic stage of the life cycle become expressed in the dikaryon when the nuclei move apart. Because the B mating-type locus is deduced to be resonsible for the expression pattern of the studied genes (SC3 and SC4, which encode hydrophobins) in dikaryotic cells, products of the B locus are believed to mark the identity of the nuclei and mediate nuclear ´ sgeirsdo´ttir et al., 1995; Schuurs et al., 1998). Work in communication (A other fungi indicates that the second mating-type locus might also be involved in nuclear communication and interaction. In Coprinus, for instance, A mating-type genes are responsible for the repression of uninucleate haploid oidia formation in dikaryons (Tymon et al., 1992; Ku¨es et al., 1994a). Illumination with blue light overrides this A-mediated repression and leads to a dedikaryotization via production of oidia in the aerial mycelium. Even though this illumination targets the A mating-type pathway, B mating-type products also play a role in modulating the negative effect of blue light on the A mating-type response (Polak et al., 1997; Kertesz-Chaloupkova´ et al., 1998; Ku¨es et al., 1998a,b). As a consequence, the dikaryotic state is partially but not totally lost in constant light. Interestingly, one of the two possible types of haploid oidia is always preferentially recovered from illuminated dikaryotic mycelium (M. Hollenstein, U. Ku¨es, and M. Aebi, unpublished observations). The contribution of the mating-type genes to this effect is not known. However, in S. commune recovery of only one nuclear type in mycelium following protoplasting of dikaryons was attributed to differential

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

199

marking of the two nuclear types by the B locus as a consequence of the linear dominance between B haplotypes (Raper, 1985). The conflicting interpretations of the involvement of particular matingtype loci in different aspects of cellular behavior in homobasidiomycetes may indicate that there is in fact cross talk between the two mating-type loci. Such speculation has been confirmed in the hemibasidiomycete U. maydis, in which the two mating-type loci mutually regulate their expression, indicating a close association between the functions of both matingtype loci. Communication between the two loci involves Prf1 (Hartmann et al., 1996; Urban et al., 1996b; Fig. 6), a homolog of the HMG box transcription factor Ste11 which mediates nutritional control of matingtype gene expression in S. pombe (Yamamoto et al., 1997; Banuett, 1998; see Section II,B,4,b). Interestingly, nutritional control systems operating via nitrogen starvation and cAMP depletion also appear to be present within the basidiomycetous yeasts Cryptococcus neoformans and Ustilago spp. (Tolkacheva et al., 1994; Ruiz-Herrera et al., 1995; Wickes et al., 1996; Alspaugh et al., 1997; Dong and Courchesne, 1998; Banuett and Herskowitz, 1994a; Gold et al., 1994, 1997; Lichter and Mills, 1997; Kronstad, 1997; Kahmann and Basse, 1998; Kronstad et al., 1998; Kru¨ger et al., 1998; Mayorga and Gold, 1998). In Ustilago a cAMP-based signal transduction system has been postulated to influence mating-type gene expression via Prf1 (Du¨rrenberger et al., 1998; Fig. 6). In dikaryons of C. cinereus cAMP production is positively controlled by light (Swamy et al., 1985a,b), and it may be that this increase in cAMP is responsible for overriding the Amediated repression of oidiation in blue light (Kertesz-Chaloupkova´ et al., 1998). As a first stage toward addressing this question, cac1 (the gene for adenylate cyclase) has recently been cloned from C. cinereus (Bottoli et al., 1998). Mating-type gene functions appear to be very conserved among the basidiomycetes, with gene products being either homeodomain transcription factors or pheromones and pheromone receptors (Table I). The best analyzed mating-type loci are those of the tetrapolar smut fungus U. maydis, the tetrapolar split gill fungus S. commune, and the tetrapolar mushroom C. cinereus. Ustilago maydis has a diallelic a locus and a multiallelic b mating-type locus with at least 25 but possibly 33 different specificities (Rowell and DeVay, 1954; Puhalla, 1970; DeVay cited in Wong and Wells, 1985; Zambino et al., 1997). In C. cinereus it is estimated that in natural populations there may be 160 different A and 80 different B specificities; the corresponding estimates for S. commune are 288 different A and 81 B specificities (Raper, 1966; Koltin et al., 1972). Due to an unfortunate historical nomenclature, the a locus of Ustilago is homologous to the multiallelic B loci of Schizophyllum and Coprinus, whereas the b locus of Ustilago is homologous to their multiallelic A loci (Table I). In order to clarify this

200

SIMON J. HISCOCK AND URSULA KU¨ES

confusing genetic nomenclature, we propose and use in this review the unifying terms matT for loci encoding the homeodomain transcription factors and matP for loci encoding pheromones and pheromone receptors. In the bipolar smut fungus U. hordei, which is closely related to the tetrapolar U. maydis, matP and matT reside within a single 400-kb chromosomal segment that is under recombinational suppression such that it behaves as a single locus; hence its classification as bipolar, referring to control of mating type by a single locus (Bakkeren and Kronstad, 1993, 1994; Kronstad and Staben, 1997; J. W. Kronstad, personal communication). A similar situation might be expected in other bipolar basidiomycetes such as R. toruloides and C. neoformans, in which mating-type-specific pheromone genes have been found in large regions of sequence dissimilarity (Akada et al., 1989; Moore and Edman, 1993). Although generally quite different in sequence [in S. commune identities between A genes are approximately 50% (Stankis et al., 1992) and those between B genes are between 15 and 90% (Vaillancourt et al., 1997)], genes of different mating types within and between species of basidiomycetes appear to have a common origin (Ku¨es and Casselton, 1992b; Kahmann and Bo¨lker, 1996; Urban et al., 1996a; Vaillancourt and Raper, 1996). Mating-type genes in basidiomycetes are therefore not truly idiomorphic (as in the ascomycetes), unless there are extra unique genes present at the locus, such as the genes lga2 and rga2 in the a2 haplotype of U. maydis which encode putative mitochondrial proteins (Urban et al., 1996a). To emphasize the common origin of highly variable but nevertheless related genes, the term ‘‘altermorph’’ has been proposed to describe the different specificity forms of mating-type genes within the basidiomycetes (Vaillancourt and Raper, 1996). Such a distinction is clearly valid because three different alleles from the A mating-type gene d1-1 of Coprinus have the same specificity (Ku¨es et al., 1994a,b,c; Ku¨es, 1995; U. Ku¨es unpublished data). a. The matT Locus Encodes Two Classes of Homeodomain Transcription Factors The b locus of U. maydis comprises the simplest form of a matT locus within the basidiomycetes, encoding two divergently transcribed genes designated bE and bW which possess hypervariable 5⬘ ends and conserved 3⬘ ends (Schulz et al., 1990; Kronstad and Leong, 1990; Gillissen et al., 1992; Fig. 7A). In C. cinereus and S. commune the situation is more complex, with several genes being present within a given haplotype and each locus consisting of two subloci (designated 움 and 웁) that show redundancy in function (Papazian, 1951; Day, 1960; Raper et al., 1960; Lukens et al., 1996; Fig. 7A). The 움 loci of C. cinereus and S. commune share a similar organization to the b locus of Ustilago, with a single pair of divergently transcribed genes (a1 and a2 in Coprinus and Z and Y in S. commune). Hybridization data for C. cinereus and sequence data for S. commune

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

201

indicate that the allelic forms of the A genes of different specificity (altermorphs) are very dissimilar in sequence, with only short stretches of homology inbetween (Ku¨es et al., 1992; Stankis et al., 1992; Gieser and May, 1994; Specht et al., 1994; May and Matzke, 1995; Pardo et al., 1996). The 웁 locus of S. commune has only been partially characterized, with the identification of just a single gene (V ) (Shen et al., 1996); however, the 웁 locus of Coprinus is well characterized, containing two functionally independent, paralogous units of divergently transcribed genes [the b gene pair (b1 and b2) and the d gene pair (d1 and d2)] with an overall structure that is similar to that of the a gene pair in the 움 locus (Ku¨es and Casselton, 1993b; Pardo et al., 1996; Fig. 7A). Homologies between genes from allelic gene pairs are between 60 and 70%, whereas homologies between genes from paralogous pairs are lower (45–50%; Gieser and May, 1994; Ku¨es et al., 1994a). Analysis of several different A haplotypes revealed that the number of altermorphs of the different gene pairs is unequal. To date, four variants of the a gene pair have been identified together with nine of the b gene pair and two of the d gene pair (Pardo et al., 1996). Shuffling of these small numbers of different forms of the three functionally independent gene pairs generates a vast number of different A specificities (Day, 1960; Ku¨es and Casselton, 1993b; May and Matzke, 1995; Lukens et al., 1996; Casselton and Olesnicky, 1998). The most obvious motifs in the proteins encoded in a matT gene pair are the homeodomains—tripartite DNA-binding motifs. These homeodomains are highly conserved within the basidiomycetes and are related to the homeodomains found within the mating-type proteins of S. cerevisiae. Two classes of homeodomain have been identified, HD1 and HD2, which share homologies respectively with the homeodomains of the 움2 and a1 matingtype proteins of S. cerevisiae (Ku¨es and Casselton, 1992b; Casselton and Ku¨es, 1994; Table I, Fig. 7B). While the HD2 motif is considered a classical homeodomain, consisting of 60 aa and the conserved W.F.N.R. sequence within the third DNA recognition helix, the HD1 motif is atypical because it has extra amino acids between the three helices and a less conserved sequence within the recognition helix (Bu¨rglin, 1994, 1997; Bharathan et al., 1997). Recent analysis of the three extra amino acids within the first and second helix of U. maydis proteins suggests that these extra amino acids have a function in cooperative DNA binding of heterodimeric complexes of the homeodomain proteins (Peltenburg and Murre, 1997). The extra amino acids between the second and third helix are only present in some of the proteins in variable numbers (Bu¨rglin, 1994), suggesting that these amino acids are probably not important for function and may be looped out in the folded domain, as found with similar atypical homeodomains from animals (Leiting et al., 1993). Protein–protein interaction between the homeodomain proteins deter-

202

SIMON J. HISCOCK AND URSULA KU¨ES

FIG. 7 The matT loci of basidiomycetes. (A) Structure of matT loci in basidiomycetes. The b locus of Ustilago maydis encodes a pair of divergently transcribed genes, bE (an HD1-type gene) and bW (an HD2-type gene). The 5⬘ ends and the promotor regions between altermorphs are very dissimilar (striped boxes), whereas approximately two-thirds of the genes are conserved at their 3⬘ ends (indicated by the gray and black boxes). Gray boxes mark the regions encoding the homeodomains. Interaction occurs between products of HD1 and HD2 genes from different (altermorphic) gene pairs (arrows; Gillissen et al., 1992). The A locus of C. cinereus contains three pairs of functionally redundant (paralogous), divergently transcribed HD1 and HD2 genes [as indicated by the archetypal A locus at the top according to Ku¨es and Casselton (1993b) and Pardo et al. (1996)]. Interactions are between products of HD1 and HD2 genes from altermorphic gene pairs (see arrows indicating all possible gene interactions between the virtual A43 and the A42 haplotype and the A42 and the A6 haplotype). Genes from altermorphic gene pairs are generally very dissimilar (open and striped boxes), with short stretches of homology between them (e.g., in the regions encoding the two different types of homeodomains; see gray boxes). Because of the high degree of redundancy in the system, some genes can be lost, as indicated for the A6 and the A42 haplotypes that are missing a copy of an a1 gene and for A6, A42, and A43 that are all missing a copy of a d2 gene. In contrast, A haplotypes can also acquire additional genes, as seen in A6 and A42 with gene e2-1 and in A42 with gene c1-1 (Ku¨es et al., 1992, 1994b; Casselton and Ku¨es, 1994; Pardo et al., 1996). (B) Protein organization of HD1 and HD2 proteins of basidiomycetes

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

203

compared to the equivalent homeodomain proteins of S. cerevisiae. HD1 proteins: C. cinereus (C. c.) a1-x, b1-x, c1-x, d1-x; S. commune (Sc. c.) Zx; U. maydis (U. m.) bEx; S. cerevisiae (Sa. c.) 움2; HD2 proteins: C. cinereus a2-x, b2-x, d2-x; S. commune Xx, Vx; U. maydis bWx; S. cerevisiae a1; x refers to altermorph number (for references of sequences see Table I). N-terminal dimerization/specificity domains are indicated by narrow striped boxes, homeodomains by dark gray boxes, (putative) dimerization domains C terminal to the homeodomains by loosely striped boxes, domains with homology to the 20-amino acid tail of 움2 by a black line, C-terminal helical regions and dimerization domains by light gray boxes, and putative transactivation domains by striped boxes [for original data, see Ku¨es and Casselton (1992c), Ho et al. (1994), Gieser and May (1994), Ku¨es et al. (1994a), Johnson (1995), Ka¨mper et al. (1995), Asada et al. (1997), Fig. 7D, and the respective references from Table I]. The letter N denotes conserved nuclear localization signals within the HD1 proteins of C. cinereus (Spit et al., 1998). Note that the dimerization domain C terminal to the homeodomain in S. commune Z proteins will interact with both Z and Y proteins, and the 400-amino acid-long dimerization

204

SIMON J. HISCOCK AND URSULA KU¨ES

mines recognition of compatible mating partners: A single homeodomain protein of either class cannot induce sexual development; instead, both classes of homeodomain protein must be present to activate sexual development. HD1 and HD2 proteins encoded within the same mating-type locus are incompatible. Only HD1 and HD2 proteins derived from compatible gene pairs from different mating types will activate sexual development (Gillissen et al., 1992; Specht et al., 1992; Ku¨es et al., 1994c; Pardo et al., 1996; Robertson et al., 1996). Interestingly, artificial fusion of normally incompatible HD1 and HD2 protein partners can overcome the need to find a heterologous protein partner for interaction in vivo (Ku¨es et al., 1994b; Asante-Owusu et al., 1996; Romeis et al. 1997). N-terminal dimerization between compatible HD1 and HD2 proteins from different mating types has been demonstrated for U. maydis, C. cinereus, and S. commune in vitro and in the yeast two-hybrid system with C-terminal-truncated mating-type proteins. Truncated HD1 and HD2 mating-type proteins from the same or from paralogous gene pairs do not interact (Banham et al., 1995; Ka¨mper et al., 1995; Magae et al., 1995). The N-terminal dimerization domains of HD1 and HD2 mating-type proteins correspond well with the regions identified in domain swap and mutational analyses as encoding matingtype specificity (Dahl et al., 1991; Yee and Kronstad, 1993, 1998; Ku¨es et al., 1994a; Banham et al., 1995; Ka¨mper et al., 1995; Wu et al., 1996; Yue et al., 1997; Fig. 7B). Strikingly, these 120 to 150-aa hypervariable N-terminal specificity regions become superfluous in HD1–HD2 fusion proteins active in regulating A-mediated development (Asante-Owusu et al., 1996). The N-

domain at the C-terminal end will interact only with Z proteins (Asada et al., 1997). (C) Model illustrating N-terminal recognition and discrimination between compatible HD1 and HD2 proteins from altermorphic gene pairs and incompatible HD1 and HD2 proteins encoded within the same gene pair. Circles, half circles, and triangles indicate hydrophobic and polar amino acids at possible contact points. A strong protein interaction will be achieved when all contact points are compatible (1). Incompatible residues at the flanks of the dimerization domain will weaken an interaction but the attracting forces might still be strong enough for proteins to dimerize (2 and 3). Incompatible residues more central to the dimerization domains together with increasing numbers of incompatible residues will eventually counteract any protein dimerization (4 and 5). For further discussion, see Kuhn and Parag (1972) and Kahmann and Bo¨lker (1996). (D) Diagram showing the similarity between the C-terminal region of the homeodomain of HD1 proteins from basidiomycetes and the C-terminal tail of 움2 from S. cerevisiae. Sequences are from Astell et al. (1981), Schulz et al. (1990), Kronstad and Leong (1990), Gillissen et al. (1992), Bakkeren and Kronstad (1993), Gieser and May (1994), Ku¨es et al. (1994a,b), Stankis et al. (1992), and Magae et al. (1995). Note that the sequence with homology to 움2 is still present in a fusion product of an HD1 and an HD2 protein of C. cinereus that is functional in the activation of A-regulated pathways despite deletion of the HD1 homeodomain sequence in the fusion product (Ku¨es et al., 1994b). The arrow indicates the point of fusion of the HD1 protein to the HD2 protein.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

205

terminal specificity regions are characterized by short elements of putative coiled-coil protein-folding patterns (Ka¨mper et al., 1995) reminiscent of those in the N-terminal dimerization domains of the 움2 and a1 proteins of S. cerevisiae (Goutte and Johnson, 1993; Ho et al., 1994; Fig. 7B). Shortening or lengthening the putative coiled-coil folding structures by increasing or reducing hydrophobicity leads to changes in the strength of the dimerization affinity. Hydrophilic and polar amino acids within these elements are believed to be contact points in the HD1–HD2 protein interaction and to be the basis of allele-specific protein recognition (Ka¨mper et al., 1995; Fig. 7C). Since mating-type specificity is determined by a noninteraction of proteins encoded within the same haplotype, recognition of specificity should not be based on amino acids mediating protein contacts but on residues acting adversely on protein dimerization (Kuhn and Parag, 1972; Kahmann and Bo¨lker, 1996; Fig. 7C). Genetical evidence suggests that there are several contact points within the N-terminal domains of the HD1 and HD2 partners and that it is the sum of the positive versus the negative amino acid interactions that will determine whether a protein interaction is compatible or incompatible (Ka¨mper et al., 1995; Yue et al., 1997; Yee and Kronstad, 1998; Fig. 7C). In U. maydis, dimerization between proteins can be abolished by deletions of 2 or 3 aa, probably as a consequence of displacement of the dimerization interface (Ka¨mper et al., 1995). Using different spacing between points of protein communication would be an easy way to exclude interactions of HD1 and HD2 products of paralogous gene pairs localized in the 움 and 웁 subloci of C. cinereus and S. commune (Banham et al., 1995; Pardo et al., 1996). Asada et al. (1997), using in vitro protein affinity assays, detected additional mating-type-independent dimerization interfaces C terminal to the homeodomains in proteins from S. commune that escaped from the yeast two-hybrid system (Magae et al., 1995). HD1 and HD2 full-length proteins encoded within the same locus or in allelic loci and proteins of the same kind (HD1 or HD2) were shown to interact via distinct domains (Asada et al., 1997; Fig. 7B). The biological role of any possible HD1–HD2 and HD2–HD2 interactions is unclear. For homologous and heterologous HD1–HD2 interactions, one can only assume that the strength of the interaction will determine whether a protein combination is active in vivo in regulating downstream functions. From computer analysis of C. cinereus proteins, a second dimerization domain was postulated to reside C terminal to the homeodomain, within a similar region of the HD1 proteins of S. commune (Ku¨es and Casselton, 1993c; Casselton and Ku¨es, 1994; Gieser and May, 1994). Interestingly, within these domains in both C. cinereus and S. commune sequences were found with similarity to the 20-aa tail of the 움2 protein of S. cerevisiae (Ku¨es et al., 1994a; Fig. 7D) which is known to interact with the homeodomain of a1 and to be essential for coordinate

206

SIMON J. HISCOCK AND URSULA KU¨ES

DNA binding (Mak and Johnson, 1993; Phillips et al., 1994; T. Li et al., 1995; see Section II,B,3,a). The Ustilago proteins, in contrast, have less homology to the 20-aa tail of 움2 in this C-terminal region but there is some similarity to the sequences from C. cinereus and S. commune (Fig. 7D). In yeast, the C-terminal dimerization domain is considered to be more important than the N-terminal domain since its fusion to a1 is enough to exert DNA binding (Stark and Johnson, 1994; Johnson, 1995; see Section II,B,3,a). Moreover, the recognition helix of the 움2 homeodomain can be mutated without the a1–움2 heterodimer losing the ability to bind to its specific DNA targets (Vershon et al., 1995; Jin et al., 1999). Similarly, the HD2 but not the HD1 motif was shown to be essential for function in C. cinereus and S. commune (Luo et al., 1994; Ku¨es et al., 1994b; Asante-Owusu et al., 1996; Wu et al., 1996). In Ustilago, however, both homeodomains are essential for function in vivo and therefore are likely to be required for DNA binding (Romeis et al., 1997; Schlesinger et al., 1997). Thus, because in Ustilago there is such low homology between the C-terminal regions of the homeodomain proteins and the 20-aa 움2 tail, and because the HD1 homeodomain is also essential for function, one might speculate that a secondary C-terminal dimerization interface has lost its function in Ustilago. The similarities between the S. cerevisiae proteins 움2 and a1 and the HD1 and HD2 proteins are striking, but the basidiomycete proteins are much longer (Fig. 7B) and may therefore harbor many more domains than their yeast counterparts. Parts, but not all, of these regions can be truncated (Specht et al., 1992; Tymon et al., 1992; Ku¨es et al., 1994a; Shen et al., 1996), and heterologous expression of C. cinereus proteins in yeast suggests that there might be transactivation domains in these extra protein regions (Banham et al., 1995). In accordance with their nuclear function as transcription factors, an essential nuclear localization signal was defined in HD1 proteins C terminal to the homeodomain. Since HD2 proteins were found to be free of any nuclear localization signals, it was concluded that it is the HD1–HD2 heterodimer that enters the nucleus (Spit et al., 1998). A similar model has been proposed for control of dikaryotic development and pathogenesis by the b proteins of Ustilago (Kronstad and Leong, 1990). Downstream genes targeted by the HD1–HD2 heterodimer have not been identified in basidiomycetes. Circumstantial evidence in Ustilago suggests that b proteins might act in some situations as inducers and in other situations as repressors of gene expression (Urban et al., 1996b). A nonessential MADS box homolog to MCM1 of S. cerevisiae isolated from Ustilago (Ucm1p) is proposed to be required for repression but not for activation by the heterologous b protein complexes (Kru¨ger et al., 1997). Robertson et al. (1996) argue that the S. commune genes will be positive transcriptional activators of development because deletion of mating-type genes does not induce any developmental phenomena. Analysis in Coprinus detected de-

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

207

velopmental functions (oidia formation) that are repressed by heterologous A products and others (clamp cell formation, chlamydospore formation, sclerotia formation, and fruit body initiation) that are induced by heterologous A products (Tymon et al., 1992; Ku¨es et al., 1994a, 1998a,b; KerteszChaloupkova´ et al., 1998; R. P. Boulianne et al., manuscript in preparation). Because it is not clear in any of the cases if the effects are direct or indirect, and as long as the specific gene targets are unknown, it will remain unclear whether heterologous matT proteins act as activators, repressors, or both. b. The matP Locus Encodes Pheromones and Pheromone Receptors As with the matT locus, the organization of the matP locus of U. maydis is the simplest among basidiomycetes (Fig. 8A). The a1 haplotype contains a gene for a pheromone precurser (mfa1) and a pheromone receptor ( pra1), whereas the a2 haplotype encodes one pheromone precursor (mfa2), one pheromone receptor ( pra2), and the two proteins associated with mitochondrial function (lga and rga). Curiously, the al haplotype also contains an inactive copy of mfa2 (Bo¨lker et al., 1992; Urban et al., 1996a; Fig. 8A). Thus, in contrast to the situation for the ascomycete yeasts, active pheromone and pheromone receptor genes are arranged within a single mating-type locus but in a nonactive combination. Pheromones of one cell type will only interact with pheromone receptors of the opposite cell type. Such an interaction initiates the mating reaction which converts yeast-like growth into the filamentous growth (Spellig et al., 1994; Snetselaar et al., 1996). The prepheromones of Ustilago Mfa1 and Mfa2 are 40 and 38 aa in length, respectively (Bo¨lker et al. 1992), whereas the mature forms of the pheromones are reduced to 13- and 9-aa residues respectively by N-terminal cleavage (Spellig et al., 1994). Like the a-factor of S. cerevisiae, both Ustilago pheromones are lipopeptides and are modified at their C-terminal CAAX motif by farnesylation (removal of the three terminal amino acids and the addition of carboxymethyl esters). These modifications increase the activity of the pheromones by a factor of 1000 (Spellig et al., 1994). Although the functional significance of these posttranslational modifications is not clear, the increase in hydrophobicity probably acts to target the pheromone to the plasma membrane (Caldwell et al., 1995). This assumption is supported by analysis of pheromone analogs with differing lipophyly in Ustilago (Koppitz et al., 1996). Functional studies with knockout strains imply that pheromones recognize and bind to their respective receptors in the opposite mating-type (Bo¨lker et al., 1992; Spellig et al., 1994). Like the receptors of S. cerevisiae and S. pombe, the pheromone receptors of U. maydis belong to the seven-transmembrane class of receptors and there is some sequence conservation between receptors from the ascomycetes and basidiomycetes. Pra1 (357 aa) and Pra2 (346 aa) are 24% identical to each other and resemble the STE3 receptor of S. cerevisiae (앑20% identity) and the M-factor recep-

208

SIMON J. HISCOCK AND URSULA KU¨ES

FIG. 8 The matP loci of basidiomycetes. (A) Organization of matP loci in basidiomycetes. Arrows indicate the direction of gene transcription. Ustilago maydis has only two forms of the a locus; a1 that contains a single pheromone gene (mfa1) and a single pheromone receptor gene ( pra1) and a2 that contains a nonfunctional copy of the mfa1 gene (mfa), a functional pheromone gene (mfa2), a pheromone receptor gene ( pra2), and two genes (lga2 and rga2) encoding putative mitochondrial proteins (Bo¨lker et al., 1992; Urban et al., 1996a). The B locus of S. commune is divided into two functionally redundant subloci (B움 and B웁) each containing one pheromone receptor gene (for the B1 haplotype: bar1 and bbr1) and three pheromone genes [for the B1 haplotype: bap1(1), bap1(2), and bbp1(3); bbp1(1), bpp1(2), and bpp1(3)] (Wendland et al., 1995; Vaillancourt et al., 1997). The B locus of C. cinereus is tripartite, but it is inherited as a single unit. Each of the three functionally redundant subunits consists of one pheromone receptor gene (for the B6 haplotype: rcb1.1, rcb2.1 and rcb3.1), and two pheromone genes (for the B6 haplotype: phb1-1.1 and phb1-1.2; phb2-1.1 and phb22.1; phb3-1.1 and phb3-2.1), (O’Shea et al., 1998). (B) Interactions between pheromones and pheromone receptors from altermorphic units as illustrated by the B6 and B42 haplotypes of C. cinereus. Different patterns and different shades of gray indicate whether the same or different genes are present within the two haplotypes. Pheromone–pheromone receptor interactions only occur between products from altermorphic subunits (dashed arrows) [gene organization after Casselton and Olesnicky (1998) and O’Shea et al. (1998)].

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

209

FIG. 8 (continued)

tor, Map3, of S. pombe (앑18% identity) (Bo¨lker et al., 1992; Tanaka et al., 1993). The pheromone receptors of Ustilago have yet to be subjected to a functional analysis, but some of the downstream elements of the signaling cascade, induced by pheromone binding, are known (Fig. 6). Signaling from activated pheromone receptors is mediated via a heterotrimeric G protein with Gpa3 as a mating-type-specific 움 subunit that interacts with the pheromone receptor (Regenfelder et al., 1997). The isolation of the fuz7 gene encoding a MEK/MAP KK (STE7 homolog) suggests that signaling follows a MAP kinase pathway (Banuett and Herskowitz, 1994a; Banuett, 1995). However, it appears that Gpa3 and Fuz7 are not part of the same pheromone response pathway, suggesting that the signaling response may be more complex than previously thought (Regenfelder et al., 1997; Kronstad and Staben, 1997). Pheromone-stimulated signaling leads to induction of genes from both mating-type loci via binding of the HMG domain transcription factor Prf1 to specific operator sites (pheromone response elements; consensus; 5⬘-ACAAAGGGA-3⬘) within the mating-type loci. Expression

210

SIMON J. HISCOCK AND URSULA KU¨ES

of mfa and pra genes is downregulated after cell fusion, whereas genes within the b (matT ) locus are upregulated (Hartmann et al., 1996; Urban et al., 1996b). Because a genes are essential not only for mating but also for maintenance of filamentous growth in culture (but curiously not in planta) (Banuett and Herskowitz, 1989, 1994b, 1996), this lower level of expression must be sufficient to perpetuate an autocrine pheromone response within the dikaryotic phase (Urban et al., 1996b). In contrast to the situation in haploid cells in which there is no b gene expression, b genes remain highly expressed in dikaryotic cells after cell fusion, indicating that they are essential for mating, morphogenesis, and pathogenesis (Puhalla, 1970; Day et al., 1971; Banuett and Herskowitz, 1994b). It is probably the high level of heterologous b proteins that keeps the level of a products low, because when haploid cells were transformed with a heterologous b gene they were unable to mate, presumably because they were unable to express the pheromone and pheromone receptor (Laity et al., 1995; Urban et al., 1996b). The complexity of the matP locus is, like that of the matT locus, far greater in C. cinereus and S. commune than in U. maydis (Figs. 8A and 8B). Classical genetic studies suggested that the B locus of S. commune has a bipartite structure with multiallelic 움 and 웁 subloci (Parag and Koltin, 1971). Cloning and sequencing revealed that the two subloci consist of three or more genes encoding pheromones and one gene encoding a pheromone receptor belonging to the seven-transmembrane class (Wendland et al., 1995; Vaillancourt et al., 1997; Fowler et al., 1998; Fig. 8A). In C. cinereus, evidence for a local separation of the B locus into two subloci was not found in genetic crosses (Day, 1960). Molecular analysis has revealed that the B locus of C. cinereus consists of three functionally identical sets of two pheromone genes and one pheromone receptor gene within a single haplotype—an arrangement even more complex than that in S. commune (Casselton and Olesnicky, 1998; O’Shea et al., 1998; Figs. 8A and 8B). Interestingly (as indicated in Fig. 8A), the direction of transcription of pheromone genes varies relative to that of the receptor genes between paralogous and between alteromorphic gene pairs (Wendland et al., 1995; Vaillancourt et al., 1997; Casselton and Olesnicky, 1998; O’Shea et al., 1998). Sequence alignments in S. commune suggest that the complexity and variability of B haplotypes results from large duplications and inversions in the past (Vaillancourt et al., 1997). Unlike matT genes, which are expressed constitutively in homobasidiomycetes (but not in the hemibasidiomycte U. maydis) (Richardson et al., 1993; Yang et al., 1995; Urban et al., 1996b), expression of pheromone genes in S. commune is extremely low prior to cellular fusion, increases dramatically at the time of or shortly after cell fusion, and then decreases again over time (Vaillancourt et al., 1997). Whether pheromone receptor

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

211

genes follow the same pattern of expression is not clear because transcript levels are too low to detect by Northern analysis with total RNA. Another interesting observation is that the increase in expression of some of the pheromone genes was seen not only in self/nonself combinations but also in self/self combinations, but at a lower level (Vaillancourt et al., 1997). This unexpected finding may explain some of the various cytological observations described for self/self and self/nonself fusions. It is thus tempting to speculate on the possibility that matP gene products might play a role in hyphal fusion in the homobasidiomycetes, probably regardless of their mating type (Hiscock et al., 1996). Heterologous transformation experiments in U. maydis and U. hordei have shown that the a locus determines interspecifc compatibility between these species (Bakkeren and Kronstad, 1996). With the cloning of B genes from S. commune and C. cinereus, it will be possible to determine whether matP locus products are also involved in interspecific recognition in homobasidiomycetes. Interestingly, transformation experiments with HD2 genes in C. cinereus and C. bilanatus have shown that the matT locus is not responsible for restricting interspecific compatibility between these two closely related species because the HD2 genes from both species can elicit A-regulated development (clamp cell formation) in either genetic background (Challen et al., 1993; Ku¨es and Challen, 1995; U. Ku¨es and M. P. Challen, unpublished results). One might expect that in more distantly related species both matT and matP locus products will play a role in species recognition. Indeed, recent transformation studies indicate that single A genes from S. commune are not active in C. cinereus, whereas pairs of compatible A genes from S. commune have been shown to be partially active in C. cinereus (Ku¨es and Challen, 1994; U. Ku¨es, unpublished data). In S. commune, not all pheromone genes activate development in all possible backgrounds, i.e., pheromone genes differ quantitatively and qualitatively in the effect they have on development (Wendland et al., 1995; Vaillancourt et al., 1997). This appears to be different from C. cinereus, in which all six different pheromone genes from the B6 haplotype were found to activate development in a B3 host (O’Shea et al., 1998). The various sequences of the immature pheromones and their predicted mature pheromones of homobasidiomycetes resemble those of U. maydis, C. neoformans, and R. toruloides, the a-factor of S. cerevisae, and the M-factor of S. pombe, all of which have a CAAX motif at their C-terminal ends. It is therefore expected that the pheromone precursors of C. cinereus and S. commune will be processed and modified at their C-terminal ends in a similar way to the lipoprotein pheromones from ascomycetes and basidiomycetous yeasts (Caldwell et al., 1995; Wendland et al., 1995; Vaillancourt and Raper, 1996; Vaillancourt et al., 1997; Casselton and Olesnicky, 1998; O’Shea et al., 1998). The pheromone receptors in C. cinereus and S. commune are closely related

212

SIMON J. HISCOCK AND URSULA KU¨ES

to the G protein-coupled STE3 receptor of S. cerevisiae, the Map3 receptor from S. pombe, and the Pra1 and Pra2 receptors of U. maydis, consistent with the presence of CAAX-modified pheromones (Wendland et al., 1995; Vaillancourt et al., 1997; O’Shea et al., 1998). A preliminary functional analysis suggested that pheromone receptors are localized within the outer hyphal membrane (Kothe, 1996). Domain swaps between the Bar1 and Bar2 receptors from different B움 haplotypes of S. commune revealed that the first and third extracellular loops contribute to pheromone interaction and specificity. Changes to the first or the third part of the receptors resulted in self-activation of the B-dependent pathway, presumably through loss of specificity (Hegner et al., 1998; E. Kothe, personal communication). Several mutants have been isolated in the past that led to a similar constitutive activation of the B-dependent pathway in self-backgrounds (Raper, 1983); these mutants await characterization. Among such mutations, three phenotypes will be expected—two showing loss of specificity and one showing ‘‘true’’ constitutive activation. The first class will show loss of specificity as a consequence of amino acid changes within the pheromone, making it more likely to bind to its own receptor. The second class of mutant will show changes within the specificity determinants of the receptors, enabling them to bind pheromones not formerly accepted. The third class of mutations will render the receptor constitutively active without the need to interact with a pheromone, as for instance if the intracellular binding region was mutated such that it could bind the heterotrimeric G protein without activation. matP genes within the homobasidiomycetes also control reciprocal migration of nuclei from one homokaryon into another (Raudaskoski, 1998). Normally, during mating a mycelium acts as both a nuclei donor and a nuclei acceptor, but mutations can lead to loss of one or the other character ( J. Raper, 1966; C. Raper, 1983). Kothe (1996) suggested that mutations within pheromone genes will result in a mycelium that accepts nuclei unilaterally, whereas mutations in the pheromone receptor gene leading to activation of the B pathway will allow the mycelium to donate but not to receive nuclei. When strains of S. commune were transformed with heterologous pheromone genes they became unilateral nuclei donors, whereas transformants with heterologous pheromone receptor genes neither donated nor accepted nuclei (Wendland, 1995; Vaillancourt et al., 1997). In C. cinereus, however, neither transformants of pheromone genes nor transformants of pheromone receptor genes accepted nuclei, but both were able to donate nuclei (O’Shea et al., 1998). However, the results, as presented, were not conclusive because about half of all wild-type isolates of C. cinereus always fail to accept nuclei (May and Taylor, 1988). Components of the downstream signaling cascade(s) have yet to be identified within homobasidiomycetes. However, progress in this direction has

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

213

been made with the recent cloning of the gene pcc1 from C. cinereus which is regulated by both B and A genes. The sequence of the deduced Pcc1 protein contains an HMG box domain with high identity to that of FPR1 (44%) from P. anserina, Mat Mc (41%) from S. pombe, and Prf1 (29%) from U. maydis (Murata et al., 1998). It remains to be determined whether Pcc1 mediates cross talk between the A and B mating-type loci of C. cinereus or whether it merely functions in the later stages of sexual developmental regulation. Progress is also being made in identifying downstream-regulated gene products in the basidiomycete yeast pathogen of humans, C. neoformans, with the cloning of the genes GPA1, which encodes the G움 subunit that presumably interacts with pheromone receptors (Tolkacheva et al., 1994; Alspaugh et al., 1997), and STE12움, encoding a mating-type-specific homolog of the STE12 transcriptional regulator from S. cerevisiae (Wickes et al., 1997). STE12움 is found only within 움 cells and regulates expression of the mating-type-encoded MF움 pheromone (Wickes et al., 1997). A curious finding for C. neoformans is that 움 cells are more virulent than a cells and under appropriate conditions will become filamentous, develop basidia, and sporulate (Kwon-Chung et al., 1992; Wickes et al., 1996).

