International Review of Cytology: A Survey of Cell Biology, Volume 153

international Review of

cyt0 I0gy VOLUME 153

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

1949-1 988 1949-1 984 19671984-1 992 1993-

ADVISORY EDITORS Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay Mark Hogarth M. Melkonian Keith E. Mostov Audrey Muggleton-Harris

Andreas Oksche Muriel J. Ord Vladimir R. Pantic M. V. Parthasarathy Lionel I. Rebhun L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Robin Wright Alexander L. Yudin

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

Jonathan Jarvik Department of Biological Sciences Carnegie Mellon University Pittsburgh, Pennsylvania

VOLUME 153

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Copyright 0 1994 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted 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.

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A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road. London NWI 7DX International Standard Serial Number: 0074-7696 International Standard Book Number: 0- 12-364556-5 PRINTED IN THE UNITED STATES OF AMERICA 94 95 9 6 9 7 98 9 9 B B 9 8 7 6 5

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CONTENTS

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

ix

Fluorescent in Sku Hybridization for the Diagnosis of Genetic Disease at Postnatal. Prenatal. and Preimplantation Stages Darren K. Griffin 1. I1. 111. IV. V. VI.

Introduction ................................................................................ Origin of Samples .......................................................................... Fluorescent in Situ Hybridization .......................................................... Sexing of Human Cells .................................................................... Further Fluorescent in Situ Hybridization Diagnoses ...................................... Concluding Remarks ....................................................................... References .................................................................................

1 3 6 21 25 33 35

Isolation and Function of Human Dendritic Cells Lisa A . Williams. William Egner. and Derek N. J. Hart 1. II. 111. IV. V. VI . VII . VIII. IX. X.

Introduction and Overview ................................................................. Blood and Bone Marrow Dendritic Cells ................................................... Nonlymphoid and Interstitial Dendritic Cells ............................................... Lymphoid Dendritic Cells .................................................................. Dendritic Cell Ontogeny: Differentiation and Migration .................................... Functional Properties of Human Dendritic Cells ........................................... Dendritic Cell Malignancies ................................................................ Role of Dendritic Cells in Transplantation. and Infectious and Autoimmune Diseases ... Clinical Applications ........................................................................ Future Dendritic Cell Research and Applications .......................................... References ................................................................................. V

41 47 53 64 68 73 85 86 90 90 91

vi

CONTENTS

Granulated Lymphoid Cells of the Pregnant Uterus: Morphological and Functional Features Chau-Ching Liu. Earl L. Parr. and John Ding-E Young I. I1. 111. IV. V. VI.

Introduction ................................................................................ Cells Associated with Decidual Tissue .................................................... Granulated Metrial Gland Cells of Rodents ................................................ Human Endometrial Granulocytes ......... .............................. Possible Functions of Uteri Concluding Remarks ...... References .................................................................................

105 106 110 117 122 126 127

The Replication Band of Ciliated Protozoa Donald E. Olins and Ada L. Olins I. Introduction ................................................................................ II. Early History: Prior to 1959 ................................................................

137 140

Functional Characteristics of Replication Bands in Cells .................................. Replication Band Ultrastructure ............................................................ Cytochemical and lmmunochemical Studies on Replication Bands ....................... Relevance to Current Models Conclusions and Speculations ..... References ......

143 148 154 161 166 168

111. IV. V. VI . VII.

Whole-Chromosome Hybridization S . D . Bouffler ......................................................................... I. rinciples of Whole-Chromosome Hybridization Techniques .......... II. Ill. Applications of Whole-Chromosome Hybridization ........................................ ............................................................ IV. ............................................................

171 174 197 219 220

Neuronal Modulation and Plasticity in Vitro Robert A . Smith and Zhi-Gang Jiang I. Introduction ................................................................................ 11. Neuronal Cell Cultures .....................................................................

233 235

CONTENTS

vii

Neurite Initiation and Elongation ............................ Synaptic Connections between Cultured Neurons ....................... Phenotypic Expression .... ....................................... Concluding Remarks ............... .................................... References ................................... ...........................

279 201

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

297

111. IV. V. VI.

This Page Intentionally Left Blank

CONTRIBUTORS

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

S. D. Bouffler (171), Biomedical Effects Department, National Radiological Protection Board, Chilton, Oxfordshire OX 1 1 ORQ, United Kingdom William Egner (41), Haematology/lmmunology Research Group, ChristchurchHospitall Christchurch, New Zealand Darren K. Griffin (1), Department of Genetics and Biometry, University College London, London NW1 2HE, United Kingdom Derek N. J. Hart (41), Haematology/lmmunology Research Group, ChristchurchHospital, Christchurch, New Zealand Zhi-Gang Jiang (233),Department of Anatomyl University of Glasgow, Glasgow, G 12 SQQ, Scotland, United Kingdom Chau-Ching Liu (105), Laboratory of Molecular Immunology and Cell Biology, The Rockefeller Universityl New York, New York 10021 Ada L. Olins (137), The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and The Biology Division, Oak Ridge National Laboratory, Oak Ridgel Tennessee 37831 Donald E. Olins (137), The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and The Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Earl L. Parr (109, Department of Anatomy, School of Medicine, Southern Illinois University, Carbondale, Illinois 6290 1 Robert A. Smith (233),Department of Anatomy, University of Glasgow, Glasgow G12 8QQ1Scotland, United Kingdom ix

X

CONTRIBUTORS

Lisa A. Williams (41),Haematology//mmunol~yResearch Groupl ChistchurchHospital Chistchurchl New Zealand John Ding-E Young (105),Laboratory of Molecular lmmunology and Cell Biology, The Rockefeller Universityl New York, New York 10021

Fluorescent in Situ Hybridization for the Diagnosis of Genetic Disease a t Postnatal, Prenatal, and Preimplantation Stages Darren K. Griffin Department of Genetics and Biometry, University College London, London NWI 2HE, United Kingdom

I. Introduction Adapted from the pioneering work of Gall and Pardue, most significantly as described by Pinkel et al. (1986), fluorescent in situ hybridization (FISH) has become one of the most powerful techniques in modem genetic research. It is principally a tool for assigning particular nucleic acid clones (“probes”) to chromosome preparations in order to map those clones to a particular chromosome region, but it has proved to be a very powerful diagnostic technique also. FISH is now very much a complementary approach to classical cytogenetics because it can be used to illuminate whole chromosomes or certain regions of chromosomes at will, and because two or more chromosomal targets can be visualized in different colors simultaneously on the same preparation. It has allowed cytogenetic analysis of metaphases that are difficult (or impossible) to analyze because of poor chromosome preparations or when the chromosome anomaly is too complex or too small to see with classic chromosome banding techniques. It also allows certain types of analysis in interphase nuclei and is hence invaluable when metaphases cannot be prepared at all. Disorders accessible to FISH diagnosis range from simple numerical chromosome changes (e.g., Down’s syndrome), to tiny deletions (such as some cases of Duchenne’s muscular dystrophy-DMD), to complex chromosomal rearrangements. Diagnosis can be made after birth (postnatally), before birth (prenatally), or before implantation of the embryo (preimplantation diagnosis). Cytogenetic diagnosis following birth is particularly useful in deciding upon future treatment of an affected individual. Often a rapid cytogenetic International Review

of Cytology. Vol. 153

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Copyright 0 1994 by Academic Press, Inc.

All rights of reproduction in MY form reserved.

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diagnosis can be essential in deciding upon immediate treatment for a newborn child or indeed upon whether to sustain life at all. In the case of trisomy 13 for instance, often a decision is made to turn off life support systems upon diagnosis because the child would have no chance of treatment or survival. Furthermore, specific chromosomal anomalies in certain tissues of an individual (e.g., bone marrow) can be diagnostic for certain cancers; perhaps the most common example of this is the Philadelphia chromosome present in chronic myeloid leukemia cells. It is in prenatal diagnosis that there has been the most interest in diagnostic cytogenetics. Amniocentesis and/or chorionic villus sampling (CVS) in the first or second trimester of pregnancy reveals the chromosomal constitution of the unborn fetus and allows the family to decide (under genetic counseling)whether to continue with the pregnancy or to terminate it if the fetus is affected. Preimplantation diagnosis (PID) is carried out on an embryo prior to implantation into the mother’s uterus. Although considerable advances have been made in prenatal diagnosis, selective abortion of affected offspring is unacceptable in some cases. The need (at least initially)to develop PID hence is not as a working alternative or replacement for prenatal diagnosis but to alleviate the suffering of many special-case families: Penketh and McLaren (1987) report one thallasemia family who had one unaffected child after seven pregnancies, four of which were terminated. Another woman whose brother died of Duchenne’s muscular dystrophy has two teenage nephews with the condition and is a carrier herself. She had two terminations in her first marriage, which broke down as a result. In her second marriage, following second trimester prenatal diagnosis, she has had three terminations of male fetuses and consequently has been advised to refrain from further conception until research in DMD has reached a more advanced stage. In some families, selective abortion is morally or religiously unacceptable. For instance, the Roman Catholic, Islamic, and Orthodox Askenazi Jewish (Penketh and McLaren, 1987; Winston, 1987) faiths all prohibit terminations of pregnancy. Clearly, families such as these would benefit from PID. It should be noted, however, that the Roman Catholic faith has not embraced this approach. There have been a multitude of developments in FISH technology and literally tens of thousands of publications involving the technique and adaptations of it. It is certainly beyond the scope of this chapter to summarize all of them. There is a gap between what is technically possible in a high-flying research laboratory and what is technically feasible (and reproducible) in the average diagnostic laboratory. With this in mind, this chapter attempts to highlight how major advances in the field have been used in diagnostic situations to suggest how (with limited resources) a diagnostic cytogenetic laboratory might harness the technology to its own ends, and to speculate on what could be possible in the future.