III. Sexual Incompatibility Systems in Nonflowering Plants A. Algae Breeding systems have been studied in detail in very few algae (Lewin, 1976; Brownlee, 1994; Goodenough et al., 1995), but in general two outbreeding strategies appear to predominate: In isogamous algae gametes are of two mating types, plus and minus, and therefore resemble ascomycetes, whereas in the more complex anisogamous and oogamous algae gametes are dimorphic and frequently differentiate into sperm and eggs which can be borne on the same plant, usually the gametophyte (monoecious), or on different plants (dioecious). Studies of sexual incompatibility in algae have focused on two main ‘‘model systems’’: Chlamydomonas (and some related Volvocales; Lewin, 1976) which typifies the isogamous plus and minus mating-type system, and Fucus which has a diplontic life cycle and well-developed oogamy. To our knowledge no self-incompatibility operates to prevent self-fertilization in monoecious Fucus individuals so we refer the reader to the key reviews on gamete recognition in these algae (Brownlee, 1994). Instead, we will concentrate on the mating-type system of Chlamydomonas which is common to many other green algae, both unicellular and multicellular, such as

214

SIMON J. HISCOCK AND URSULA KU¨ES

Volvox, Eudorina, Cosmarium, Closterium, and Ulva, and probably also occurs in isogamous brown and red algae (Lewin, 1976). Chlamydomonas reinhardtii is a unicellular biflagellate green alga with a haplontic life cycle—its haploid cells usually reproduce asexually by mitosis. However, in response to nitrogen starvation and a blue light signal (Beck and Haring, 1996) haploid cells differentiate into gametes of two mating types, plus and minus, and adhere to one another via their flagellae (Fig. 9). They then proceed to fuse to form diploid zygotes which mature into stress-resistant zygospores. Two independent recognition systems regulate this mating process. The first is mediated by the flagellar agglutinins, which are hydroxyproline-rich glycoproteins similar to higher plant extensins (Collin-Osdoby and Adair, 1985) which bind to each other in a complementary manner, plus to minus, to mediate flagellar agglutination. Agglutination induces a rise in intracellular cAMP (Quarmby, 1994) which activates

FIG. 9 Mating-type-specific behavior in Chlamydomonas reinhardtii. Under conditions of nitrogen starvation and in response to a blue light signal, cells of opposite mating-type (mt⫹ and mt⫺) ‘‘agglutinate’’ via mating-type-specific agglutinins on their flagellae. This is followed by the production of cellular outgrowths specific to mating type—the ⫹ and ⫺ mating structures. Cell fusion is mediated by another set of mating-type-specific agglutinins (the ⫹ and ⫺ fringe proteins) that are borne on their respective mating structures.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

215

the second recognition system, again mediated by complementary surface glycoproteins. These glycoproteins, known as ‘‘fringe’’ proteins, are unrelated to the flagellar agglutinins (Ferris et al., 1996). Plus and minus fringe proteins are associated with specific localized regions of the plasma membrane known as ‘‘mating structures’’ which form between the flagellae at the site of closest contact between the gametes in response to elevated cAMP levels (van den Ende, 1992; Quarmby, 1994). The cAMP stimulus induces the plus mating structure to extrude a fertilization tubule coated in plus fringe protein which, following contact with the minus mating structure, adheres to the minus fringe protein and thereby mediates cell fusion, which is followed eventually by nuclear fusion and spore formation (Forest, 1987; Goodenough et al., 1982; Goodenough, 1991; van den Ende, 1992; Brownlee, 1994). Under favorable conditions the zygospore undergoes meiosis and germination to produce four haploid cells, two plus and two minus (Goodenough, 1991). Mating type is controlled by a single locus, mt, which exists in two forms, mt⫹ and mt⫺. Both the mt⫹ and mt⫺ haplotypes have been cloned and shown to cover an extensive chromosomal region of about 1 Mb, which is under recombinational suppression (Ferris and Goodenough, 1994). The mt locus consists of three domains: the centromere-proximal (C) domain and the telomere-proximal (P) domain are homologous between haplotypes, whereas the central rearranged (R) domain of 앑200 kb is highly nonhomologous between haplotyptes, containing inversions, deletions, transpositions, insertions, and large stretches of DNA that are unique to each mt haplotype. It is within the nonhomologous R domain that the mating-type-specific genes reside (Ferris and Goodenough, 1994). Two mating-type-specific genes have been cloned: fus1 (Ferris et al., 1996) and mid (Ferris and Goodenough, 1997). fus1 is unique to the mt⫹ haplotype and encodes a novel single-pass membrane-spanning glycoprotein, the plus fringe protein, which is essential for fusion between mt⫹ and mt⫺ cells, presumably through its interaction with the corresponding minus fringe glycoprotein (Ferris et al., 1996). mid is a gene unique to the R domain of the mt⫺ locus and encodes a putative transcription factor with a leucine zipper motif. It is proposed that the mid transcription factor is responsible for switching on the minus differentiation program while switching off the plus program during gamete differentiation (Ferris and Goodenough, 1997). Recent work has shown that mid negatively regulates expression of the gsp1, a gene expressed specifically in mtt gametes. Interestingly, gsp1 contains an atypical homeodomain which shares significant identity with the 움2/HD1 fungal motifs (Kurvari et al., 1998). A number of other genes are known to be tightly linked to the mt locus, some associated with mating-type determination and recognition and some not (housekeeping genes). Of those genes involved in mating, sag1 and the

216

SIMON J. HISCOCK AND URSULA KU¨ES

unlinked sag2 are required for production of the plus agglutinin, whereas sad1 is required for minus agglutinin production, and gam1, which is expressed only in minus cells (like mid ), is involved in membrane fusion (Goodenough et al., 1995). Interestingly, as with certain fungi (Ro¨hr et al., 1998), genes present at the mt locus also control the inheritance of organelles in Chlamydomonas which, being isogamous, receives an equal number of mitochondria and chloroplasts from each gamete. Curiously, the meiotic products inherit their mitochondria from the minus parent and chloroplasts from the plus parent, with mitochondrial genomes from the plus parent and chloroplast genomes from the minus parent being selectively destroyed in the zygote (Goodenough et al., 1995; Ro¨hr et al., 1998). One current model to explain this phenomenon is that specific mt-encoded methylating enzymes protect the respective plus chloroplast genomes and minus mitochondrial genomes from destruction by specific nucleases, which may be mt-specific, in the zygote. An mt⫺specific gene ezy1, located in the C domain of both mt⫹ and mt⫺ haplotypes, has been proposed to encode a nuclease or nuclease activator responsible for degrading the minus chloroplast genome (Goodenough et al., 1995). The vast mt locus therefore appears to consist of an array of different genes with a diversity of function, including flagellar adhesion, cell fusion, organelle inheritance, sporulation, and metabolism, with the genes specific to mating type clustered in the genetically complex and idiomorphic R domain.

B. Bryophytes and Pteridophytes In recent years, increasingly more genetic data have become available on the breeding systems of bryophytes and pteridophytes, and it has become clear that the breeding systems of these nonseed plants are very similar to those of the seed plants, even though their predominantly homosporous life cycles with a free-living gametophyte are very different. An important finding of these studies is that outcrossing rates in bryophytes and pteridophytes are often much higher than was previously thought, apparently as a consequence of widespread dioecy and self-sterility (Cruden and Lloyd, 1995). Some evidence suggests that self-sterility arises from systems of self-incompatibility [Lazarenko and Lesnyak (1972) and Ashton and Cove (1976) as cited in Wyatt and Anderson (1984); McLetchie (1992) and Masuyama (1979, 1986) as cited in Cruden and Lloyd (1995)], whereas other evidence points to inbreeding depression resulting from the expression of sporophytically lethal alleles in zygotes as a consequence of self-fertilization (Soltis and Soltis, 1992; Husband and Schemske, 1996). In an extensive study of mating behavior in Pteridium aquilinum (Bracken) Wilkie (1956) proposed that outcrossing was promoted by a self-incompatibilty (SI) sys-

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

217

tem controlled by a single multialleleic locus. This finding, which represents the only genetic evidence for the existance of SI in ferns or bryophytes, was later confirmed by Lovis (1977), who did not find the phenomenon to be widespread in Pteridium; other studies totally disputed the evidence for an SI system and advocated inbreeding depression as the major cause of self-sterility (Klekowski, 1972; Verma and Cheema, 1987; Soltis and Soltis, 1992). It is interesting to speculate as to where an SI system (if indeed any exist) would operate in these homosporous plants. The obvious site of recognition (leading to rejection or acceptance) would be at the level of the oogamous gametes. It is also possible, however, that the cells of the archegonium, the female reproductive structure containing the egg, might be able to discriminate self-sperm from non-self-sperm in an analogous way to that in which the hollow transmitting tissue of certain angiosperm pistils discriminates (and rejects) self-pollen tubes. Clearly, these phenomona need more detailed investigation.

C. Gymnosperms Almost nothing is known about the genetic control of gymnosperm breeding systems. Dioecy ensures outcrossing in, for instance, cycads, ginkgo, and the gnetales, but most conifers are monoecious and therefore liable to selfpollination. Early studies of self-pollination in conifers revealed no apparent barriers to self-fertilization (Plym-Forshell, 1974; Mergen et al., 1965), so conifers are usually considered to be self-compatible (Hagman, 1975; Zavada and Taylor, 1986; Sage et al., 1994) even though lowered seed set generally results from self-pollinations (Owens et al., 1990, 1991). This reduced seed set after self-pollination has been attributed to postzygotic events, such as embryo abortion, as a consequence of inbreeding depression arising from the expression of recessive sporophytic lethal alleles (Koski, 1973; Bishir and Namkoong, 1987). Evidence for the existence of any prezygotic barriers to selfing in conifers was lacking until a recent detailed ultrastructural study of cross- and self-pollinations in Picea (white spruce) indicated that self-pollen tubes often ceased growing within the nucellus (nutritive sporophytic tissue surrounding the female gametophyte) of the ovule before reaching the archegonia (Runions and Owens, 1998). This observation suggests that self-incompatibility, in a form analogous to lateacting SI in angiosperms (see Section IV,A), may exist in conifers. If confirmed, this finding could have profound implications for the evolution of self-incompatibility systems, which are generally assumed to be a unique adaptive innovation of the angiosperms (Whitehouse, 1950; see Section IV,A). Interestingly, prezygotic incompatibility has been widely reported in studies of interspecific crosses in pines (Hagman, 1975) in which the

218

SIMON J. HISCOCK AND URSULA KU¨ES

pollen ‘‘inhibition reaction’’ is apparently also centered in the nucellus. Clearly, the reproductive biology of monoecious gymnosperms merits more extensive ultrastructural investigations akin to those of Runions and Owens (1998).

IV. Self-Incompatibility Systems in Flowering Plants A. The Distribution and Genetic Basis of Self-Incompatibility Self-incompatibility, defined as ‘‘the inability of a plant producing functional gametes to set seed upon selfing’’ (Brewbaker, 1957), was first described extensively by Darwin (1876), although its discovery can probably be attributed to Kolreuter as early as 1764 (Richards, 1997). SI is a prezygotic barrier to seed formation, with pollen development being inhibited usually on or within the pistil before the pollen tube reaches the ovule. In recent years, however, many so-called ‘‘late-acting’’ systems of SI have been reported which show ovarian or ovular inhibition of pollen tubes (Seavey and Bawa, 1986; Sage et al., 1994). Postzygotic incompatibility phenomena, such as zygote breakdown and embryo abortion, are usually considered to be (as in gymnosperms) consequences of inbreeding depression (Mulcahy and Mulcahy, 1983; Krebs and Hancock, 1991) rather than the function of a discreet genetic incompatibility system. There is little doubt that SI is the most widespread and most important outbreeding mechanism in flowering plants, and although estimates of its extent are difficult and prone to variability, SI is believed to be present in at least half of the 250,000 or so species of angiosperms (East, 1940; Fryxel, 1957; Darlington and Mather, 1949; Brewbaker, 1959; Richards, 1997; Fig. 10). SI involves cells of the diploid sporophyte pistil (carpel) recognizing and actively rejecting genetically related haploid male gametophytes (pollen grains) that are derived from itself or from closely related sporophytes. Only pollen grains that display species-specific recognition factors and which are not recognized as ‘‘self’’ (or closely related) during this pollen–pistil interaction will undergo a ‘‘compatible’’ developmental pathway leading to fertilization and production of seed (Heslop-Harrison, 1975). The fact that no SI systems have been demonstrated conclusively at a genetic level to occur in gymnosperms (which do not possess carpels to protect their ‘‘naked’’ ovules) suggests, as was first postulated by Whitehouse (1950), that the evolution of the carpel (pistil) was fundamentally associated with the evolution of SI in early angiosperms. SI systems can be divided into two groups based on the genetic control of the incompatibility phenotype of the pollen: In gametophytic SI (GSI),

FIG. 10 The distribution and diversity of self-incompatibility systems within the major clades of flowering plants (phylogenetic tree based on Chase et al., 1993). 1, de Nettancourt (1977); 2, Kowyama et al. (1994); 3, Seavey and Bawa (1986); 4, Xue et al. (1996); 5, Kohn and Barrett (1992); 6, Fryxell (1957); 7, Levin (1993); 8, Goodwillie (1997); 9, Lundqvist (1995); 10, Lundqvist et al. (1973); 11, Barrett and Cruzan (1994); 12, Cope (1962); 13, Barrett and Richards (1990); 14, Thompson (1979); 15, Lawrence (1996); 16, Lundqvist (1956); 17, Hayman (1956); 18, East (1940); 19, Dressler (1993); 20, Lundqvist (1991); 21, Sage et al. (1994).

220

SIMON J. HISCOCK AND URSULA KU¨ES

the incompatibility phenotype of the pollen is determined by its own haploid genome (East and Mangelsdorf, 1925), whereas in sporophytic SI (SSI) the diploid parent plant (sporophyte) determines the incompatibility phenotype of the pollen (Hughes and Babcock, 1950; Bateman, 1952, 1954, 1955). Generally, GSI is controlled by a single locus—S (self-incompatibility)— which has multiple alleles. These alleles act in opposition such that when an allele is present in pollen and pistil the pollen is rendered incompatible (Fig. 11A). S alleles therefore must act in a codominant manner in the pistil and will reject any pollen that expresses an S allele in common with that pistil—a feature which allows the regulation of matings between closely related individuals (siblings) and the prevention of selfing (Lewis, 1979a). GSI is typically found in the Solanaceae, Rosaceae, Scrophulariaceae, and Papaveraceae (Fig. 10). SSI is also typically controlled by a single multiallelic S locus, and because expression of the S alleles is sporophytic with respect to pollen, the phenotype of the pollen will appear diploid for S alleles (Fig. 11B). Sporophytic control of SI increases the efficiency with which sibling matings can be regulated and also permits linear dominance relationships between S alleles to exist in pollen and pistil (Thompson and Taylor, 1966; Kowyama et al., 1994; Hatakeyama et al., 1998). SSI is characteristic of the Brassicaceae, Asteraceae, and Convolvulaceae (Fig. 10). GSI and SSI systems have also been characterized that are controled by more than one locus (Franklin et al., 1995; de Nettancourt, 1997) (Fig. 10): In the grasses (Poaceae), GSI is controlled by multiallelic loci S and Z (Lundqvist, 1955, 1956; Hayman, 1956) such that compatibility is only possible if pollen and pistil possess different alleles at both loci. In this system of GSI four pollination scenarios are possible because if two plants which share one S allele and one Z allele in common cross, the outcome will be three-quarters compatible (Lewis, 1979a,b; Hiscock et al., 1994). Such a system is therefore less efficient at restricting sib matings than the single locus system of GSI, a factor which Lewis (1979a,b) suggests is responsible for the relative infrequency of multilocus systems of SI. Even so, a number of multilocus systems do appear to exist. For example, GSI in Lilium martagon is controled by three loci (Lundqvist, 1991), whereas in Ranunculus and Beta GSI is controlled by four multialleleic loci (Lundqvist, 1990b; Lundqvist et al., 1973). Interestingly, only one system of SSI has been suggested to have multilocus control—Eruca sativa, in which three or four loci have been implicated (Verma et al., 1977). In plants with ‘‘heteromorphic’’ SI (distyly and tristyly), incompatibility is controlled sporophytically (Ganders, 1979; Gibbs, 1986; Barrett and Richards, 1990; Kohn and Barrett, 1992; Barrett and Cruzan, 1994). The single S locus is diallelic (S and s) and controls not only the physiological system of SI but also many developmental pathways that produce differences in

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

221

FIG. 11 Genetic control of self-incompatibility systems: (A) gametophytic SI; (B) sporophytic SI. (In this example, both GSI and SSI are under the control of a single multiallelic S locus.) (A) In GSI the SI phenotype of the pollen is determined by its own haploid genome. When an S allele in the pollen is matched with either of the two codominant S alleles in the pistil the pollen is rendered incompatible and inhibition usually takes place in the style. Note that 50% of the pollen from an S1S2 plant is incompatible with the S1S3 pistil. (B) In SSI the SI phenotype of the pollen is determined by the diploid genome of the parent plant (sporophyte) and the incompatibility response usually occurs at the stigma surface. Note that if S alleles are codominant all pollen derived from the S1S2 plant is incompatible on the S1S3 stigma, indicating the more efficient control over sib matings provided by SSI. Italic text indicates genotype and Roman text indicates phenotype.

floral morphology, such as pistil height, anther position, pollen size, and form, and stigma form. For this reason, the S locus is frequently described as a ‘‘supergene’’ (Richards, 1997). In distylous plants such as Primula and Oxalis, flowers exist in two forms—‘‘pin’’ and ‘‘thrum.’’ Pin plants have

222

SIMON J. HISCOCK AND URSULA KU¨ES

the genotype ss and thrum plants are Ss, so with sporophytic determination in the pollen the polymorphism will remain balanced at approximately 50% pin and 50% thrum within a population. Tristyly represents a modification of this system through the influence of an epistatic modifier locus, M, which, like S is diallelic. Tristylous plants such as Lythrum show three style length morphs—long (mmss), mid (Mmss), and short (mmSs or MmSs)—which, because of sporophytic control of SI in the pollen, are maintained in approximately equal numbers within a population. Control over sib mating by distylous and tristylous species is fairly ineffectual: Distyly (like dioecy) imposes no restriction on sib mating, whereas tristyly imposes a slight restriction on sib mating because the number of mating types is increased from two to three (Lewis, 1979a). The genetic basis of late-acting SI is unknown (Seavey and Bawa, 1986; Sage et al., 1994) because many of the species exhibiting this type of SI are woody plants with long generation times that make genetic studies impractical. In Theobroma cacao, however, Cope (1962) proposed that ovular arrest of pollen is under joint gametophytic and sporophytic control, whereas for Acacia retinoides Knox and Kenrick (1983) proposed gametophytic control to explain pollen tube arrest in the nucellus. There is no indication as to how many loci and alleles are involved in these SI systems.

B. The Number of S Alleles in Populations and Species Wright (1939) predicted that the number of S alleles in a population with single locus, multiallelic SI would be potentially very high based on the assumption that S alleles are maintained in a population by strong negative frequency-dependent selection. That is, any new allele (arising from migration or mutation) will be at an instant advantage within the population (because the individual that possesses this allele will be able to mate with all other individuals in the population) and will spread rapidly until it reaches equilibrium with the other S alleles. Wright’s prediction has been borne out by empirical studies which have shown the number of S alleles present in populations of species with GSI and SSI to be between 20 and 50 (Lawrence, 1996). However, an extraordinarily high number of S alleles has been shown to exist in populations of the clovers Trifolium repens (앑100) and T. pratense (앑200) [Lawrence (1996) based on Atwood et al. (1942, 1944); Williams and Williams (1947) as cited in Lawrence (1996)]. Such numbers appear to be exceptional and are probably a consequence of special genetical and ecological circumstances that are unique to these species of clover (discussed by Lawrence, 1996) because all S allele estimates for other species are far lower: 45 for Phlox drummondii (Levin, 1993) and Oenothera organensis (Emerson, 1939), 44 for Physalis crassifolia (Richman

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

223

et al., 1996b), 40–45 for Papaver rhoeas (O’Donnell and Lawrence, 1984; Lawrence et al., 1993), and 14 for Solanum carolinense (Richman et al., 1995). Similar numbers of S alleles have also been predicted for populations of species with SSI, i.e., 22 for Iberis amara (Bateman, 1954), 20–30 for Brassica campetris (Nou et al., 1993), 22 for Raphanus raphanistrum (Karron et al., 1990), and 52 for Sinapis arvensis (Stevens and Kay, 1989). Most estimates of the total number of S alleles occurring in a species as a whole are simply reflections of the number of S alleles estimated for a particular population because few studies have cross-classified alleles between different populations. In one such rare study, Lane and Lawrence (1993) predicted 66 S alleles to be present in Papaver rhoeas based on cross-compatibilities between Spanish and British populations. This figure is very similar to the number of S alleles (앑70) shown to be present in cultivar stocks of Brassica oleracea (Ockendon, 1974). What is clear from these limited studies is that the numbers of S alleles present in populations, and therefore presumably in species, are (with the exception of Trifolium) very similar, suggesting that S allele number is subject to dynamic restraint (Franklin et al., 1995) which Lawrence et al. (1993) attribute to the negative relationship between the selective advantage of a new allele and the number of alleles already present in the population.

C. The Site of the Self-Incompatibility Response Figure 10 summarizes the sites of pollen inhibition for the various types of SI described in Section IV,A. As a general rule, it can be seen that in species with SSI, self-recognition and pollen inhibition take place on the stigma surface, whereas in species with GSI, self-recognition and the incompatibility reaction occur after pollen tube penetration of the stigma, within the transmitting tissue of the stigma and style (Brewbaker, 1959; de Nettancourt, 1977). Exceptions occur in heteromorphic SSI, in which pollen tubes can be arrested on the stigma surface or within the stigma or style (Shivanna et al., 1981); in the grasses, in which the GSI reaction occurs very rapidly at the stigma surface (Heslop-Harrison, 1979, 1982); and in Papaver which, lacking a style, also exhibits a stigmatic GSI response (Franklin-Tong et al., 1992). A vast number of detailed ultrastructural and cytological studies have described features of the incompatible vs compatible pollen–pistil interaction for the major SI systems in various species, and we draw the readers’ attention to the following: for SSI in the Brassicaceae, see Dickinson and Lewis (1973a,b), Elleman and Dickinson (1986, 1990, 1994), and Elleman et al. (1988); for hetermorphic SSI, see Shivanna et al. (1981), Shivanna and Johri (1985), and Wong et al. (1994); for GSI in the Solanaceae, see de Nettancourt et al. (1973, 1974), de Nettancourt (1977), Herrero

224

SIMON J. HISCOCK AND URSULA KU¨ES

and Dickinson (1980, 1981), and Lush and Clarke (1997); for GSI in Oenothera, see Dickinson and Lawson (1975); for GSI in the grasses, see HeslopHarrison (1979, 1982) and Heslop-Harrison and Heslop-Harrison (1981); and for GSI in Papaver, see Elleman et al. (1992).

D. Characterization of S Genes Classical genetic studies on SI predicted a tripartite structure for the S locus, with each S allele consisting of a specificity part, a pistil part, and a pollen part (Lewis, 1960, 1965; Pandey, 1975). The specificity part was presumed to encode an allele-specific polypeptide subunit that would unite with a pistil-specific subunit in the pistil and with a pollen-specific subunit in the pollen; if specificity subunits were the same in pollen and pistil then the two polypeptides would dimerize to form an active inhibitory complex that would cause the arrest of further pollen tube development (Pandey, 1975). This dimer hypothesis first proposed by Lewis (1965) became a unifying mechanism for all forms of SI and made the prediction that the pollen and pistil S gene products would be essentially the same. Extensive molecular studies of S genes during the past 15 years, however, have dramatically changed this classical perception of the S locus and the relationships between the various SI systems by revealing that male and female S gene products are almost certainly not the same and that different SI systems are unrelated at a molecular level (Fig. 10) (Franklin et al., 1995; Hiscock et al., 1996; Kao and McCubbin, 1996; Newbigin, 1996; Nasrallah, 1997; de Nettancourt, 1997). Molecular studies have also shown that S genes, like the mating-type genes of fungi and algae, are highly polymorphic and represent some of the most polymorphic of all plant genes. No single S locus or SI system has been fully characterized yet, mainly because of the failure to identify S gene products in pollen. Nevertheless, pistilexpressed S genes are well characterized for three distinct SI systems: GSI in the Solanaceae, GSI in the Papaveraceae, and SSI in the Brassicaceae. 1. Pistil-Expressed S Genes Associated with GSI Style-specific glycoproteins associated with GSI were first identified in Nicotiana alata (Solanaceae) (Bredemeijer and Blass, 1981) and subsequent cloning and characterization of cDNAs for many S-associated glycoproteins revealed them to be ribonucleases (Anderson et al., 1986, McClure et al., 1989, 1990). So-called S-RNases have now been characterized from many species from Solanaceous genera, particulary Petunia (Ai et al., 1990), Lycopersicon (Mau et al., 1986; Tsai et al., 1992), and Solanum (Xu et al., 1990; Kaufmann et al., 1991). All are basic proteins with molecular masses

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

225

ranging from 23 to 34 kDa that accumulate in tissues of the stigma and style late during floral development; maximal S-RNase gene expression coincides with the onset of SI (Cornish et al., 1987; Anderson et al., 1989). Sequence comparisons between S-RNase alleles for many Solanaceous species have revealed important features and a common structure (Haring et al., 1990; Ioerger et al., 1990, 1991; Singh and Kao, 1992; Tsai et al., 1992; Newbigin et al., 1993; Fig. 12A). The overall sequence similarity between alleles is low (approximately 50%; Haring et al., 1990), and often alleles are more similar between species than within species (Ioerger et al., 1990) indicating that most S allele diversification occurred prior to speciation. All S-RNases possess a signal peptide sequence (which presumably directs their secretion into the extracellular matrix of the pistil), five conserved domains (C1–C5), and several hypervariable domains (Tsai et al., 1992; Fig. 12A). Two of the conserved domains, C2 and C3, share high homology with conserved regions flanking the active site of fungal RNases, and each contains a histidine residue that in fungal RNases is essential for enzymic activity (Kawata et al., 1990). Evidence from a Lycopersicon S-RNase mutant confirmed that at least one of the histidines (that from the C2 region; Fig. 12A) is essential for ribonuclease activity (Royo et al., 1994). Furthermore, lack of ribonuclease activity in this S-RNase was sufficient to impart a self-compatible phenotype upon the mutant plant (Royo et al., 1994). Similar results have been obtained from site-directed mutagenesis of a histidine residue on the S3-RNase from Petunia inflata (Huang et al., 1994) which produced an RNase lacking enzymic activity that could not direct rejection of S3 pollen when transformed into an S1S2 plant. These biochemical data confirmed the essential role of S-RNases in the GSI response in the Solanaceae which had previously been demonstrated by transformation experiments inducing loss and gain of S-RNase function: Lee et al. (1994) transformed P. inflata (S2S3) with an antisense construct of its S3-RNase and showed that the consequent reduction in synthesis of the S3-RNase (but not the S2-RNase) in the styles of transformants was accompanied by loss of the ability of those styles to reject S3 pollen. In addition to this loss of function experiment, Lee et al. (1994) also performed a gain of function experiment by transforming an S1S2 P. inflata plant with an S3-RNase construct and showed that the acquired ability of the plant to synthesize the new S3-RNase was accompanied by acquisition of an ability to reject S3 pollen. In a similar set of experiments, Murfett et al. (1994) transformed a self-compatible hybrid of Nicotiana langsdorfii ⫻ N. alata with an S2-RNase from N. alata and showed that plants expressing the S2-RNase were able to reject S2 pollen from N. alata. Together, these data demonstrated conclusively that stylar S-RNases are required for fully functional GSI. The hypervariable domains of S-RNases have been proposed to be the sites at which S allele specificity resides (Ioerger et al., 1990). Recent data

FIG. 12 Gametophytic S-RNases. (A) Generalized schematic representation of an S-RNase present in species from the Solanaceae. C1–C5 represent regions of conserved amino acid sequence; HVa and HVb represent the two major hypervariable domains; His38 and His103 indicate the positions of the two active site histidines essential for RNase activity. (B) Phylogenetic tree of angiosperm S-RNases using the fungal RNase T2 as an outgroup (adapted from Xue et al., 1996). S-RNases from the Solanaceae (Sol), Scrophulariaceae (Scr), and Rosaceae (Ros) from three distinct groups, whereas S-like RNases from Arabidopsis (and other species not included) form a separate group. Note that within the Solanaceous group S-RNase alleles from different species tend not to form species-specific clusters indicating that most of the allelic diversification occurred prior to speciation events involving these genera. Letters refer to the genera from which the sequences were obtained (for detailed explanation see Xue et al. 1996). N, Nicotiana; P, Petunia; S, Solanum; L, Lycopersicon; A, Antirrhinum; M, Malus; Atrns1 and Atrns3, Arabidopsis thaliana S-like RNase sequences; T2, RNase sequence from Aspergillus oryzae.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

227

from Solanum (Matton et al., 1997) and Nicotiana (Zurek et al., 1997) support this prediction, although whether S specificity is determined solely by the hypervariable domains remains controversial (Verica et al., 1998; Matton et al., 1998). Matton et al. (1997) clearly demonstrated that in Solanum chacoense it is possible to convert an S11 RNase into an S13 RNase by replacing the two hypervariable regions of the S11 RNase with those of the S13 RNase; however, as Verica et al. (1998) argue, because these two S-RNases are very similar in amino acid sequence (differing in only 10 of 190 aa), the role of amino acids outside of the hypervariable regions which are the same in the S11 and S13 S-RNases but which regularly differ in other, more divergent, S-RNase alleles cannot be ascertained. Indeed, domain swap experiments carried out on two dissimilar S-RNase genes (Sa2 and Sc10) from N. alata (Zurek et al., 1997) showed that exchange of hypervariable regions was not only insufficient to change S specificity but also eliminated the ability of the transformed plants to reject either Sa2 or Sc10 pollen; these findings prompted Zurek et al. (1997) to suggest that amino acids throughout the S-RNase molecule were required for S allele specificity. Stylar RNases associated with GSI are not found exclusively in the Solanaceae; similar S-RNases associated with GSI have been identified and characterized in the Rosaceae [Pyrus sp. (Ishimizu et al., 1998; Norioka et al., 1996; Sassa et al., 1996), Malus ⫻ domestica (Broothaerts et al., 1995), and Prunus sp. (Tao et al., 1997; Burgos et al., 1998)] and from the Scrophulariaceae [Antirrhinum hispanicum (Xue et al., 1996)]. As would be expected, sequence similarity between these S-RNases and those from the Solanaceae is low and the S-RNases can be categorized into three family-specific groups (Fig. 12B); however, the general structure is similar—signal peptide, conserved regions (four instead of the five seen in the Solanaceae), and hypervarible regions (Fig. 12A). In Papaver rhoeas (Papaveraceae), which, like species from the Solanaceae, Rosaceae, and Scrophulariaceae, has a single locus multiallelic system of GSI, the pistil S gene product is not an RNase (Franklin-Tong and Franklin, 1992; Foote et al., 1994). To date, four pistil-specific S alleles have been cloned (Foote et al., 1994; Walker et al., 1996) and shown to encode small (앑14–16 kDa) secreted, hydrophylic glycoproteins with no significant homology to any known proteins. Poppy does not have a style so the S glycoprotein accumulates in the cells of the stigmatic rays where S glycoprotein gene expression occurs specifically just prior to the onset of SI (Franklin et al., 1995). As with S-RNases, the poppy S glycoprotein alleles are highly polymorphic, showing only 앑55% sequence identity, and have characteristic conserved and hypervariable domains (Walker et al., 1996). The cloned S1 gene has been expressed in Escherichia coli and the expressed protein, which is unglycosylated and larger than the plant-produced protein, shown

228

SIMON J. HISCOCK AND URSULA KU¨ES

to be capable of inducing the S-specific inhibition of pollen tube growth in an in vitro bioassay (Foote et al., 1994). This inhibition mirrors that produced by S glycoprotein purified from the plant, indicating that glycosylation is not required for recognition and that there is some structural flexibility in the biologically active molecule. To date, no other pistil-expressed S genes have been cloned from species with GSI, although attempts have been made to identify pistil-specific S sequences in the grass Secale cereale: Wehling et al. (1994) used degenerate PCR with primers to Brassica stigma-specific SLG (S locus glycoprotein) gene sequences on genomic DNA from S. cereale to obtain amplification products, but these sequences were neither characterized nor shown to be stigma specific. 2. Pistil-Expressed S Genes Associated with SSI Stigma-specific glycoproteins which segregate with defined S alleles in Brassica oleracea and B. campestris have been known for some time (Nasrallah and Wallace, 1967; Nasrallah et al., 1970, 1972; Nishio and Hinata, 1977; Hinata and Nishio, 1978). These S locus glycoproteins (SLGs) are basic proteins of 앑55–65 kDa which localize specifically to the epidermal cells of the stigma, being synthesized by these papillar cells just prior to the onset of SI and accumulating in the cell wall (Nasrallah et al., 1985a; Kandasamy et al., 1989). SLG genes were subsequently cloned for many S alleles of B. oleracea (Nasrallah et al., 1985b, 1987, 1988; Trick and Flavell, 1989; Chen and Nasrallah, 1990; Dwyer et al., 1991) and B. campestris (Takayama et al., 1986; Isogai et al., 1988; Kusaba et al., 1997) and shown to encode highly polymorphic proteins with an N-terminal signal sequence, many variable N-glycosylation sites, three hypervariable domains, conserved domains, and 12 highly conserved cysteine residues [see Trick and Heizmann (1992) and Dickinson et al. (1992) for detailed comparisons of SLG sequences]. Sequence analyses of SLGs do not predict a biochemical function for these proteins. Comparative studies of SLG sequences have defined two distinct classes of S allele products which reflect their position in the dominance hierarchy (Nasrallah et al., 1991). Class I SLGs are all dominant alleles and share ⬎80% amino acid sequence identity, whereas class II SLGs are all pollen-recessive alleles and share only 65–70% homology with class I alleles at the amino acid level. There is again ⬎80% amino acid identity among different class II alleles. That SLG expression is required for functional SI has been demonstrated by analyses of self-compatible (SC) mutants which show dramatically reduced levels of SLG expression (Nasrallah et al., 1992) and by transgenic experiments which show that cosuppression of SLG by introduced SLG constructs is accompanied by breakdown of SI (Toriyama et al., 1991;

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

229

Conner et al., 1997). Recently, the first SLG genes have been isolated from SI lines of radish (Raphanus sativus, a close relative of Brassica which also possesses SSI) using PCR with degenerate primers designed to the conserved regions of Brassica SLGs (Niikura and Matsuura, 1997; Sakamoto et al., 1998). The eight R. sativus SLG sequences showed 85–92% nucleotide homology to the SLG6-gene from B. oleracea and the eight deduced amino acid sequences contained all 12 conserved cysteins and the three hypervariable regions characteristic of Brassica SLGs, suggesting that these SLG homologs perform an equivalent function to those of Brassica. In addition to SLG, a related gene, the S receptor kinase (SRK ) gene, also resides at the S locus in Brassica spp. (Stein et al., 1991; Goring and Rothstein, 1992; Suzuki et al., 1995), the two genes being separated by approximately 200–300 kb (Boyes and Nasrallah, 1993; Fig. 13). Because of this complexity at the S locus in Brassica, allelic forms of S are regularly referred to as haplotypes (Nasrallah and Nasrallah, 1993). SRK encodes a functional transmembrane receptor serine/threonine kinase that spans the plasma membrane of the stigmatic papillar cells (Stein et al., 1991, 1996; Stein and Nasrallah, 1993; Goring and Rothstein, 1992; Delorme et al., 1995). The extracellular, putative receptor domain of SRK, which projects into the cell wall, shares 앑90% amino acid sequence identity with the corresponding SLG and is similarly polymorphic (Stein et al., 1991; Yamakawa et al., 1995; Kusaba et al., 1997; Nasrallah, 1997). This extracellular domain is encoded by the first exon of the SRK gene, whereas the remainder of the protein is encoded by a further six exons which produce a transmem-

FIG. 13 Schematic representation of the Brassica S locus. SLG, S locus glycoprotein gene; SRK, S receptor kinase gene; SLA, S locus anther gene; SLL1, S locus linked one gene; SLL2, S locus linked two gene; see text for explanation.

230

SIMON J. HISCOCK AND URSULA KU¨ES

brane domain and 11 subdomains that are conserved among serine/threonine kinases (Stein et al., 1991; Nasrallah and Nasrallah, 1993; Yamakawa et al., 1995). The apparent high degree of similarity between certain SLGs and the extracellular domain of the corresponding SRK (particularly for the S6 class I haplotype and the S2 class II haplotype; Nasrallah and Nasrallah, 1993) suggests that there has been a close coevolution of these two sequences within the same locus. It has therefore been suggested that SLG may have evolved from SRK by gene duplication, with sequence similarity between the gene pairs being maintained through gene conversion (Tantikanjana et al., 1993). However, a recent survey of all SLG and SRK sequences currently available (Kusaba et al., 1997) revealed that SLG and SRK sequences from the same haplotype can be more divergent than was previously thought, such that some SLGs share more amino acid sequence homology with SRKs from other S haplotypes than they do with their own S haplotypic partner. Interestingly, in some class II haplotypes alternative truncated forms of the SRK have been identified which encode either a membrane-anchored form of SRK which lacks a kinase domain or an apparently secreted form of the SRK which lacks both a kinase domain and a transmembrane domain (Tantikanjana et al., 1993; Giranton et al., 1995). The role, if any, of these variants of SRK in the SSI response is unknown. Like SLG, SRK is expressed predominantly in stigmatic papillae just prior to the onset of SI, but expression levels of SRK are considerably (140–180 times) lower than expression levels of SLG (Stein et al., 1991). Curiously, very low levels of SRK expression can also be detected in developing anthers at the binucleate stage of pollen development, although a mature SRK protein has never been detected in pollen (Delorme et al., 1995; Stein et al., 1996). Mutations affecting the correct expression or structure of the SRK gene have been shown to be associated SC phenotypes (Goring et al., 1993; Nasrallah et al., 1994a) providing strong (but indirect) evidence that SRK is essential for functional SSI. In an attempt to prove this requirement of SRK for SSI, attempts were made to transform a SC line of Brassica with a chimeric SRK gene and to transform a SI line with an antisense SRK construct (Conner et al., 1997); however, because these constructs were poorly expressed in the transgenic plants and the expression of endogenous S genes and related genes was also reduced as a consequence of the insertion of the transgenes, these gain of function and loss of function experiments proved inconclusive. Attempts to transform SC B. napus with an SRK construct yielded similar results (Stahl et al., 1998). Because both SRK and SLG appear to be essential requirements for SSI in Brassica (however, see Gaude et al., 1995) it has been proposed, on the basis of analogy with receptor kinase activation mechanisms in animal cells, that

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

231

SRK and SLG form a receptor complex with an unidentified pollen S ligand to initiate the inhibition response during SSI (see Section IV,E,3). SLGs and SRKs are known to be merely part of a large multigene family, the members of which are not exclusive to Brassica nor indeed to the Brassicaceae (Umbach et al., 1990; Dwyer et al., 1992, 1994; Tobias et al., 1992; Walker, 1993; Yamakawa et al., 1994; Braun and Walker, 1996; Tantikanjana et al., 1996). In Brassica the best characterized members of this family are the SLRs (S locus-related glycoprotein genes) which are not linked to the S locus but show varying degrees of homology to class I and II SLGs, particularly conservation of the 12 conserved cysteine residues (Trick and Heizmann, 1992). For instance, SLR-1 (Lalonde et al., 1989; Trick and Flavell, 1989) shares about 33% amino acid identity with class I and class II SLGs, respectively, while SLR-2, which is linked to SLR-1 (Boyes et al., 1991), shares approximately 79% amino acid identity with class II SLGs and approximately 45 and 33% amino acid identity with class I SLGs and SLR1, respectively. Interestingly, the expression patterns of SLR-1 and SLR-2 mirror those of SLG in terms of tissue specificity and developmental timing, suggesting that these genes could play a role in pollen–stigma interactions (Trick and Heizmann, 1992). Indeed, recent studies indicate that SLR-1, but not SLR-2, in concert with SLG, is involved in mediating pollen adhesion to the stigma during compatible pollinations (Luu et al., 1997, 1999). Homologs of SLR-1 and SLR-2 have been detected in other members of the Brassicaceae (Umbach et al., 1990; Dwyer et al., 1992, 1994; Tantikanjana et al., 1996; Sakamoto et al., 1998) including Arabidopsis, from which a number of these genes have been cloned and characterized, although their cellular functions remain to be determined (Dwyer et al., 1994). Markedly different from SLR-1 and SLR-2, SLR-3 (Cock et al., 1995) appears to encode an extensively modified SRK that lacks both a kinase domain and a transmembrane domain and is apparently secreted (like an SLG), but unlike SLG or SRK, SLR-3 is expressed at varying levels in all aerial tissues of the plant (Cock et al., 1995). SRK homologs have also been identified in a number of different species within and outside the Brassicaceae and have been classified as the Sdomain class of receptor-like kinases (RLKs) in plants (Braun and Walker, 1996). In addition to SRK, members include the first such putative kinase gene to be identified, ZmPK1 from maize (Walker and Zang, 1990); ARK1, ARK2, and ARK3 from Arabidopsis (Tobias et al., 1992; Tobias and Nasrallah, 1996; Dwyer et al., 1994); and OsPK10 from rice (Zhao et al., 1994). Apart from SRK all S-domain RLKs are expressed in vegetative tissues, in which they have been proposed to play a role in cell signaling during development (Tobias and Nasrallah, 1996). Until recently, only SRK could be shown to have a defined cellular function, but characterization of the SRF2 gene from B. oleracea (Pastuglia et al., 1997a) and detection of its

232

SIMON J. HISCOCK AND URSULA KU¨ES

expression in response to bacterial infection and wounding suggest that this gene and possibly other S-domain RLKs may play a role in plant responses to pathogen attack and mechanical wounding. In an attempt to identify genes associated with SSI in Ipomoea (Convolvulaceae) Kowyama et al. (1995) used degenerate PCR primers designed to the conserved regions of Brassica SLGs and SRKs to isolate S-like mRNAs from stigmas and anthers of I. trifida. Four classes of S-like genes were identified (IPG1–IPG4) which shared 40–46% deduced amino acid similarity to SLGs and the extracellular domain of SRKs from Brassica. Unfortunately, RFLP analysis revealed none of these sequences to be linked to the S locus of I. trifida, suggesting that these genes are not fundamental to SSI in this species (Kakeda and Kowyama, 1996). Such a finding is not altogether unexpected given the phylogenetic distance between the Brassicaceae and the Convolvulaceae; indeed, the Convolvulaceae is more closely related to the Solanaceae than to the Brassicaceae (Chase et al., 1993). Further characterization of IPG1 showed it to encode the extracellular domain of an S-like receptor kinase, IRK1 (Kowyama et al., 1996), with 50 and 53% amino acid identity to SRK6 and ARK1, respectively. IRK1 is expressed in reproductive tissues as well as in petals, leaves, and roots, suggesting that, like other RLKs, it has a more general cellular role.