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II. Origin of Samples

In this section, sampling and preparation of material for FISH studies are reviewed. Postnatal and prenatal sampling have been covered in great detail by many other authors and so are only briefly summarized here. However, since preimplantation diagnosis is a more recent development, it is covered in more detail. A. Postnatal Samples

Postnatal samples can be taken from blood, skin, bone marrow, lymph nodes, or directly from solid tumors. Blood samples are usually taken from peripheral veins and lymphocytes are stimulated into a dividing state using a mitogen. Metaphase arrest precedes hypotonic swelling of the cells and is followed by fixation and spreading onto glass slides. In the case of the other tissues, cells can be induced to divide (if necessary) by the culture techniques used to prepare chromosomes. The techniques used for hypotonic swelling, fixation, and spreading onto glass are similar to those for blood culture.

6. Prenatal Samples

Prenatal sampling involves removing samples of the chorionic villus, amniotic fluid, or fetal blood with a needle or catheter. In order to prepare chromosomes, cells from the amniotic fluid need to be cultured over 2 weeks. CVSs can also be cultured but an advantage of this sample is that chromosomes can be obtained directly without culturing, and hence a diagnosis can be achieved with speed. Direct CVS chromosome preparations are derived from the outer epithelial layer of the villus and, in general, give metaphases. Chromosome spreads can be difficult to analyze completely, however, and therefore in most diagnostic labs are merely homogeneously stained and analyzed for sex and aneuploidy alone. Cultured CVS preparations are derived from the inner mesenchymal core of the villus, and chromosome spreads prepared from this area are of much better quality. It is because of this that it is often recommended that laboratories use both methods so that a confident diagnosis can be made for each patient. CVS is usually routinely performed at 8-9 weeks of gestation whereas amniocentesis is routinely performed at 15- 16 weeks. Brambati and Lucia (1990) have reported CVS taken as early as the 6th week of gestation, and Smith et al. (1990) report that amniocentesis can be performed at

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DARREN K. GRIFFIN

11 weeks (bringing it into the first trimester). Rapid results mean that therapeutic abortion can be offered quickly, which ultimately leads to less trauma for families having to make the difficult decision of whether to proceed with a termination. Hence, CVS gained rapid popularity following its development. Various reports have appeared of more rapid approaches to both amniocentesis (as early as 11 weeks; Smith et al. (1990) and CVS (as early as the 6th week; Brambati and Lucia, 1990). Recent studies have, however, indicated that both early amniocentesis and early CVS may be potentially damaging to the fetus and most clinics have reverted to their original approaches. The great advantage of an earlier result associated with CVS is balanced by the fact that CVS reveals mosaic results more often than amniocentesis since amniocentesis analyzes cells shed directly from the fetus. Furthermore, because amniocyte chromosomes are analyzed more easily than those from direct CVS, the former are more likely to reveal more subtle chromosomal abnormalities (Lilford, 1991). As with postnatal material, cells taken for prenatal sampling are swelled in hypotonic solution, fixed, and spread onto glass slides. C. Preirnplantation Embryos

Prospectively, material from preimplantation embryos can be sampled in one of three ways. Each can involved either in vitro fertilized (IVF) embryos (Steptoe and Edwards, 1978)or embryos fertilized normally and flushed from the mother’s uterus (uterine lavage) (Buster et al., 1985): These methods are (1) removal and diagnosis of a polar body; (2) removal and diagnosis of trophectoderm (TE) material; (3) removal and diagnosis of single blastomeres at early cleavage stages. It is the latter technique that has aroused the most interest in this field (reviewed in Adinolfi and Polani, 1989). Polar-body (PB) biopsy involves physical removal of a polar body (usually the second polar body appearing postfertilization)from an IVF embryo at the 2-cell stage. This could be used for either DNA or chromosomal analyses. The technique of course only assesses the contribution of the maternal genome, but in the case of recessive disorders, the contribution of only one of the parents need be controlled. Genetic analysis is further clouded by the process of meiotic “crossing over” where the homologous chromosomes exchange genetic material at metaphase I. In the case of chromosome analysis, assessing the contribution of only one parent is obviously limiting despite the fact that 80% of all Down’s syndrome cases arise from an extra chromosome 21 donated by the mother. The second approach involves biopsy of material from the trophectoderm when the embryo is at the blastocyst stage. The first report of

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5

preimplantation diagnosis (diagnosing sex in rabbits) used this approach (Gardner and Edwards, 1968). Micromanipulation techniques were used to remove the outer layer of TE. Sexing was achieved by detecting Barr bodies (inactive X chromosomes visible only in female interphases). Embryos were transferred into the mother’s uterus and sex was confirmed later in gestation or at birth. TE biopsy at the blastocyst stage has some attractive features: (1) The maximum number of cells are available for diagnosis, making any test more reliable than if fewer cells were available. (2) TE cells are extraembryonic and contribute only to the tissues surrounding the fetus (Handyside and Delhanty, 1993). The major problems with applying this strategy clinically (at least using IVF) are (1) the fact that only half the embryos survive to this stage in culture, (2) the unexpectedly low pregnancy rate reported in some (but not all) clinics following IVF and embryo transfer at this stage (Dawson et al., 1988; Bolton et al., 1991), and (3) the theory that removal of a substantial proportion of TE may affect implantation of the embryo. Cleavage-stage biopsy involves removal of one or more blastomeres when the embryo is at the 4-16-cell stage. Diagnosis can be made on the biopsied cell(s) and the remainder implanted into the mother’s uterus if necessary. At these early cleavage stages, mammalian blastomeres remain totipotent and preimplantation development is not adversely affected by biopsy at the &cell stage. This was confirmed by Hardy et al. (1990), who took embryos with one and two cells biopsied from them and found that glucose and pyruvate uptake only decreased in proportion to the reduction in cellular mass, and furthermore over half (the usual proportion) of the embryos hatched out of the zona pellucida in vitro. The technique (e.g., Handyside et al., 1990) involves placing the embryo in a drop of medium under oil and placing it under a dissecting microscope for micromanipulation. The embryo is immobilized by suction on a holding pipette; a small hole is drilled in the zona pellucida using a tiny pipette and a stream of acid tyrodes which dissolves the zona; and a second larger pipette is then pushed into the hole to remove one or two cells. The remaining embryo is quickly returned to culture and the biopsied cell($ are prepared for analysis. The technique for biopsy of a cleavage-stageembryo is illustrated in Fig. 1. Because of the problems associated with PB and TE biopsies, at present, cleavage-stage biopsy remains the only clinically applicable approach. Indeed, the first preimplantation diagnosis to be performed in a clinical situation used cleavage-stage biopsy followed by a polymerase chain reaction (PCR)-basedassay to determine the sex of the biopsied cell. Families at risk of transmitting sex-linked disorders to their male offspring were treated in this way and female live births have ensued (Handyside et al., 1990).

DARREN K. GRIFFIN

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CELL HUMAN EMBRYO

/

\\ /

HOLDINGPlPFlTE I STABILIZES EMBRYO

DRILLING P l m E MAKES A HOLE IN ZONA WITH A STREAM0F"ACID TYRODES"

ASPIRATION PlPmE TO REMOVE BLASTOMERE

/I

'/

\\

\\

FIG. 1 Biopsy of a human embryo at cleavage stage.

In order to prepare material biopsied from preimplantation embryos for FISH analysis, standard cytogenetic procedures need to be adapted. The cells need to be swelled, fixed, and spread onto glass slides as with other material, but because there is only one cell (or, in some cases, a few cells), the cell needs to be accurately watched when it is moved from one solution to the next and its position on the slide carefully noted (Griffin et al., 1991, 1992).

111. Fluorescent in Situ Hybridization A. Basic Principles

In situ hybridization (ISH) was first described by Gall and Pardue (1969) and involved "formation and detection of RNA-DNA hybrids in cytologi-

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cal preparations” to detect radioactively labeled rRNA genes in Xenupus tissue sections by autoradiographic means. Techniques incorporating DNA-DNA hybrids and using chromosomal preparations soon followed and the original approach remains remarkably unchanged to the present. Many cloned genes have been mapped directly to a particular chromosome band by using this approach (Malcolm et al., 1986). Isotopic ISH typically results in a scatter of silver grains over the chromosome spreads. These need to be analyzed statistically by examining 50-100 metaphases producing a mode of hybridization signals. Also, chromosomes need to be banded, either pre- or posthybridization, in order to achieve accurate cytogenetic assignment. Autoradiographic exposure takes about 2 weeks and the hazards associated with the handling of radioactive isotopes are well documented. The application of fluorescent ISH to chromosomes was first described by Rudkin and Stollar (1977), who detected rRNA genes in Drusuphilia by using a fluorescent antibody to DNA-RNA hybrids as the detection system. Van Prooijen-Knegt et al. (1982) first visualized 18s and 28s rRNA genes on human metaphase spreads, again by forming RNA-DNA hybrids and detecting via a fluorescent RNA-DNA antiserum. Detection using RNA-DNA hybrids, however, proved to be insensitive for most applications in comparison with isotopic ISH and it became clear that a totally different approach would have to be developed. Pinkel et al. (1986) developed a FISH system that involved directly labeling DNA probes with vitamin H (biotin) molecules. DNA can be labeled by nick labeling, random priming of oligonucleotides, or photoactivated labeling. Of the three methods, nick labeling (or nick translation as it is often referred to) is the most common. DNA-DNA hybrids are formed between the labeled probe and chromosomal DNA. Biotin detection is facilitated by fluorescently labeled avidin molecules which show an extremely high affinity for biotin. Furthermore, the fluorescent signal can be amplified by biotinylated antiavidin followed by a second layer of fluorescent avidin. This method has since proved to be the most widely applicable of all FISH approaches and hence the vast majority of advances in this field have incorporated it. Briefly, cytogenetic preparations are made by routine protocols. Exogenous RNA and protein are enzymatically removed. The chromosomal and labeled probe DNA is denatured and allowed to hybridize in situ. Fluorescence is detected with a fluorescein-avidin conjugate. Sequential layers of biotin-antiavidin conjugate followed by a second layer of avidinfluorescein facilitate signal amplification. This amplification step is referred to as the “Pinkel sandwich” (Trask, 1991). Finally, the chromosomes are stained with a fluorescent total DNA counterstain for relocation purposes. Figure 2 demonstrates how successive layers lead to signal