3. The Pollen S Component of GSI and SSI Remains Elusive The greatest handicap to elucidation of the cell signaling and molecular recognition pathways leading to GSI and SSI has been the failure to identify the pollen S component(s). Ironically, the first pollen-expressed S gene to be identified that fulfills the criteria for a male determinant of GSI was cloned from a grass, Phalaris coerulescens, in which GSI is controlled by two loci, S and Z (see Section IV,A), but no pistil-expressed S or Z genes have been cloned. This gene, Bm2, encodes a functional pollen-specific thioredoxin-H that segregates with the S2 allele of P. coerulescens (Li et al., 1994, 1995). Sequencing of other allelic forms of Bm2 from S1 and S4 homozygotes revealed the predicted protein to have a variable N terminus and a conserved C terminus (thioredoxin domain), and it was proposed that S specificity may reside in the N-terminal region (Li et al., 1994)—a prediction supported by analysis of S-type thioredoxin sequences from other SI species of grass (Li et al., 1997). The finding that a SC mutant line of P. coerulescens shows significantly reduced thioredoxin activity suggests that the catalytic activity of the S-thioredoxin could be required for the GSI response (Li et al., 1996). Unfortunately, because pistil-expressed S and Z genes have not been identified in P. coerulescens it is not possible to speculate how S-thioredoxin activity might participate in the SI response.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

233

The finding that in N. alata low levels of S-RNase transcript were detectable in pollen (Dodds et al., 1993) prompted renewed interest in the dimer hypothesis as a mechanistic model for GSI (Lewis, 1960, 1965). However, attempts to detect the mature S-RNase protein in pollen proved fruitless (Dodds et al., 1996, 1997), supporting the current opinion that the S determinants of pollen and pistil are different molecules. The male determinant of the Solanaceous type of GSI is predicted to be either an S-RNase translocator situated in the plasma membrane of the pollen tube that specifically internalizes its allelic S-RNase counterpart or an inhibitor of SRNases that is unable to inhibit its own S-RNase counterpart (Thompson and Kirch, 1992; Dodds et al., 1996; Kao and McCubbin, 1996; Newbigin, 1996). Differential display has been used to identify pollen-expressed genes linked to the S locus of N. alata (Li et al., 1998); the search was not comprehensive but three genes were discovered—48A, 133G, and 167A. All are linked to the S locus and are expressed in mature pollen during anther development. The most tightly linked of the three genes is 48A and, intriguingly, each of the N. alata S alleles tested is associated with a different 48A RFLP. A possible role for 48A in GSI is currently being investigated (E. Newbigin, personal communication). In an alternative approach to identifying the pollen S component in N. alata, Golz et al. (1997) generated pollen-part mutants (PPMs). PPMs are plants that are self-compatible but can still reject pollen carrying either S allele present in the unmutated progenitor plant, indicating that the PPM does not affect expression of stylar S genes. Conversely, pollen from a PPM is not rejected by incompatible styles such as those of an unmutated progenitor plant, indicating that the PPM only affects expression of the S locus in pollen. Earlier studies of PPMs in Petunia and Nicotiana revealed an association between this phenotype and the production of pollen containing two S alleles instead of one. In many cases, the additional S allele appeared to be on a small additional chromosome—a ‘‘centric fragment’’ (Pandey, 1965). The role of the extra S allele in producing the PPM phenotype remained unresolved mainly because laborious pollination checks were the only way to assess which alleles were present in the plants. Golz et al. (1997) are using molecular markers linked to the S locus to reassess the role of duplicated S alleles in the PPM phenotype. By following the PPM phenotype in lines of mutant N. alata plants, it has become clear that PPM plants always carry an extra S allele. These data are consistent with the pollen S gene product being an RNase inhibitor that inactivates S-RNases encoded by different S alleles but not the S-RNase encoded by the same S allele (Dodds et al., 1997; see Section IV,E,1). Moreover, using molecular markers on either side of the S locus Golz et al. (1997) showed how much of the S-bearing chromosome was duplicated in their mutant lines. Such an approach offers great hope for eventually identifying the elusive pollen S gene product.

234

SIMON J. HISCOCK AND URSULA KU¨ES

The male determinant of GSI in the poppy system is also an enigma, although physiological and biochemical studies of the SI response predict the protein to be a membrane-bound receptor that activates a Ca2⫹mediated signaling cascade (Franklin et al., 1995; Rudd et al., 1996; see Section IV,E,2). To date, the search for this receptor has identified a membrane-associated glycoprotein, SBP (S-binding protein), in pollen that binds tightly but nonspecifically to stigmatic S glycoproteins (Hearn et al., 1996). It is speculated that SBP may function as an accessory receptor that presents the S glycoprotein to the S receptor (Hearn et al., 1996; F. C. H. Franklin, personal communication). In Brassica, weak SLG and SRK expression has often been detected in anther tapetum tissue and in pollen (Guilluy et al., 1991; Stein et al., 1991), and the SLG promoter is known to be active in these tissues (Sato et al., 1991). However, SLG and SRK proteins have never been detected in pollen or anthers, indicating that, as with GSI, the male determinant is almost certainly encoded by a different gene. Chromosome walking between SLG and SRK has identified many pollen-expressed and anther-expressed genes at the Brassica S locus (Boyes and Nasrallah, 1995; Yu et al., 1996), but none of these genes have proved likely candidates for the male determinant because either they lack sufficient polymorphism between S alleles for an S determinant or they are dispensible for SSI (Pastuglia et al., 1997b). SLA (S locus anther) was isolated from the S2 haplotype of B. oleracea and shown to be expressed exclusively in anthers (Boyes and Nasrallah, 1995). It was speculated that the apparent absence of sequences homologous to SLA in other haplotypes was due to it being extremely polymorphic and possibly the male determinant of SSI, and this notion was supported by the finding that a SC line of B. napus possessed a disfunctional SLA gene containing a large insertion (Boyes and Nasrallah, 1995). However, SLA sequences containing this insertion have been detected in other lines of B. oleracea that are both SC and SI, indicating that this gene is not required for SSI (Pastuglia et al., 1997b). Two other anther-expressed genes (SLL1 and SLL2) have been identified in the region between SLG and SRK in an SI line of B. napus (Yu et al., 1996). SLL1 encodes a small peptide (앑2 or 3 kDa) and is expressed specifically in anthers of SI plants, but its sequence was found to be identical in many different SI lines, suggesting that it is unlikely to be the male determinant. SLL2 is also an unlikely candidate for the male determinant because it exists in other parts of the genome and is expressed in anthers and stigmas of both SI and SC plants (Yu et al., 1996). A recent chromosome walk away from SLG in B. campestris identified a homolog of the proteolytic subunit of the Clp protease (ClpP ), but the expression of this gene in most tissue types (including anthers and stigmas) suggests that its function is a general one and not specific to SSI (Letham and Nasrallah, 1998).

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

235

The only fact that is certain about the male determinant of SSI in Brassica is that it is located within the pollen coating (Stephenson et al., 1998). Meticulous micromanipulation experiments involving the transter of pollen coatings from one haplotype to another revealed that the S phenotype of the pollen could be changed by supplementing its coating with an excess of coating extracted from another S haplotype (Stephenson et al., 1998) and so confirmed the earlier findings and predictions of Heslop-Harrison et al. (1974, 1975) and Dickinson and Lewis (1973a,b) that in SSI species the male determinant resides in the pollen coating. Fractionation of the proteins contained in the pollen coating has identified an ‘‘active fraction’’ of 앑12 polypeptides with molecular weights between 7 and 10 kDa that are responsible for changing the S phenotype of the pollen (Stephenson et al., 1998; Dickinson et al., 1998). Many of the polypeptides from this fraction have been characterized and shown to be members of a multigene family of defensin-like proteins termed PCPs (Stanchev et al., 1996; Doughty et al., 1998). One defensin-like PCP, PCP-A1 (Dickinson et al., 1998; Doughty et al., 1998), has been shown to interact in vitro with SLGs but in a nonS-specific manner (Doughty et al., 1993), whereas another PCP has been demonstrated to interact with SLR1 (Hiscock et al., 1995b). Despite its strong interaction with SLG, PCP-A1 has not been conclusively demonstrated to interact with SRK (Doughty et al., 1998). Because PCP-A1 is not significantly polymorphic between haplotypes and is not linked to the S locus, it is unlikely to be the male determinant of SSI but it remains possible that PCP-A1, by virtue of its ability to interact with SLG, could act as a cofactor in the interaction between the pollen S ligand and the SLG/SRK receptor complex (see Section IV,E,3). Interestingly, PCP-A1 is expressed gametophytically in pollen just prior to anthesis. This important finding indicates that pollen coat proteins (including the male S determinant), which were previously assumed to be derived from sporophytic expression in the tapetum, can be synthesized in maturing pollen and secreted into the anther loculus before their incorporation into the pollen coat. Thus, SSI might simply be a consequence of the early gametophytic expression of the male S component in the pollen rather than in the diploid tapetum—‘‘pseudosporophytic SI’’ (Doughty et al., 1998). Continued analysis of the active fraction of pollen coat PCPs will no doubt eventually identify the male determinant of SSI. E. Cellular Mechanisms of GSI and SSI: Current Models 1. GSI Involving S-RNases There is little doubt that pistil-expressed S-RNases are central to the incompatibility response in species from the Solanaceae (Lee et al., 1994; Murfett

236

SIMON J. HISCOCK AND URSULA KU¨ES

et al., 1994; Huang et al., 1994) and probably also in species from the Rosaceae (Sassa et al., 1996; Norioka et al., 1996) and Scrophulariaceae (Xue et al., 1996). A cytotoxic model of GSI has been proposed to explain these findings (Lee et al., 1994; Murfett et al., 1994; Dodds et al., 1996, 1997; Kao and McCubbin, 1996). This model predicts that S-RNases enter incompatible pollen tubes and degrade ribosomal RNA (which is apparently not synthesized in pollen tubes; Mascarenhas, 1993), thus preventing protein synthesis and causing cessation or retardation of pollen tube growth. Although some ultrastructural evidence suggests that protein synthesis may be affected during an incompatible response in Nicotiana (de Nettancourt et al., 1973, 1974) and that rRNA is degraded in incompatible pollen tubes of N. alata in vivo (McClure et al., 1990; Gray et al., 1991), it has yet to be conclusively demonstrated that S-RNases enter pollen tubes in vitro and that degradation of rRNA is the primary cause of pollen tube arrest (Kao and McCubbin, 1996; Dodds et al., 1997). In a recent study of the growth of N. alata pollen tubes through compatible and incompatible styles it was demonstrated that growth of incompatible pollen tubes is rarely fully arrested and, importantly, ‘‘incompatible’’ pollen tubes could even reinitiate growth if an incompatible style was grafted onto a compatible one (Lush and Clarke, 1997). How can such observations be accommodated by the cytotoxic model? Dodds et al. (1997) cite new evidence that rRNA synthesis does take place in N. alata pollen tubes, contrary to previous reports in other species (Mascarenhas, 1993), and suggest that the observations of Lush and Clarke (1997) can be explained in terms of differential rates of rRNA degradation vs rRNA synthesis in incompatible pollen tubes. Such a prediction is made feasable by studies of gametophytic selection which clearly show that in a population of pollen tubes (male gametophytes) from a given individual some male gametophytes are inherently ‘‘fitter’’ than others in terms of growth rate (Hormazo and Herrero, 1992). A further finding, the significance of which to the cytotoxic model (if any) is unclear, is that N. alata style S-RNases can be phosphorylated nonspecifically in vitro by a soluble protein kinase (Nak-1) from pollen (Kunz et al., 1996). Nak-1, which shares a number of biochemical similarities with calcium-dependent protein kinases (CDPKs), cannot phosphorylate S-RNases from Lycopersicon peruvianum, but its ability to phosphorylate S-RNases of N. alata in vivo is not known. Given the general acceptance of the cytotoxic model of GSI, an explanation of how S specificity and allelic recognition is determined is required. Section IV,D,3 described the two current models for recognition during the GSI response in the Solanaceae: either S-specific uptake of the S-RNase into the pollen or S-directed inactivation of S-RNases in the pollen after nonspecific uptake. The S-specific translocator model has gained support from some reviewers (Dickinson, 1994; Franklin et al., 1995; McCormick,

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

237

1998), and the finding that a mutant S3-RNase from P. inflata that lacks RNase activity has an allele-specific dominant negative effect on SI (McCubbin et al., 1997) is consistent with such a model because the partial loss of SI observed when P. inflata was transformed with this mutant S3-RNase could be due to fewer S3-RNases being taken up by the pollen tube as a consequence of competition between the wild-type S3-RNase and the mutant S3-RNase for binding to the translocator. Despite these observations, it is the more intricate inhibitor model that is supported by studies of pollen part mutants (PPMs) (Dodds et al., 1997; Golz et al., 1998; E. Newbigin, personal communication; see Section IV,D,3). All PPMs possess an extra S allele and produce pollen that carries two different S alleles. If the pollen S gene product is indeed an S-RNase inhibitor, then pollen expressing two different inhibitors should be able to grow through any style because it can inactivate any S-RNase. Pollination studies of PPMs have confirmed this prediction ( J. Golz and E. Newbigin, personal communication). Furthermore, the well-known occurrence of a breakdown of GSI in tetraploids (Lewis, 1947, 1949a, Chawla et al., 1997), which produce diploid pollen, is consistent with the inhibitor model. To explain the dominant negative effect of the mutant P. inflata S-RNase on weakening SI, one might assume (as suggested by McCubbin et al., 1997) competition between the two S3-RNase variants for binding to the RNA substrate within the pollen tube, thus preventing continuous RNA degredation by the functional S3-RNase. Assuming the inhibitor model is correct, the question arises as to how a single S-RNase inhibitor is able to inactivate all S-RNases except the one with which it is allelic (Fig. 14). This can be envisaged if the S specificity is achieved through the lack of a complementary S-RNase recognition domain in the inhibitor or on the S-RNase (Fig. 14), such that in an incompatible interaction with the allelic S-RNase evades detection by the inhibitor in a ‘‘stealth-like’’ manner. Current opinion on which regions of the SRNase determine S specificty are unresolved (see Section IV,D,1), but comparisons between S-RNase sequences indicate that a number of hypervariable regions and hypervariable individual amino acids may be involved (Zurek et al., 1997), providing a potentially very large number of different binding sites for the S-RNase inhibitors, as is predicted by the model. 2. GSI in Papaver rhoeas Study of the physiology and biochemistry of GSI in the pollen of P. rhoeas has been greatly facilitated by the development of a unique in vitro bioassay (Franklin-Tong et al., 1988). Poppy pollen can be grown in germination media and when it is challenged by the addition of naturally purified or recombinant stigmatic S glycoprotein of corresponding allelotype pollen tube growth is arrested and the pollen eventually dies (Franklin-Tong et

238

SIMON J. HISCOCK AND URSULA KU¨ES

FIG. 14 Hypothetical cytotoxic model for the mechanism of GSI in Nicotiana. The pollen S gene product is proposed to be an inhibitor of S-RNases that is unable to inhibit the S-RNase with which it is allelic (see Section IV, D, 3). Stylar S-RNases enter pollen tubes freely and in a compatible interaction are inhibited by nonallelic inhibitor molecules. In an incompatible interaction an S-RNase that is allelic to the inhibitor is not inhibited and degrades rRNA in the pollen at a rate greater than that at which the pollen tube can synthesize it (Dodds et al., 1997).

al., 1988, 1995; Franklin, 1995). Such a system allows intracellular responses of the pollen to be studied much more effectively than for pollen growing in vivo. These studies have provided strong evidence that GSI in poppy is mediated by a Ca2⫹-dependent signal transduction pathway (Franklin-Tong et al., 1994). Rapid transient increases in cytosolic calcium have been observed in pollen tubes following challenge by their allelic S glycoproteins which is followed shortly by cessation of pollen tube growth (Franklin-Tong et al., 1993, 1996), while microinjection of calcium into pollen tubes or release of caged calcium within pollen tubes mirrors this response (Franklin-Tong et al., 1993, 1995). A recent study using ratio imaging of intracellular free calcium in pollen tubes challenged by S glycoprotein demonstrated that the

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

239

tip-focused calcium gradient observed in normally growing pollen, which is essential for continuous growth (Mahlo et al., 1994), becomes dissipated such that calcium levels fall at the tip and increase rapidly in the subapical regions (Franklin-Tong et al., 1997). It is not resolved whether such spatial changes in [Ca2⫹]i are a cause or a consequence of pollen tube arrest. The SI response in pollen tubes has also been shown to involve the rapid phosphorylation and dephosphorylation of a set of pollen proteins (Franklin-Tong et al., 1992). Two of the proteins showing increased levels of phosphorylation after challenge by stigmatic S protein have been partially characterized. p26.1, a cytosolic 26-kDa protein, is hyperphosphorylated within 90 sec of challenge by S glycoprotein and this hyperphosphorylation appears to be calcium and calmodulin dependent (Rudd et al., 1996), suggesting that an initial stage of the GSI response may be the activation of Ca2⫹-dependent CaM protein kinase by the calcium transient of a Ca2⫹dependent CaM protein kinase (Fig. 15). The second partially characterized phosphoprotein p68 (a 68-kDa protein) is phosphorylated downstream of p26.1 approximately 240–400 sec after challenge by S glycoprotein and, surprisingly, its phosphorylation is calcium independent, implying that a different kinase is responsible for the phosphorylation of this protein (Rudd et al., 1997). A picture thus emerges of a pollen response pathway involving an initial Ca2⫹-dependent signaling pathway followed by or activating a calcium-independent signaling pathway (Fig. 15). The target(s) of the second-phase signaling pathway is/are possibly transcription factors that mediate the observed alterations in specific pollen gene expression later in the incompatibility response (Franklin-Tong et al., 1992). This altered gene expression is thought to be responsible for the ultimate ‘‘death’’ of the pollen tube (Rudd et al., 1997). The primary activator of the pollen signaling pathway that leads to tube arrest and cell death must be the male determinant of SI, and although this molecule has yet to be identified, it is postulated to be a receptor molecule spanning the plasma membrane of the pollen tube (Franklin et al., 1995; Rudd et al., 1997). The class of receptor involved is a matter for speculation; receptor kinases and G protein-linked seven-bypass receptors usually activate Ca2⫹-based signaling systems in animal cells, so the receptor is likely to belong to one of these classes of protein. Such a receptor would then interact with the inositol-triphosphate signaling pathway that is known to be involved in the induction of elevated [Ca2⫹]i in pollen tubes prior to their arrest (Franklin-Tong et al., 1996). The identification of SBP (Sbinding protein), a heavily glycosylated protein in pollen that binds nonspecifically to S glycoproteins (Hearn et al., 1996), has led to the proposal that SBP acts as a ‘‘ligand presenter’’ that shuttles the stigmatic S glycoprotein to the pollen S receptor (Fig. 15), implying that SBP may be part of a receptor complex with the S-glycoprotein and the S receptor (Hearn et

240

SIMON J. HISCOCK AND URSULA KU¨ES

FIG. 15 Hypothetical model for the mechanism of GSI in Papaver rhoeas. Inhibition of growing pollen tubes takes place at the stigma surface and is mediated by a Ca2⫹-based signaling system. The identity of the pollen S gene product is unknown but it is proposed to encode a receptor (see text). The receptor is thought to bind its allelic stigmatic S glycoprotein, possibly through the mediation of SBP, a heavily glycosylated pollen protein that binds S glycoproteins nonspecifically. Binding of the S glycoprotein to the S receptor then initiates an increase in cytosolic free calcium which leads to the hyperphosphorylation of the pollen protein p26.1. This is followed by the calcium-independent hyperphosphorylation of another pollen protein, p68, which is followed by altered patterns of gene expression in the pollen and subsequent pollen tube arrest and death of the pollen tube.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

241

al., 1996; F. C. H. Franklin, personal communication Fig. 15). Interestingly, a pollen protein (PCP-A1) from Brassica which binds stigmatic SLGs in a similarly nonspecific manner has been proposed to fulfill a similar role to SBP in the Brassica SSI response, which, as with poppy, takes place at the stigma surface (see Section IV,E,3). 3. SSI in Brassica As outlined in Section IV,D,2, the current model for the mechanism of SSI in Brassica proposes that SLG and SRK form a receptor complex with the unidentified pollen S determinant (ligand) which then triggers a signal transduction system in the stigmatic papillar cell that results in localized inhibition of pollen tube development, prior to, or just after, germination (Nasrallah and Nasrallah, 1993; Nasrallah et al., 1994b; Dickinson, 1995, Nasrallah, 1997). Activation of SRK is thought (by analogy to the behavior of animal receptor kinases) to involve dimerization between SRKs in the presence of the pollen ligand and possibly SLG, leading to autophosphorylation on serine and threonine residues which provide interaction sites for other downstream signaling molecules that mediate pollen inhibition. Interestingly, this inhibition response appears, like the stylar inhibition of pollen tube growth in Nicotiana, to be biostatic in that it can be overcome by transferring pollen grains to a compatible stigma (Kroh, 1966). This fact must therefore be accommodated by contemporary models of SSI. We speculated in Section IV,D,3 as to the identity of the pollen S ligand and concluded that currently there are no suitable candidates, even though the elusive molecule is known to reside in a fraction of 앑12 pollen coat proteins (PCPs) with molecular weights between 7 and 10 kDa (Stephenson et al., 1997). One of these PCPs, PCP-A1, which interacts with SLGs in a non-allele-specific manner, may function as a cofactor in the receptor complex perhaps to stabilize the interaction between SLG and the pollen ligand prior to and during the interaction with SRK (Fig. 16) (Dickinson et al., 1998; Doughty et al., 1998). As such, PCP-A1 may play a similar role to that postulated for SBP in poppy (see Section IV,D,3). A clue to the nature and specificity of the interaction between SRK, SLG, and the pollen S ligand has been revealed by detailed sequence comparisons between the hypervariable regions of SLGs and SRKs (Kusaba et al., 1997). Surprisingly, this study revealed remarkable sequence divergence within these hypervariable regions of SLG and SRK pairs from the same haplotype, indicating that if S specificity does indeed reside within these hypervariable regions then SLG and SRK may bind different domains of the pollen ligand (Kusaba et al., 1997; Fig. 16). Signaling events downstream of SRK activation have also been the subject of recent investigation. Experimental strategies to identify interactors

242

SIMON J. HISCOCK AND URSULA KU¨ES

FIG. 16 Hypothetical model for SSI in Brassica. The pollen S gene product (unidentified) resides in the pollen coating and is proposed to bind the stigmatic S proteins SRK and SLG, probably via two different binding domains (see text for explanation). Concerted binding leads to the formation of a receptor complex that allows activation of SRK by dimerization and autophosphorylation on serine and threonine residues. PCP-A1, a pollen coating protein known to bind SLGs nonspecifically in vitro, may help to stabilize this interaction. Phosphorylated serines and threonines are expected to be targets for the binding of downstream signaling molecules—possibly THL-1 (a thioredoxin) or ARC1 (an arm repeat protein). The subsequent signaling cascade is thought to involve phosphorylation of stigmatic proteins, one of which may be the aquaporin MOD that has been shown to be required for the S response.

with the kinase domain of SRK using the yeast two-hybrid system have identified a number of potential interactors: THL-1 and THL-2 are both thioredoxin-H-like molecules (Bower et al., 1996), whereas ARC1 is an arm repeat-containing protein with homology to 웁-catenins and pendulins

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

243

(Gu et al., 1998). THL-1 and THL-2 both interact specifically (but phosphorylation independently) with the kinase domain of SRK-910 from B. napus. Neither molecule interacts with the kinase domain of RLK4 and RLK5, which belong to the leucine-rich repeat class of RLK (see Section IV,D,2); however, both THL-1 and THL-2 are expressed in a variety of tissues suggesting that they are not involved specifically in SSI (Bower et al., 1996). Interestingly, it is known from animal systems that thioredoxins can act as negative regulators of protein kinases (Saitoh et al., 1998), suggesting that the phosphorylation-independent binding of THL-1 and THL-2 to the kinase domain of SRK-910 could be a feature of its inactive state. A much more likely candidate for an SSI-dependent kinase interactor is ARC1, which interacts specifically with the kinase domains of SRK-910 and SRKA14 in a phosphorylation-dependent manner and is expressed exclusively in stigmas (Gu et al., 1998). In animal systems arm repeat-containing proteins participate in protein–protein interactions involved in signaling and 웁-catenins mediate cadherin-directed cell adhesion (Gu et al., 1998). Although the plant extracellular matrix (ECM) is very different to the animal ECM, a role for ARC1 in controlling adhesion between pollen and stigma would be compatible with a number of mechanistic models involving differential adhesion of self- and non-self-pollen during the Brassica SSI response (Stead et al., 1980; Nasrallah et al., 1994b). A further protein, KAPP (kinaseassociated protein phosphatase; Stone et al., 1994), which interacts with the kinase domains of many RLKs, was also shown to interact with the kinase domain of SRK-A14 (Braun et al., 1997), but the nonspecific nature of this interaction suggests that KAPP, like THL-1 and THL-2, may only play a general regulatory role in SRK activity. Evidence that the stigmatic signaling pathway could involve a phosphorylation cascade has come from studies in which Brassica stigmas were allowed to imbibe okadeic acid (an inhibitor of type 1 and type 2A serine/threonine phosphatases) prior to pollination. Scutt et al., (1993) showed that okadeic acid treatment overcame the SSI response, whereas two subsequent studies showed okadeic acid to eliminate all stigmatic receptivity toward pollen (Rundle et al., 1993; Kandasamy et al., 1993). Interestingly, receptivity of Arabidopsis stigmas to pollen was unaffected by okadeic acid treatment, indicating that the signaling system affected by this drug in Brassica is not operating in SC Arabidopsis (Kandasamy et al., 1993). Whatever form the subsequent signal tranduction cascade takes after kinase activation, one potential target is an aquaporin-like protein (MOD) which is situated in the plasma membrane of stigmatic papillae (Ikeda et al., 1997), and aquaporins are known to be regulated by differential phosphorylation ( Johansson et al., 1998). MOD was identified through characterization of the recessive mod mutation that generates a SC phenotype in B. campestris but which is not linked to the S locus (Hinata et al.,

244

SIMON J. HISCOCK AND URSULA KU¨ES

1983). That a water channel protein should be implicated in SSI is consistent with findings that a highly regulated flow of water from stigma to pollen is essential for compatibility (Sarker et al. 1988; Dickinson, 1995); how the lack of a water channel can lead to self-compatibility needs careful explanation. Ikeda et al. (1997) make two suggestions: either activation of MOD by SRK increases water flow away from the pollen and back into the stigma, which seems very unlikely given the osmotic potential differential between highly dehydrated pollen grains and the relatively turgid papillar cells, or that the MOD channel opens in response to SRK and specifically allows the passage of inhibitory substances into the pollen during hydration. The latter hypothesis would seem the most logical and is supported by the earlier work of Ferrari and Wallace (1977) which suggested that at least part of the SSI response was centered in the pollen. Indirect support for this hypothesis is derived from the finding that stigmas of Brassica produce volatile substances that are inhibitory to pollen growth (Hodgkin and Lyon, 1984, 1986) and that there appears to be differential phosphorylation of certain proteins in pollen during incompatible vs compatible pollen–stigma interactions (Hiscock et al., 1995a), suggesting that intracellular signaling occurs in pollen as well as in papillar cells during the SSI response. The fact that there is no clear inhibition phenotype for SSI in Brassica—pollen can be inhibited before hydration, during hydration, after complete hydration, following germination of the pollen tube, or even after penetration of the stigma and entry into the cell wall (Dickinson and Lewis, 1973b)— indicates that there may be more than one way to inhibit a Brassica pollen tube.

F. Evidence for the Involvement of S Genes in Interspecific Incompatibility Indirect evidence for the involvement of S genes in interspecific incompatibility originally came from studies of the relationship between SI and the phenomenon of unilateral interspecific incompatibility (UI; Harrison and Darby, 1955; Lewis and Crowe, 1958; Hiscock et al., 1998). UI most commonly occurs in crosses between related SI species and SC species where the cross only succeeds if the SC species is the female parent and the SI species the male parent. In an extensive survey of UI in angiosperms Lewis and Crowe (1958) showed that the SI ⫻ SC UI ‘‘rule’’ was true for many families of dicots and for monocots (Poaceae) and was associated with GSI (as determined by one or two loci), homomorphic SSI, and heteromorphic SSI. The strong correlation between the presence of SI in one partner and its ability to inhibit SC pollen led Lewis and Crowe (1958) to propose that UI is controlled by the S locus. This hypothesis was further developed and

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

245

greatly expanded by Pandey (1968, 1969, 1973, 1979, 1981), who developed the concept of the ‘‘S gene complex’’ and proposed that this complex encodes two specificities—a primary specificity element which is responsible for controlling interspecific recognition/incompatibility and a secondary specificity element which controls intraspecific SI. He further proposed that the primary specificity had evolved first in gymnosperms as a mechanism to limit the number of futile interspecies pollinations incurred by plants as a consequence of wind pollination. Secondary specificity (SI) evolved later, probably in the protoangiosperms, by duplication and modificational finetuning of this male/female recognition system such that the early angiosperms were endowed with the ability to recognize and reject self-pollen as well as pollen from related species. Many authors have highlighted exceptions to the UI rules of Lewis and Crowe (1958) as evidence against a mechanistic model for UI based on control by the S locus and to promote an alternative model—that of ‘‘incongruity’’ (Hogenboom, 1975; Mutschler and Leidl, 1994; Kandasamy et al., 1994; Leidl et al., 1996), which proposes that all forms of interspecific incompatibility are manifestations of the degree of evolutionary divergence of the two species involved and as such are under polygenic control. Such a model, while undoubtedly holding true for wide intergeneric and interfamilial crosses, fails to account satisfactorily for the high incidence of UI in crosses between closely related species and genera (McGuire and Rick, 1954; Lewis and Crowe, 1958; Martin, 1964; 1967, 1968; Pandey, 1979, 1981; Sampson, 1962; Hiscock and Dickinson, 1993; Hiscock et al., 1998). The striking similarity between the SI response and the UI response in Brassica (Hiscock and Dickinson, 1993; Hiscock et al., 1998) provides further evidence for participation of elements of the SI rejection pathway in UI because UI, like SI, is absent in immature stigmas and is overcome by the protein synthesis inhibitor cycloheximide. Because no system of SI has been characterized fully, it is difficult to test the role of the S locus in UI at a molecular level. Nevertheless, some molecular evidence for the involvement of S genes in UI has been forthcoming, particularly from work on species from the Solanaceae. RFLP mapping studies in Lycopersicon pennellii indicate that the UI function of the pollen is determined by three major loci, one of which maps to, or very near, the S locus (Chetelat and De Verna, 1991), whereas more direct evidence for the involvement of S-RNases in UI has come from genetic transformation studies in Nicotiana species (Murfett et al., 1996). Murfett et al., (1996) focused on a SC race of N. alata (a species which is normally strongly SI) that differed from the SI race only by its inability to synthesize S-RNase. SI ⫻ SC interrace crosses were unilaterally incompatible and the SI race was able to resist pollination by pollen from SC species such as N. plumbaginifolia, while the SC race accepted such pollen. To test whether S-RNase synthesis was the absolute

246

SIMON J. HISCOCK AND URSULA KU¨ES

requirement for inhibition of pollen from N. plumbaginifolia, it was necessary to introduce an S-RNase construct into the SC N. alata. Since N. alata is not readily transformable, the S-RNase construct was introduced through introgression from a N. plumbaginifolia S-RNase transformant. The N. alata ⫻ N. plumbaginifolia hybrid was capable of rejecting pollen from SC N. plumbaginifolia and also pollen from SC N. tabacum, indicating a direct relationship between the presence of an active S-RNase in a style and the ability of that style to reject pollen from SC species. Interestingly, the N. plumbaginifolia primary transformants, while not capable of rejecting pollen from SC N. plumbaginifolia, were capable of rejecting pollen from SC N. tabacum, indicating that UI in Nicotiana must operate through more than one pathway and that dependence of the different pathways on the S-RNase varies. While these data do not support an absolute role of the S locus in UI, as proposed by Lewis and Crowe (1958) and Pandey (1981), they do demonstrate conclusively that at least one S gene product plays a crucial role in certain UI pathways in Nicotiana.

V. Evolution of Mating-Type and Self-Incompatibility Systems The evolution of sexual reproduction is inextricably linked with the evolution of mechanisms that control/restrict the breeding possibilities of the sexual cells produced by meiosis. It can easily be argued that the restriction imposed on selfing will promote outcrossing and so maximize the benefits of meiosis; however, the almost universal regulation of mating by just two mating types, or two sexes, cannot be accounted for purely on the basis of the advantages of inbreeding avoidance and outcrossing (Hurst, 1996). Indeed, there is theoretical and empirical data to suggest that the evolution of two mating types and two sexes arose as the most effective means of controlling the uniparental inheritance of selfish cytoplasmic organelles that would otherwise be in direct conflict within the zygote (Hoekstra, 1987; Hurst and Hamilton, 1992; Hurst, 1996; Partridge and Hurst, 1998). As a bonus, binary control of mating also imposes a modicum of control over selfing (33% reduction in selfing in bipolar fungi and Chlamydomonas), the benefits of which, in terms of mating potential and outcrossing, are greatly increased if dispersal is efficient and gametes and individuals can mix freely. The reproductive efficiency of a binary system is therefore dependent on the costs of finding a mate being low; if the costs of mate finding are high, more mating-type specificites would be expected to arise (Hurst, 1996). Thus, the more complex multiple mating-type systems of protozoa and basidiomycetes might be interpreted as having evolved from the ancestral

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

247

binary mating-type systems primarily to increase the efficiency of finding a mate. The consumate increase in outcrossing efficiency provided by multiple mating types in terms of their partial control of sib matings (Lewis, 1979a,b; Ku¨es and Casselton, 1993b; Hiscock et al., 1994) would have reinforced the selective value of the multiple mating-type system and favored the continued evolution of new mating-type specificities by frequency-dependent selection (Wright, 1939). The analogous multiallelic self-incompatibility systems of hermaphrodite angiosperms can similarly be interpreted as having evolved primarily as mechanisms to promote mate choice and outcrossing.

A. Origins of Mating-Type Loci and Self-Incompatibility Loci The products of genes present at mating-type loci and sex-determining loci clearly control cell and nuclear fusion and the uniparental inheritance of cytoplasmic organelles either directly or indirectly (Hurst, 1996; Ro¨hr et al., 1998), but what functions did the genes at these loci perform before their recruitment to the sexual cycle? Work on the protozoan ciliate E. raikovi suggests that the pheromone-mediated mating-type system evolved through the diversification and elaboration of an extant autocrine signaling system involved in asexual reproduction. Evidence is derived from the finding that mating-type pheromones are able to promote mitogenic proliferation (vegetative reproduction) of the cells from which they originate (Vallesi et al., 1995). Mitogenic activity associated with secreted proteins has also been observed in slime molds (Whitbread et al., 1991) but not specifically with any fungal mating-type pheromones. However, one could speculate that pheromone signaling systems, associated with mating type in higher fungi, were also recruited from a previous autocrine signaling system functional in asexual reproduction (Hiscock et al., 1996). It is thus interesting to note that in some bipolar ascomycetes, mating-type loci have also been shown to control vegetative fusion between mycelia—fusions only taking place between hyphae of the same mating type—contrasting with sexual fusions which only occur between hyphae of different mating type (Glass and Kuldau, 1992) and raising the question as to which function arose first—vegetative or sexual? The unusual idiomorphic nature of mating-type loci in ascomycetes and Chlamydomonas has prompted diverse speculation as to the origins of these accutely nonallelic loci (Sogin, 1991, Goodenough et al., 1995). Bell (1993) suggests that the idiomorphic nature of these genes indicates that they arose independently of one another and are the remnants of ultraselfish parasitic elements able to direct cell fusion (and therefore promote their own spread) that became ‘‘tamed’’ into regulating the sexual cycle. This hypothesis is supported by the retrovirus-like transposable nature of idio-

248

SIMON J. HISCOCK AND URSULA KU¨ES

morphic genes during mating-type switching in ascomycetes (see Section II,B,4). Interestingly, transposon-like behavior has also been demonstrated for the mid gene from the mt-locus of C. reinhardtii, while characterization of both mid and fus1 (specific to the mt⫹ locus) has revealed that these genes lack the codon bias characteristic of all other nuclear genes from C. reinhardtii and other members of the Volvocales, further emphasizing the unusual genetic nature of these mating-type genes (Ferris and Goodenough, 1997). Despite the clearly unusual genetic nature of idiomorphic matingtype loci, a more conventional, in situ origin is also possible: Given that these loci frequently encode transcription factors which, despite being a very diverse and heterogeneous class of proteins, can participate in the regulation of similar, complementary developmental pathways, it could be speculated that two very different transcription factors might have been ‘‘recruited’’ to mating-type function by virtue of a shared ability to participate in, for instance, the processes regulating organelle inheritance. Indeed, in Ustilago, genes with mitochondrial function (lga2 and rga2) appear to have been recruited to the mating-type loci, whereas in higher basidiomycetes a mitochondrial endopeptidase gene (MIP, 움-fg) is also present at the mating-type locus (Ro¨hr et al., 1998). Similarly, in Chlamydomonas the idiomorphic region of the mt- locus contains the transcription regulator gene mid and also specifically directs organelle inheritance (Goodenough et al., 1995). In contrast to the situation for SI in angiosperms, mating type in fungi and probably also in algae (Chlamydomonas) appears to be under the control of mechanistically very similar sets of molecules—transcription factors and pheromones and pheromone receptors. This may reflect the evolutionary age of mating-type control systems, with binary mating types of fungi and algae being of ancient origin and present in the most basal lineages, suggesting the possibility of monophyly. However, the relatively recent origin of a molecular diversity of SI systems scattered throughout different angiosperm groups supports a view that SI has arisen many times during angiosperm evolution and is therefore polyphyletic (Charlesworth and Charlesworth, 1979, Charlesworth, 1995; Hiscock et al., 1996; Barrett, 1998). Therefore, does the apparent mechanistic and partial molecular conservation between the various mating-type systems in ascomycetes and basidiomycetes indicate a monophyletic origin of mating types? If so, what was or is the ancestral mating-type system? Given the apparent diverse phylogenies of the fungi sensu lato (Bowman et al., 1992; Radford, 1993; Natvig and May, 1996), suppose that the mating-type system of the diploid yeast, S. cerevisiae, represents the closest approximation to the ancestral mating-type condition. We suggest this because all other mating-type systems in the ascomycetes and basidiomycetes share specific classes of molecules in common with S. cerevisiae (Table I). For instance, 움1 homologs

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

249

are encoded by the MAT loci of filamentous ascomycetes and the fission yeast S. pombe, whereas 움2 homologs are encoded by the MAT loci of S. pombe and the basidiomycetes, but a1 homologs are only found in basidiomycetes. Agreeably, the phylogenies of these quite distant groups of fungi are not well designated (Bowman et al., 1992; Radford, 1993), and it is very unlikely that S. cerevisiae represents a basal lineage, so the molecular similarities might represent convergence through independent acquisition of the same class of transcription factor by different fungal clades (Bell, 1993). However, the striking conservation of these specific classes of matingtype protein between disparate fungal lineages needs explanation. Current molecular phylogenies indicate that the ancestor of S. cerevisiae must have been a haploid, so one might expect that the ancestral mating-type system may have become obscured (through modification) during the transition from a haplontic to a diplontic life cycle. Further modification might also have arisen as a consequence of the apparent complete duplication of the S. cerevisiae genome during its early evolution followed by its subsequent erosion, through deletions, to its current status (Wolfe and Shields, 1997; Seoighie and Wolfe, 1998). Thus, the common ancestor of S. cerevisiae and the later lineages that gave rise to the haplontic yeast S. pombe, filamentous ascomycetes, and basidiomycetes might well have contained a prototype version of this highly adapted mating-type system that was functional in a haplont. In the haplontic S. pombe, mating type is controlled in a very similar manner to that of S. cerevisiae and certain elements are common to both systems: Like S. cerevisiae, S. pombe contains two silenced loci containing copies of the two mating-type idiomorphs which can only be expressed when translocated to an active locus (see Section II,B,4,a). The P idiomorph of S. pombe is closely related to the Mat움 idiomorph of S. cerevisiae, whereas the M idiomorph contains genes which are clearly unrelated to any S. cerevisiae mating-type genes. Thus, a1 was either not acquired by S. pombe or has been lost after its divergence from the common ancestor with S. cerevisiae. Instead, the additional Mm function of S. pombe permits autocrine pheromone reactions to occur after cellular fusion, which ensures that no mitotic divisions take place in the diploid state and that meiosis proceeds directly after cell fusion (see Section II,B,4,b). The system in S. pombe is in turn very closely related to the mating-type systems of filamentous ascomycetes (see Sections II,B,4,b and II,B,4,c). In the simplest mating-type system of for instance C. heterostrophus, neither idiomorph contains a homeodomain gene, indicating that this lineage either did not acquire such a gene or that the homeodomain genes have been lost. Despite lacking the homeodomain genes, the presence of an 움1 domain gene at one idiomorph and an HMG box gene at the other idiomorph are characteristics reminiscent of S. pombe. Strikingly, the 움1 domain proteins and the HMG box proteins of S. pombe and C. heterostrophus, and the

250

SIMON J. HISCOCK AND URSULA KU¨ES

equivalent proteins from Podospora and Neurospora, all determine cell fusion, whereas the extra mating-type proteins in S. pombe (Pm and Mm) and Podospora and Neurospora are all proteins acting after fertilization. In P. anserina, N. crassa, and other ascomycetes the complexity of the mating system has been increased over time with the acquisition of another HMG box gene and a gene encoding an uncharacterized protein at the idiomorphic locus that encodes the 움1 protein (see Section II,B,4,c). Regarding the basidiomycetes, the presence of complete HD1–HD2 gene pairs at the matT loci strongly suggests that basidiomycete mating-type loci share a common ancestor with those of S. cerevisiae. In basidiomycetes it might be speculated that the bipolar idiomorphic mating system present in the ascomycetes has been modified into a multiple mating-type system based on an allelic series of genes, possibly as a consequence of selection for greater mating potential (Hurst, 1996). To establish a functional mating system based on pairs of HD1–HD2 genes present in the same haplotype would have required repeated silencing and activation events to eliminate compatible interactions between gene pairs within the same haplotype and to establish compatible interactions between gene pairs from different haplotypes. Increased outcrossing rates coupled with immediate reduction in costs of finding a mate probably drove selection to favor the acquisition of increasingly more haplotypes (as well as the modification of allelic gene forms into altermorphs) until an equilibrium was established (see Section II,B,5,a). In basidiomycetes a further allelic series of mating-type genes, encoding pheromones and their receptors, has also been acquired and elaborated. In tetrapolar species, such as C. cinereus and S. commune, these genes form a separate unique locus—the B mating-type locus which is situated on a separate chromosome from the A locus (North, 1990; Raper, 1990). The genes encoding these pheromones and pheromone receptors must represent the descendents of the once independent pheromone and pheromone receptor genes that direct mating under the control of the idiomorphic mat loci in the ascomycetes. As such, they offer an example of the direct recruitment and installation of originally independent genes to mating-type status. In contrast to the situation in fungi, in which similar classes of molecule control mating type, SI in flowering plants is mediated by a diversity of signaling molecules. RNases, Ca2⫹-signaling involving protein kinases, and possibly thioredoxins participate in various forms of GSI, whereas a receptor kinase mediates SSI in Brassica and recent molecular studies suggest other molecules mediate SSI in the Convolvulaceae (Y. Kowyama, personal communication) and Asteraceae (S. J. Hiscock, unpublished results). This molecular diversity combined with the scattered phylogenetic distribution of SI systems among flowering plant families (Fig. 10) clearly points to a polyphyletic origin of SI systems (Charlesworth and Charlesworth, 1979;

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

251

Newbigin, 1996; Hiscock et al., 1996). It has been suggested that certain SI systems (notably, the S-RNase and S-receptor kinase systems) may have evolved through recruitment of molecules involved in host–pathogen defense responses (Hodgkin et al., 1988; Dickinson, 1994). Such speculation is supported by the finding that the expression of a gene (NE ) encoding an S-like RNase from tobacco is upregulated in response to infection by pathogenic Phytophthora sp. (Galiana et al., 1997) and that the S receptor kinase of Brassica belongs to a family of S-like receptor kinases, one member of which appears to be involved in cellular responses to pathogen attack and wounding (Pastuglia et al. 1997a). Indeed, Brassica SRK and SLG are just part of a superfamily of S-like genes present within monocots and dicots which are expressed predominantly in vegetative tissues (Dwyer et al., 1994; Braun and Walker, 1996; Pastuglia et al., 1997a). These S-like genes have been implicated in various cellular and developmental roles outside of reproduction and so could represent ancestral lineages from which the Brassica SRK/SLG system was derived. Interestingly, in Arabiodopsis, which is self-compatible, there is no evidence for the existence of closely linked pairs of SRK-like and SLG-like genes (Dwyer et al., 1994) and the genomic region homeologous to the S locus has been deleted (Conner et al., 1998). Therefore, it is likely that the functional pairing of SLG and SRK, probably as a consequence of duplication of SRK and subsequent deletion of the transmembrane domain and kinase domain to form SLG (Tantikanjana et al., 1993), was an event unique to the functional evolution of this SI system. Interestingly, however, alternative splicing of ARK1 transcripts has been shown to produce a functional receptor and a secreted form of the extracellular domain, analogous to SRK and SLG, which may function together in a developmental signaling pathway (Tobias and Nasrallah, 1996). The functional requirement of alternative transcripts of an S-domain kinase may reflect an initial stage in the evolution of the SRK/SLG signaling unit, subsequently elaborated by duplication. In principle, any molecule with appropriate function could be recruited into SI provided it somehow became linked to the sexual cycle. Thus, any group of signaling molecules, be they involved in pathogen defense, development, or environment-induced responses, could have given rise to an SI system if they acquired the necessary pollen and pistil specificity elements. Such specificities might arise through virtue of a role in the control of developmental processes in male and female reproductive tissues or through a role in the control of the general complementary recognition processes that allow stigmas to discriminate pollen, particularly those involved in interspecific recognition. Indeed, Pandey (1979, 1980, 1981) proposed that SI evolved through elaboration of ancient interspecific incompatibility systems that developed in the gymnosperm-like ancestors of the angiosperms as a means to protect their ovules from contamination by

252

SIMON J. HISCOCK AND URSULA KU¨ES

rogue pollens dispersed through the nondiscriminatory agency of wind pollination. The widespread occurrence of interspecific incompatibility in gymnosperms (Hagman, 1975) and the recent report of a putative SI system in pine (Runions and Owens, 1998) lend some support to this hypothesis, and a growing body of evidence clearly demonstrates a role for S gene products, most notably S-RNases (Murfett et al., 1996), in interspecific incompatibility phenomena (Hiscock et al., 1998). However, whether the role of S genes in interspecific incompatibilities is an ancestral or a derived condition remains to be determined. It is very unlikely that the roots of SI evolution will be found by studying the reproductive systems of extant gymnosperms, but given the widely held belief that part of the success story of the angiosperms is attributable to the early acquisition of SI (Whitehouse, 1950), at least one extant system of SI, probably of the late-acting type (Sage et al., 1994), might have its origins in the gymnosperms and the naked ovules of gymnosperms will be more amenable to pollination studies of this form of self-sterility than the enclosed ovules of angiosperms. Population genetic theorists have addressed the conditions necessary for the evolution of SI from a SC ancestral population with no taxonomic rank and have generally shown that inbreeding depression and selfing in the SC population are necessary to maintain the invasion of SI mutants (Charlesworth and Charlesworth, 1979; Uyenoyama, 1991). Uyenoyama (1991) proposed that SI evolved as a means for preferential investment of the maternal plant in fitter offspring and suggested that SI evolves slowly from SC by gradually increasing in strength as a consequence of the action of modifier loci. Indeed, there is much evidence for the existence of such modifier loci (de Nettancourt, 1977; Hinata et al., 1983; Nasrallah, 1989; Nasrallah et al., 1992; Ai et al., 1991), which have also been proposed to be important in maintaining a flexibility within the breeding system of natural populations by allowing weakening of SI under certain selection pressures whilst allowing reversion to full SI when selection once again favors SI (Charlesworth et al., 1990; Levin, 1996). Likewise in fungi there is much flexibility within the breeding systems of natural populations, with individuals being heterothallic, homothallic, or pseudohomothallic, and switching from one reproductive mode to another can take place (Coppin et al., 1997) although, as in flowering plants, evolution of SC from SI is theoretically and empirically the most likely event (Nauta and Hoekstra, 1992; Coppin et al., 1997). Indeed, in fungi and angiosperms self-compatibility (homothallism in fungi) has evolved many times in originally self-incompatible (heterothallic) lineages, and so is considered a derived condition (Charlesworth and Charlesworth, 1979; Jarne and Charlesworth, 1993; Nauta and Hoekstra, 1992; Coppin et al., 1997; Turgeon, 1998, personal communication. Nevertheless, in fungi there is also evidence for reversion to heterothallism from homothallism (Harrington and McNew, 1997; Geiser et al., 1998) (an event

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

253

equivalent to SC reverting to SI in flowering plants—an event never documented and thought most unlikely), but this phenomenon is probably a consequence of genetic events akin to mating-type switching. What was the identity of the theoretical SC populations from which SI angiosperms arose? A polyphyletic origin of SI is generally inconsistent with an origin of SI in the gymnosperms or protoangiosperms (unless we consider specifically late-acting SI), so we must examine the evolution of most SI systems within angiosperm lineages. Sequence analyses of S alleles predict an ancient origin of GSI and SSI: The S-RNase system of GSI present in the Solanaceae, Scrophulariaceae, and Rosaceae is predicted to be approximately 70 million years old (Xue et al., 1996) and the SSI system of Brassica to be at least 50 million years old (Uyenoyama, 1995). As more S allele sequences become available from species within these families, and from other families with GSI and SSI, more insights into the evolution of SI systems will become possible and questions such as the homology of the three S-RNase systems can be addressed. In a recent extensive analysis of S-RNase gene sequences, Richman et al. (1996c) provided strong support for homology between the Solanaceous and Scrophulariaceous S-RNase sequences but were unable to infer homology or convergence for these sequences and those from the Rosaceae. They predicted that if the three S-RNase lineages are homologous, then because of the taxonomic distance between the Solanaceae and Scrophulariaceae and the Rosaceae S-RNasebased systems of SI must be present in many other dicot families that share a common lineage. With this point in mind, it has been estimated that the divergence of the Rosaceae and Brassicaceae took place ⬍70 million years ago (Chase et al., 1993), so if the S-RNase systems of the Rosaceae, Scrophulariaceae, and Solanaceae are homologous, one might predict that the Brassicaceae would contain the remnants of a gametophytic S-RNase system underlying the SSI system of SLGs and SRKs. A simpler alternative is that the S-RNase systems represent convergence. However, genetic evidence suggests that in Brassica there is a cryptic gametophytic system of SI operating beneath the sporophytic SI system (Zuberi and Lewis, 1988; Lewis et al., 1988; Lewis, 1994), indicating that a sporophytic system of SI may have superceded an ancestral gametophytic system. The nature of the gametophytic gene products is unknown. Interestingly, genetic evidence from the Caryophyllaceae suggests that SI systems may have mixed gametophytic and sporophytic determinants (Lundqvist, 1990, 1994, 1995), which would make it difficult to determine whether one system was in the process of superceding the other or merely part of it. In this context the first evidence that two genetically different SI systems can be present in the same family, the Polemoniaceae [GSI in Phlox drummondiae (Levin, 1993) and SSI in Linanthus parviflorus (Goodwillie, 1997)], is very important because it indicates that selection may favor the transition from one SI system to

254

SIMON J. HISCOCK AND URSULA KU¨ES

another under different population or environmental conditions. It is usually assumed that it is more likely for SSI to evolve from GSI (by earlier expression of the pollen S component) than vice versa (de Nettancourt, 1977) and it might be supposed that selection will tend to favor evolution from GSI to SSI because SSI is a more efficient system at regulating sib matings and restricting biparental inbreeding depression than is GSI (Lewis, 1979a; Hiscock et al., 1994). The apparently recent transition from GSI to SSI in Linanthus (Goodwillie, 1997) suggests that selection for SSI may indeed be very strong, such that the evolution of SSI could be swift and complete even after the divergence of a particular family lineage—a fact formerly thought to be very unlikely, given the ancient nature of extant S alleles (Uyenoyama, 1995; Clark and Kao, 1996; Nasrallah, 1997). The finding of GSI in P. drummondiae and SSI in L. parviflorus is even more surprising given that Phlox and Linanthus are sister genera and therefore closely related (Goodwillie, 1997). This suggests that the tempo of evolution of SI systems can increase depending on different selection pressures and that only by studying SI at a molecular level in diverse families and natural populations can we begin to understand its evolution and evolutionary flexibility.

B. Diversification of Mating-Type Altermorphs and S Alleles It is estimated that with unrestricted spore dispersal the multialleleic mating-type systems of basidiomycete fungi approach, if not equal, the outcrossing efficiency of multiallelic SI systems in flowering plants (Lewis, 1979a; Hiscock et al., 1994). The evolution and maintenance of allelic diversity is essential to the effectiveness of the outbreeding system and in both basidiomycetes and angiosperms this extreme polymorphism is expected to be promoted and maintained by frequency-dependent selection (Wright, 1939; Charlesworth and Charlesworth, 1979; Vekemans and Slatkin, 1994; Zambino et al., 1997; Badrane and May, 1999; G. May et al., manuscript in preparation). Thus, any new allelic (altermorphic) specificity that arises within a population will be at an immediate advantage because it will permit mating with all other mating types and will spread quickly within the population until it reaches an equilibrium with the other alleles (Wright, 1939). However, the strength of selection declines with the increased number of alleles leaving the extreme polymorphism of most systems unexplained by simple frequency-dependent selection models (G. May et al., manuscript in preparation). Analyses of S allele sequences from the Solanaceae and Brassicaceae revealed that the majority of S allele diversification in these families took place shortly after their inception because many alleles tend to be more similar between species and genera than they are within species, indicating that allelic diversification occurred prior to

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

255

speciation (Ioerger et al., 1990; Dwyer et al., 1991; Clark and Kao, 1991; Uyenoyama, 1995; Sakamoto et al., 1998). Similar studies in fungi have also revealed that most mating type altermorph polymorphisms are extremely old (Zambino et al., 1997; Badrane and May, 1999; G. May et al., manuscript in preparation) and reflect data from similar highly polymorphic vegetative incompatibility systems in fungi (Wu et al., 1998) and genetic systems in animals, such as the MHC system (Ayala et al., 1994; Ayala Escalante, 1996). Notwithstanding the apparent age of S sequence polymorphisms, because such sequence data are available for a limited number of (mainly cultivated) species from just a few families of angiosperms and detailed phylogenetic analysis of mating-type altermorph sequences is in its infancy, one cannot rule out the possibility of recent bursts of allelic divergence in natural populations perhaps as a consequence of population size fluctuations, particularly population ‘‘bottlenecks’’ as might be found in small founder populations (Richman and Kohn, 1996; Richman et al., 1996c; Newbigin, 1996). It might also be predicted that reduced population size has also been an important catalyst for diversification of certain matingtype altermorphs in fungi (Zambino et al., 1997). As more S alleles and mating-type altermorphs are sequenced it will become possible to address two fundamental questions concerning their diversification: How is the extreme allelic/altermorphic diversity generated? And what is the frequency with which new allelic/altermorphic specificities are generated? Clues to the mode of altermorphic diversification in fungi have come from sequence analysis of A mating-type gene pairs in natural populations of C. cinereus, in which mating-type diversity appears to have arisen by repeated mutations resulting in amino acid substitutions at the N-terminal regions of the A proteins (Badrane and May, 1998) and also as a consequence of recombination in regions separating the two A matingtype subloci (May and Matzke, 1995; Lukens et al., 1996). Evidence from U. maydis also suggests that recombination between b altermorphs has played a key role in the recent diversification of these altermorphs because homologous recombination events have been detected between short stretches of homologous sequence at the 5⬘ specificity-determining ends of altermorphic gene pairs ( J. Ka¨mper, personal communication). Similar recombination events, involving short stretches of homology in paralogous gene pairs of more complex mating-type loci, might result in the exchange of regions responsible for gene specificity, which could account for the rare cases of changes in mating-type specificity (mating-type switching) observed in the mushroom Agrocybe aegerita. This phenomenon was previously considered analogous to mating-type switching in yeasts (Labare`re and Noe¨l, 1992; see Sections II,B,4,a and II,B,4,b). There is also evidence from Coprinus that diversification has occurred through duplication of gene pairs. Importantly, this process may still be ongoing in Coprinus because, in

256

SIMON J. HISCOCK AND URSULA KU¨ES

addition to the three active paralogous pairs of HD1–HD2 genes that may be present at the A locus (all three complete pairs of genes have only been identified for one A haplotype), additional HD1 or HD2 genes have occasionally been observed in certain A haplotypes (Fig. 7A). Notable examples are the untranscribed HD2 gene e1-2 at the 움 sublocus of A6 and A42 and the HD1 gene c1-1 of A42; neither of these genes appear to have any partner in any other A haplotype (Ku¨es et al., 1992, Ku¨es and Casselton, 1993b; Casselton and Ku¨es, 1994; Pardo et al., 1996). The unusually high sequence homology between e1-2 and a1-2 suggests that e1-2 is a duplicated version of a1-2, while other evidence indicates that c1-1 may have arisen via a duplication of the d locus (Casselton and Ku¨es, 1994; U. Ku¨es, unpublished data). To date, just two different d loci have been identified—one contains only a single HD1 gene (d1-1) whereas the other locus contains a single HD2 gene (d2-2; Pardo et al., 1996). Because c1-1 has only been observed in the presence of d1-1, it is conceivable that c1-1 is the original partner (d1-2) of d2-2. ‘‘Footprints’’ of c1-1 and other related sequences within A43 suggest that the d-pair region is a hot spot for insertions and deletions of the third gene pair (Ku¨es et al., 1994b; Ku¨es, 1995; U. Ku¨es, unpublished data). Thus, in fungi we can see potential mechanisms for accelerated generation of altermorphic diversity. In angiosperms fewer insights into the mechanism(s) of allelic diversification have been forthcoming. Nevertheless, a number of mechanisms for the generation of S allele diversity have been proposed, such as mutations (Lewis, 1948, 1949b), homologous recombination (Fisher, 1961), and gene conversion (Ebert et al., 1989; Kaufmann et al., 1991). In an attempt to generate new S specificities by mutagenesis Lewis (1948, 1949b, 1951) and Pandey (1965) could only generate SC mutations affecting either pollen or pistil but never recovered any new S alleles. Similar attempts to generate new mating-type specificities in fungi (Raper, 1966; Raper, 1983; Casselton and Ku¨es, 1994; Kahmann et al. 1995) also proved fruitless. Clark and Kao (1994) stress that because most of the S allele sequences currently available appear to be extremely ancient, it is almost impossible to determine whether recombination has played a role in their diversification because any past recombination events will have been obscured by recent point mutations. However, in a recent detailed analysis of the number of substitutions in the hypervariable regions of 42 SLGs and 7 SRKs from Brassica, Kusaba et al. (1997) showed that extensive intragenic recombination has occurred within these genes and this recombination, together with single base pair substitutions, insertions, and deletions, can account for all the observed polymorphism between these genes. Thus, recombination appears to have been an important element for generating diversity among S alleles and mating-type altermorphs.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

257

The inability to generate new S specificties by mutagenesis and the apparently very low generation time of new alleles in populations (Xue et al., 1996) can be explained if one assumes that more than one mutation or recombination event is needed to change S specificity, which would be expected if the male and female S products are derived from different genes. Mutation of the male or female S component might be predicted to change the selection pressure on mutations to the corresponding male or female component. Thus, in the S-RNase system, assuming the cytotoxic model and the pollen component to be an S-RNase inhibitor (see Section IV,E,I), it is possible to imagine a constant war of attrition between the pollen anti-S-RNase defenses and the ‘‘stealth’’ capability of specific S-RNases, like the gene-for-gene interactions in host–parasite interactions. Selection for S-RNase inhibitors that acquire an extra recognition element allowing them to overcome the stealth capability of their allelic S-RNases would be at an immediate advantage and would spread rapidly within a population. Plants with the new S-RNase inhibitor, however, would be self-compatible and carry a redundant S-RNase. One might then imagine selection for a mutant S-RNase with a new stealth element (allowing it to evade detection by the new inhibitor) as a consequence of the increase in inbreeding depression in the progeny of individuals derived from the SC pollen mutant. Such a scenario could eventually lead to the full evolution of a new S-allelic specificity. Xue et al. (1996) estimated that in the Solanaceae and Scrophulariaceae such new alleles have arisen at a rate of one allele every million years since the divergence of these families 앑40 million years ago, which is in accordance with previous theoretical estimates for the evolution of gametophytic S alleles (Vekemans and Slatkin, 1994). However, recent evidence from analyses of S-RNase sequences obtained from natural populations of Solanaceous species suggests that in certain circumstances S allele diversification may have proceeded more rapidly than in the estimates of Xue et al. (1996). In an illuminating series of papers, Richman and coworkers (Richman et al., 1995, 1996a,b,c; Richman and Kohn, 1996) describe the use of PCR methodology to sample S-RNases from wild populations of Solanum carolinense and Physalis crassifolia. Surprisingly, the S-RNase sequences from P. crassifolia were found to cluster into two divergent groups nested within the rest of the Solanaceous S alleles included in their phylogenetic analysis of 63 S alleles (Richman and Kohn, 1996; Richman et al., 1996c). The finding of relatively large numbers of S alleles specific to a single species indicates that these alleles probably arose much more recently than other Solanaceous S alleles which are characteristically scattered between different species with only limited species-specific clustering (Clark and Kao, 1994). Indeed, Richman and Kohn (1996) speculated that past population bottlenecks might have increased the tempo of S allele evolution in this species. It will therefore be extremely interesting

258

SIMON J. HISCOCK AND URSULA KU¨ES

to compare these P. crassifolia S-RNase sequences (especially the cluster of 24 alleles) in detail because any recent recombination events or other structural changes to these sequences will be apparent and will surely yield important insights into the mechanism(s) of allelic diversification.

VI. Prospect Clearly, more is known about the control of mating type at the molecular level in fungi, particularly ascomycetes and basidiomycetes, than is known about the control of mating type in algae or the control of self-incompatibility in flowering plants. However, the cell biology of SI is more clearly understood than is the cell biology of mating-type determination in filamentous ascomycetes and basidiomycetes. Major future goals in mating-type research will thus be the localization of the pheromone receptors and transcription factors in these fungi, which will give insights into the role of pheromones in directed cell growth (including homing responses), hyphal and cell fusions, and the role of transcription factors in nuclear marking during mating-type determination. The major goal of SI research is undoubtedly to discover the nature of various pollen S components which will allow current mechanistic models of SI to be tested, particularly regarding the controversy surrounding the workings of the cytotoxic model of GSI in species in which the stylar S product is an RNase. Likewise, in the sporophytic system of Brassica, identification of the pollen-coating borne S component will allow its site of expression to be determined and will confirm or refute predictions of pseudosporophytic control of SSI (see section IV,D,3). Molecular-based investigations of SSI in families other than the Brassicaceae, such as those in progress in the Convolvulaceae and Asteraceae, will also shed important insights into the molecular diversity of SSI. In mating-type research rapid progress is being made toward identification of downstream components of the pheromone signaling system, mainly by virtue of the apparent conservation of such signaling cascades between S. cerevisiae and S. pombe for which many components have already been characterized, and the systems found in other fungi. It will also be important to build on knowledge of how molecular dialogue takes place between the matP genes and the matT genes in basidiomycetes and to establish the nature of the genes targeted by mating-type-specific transcription factors. Further exciting insights into the control of mating and signal transduction in fungi will surely come from studies of how particular environmental stimuli, such as nitrogen starvation and light, are perceived and exert their effects on the sexual cycle via interaction with components of the matingtype system.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

259

Studies of mating type in Chlamydomonas are providing important insights into how sexual reproduction may have evolved and are confirming theoretical predictions about the origins of binary idiomorphic mating-type systems. Exciting questions as to how these mat loci control organelle inheritance and cell and nuclear fusion will no doubt be addressed as more genes from the mat loci are characterized. In a broader evolutionary context, as more sequence data become available for S genes and mating-type genes from closely related and distantly related species, particulary from natural populations, more insights into the evolution of these genes will be possible. Studies of mating-type sequence diversity among basidiomycetes such as Ustilago and Coprinus (see Section V,B) have already shown that regions of these genes are highly mutable and can undergo recombination and duplication readily, indicating that despite the apparent ancient nature of mating-type altermorphs, mechanisms for the generation of new mating-type specificities are present, suggesting that the tempo of altermorph diversification does not adhere to a ‘‘molecular clock’’ and can speed up or slow down. Similar data have been forthcoming from studies of S allele sequence diversity in the wild Solanaceous species Physalis, in which S allele diversification has apparently occurred relatively recently, and analyses of Brassica S allele sequences have implicated localized intragenic recombination as a possible mechanism for allelic diversification (see Section V,B). More studies of this nature are essential before we can begin to unravel the molecular complexities intrinsic to the evolution of multiple mating-type systems in fungi and multiallelic self-incompatibility systems in flowering plants.

Acknowledgments We thank Hassan Badrane, Deborah Bell-Pedersen, Evelyne Coppin, Robert Debuchy, Hugh Dickinson, James Doughty, Chris Franklin, Louise Glass, John Golz, Jo¨rg Ka¨mper, Jim Kronstad, Erika Kothe, Yasuo Kowyama, Georgiana May, Ed Newbigin, and Gillian Turgeon for communication of results prior to publication and for reprints of their work; Rob Boulianne, Georgiana May, Suzanne O’Shea, and Yan Wong for discussions and critical reading of the manuscript; Johannes Wo¨stemeyer for helpful comments on the manuscript and for supplying Fig. 3; Paresh Shah for translating French texts into English; and our current and former colleagues in the labs for their work and support. S. J. H. is a BBSRC David Phillips Fellow. Work by U. K. was supported by a Violette and Samuel Glasstone Fellowship from the University of Oxford, by the Swiss National Foundation (Grant 31-46⬘940.96 to U. K. and M. Aebi), and by grants from the ETH Zu¨rich.

References Abe, K., Kusaka, I., and Fukui, S. (1975). Morphological change in the early stages of the mating process of Rhodosporidium toruloides. J. Bacteriol. 122, 710–718.

260

SIMON J. HISCOCK AND URSULA KU¨ES

Ahmad, S. S., and Miles, P. G. (1970). Hyphal fusions in the wood-rotting fungus Schizophyllum commune. 1. Effects of incompatibility factors. Genet. Res. 15, 19–28. Ai, Y., Singh, A., Coleman, C. E., Ioerger, T. R., Kheyr-Pour, A., and Kao, T.-H. (1990). Self-incompatibility in Petuania inflata: Isolation and characterization of cDNAs encoding S-allele-associated proteins. Sex. Plant Reprod. 3, 130–138. Akada, R., Minomi, K., Kai, J., Yamashita, I., Miyakawa, T., and Fukui, S. (1989). Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides. Mol. Cell. Biol. 9, 3491–3498. Albert, H. H., and Schenck, S. (1996). PCR amplification from a homolog of the bE matingtype gene as a sensitive assay for the presence of Ustilago scitaminea DNA. Plant Dis. 80, 1189–1192. Alspaugh, J. A., Perfect, J. R., and Heitman, J. (1997). Cryptococcus neoformans mating and virulence are regulated by the G-protein 움 subunit GPA1 and cAMP. Genes Dev. 11, 3206–3217. Anderson, D. H., Weiss, M. S., and Eisenberg, D. (1997). Charges, hydrogen bonds, and ˚ resolution refined structure of the mating pheromone Er-1 correlated motions in the 1 A from Euplotes raikovi. J. Mol. Biol. 273, 479–500. Anderson, M. A., Cornish, E. C., Mau, S.-L., Williams, E. G., Hoggart, R., Atkinson, A., Bonig, I., Grego, B., Simpson, R., Roche, P. J., Haley, J. D., Penschow, J. D., Niall, H. D., Tregear, G. W., Cochlan, J. P., Crawford, R. J., and Clarke, A. E. (1986). Cloning of a cDNA for a stylar glycoprotein associated with expression of self-incompatibility in Nicotiana alata. Nature 321, 38–44. Anderson, M. A., McFadden, G. I., Bernatzky, R., Atkinson, A., Orpin, T., Dedman, H., Tregear, G. W., Fernley, R., and Clarke, A. E. (1989). Sequence variability of three alleles of the self-incompatibility gene of Nicotiana alata. Plant Cell 1, 483–491. Aono, T., Yanai, H., Miki, F., Davey, J., and Shimoda, C. (1994). Mating pheromone-induced expression of the mat1-Pm gene of Schizosaccharomyces pombe: Identification of signalling components and characterization of signalling components and characterization of upstream controlling elements. Yeast 10, 757–770. Arcangioli, B. (1998). A site- and strand-specific DNA break confers asymmetric switching potential in fission yeast. EMBO J. 17, 4503–4510. Arie, T., Christiansen, S. K., Yoder, O. C., and Turgeon, B. G. (1997). Efficient cloning of ascomycete mating type genes by PCR amplification of the conserved MAT HMG box. Fungal Genet. Biol. 21, 118–130. Arnaise, S., Zickler, D., and Glass, N. L. (1993). Heterologous expression of mating-type genes in filamentous fungi. Proc. Natl. Acad. Sci. USA 90, 6616–6620. Arnaise, S., Debuchy, R., and Picard, M. (1997). What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina. Mol. Gen. Genet. 256, 169–178. Asada, Y., Yue, C., Wu, J., Shen, G.-P., Novotny, C. P., and Ullrich, R. C. (1997). Schizophyllum commune A움 mating-type proteins, Y and Z, form complexes in all combinations in vitro. Genetics 147, 117–123. Asante-Owusu, R. N., Banham, A. H., Bo¨hnert, H. U., Mellor, E. J. C., and Casselton, L. A. (1996). Heterodimerization between two classes of homeodomain proteins in the mushroom Coprinus cinereus brings together potential DNA-binding and activation domains. Gene 172, 25–31. ´ sgeirsdo´ttir, S. A., van Wetter, M. A., and Wessels, J. G. H. (1995). Differential expression A of genes under control of the mating-type genes in the secondary mycelium of Schizophyllum commune. Microbiology 141, 1281–1288. Astell, C. R., Ahlstrom-Jonasson, L., Smith, M., Tatchell, K., Nasmyth, K. A., and Hall, B. D. (1981). The sequence of the DNAs coding for the mating-type loci of Saccharomyces cerevisiae. Cell 27, 15–23.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

261

Ayala, F. J., and Escalante, A. A. (1996). The evolution of human populations: A molecular perspective. Mol. Phylogenet. Evol. 5, 188–201. Ayala, F. J., Escalante, A., O’Huigin, C., and Klein, J. (1994). Molecular genetics of speciation and human origins. Proc. Natl. Acad. Sci. USA 91, 6787–6794. Baasiri, R. A., Lu, X., Rowley, P. S., Turner, G. E., and Borkovich, K. A. (1997). Overlapping functions for two G protein 움 subunits in Neurospora crassa. Genetics 147, 137–145. Badrane, H., and May, G. (1999). The divergence–homogenization duality in the evolution of the b1 mating type of Coprinus cinereus. Submitted for publication. Bailey, J. (1995). Plasmodium development in the myxomycete Physarum polycephalum, genetic control and cellular events. Microbiology, 141, 2355–2365. Bakkeren, G., and Kronstad, J. W. (1993). Conservation of the b mating-type gene complex among bipolar and tetrapolar smut fungi. Plant Cell 5, 123–136. Bakkeren, G., and Kronstad, J. W. (1994). Linkage of mating-type loci distinguishes bipolar from tetrapolar mating in basidiomycetous smut fungi. Proc. Natl. Acad. Sci. USA 91, 7085– 7089. Bakkeren, G., and Kronstad, J. W. (1996). The pheromone cell signalling components of the Ustilago a mating type loci determine intercompatibility between species. Genetics 143, 1601–1613. Bandoni, R. J. (1963). Conjugation in Tremella mesenterica. Can. J. Bot. 41, 467–474. Bandoni, R. J. (1965). Secondary control of conjugation in Tremella mesenterica. Can. J. Bot. 43, 627–630. Banham, A. H., Asante-Owusu, R. N., Go¨ttgens, B., Thompson, S. A. J., Kingsnorth, C. S., Mellor, E. J. C., and Casselton, L. A. (1995). An N-terminal dimerization domain permits homeodomain proteins to choose compatible partners and initiate sexual development in the mushroom Coprinus cinereus. Plant Cell 7, 773–783. Banuett, F. (1995). Genetics of Ustilago maydis, a fungal pathogen that induces tumors in maize. Annu. Rev. Genet. 29, 179–208. Banuett, F. (1998). Signalling in the yeasts: An informational cascade with links to the filamentous fungi. Microbiol. Mol. Biol. Rev. 62, 249–274. Banuett, F., and Herskowitz, I. (1989). Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis. Proc. Natl. Acad. Sci. USA 86, 5878–5882. Banuett, F., and Herskowitz, I. (1994a). Identification of Fuz7, a U. maydis MEK/MAPKK homologue required for a-locus-dependent and -independent processes in the fungal life cycle. Genes Dev. 8, 1367–1378. Banuett, F., and Herskowitz, I. (1994b). Morphological transitions in the life cycle of Ustilago maydis and their genetic control by the a and b loci. Exp. Mycol. 18, 247–266. Banuett, F., and Herskowitz, I. (1996). Discrete developmental stages ensuring teliospore formation in the corn smut fungus, Ustilago maydis. Development 22, 2965–2976. Bardwell, L., Cook, J. G., Inouye, C. J., and Thorner, J. (1994). Signal propagation and regulation in the mating pheromone response pathway of the yeast Saccharomyces cerevisiae. Dev. Biol. 166, 363–379. Barrett, S. C. H. (1998). The evolution of mating strategies in flowering plants. Trends Plant Sci. 3, 335–341. Barrett, S. C. H., and Cruzan, M. B. (1994). Incompatibility in heterostylous plants. In ‘‘Genetic Control of Self-Incompatibility and Reproductive Development in Flowering Plants’’ (E.G. Williams, A. E. Clarke and R. B. Knox, Eds.), pp. 189–119, Kluwer, Dordrecht. Barrett, S. C. H., and Richards, J. H. (1990). Heterostyly in tropical plants. Mem. N. Y. Bot. Gard. 55, 35–61. Barton, N. H., and Charlesworth B. (1998). Why sex and recombination? Science 281, 1986– 1990.

262

SIMON J. HISCOCK AND URSULA KU¨ES

Bateman, A. J. (1952). Self-incompatibility systems in angiosperms. I. Theory, Heredity 6, 285–310. Bateman, A. J. (1954). Self-incompatibility systems in angiosperms. II. Iberis amara. Heredity 8, 305–332. Bateman, A. J. (1955). Self-incompatibility systems in angiosperms. III. Cruciferae. Heredity 9, 52–68. Bauch, R. (1932). Untersuchungen u¨ber die Entwicklungsgeschichte und Sexualphysiologie der Ustilago bromivora und Ustilago grandis. Z. Bot. 17, 129–177. Beadle, G. W., and Coonradt, V. L. (1944). Heterocaryosis in Neurospora crassa. Genetics 29, 291–308. Beatty, N. P., Smith, M. L., and Glass, N. L. (1994). Molecular characterization of matingtype loci in selected homothallic species of Neurospora, Gelasinospora and Anxiella. Mycol. Res. 98, 1309–1316. Beck, C. F., and Haring, M. A. (1996). Gametic differentiation in Chlamydomonas. Int. Rev. Cytol. 168, 259–302. Beckerman, J. L., Naider, F., and Ebbole, D. J. (1997). Inhibition of pathogenicity of the rice blast fungus by Saccharomyces cerevisiae 움-factor. Science 276, 1116–1119. Bell, G. (1993). The sexual nature of the eukaryotic genome. J. Hered. 84, 351–359. Bender, A., and Sprague, G. F., Jr. (1989). Pheromones and pheromone receptors are the primary determinants of mating specificity in the yeast Saccharomyces cerevisiae. Genetics 121, 463–476. Berkower, C., and Michaelis, S. (1991). Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily. EMBO J. 10, 3777– 3785. Berteaux-Lecellier, V., Zickler, D., Debuchy, R., Panvier-Adoutte, A., Thompson-Coffe, C., and Picard, M. (1998). A homologue of the yeast SHE4 gene is essential for the transition between the syncytical and cellular stages during sexual reproduction of the fungus Podospora anserina. EMBO J. 17, 1248–1258. Betterley, D. A., and Collins, O. R. (1983). Reproductive systems, morphology, and genetic diversity in Didymium iridis (myxomycetes). Mycologia 75, 1044–1063. Bharathan, G., Janssen, B. J., Kellogg, E. A., and Sinha, N. (1997). Did homeodomain proteins duplicate before the origin of angiosperms, fungi, and metazoa? Proc. Natl. Acad. Sci. USA 94, 13749–13753. Bishir, J., and Namkoong, G. (1987). Unsound seeds in conifers: Estimation of number of lethal alleles and the magnitudes of effects associated with maternal parent. Silvae Genet. 36, 180–185. Bistis, G. N. (1956). Sexuality in Ascobolus stercorarius. I. Morphology of the ascogonium; Plasmogamy; Evidence for a sexual hormonal mechanism. Am. J. Bot. 6, 389–394. Bistis, G. N. (1981). Chemotrophic interactions between trichogynes and conidia of opposite mating type in Neurospora crassa. Mycologia 73, 959–975. Bistis, G. N. (1983). Evidence for diffusible, mating-type-specific trichogyne attractants in Neurospora crassa. Exp. Mycol. 7, 292–295. Bistis, G. N. (1998). Physiological heterothallism and sexuality in Euascomycetes, a partial history. Fungal Genet. Biol. 23, 213–22. Blakeslee, A. F. (1904). Sexual reproduction in the Mucorineae. Proc. Am. Acad. Arts Sci. 40, 205–319. Blinder, D., Bouvier, S., and Jenness, D. D. (1989). Constitutive mutants in the yeast pheromone response: Ordered function of the gene products. Cell 56, 479–486. Bo¨lker, M., and Kahmann, R. (1993). Sexual pheromones and mating response in fungi. Plant Cell 5, 1461–1469. Bo¨lker, M., Urban, M., and Kahmann, R. (1992). The a mating type locus of U. maydis specifies cell signalling components. Cell 68, 441–450.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

263

Boone, C., Davis, N. G., and Sprague, G. F., Jr. (1993). Mutations that alter the third cytoplasmic loop of the a-factor receptor lead to a constitutive and hypersensitive phenotype. Proc. Natl. Acad. Sci. USA 90, 9921–9925. Bottoli, A. P. F., Kertesz-Chaloupkova´, K., Boulianne, R. P., Grando, J. D., Aebi, M., and Ku¨es, U. (1998). Rapid isolation of genes from an indexed library of C. cinereus in a novel pab1⫹-cosmid. J. Microbiol. Methods 35, 129–141. Bower, M. S., Matias, D. D., Fernandes-Carvalho, E., Mazzurco, M., Gu, T., Rothstein, S. J., and Goring, D. R. (1996). Two members of the thioredoxin-h family interact with the kinase domain of Brassica S locus receptor kinase. Plant Cell 8, 1641–1650. Bowman, B. H., Taylor, J. W., Brownlee, A. G., Lee, J., Lu, S.-D., and White, T. J. (1992). Molecular evolution of the fungi: Relationship of the Basidiomycetes, Ascomycetes, and Chytridiomycetes. Mol. Biol. Evol. 9, 285–296. Boyes, D. C., and Nasrallah, J. B. (1993). Physical linkage of SLG and SRK genes at the selfincompatibility locus of Brassica oleraceae. Mol. Gen. Genet. 236, 369–373. Boyes, D. C., and Nasrallah, J. B. (1995). An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts. Plant Cell 7, 1283–1294. Boyes, D. C., Chen, C. H., Tantikanjana, T., Esch, J. J., and Nasrallah, J. B. (1991). Isolation of a second S-locus related cDNA from Brassica oleracea: Genetic relationships between the S-locus and two related loci. Genetics 127, 221–228. Braun, D. M., and Walker, J. C. (1996). Plant transmembrane receptors: New pieces in the signalling puzzle. Trends Biochem. Sci. 21, 70–73. Braun, D. M., Stone, J. M., and Walker, J. C. (1997). Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor-like protein kinases: Implications for transmembrane signalling in plants. Plant J. 12, 83–95. Bredemeijer, M. M., and Blass, C. (1981). S-specific proteins in styles of self-incompatible Nicotiana alata. Theor. Appl. Genet. 59, 185–190. Brewbaker, J. L. (1957). Pollen cytology and self-incompatibility systems in plants. J. Hered. 48, 271–277. Broothaerts, W., Janssens, G. A., Proost, P., and Broekaert, W. F. (1995). cDNA cloning and molecular analysis of two self-incompatibility alleles from apple. Plant Mol. Biol. 27, 499–511. Brownlee, C. (1994). Signal transduction during fertilization in algae and vascular plants. New Phytol. 127, 399–423. Bu¨ku¨s¸og˘lu, G., and Jenness, D. D. (1996). Agonist-specific conformational changes in the yeast 움-factor pheromone receptor. Mol. Cell. Biol. 16, 4818–4823. Bu¨rglin, T. R. (1994). A comprehensive classification of homeobox genes. In ‘‘Guidebook to the Homeobox Genes’’ (D. Duboule, Ed.), pp. 25–72. Oxford Univ. Press, Oxford. Bu¨rglin, T. R. (1997). Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals. Nucleic Acids Res. 25, 4173–4180. Burgos, L., Perez-Tornero, O., Ballester, J., and Olmos, E. (1998). Detection and inheritance of stylar ribonucleases associated with incompatibility alleles in apricot. Sex. Plant Reprod. 11, 153–158. Cabib, E., Drgonova´, J., and Drgon, T. (1998). Role of small G proteins in yeast cell polarization and wall biosynthesis. Annu. Rev. Biochem. 67, 307–333. Caldwell, G. A., Naider, F., and Becker, J. M. (1995). Fungal lipopeptide mating pheromones, a model system for the study of protein prenylation. Microbiol. Rev. 59, 406–422. Casselton, L. A. (1978). Dikaryon formation in higher basidiomycetes. In ‘‘The Filamentous Fungi’’ ( J. E. Smith and D. R. Barry, Eds.), pp. 275–297. Arnold, London. Casselton, L. A., and Ku¨es, U. (1994). Mating type genes in homobasidiomycetes. In ‘‘The Mycota. I. Growth, Differentiation and Sexuality’’ ( J. G. H. Wessels and F. Meinhardt, Eds.), pp. 307–322. Springer-Verlag, Berlin.