n

DARREN K. GRIFFIN

8

0 BIOTIN MOLECULE

CROSS SECTION THROC'QH 4 CHROMOSOME

A

CHROMOSOMAL DNA

@ FLUORESCEIN MOLECULE

ENZYMATIC REMOVAL OF CONTAMINATING RNA AND PROTEIN

9

DNA STRANDS

AVIDIN MOLECULE

,/

AVTN MOLECULE

APPLICATION

OF BIOTINYLATED

PROBE AND SEPARATION OF BOTH CHROMOSOMAL AND PROBE DNA STRANDS

.1

8

I

"f

IN-SITU HYBRIDAZATION OF PROBE FOLLOWED BY EXCESS BEING WASHED OFF

ADDITIONOF AVIDIN-FLUORESCEIN CONJUGATE

f

' I

1

ADDITION OF BIOTINANTI AVIDIN CONJUGATE

/

ADDITION OF AVIDIN-FLUORESCEIN CONJUGATE (2nd LAYER)

ADDITION OF FLUORESCENT COUNTERSTAW

FIG. 2 Diagrammatic representation of the FISH protocol. (Griffin. 1992.)

FLUORESCENT IN SITU HYBRIDIZATION FOR DIAGNOSING GENETIC DISEASE

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amplification. In fact, four biotin molecules bind to each avidin molecule. Figure 3 is a flow diagram of how a FISH protocol may be performed in the laboratory. The chromosomes appear in one color and the area of the chromosome to which the probe hybridizes (the target sequence) is marked by a second fluorescent color. A recent innovation in FISH technology has allowed Pinkel amplification in one detection layer. In this method, fluorescein-labeled avidin is preincubated with fluorescein-labeledantiavidin. The two conjugates form

CHROhfOSOhfES (AND INTERPHASES) PREPARED BY STANDARD ('YTOGENWIC MkTHODO1.OGY

* * * * *

(CIIROIIOSOMES BANDED AND PHOTOGRAPHED)

(1X:STAINI:D AND) DEHYDRATED RN APC TRtAThlENT PROBE LABELED .AND PLRIFTED

PROTbIN ASE TRE4TMENT

))r DISSOLVED IN HYBRIDIZATION MIX

FIXITION AND DEWDRATION

* * * * * * * * *

PROBE APPLIED TO SLIDE AND SEALED UNDER A COVERSLIP DENATLIRhTION OF PROBE .AND CHROMOSOMAL DNA

IN SITU HYBRIDIZATION POST HYBRIDIZATION WASHES FLLIOROCHROME BLOCKING STEP INCL'BATION WITH FLL'OROCHROME WASHES

(.AMPLIRC.ATION STEP WITH WASHES) MOI'NTED IN ANTI-FADE MEDILJMCONTAINING FLIIORESCENT CO1"TERSTAIN

\'IEWED I'NDER A . MICROSCOPE EQLIIPPED WITH APPROPRIATE FILTERS

FIG. 3 Flow diagram representing FISH methodology.

DARREN K. GRIFFIN

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a complex which is then applied to the biotinylated probe. This is illustrated in Fig. 4. When detecting probes hybridized in situ, two molecules are important-first the hapten and second, the fluorochrome. A hapten or “reporter molecule” is the name given to the molecule that labels the DNA. Biotin is not the only hapten available but is the most common. Also used are digoxigenin, 2-acetylaminofluorene (AAF), the sulfone radical, and mercury. Digoxigenin is a steroid derived from Digitalis plants and can be introduced into a probe in ways similar to biotin. It can be detected by fluorescent-conjugated antibodies to it or to digoxin, from which it is derived. Signal amplification can be achieved using secondary and tertiary fluorescent antibodies. AAF can be used to label a probe because it (a carcinogen) binds covalently in the C8 (8th carbon atom) position of guanosine residues in DNA or RNA. It is detected by fluorescent antibodies (Landegent et al., 1984; Tchen et al., 1984). Labeling with sulfone radicals (sulfonation/transamination)relies on the fact that bisulfite reacts reversibly with C5-C6 double bond of cytidine residues in DNA to give 5,6-dihydrocytidine-6-sulfonate. Detection can be achieved using fluorescent antibodies. Mercury modification is one of the oldest of the nonradioactive approaches. The probe is chemically mercurated at the C5 (5th carbon atom) position of the pyrimidines. To facilitate detection, a mercury binding ligand is attached that carries a sulfydryl group on one end and

CHROMOSOME

ADDITION OF AVIDIN-FLUORESCEIN CONJUGATE AND ANTI-AVIDIN-FLUORESCEIN CONJUGATES EXPOSED TO EACH OTHER

I CONJUGATES FORM

I

J.

ADDITION OF CONJUGATE COMPLEX

.1

ACoMPLEX

t

FIG. 4 Diagrammatic representation of one-step “Pinkel sandwich.”