264

SIMON J. HISCOCK AND URSULA KU¨ES

Casselton, L. A., and Olesnicky, N. S. (1998). Molecular genetics of mating recognition in basidiomycetes fungi. Microbiol. Mol. Biol. Rev. 62, 55–70. Challen, M. P., Elliott, T. J., Ku¨es. U., and Casselton, L. A. (1993). Expression of A mating type genes of Coprinus cinereus in a heterologous basidiomycetes host. Mol. Gen. Genet. 241, 474–478. Chang, M. T., and Raper, K. B. (1981). Mating types and macrocyst formation in Dictyostelium. J. Bacteriol. 147, 1049–1053. Chang, S., and Staben, C. (1994). Directed replacement of mtA by mta-1 effects a matingtype switch in Neurospora crassa. Genetics 138, 75–81. Charlesworth, D. (1995). Multi-allelic self-incompatibility polymorphisms in plants. Bioessays 17, 31–38. Charlesworth, D., and Charlesworth, B. (1979). The evolution and breakdown of S-allele systems. Heredity 43, 41–55. Charlesworth, D., Morgan, M. T., and Charlesworth, B. (1990). Inbreeding depression with heterozygote advantage and its effect on selection of modifiers changing the outcrossing rate. Evolution 44, 870–888. Chase, M. W., Soltis, D. E., Olmstead, R. G., Morgan, D., Les, D. H., Mishler, B. D., Duvall, M. R., Price, R. A., Hills, H. G., Qiu, Y. L., Kron, K. A., Rettig, J. H., Conti, E., Palmer, J. D., Manhart, J. R., Systma, K. J., Michaels, H. J., Kress, W. J., Karol, K. G., Clark, W. D., Hedre´n, M., Gaut, B. S., Jansen, R. K., Kim, K.-J., Wimpee, C. F., Smith, J. F., Furnier, G. R., Strauss, S. H., Xiang, Q.-Y., Plunkett, G. M., Soltis, P. S., Swensen, S., Williams, S. E., Gadek, P. A., Quinn, C. J., Eguiartre, L. E., Golenberg, E., Learn, G. H., Jr., Graham, S. W., Barrett, S. C. H., Dayanandan, S., and Albert, V. A. (1993). Phylogenies of seed plants: An analysis of nucleotide sequences from the plastid gene rbcL. Ann. Mo. Bot. Gard. 80, 528–580. Chawla, B., Bernatzky, R., Liang, W., and Marcotrigiano, P. (1997). Breakdown of selfincompatibility in tetraploid Lycopersicon peruvianum—Inheritance and expression of Srelated proteins. Theor. Appl. Genet. 95, 992–996. Chen, C. H., and Nasrallah, J. B. (1990). A new class of S-sequences defined by a pollen recessive self incompatibility allele of Brassica oleracea. Mol. Gen. Genet. 222, 241–248. Chen, P., Sapperstein, S. K., Choi, J. C., and Michaelis, S. (1997). Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor. J. Cell Biol. 136, 251–269. Chen, Q., and Konopka, J. B. (1996). Regulation of the 움-factor receptor by phosphorylation. Mol. Cell. Biol. 16, 247–257. Chetelat, R. T., and De Verna, J. W. (1991). Expression of unilateral incompatibility in pollen of Lycopersicon pennellii. Theor. Appl. Genet. 82, 704–712. Choi, W., and Dean, R. A. (1997). The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 9, 1973–1983. Christensen, P. U., Davey, J., and Nielsen, O. (1997a). The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secrection of M-factor. Mol. Gen. Genet. 255, 226–236. Christensen, P. U., Davis, K., Nielsen, O., and Davey, J. (1997b). Abc1: A new ABC transporter from the fission yeast Schizosaccharomyces pombe. FEMS Microbiol. Lett. 147, 97–102. Christiansen, S. K., Wirsel, S., Yun, S.-H., Yoder, O. C., and Turgeon, B. G. (1998). The two Cochliobolus mating type genes are conserved among species but one of them is missing in C. victoriae. Mycol. Res. 102, 919–929. Cisar, C. R., and TeBeest, D. O. (1999). Mating system of the filamentous ascomycete, Glomerella cingulata. Curr. Genet. 294, 127–133. Cisar, C. R., TeBeest, D. O., and Spiegel, F. W. (1994). Sequence similarity of mating-type idiomorphs, a method which detects similarity among the Sordariaceae fails to detect similar sequences in other filamentous ascomycetes. Mycologia 86, 540–546.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

265

Clark, A. G., and Kao, T.-H. (1991). Excess nonsynonymous substitution at shared polymorphic sites among self-incompatibility alleles of the Solanaceae. Proc. Natl. Acad. Sci. USA 88, 9823–9827. Clark, A. G., and Kao, T.-H. (1994). Self-incompatibility: Theoretical concepts and evolution. In ‘‘Genetic Control of Self-Incompatibility and Reproductive Development in Flowering Plants’’ (E. G. Williams, A.E. Clarke, and R. B. Knox, Eds.), pp. 220–242, Kluwer, Dordrecht. Clark, J. D., and Haskins, E. F. (1998). Heterothallic mating systems in the Echinostelium minutum complex. Mycologia 90, 382–388. Cock, J. M., Stanchev, B., Delorme, V., Croy, R. D., and Dumas, C. (1995). SLR3: A modified receptor kinase gene that has been adapted to encode a putative secreted glycoprotein similar to the S locus glycoprotein. Mol. Gen. Genet. 248, 151–161. Collin-Osdoby, P., and Adair, W. S. (1985). Characterization of the Chlamydomonas minus agglutinin. J. Cell Biol. 101, 1144–1152. Collins, O. R. (1980). Apomictic–heterothallic conversion in a myxomycete, Didymium iridis Funi. Mycologia 72, 1109–116. Connor, J. A., Tantikanjana, T., Stein, J. C., Kandasamy, M. K., Nasrallah, J. B., and Nasrallah, M. E. (1997). Transgene-induced silencing of S-locus genes and related genes in Brassica. Plant J. 11, 809–823. Conner, J. A., Conner, P., Nasrallah, M. E., and Nasrallah, J. B. (1998). Comparative mapping of the Brassica S locus region and its homeolog in Arabidopsis: Implications for the evolution of mating systems in the Brassicaceae. Plant Cell 10, 801–812. Cope, F. W. (1962). The mechanism of pollen incompatibility in Theobroma cacao L. Heredity 17, 157–182. Coppin, E., Arnaise, S., Contamine, V., and Picard, M. (1993). Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation. Mol. Gen. Genet. 241, 409–414. Coppin, E., Debuchy, R., Arnaise, S., and Picard, M. (1997). Mating types and sexual development in filamentous ascomycetes. Microbiol. Mol. Biol. Rev. 61, 411–428. Cornish, E. C., Pettit, J. M., Bonig, I., and Clarke, A. E. (1987). Developmentally controlled expression of a gene associated with self-incompatibility in Nicotiana alata. Nature 326, 99–102. Crow, J., and Kimura, M. (1965). Evolution in sexual and asexual populations. Am. Nat. 99, 439–450. Cruden, R. W., and Lloyd, R. M. (1995). Embryophytes have equivalent sexual phenotypes and breeding systems: Why not a common terminology to describe them? Am. J. Bot. 82, 816–825. Czempinski, K., Kruft, V., Wo¨stemeyer, J., and Burmester, A. (1996). 4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, and enzyme of the sexual hormone pathway, purification, and cloning of the corresponding gene. Microbiology, 142, 2647–2654. Dahl, M., Bo¨lker, M., Gillissen, B., Schauwecker, F., Schroer, B., and Kahmann, R. (1991). The b locus of Ustilago maydis, molecular analysis of allele specificity. In ‘‘Advances in Molecular Genetics of Plant–Microbe Interactions, Vol. 1’’ (H. Hennecke and P. D. S. Verma, Eds.), pp. 264–271. Kluwer, Dordrecht. Darlington, C. D., and Mather, K. (1949). ‘‘The Elements of Genetics.’’ Allen & Unwin, London. Darwin, C. (1876). ‘‘Effects of Cross and Self Fertilisation in the Vegetable Kingdom.’’ Murray, London. Davey, J. (1992). Mating pheromones of the fusion yeast Schizosaccharomyces pombe: Purification and structural characterisation of M-factor and isolation and structural characterisation of two genes encoding the pheromone. EMBO J. 11, 951–960.

266

SIMON J. HISCOCK AND URSULA KU¨ES

Davey, J., and Nielsen, O. (1994). Mutations in cyr1 and pat1 reveal pheromone-induced G1 arrest in the fission yeast Schizosaccharomyces pombe. Curr. Genet. 26, 105–112. Davey, J., Davis, K., Imai, Y., Yamamoto, M., and Mathews, G. (1994). Isolation and characterisation of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe. EMBO J. 13, 5910–5921. Davis, K., and Davey, J. (1997). G-protein-coupled receptors for peptide hormones in yeast. Biochem. Soc. Trans. 25, 1015–1021. Day, A. W. (1972). Dominance at a fungus mating type locus. Nature New Biol. 237, 282–283. Day, A. W. (1976). Communication through fimbriae during conjugation in a fungus. Nature 262, 583–584. Day, P. R. (1960). The structure of the A mating-type locus in Coprinus lagopus. Genetics 45, 641–650. Day, P. R., Anagnostakis, S. L., and Puhalla, J. E. (1971). Pathogenicity resulting from mutation at the b locus of Ustilago maydis. Proc. Natl. Acad. Sci. USA 68, 533–535. Debuchy, R., and Coppin, E. (1992). The mating types of Podospora anserina, functional analysis and sequence of the fertilzation domains. Mol. Gen. Genet. 233, 113–121. Debuchy, R., Arnaise, S., and Lecellier, G. (1993). The mat- allele of Podospora anserina contains three regulatory genes required for the development of fertilized female organs. Mol. Gen. Genet. 241, 667–673. Delorme, V., Giranton, J.-L., Hatzfeld, Y., Friry, A., Heizmann, P., Ariza, M. J., Dumas, C., Gaude, T., and Cock, J. M. (1995). Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: Identification of a membrane-localized protein encoded by the S locus receptor kinase gene. Plant J. 7, 429–440. de Nettancourt, D. (1977). ‘‘Incompatibility in Angiosperms.’’ Springer-Verlag, Berlin. de Nettancourt, D. (1997). Incompatibility in Angiosperms. Sex. Plant Reprod. 10, 185–199. de Nettancourt, D., Devreux, M., Bozzini, A., Cresti, M., Pacini, E., and Sarfatti, G. (1973). Ultrastructural aspects of the self-incompatibility mechanism in Lycopersicon peruvianum Mill. J. Cell Sci. 12, 403–419. de Nettancourt, D., Devreux, M., Laneri, U., Cresti, M., Pacini, E., and Sarfatti, G. (1974). Genetical and ultrastructural aspects of self and cross incompatibility in interspecific hybrids between self-compatible Lycopersicum esculentum and self-incompatible Lycopersicum peruvianum. Theor. Appl. Genet. 44, 278–288. Dickinson, H. G. (1994). Simply a social disease? Nature 367, 517–518. Dickinson, H. G. (1995). Dry stigmas, water and self-incompatibility in Brassica. Sex. Plant Reprod. 8, 1–10. Dickinson, H. G., and Lawson, J. (1975). Pollen tube growth in the stigma of Oenothera organensis following compatible and incompatible intraspecific pollinations. Proc. R. Soc. London B 188, 327–344. Dickinson, H. G., and Lewis, D. (1973a). Cytochemical and ultrastructural differences between intraspecific pollinations in Raphanus. Proc. R. Soc. London, B 183, 21–38. Dickinson, H. G., and Lewis, D. (1973b). The formation of the tryphine coating the pollen grains of Raphanus and its properties relating to the self-incompatibility system. Proc. R. Soc. London B 184, 149–165. Dickinson, H. G., Crabbe, M. J. C., and Gaude, T. (1992). Sporophytic self-incompatibility systems: S-gene products. Int. Rev. Cytol. 140, 525–561. Dickinson, H. G., Doughty, J., Hiscock, S. J., Elleman, C. J., and Stephenson, A. G. (1998). Pollen–stigma interactions in Brassica. In ‘‘Control of Plant Development: Genes and Signals’’ (A. Greenland, E. Meyerowitz, and M. Steer, Eds.), Society for Experimental Biology symposium series, pp. 51–57. Cambridge University Press, Cambridge, U.K. Dickinson, S. (1927). Experiments on the physiology and genetics of the smut fungi—Hyphalfusion. Proc. R. Soc. Series B 101, 126–136.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

267

Dodds, P. N., Bonig, I., Du, H., Rodin, J., Anderson, M. A., Newbigin, E., and Clarke, A. E. (1993). The S-RNase gene of Nicotiana is expressed in developing pollen. Plant Cell 5, 1771–1782. Dodds, P. N., Clarke, A. E., and Newbigin, E. (1996). A molecular perspective on pollination in flowering plants. Cell 85, 141–144. Dodds, P. N., Clarke, A. E., and Newbigin, E. (1997). Molecules involved in self-incompatibility in flowering plants. In ‘‘Plant Breeding Reviews, 15’’ ( J. Janick, Ed.), pp. 19–42. Wiley, New York. Dolan, J. W., and Fields, S. (1991). Cell-type-specific transcription in yeast. Biochim. Biophys. Acta 1088, 155–169. Dong, H., and Courchesne, W. (1998). A novel quantitative mating assay for the fungal pathogen Cryptococcus neoformans provides insight into signalling pathways responding to nutrients and temperature. Microbiology 144, 1691–1697. Dooijes, D., van de Wetering, M., Knippels, L., and Clevers, H. (1993). The Schizosaccharomyces pombe mating-type gene mat-Mc encodes a sequence-specific DNA-binding high mobility group box protein. J. Biol. Chem. 268, 24813–24817. Doughty, J., Hedderson, F., McCubbin, A., and Dickinson, H. G. (1993). Interaction between a coating-borne peptide of the Brassica pollen grain and stigmatic S(incompatibility)-locusspecific glycoproteins. Proc. Natl. Acad. Sci. USA 90, 467–471. Doughty, J., Dixon, S., Hiscock, S. J., Willis, A. C., Parkin, I. A. P., and Dickinson, H. G. (1998). PCP-A1, a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression. Plant Cell 10, 1333–1347. Dressler, R. A. (1993). ‘‘Phylogeny and Classification of the Orchid Family.’’ Dioscorides Press, Portland, OR. Du¨rrenberger, F., Wong, K., and Kronstad, J. W. (1998). Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustligo maydis. Proc. Natl. Acad. Sci. USA 95, 5684–5689. Dwyer, K. G., Balent, M. A., Nasrallah., J. B., and Nasrallah, M. E. (1991). DNA sequences of self-incompatibility genes from Brassica campestris and B. oleracea: Polymorphism predating speciation. Plant Mol. Biol. 16, 481–486. Dwyer, K. G., Lalonde, B. A., Nasrallah, J. B., and Nasrallah, M. E. (1992). Structure and expression of AtS1, and Arabidopsis thaliana gene homologous to the S-locus related genes of Brassica. Mol. Gen. Genet. 231, 442–448. Dwyer, K. G., Kandasamy, M. K., Mahosky, D. I., Acciai, J., Kudish, B. I., Miller, J. E., Nasrallah, M. E., and Nasrallah, J. B. (1994). A superfamily of S locus-related sequences in Arabidopsis: Diverse structures and expression patterns. Plant Cell 6, 1829–1843. Dyer, S., Nicholson, P., Rezanoor, H. N., and Peberdy, J. F. (1993). Two-allele heterothallism in Tapesia yallundae, the teleomorph of the cereal eyespot pathogen Pseudocercosporella herpotrichoides. Physiol. Mol. Plant Pathol. 43, 403–414. East, E. M. (1940). The distribution of self-sterility in flowering plants. Proc. Am. Philos. Soc. 82, 449–518. East, E. M., and Mangelsdorf, A. J. (1925). A new interpretation of the heredity behaviour of self-sterile plants, Proc. Natl. Acad. Sci. USA 2, 166–183. Ebert, P. R., Anderson, M. A., Bernatzky, R., Altschuler, M., and Clarke, A. E. (1989). Genetic polymorphism of self-incompatibility in flowering plants. Cell 56, 255–262. Egel, R., Nielsen, O., and Weilguny, D. (1990). Sexual differentiation in fission yeast. Trends Genet. 6, 369–373. Elion, E. A. (1995). Ste5: A meeting place for MAP kinases and their associates. Trends Cell Biol. 5, 322–327. Elleman, C. J., and Dickinson, H. G. (1986). Pollen–stigma interactions in Brassica IV. Structural reorganisation in the pollen grains during hydration. J. Cell Sci. 80, 141–157.

268

SIMON J. HISCOCK AND URSULA KU¨ES

Elleman, C. J., and Dickinson, H. G. (1990). The role of the exine coating in pollen–stigma interactions in Brassica oleracea L. New Phytol. 114, 511–518. Elleman, C. J., and Dickinson, H. G. (1994). Pollen–stigma interaction during self-incompatibility in Brassica oleracea. In ‘‘Genetic Control of Self-Incompatibility and Reproductive Development in Flowering Plants’’ (E. G. Williams, A. E. Clarke, and R. B. Knox, Eds.), pp. 67–87, Kluwer, Dordrecht. Elleman, C. J., Willson, C. E., Sarker, R. H., and Dickinson, H. G. (1988). Interaction between the pollen tube and stigmatic cell wall following pollination in Brassica oleracea. New Phytol. 109, 111–117. Elleman, C. J., Franklin-Tong, V., and Dickinson, H. G. (1992). Pollination in species with dry stigmas: The nature of the early stigmatic response and the pathway taken by pollen tubes. New Phytol. 121, 413–424. Elliott, C. G. (1994). ‘‘Reproduction in Fungi. Genetical and Physiological Aspects.’’ Chapman & Hall, London. Emerson, S. (1939). A preliminary survey of the Oenothera organensis population. Genetics 24, 524–537. Esser, K. (1959). Die Inkompatibilita¨tsbeziehungen zwischen geographischen Rassen von Podospora anserina (Ces.) Rehm. II. Die Wirkungsweise der Inkompatibilita¨tsgene. Z. Vererbungsl. 90, 29–52. Esser, K., and Straub, J. (1958). Genetische Untersuchungen an Sordaria macrospora Auersw., Kompensation und Induktion bei genbedingten Entwicklungsdefekten. Z. Vererbungsl. 89, 729–746. Evangelista, M., Blundell, K., Longtine, M. S., Chow, C. J., Adames, N., Pringle, J. R., Peter, M., and Boone, C. (1997). Bn1p, a yeast formin linking Cdc42p and the actin cytoskeleton during polarized morphogenesis. Science 276, 118–122. Fabritius, A.-L., and Judelson, H. S. (1997). Mating type loci segregate aberrantly in Phytophthora infestans but normally in Phythophthora parasitica: Implications for models of mating type determination. Curr. Genet. 32, 60–65. Faretra, F., and Pallastro, S. (1996). Genetic studies of the phytopathogenic fungus Botryotinia fuckeliana (Botrytis cinerea) by analysis of ordered tertrads. Mycol. Res. 100, 620–624. Faretra, F., Antonacci, E., and Pallostro, S. (1988). Sexual behaviour and mating system of Botrytis fuckeliana, teleomorph of Botrytis cinerea. J. Gen. Microbiol. 134, 2443–2550. Ferrari, T. E., and Wallace, D. H. (1977). A model for self-recognition and regulation of the incompatibility response in pollen. Theor, Appl. Genet. 50, 211–225. Ferreira, A. V.-B., Saupe, S., and Glass, N. L. (1996). Transcriptional analysis of the mtA idiomorph of Neurospora crassa identifies two genes in addition to mtA-1. Mol. Gen. Genet. 250, 767–774. Ferreira, A. V.-B., An, Z., Metzenberg, R. L., and Glass, N. L. (1998). Characterization of mat A-2, mat A-3 and ⌬matA mating-type mutants of Neurospora crassa. Genetics 148, 1069– 1079. Ferris, P. J., and Goodenough, U. W. (1994). The mating type locus of Chlamydomonas reinhardtii contains highly rearranged DNA sequences. Cell 76, 1135–1145. Ferris, P. J., and Goodenough, U. W. (1997). Mating type in Chlamydomonas is specified by mid, the minus-dominance gene. Genetics 146, 859–869. Ferris, P. J., Woessner, J. P., and Goodenough, U. W. (1996). A sex recognition glycoprotein is encoded by the plus mating type gene fus1 of Chlamydomonas reinhardtii. Mol. Biol. Cell 7, 1235–1248. Fisher, R. A. (1961). A model for the generation of self-sterility alleles. J. Theor. Biol. 1, 411–414. Flegel, T. W. (1981). The pheromonal control of mating in yeast and its phylogenetic implication, a review. Can. J. Microbiol. 27, 373–389.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

269

Foote, H. C. C., Ride, J. P., Franklin-Tong, V. E., Walker, E. A., Lawrence, M. J., and Franklin, F. C. H. (1994). Cloning and expression of a novel self-incompatibility (S-) gene from Papaver rhoeas L. Proc. Natl. Acad. Sci. USA 91, 2265–2269. Forest, C. L. (1987). Genetic control of plasma membrane adhesion and fusion in Chlamydomonas gametes. J. Cell Sci. 88, 613–621. Fowler, T. J., Mitton, M., and Raper, C. A. (1998). Gene mutations affecting specificity of pheromone/receptor mating interactions in Schizophyllum commune. In ‘‘Proceedings of the Fourth Meeting on the Genetics and Cellular Biology of Basidiomycetes’’ (L. J. L. D. Van Griensven and J. Visser, Eds.), pp. 130–134. Mushroom Experimental Station, Horst, the Netherlands. Franklin, F. C. H., Lawrence, M. J., and Franklin-Tong, V. E. (1995). Cell and molecular biology of self-incompatibility in flowering plants. Int. Rev. Cytol. 158, 1–64. Franklin-Tong, V. E., and Franklin, F. C. H. (1992). Gametophytic self-incompatibility in Papaver rhoeas. Sex. Plant Reprod. 5, 1–8. Franklin-Tong, V. E., Lawrence, M. J., and Franklin, F. C. H. (1988). An in vitro bioassay for the stigmatic product of the self-incompatibility gene in Papaver rhoeas L. New Phytol. 110, 109–118. Franklin-Tong, V. E., Thorlby, G. J., Lawrence, M. J., and Franklin, F. C. H. (1992). Recognition, signals and pollen responses in the incompatibility reaction in Papaver rhoeas. In ‘‘Angiosperm Pollen and Ovules: Basic and Applied Aspects’’ (D. J. Mulcahy, G. BergaminiMulcahy, and E. Ottaviano, Eds.), pp. 84–93. Springer, New York. Franklin-Tong, V. E., Ride, J. P., Read, D. R., Trewavas, A. J., and Franklin, F. C. H. (1993). The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium. Plant J. 4, 163–177. Franklin-Tong, V. E., Lawrence, M. J., and Franklin, F. C. H. (1994). The molecular and cellular biology of gametophytic self-incompatibility in Papaver rhoeas. In ‘‘Genetic Control of Self-Incompatibility and Reproductive Development in Flowering Plants’’ (E. G. Williams, A. E. Clarke, and R. B. Knox, Eds.), pp. 42–64. Kluwer, Dordrecht. Franklin-Tong, V. E., Ride, J. P., and Franklin, F. C. H. (1995). Recombinant stigmatic selfincompatibility (S-) protein elicits a Ca2⫹ transient in pollen of Papaver rhoeas. Plant. J. 8, 299–307. Franklin-Tong, V. E., Drøbak, B. K., Allan, A. C., Watkins, P. A. C., and Trewavas, A. J. (1996). Growth of pollen tubes of Papaver rhoeas is regulated by a slow-moving calcium wave propogated by inositol 1,4,5-triphosphate. Plant Cell 8, 1305–1321. Franklin-Tong, V. E., Hackett, G., and Hepler, P. K. (1997). Ratio imaging of Ca2⫹i in the self-incompatibility response in pollen tubes of Papaver rhoeas. Plant J. 12, 1375–1386. Fries, N. (1981). Recognition reactions between basidiospores and hyphae in Leccinum. Trans. Br. Mycol. Soc. 77, 9–14. Fryxell, P. A. (1957). Mode of reproduction in higher plants. Bot. Rev. 23, 135–233. Fujino, M., Kitada, C., Sakagami, Y., Isogai, A., Tamura, S., and Suzuki, A. (1980). Biological activity of synthetic analogs of Tremerogen A-10. Naturwissenschaften 67, 406–408. Galiana, E., Bonnet, P., Conrod, S., Keller, H., Panabieres, F., Ponchet, M., Poupet, A., and Ricci, P. (1997). RNase activity prevents the growth of a fungal pathogen in tobacco leaves and increases upon induction of systemic acquired resistance with elicitin. Plant Physiol. 115, 1557–1567. Ganders, F. R. (1979). The biology of heterostyly. N. Z. J. Bot. 17, 607–635. Gao, S., and Nuss, D. L. (1996). Distinct roles for two G protein 움 subunits in fungal virulence, morphology, and reproduction revealed by targeted gene disruption. Proc. Natl. Acad. Sci. USA 93, 14122–14127. Gaude, T., Rougier, M., Heizmann, P., Ockendon, D. J., and Dumas, C. (1995). Expression level of the SLG gene is not correlated with the self-incompatibility phenotype in class II S haplotypes of Brassica oleracea. Plant Mol. Biol. 27, 1003–1014.

270

SIMON J. HISCOCK AND URSULA KU¨ES

Geiser, D. M., Frisvad, J. C., and Taylor, J. W. (1998). Evolutionary relationships in Aspergillus section Fumigati inferred from partial 웁-tubulin and hydrophobin sequences. Mycologia 90, 831–845. Gibbs, P. E. (1986). Do homomorphic and heteromorphic self-incompatibility systems have the same sporophytic mechanism? Plant Syst. Evol. 154, 285–323. Gieser, P. T., and May, G. (1994). Comparison of two b1 alleles from within the A matingtype of the basidiomycete Coprinus cinereus. Gene 146, 167–176. Giesy, R. M., and Day, P. R. (1965). The septal pores of Coprinus lagopus in relation to nuclear migration. Am. J. Bot. 52, 287–293. Gillissen, B., Bergemann, J., Sandmann, C., Schroeer, B., Bo¨lker, M., and Kahmann, R. (1992). A two-component regulatory system for self/non-self recognition in Ustilago maydis. Cell 68, 647–657. Giranton, J.-L., Ariza, M. J., Dumas, C., Cock, J. M., and Gaude, T. (1995). The S locus receptor kinase gene encodes a soluble glycoprotein gene corresponding to the SRK extracellular domain. Plant J. 8, 827–834. Glass, N. L., and Kuldau, G. A. (1992). Mating type and vegetative incompatibility in filamentous ascomycetes. Annu. Rev. Phytopathol. 30, 201–224. Glass, N. L., and Lee, L. (1992). Isolation of Neurospora crassa A mating-type mutants by repeat induced point (RIP) mutation. Genetics 132, 125–133. Glass, N. L., and Nelson, M. A. (1994). Mating type genes in mycelial ascomycetes. In ‘‘The Mycota I. Growth, Differentiation and Sexuality’’ ( J. G. H. Wessels and F. Meinhardt, Eds.), pp. 295–306. Springer-Verlag, Berlin. Glass, N. L., and Smith, M. L. (1994). Structure and function of a mating-type gene from the homothallic species Neurospora africana. Mol. Gen. Genet. 244, 401–409. Glass, N. L., Vollmer, S. J., Staben, C., Grotelueschen, J., Metzenberg, R. L., and Yanofsky, C. (1988). DNAs of the two mating-type alleles of Neurospora crassa are highly dissimilar. Science 241, 570–573. Glass, N. L., Groteluschen, J., and Metzenberg, R. L. (1990a). Neurospora crassa A matingtype region. Proc. Natl. Acad. Sci. USA 87, 4912–4916. Glass, N. L., Metzenberg, R., and Raju, N. B. (1990b). Homothallic Sordariaceae from nature, the absence of strains containing only the a mating type sequence. Exp. Mycol. 14, 274–289. Gold, S. E., Duncan, G. A., Barrett, K. J., and Kronstad, J. W. (1994). cAMP regulates morphogenesis in the fungal pathogen Ustilago maydis. Genes Dev. 8, 2805–2816. Gold, S. E., Brogdon, S. M., Mayorga, M. E., and Kronstad, J. W. (1997). The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize. Plant Cell 9, 1585–1594. Goldstein, D. B. (1992). Heterozygote advantage and the evolution of a dominant diploid phase. Genetics 132, 1195–1198. Golz, J., Su, V., and Newbigin, E. (1997). A mutational analysis of self-incompatibility in Nicotiana alata. Fifth International Congress on Plant Molecular Biology, Singapore. [Abstract 40] Gooday, G. W. (1994). Hormones in mycelial fungi. In ‘‘The Mycota. Vol I. Growth, Differentiation and Sexuality’’ ( J. G. H. Wessels and F. Meinhardt, Eds.), pp. 401–412. SpringerVerlag, Berlin. Goodenough, U. V. (1991). Chlamydomonas mating interactions. In ‘‘Microbial Cell–Cell Interactions’’ (M. Dworkin, Ed.), pp. 71–112. Am. Soc. Microbiol., Washington, DC. Goodenough, U. V., Detmers, P. A., and Hwang, C. (1982). Activation for cell fusion in Chlamydomonas: Analysis of wild-type gametes and non-fusing mutants. J. Cell Biol. 92, 378–386. Goodenough, U. V., Ambrust, E. V., Campbell, A. M., and Ferris, P. J. (1995). Molecular genetics of sexuality in Chlamydomonas. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 21–44.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

271

Goodwillie, C. (1997). The genetic control of self-incompatibility in Linanthus parviflorus (Polemoniaceae). Heredity 79, 424–432. Goring, D. R., and Rothstein, S. J. (1992). The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase. Plant Cell 4, 1273–1281. Goring, D. R., Glavin, T. L., Schafer, V., and Rothstein, S. J. (1993). An S receptor kinase gene in self-compatible Brassica napus has a 1-bp deletion. Plant Cell 5, 531–539. Goutte, C., and Johnson, A. D. (1993). Yeast a1 and 움2 homeodomain proteins form a DNAbinding activity with properties distinct to those of either protein J. Mol. Biol. 233, 359–371. Gray, J. E., McClure, B. A., Bonig, I., Anderson, M. A., and Clarke, A. E. (1991). Action of the style product of the self-incompatibility gene of Nicotiana alata (S-RNase) on in vitro-grown pollen tubes. Plant Cell 3, 271–283. Griffiths, A. J. F. (1982). Null mutants of the A and a mating type alleles of Neurospora crassa. Can. J. Genet. Cytol. 24, 167–176. Griffiths, A. J. F., and Delange, A. M. (1978). Mutations of the a mating type in Neurospora crassa. Genetics 88, 239–254. Gu, T., Mazzurco, M., Sulaman, W., Matias, D. D., and Goring, D. R. (1998). Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase. Proc. Natl. Acad. Sci. USA 95, 382–387. Guilluy, C.-M., Trick, M., Heizmann, P., and Dumas, C. (1991). PCR detection of transcripts homologous to the self-incompatibility gene in anthers of Brassica. Theor. Appl. Genet. 82, 466–472. Haber, J. E. (1998). A locus control region regulates yeast recombination. Trends Genet. 14, 317–321. Hagen, D. C., McCaffrey, G., and Sprague, G. F. (1986). Evidence that yeast STE3 genes encode a receptor for the peptide pheromone a-factor: Gene sequence and implication for the structure of the presumed receptor. Proc. Natl. Acad. Sci. USA 83, 1418–1422. Hagman, M. (1975). Incompatibility in forest trees. Proc. R. Soc. London B 188, 313–326. Ha¨nfler, J., Teepe, H., Weigel, C., Kruft, V., Lurz, R., and Wo¨astemeyer, J. (1992). Circular extrachromosomal DNA codes for a surface protein in the (⫹) mating type of the zygomycete Absidia glauca. Curr. Genet. 22, 319–325. Haring, V., Gray, J. E., McClure, B. A., Anderson, M. A., and Clarke, A. E. (1990). Selfincompatibility: A self-recognition system in plants. Science 250, 937–941. Harrington, T. C., and McNew, D. L. (1997). Self-fertility and uni-directional mating-type switching in Ceratocystis coerulescens, a filamentous ascomycte. Curr. Genet. 32, 52–59. Harrison, B. J., and Darby, L. A. (1955). Unilateral hybridisation. Nature 176, 952. Hartmann, H. A., Kahmann, R., and Bo¨lker, M. (1996). The pheromone response factor coordinates filamentous growth and pathogenicity in Ustilago maydis. EMBO J. 15, 1632– 1641. Hatakeyama, K., Watanabe, M., Takasaki, T., Ojima, K., and Hinata, K. (1998). Dominance relationships between S-alleles in self-incompatible Brassica campestris L. Heredity 80, 241–247. Hawksworth, D. L., Kirk, P. M., Sutton, B. C., and Pegler, D. N. (1985). ‘‘Ainsworth & Bisby’s Dictionary of the Fungi,’’ Int. Mycol. Inst., 8th ed. CAB International, Wallingford, UK. Hayman, D. L. (1956). The genetic control of incompatibility in Phalaris coerulescens Desf. Aust. J. Biol. Sci. 9, 321–331. Hearn, M. J., Franklin, F. C. H., and Ride, J. P. (1996). Identification of a membrane glycoprotein in pollen of Papaver rhoeas which binds stigmatic self-incompatibility (S-) proteins. Plant J. 9, 467–475. Hegner, J., Lengeler, K. B., Gola, S., and Kothe, E. (1998). Regulation of sexual development in Schizophyllum commune. In ‘‘Proceedings of the Fourth Meeting on the Genetics and Cellular Biology of Basidiomycetes’’ (L. J. L. D. Van Griensven and J. Visser, Eds.), pp. 125–129. Mushroom Experimental Station, Horst, the Netherlands.

272

SIMON J. HISCOCK AND URSULA KU¨ES

Hennebert, G. L., Pascal, S., and Cosyns, M. (1994). Interactions d’incompatibilite´ entre homocaryons du basidiomyce`te bifactoriel Lenzites betulinus. Une phe´romone de re´pulsion sexuelle? Cryptogamie, Mycol. 15, 83–116. Herrero, M., and Dickinson, H. G. (1980). Pollen tube growth following compatible and incompatible intraspecific pollinations in Petunia hybrida. Planta 148, 217–221. Herrero, M., and Dickinson, H. G. (1981). Pollen tube development in Petunia hybrida following compatible and incompatible intraspecific matings. J. Cell Sci. 47, 365–383. Herskowitz, I. (1989). A regulatory hierachy for cell specialisation in yeast. Nature 342, 749–757. Herskowitz, I. (1995). MAP kinase pathways in yeast, for mating and more. Cell 80, 187–197. Herskowitz, I., Rine, J., and Strathern, J. (1992). Mating-type determination and interconversion. In ‘‘The Molecular and Cellular Biology of the Yeast Saccharomyces’’ ( J. R. Broach, J. R. Pringle, and E. W. Jones, Eds.), pp. 583–656. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Heslop-Harrison, J. (1975). Incompatibility and the pollen–stigma interaction. Annu. Rev. Plant Physiol. 26, 403–425. Heslop-Harrison, J. (1979). Aspects of the structure cytochemistry and germination of the pollen of rye (Secale cereale L.) Ann. Bot. 44(Suppl. 1), 1–47. Heslop-Harrison, J. (1982). Pollen–stigma interaction and cross-incompatibility in the grasses. Science 215, 1358–1364. Heslop-Harrison, J., and Heslop-Harrison, Y. (1981). The pollen–stigma interaction in the grasses. 2. Pollen tube penetration and the stigma response in Secale. Acta Bot. Neerl. 30, 289–307. Heslop-Harrison, J., Knox, R. B., and Heslop-Harrison, Y. (1974). Pollen-wall proteins: Exineheld fractions associated with the incompatibility response in Cruciferae. Theor. Appl. Genet. 44, 133–177. Heslop-Harrison, J., Knox, R. B., Heslop-Harrison, Y., and Mattsson, O. (1975). Pollen wall proteins: Emission and role in incompatibility responses. In ‘‘Biology of the Male Gamete’’ ( J. G. Duckett and P. A. Racey, Eds.), pp. 165–202. Linnean Society of London Academic Press, London. Heslot, H. (1958). Contribution a` l’e´tude cytoge´ne´tique et ge´ne´tique des Sordariace´es. Rev. Cytol. Biol. Veg. 19(Suppl. 2), 1–235. Hicke, L., and Riezman, H. (1996). Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 84, 277–287. Hinata, K., and Nishio, T. (1978). S-allele specificity of stigma proteins in Brassica oleracea and B. campestris. Heredity 41, 93–100. Hinata, K., Okasaki, K., and Nishio, T. (1983). Gene analysis of self-compatibility in Brassica campestris var. yellow sarson (a case of recessive epistatic modifier). Proceedings of the 6th International Rapeseed Conference, Paris, Vol. 1, pp. 354–359. Hiscock, S. J., and Dickinson, H. G. (1993). Unilateral incompatibility within the Brassicaceae—Further evidence for an involvement of the self-incompatibility (S )-locus. Theor. Appl. Genet. 86, 744–753. Hiscock, S. J., Ku¨es, U., and Stahl, U. (1994). Sexual reproduction in plants: Self-incompatibility as a mechanism promoting outbreeding and gene flow. Prog. Bot. 56, 276–300. Hiscock, S. J., Doughty, J., and Dickinson, H. G. (1995a). Synthesis and phosphorylation of pollen proteins during the pollen–stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L. Sex. Plant Reprod. 8, 345–353. Hiscock, S. J., Doughty, J., Willis, A. C., and Dickinson, H. G. (1995b). A 7-kDa pollen coating-borne peptide from Brassica napus interacts with S-locus glycoprotein and S-locusrelated glycoprotein. Planta 196, 367–374. Hiscock, S. J., Ku¨es, U., and Dickinson, H. G. (1996). Molecular mechanisms of self-incompatibility in flowering plants and fungi—different means to the same end. Trends Cell Biol. 6, 421–428.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

273

Hiscock, S. J., Doughty, J., and Dickinson, H. G. (1998). Unilateral incompatibility and the S (self-incompatibility) locus. In ‘‘Reproductive Biology in Systematics, Conservation and Economic Botany’’ (S. J. Owens and P. J. Rudall, Eds.), pp. 31–46. Royal Botanic Gardens, Kew, Richmond, Surrey, U.K. Ho, C.-Y., Adamson, R. S., Hodges, R. S., and Smith, M. (1994). Heterodimerization of the yeast MATa1 and MAT움2 proteins is mediated by two leucine zipper-like coiled-coil motifs. EMBO J. 13, 1403–1413. Hodgkin, T., and Lyon, G. D. (1984). Pollen germination inhibitors in extracts of Brassica oleracea stigmas. New Phytol. 96, 293–298. Hodgkin, T., and Lyon, G. D. (1986). The effect of Brassica oleracea stigma extracts on the germination of B. oleracea pollen in a thin layer chromatographic bioassay. J. Exp. Bot. 37, 406–411. Hodgkin, T., Lyon, G. D., and Dickinson, H. G. (1988). Recognition in flowering plants: A comparison of the Brassica self-incompatibility system and plant pathogen interactions. New Phytol. 110, 557–569. Hoekstra, R. F. (1987). The evolution of sexes. In ‘‘The Evolution of Sex and Its Consequences’’ (S. C. Stearns, Ed.), pp. 59–91. Birkha¨user, Basel. Hogenboom, N. G. (1975). Incompatibility and incongruity: Two different mechanisms for the nonfunctioning of intimate partner relationships. Proc. R. Soc. London B 188, 361–375. Hormazo, J. I., and Herrero, M. (1992). Pollen selection. Theor. Appl. Genet. 83, 663–672. Horwitz, B. A., Sharon, A., Lu, S.-W., Ritter, V., Sandrock, T. M., Yoder, O. C., and Turgeon, B. G. (1999). A G protein alpha subunit from Cochliobolus heterostrophus involved in mating and appressorium formation. Fungal Genet. Biol. 26, 19–32. Huang, S., Lee, H.-S., Karunanadaa, B., and Kao, T.-H. (1994). Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen. Plant Cell 6, 1021–1028. Hughes, M. B., and Babcock, E. B. (1950). Self-incompatibility in Crepis foetida L. subsp. rhoeadifolia Bieb. Schinz et Keller. Genetics 35, 570–588. Hurst, L. D. (1996). Why are there only two sexes? Proc. R. Soc. London B 263, 415–422. Hurst, L. D., and Hamilton, W. D. (1992). Cytoplasmic fusion and the nature of sexes. Proc. R. Soc. London B 47, 189–194. Husband, B. C., and Schemske, D. W. (1996). Evolution of the magnitude and timing of inbreeding depression in plants. Evolution 50, 54–70. Ikeda, S., Nasrallah, J. B., Dixit, R., Preiss, S., and Nasrallah, M. E. (1997). An aquaporinlike gene required for the Brassica self-incompatibility response. Science 276, 1564–1566. Imai, Y., and Yamamoto, M. (1992). Schizosaccharomyces pombe sxa1⫹ and sxa2⫹ encode putative proteases involved in the mating response. Mol. Cell. Biol. 12, 1827–1834. Imai, Y., and Yamamoto, M. (1994). The fission yeast mating pheromone P-factor: Its molecular structure, gene structure and physiological activities to induce gene expression and G1 arrest in the mating partner. Genes Dev. 8, 328–338. Ioerger, T. R., Clark, A. G., and Kao, T.-H. (1990). Polymorphism at the self-incompatibility locus in the Solanaceae predates speciation. Proc. Natl. Acad. Sci. USA 87, 9732–9735. Ioerger, T. R., Golke, J. R., Xu, B., and Kao, T.-H. (1991). Primary structural features of the self-incompatibility protein in the Solanaceae. Sex. Plant Reprod. 4, 81–87. Ishibashi, Y., Sakagami, Y., Isogai, A., Suzuki, A., and Bandoni, R. J. (1983). Isolation of tremerogenes A-9291-I and A-9291-II, novel sex hormones of Tremella brasiliensis. Can. J. Biochem. Cell Biol. 61, 796–801. Ishibashi, Y., Sakagami, Y., Isogai, A., and Suzuki, A. (1984). Structures of tremerogens A9291-I and A9291-VIII, peptidyl sex hormones of Tremella brasiliensis. Biochemistry 23, 1399–1404. Ishimizu, T., Shinkawa, T., Sakiyama, F., and Norioka, S. (1998). Primary structural features of Rosaceous S-RNases associated with gametophytic self-incompatibility. Plant Mol. Biol. 37, 931–941.

274

SIMON J. HISCOCK AND URSULA KU¨ES

Isogai, A., Takayama, S., Shiozawa, H., Tsukamoto, C., Kanbara, T., Hinata, K., Okazaki, K., and Suzuki, A. (1988). Existence of a common glycoprotein homologous to S-glycoproteins in two self-incompatible homozygotes of Brassica campestris. Plant Cell Physiol. 29, 1331–1336. Ito, S., Matsui, Y., Toh-e, A., Harashima, T., and Inoue, H. (1997). Isolation and characterisation of the krev-1 gene, a novel member of ras superfamily in Neurospora crassa, involvement in sexual cycle progression. Mol. Gen. Genet. 255, 429–437. Ivey, F. D., Hodge, P. N., Turner, G. E., and Borkovich, K. A. (1996). The G움i homologue gna-1 controls multiple differentiation pathways in Neurospora crassa. Mol. Cell. Biol. 7, 1283–1297. Iwasa, M., Tanabe, S., and Kamada, T. (1998). The two nuclei in the dikaryon of the homobasidiomycete Coprinus cinereus change position after each conjugate division. Fungal Genet. Biol. 23, 110–116. Jackson, C. L., and Hartwell, L. H. (1990). Courtship in S. cerevisiae, both cell types choose mating partners by responding to the strongest pheromone signal. Cell 63, 1039–1051. Jarne, P., and Charlesworth, D. (1993). The evolution of the selfing rate in functionally hermaphrodite plants and animals. Annu. Rev. Ecol. Syst. 24, 441–466. Jin, Y., Zhong, H., and Vershon, A. K. (1999). The yeast a1 and 움2 homeodomain proteins do not contribute equally to heterodimeric DNA binding. Mol. Cell. Biol. 19, 585–593. Johansson, I., Karlsson, M., Shukla, V. K., Chrispeels, M. J., Larsson, C., and Kjellbom, P. (1998). Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation. Plant Cell 10, 451–459. Johnson, A. D. (1995). Molecular mechanisms of cell-type determination in budding yeast. Curr. Opin. Genet. Dev. 5, 552–558. Johnson, P. R., Swanson, R., Rakhilina, L., and Hochstrasser, M. (1998). Degradation signal masking by heterodimerization of MAT움2 and MATa1 blocks their mutual destruction by ubiquitin–proteasome pathway. Cell 94, 217–227. Judelson, H. S. (1996a). Chromosomal heteromorphism linked to the mating type locus of the oomycete, Phythophthora infestans. Mol. Gen. Genet. 239, 241–250. Judelson, H. S. (1996b). Physical and genetic variability at the mating type locus of the oomycete, Phythophthora infestans. Genetics 144, 1005–1013. Judelson, H. S. (1997). The genetics and biology of Phytophthora infestans: Modern approaches to a historical challenge. Fungal Genet. Biol. 22, 65–76. Judelson, H. S., Spielman, L. J., and Shattock, R. C. (1995). Genetic mapping and nonMendelian segregation of mating type loci in the oomycete, Phytophthora infestans. Genetics 141, 503–512. Jurand, M. K., and Kemp, R. F. O. (1973). An incompatibility system determined by three factors in a species of Psathyrella (Basidiomycetes). Genet. Res. Cambridge 22, 125–134. Kahmann, R., and Basse, C. (1998). Signalling and development in pathogenic fungi—New strategies for plant protection? Trends Plant Sci. 2, 366–368. Kahmann, R., and Bo¨lker, M. (1996). Self/nonself recognition in fungi, old mysteries and simple solutions. Cell 85, 145–148. Kahmann, R., Romeis, T., Bo¨lker, M., and Ka¨mper, J. (1995). Control of mating and development in Ustilago maydis. Curr. Opin. Genet. Dev. 5, 559–564. Kakeda, K., and Kowyama, Y. (1996). Sequences of Ipomoea trifida cDNAs related to the Brassica S-locus genes. Sex. Plant Reprod. 9, 309–310. Kamiya, Y., Sakurai, A., Tamura, S., Takahashi, N., Abe, K., Tsuchiya, E., Fukui, S., Kitada, C., and Fujino, M. (1978). Structure of rhodotorucine A, a novel lipopeptide inducing mating tube formation in Rhodosporium toruloides. Biochem. Biophys. Res. Commun. 83, 1077–1083. Kamiya, Y., Sakurai, A., Tamura, S., and Takahashi, N. (1979). Structure confirmation of Strans-trans-farnesylcysteine in rhodotorucine A by application of sulfonium salt cleavage reaction. Agric. Biol. Chem. 43, 1049–1053.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

275

Ka¨mper, J., Reichmann, M., Romeis, T., Bo¨lker, M., and Kahmann, R. (1995). Multiallelic recognition, nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis. Cell 81, 73–83. Kandasamy, M. K., Paolillo, D. J., Faraday, C. D., Nasrallah, J. B., and Nasrallah, M. E. (1989). The S-locus-specific glycoproteins of Brassica accumulate in the cell wall of the developing stigmatic papillae. Dev. Biol. 134, 462–472. Kandasamy, M. K., Thorsness, M. K., Rundle, S. J., Goldberg, M. L., Nasrallah, J. B., and Nasrallah, M. E. (1993). Ablation of papillar cell function in Brassica flowers results in the loss of stigma receptivity to pollination. Plant Cell 5, 263–275. Kandasamy, M. K., Nasrallah, J. B., and Nasrallah, M. E. (1994). Pollen–pistil interactions and developmental regulation of pollen tube growth in Arabidopsis. Development 120, 3405– 3418. Kang, S. C., Chumley, F. G., and Valent, B. (1994). Isolation of the mating-type genes of the phytopathogenic fungus Magnaporthe grisea using genomic subtraction. Genetics 138, 289–296. Kao, T.-H., and McCubbin, A. G. (1996). How flowering plants discriminate between self and non-self pollen to prevent inbreeding. Proc. Natl. Acad. Sci. USA 93, 12059–12065. Karron, J. D., Marshall, D. L., and Oliveras, D. M. (1990). Numbers of sporophytic selfincompatibility alleles in wild radish. Theor. Appl. Genet. 79, 457–460. Kasahara, S., and Nuss, D. L. (1997). Targeted disruption of a fungal G-protein 웁 subunit gene results in increased vegetative growth but reduced virulence. Mol. Plant Microbe Interact. 10, 984–993. Kaufmann, H., Salamini, F., and Thompson, R. D. (1991). Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum. Mol. Gen. Genet. 226, 457–466. Kawamukai, M., Ferguson, M., Wigler, M., and Young, D. (1991). Genetic and biochemical analysis of the adenynyl cyclase of Schizosaccharomyces pombe. Cell Regul. 2, 155–164. Kawano, S., Tsuneyoski, K., and Anderson, R. W. (1987). A third multiallelic mating type locus in Physarum polycephalum. J. Gen. Microbiol. 133, 2529–2546. Kawano, S., Takano, H., and Kuroiwa, T. (1995). Sexuality of mitochondria, fusion, recombination, and plasmids. Int. Rev. Cytol. 161, 49–110. Kawata, Y., Sakiyama, F., Hayashi, F., and Kyogoku, Y. (1990). Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae. Eur. J. Biochem. 176, 683–697. Kellner, M., Burmester, A., Wo¨stemeyer, A., and Wo¨stemeyer, J. (1993). Transfer of genetic information from the mycoparasite Parasitella parasitica to its host Absidia glauca. Curr. Genet. 23, 334–337. Kelly, M., Burke, J., Smith, M., and Beach, D. (1988). Four mating-type genes control sexual differentiation in the fission yeast. EMBO J. 7, 1537–1547. Kemp, R. F. O. (1974). Bifactorial incompatibility in the two-spored basidiomycetes Coprinus sassii and Coprinus bilanatus. Trans. Br. Mycol. Soc. 62, 547–555. Kemp, R. F. O. (1975). Breeding biology of Coprinus species in the section Lanatuli. Trans. Br. Mycol. Soc. 65, 375–388. Kemp, R. F. O. (1977). Oidial homing and the taxonomy and speciation of basidiomycetes with special reference to the genus Coprinus. In ‘‘The Species Concept in Hymenomycetes’’ (H. Cle´menc¸on, Ed.), pp. 259–276. Cramer, Vaduz, Switzerland. Kertesz-Chaloupkova´, K., Walser, P. J., Granado, J. D., Aebi, M., and Ku¨es, U. (1998). Blue light overrides repression of asexual sporulation by mating type genes in the basidiomycete Coprinus cinereus. Fungal Genet. Biol. 23, 95–109. Kirouac-Brunet, J., Masson, S., and Pallotta, D. (1981). Multiple alleles at the matB locus in Physarum polycephalum. Can. J. Gen. Cytol. 23, 9–16. Kitamura, K., and Shimoda, C. (1991). The Schizosaccharomyces pombe mam2 gene encodes a putative pheromone receptor which has significant homology with the Saccharomyces cerevisiae STE2 protein. EMBO J. 10, 3743–3751.

276

SIMON J. HISCOCK AND URSULA KU¨ES

Kjaerulff, S., Davey, J., and Nielsen, O. (1994). Analysis of the structural gene encoding the M-factor in the fission yeast Schizosaccharomyces pombe: Identification of a third gene mfm3. Mol. Cell. Biol. 14, 3895–3905. Kjaerulff, S., Dooijes, D., Clevers, H., and Nielsen, O. (1997). Cell differentiation by interaction of the two HMG-box proteins: Mat1-Mc activates M cell-specific genes in S. pombe by recruiting the ubiquitous transcription factor Ste11 to weak binding sites. EMBO J. 16, 4021– 4033. Klar, A. J. S. (1992). Fission yeast cell type. In ‘‘The Molecular and Cellular Biology of the Yeast Saccharomyces’’ ( J. R. Broach, J. R. Pringle, and E. W. Jones, Eds.), pp. 745–777. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Klar, A. J. S. (1993). Lineage-dependent mating-type transposition in fission and budding yeast. Curr. Biol. 3, 745–751. Klar, A. J., Ivanova, A. V., Dalgaard, J. Z., Bonaduce, M. J., and Grewal, S. I. (1998). Multiple epigenetic events regulate mating-type switching of fission yeast. Novartis Found. Symp. 214, 87–99. Klekowski, E. J. (1972). Evidence against genetic self-incompatibility in the homosporous fern Pteridium aquilinum. Evolution 26, 66–73. Kniep, H. (1928). ‘‘Die Sexualita¨t der niederen Pflanzen.’’ Fischer, Jena. Knox, R. B., and Kenrick, J. (1983). Polyad function in relation to the breeding system of Acacia. In ‘‘Pollen: Biology and Implications for Plant Breeding’’ (D. Mulcahy and E. Ottaviano, Eds.), pp. 411–417. Elsevier, New York. Kohn, J. R., and Barrett, S. C. H. (1992). Experimental studies on the functional significance of heterostyly. Experientia 46, 43–55. Koltin, Y., Stamberg, J., and Lemke, P. A. (1972). Genetic structure and evolution of the incompatibility factors in higher fungi. Bacterial Rev. 36, 115–124. Komachi, K., and Johnson, A. D. (1997). Residues in the WD repeats of Tup1 required for interaction with 움2. Mol. Cell. Biol. 17, 6023–6028. Konopka, J. B., Margarit, S. M., and Dube, P. (1996). Mutation of Pro-258 in transmembrane domain 6 constitutively activates the G protein-coupled 움-factor receptor. Proc. Natl. Acad. Sci. USA 93, 6764–6769. Koppitz, M., Spellig, T., Kahmann, R., and Kessler, H. (1996). Lipoconjugates, structure– activity studies for pheromone analogues of Ustilago maydis with varied lipophilicity. Int. J. Peptide Protein Res. 48, 377–390. Koski, V. (1973). On self-pollination, genetic load and subsequent inbreeding in some conifers. Commun. Inst. Forest. Fenniae 78.10, 1–40. Kothe, E. (1996). Tetrapolar fungal mating types, sexes by the thousands. FEMS Microbiol. Rev. 18, 65–87. Kothe, G. O., and Free, S. J. (1998). The isolation and characterization of nrc-1 and nrc-2, two genes encoding protein kinases that control growth and development in Neurospora crassa. Genetics 149, 117–130. Kowyama, Y., Takahasi, H., Muraoka, K., Tani, T., Hara, K., and Shiotani, I. (1994). The number, frequency and dominance relationships of S-alleles in diploid Ipomoea trifida. Heredity 73, 275–283. Kowyama, Y., Kakeda, K., Nakano, R., and Hattori, T. (1995). SLG/SRK-like genes are expressed in the reproductive tissues of Ipomoea trifida. Sex. Plant Reprod. 8, 333–338. Kowyama, Y., Kakeda, K., Kondo, K., Imada, T., and Hattori, T. (1996). A putative receptor kinase gene in Ipomoea trifida. Plant Cell Physiol. 37, 681–685. Krebs, S. L., and Hancock, J. F. (1991). Embryonic genetic load in the highbush blueberry, Vaccinium corymbossum (Ericaceae). Am. J. Bot. 78, 1427–1437. Kroh, M. (1966). Reaction of pollen after transfer from one stigma to another (contribution of the character of the incompatibility mechanism in Cruciferae). Zu¨chter 36, 185–189.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

277

Kronstad, J. W. (1997). Virulence and cAMP in smuts, blasts and blights. Trends Plant Sci. 2, 193–199. Kronstad, J. W., and Leong, S. A. (1990). The b mating-type locus of Ustilago maydis contains variable and constant regions. Genes Dev. 4, 1384–1395. Kronstad, J. W., and Staben, C. (1997). Mating type in filamentous fungi. Annu. Rev. Genet. 31, 245–276. Kronstad, J., De Maria, A. D., Funnell, D., Laidlaw, R. D., Lee, N., de Sa, M. M., and Ramesh, M. (1998). Signalling via cAMP in fungi: Interconnections with mitogen-activated protein kinase pathways. Arch. Microbiol. 170, 395–404. Kru¨ger, J., Aichinger, C., Kahmann, R., and Bo¨lker, M. (1997). A MADS-box homologue in Ustilago maydis regulates the expression of pheromone-inducible genes but is nonessential. Genetics 147, 1643–1652. Kru¨ger, J., Loubradou, G., Regenfelder, E., Hartmann, A., and Kahmann, R. (1998). Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis. Mol. Gen. Genet. 260, 193–198. Ku¨es, U. (1995). Evolution of A factor specificity genes in Coprinus cinereus. Fungal Genet. Newslett. 42A, 67. Ku¨es, U., and Casselton, L. A. (1992a). Fungal mating type genes—Regulators of sexual development. Mycol. Res. 96, 993–1006. Ku¨es, U., and Casselton, L. A. (1992b). Homeodomains and regulation of sexual development in basidiomycetes. Trends Genet. 8, 154–155. Ku¨es, U., and Casselton, L. A. (1992c). Molecular and functional analysis of the A mating type genes of Coprinus cinereus. In ‘‘Genetic Engineering. Principles and Methods. Vol. 14’’ ( J. K. Setlow, Ed.), pp. 251–268. Plenum, New York. Ku¨es, U., and Casselton, L. A. (1993a). Regulation of fungal development by mating type genes. In ‘‘The Eukaryotic genome. Organisation and Regulation’’ (P. M. A. Broda, S. G. Oliver, and P. F. G. Sims, Eds.), Society for General Micobiology Symposium 50, pp. 185–210. Cambridge Univ. Press, Cambridge, UK. Ku¨es, U., and Casselton, L. A. (1993b). The origin of multiple mating types in mushrooms. J. Cell Sci. 104, 227–230. Ku¨es, U., and Challen, M. P. (1994). HD1 and HD2 genes in the multiple A mating type factor of higher basidiomycetes. BioEngineering 3/94, 64. Ku¨es, U., and Challen, M. P. (1995). Molecular characterization of the A1 mating type factor of Coprinus bilanatus. Fungal Genet. Newslett. 42A, 66. Ku¨es, U., and Stahl, U. (1992). Sexual reproduction—A tool for outcrossing and recombination of genetic material. Prog. Bot. 54, 277–294. Ku¨es, U., Richardson, W. V. J., Tymon, A. M., Mutasa, E. S., Go¨ttgens, B., Gaubatz, S., Gregoriades, A., and Casselton, L. A. (1992). The combination of dissimilar alleles of the A움 and A웁 gene complexes, whose proteins contain homeodomain motifs, determine sexual development in the mushroom Coprinus cinereus. Genes Dev. 4, 568–577. Ku¨es, U., Asante-Owusu, R. N., Mutasa, E. S., Tymon, A. M., Pardo, E. H., O’Shea, S. F., and Casselton, L. A. (1994a). Two classes of homeodomain proteins specify the multiple A mating types of the mushroom Coprinus cinereus. Plant Cell 6, 1467–1475. Ku¨es, U., Go¨ttgens, B., Stratmann, R., Richardson, W. V. J., O’Shea, S. F., and Casselton, L. A. (1994b). A chimeric homeodomain protein causes self-compatibility and constitutive sexual development in the mushroom Coprinus cinereus. EMBO J. 13, 4054–4059. Ku¨es, U., Tymon, A. M., Richardson, W. V. J., May, G., Gieser, P. T., and Casselton, L. A. (1994c). A mating-type factors of Coprinus cinereus have variable numbers of specificity genes encoding two classes of homeodomain proteins. Mol. Gen. Genet. 245, 45–52. Ku¨es, U., Granado, J. D., Hermann, R., Boulianne, R. P., Kertesz-Chaloupkova´, K., and Aebi, M. (1998a). The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus. Mol. Gen. Genet. 260, 81–91.

278

SIMON J. HISCOCK AND URSULA KU¨ES

Ku¨es, U., Granado, J. D., Kertesz-Chaloupkova´, K., Walser, P. J., Hollenstein, M., Polak, E., Liu, Y., Boulianne, R. P. Bottoli, A. P. F., and Aebi, M. (1998b). Mating types and light are major regulators of development in Coprinus cinereus. In ‘‘Proceedings of the Fourth Meeting on the Genetics and Cellular Biology of Basidiomycetes’’ (L. J. L. D. Van Griensven and J. Visser, Eds.), pp. 113–118. Mushroom Experimental Station, Horst, the Netherlands. Kuhlmann, H.-W., Bru¨nen-Nieweler, C., and Heckmann, K. (1997). Pheromones of the ciliate Euplotes octocarinatus not only induce conjugation but also function as chemoattractants. J. Exp. Zool. 277, 38–48. Kuhn, J., and Parag, Y. (1972). Protein-subunit aggregation model for self-incompatibility in higher fungi. J. Theor. Biol. 35, 77–91. Kunz, C., Chang, A., Faure, J. D., Clarke, A. E., Polya, G. M., and Anderson, M. A. (1996). Phosphorylation of style S-RNases by Ca2⫹-dependent protein kinases from pollen tubes. Sex. Plant Reprod. 9, 25–34. Kurjan, J. (1992). Pheromone response in yeast. Annu. Rev. Biochem. 61, 1097–1129. Kurjan, J. (1993). The pheromone response pathway in Saccharomyces cerevisiae. Annu. Rev. Genet. 27, 147–179. Kurvari, V., Grishin, N. V., and Snell, W. J. (1998). A gamete-specific, sex-limited homeodomain protein in Chlamydomonas. J. Cell Biol. 143, 1971–1980. Kusaba, M., Nishio, T., Satta, Y., Hinata, K., and Ockendon, D. (1997). Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleraceae and Brassica campestris: Implications for the evolution and recognition mechanism. Proc. Natl. Acad. Sci. USA 94, 7673–7678. Kwon-Chung, K. J., Edman, J. C., and Wickes, B. L. (1992). Genetic association of mating types and virulence in Cryptococcus neoformans. Infect. Immun. 60, 602–605. Labare`re, J., and Noe¨l, T. (1992). Mating type switching in the tetrapolar basidiomycete Agrocybe aegerita. Genetics 131, 307–319. Laity, C., Giasson, R., Campbell, R., and Kronstad, J. W. (1995). Heterozygosity at the b mating type locus attenuates fusion in Ustilago maydis. Curr. Genet. 27, 451–455. Lalonde, B. A., Nasrallah, M. E., Dwyer, K. G., Chen, C. H., Barlow, B., and Nasrallah, J. B. (1989). A highly conserved Brassica gene with homology to the S-locus-specific glycoprotein structural gene. Plant Cell 1, 249–258. Lane, M. D., and Lawrence, M. J. (1993). The population genetics of the self-incompatibility polymorphism in Papaver rhoeas. VII. The number of S-alleles in the species. Heredity 71, 596–602. Lawrence, M. J. (1996). Number of incompatibility alleles in clover and other species. Heredity 76, 610–615. Lawrence, M. J., Lane, M. D., O’Donnell, S., and Franklin-Tong, V. E. (1993). The population genetics of the self-incompatibility polymorphism in Papaver rhoeas. V. Cross-classification of the S-alleles of samples from three natural populations. Heredity 71, 581–590. Leary, J. V., and Ellingboe, A. H. (1970). The kinetics of initial nuclear exchange in compatible and noncompatible matings of Schizophyllum commune. Am. J. Bot. 57, 19–23. Leberer, E., Thomas, D. Y., and Whiteway, M. (1997). Pheromone signalling and polarized morphogenesis in yeast. Curr. Opin. Genet. Dev. 7, 59–66. Lee, H.-S., Huang, S., and Kao, T.-H. (1994). S proteins control rejection of incompatible pollen in Petunia inflata. Nature 367, 560–563. Leeuw, T., Fourest-Lieuvin, A., Wu, C., Chenevert, J., Clark, K., Whiteway, M., Thomas, D. Y., and Leberer, E. (1995). Pheromone response in yeast: Association of Bem1p with proteins of the MAP kinase cascade and actin. Science 270, 1210–1213. Leidl, B. E., McCormick, S., and Mutschler, M. A. (1996). Unilateral incongruity in crosses involving Lycopersicon pennellii and L. esculentum is distinct from self-incompatibility in expression, timing and location. Sex. Plant Reprod. 9, 299–308.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

279

Leiting, B., De Francesco, R., Tomei, L., Cortese, R., Otting, G., and Wu¨thrich, K. (1993). The three-dimensional NMR-solution structure of the polypeptide fragment 195–286 of the LFB1/HNF1 transcription factor from rat liver comprises a nonclassical homeodomain. EMBO J. 12, 1797–1803. Letham, D. L. D., and Nasrallah, J. B. (1998). A ClpP homolog linked to the Brassica selfincompatibility (S ) locus. Sex. Plant Reprod. 11, 117–119. Leubner-Metzger, G., Horwitz, B. A., Yoder, O. C., and Turgeon, B. G. (1997). Transcripts at the mating type locus of Cochliobolus heteostrophus. Mol. Gen. Genet. 256, 661–673. Levin, D. A. (1993). S-gene polymorphism in Phlox drummondii. Heredity 71, 193–198. Levin, D. A. (1996). The evolutionary significance of pseudo-self-fertility. Am. Nat. 148, 321–332. Lewin, R. A. (1976). ‘‘The Genetics of Algae,’’ Botanical monographs Vol. 12. Blackwell Sci., Oxford. Lewis, D. (1947). Competition and dominance of incompatibility alleles in diploid pollen. Heredity 1, 85–108. Lewis, D. (1948). Mutation of the incompatibility gene. I. Spontaneous mutation rate. Heredity 2, 219–236. Lewis, D. (1949a). Incompatibility in flowering plants. Biol. Rev. 24, 472–496. Lewis, D. (1949b). Mutation of the incompatibility gene. II. Induced mutation rate. Heredity. 3, 339–355. Lewis, D. (1951). Structure of the incompatibility gene. III. Types of spontaneous and induced mutation. Heredity 5, 399–414. Lewis, D. (1960). Genetic control of specificity and activity of the S antigen in plants. Proc. R. Soc. London B 151, 468–477. Lewis, D. (1965). A protein dimer hypothesis on incompatibility. In ‘‘Genetics Today, Proceedings of the XI International Congress on Genetics 1963’’ (S. J. Geerts, Ed.), pp. 657–663. Pergamon, Oxford. Lewis, D. (1979a). ‘‘Sexual Incompatibility in Plants.’’ Arnold, London. Lewis, D. (1979b). Genetic versatility of incompatibility in plants. N. Z. J. Bot. 17, 637–644. Lewis, D. (1994). Gametophytic–sporophytic incompatibility. In ‘‘Genetic Control of SelfIncompatibility and Reproductive Development in Flowering Plants’’ (E. G. Williams, A. E. Clarke, and R. B. Knox, Eds.), pp. 88–101. Kluwer, Dordrecht. Lewis, D., and Crowe, L. K. (1958). Unilateral interspecific incompatibility in flowering plants. Heredity 12, 233–256. Lewis, D., Verma, S. C., and Zuberi, M. I. (1988). Gametophytic–sporophytic incompatibility in the Cruciferae, Raphanus sativus. Heredity 61, 355–366. Li, J., Kusaba, M., Newbigin, E., and Clarke, A. E. (1988). Identifying pollen-expressed genes linked to the S-locus of Nicotiana alata. XVIII International Congress on Genetics, Beijing, China. [Abstract]. Li, P., and McLeod, M. (1996). Molecular mimicry in development: Identification of ste11⫹ as a substrate and mei3⫹ as a pseudosubstrate inhibitor of ran1⫹ kinase. Cell 87, 869–880. Li, T., Stark, M. R., Johnson, A. D., and Wolberger, C. (1995). Crystal structure of the MATa1/ MAT움2 homeodomain heterodimer bound to DNA. Science 270, 262–269. Li, X., Nield, J., Hayman, D., and Langridge, P. (1994). Cloning a putative self-incompatibility gene from the pollen of the grass Phalaris coerulescens. Plant Cell 6, 1923–1932. Li, X., Nield, J., Hayman, D., and Langridge, P. (1995). Thioredoxin activity in the C terminus of Phalaris S protein. Plant J. 7, 133–138. Li, X., Nield, J., Hayman, D., and Langridge, P. (1996). A self-fertile mutant of Phalaris produces an S protein with reduced thioredoxin activity. Plant J. 10, 505–513. Li, X., Peach, N., Nield, J., Hayman, D., and Langridge, P. (1997). Self-incompatibility in the grasses: Evolutionary relationship of the S gene from Phalaris coerulescens to homologous sequences in other grasses. Plant Mol. Biol. 34, 223–232.

280

SIMON J. HISCOCK AND URSULA KU¨ES

Lichter, A., and Mills, D. (1997). Fil1, a G-protein 움-subunit that acts upstream of cAMP and is essential for dimorphic switching in haploid cells of Ustilago hordei. Mol. Gen. Genet. 256, 426–435. Liu, S., and Dean, R. A. (1997). G protein 움 subunit genes control growth, development, and pathogenicity of Magnaporthe grisea. Mol. Plant Microbe Interact. 10, 1075–1086. Lovis, J. D. (1977). Evolutionary patterns and processes in ferns. Adv. Bot. Res. 4, 229–415. Luginbu¨hl, P., Ottinger, M., Mronga, S., and Wu¨thrich, K. (1994). Structure comparison of the pheromones Er-1, Er-10, and Er-2 from Euplotes raikovi. Protein Sci. 3, 1537–1546. Luginbu¨hl, P., Wu, J., Zerbe, O., Ortenzi, C., Luporini, P., and Wu¨thrich, K. (1996). The NMR solution structure of the pheromone Er-11 from the ciliated protozoan Euplotes raikovi. Protein Sci. 5, 1512–1522. Lukens, L., Yicun, H., and May, G. (1996). Correlation of genetic and physical maps at the A mating type locus of Coprinus cinereus. Genetics 144, 1471–1477. Lundqvist, A. (1955). Studies of self-sterility in rye, Secale cereale L. Hereditas 40, 278–294. Lundqvist, A. (1956). Self-incompatibility in rye I. Genetic control in the diploid. Hereditas 42, 293–348. Lundqvist, A. (1990a). One-locus sporophytic S-gene system with traces of gametophytic pollen control in Cerastium arvense ssp. strictum (Carophyllaceae). Hereditas 113, 203–215. Lundqvist, A. (1990b). Variability within and among populations in the 4-gene system for the control of self-incompatibility in Ranunculus polyanthemos. Hereditas 113, 47–61. Lundqvist, A. (1991). Four-locus S-gene control of self-incompatibility made probable in Lilium martagon (Liliaceae). Hereditas 114, 57–63. Lundqvist, A. (1994). ‘‘Slow’’ and ‘‘quick’’ S-alleles without dominance interaction in the sporophytic one-locus self-incompatibility systen of Stellaria holostea (Carophyllaceae). Hereditas 120, 191–202. Lundqvist, A. (1995). Concealed genes for self-incompatibility in the carnation family Carophyllaceae? Hereditas 122, 85–89. Lundqvist, A., Østerbye, V., Larsen, K., and Linde-Laursen, I. (1973). Complex self-incompatibility systems in Ranunculus acris L. and Beta vulgaris L. Hereditas 74, 161–168. Luo, Y. H., Ullrich, R. C., and Novotny, C. P. (1994). Only one of the paired Schizophyllum commune A움 mating-type putative homeobox genes encodes a homeodomain essential for A움 regulated development. Mol. Gen. Genet. 244, 318–324. Luporini, P., Vallesi, A., Miceli, C., and Bradshaw, R. A. (1995). Chemical signalling in ciliates. J. Eukaryote Microbiol. 42, 208–212. Luporini, P., Miceli, C., Ortenzi, C., and Vallesi, A. (1996). Ciliate pheromones. Prog. Mol. Subcell. Biol. 17, 80–104. Lush, M. W., and Clarke, A. E. (1997). Observations on pollen tube growth in Nicotiana alata and their implications for the mechanism of self-incompatibility. Sex. Plant Reprod. 10, 27–35. Luu, D.-T., Heizmann, P., Dumas, C., Trick, M., and Cappadocia, M. (1997). Involvement of SLR1 genes in pollen adhesion to the stigma surface in Brassicaceae. Sex. Plant Reprod. 10, 227–235. Luu, D.-T., Marty Mazars, D., Trick, M., Dumas, C., and Heizmann, P. (1999). Pollen-stigma adhesion in Brassica spp. involves SLG and SLR-1 glycoprotein. Plant Cell 11, 251–262. Mach, K., Cheng, Q.-C., and Albright, C. F. (1998). ras1 and pat1 alleles interact to quantitatively and qualitatively alter conjugation in fission yeast. Curr. Genet. 34, 172–182. Madden, K., and Snyder, M. (1998). Cell polarity and morphogenesis in budding yeast. Annu. Rev. Microbiol. 52, 687–744. Maeda, T., Watanabe, Y., Kunitomo, H., and Yamamoto, M. (1994). Cloning of the pka1 gene encoding the catalytic subunit of the cAMP-dependent protein kinase in Schizosaccharomyces pombe. J. Biol. Chem. 269, 9632–9637. Magae, Y., Novotny, C., and Ullrich, R. (1995). Interaction of the A움 Y mating-type and Z mating-type homeodomain proteins of Schizophyllum commune detected by the two-hybrid system. Biochem. Biophys. Res. Commun. 211, 1071–1076.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

281

Mahlo, R., Read, N. D., Pais, M. S., and Trewavas, A. J. (1994). Role of cytosolic free calcium in reorientation of pollen tube growth. Plant J. 5, 331–341. Mak, A., and Johnson, A. D. (1993). The carboxy-terminal tail of the homeodomain protein 움2 is required for function with a second homeodomain protein. Genes Dev. 7, 1862–1870. Marsh, L., and Rose, M. D. (1997). The pathway of cell and nuclear fusion during mating in S. cerevisiae. In ‘‘The Molecular and Cellular Biology of the Yeast Saccharomyces Vol. 3. Cell Cycle and Cell Biology’’ ( J. R. Pringle, J. R. Broach, and E. W. Jones, Eds.), pp. 827–888. Cold Spring Harbor Laboraty Press, Cold Spring Harbor, NY. Martin, F. W. (1964). The inheritance of unilateral incompatibility in Lycopersicon hirsutum. Genetics 50, 459–469. Martin, F. W. (1967). The genetic control of unilateral incompatibility between two tomato species. Genetics 56, 391–398. Martin, F. W. (1968). The behaviour of Lycopersicon incompatibility alleles in an alien genetic milieu. Genetics 60, 101–109. Martinez-Espinoza, A. D., Gerhardt, S. A., and Sherwood, J. W. (1993). Morphological and mutational analysis of mating in Ustilago hordei. Exp. Mycol. 17, 200–214. Mascarenhas, J. P. (1993). Molecular mechanisms of pollen tube growth and differentiation. Plant Cell 5, 1303–1314. Mathieson, M. J. (1952). Ascospore dimorphism and mating type in Chromocrea spinulosa (Fuckel) Petch n. comb. Ann. Bot. 16, 449–466. Matton, D. P., Maes, O., Laublin, G., Xike, Q., Bertrand, C., Morse, D., and Cappadocia, M. (1997). Hypervariable domains of self-incompatible RNases mediate allele-specific pollen recognition. Plant Cell 9, 1757–1766. Matton, D. P., Morse, D., and Cappadocia, M. (1998). Reply. Plant Cell 10, 316–317. Mau, S.-L., Williams, E. G., Atkinson, A., Cornish, E. C., Grego, B., Simpson, R., Kheyr-Pour, A., and Clarke, A. E. (1986). Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility. Planta 169, 184–191. May, G., and Matzke, E. (1995). Recombination and variation at the A mating-type of Coprinus cinereus. Mol. Biol. Evol. 12, 794–802. May, G., and Taylor, J. W. (1988). Patterns of mating and mitochondrial DNA inheritance in the agaric basidiomycete Coprinus cinereus. Genetics 118, 213–220. May, G., Le Chevanton, L., and Pukkila, P. J. (1991). Molecular analysis of the Coprinus cinereus mating type A factor demonstrated an unexpectedly complex structure. Genetics 128, 529–538. Maynard Smith, J. (1978). ‘‘The Evolution of Sex.’’ Cambridge Univ. Press, New York. Mayorga, M.E., and Gold, S. E. (1998). Characterization and molecular genetic complementation of mutants affecting dimorphism in the fungus Ustilago maydis. Fungal Genet. Biol. 24, 364–376. McClure, B. A., Haring, V., Ebert, P. R., Anderson, M. A., Simpson, R. J., Sakiyama, F., and Clarke, A. E. (1989). Style self-incompatibility gene products of Nicotiana alata are ribonucleases. Nature 342, 955–957. McClure, B. A., Gray, J. E., Anderson, M. A., and Clarke, A. E. (1990). Self-incompatibility in Nicotiana alata involves degradation of pollen rRNA. Nature 347, 757–760. McCormick, S. (1998). Self-incompatibility and other pollen–pistil interactions. Curr. Opin. Plant Biol. 1, 18–25. McCubbin, A. G., Chung, Y.-Y., and Kao, T.-H. (1997). A mutant S3 RNase of Petunia inflata lacking RNase activity has an allele-specific dominant negative effect on self-incompatibility interactions. Plant Cell 9, 85–95. McGuire, D. C., and Rick, C. M. (1954). Self-incompatibility in species of Lycopersicon Sect. Eriopersicon and hybrids with L. esculentum. Hilgardia 23, 101–124. McLeod, M., Stein, M., and Beach, D. (1987). The product of the mei3⫹ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast. EMBO J. 6, 729–736.