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a hapten on the other. This secondary hapten can be trinitrophenol (TNP) or biotin, both of which can be detected by fluorescent conjugates, or a fluorescent material itself (Hopman et al., 1986; Raap et al., 1990). Of all these reporter molecules, biotin and digoxigenin are thought to be the most sensitive ( J. Wiegant, personal communication). Figure 5 shows immunocytochemical detection of digoxigenin. A fluorochrome is the fluorescent material which, when conjugated to a molecule which will bind to the hapten (such as avidin or an antibody), facilitates detection of the probe. By definition, a fluorochrome is a material which will become excited by light of one wavelength and then emit light of a higher wavelength. Thus, specially adapted microscopes can detect fluorochromes. Fluorescein isothiocyanate (FITC), which excites in the blue range and emits in the green, is the most commonly used. Also common is tetramethylrhodamine isothiocyanate (TRITC), which excites in the green range and emits in the red; Texas red (or sulforhodamine 101), which excites in the green and gives a very deep red color; and 7amino-4-methyl coumarin-3 acetic acid (AMCA) (Khalfan et al., 1986), which excites in the ultraviolet and emits in the blue range. Two fluorescent DNA counterstains for chromosome location are commonly used-6diamino phenylindole dihydrochloride (DAPI), which absorbs ultraviolet and emits blue, and PI, which absorbs in violet-green wavelengths and emits red. A combination of both counterstains can be used with FITC preparations (hence red chromosomes and yellow/green signals can be viewed simultaneously) but propidium iodide cannot be used with TRITC

I

ANT-DIGOSIGENIN ANTIBODY

PROBE

1 CHROMOSOME

FIG. 5

Fluorescent detection of a digoxigenin-labeledprobe.

DARREN K. GRIFFIN

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or Texas red because it drowns out the signal. Figure 6 shows the light path a fluorescent microscope when it is exciting and detecting FITC. Recently, labeling probes directly with the fluorochrome has become possible. The labeling is achieved by means similar to those for biotin and digoxigenin; signal amplification, if necessary, can be achieved by using antifluorochrome antibodies. The applicability of this approach has been assessed by Wiegant et al. (1991), who found that probes 50-100 kb in length could be detected when the signal was unamplified, whereas probes 1-5 kb could be detected when they were amplified. These figures

BARRIER. ALTER

RLTER COMBINATION BLCXX

DICHROIC MIRROR

MERCURY VAPOR

ExmER HLTER

+t

LAMP

LIGHT OF ALL WAVELENGTHS

II) +

BLUELIGHT GREWlLIGHr

-0BJEXTIVE

' - 4 4 4 ,d

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FIG. 6 Light path of a fluorescent microscope detecting fluorescein. 1. Light of all wavelengths emitted from lamp. 2. Exciter filter lets through only blue light. 3. Dichroic mirror reflects blue light. 4. Fluorescein is ecited by blue light and emits green. 5 . Dichroic mirror lets through green light. 6. Banier filter lets through only green light. 7. Green signal is viewed. (Grimn, 1992.)

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FLUORESCENT IN SITU HYBRIDIZATION FOR DIAGNOSING GENETIC DISEASE

are comparable to amplified and unamplified signals using the biotin-avidin-fluorochrome system. This strategy also has the advantage that a low background yield is obtained. Given that a number of probe labels and fluorochromes exist, the possibility of multiple labeling (i.e., simultaneous hybridization and detection of two or more probes on the same cytological preparation)arises. Applications of this are discussed in subsequent sections. Figure 7 illustrates the principle of dual labeling. DNA probes contain two elements-namely, the insert (i.e., the human DNA complementary to the target sequence on the chromosome) and the vector (i.e., unrelated DNA into which the insert is cloned and through which the DNA can be replicated). Four types of probes are common-plasmid probes, which have inserts ranging from 500 base pairs to around 6-kb and vectors of, on average 2 kb; phage clones with inserts of 3-20 kb and vectors of around 40 kb; cosmids with inserts of 40 kb and vectors of 5-6 kb; and finally, yeast artificial chromosomes (YACs) with inserts of 300+ kb and vectors of 7-8 kb. Throughout the whole genome, however, are interspersed repeated sequences of which the Alu elements are the most common. Many probes (invariable YACs and cosmids, frequently phages, and occasionally plasmids) contain these and other repeats between the unique sequences. If these probes were applied directly onto chromosomes via FISH, the whole chromosome complement would light up because these repeats would find Q biotin molecule

digoxignin molecule

w

A A

avidln molecule

dipxienin molecule

.......

ga

I FIG. 7 Dual detection of biotin and digoxigenin-labeledprobes in red and green.

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DARREN K. GRIFFIN

complementary sequences all over the genome. In order to inactivate this and allow the unique portion of the probe to find its complementary sequence, the probe must be preannealed, with total unlabeled DNA in excess. Such an approach was first described for FISH by Lichter et al. (1988) and Cremer et al. (1988a) and is known as competitive in situ suppression or CISS. Chromosomal DNA is denatured separately; then the preannealed probe is applied, leaving unique sequences free to hybridize.