282

SIMON J. HISCOCK AND URSULA KU¨ES

Meinhardt, F., and Esser, K. (1990). Sex determination and sexual differentiation in filamentous fungi. Crit. Rev. Plant Sci. 9, 329–341. Meland, S., Johansen, S., Johansen, T., Haugli, K., and Haugli, F. (1991). Rapid disappearance of one parental mitochondrial genotype after isogameous mating in the myxomycete Physarum polycephalum. Curr. Genet. 19, 55–60. Mergen, F., Burley, J., and Furnival, G. M. (1965). Embryo and seedling development in Picea glauca (Moench) Vos after self, cross, and wind pollination. Silvae Genet. 14, 188–194. Metzenberg, R. L., and Glass, N. L. (1990). Mating-type and mating strategies in Neurospora. Bioessays 12, 53–59. Metzenberg, R. L., and Randall, T. A. (1995). Mating type in Neurospora and closely related ascomycetes. Some current problems. Can. J. Bot. 73, S258–S265. Miceli, C., La Terza, A., Bradshaw, R. A., and Luporini, P. (1992). Identification and structural characterization of a cDNA clone encoding a membrane-bound form of the polypeptide pheromone Er-1 in the ciliate protozoan Euplotes raikovi. Proc. Natl. Acad. Sci. USA 89, 1988–1992. Michod, R. E., and Levin, B. R. (Eds.) (1988). ‘‘The Evolution of Sex: An Examination of Current Ideas.’’ Sinauer, Sunderland, MA. Mochizuki, N., and Yamamoto, M. (1992). Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast. Mol. Gen. Genet. 233, 17–24. Moore, T. D. E., and Edman, J. C. (1993). The a mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol. Cell. Biol. 13, 1962–1970. Mulcahy, D. L., and Mulcahy, G. B. (1983). Gametophytic self-incompatibility reexamined. Science 220, 1247–1251. Mullins, J. T. (1994). Hormonal control of sexual dimorphism. In ‘‘The Mycota. Vol. I. Growth, Differentiation and Sexuality’’ ( J. G. H. Wessels and F. Meinhardt, Eds.), pp. 413–424. Springer-Verlag, Berlin. Murata, Y., Fujii, M., Zolan, M. E., and Kamada, T. (1998). Molecular analysis of pcc1, a gene that leads to A-regulated sexual morphogenesis in Coprinus cinereus. Genetics 149, 1753–1761. Murfett, J., Atherton, T. L., Mou, B., Gasser, C. S., and McClure, B. A. (1994). S RNase expressed in transgenic Nicotiana causes S allele-specific pollen rejection. Nature 367, 563–566. Murfett, J., Strabala, T. J., Zurek, D. M., Mou, B., Beecher, B., and McClure, B. A. (1996). S-RNase and interspecific pollen rejection in the genus Nicotiana: Multiple pollen-rejection pathways contribute to unilateral incompatibility between self-incompatible and selfcompatible species. Plant Cell 8, 943–958. Mutschler, M. A., and Leidl, B. E. (1994). Interspecific crossing barriers in Lycopersicon and their relationship to self-incompatibility. In ‘‘Genetic Control of Self-Incompatibility and Reproductive Development in Flowering Plants’’ (E. G. Williams, A. E. Clarke, and R. B. Knox, Eds.), pp. 164–188. Kluwer, Dordrecht. Nadin-Davis, S. A., and Nasim, A. (1990). Schizosaccharomyces pombe ras1 and byr1 are functionally related genes of the ste family that affect starvation-induced transcription of mating-type genes. Mol. Cell. Biol. 10, 549–560. Nakayama, N., Miyajima, A., and Arai, K. (1985). Nucleotide sequence of STE2 and STE3, cell-type specific sterile genes from Saccharomyces cerevisiae. EMBO J. 4, 2643–2648. Nasrallah, J. B. (1997). Evolution of the Brassica self-incompatibility locus: A look into Slocus gene polymorphisms. Proc. Natl. Acad. Sci. USA 94, 9516–9519. Nasrallah, J. B., and Nasrallah, M. E. (1993). Pollen–stigma signalling in the sporophytic selfincompatibility response. Plant Cell 5, 1325–1335. Nasrallah, J. B., Doney, R. C., and Nasrallah, M. E. (1985a). Biosynthesis of glycoproteins involved in the pollen–stigma interaction of incompatibility in developing flowers of Brassica oleracea L. Planta 165, 100–107.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

283

Nasrallah, J. B., Kao, T.-H., Goldberg, M. L., and Nasrallah, M. E. (1985b). A cDNA clone encoding an S-locus specific glycoprotein from Brassica oleracea. Nature 318, 263–267. Nasrallah, J. B., Kao, T.-H., Chen, C. H., Goldberg, M. L., and Nasrallah, M. E. (1987). Amino-acid sequence of glycoproteins encoded by three alleles of the S locus of Brassica oleracea. Nature 326, 617–619. Nasrallah, J. B., Yu, S. M., and Nasrallah, M. E. (1988). Self-incompatibility genes of Brassica oleracea: Expression, isolation and structure. Proc. Natl. Acad. Sci. USA 85, 5551–5555. Nasrallah, J. B., Nishio, T., and Nasrallah, M. E. (1991). The self-incompatibility genes of Brassica: Expression and use in genetic ablation of floral tissues. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 393–422. Nasrallah, J. B., Rundle, S. J., and Nasrallah, M. E. (1994a). Genetic evidence for the requirement of the Brassica S-locus receptor kinase gene in the self-incompatibility response. Plant J. 5, 373–384. Nasrallah, J. B., Stein, J. C., Kandasamy, M. K., and Nasrallah, M. E. (1994b). Signalling the arrest of pollen tube development in self-incompatible plants. Science 266, 1505–1508. Nasrallah, M. E. (1989). The genetics of self-incompatibility reactions in Brassica and the effects of suppressor genes. In ‘‘Plant Reproduction: From Floral Induction to Pollination’’ (E. Lord and G. Bernier, Eds.), Am. Soc. Plant Physiologists Symp. Ser. 1, pp. 146–155. Am. Soc. Plant Physiologists, Rockville, MD. Nasrallah, M. E., and Wallace, D. H. (1967). Immunogenetics of self-incompatibility in Brassica oleracea L. Heredity 22, 519–527. Nasrallah, M. E., Barber, J. T., and Wallace, D. H. (1970). Self-incompatibility proteins in plants: Detection, genetics, and possible mode of action. Heredity 25, 23–27. Nasrallah, M. E., Wallace, D. H., and Savo, R. M. (1972). Genotype, protein, phenotype relationships in self-incompatibility of Brassica. Genet. Res. 20, 151–160. Nasrallah, M. E., Kandasamy, M. K., and Nasrallah, J. B. (1992). A genetically defined transacting locus regulates S-locus function in Brassica. Plant J. 2, 497–506. Natvig, D. O., and May, G. (1996). Fungal evolution and speciation. J. Genet. 75, 441–452. Nauta, M. J., and Hoekstra, R. F. (1992). Evolution of reproductive systems in filamentous ascomycetes. I. Evolution of mating types. Heredity 68, 405–410. Neiman, A. M., Stevenson, J., Xu, H. P., Sprague, G. F., Jr., Herskowitz, I., Wigler, M., and Marcus, S. (1993). Functional homology of protein kinases required for sexual activation in Schizosaccharomyces pombe and Saccharomyces cerevisiae suggests a conserved signal transduction module in eukaryotic organisms. Mol. Biol. Cell 4, 107–120. Nelson, M. A. (1996). Mating systems in ascomycetes, a romp in the sac. Trends Genet. 12, 69–74. Nelson, M. A., and Metzenberg, R. L. (1992). Sexual development genes of Neurospora crassa. Genetics 132, 149–162. Nelson, M. A., Kang, S., Braun, E. L., Crawford, M. E., Dolan, P. L., Leonard, P. M., Mitchell, J., Armijo, A. M., Bean, L., Blueeyes, E., Chushing, T., Errett, A., Fleharty, M., Gorman, M., Judson, K., Miller, R., Ortega, J., Pavolova, I., Perea, S., Todisco, S., Trujillo, R., Valentine, J., Wells, A., Werner-Washburne, M., Yazzie, S., and Natvig, D. O. (1997a). Expressed sequences from conidial, mycelial, and sexual stages of Neurospora crassa. Fungal Genet. Biol. 21, 348–363. Nelson, M. A., Merino, S. T., and Metzenberg, R. L. (1997b). A putative rhamnogalacturonase required for sexual development of Neurospora crassa. Genetics 146, 531–540. Nern, A., and Arkowitz, R. A. (1998). A GTP-exchange factor required for cell orientation. Nature 391, 195–198. Newbigin, E. (1996). The evolution of self-incompatibility: A molecular voyeur’s perspective. Sex. Plant Reprod. 9, 357–361. Newbigin, E., Anderson, M. A., and Clarke, A. E. (1993). Gametophytic self-incompatibility systems. Plant Cell 5, 1315–1324.

284

SIMON J. HISCOCK AND URSULA KU¨ES

Nielsen, O. (1993). Signal transduction during mating and meiosis in S. pombe. Trends Cell Biol. 3, 60–65. Nielsen, O., and Davey, J. (1995). Pheromone communication in the fission yeast Schizosaccharomyces pombe. Sem. Cell Biol. 6, 95–104. Nielsen, O., and Egel, R. (1990). The pat1 protein kinase controls transcription of the matingtype genes in fission yeast. EMBO J. 9, 1401–1406. Nielsen, O., Davey, J., and Egel, R. (1992). The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription. EMBO J. 11, 1391–1395. Nielsen, O., Friis, T., and Kjærulff, S. (1996). The Schizosaccharomyces pombe map1 gene encodes an SRF/MCM1-related protein required for P-cell specific gene expression. Mol. Gen. Genet. 253, 387–392. Niikura, S., and Matsuura, S. (1997). Genomic sequence and expression of S-locus gene in radish (Raphanus sativus L). Sex. Plant Reprod. 10, 250–252. Nishio, T., and Hinata, K. (1977). Analysis of S-specific proteins in stigma of Brassica oleracea L. by isoelectric focusing. Heredity 38, 391–396. Norioka, N., Norioka, S., Ohnishi, Y., Ishimizu, T., Oneyama, C., Nakanishi, T., and Sakiyama, F. (1996). Molecular cloning and nucleotide sequences of cDNAs encoding S-allele-specific stylar RNases in a self-incompatible cultivar and its self-compatible mutant of Japanese Pear, Pyrus pyrifolia. J. Biochem. 120, 335–345. North, J. (1990). Coprinus cinereus. In ‘‘Genetic Maps. Locus Maps of Complex Genomes. Fifth Edition’’ (S. J. O’Brien, Ed.), pp. 3.76–3.81. Cold Spring Harbor Laboratory Press., Cold Spring Harbor, NY. Nou, I. S., Watanabe, M., Isogai, A., and Hinata, K. (1993). Comparison of S-alleles and Sglycoproteins between two wild populations of Brassica campestris in Turkey and Japan. Sex. Plant Reprod. 6, 79–86. Obara, T., Nakafuku, M., Yamamoto, M., and Kaziro, Y. (1991). Isolation and characterization of a gene encoding a G-protein 움 subunit from Schizosaccharomyces pombe, involvement in mating and sporulation pathways. Proc. Natl. Acad. Sci. USA 88, 5877–5881. Ockendon, D. J. (1974). Distribution of self-incompatibility alleles and breeding structure of open-pollinated cultivars of Brussels sprouts. Heredity 33, 159–171. O’Day, D. H., Rama, R. R., and Lydan, M. A. (1987). Gamete formation reflects the pheromonal hierarchy of Dictyostelium giganteum. Experientia 43, 619–621. O’Day, D. H., Lewis, K. E., and Lydan, M. A. (1995). Amoedoid gametes and fertilization in Dictyostelium. Gamete and pronuclear fusion are mediated by calmodulin and its binding proteins. Me´m. Mus. Natl. History Nat. 166, 23–36. O’Donnell, S., and Lawrence, M. J. (1984). The population genetics of the self-incompatibility polymorphism in Papaver rhoeas. IV. The estimation of the number of alleles in a population. Heredity 53, 495–507. Oehlen, B., and Cross, F. R. (1994). Signal transduction in the budding yeast Saccharomyces cerevisiae. Curr. Opin. Cell. Biol. 6, 836–841. Okazaki, N., Okazaki, K., Watanabe, Y., Kato-Hayashi, M., Yamamoto, M., and Okayams, H. (1998). Novel factor highly conserved among eukaryotes controls sexual development in fission yeast. Mol. Cell. Biol. 18, 887–895. Olsson, S., and Gray, S. N. (1998). Patterns and dynamics of 32P-phosphate and labelled 2-aminoisobutyric acid (14C-AIB) translocation in intact basidiomycete mycelia. FEMS Microbiol. Ecol. 26, 109–120. O’Shea, S. F., Chaure, P. T., Halsall, J. R., Olesnicky, N. S., Leibbrandt, A., Connerton, I. F., and Casselton, L. A. (1998). A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus. Genetics 148, 1081–1090. Otto, S. P., and Goldstein, D. B. (1992). Recombination and the evolution of diploidy. Genetics 131, 745–751. Owens, J. N., Colangeli, A. M., and Morris, S. J. (1990). The effect of self-, cross-, and no pollination on ovule, embryo, seed and cone development in western red cedar (Thuja plicata). Can. J. Forest Res. 20, 66–75.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

285

Owens, J. N., Colangeli, A. M., and Morris, S. J. (1991). Factors affecting seed set in Douglas fir (Pseudotsuga menziesii). Can. J. Bot. 69, 229–238. Pandey, K. K. (1965). Centric chromosome fragments and pollen-part mutation of the incompatibility gene. Nature 206, 792–795. Pandey, K. K. (1968). Compatibility relationships in flowering plants: Role of the S-gene complex. Am. Nat. 102, 475–489. Pandey, K. K. (1969). Elements of the S-gene complex. V. Interspecific cross-compatibility relationships and theory of the evolution of the S-complex. Genetica 40, 447–474. Pandey, K. K. (1973). Phases in S-gene expression, and S-allele interaction in the control of interspecific incompatibility. Heredity 31, 381–400. Pandey, K. K. (1975). Model for incompatibility determination in flowering plants. Incompatibility Newslett. 6, 70–73. Pandey, K. K. (1979). The genus Nicotiana: Evolution of incompatibility in flowering plants. Linnean Soc. Symp. Ser. 7, 421–434. Pandey, K. K. (1980). Evolution of incompatibility systems in plants: Origin of ‘‘independent’’ and ‘‘complimentary’’ control of incompatibility in angiosperms. New Phytol. 84, 381–400. Pandey, K. K. (1981). Evolution of unilateral incompatibility in flowering plants: Further evidence in favour of twin specificities controlling intra- and interspecific incompatibility. New Phytol. 89, 705–728. Papazian, H. P. (1951). The incompatibility factors and a related gene in Schizophyllum commune. Genetics 36, 441–459. Parag, Y., and Koltin, Y. (1971). The structure of the incompatibility factors of Schizophyllum commune, constitution of the three classes of B factors. Mol. Gen. Genet. 112, 43–48. Pardo, E. H., O’Shea, S. F., and Casselton, L. A. (1996). Multiple versions of the A mating type locus of Coprinus cinereus are generated by three paralogous pairs of multiallelic homeobox genes. Genetics 144, 87–94. Partridge, L., and Hurst, L.D. (1998). Sex and conflict. Science 281, 2003–2008. Pastuglia, M., Roby, D., Dumas, C., and Cock, J. M. (1997a). Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea. Plant Cell 9, 49–60. Pastuglia, M., Ruffio-Chable, V., Delorme, V., Gaude, T., Dumas, C., and Cock, J. M. (1997b). A functional S locus anther gene is not required for the self-incompatibility response in Brassica oleracea. Plant Cell 9, 2065–2076. Peltenburg, L. T. C., and Murre, C. (1997). Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins. Development 124, 1089–1098. Perkins, D. D. (1987). Mating-type switching in filamentous Ascomycetes. Genetics 115, 215–216. Philley, M. L., and Staben, C. (1994). Functional analyses of Neurospora crassa MT a-1 mating type polypeptide. Genetics 137, 715–722. Phillips, C. L., Vershon, A. K., Johnson, A. D., and Dahlquist, F. W. (1991). Secondary structure of the homeodomain of the yeast 움2 repressor determined by NMR spectroscopy. Genes Dev. 5, 764–772. Phillips, C. L., Stark, M. R., Johnson, A., and Dahlquist, F. W. (1994). Heterodimerization of the yeast homeodomain transcriptional regulators 움2 and a1 induces an interfacial helix in 움2. Biochemistry 33, 9294–9302. Picard, M., Debuchy, R., and Coppin, E. (1991). Cloning the mating types of the heterothallic fungus Podospora anserina; Developmental features of haploid transformants carrying both mating types. Genetics 128, 539–547. Plym-Forshell, C. (1974). Seed development after self-pollination and cross-pollination of Scots pine, Pinus sylvestris L. Stud. Forest. Suec. 118, 1–37. Po¨ggeler, S., Rish, S. Ku¨ck, U., and Osiewacz, H. D. (1997). Mating-type genes from the homothallic fungus Sordaria macrospora are functionally expressed in a heterothallic ascomycete. Genetics 147, 567–580.

286

SIMON J. HISCOCK AND URSULA KU¨ES

Polak, E., Hermann, R., Ku¨es, U., and Aebi, M. (1997). Asexual sporulation in Coprinus cinereus: Structure and development of oidiophores and oidia in an Amut Bmut homokaryon. Fungal Genet. Biol. 22, 112–126. Puhalla, J. E. (1969). The formation of diploids of Ustilago maydis on agar medium. Phytopathology 59, 1771–1772. Puhalla, J. E. (1970). Genetic studies of the b incompatibility locus of Ustilago maydis. Genet. Res. Camb. 16, 229–232. Quarmby, L. M. (1994). Signal transduction in the sexual life of Chlamydomonas. Plant Mol. Biol. 26, 1271–1287. Quinby, G. E., and Deschenes, R. J. (1997). An amino terminal prosequence is required for efficient synthesis of S. cerevisiae a-factor. Biochim. Biophys. Acta 1356, 23–34. Radford, A. (1993). A fungal phylogeny based upon orotidine 5⬘-monophosphate decarboxylase. J. Mol. Evol. 36, 389–395. Raffioni, S., Miceli, C., Vallesi, A., Chowdhury, S. K., Chait, B. T., Luporini, P., and Bradshaw, R. A. (1992). Primary structure of Euplotes raikovi pheromones; Comparison of five sequences of pheromones from cells with variable mating interactions. Proc. Natl. Acad. Sci. USA 89, 2071–2075. Raju, N. B. (1992). Genetic control of the sexual cycle in Neurospora. Mycol. Res. 96, 241–262. Randall, T. A., and Metzenberg, R. L. (1998). The mating type locus of Neurospora crassa: Identification of an adjacent gene and characterization of transcripts surrounding the idiomorphs. Mol. Gen. Genet. 259, 615–621. Raper, C. A. (1983). Controls for development and differentiation in basidiomycetes. In ‘‘Secondary Metabolism and Differentiation in Fungi’’ ( J. W. Bennett and A. Ciegler, Eds.), pp. 195–238. Dekker, New York. Raper, C. A. (1985). B-mating-type genes influence survival of nuclei separated from heterokaryons of Schizophyllum. Exp. Mycol. 9, 149–160. Raper, C. A. (1990). Schizophyllum commune. In ‘‘Genetic Maps. Locus Maps of Complex Genomes. Fifth Edition’’ (S. J. O’Brien, Ed.), pp. 3.90–3.91. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Raper, J. R. (1966). ‘‘Genetics of Sexuality in Higher Fungi.’’ Ronald Press, New York. Raper, J. R., Baxter, M. G., and Ellingboe, A. H. (1960). The genetic structure of the incompatibility factors of Schizophyllum commune, the A factor. Proc. Natl. Acad. Sci. USA 46, 833–842. Raudaskoski, M. (1998). The relationship between B-mating-type genes and nuclear migration in Schizophyllum commune. Fungal Genet. Biol. 24, 207–227. Rayner, A. D. M. (1991). The challenge of the individualistic mycelium. Mycologia 83, 48–71. Rayner, A. D. M., Griffith, G. S., and Ainsworth, A. M. (1994). Mycelial interconnectedness. In ‘‘The Growing Fungus’’ (N. A. R. Gow and G. M. Gadd, Eds.), pp. 21–40. Chapman & Hall, London. Regenfelder, E., Spellig, T., Hartmann, A., Lauenstein, S., Bo¨lker, M., and Kahmann, R. (1997). G proteins in Ustilago maydis, transmission of multiple signals? EMBO J. 16, 1934–1942. Richards A. J. (1997). ‘‘Plant Breeding Systems.’’ Allen & Unwin, London. Richardson, W. V. J., Ku¨es, U., and Casselton, L. A. (1993). The A mating type genes of the mushroom Coprinus cinereus are not differentially transcribed in monokaryons and dikaryons. Mol. Gen. Genet. 238, 304–307. Richman, A. D., and Kohn, J. R. (1996). Learning from rejection: The evolutionary biology of single-locus incompatibility. Trends Ecol. Evol. 11, 497–502. Richman, A. D., Kao, T.-H., Schaeffer, S. W., and Uyenoyama, M. K. (1995). S-allele sequence diversity in natural populations of Solanum carolinense (horsenettle). Heredity 75, 405–415. Richman, A. D., Broothaerts, W., and Kohn, J. R. (1996a). Self-incompatibility RNases from three plant families: Homology or convergence? Am. J. Bot. 84, 912–917. Richman, A. D., Uyenoyama, M. K., and Kohn, J. R. (1996b). S-allele diversity in a natural population of Physalis crassifolia (Solanaceae) (ground cherry) assessed by RT-PCR. Heredity 76, 497–505.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

287

Richman, A. D., Uyenoyama, M. K., and Kohn, J. R. (1996c). Allelic diversity and gene geneology at the self-incompatibility locus in the Solanaceae. Science 273, 1212–1216. Robertson, C. I., Bartholomew, K. A., Novotny, C. P., and Ullrich, R. C. (1996). Deletion of the Schizophyllum commune A움 locus. The roles of A움 Y and Z mating-type genes. Genetics 144, 1437–1444. Ro¨hr, H., Ku¨es, U., and Stahl, U. (1998). Organelle DNA of plants and fungi: Inheritance and recombination. Prog. Bot. 60, 39–87. Romeis, T., Ka¨mper, J., and Kahmann, R. (1997). Single chain fusions of two unrelated homeodomain proteins trigger pathogenecity in Ustilago maydis. Proc. Natl. Acad. Sci. USA 94, 1230–1234. Roth, A. F., and Davis, N. G. (1996). Ubiquitination of the yeast a factor receptor. J. Cell Biol. 134, 661–674. Rowell, J. B., and DeVay, J. E. (1954). Genetics of Ustilago zeae in relation to basic problems of its pathogenicity. Phytopathology 44, 356–362. Royo, J., Kunz, C., Lewis, I., Anderson, M. A., Clarke, A. E., and Newbigin, E. (1994). Loss of a histidine residue at the active site of the S locus ribonuclease is associated with selfcompatibility in Lycopersicon peruvianum. Proc. Natl. Acad. Sci. USA 91, 6511–6514. Rudd, J. J., Franklin, F. C. H., Lord, J. M., and Franklin-Tong, V. E. (1996). Increased phosphorylation of a 26-kD pollen protein is induced by the self-incompatibility response in Papaver rhoeas. Plant Cell, 8, 713–724. Rudd, J. J., Franklin, F. C. H., and Franklin-Tong, V. E. (1997). Ca2⫹-independent phosphorylation of a 68 kDa pollen protein is stimulated by the self-incompatibility response in Papaver rhoeas. Plant J. 12, 507–514. Ruiz-Herrera, J., Leo´n, C. G., Guevara-Olvera, L., and Ca´rabez-Trejo, A. (1995). Yeastmycelial dimorphism of haploid and diploid strains of Ustilago maydis. Microbiology 141, 695–703. Rundle, S. J., Nasrallah, M. E., and Nasrallah, J. B. (1993). Effects of inhibitors of protein serine/threonine phosphatases on pollination in Brassica. Plant Physiol. 103, 1165–1171. Runions, C. J., and Owens, J. N. (1998). Evidence of pre-zygotic self-incopatibility in a conifer. In ‘‘Reproductive Biology in Systematics, Conservation and Economic Botany’’ (S. J. Owens and P. J. Rudall, Eds.), pp. 255–264. Royal Botanic Gardens, Kew, Richmond, Surrey, U.K. Sage, T. L., Bertin, R. I., and Williams, E. G. (1994). Ovarian and other late-acting selfincompatibility systems. In ‘‘Genetic Control of Self-Incompatibility and Reproductive Development in Flowering Plants’’ (E. G.) Williams, A. E. Clarke, and R. B. Knox, Eds.), pp. 116–140. Kluwer, Dordrecht. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawata, Y., Kawabata, M., Miyazono, K., and Ichijo, H. (1998). Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J. 17, 2596–2606. Sakagami, Y., Isogai, A., Suzuki, A., Tamura, S., Kitada, C., and Fujino, M. (1979). Structure of tremerogen A-10, a peptidal hormone inducing conjugation tube formation in Tremella mesenterica. Agric. Biol. Chem. 43, 2643–2645. Sakagami, Y., Yoshida, M., Isogai, A., and Suzuki, A. (1981a). Peptidal sex pheromones inducing conjugation tube formation in compatible mating-type cells of Tremella mesenterica. Science 212, 1525–1527. Sakagami, Y., Yoshida, M., Isogai, A., and Suzuki, A. (1981b). Structure of tremerogen a13, a peptidal sex hormone of Tremella mesenterica. Agric. Biol. Chem. 45, 1045–1047. Sakamoto, K., Kusaba, M., and Nishio, T. (1998). Polymorphism of the S-locus glycoprotein gene (SLG ) and the S-locus related gene (SLR1) in Raphanus sativus L. and selfincompatible ornamental plants in the Brassicaceae. Mol. Gen. Genet. 258, 397–403. Sampson, D. R. (1962). Intergeneric pollen–stigma incompatibility in the Cruciferae. Can. J. Genet. Cytol. 4, 38–49. Sarker, R. H., Elleman, C. J., and Dickinson, H. G. (1988). Control of pollen hydration in Brassica requires continued protein synthesis, and glycosylation is necessary for intraspecific incompatibility. Proc. Natl. Acad. Sci. USA 85, 4340–4344.

288

SIMON J. HISCOCK AND URSULA KU¨ES

Sassa, H., Nishio, T., Kowyama, Y., Hirano, H., Koba, T., and Ikehashi, H. (1996). Selfincompatibility (S)-alleles of the Rosaceae encode members of a distinct class of the T-2/ S ribonuclease superfamily. Mol. Gen. Genet. 250, 547–557. Sato, T., Thorsness, M. K., Kandasamy, M. K., Nishio, T., Hirai, M., Nasrallah, J. B., and Nasrallah, M. E. (1991). Activity of an S-locus gene promoter in pistils and anthers of transgenic Brassica. Plant Cell 3, 867–876. Saupe, S., Stenberg, L., Shiu, K. T., Grifths, A. J. F., and Glass, N. L. (1996). The molecular nature of mutations in the mtA-1 gene of the Neurospora crassa A idiomorph and their relation to mating-type function. Mol. Gen. Genet. 250, 155–122. Schlesinger, R., Kahmann, R., and Ka¨mper, J. (1997). The homeodomain of the heterodimeric bE and bW proteins of Ustilago maydis are both critical for function. Mol. Gen. Genet. 254, 514–519. Schmidt, H., and Gutz, H. (1994). The mating-type switch in yeasts. In ‘‘The Mycota. I. Growth, Differentiation and Sexuality’’ ( J. G. H. Wessels and F. Meinhardt, Eds.), pp. 283–294. Springer-Verlag, Berlin. Schrick, K., Garvik, B., and Hartwell, L. H. (1997). Mating in Saccharomyces cerevisiae: The role of the pheromone response signal transduction pathway in the chemotrophic response to pheromone. Genetics 147, 19–32. Schulz, B., Banuett, F., Dahl, M., Schlesinger, R., Scha¨fer, W., Martin, T., Herskowitz, I., and Kahmann, R. (1990). The b alleles of U. maydis whose combinations program pathogenic development, code for polypeptides containing a homeodomain-related motif. Cell 60, 295–306. Schuurs, T. A., Dalstra, H. J. P., Scheer, J. M. J., and Wessels, J. G. H. (1998). Positioning of nuclei in the secondary mycelium of Schizophyllum commune in relation to differential gene expression. Fungal Genet. Biol. 23, 150–161. Scutt, C., Fordham-Skelton, A. P., and Croy, R. R. D. (1993). Okadaic acid causes breakdown of self-incompatibility in Brassica oleracea: Evidence for the involvement of protein phosphatases in signal transduction. Sex. Plant Reprod. 6, 282–283. Seavey, S. R., and Bawa, K. S. (1986). Late-acting self-incompatibility in angiosperms. Bot. Rev. 52, 195–218. Segall, J. E. (1993). Polarization of yeast cells in spatial gradients of 움 mating factor. Proc. Natl. Acad. Sci. USA 90, 8322–8336. Sen, M., Shah, A., and Marsh, L. (1997). Two types of a-factor receptor determinants for pheromone specificity in the mating-incompatible yeasts S. cerevisiae and S. kluyveri. Curr. Genet. 31, 235–240. Seoighe, C., and Wolfe, K. H. (1998). Extent of genomic rearrangement after genome duplication in yeast. Proc. Natl. Acad. Sci. USA 95, 4447–4452. Sharon, A., Yamaguchi, K., Christiansen, S., Horwitz, B. A., Yoder, O. C., and Turgeon, B. G. (1996). An asexual fungus has the potential for sexual development. Mol. Gen. Genet. 251, 60–68. Shen, G.-P., Park, D.-C., Ullrich, R. C., and Novotny, C. P. (1996). Cloning and characterization of a Schizophyllum gene with A웁6 mating-type activity. Curr. Genet. 29, 136–142. Shivanna, K. R., and Johri, B. M. (1985). ‘‘The Angiosperm Pollen: Structure and Function.’’ Wiley Eastern, Delhi. Shivanna, K. R., Heslop-Harrison, J., and Heslop-Harrison, Y. (1981). Heterostyly in Primula. 2. Sites of pollen inhibition, and effect of pistil constituents on compatible and incompatible pollen tube growth. Protoplasma 107, 319–337. Sicari, L. M., and Ellingboe, A. H. (1967). Microscopical observations of initial interactions in various matings of Schizophyllum commune and of Coprinus cinereus. Am. J. Bot. 54, 437–439. Singh, G., and Ashby, A. M. (1998). Cloning of the mating type loci from Pyrenopeziza brassicae reveals the presence of a novel mating type gene within a discomycete MAT1-2 locus encoding a putative metallothionein-like protein. Mol. Microbiol. 30, 799–806.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

289

Singh, A., and Kao, T.-H. (1992). Gametophytic self-incompatibility: Biochemical, molecular genetic, and evolutionary aspects. Int. Rev. Cytol. 140, 449–483. Smith, D. L., and Johnson, A. D. (1992). A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomain of an 움2 dimer. Cell 68, 133–142. Smith, D. L., Desai, A. B., and Johnson, A. D. (1995a). DNA bending by the a1 and 움2 homeodomain proteins from yeast. Nucleic Acids Res. 23, 1239–1243. Smith, D. L., Redd, M. J., and Johnson, A. D. (1995b). The tetratricopeptide repeats of Ssn6 interact with the homeodomain of 움2. Genes Dev. 9, 2903–2910. Smythe, R. (1973). Hyphal fusions in the basidiomycete Coprinus lagopus sensu Buller. I. Some effects of incompatibility factors. Heredity 31, 107–111. Snetselaar, K. M. (1993). Microscopic observation of Ustilago maydis mating interactions. Exp. Mycol. 17, 345–355. Snetselaar, K. M., Bo¨lker, M., and Kahmann, R. (1996). Ustilago maydis mating hyphae orient their growth toward pheromone sources. Fungal Genet. Biol. 20, 299–312. Sogin, M. L. (1991). Early evolution and the origins of eukaryotes. Curr. Opin. Genet. Dev. 1, 457–463. Soltis, D. E., and Soltis, P. A. (1992). The distribution of selfing rates in homosporous ferns. Am. J. Bot. 79, 97–100. Specht, C. A., Stankis, M. M., Giasson, L., Novotny, C. P., and Ullrich, R. C. (1992). Functional analysis of the homeodomain-related proteins of the A움 locus of Schizophyllum commune. Proc. Natl. Acad. Sci. USA 89, 7174–7178. Specht, C. A., Stankis, M. M., Novotny, C. P., and Ullrich, R. C. (1994). Mapping the heterogeneous DNA region that determines the nine A움 mating-type specificies of Schizophyllum commune. Genetics 137, 709–714. Spellig, T., Bo¨lker, M., Lottspeich, F., Frank, R. W., and Kahmann, R. (1994). Pheromones trigger filamentous growth in Ustilago maydis. EMBO J. 13, 1620–1627. Spit, A., Hyland, R. H., Mellor, E. J. C., and Casselton, L. A. (1998). A role for heterodimerization in nuclear localisation of a homeodomain protein. Proc. Natl. Acad. Sci. USA 95, 6228– 6233. Sprague, G. F., Jr., and Thorner, J. W. (1992). Pheromone response and signal transduction during the mating process of Saccharomyces cerevisiae. In ‘‘The Molecular and Cellular Biology of the Yeast Saccharomyces. Gene Expression’’ (E. W. Jones, J. R. Pringle, and J. R. Broach, Eds.), pp. 657–744. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Staben, C., (1996). The mating-type locus of Neurospora. J. Genet. 75, 341–350. Staben, C., and Yanofsky, C. (1990). Neurospora crassa mt a mating-type region. Proc. Natl. Acad. Sci. USA 87, 4917–4921. Stahl, R. J., Arnoldo, M., Glavin, T. L., Goring, D. R., and Rothstein, S. J. (1998). The selfincompatibility phenotype in Brassica is altered by transformation of a mutant S locus receptor kinase. Plant Cell 10, 209–218. Stanchev, B. S., Doughty, J., Scutt, C. P., Dickinson, H. G., and Croy, R. R. D. (1996). Cloning of PCP1, a member of a family of pollen coat protein (PCP) genes, from Brassica oleracea encoding novel cysteine-rich proteins involved in pollen–stigma interactions. Plant J. 10, 303–313. Stankis, M. M., Specht, C. A., Yang, H., Giasson, L., Ullrich. R. C., and Novotny, C. P. (1992). The A움 mating type locus of Schizophyllum commune encodes two dissimilar multiallelic homeodomain proteins. Proc. Natl. Acad. Sci. USA 89, 7169–7173. Stark, M. R., and Johnson, A. D. (1994). Interaction between two homeodomain proteins is specified by a short C-terminal tail. Nature 371, 429–432. Stead, A. D., Roberts, I. N., and Dickinson, H. G. (1980). Pollen–stigma interaction in Brassica oleracea: The role of stigmatic proteins in pollen grain adhesion. J. Cell Sci. 42, 417–423.