6. Types and Applications

1. Tandem Repetitive Probes Classical satellite, and a-satellite sequences have been studied using FISH. Both these types of DNA contain arrays of highly repeated DNA sequences. Hence probes for them have large areas of target sequence on the chromosome on which to hybridize and thus produce large and bright FISH signals. Classical satellite DNA sequences include the large C-bands on chromosomes 1,9, 16, and Y. The majority of chromosomespecific probes, however, consist of a-satellite or alphoid DNA. Alphoid repeats constitute a group of related, highly divergent sequences, each approximately 171 kb in length. These sequences show 20-40% divergence from one another. Arrays of alphoid repeats are found exclusively around the centromeric regions of all the chromosomes. Tandem arrays of these units show chromosome-specific, higher order repeat units; this is why probes for these regions generally recognize only one chromosome. Figure 8 shows this hierarchical order of repeat units for chromosomes X, 7, and 10. Since these tandem repeat sequences are largely chromosome specific and since they are generally cloned into plasmid vectors with inserts of around 2 kb, CISS is not necessary prior to FISH; however, high stringency conditions are often needed to maintain chromosomal specificity. It is in the exploitation of classical-satellite and a-satellite DNA that there has been much interest in FISH. When conditions are ideal, these probes brightly light up specific regions (usually centromeres) of human chromosomes. Signals are large enough to be seen and counted in interphase nuclei. Probes are now available commercially for nearly all the human chromosomes, including both sex chromosomes, although some probes detect more than one chromosome simultaneously. A notable example of this is chromosomes 21 and 13. Probe L1.26 (Devilee et al., 1986)recognizes the centromeres of chromosomes 13and 21. These probes have a multitude of applications, chiefly in fields where analyzable metaphases cannot always be obtained in cytogenetic preparations. Such fields

15

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include cancer cytogenetics and prenatal and preimplantation diagnoses. Detection of chromosomes in interphase nuclei is referred to as “interphase cytogenetics.” Figure 9 is a FISH preparation showing the detection of a centromeric probe specific for chromosome 18 in a metaphase and an interphase nucleus. The applications of these probes in postnatal, prenatal, and preimplantation diagnosis are discussed in subsequent sections. 2. Chromosome Painting

By using a collection of probes or a ‘‘library’’ specific for a particular chromosome, it is possible to light up or “paint” a whole chromosome along its length. Since many of the clones in the library contain inter-

16

DARREN K. GRIFFIN

FIG. 9 Detection of the alphoid probe for chromosome 18 using FISH.

spersed repeats, CISS generally needs to be applied. Julien et al. (1986) were the first to report the painting of chromosome 21 by creating chromosome 21 probes using a dual laser flow cytometer and subsequently detecting trisomy 21 in prenatal samples. Cremer et al. (1988a)and Lichter et al. (1988) (accompanying papers) describe the delineation of chromosomespecific libraries, subsequent chromosome painting, and detection of chromosome I , 4,7, 18, and 22 aberrations in tumor cells. Flow-sorted human chromosome libraries were used here also. Pinkel et al. (1988) detected trisomy 21 in interphase nuclei and chromosome 4 translocations using libraries obtained from the American Type Culture Collection which were cloned into “Bluescribe” plasmid vectors. An improvement in selecting library clones was demonstrated by Fuscoe et al. (1989), who subcloned a chromosome 21-specific library into “Bluescribe” and selectively picked unique sequence inserts. The result was more intense signals when the library of clones was put through CISS and FISH. Very intense chromosome painting was also reported by Jauch et al. (1990), who applied their studies to human sex chromosomes and amplified libraries in their original phage vectors. Currently, chromosome painting libraries are available for all human chromosomes. Recent innovations (e.g., Telenius et al., 1992) have generated chromosome painting probes by flow sorting whole human chromosomes and subsequently amplifying the resulting DNA by a PCR protocol designed to amplify total DNA [degenerate oligonucleotide-primed (DOP) PCR]. These paints tend to give brighter signals than those isolated from cloned libraries. The use of DOP-PCR to amplify the sorted chromosomes allows large quantities of DNA to be generated and these probes are now marketed commercially. Figure 10 shows the painting of chromosome 5 in a preparation of human lymphocytes.

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FIG. 10 Chromosome painting (chromosome 5).