290

SIMON J. HISCOCK AND URSULA KU¨ES

Stefan, C. J., and Blumer, K. J. (1994). The third cytoplasmic loop of a yeast G-proteincoupled receptor controls pathway activation, ligand discrimination, and receptor internalization. Mol. Cell. Biol. 14, 3339–3349. Stein, J. C., and Nasrallah, J. B. (1993). A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L., encodes a functional serine/threonine kinase. Plant Physiol. 101, 1103–1106. Stein, J. C., Howlett, B., Boyes, D. C., Nasrallah, M. E., and Nasrallah, J. B. (1991). Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc. Natl. Acad. Sci. USA 88, 8816–8820. Stein, J. C., Dixit, R., Nasrallah, M. E., and Nasrallah, J. B. (1996). SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco. Plant Cell 8, 429–445. Stephenson, A., Doughty, J., Dixon, S., Elleman, C. J., Hiscock, S. J., and Dickinson, H. G. (1997). The male determinant of sporophytic self-incompatibility in Brassica is located in the pollen coating. Plant J. 12, 1351–1359. Stettler, S., Warbrick, E., Prochnik, S., Mackie, S., and Fantes, P. (1996). The wis1 signal transduction pathway is required for expression of cAMP-repressed genes in fission yeast. J. Cell Sci. 109, 1927–1935. Stevens, J. P., and Kay, Q. O. N. (1989). The number, dominance relationships and frequencies of self-incompatibility alleles in a natural population of Sinapis arvensis L. in South Wales. Heredity 62, 199–205. Stewart, A. E., Raffioni, S., Chaudhary, T., Chait, B. T., Luporini, P., and Bradshaw, R. A. (1992). The disulphide bond pairing of the pheromones Er-1 and Er-2 of the ciliated protozoan Euplotes raikovi. Protein Sci. 1, 777–785. Stone, J. M., Collinge, M. A., Smith, R. D., Horn, M. A., and Walker, J. C. (1994). Interaction of a protein phosphatase with an Arabidopsis serine–threonine kinase. Science 266, 793–795. Sugimoto, A., Iino, Y., Maeda, T., Watanabe, Y., and Yamamoto, M. (1991). Schizosaccharomyces pombe ste11⫹ encodes a transcription factor with an HMG motif that is a critical regulator of sexual development. Genes Dev. 5, 1990–1999. Suzuki, G., Watanabe, M., Toriyama, K., Isogai, A., and Hinata, K. (1995). Molecular cloning of members of the S-multigene family in self-incompatible Brassica campestris L. Plant Cell Physiol. 36, 1273–1280. Swamy, S., Uno, I., and Ishikawa, T. (1985a). Regulation of cyclic AMP metabolism by the incompatibility factors in Coprinus cinereus. J. Gen. Microbiol. 131, 3211–3217. Swamy, S., Uno, I., and Ishikawa, T. (1985b). Regulation of cyclic AMP-dependent phosporylation of cellular proteins by the incompatibility factors of Coprinus cinereus. J. Gen. Appl. Microbiol. 31, 339–346. Swiezynski, K. M., and Day, P. R. (1960a). Heterokaryon formation in Coprinus lagopus. Genet. Res. Camb. 1, 114–128. Swiezynski, K. M., and Day, P. R. (1960b). Migration of nuclei in Coprinus lagopus. Genet. Res. Camb. 1, 129–139. Takayama, S., Isogai, A., Tsukamoto, C., Ueda, Y., Hinata, K., Okazaki, K., Koseki, K., and Suzuki, A. (1986). Structure of carbohydrate chains of S-glycoproteins in Brassica campestris associated with self-incompatibility. Agric. Biol. Chem. 50, 1673–1676. Tam, A., Nouvet, F. J., Fujimura-Kamada, K., Slunt, H., Sisodia, S. S., and Michaelis, S. (1998). Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing. J. Cell Biol. 142, 635–649. Tan, S., and Richmond, T. J. (1998). Crystal structure of the yeast Mat움2/MCM1/DNA ternary complex. Nature 391, 660–666. Tanabe, S., and Kamada, T. (1994). The role of astral microtubules in conjugate division in the dikaryon of Coprinus cinereus. Exp. Mycol. 18, 338–348.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

291

Tanaka, K., Davey, J., Imai, Y., and Yamamoto, M. (1993). Schizosaccharomyces pombe map3⫹ encodes a putative M-factor receptor. Mol. Cell. Biol. 13, 80–88. Tantikanjana, T., Nasrallah, M. E., Stein, J. C., Chen, C.-H. and Nasrallah, J. B. (1993). An alternative transcript of the S locus glycoprotein gene in a class II pollen-recessive selfincompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein. Plant Cell 5, 657–666. Tantikanjana, T., Nasrallah, M. E., and Nasrallah, J. B. (1996). The Brassica S gene family: Molecular characterization of the SLR2 gene. Sex. Plant Reprod. 9, 107–116. Tao, R., Yamane, H., Sassa, H., Mori, H., Gradziel, T. M., Dandekar, A. M., and Sugiura, A. (1997). Identification of stylar RNases associated with self-incompatibility in almond (Prunus dulcis). Plant Cell Physiol. 38, 304–311. Thompson, M. M. (1979). Genetics of incompatibility in Corylus avellana L. Theor. Appl. Genet. 54, 113–116. Thompson, K. F., and Taylor, J. P. (1966). Non-linear dominance relationships between Salleles. Heredity 21, 345–364. Thompson, R. D., and Kirch, H.-H. (1992). The S locus of flowering plants: When self-rejection is self-interest. Trends Genet. 8, 381–387. Thompson-Coffe, C., and Zickler, D. (1994). How the cytoskeleton recognizes and sorts nuclei of opposite mating type during the sexual cycle in filamentous ascomycetes. Dev. Biol. 165, 257–271. ¨ ber Zustandekommen und Erhaltung der Dikaryophase von Ustilago Thren, R. (1940). U nuda ( Jensen) Kellerm. et Sw. und Ustilago tritici (Persoon) Jensen. Z. Bot. 36, 449–498. Tobias, C. M., and Nasrallah, J. B. (1996). An S-locus-related gene in Arabidopsis encodes a functional kinase and produces two classes of transcripts. Plant J. 10, 523–531. Tobias, C. M., Howlett, B., and Nasrallah, J. B. (1992). An Arabidopsis thaliana gene with sequence similarity to the S-locus receptor kinase of Brassica oleracea. Plant Physiol. 99, 284–290. Toda, T., Shimanuki, M., and Yanagida, M. (1991). Fission yeast genes that confer resistance to stauroporine encode an AP-1-like transcription factor and a protein kinase related to mammalian ERK1/MAP2 and budding yeast Fus3 and Kss1 kinases. Genes Dev. 5, 60–73. Tolkacheva, T., McNamara, P., Piekarz, E., and Courchesne, W. (1994). Cloning of a Cryptococcus neoformans gene, GPA1, encoding a G-protein 움-subunit homolog. Infect. Immun. 62, 2849–2856. Toriyama, K., Stein, J. C., Nasrallah, M. E., and Nasrallah, J. B. (1991). Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype. Theor. Appl. Genet. 81, 769–776. Trick, M., and Flavell, R. B. (1989). A homozygous S-genotype of Brassica oleracea expresses two S-like genes. Mol. Gen. Genet. 218, 212–217. Trick, M., and Heizmann, P. (1992). Sporophytic self-incompatibility systems: Brassica S gene family. Int. Rev. Cytol. 140, 485–524. Truehart, J., and Herskowitz, I. (1992). The a locus governs cytoduction in Ustilago maydis. J. Bacteriol. 174, 7831–7833. Tsai, D.-S., Lee, H.-S., Post, L. C., Krelling, K. M., and Kao, T.-H. (1992). Sequence of an S-protein of Lycopersicon peruvianum and comparison with other solanaceous S-proteins. Sex. Plant Reprod. 5, 256–263. Turgeon, B. G. (1998). Application of mating type gene technology to problems in fungal biology. Annu. Rev. Phytopathol. 36, 115–137. Turgeon, B. G., Bohlmann, H., Ciuffetti, L. M., Christiansen, S. K., Yang, G., Scha¨fer, W., and Yoder, O. C. (1993a). Cloning and analysis of the mating-type genes from Cochliobolus heterostrophus. Mol. Gen. Genet. 238, 270–284. Turgeon, B. G., Christiansen, S. K., and Yoder, O. C. (1993b). Mating type genes in Ascomyctes and their imperfect relatives. In ‘‘The Fungal Holomorph; Mitotic, Meiotic and Pleomorphic

292

SIMON J. HISCOCK AND URSULA KU¨ES

Speciation in Fungal Systematics’’ (D. R. Reynolds and J. W. Taylor, Eds.), pp. 199–215. CAB International Wallingford, UK. Turgeon, B. G., Sharon, A., Wirsel, S., Christiansen, S. K., Yamaguchi, K., and Yoder, O. C. (1995). Structure and function of mating type genes in Cochliobolus spp. and asexual fungi. Can. J. Bot. 73, S778– S783. Tymon, A. M., Ku¨es, U., Richardson, W. V. J., and Casselton, L. A. (1992). A fungal mating type protein that regulates sexual and asexual development contains a POU-related domain. EMBO J. 11, 1805–1813. Uhm, J. Y., and Fujii, H. (1983a). Ascospore dimorphism in Sclerotinia trifolorium and cultural characters of strains from different-sized spores. Phytopathology 73, 565–569. Uhm, J. Y., and Fujii, H. (1983b). Heterothallism and mating-type mutation in Sclerotinia trifolium. Phytopathology 73, 569–572. Umbach, A. L., Lalonde, B. A., Kandasamy, M. K., Nasrallah, J. B., and Nasrallah, M. E. (1990). Immunodetection of protein glycoforms encoded by two independent genes of the self-incompatibility multigene family of Brassica. Plant Physiol. 93, 739–747. Urban, M., Kahmann, R., and Bo¨lker, M. (1996a). The biallelic a mating type locus of Ustilago maydis: Remnants of an additional pheromone gene indicate evolution from a multiallelic ancestor. Mol. Gen. Genet. 250, 414–420. Urban, M., Kahmann, R., and Bo¨lker, M. (1996b). Identification of the pheromone response element in Ustilago maydis. Mol. Gen. Genet. 251, 31–37. Urushihara, H. (1996). Choice of partners; Sexual cell interaction in Dictyostelium discoideum. Cell Struct. Funct. 21, 231–236. Urushihara, H., and Aiba, K. (1996). Protease-sensitive component(s) on the cell surface prevents self-fusion in a bisexual strain of Dictyostelium discoideum. Cell Struct. Funct. 21, 41–46. Uyenoyama, M. K. (1991). On the evolution of genetic incompatibility systems. VI. A threelocus modifier model for the origin of gametophytic self-incompatibility. Genetics 128, 453–469. Uyenoyama, M. K. (1995). A generalized least squares estimate for the origin of sporophytic self-incompatibility. Genetics 139, 975–992. Vaillancourt, L. J., and Raper, C. A. (1996). Pheromones and pheromone receptors as matingtype determinants in basidiomycetes. In ‘‘Genetic Engineering. Principles and Methods. Vol. 18’’ ( J. K. Setlow, Ed.), pp. 219–247. Plenum, New York. Vaillancourt, L. J., Raudaskoski, M., Specht, C. A., and Raper, C. A. (1997). Multiple genes encoding pheromones and a pheromone receptor define the B웁1 mating-type specificity in Schizophyllum commune. Genetics 146, 541–551. Vallesi, A., Giuli, G., Bradshaw, R. A., and Luporini, P. (1995). Autocrine mitogenic activity of pheromones produced by the protozoan ciliate Euplotes raikovi. Nature 376, 522–524. van den Ende, H. (1992). Sexual signalling in Chlamydomonas. In ‘‘Perspectives in Plant Cell Recognition’’ ( J. A. Callow and J. R. Green, Eds.), pp 1–17. Cambridge Univ. Press, Cambridge, UK. van Heeckeren, W. J., Dorris, D. R., and Struhl, K. (1998). The mating-type proteins of fission yeast induce meiosis by directly activating mei3 transcription. Mol. Cell. Biol. 18, 7317–7326. Vekemans, X., and Slatkin, M. (1994). Gene and allelic genealogies at a gametophytic selfincompatibility locus. Genetics 137, 1157–1165. Verica, J. A., McCubbin, A. G., and Kao, T.-H. (1998). Are the hypervariable regions of S RNases sufficient for allele-specific recognition of pollen? Plant Cell 10, 314–316. Verma, S. C., and Cheema, H. K. (1987). Evidence of self-incompatibility in a population of bracken fern from Britain. Phytomorphology 37, 317–321. Verma, S. C., Malik, R., and Dhir, I. (1977). Genetics of the incompatibility system in the crucifer Eruca sativa L. Proc. R. Soc. London B 196, 131–159.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

293

Vershon, A. K., and Johnson, A. D. (1993). A short, disordered protein region mediates interactions between the homeodomain of the yeast 움2 protein and the MCM1 protein. Cell 72, 105–112. Vershon, A. K., Jin, Y., and Johnson, A. D. (1995). A homeodomain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression. Genes Dev. 15, 182–192. Waddell, D. R., and Duffy, K. T. I. (1986). Breakdown of self/nonself recognition in cannibalistic strains of the predatory slime mold, Dictyostelium caveatum. J. Cell Biol. 102, 298–305. Walker, E. A., Ride, J. P., Kurup, S., Franklin-Tong, V. E., Lawrence, M. J., and Franklin, F. C. H. (1996). Molecular analysis of two functional homologues of the S3 allele of the Papaver rhoeas incompatibility gene isolated from different populations. Plant Mol. Biol. 30, 983–994. Walker, J. C. (1993). Receptor-like protein kinase genes of Arabidopsis thaliana. Plant J. 3, 451–456. Walker, J. C., and Zhang, R. (1990). Relationship of a putative receptor protein kinase from maize to the S-locus glycoproteins of Brassica. Nature 345, 743–746. Wang, S.-H., Xue, C.-B., Nielsen, O., Davey, J., and Naider, F. (1994). Chemical synthesis of the M-factor mating pheromone from Schizosaccharomyces pombe. Yeast 10, 595–601. Wang, Y., Xu, H.-P., Riggs, M., Rodgers, L., and Wigler, M. (1991). byr2, a Schizosaccharomyces pombe gene encoding a protein kinase capable of partial suppression of the ras1 mutant phenotype. Mol. Cell. Biol. 11, 3554–3563. Watanabe, Y., Shinozaki-Yanaba, S., Chikashige, Y., Hiraoka, Y., and Yamamoto, M. (1997). Phosphorylation of RNA-binding protein controls cell cycle switch from mitotic to meiotic in fission yeast. Nature 386, 187–190. Wehling, P., Hackauf, B., and Wricke, G. (1994). Identification of self-incompatibility-related PCR fragments in rye (Secale cereale L.) by denaturing gradient gel electrophoresis. Plant J. 5, 891–893. Weiss, M. S., Anderson, D. H., Raffioni, S., Bradshaw, R. A., Ortenzi, C., Luporini, P., and Eisenberg, D. (1995). A cooperative model for receptor recognition and cell adhesion; ˚ crystal structure of the pheromone ErEvidence from the molecular packing in the 1.6-A 1 from the ciliated protozoan Euplotes raikovi. Proc. Natl. Acad. Sci. USA 92, 10172–10176. Wendland, J., Vaillancourt, L. J., Hegner, J., Lengeler, K. B., Laddison, K. J., Specht, C. A., Raper, C. A., and Kothe, E. (1995). The mating-type locus B움1 of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes. EMBO J. 14, 5271– 5278. Wheeler, H. (1950). Genetics of Glomerella. VIII. A genetic basis for the occurrence of minus mutants. Am. J. Bot. 37, 304–312. Whitbread, J. A., Sims, M., and Katz, E. R. (1991). Evidence for the presence of a growth factor in Dictyostelium discoideum. Dev. Genet. 12, 78–81. Whitehouse, H. L. K. (1949a). Heterothallism and sex in fungi. Biol. Rev. 24, 411–447. Whitehouse, H. L. K. (1949b). Multiple allelomorph heterothallism in the fungi. New Phytol. 48, 212–244. Whitehouse, H. L. K. (1950). Multiple allelomorph incompatibility of pollen and style in the evolution of angiosperms. Ann. Bot. N. S. 14, 198–216. Whiteway, M., Hougan, L., Dignard, D., Thomas, D. Y., Bell, L., Saari, G. C., Grant, F. J., O’Hara, P., and MacKay, V. L. (1989). The STE4 and STE18 genes of yeast encode potential 움 and 웂 subunits of the mating factor receptor-coupled G protein. Cell 56, 467–477. Wickes, B. L., Mayorga, M. E., Edman, U., and Edman, J. C. (1996). Dimorphism and haploid fruiting in Cryptococcus neoformans: Association with the 움-mating type. Proc. Natl. Acad. Sci. USA 9, 7327–7331. Wickes, B. L., Edman, U., and Edman, J. C. (1997). The Cryptococcus neoformans STE12움 gene: A putative Saccharomyces cerevisiae STE12 homologue that is mating type specific. Mol. Microbiol. 26, 951–960.

294

SIMON J. HISCOCK AND URSULA KU¨ES

Wigler, M., Field, S., Powers, S., Broek, D., Toda, T., Cameron, S., Nikawa, J., Michaeli, T., Colicelli, J., and Ferguson, K. (1988). Studies of RAS functions in the yeast S. cerevisiae. Cold Spring Harbor Symp. Quant. Biol. 53, 649–655. Wilkie, D. (1956). Incompatibility in bracken. Heredity 10, 247–336. Willer, M., Hoffman, L., Styrarsdottir, U., Egel, R., Davey, J., and Nielsen, O. (1995). Twostep activation of meiosis by the mat1 locus in Schizosaccharomyces pombe. Mol. Cell. Biol. 15, 4964–4970. Williams, G. C. (1975). ‘‘Sex and Evolution.’’ Princeton Univ. Press, Princeton, NJ. Wilmsen Thornhill, N. (1993). ‘‘The Natural History of Inbreeding and Outbreeding. Theoretical and Empirical Perspectives.’’ Univ. of Chicago Press, Chicago. Wirsel, S., Turgeon, B. G., and Yoder, O. C. (1996). Deletion of the Cochliobolus heterostrophus mating-type (MAT ) locus promotes the function of MAT transgenes. Curr. Genet. 29, 241–249. Wirsel, S., Horwitz, B., Yamaguchi, K., Yoder, O. C., and Turgeon, B. G. (1998). Single mating type-specific genes and their 3⬘ UTRs control mating and fertility in Cochliobolus heterostrophus. Mol. Gen. Genet. 259, 272–281. Wittenberg, C., and Reed, S. I. (1996). Plugging it in: Signalling circuits and the yeast cell cycle. Curr. Opin. Cell Biol. 8, 223–230. Wolberger, C. (1996). Homeodomain interactions. Curr. Opin. Struct. Biol. 6, 62–68. Wolberger, C., Vershon, A. K., Liu, B., Johnson, A. D., and Pabo, C. O. (1991). Crystal structure of a MAT움2 homeodomain–operator complex: Implications for a general model of homeodomain–DNA interactions. Cell 67, 517–528. Wolfe, K. H., and Shields, D. C. (1997). Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387, 708–713. Wong, G. J., and Wells, K. (1985). Modified bifactorial incompatibility in Tremella mesenterica. Trans. Br. Mycol. Soc. 84, 95–109. Wong, K. C., Watanabe, M., and Hinata, K. (1994). Fluorescence and scanning electron microscopic study on self-incompatibility in distylous Averrhoa carambola L. Sex. Plant Reprod. 7, 116–121. Wo¨stemeyer, J., Wo¨stemeyer, A., Burmester, A., and Czempinski, K. (1995). Relationship between sexual processes and parasitic interactions in the host–pathogen system Absidia glauca–Parasitella parasitica. Can. J. Bot. 73(Suppl. 1), S243–S250. Wright, S. (1939). The distribution of self-sterility alleles in populations. Genetics 24, 538–552. Wu, C., Weiss, K., Yang, C., Harris, M. A., Tye, B. K., Newlon, C. S., Simpson, R. T., and Haber, J. E. (1998). Mcm1 regulates donor preference controlled by the recombination enhance in Saccharomyces mating-type switching. Genes Dev. 12, 1726–1737. Wu, J., Ullrich, R. C., and Novotny, C. P. (1996). Regions in the Z5 mating gene of Schizophyllum commune involved in Y–Z binding and recognition. Mol. Gen. Genet. 252, 739–745. Wu, J., Saupe, S. J., and Glass, N. L. (1998). Evidence for balancing selection operating at the het-c heterokaryon incompatibility locus in a group of filamentous fungi. Proc. Natl. Acad. Sci. USA 95, 12398–12403. Wyatt, R., and Anderson, L. E. (1984). Breeding systems in bryophytes. In ‘‘The Experimental Biology of Bryophytes. An International Series of Monographs’’ (A. F. Dyer and J. G. Duckett, Eds.), pp. 39–64. Academic Press, London. Xu, B., Mu, J., Nevins, D., Grun, P., and Kao, T.-H. (1990). Cloning and sequencing of cDNAs encoding two self-incompatibility associated proteins in Solanum chacoense. Mol. Gen. Genet. 224, 341–346. Xu, J.-R., and Hamer, J. E. (1996). MAP kinase and cAMP signalling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea. Genes Dev. 10, 2696–2706. Xue, Y., Carpenter, R., Dickinson, H. G., and Coen, E. S. (1996). Origin of allelic diversity in Antirrhinum S-locus RNases. Plant Cell 8, 805–814.

SEXUAL INCOMPATIBILITY IN PLANTS AND FUNGI

295

Yamakawa, S., Shiba, H., Watanabe, M., Shiozawa, H., Takayama, S., Hinata, K., Isogai, A., and Suzuki, A. (1994). The sequences of S-glycoproteins involved in self-incompatibility of Brassica campestris and their distribution among Brassicaceae. Biosci. Biotech. Biochem. 58, 921–925. Yamakawa, S., Watanabe, M., Hinata, K., Suzuki, A., and Isogai, A. (1995). The sequences of S-receptor kinases (SRK) involved in self-incompatibility and their homologies to the S-locus glycoproteins of Brassica campestris. Biosci. Biotech. Biochem. 59, 161–162. Yamamoto, M. (1996a). Regulation of meiosis in fission yeast. Cell Struct. Funct. 21, 431–436. Yamamoto, M. (1996b). The molecular mechanism of meiosis in fission yeast. Trends Biochem. Sci. 21, 18–22. Yamamoto, M., Imai, Y., and Watanabe, Y. (1997). Mating and sporulation in Schizosaccharomyces pombe. In ‘‘The Molecular and Cellular Biology of the Yeast Saccharomyces. Cell Cycle and Cell Biology, Vol. 3’’ ( J. R. Pringle, J. R. Broach, and E. W. Jones, Eds.), pp. 1037–1106. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Yang, H., Shen, G. P., Park, D. C., Novotny, C. P., and Ullrich, R. C. (1995). The A움 matingtype transcripts of Schizophyllum commune. Exp. Mycol. 19, 16–25. Yee, A. R., and Kronstad, J. W. (1993). Construction of chimeric alleles with altered specificity at the b incompatibility locus of Ustilago maydis. Proc. Natl. Acad. Sci. USA 90, 664–668. Yee, A. R., and Kronstad, J. W. (1998). Dual sets of chimeric alleles identify specificity sequences for the bE and bW mating and pathogenicity genes of Ustilago maydis. Mol. Cell. Biol. 18, 221–232. Youngman, P. J., Anderson, R. W., and Holt, C. E. (1981). Two multiallelic mating compatibility loci separately regulate zygote formation and zygote differentiation in the myxomycete Physarum polycephalum. Genetics 97, 513–530. Yu, K., Schafer, U., Glavin, T. L., Goring, D. R., and Rothstein, S. J. (1996). Molecular characterization of the S locus in two self-incompatible Brassica napus lines. Plant Cell 8, 2369–2380. Yue, C., Osier, M., Novotny, C. P., and Ullrich, R. C. (1997). The specificity determinant of the Y mating-type proteins of Schizophyllum commune is also essential for Y–Z protein binding. Genetics 145, 253–260. Zambino, P., Groth, J. V., Lukens, L., Garton, J. R., and May, G. (1997). Variation at the b mating type locus of Ustilago maydis. Phytopathology 87, 1233–1239. Zavada, M. S., and Taylor, T. N. (1986). The role of self-incompatibility and sexual selection in the gymnosperm–angiosperm transition: A hypothesis. Am. Nat. 128, 538–550. Zhang, L., Churchill, A. C. L., Kazmierczak, P., Kim, D., and Van Alfen, N. K. (1993). Hypovirulence-associated traits, induced by a mycovirus of Cryphonectria parasitica, mimicked by targeted inactivation of a host gene. Mol. Cell. Biol. 13, 7782–7793. Zhang, L., Baasiri, R. A., and Van Alfen, N. K. (1998). Viral expression of fungal pheromone precursor gene expression. Mol. Cell. Biol. 18, 953–959. Zhao, Y., Feng, X.-H., Watson, J. C., Bottino, P. J., and Kung, S.-D. (1994). Molecular cloning and biochemical characterization of a receptor-like serine/threonine kinase from rice. Plant Mol. Biol. 26, 791–803. Zickler, D., Arnaise, S., Coppin, E., Debuchy, R., and Picard, M. (1995). Altered matingtype identity in the fungus Podospora anserina leads to selfish nuclei, uniparental progeny, and haploid meiosis. Genetics 140, 493–503. Zickler, H. (1952). Zur Entwicklungsgeschichte des Askomyzeten Bombardia lunata. Arch. Protistenkd. 98, 1–70. Zuberi, M. I., and Lewis, D. (1988). Gametophytic–sporophytic incompatibility in the Cruciferae, Brassica campestris. Heredity 61, 367–377. Zurek, D. M., Mou, B., Beecher, B., and McClure, B. (1997). Exchanging domains between S-RNases from Nicotiana alata disrupts pollen recognition. Plant J. 11, 797–808.

This Page Intentionally Left Blank

INDEX

A

Bombardia lunata, mating type determination, 190–195 Brassica, SSI, 228–232, 241–244 Bryophytes, sexual incompatibility, 216–217

Acetylation, histones, 84–87 ACh, see Nicotinic acetylcholine receptors Adulthood, effects of gonadal steroids, 37–44 Albino callus culture, for ptDNA, 154–155 Algae green, plastid division, 134–135 life cycles, 167, 169–170 sexual incompatibility, 213–216 Altermorphs, mating type, 254–258 Ascobolus stercorarius, mating type determination, 190–195 Ascomycetes, mating type determination diplotonic yeast, 181–187 filamentous ascomycetes, 190–195 haplotonic yeast, 187–190 Ascomycota, see Fungi Autonomic ganglia neuron morphology, 6–7 neurotransmitters, 7–9 peripheral nervous system, 9–11 types, 2–6 Autonomic reflex, action of gonadal steroids, 42–44 Axotomy, nerve injury response, 47–48

C Cambrian explosion, sex evolution, 165–167 Carboxy-terminal domain, RNA polymerase II, 96–98 Catecholamine analog 6-OHDA, 28 fluorescence, 18 Cell cycle and plastid division, 135–136 ptDNA synthesis, 139–140 ptDNA variation, 133–134 role in chromatin control, 87–91 Cell differentiation ptDNA, 137–138 in tissues, 136 Cell lines, albino callus, for ptDNA, 154–155 Cell multiplication, ptDNA, 137–138 Cells, plastid position, 143–146 Cell size effects of mutation, 139 mature cells, 137 multiplying and differentiating cells, 137–138 ChAT, see Choline acetyltransferase Chlamydomonas DNA replication, 148–149 ptDNA variation, 133–134

B Basidiomycetes, mating type determination homeodomain TF, 200–201, 204–207 matP locus, 207, 209–213 role of pheromones, 195–200 Basidiomycota, see Fungi

297

298

INDEX

Chlamydomonas reinhardtii, sexual incompatibility, 213–216 Choline acetyltransferase, pelvic ganglia, 26–27, 29 Chromatin control, cell cycle role, 87–91 EGA regulation, 93–94 nonhistone, changes, 87 remodeling, 92 repackaging, 77–78 Chytridiomycota, see Fungi Cleavage, EGA cell cycle role, 87–91 histone acetylation changes, 84–87 histone array changes, 82–84 nonhistone chromatin proteins, 87 Cochliobolus heterostrophus, mating type determination, 191–193 Coprinus cinereus mating type determination, 196 matP locus, 210–213 Cryphonectria parasitica, mating type determination, 190–191 CTD, see Carboxy-terminal domain Culture, albino callus, 154–155 Cyanidium plastid division, 134–135 plastids, 130, 132 Cytochalasin B, role in plastid division, 134–135 Cytokinesis, ptDNA distribution, 141–143 Cytoplasm, role in EGA effect of translational control, 105–106 maternal mRNA role, 101–105 protein synthesis role, 99–101 TF content changes, 95–96 TF posttranslational activation, 96–99 transcriptional state conversion, 94–95 transplanted nuclei reprogramming, 106–109

D Deafferentation, nerve injury responses, 44–47 Dendritic arbor, pelvic ganglia, 24–25 Development effects of gonadal steroids, 37–44 ptDNA, variation, 133

Diplotonic yeast, mating type determination, 181–187 Division, plastid apparatus, 134–135 rapid division, 135–136 tissue differentiation, 136 DNA methylation changes, 92–93 plastid, see Plastid DNA replication, role in EGA, 88–91 DNA-dependent protein kinase, Sp1 and TBP phosphorylation, 98–99 Dye-filling analysis, pelvic ganglia, 24

E EGA, see Embryonic genome activation Electrophysiology, pelvic ganglia, 19–20, 30–33 Embryo, cultured, cleavage arrest, 91 Embryonic genome activation cell cycle role, 87–91 chromatin regulation, 93–94 chromatin remodeling, 92 DNA methylation changes, 92–93 histone acetylation changes, 84–87 histone array changes, 82–84 model for control, 109–111 nonhistone chromatin proteins, 87 pronucleus formation chromatin repackaging, 77–78 pronuclei differences, 79–81 sperm nuclear envelope replacement, 74–77 role of cytoplasm effect of translational control, 105–106 maternal mRNA role, 101–105 protein synthesis role, 99–101 TF content changes, 95–96 TF posttranslational activation, 96–99 transcriptional state conversion, 94–95 transplanted nuclei reprogramming, 106–109 Enteric ganglia connection with pelvic ganglia, 34–37 features, 2–6 in peripheral nervous system, 10–11 Enteric ganglion cells, neurotransmitters, 9 Envelope, nuclear, sperm, replacement, 74–77

299

INDEX Evolution mating type systems, 246–247 sex, 165–167 SI systems, 246–247 mating type altermorphs, 254–258 origin, 247–254 S alleles, 254–258

F Flowering plant, SI systems distribution and genetic basis, 218, 220–222 GSI-associated S genes, 228–232 GSI in Papaver rhoeas, 237–239, 241 GSI in S-RNase, 235–237 pollen S component of GSI and SSI, 232–235 S allele number, 222–223 S gene role, 244–246 site of response, 223–224 SSI-associated S genes, 224–225, 227– 228 SSI in Brassica, 241–244 Fluorescence, catecholamine, 18 Funaria, plastid division, 134–135 Fungi life cycles, 167, 169–170 mating type, genetic control, 170–171 mating type determination, 171, 178–179 basidiomycetes homeodomain TF, 200–201, 204–207 matP locus, 207, 209–213 role of pheromones, 195–200 diplotonic yeast, 181–187 filamentous ascomycetes, 190–195 haplotonic yeast, 187–190 in oomycetes, 180 in zygomycetes, 180–181

G Gametic self-incompatibility associated pistil-expressed S genes, 224–225, 227–228 Papaver rhoeas, 237–239, 241 phenotype, 218, 220 pollen S component, 232–235 S-RNases, 235–237

Ganglia autonomic features, 2–6 neuron morphology, 6–7 neurotransmitters, 7–9 peripheral nervous system, 9–11 types, 2–6 enteric connection with pelvic ganglia, 34–37 features, 2–6 in peripheral nervous system, 10–11 lower bowel, 36–37 parasympathetic features, 2–6 pelvic ganglia, 18–21 paravertebral, features, 3–5 pelvic, see Pelvic ganglia preganglionic neurons, pelvic ganglia, 28–30 prevertebral, features, 3–5 sensory connection with pelvic ganglia, 33–34 in peripheral nervous system, 9–10 sympathetic features, 2–6 pelvic ganglia, 16, 18–21 Ganglion cells, neurotransmitters, 7–9, 25–28 Genes basis of SI systems, 218, 220–222 embryonic, early expression, 105–106 mating type, origin, 247–254 matP, 207, 209–213 matT, 200–201, 204–207 mt, 215–216 S diversification, 254–258 number in populations and species, 222–223 pistil-expressed gene association with GSI, 224–225, 227–228 association with SSI, 228–232 pollen component, GSI and SSI, 232–235 role in interspecific incompatibility, 244–246 self-incompatibility, origin, 247–254 Genetic control, fungi mating type, 170–171 Genetics, ptDNA exchange methods, 151–152 genome multiplicity, 153

300

INDEX

Genetics (continued) sorting, 151 uniparental inheritance, 150–151 Genome complexes multigenome, plastid, 146, 148 ptDNA developmental variation, 133 mature plastids, 129–130, 132 variation with cell cycle, 133–134 Genome multiplicity, ptDNA, 153 Gonadal steroid effects on autonomic reflexes, 42–44 on pelvic autonomic neurons, 38–42 on sexual dimorphism, 37–38 GSI, see Gametic self-incompatibility Gymnosperms, sexual incompatibility, 217–218

H Haplotonic yeast, mating type determination, 187–190 Histones, EGA acetylation changes, 84–87 array change, 82–84 nonhistone chromatin, 87

I Intestine innervation, 35 vasoactive peptide in autonomic ganglia, 3, 5 ganglion cells, 27 pelvic ganglia, 13

evolution, 246–247 fungi basidiomycetes homeodomain TF, 200–201, 204– 207 matP locus, 207, 209–213 role of pheromones, 195–200 determination, 171, 178–179 diplotonic yeast, 181–187 filamentous ascomycetes, 190–195 haplotonic yeast, 187–190 in oomycetes, 180 in zygomycetes, 180–181 genetic control, 170–171 locus origin, 247–254 Meiosis, sex evolution, 165–167 Membranes, pelvic ganglia, 30–33 Messenger RNA, role in EGA, 101–105 Metabolic pathways, ptDNA, 150 Methylation, DNA, 92–93 Microelectrodes, intracellular recordings, 19–20 Models for EGA control, 109–111 GSI in Papaver rhoeas, 237–239, 241 GSI in S-RNase, 235–237 ptDNA, 153–154 remodeling, chromatin, 92 Morphology neuron, 6–7 pelvic neurons, 23–25 Moss, plastid division, 134–135 Multigenome complex, plastid, 146, 148 Mutation, effect on plastid DNA, 139

N L Large intestine, innervation, 35 Life cycles algae, 167, 169–170 fungi, 167, 169–170 Lower bowel, ganglia location, 36–37

M Mating types algae, determination, 213–216 altermorphs, 254–258

Nerve damage, see Nerve injury response Nerve growth factor, secretion from urinary bladder, 46 Nerve injury response axotomy, 47–48 damage types, 48 deafferentation, 44–47 Nervous system connection with pelvic ganglia, 33–34 parasympathetic, supply, 15–16 peripheral enteric ganglia, 10–11 sensory ganglia, 9–10

301

INDEX Neurons morphology, 6–7 pelvic autonomic, effects of circulating gonadal steroids, 38–42 pelvic ganglia morphology, 23–25 preganglionic neurons, 28–30 steroid hormone sensitivity, 50 Neurospora crassa, mating type determination, 190–195 Neurotransmitters enteric ganglion cells, 9 parasympathetic ganglion cells, 8–9 pelvic ganglia, 25–30 preganglionic neurons, 28–30 sympathetic ganglion cells, 7–8 NGF, see Nerve growth factor Nicotiana, GSI, 224–225, 227–228 Nicotinic acetylcholine receptors pelvic ganglia, 13 in sympathetic ganglia, 3 sympathetic ganglion cells, 7–9 VIP, 7–9 Nonflowering plant, sexual incompatibility, 213–216 Nuclear envelope, sperm, replacement, 74–77 Nuclear ploidy effects of mutation, 139 mature cells, 137 multiplying and differentiating cells, 137–138 Nucleoids, mature plastids, 129–130, 132 Nucleus, transplanted, reprogramming, 106–109

O Ochromonas, plastid positioning, 145–146 Oomycetes, mating type determination, 180 Outbreeding, evolution, 165–167

P Papaver rhoeas, GSI, 237–239, 241 Parasympathetic ganglia features, 2–6 pelvic ganglia, 18–21

Parasympathetic ganglion cells, neurotransmitters, 8–9 Parasympathetic nerve supply, pelvic ganglia, 15–16 Paravertebral ganglia, features, 3–5 PCC, see Premature chromatin condensation Pelvic autonomic pathways axotomy, 47–48 effects of circulating gonadal steroids, 38–42 gonadal steroid action on autonomic reflexes, 42–44 nerve damage types, 48 sexual dimorphism, 37–38 steroid hormone sensitivity, 50 Pelvic ganglia anatomy and physiology, 11–16 connection with enteric ganglia, 34–37 connection with sensory ganglia, 33–34 deafferentation, 44–47 ganglion cells, 25–28 membrane properties, 30–33 mixed sympathetic and parasympathetic ganglia, 18–21 neuron morphology, 23–25 preganglionic neurons, 28–30 Peptides, vasoactive intestinal, 3, 5 Peripheral nervous system enteric ganglia, 10–11 sensory ganglia, 9–10 Pheromone receptor, matP locus encoding, 207, 209–213 Pheromones matP locus encoding, 207, 209–213 role in mating type determination in basidiomycetes, 195–200 Phytophthora, mating type determination, 180 Pistil, expressed S genes association with GSI, 224–225, 227–228 association with SSI, 228–232 Plant flowering, see Flowering plant nonflowering, sexual incompatibility, 213–216 Plastid cell positioning, 143–145 definition, 128–129 division apparatus, 134–135

302 Plastid (continued) rapid division, 135–136 tissue differentiation, 136 other positioning, 145–146 Plastid DNA albino callus cultures, 154–155 distribution at cytokinesis, 141–143 effects of mutation, 139 exchange methods, 151–152 genome complexes developmental variation, 133 mature plastids, 129–130, 132 variation with cell cycle, 133–134 genome multiplicity, 153 mature cells, 137 metabolic pathways, 150 models, 153–154 multigenome complex, 146, 148 multiplying and differentiating cells, 137–138 mutant plastids, 149–150 origin of replication, 149 replication, 148–149 sorting, 151 synthesis cell cycle, 139–140 cell measurement, 140–141 uniparental inheritance, 150–151 Plastome definition, 128 effects of mutation, 139 mature cells, 137 multiplying and differentiating cells, 137–138 Podospora anserina, mating type determination, 190–195 Pollen, S component, GSI and SSI, 232–235 Population, S allele number, 222–223 Preganglionic neurons, pelvic ganglia, 28–30 Premature chromatin condensation, transplanted nuclei, 107 Prevertebral ganglia, features, 3–5 Pronucleus, EGA chromatin repackaging, 77–78 pronuclei differences, 79–81 sperm nuclear envelope replacement, 74–77 Pronucleus envelope, permeable, in replacement, 74–77 Protein kinase, DNA-dependent, Sp1 and TBP phosphorylation, 98–99

INDEX Proteins ptDNA-associated DNA replication, 148–149 metabolic pathways, 150 multigenome complex, 146, 148 mutant plastids, 149–150 origin of replication, 149 Sp1 protein, 98–99 synthesis, role in EGA, 99–101 TATA binding protein, 98–99 TRC, 107 Protozoa, mating type determination, 171, 178–179 ptDNA, see Plastid DNA Pteridium aquilinum, sexual incompatibility, 216–217 Pteridophytes, sexual incompatibility, 216–217 Pyramimonas, plastid division, 134–135 Pythium, mating type determination, 180

R Remodeling, chromatin, 92 Replication DNA, role in EGA, 88–91 ptDNA, 148–149 RNA, messenger, role in EGA, 101– 105 RNA polymerase II, regulation, 96–98 RNase association with GSI, 227 S-RNase, 235–237

S Saccharomyces cerevisiae, mating type determination, 181–187 Saccharomyces pombe, mating type determination, 187–190 Schizophyllum commune mating type determination, 196 matP locus, 210–212 Self-incompatibility system evolution, 246–247 locus origin, 247–254 mating type altermorphs and S alleles, 254–258

303

INDEX flowering plant distribution and genetic basis, 218, 220–222 S allele number, 222–223 S gene role in interspecific incompatibility, 244–246 site of response, 223–224 GSI associated pistil-expressed S genes, 224–225, 227–228 Papaver rhoeas, 237–239, 241 phenotype, 218, 220 pollen S component, 232–235 S-RNases, 235–237 SSI associated pistil-expressed S genes, 228–232 in Brassica, 241–244 phenotype, 220 pollen S component, 232–235 Sensory ganglia connection with pelvic ganglia, 33–34 in peripheral nervous system, 9–10 Sex, evolution, 165–167 Sexual dimorphism, pelvic autonomic pathways, 37–38 Sexual incompatibility bryophytes, 216–217 gymnosperms, 217–218 in nonflowering plants, algae, 213–216 pteridophytes, 216–217 SI system, see Self-incompatibility system Slime mold, heterothallic, mating type determination, 171, 178–179 Small intestine, innervation, 35 Sorting, ptDNA, 151 Sp1 protein, posttranslational regulation, 98–99 Species, S allele number, 222–223 Sperm, nuclear envelope, replacement, 74–77 Sporophytic self-incompatibility associated pistil-expressed S genes, 228–232 in Brassica, 241–244 phenotype, 220 pollen S component, 232–235 SSI, see Sporophytic self-incompatibility Steroid hormones, sensitivity in pelvic autonomic pathways, 50 Sympathetic ganglia features, 2–6 pelvic ganglia, 16, 18–21

Sympathetic ganglion cells, neurotransmitters, 7–8 Synaptology, pelvic ganglia, 25

T TATA binding protein, posttranslational regulation, 98–99 TBP, see TATA binding protein TF, see Transcription factors Tissues, differentiation, 136 Transcription, state conversion, 94–95 Transcription factors homeodomain, matT locus, 200–201, 204–207 role in EGA maternal mRNA role, 101–105 protein synthesis role, 99–101 TF content changes, 95–96 TF posttranslational activation, 96–99 Translation control, effect on early EGA, 105–106 posttranslation, TF activation RNA polymerase II, 96–98 Sp1 regulation, 98–99 TBP regulation, 98–99 TRC protein, expression, 107 Trebouxia, plastid division, 134–135

U Uniparental inheritance, ptDNA, 150–151 Urinary bladder, NGF secretion, 46 Ustilago maydis, matP locus, 207, 209–211, 213

V Vasoactive intestinal peptide in autonomic ganglia, 3, 5 ganglion cells, 27 pelvic ganglia, 13 Vaucheria, plastids, 130 VIP, see Vasoactive intestinal peptide

Z Zygomycetes, mating type determination, 180–181 Zygomycota, see Fungi

This Page Intentionally Left Blank