3. Detection of a Single Locus In the field of gene mapping, it is in the direct visualization of large probes on chromosomes-namely phages, YACs, and more commonly cosmids-that most progress has been made. Mapping of longer sequences by FISH invariably requires the employment of CISS. However, as will be described in subsequent sections, such probes also have diagnostic uses. Lichter et al. (1990b) were the first to describe the FISH mapping of cosmids on chromosome 11. Biotin and digoxigenin labels were employed. Since the realization that cosmids could be mapped using FISH, most of the interest in this field has been in that direction. Signals are typically visible on both chromatids of both homologs. When conditions are optimal, a cosmid signal can be visualized in the interphase nucleus as well as on the metaphase chromosome. Using single-color FISH, signals between 25 and at least 250 kb can be resolved in interphase nuclei. Furthermore, the distance between two clones can be ascertained solely by measuring the distance between them in an interphase nucleus with an accuracy of within 40 kb (Trask et al., 1990; Bentley-Lawrence et al., 1990). Using dual-color fluorescence, signals 50 kb apart can be resolved at interphase and signals 3 megabases apart can be resolved at metaphase. Clones which are close together can be ordered with respect to one another by examining interphase nuclei. This is achieved by varying the hapten (usually biotin and digoxigenin are used) with which each of three or more probes is labeled (e.g., labeling two probes with biotin and the other with digoxigenin). The probes can be simultaneously hybridized on

DARREN K. GRIFFIN

18

the same preparation and then detected with red and green fluorochromes respectively. Simply scoring the order of red and green dots in a series of experiments where different combinations of haptens are used thus gives a physical order of clones. Such an approach is referred to as “interphase mapping” (Trask, 1991) and can be used not only for ordering genes but also, when the usual gene order is known, for detecting subtle rearrangements, deletions, or duplications that lead to genetic conditions. The mapping of yeast artificial chromosomes is also well documented in the literature. Being of very large insert size, YACs tend to give large signals; however, nonspecific background signals on unrelated chromosomes can be a problem when hybridizing if conditions are suboptimal (Rietman et al., 1989; Wada er al., 1990). YACs can be used to narrow down the molecular location of chromosomal breakpoints in translocations. A split YAC signal that is visible on derivative translocation chromosomes indicates that the breakpoint must be encompassed within the few hundred kilobases to which that YAC hybridizes (Rowley et al., 1990). YAC signals, like cosmids, are visible in the interphase nucleus. Figure 11 shows a YAC containing DNA from the gene adenomatous polyposis coli hybridizing to chromosome 5 . 4. Total Genomic Probes

The use of total genomic probes has been largely exploited in screening somatic cell hybrids. Total human DNA can be used as a probe on meta-

FIG. 11

FISH detection of a YAC containing the adenomatous polyposis coli gene.

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phases of hybrids to determine how many human pieces are present, whereas total hybrid DNA can be probed on to human spreads to determine the chromosomal nature of these pieces. As there are, to my knowledge, no living human-animal hybrids, it is difficult to envisage how total genomic probes could be used for diagnostic purposes and thus they will be discussed no further. 5. Further Developments

A number of areas of research involving FISH technology are rapidly becoming apparent.

MulticolorFISH Nederlof et al. (1989) were the first to achieve triple hybridization and detection using centromere-specific probes. Biotin, AAF, and mercury labeling were employed, coupled with FITC, TRITC, and AMCA fluorescent detection systems. Although it was a significant breakthrough, three-color fluorescence has limited usefulness because the DNA usually..needs to be counterstained fluorescently for location purposes, thus using up the spectra of one fluorochrome. Reid et al. (1992) demonstrated for the first time the simultaneous detection of probes for chromsomes 21, 18, 13, X and Y, each in a different color on the same preparation. The probes used were contig cosmid clones, and five-color detection was facilitated by labeling each probe with either one or a different combination of two labels, detecting them with appropriate fluorochromes, analyzing each digitally for the presence of one, two, or three colors and then assigning each a pseudocolor using computer software. For instance, with two fluorochromes, FITC and Texas red, a computer linked to an image analysis system could assign a Texas red image a red pseudocolor, an FITC image a green pseudocolor, and an image fluorescing both FITC and Texas red, a blue pseudocolor. Thus from a two-color system, three-color fluorescence can be obtained. Hence with a three-color detection system (red, green, and blue or infrared, red, and green), seven-color fluorescence could theoretically be achieved. Seven-colorfluorescencewould be invaluable in clarifyingcomplex karyotypes, whether using chromosome-specificcentromeric probes or chromosome painting. A further application would be the ability to order seven cosmids in relation to each other. This technology can be taken a step further by using “ratioing” of certain fluorochromes, for instance, distinguishing probes labeled with a I : 1 ratio of red to green from those labeled with a 2 : 1 ratio. In such a way, it is easy to envisage a large number of chromosomes or chromosome regions illuminated in different colors. Indeed, multiple-color chromosome painting has been described by Dauwerse et al. (1992), who performed 6-, Q.

DARREN K. GRIFFIN 20 7-, 9-, and 12-color chromosome detection on the same metaphase using

different ratios of red, green, and blue. Furthermore, pictures were generated by normal photography and not computer-aided image analysis.

b. Application of PCR Technology to FISH Another possible direction of investigation is to combine FISH and PCR technology. A small oligonucleotide could be annealed to a chromosome (as in PCR) and a string of nucleotides “zipped” on after it (as in PCR). This approach is referred to as “primed in situ DNA synthesis” or “PRINS.” Gosden et al. (1991) have described the use of this approach incorporatinga biotinylated deoxyuridine triphosphate (dUTP) in the nucleotide mix, followed by detection with fluorescent avidin. They detected human satellite sequences and Alu sequences and also telomere-specific sequences in Tetrahymena and Trypanosoma. I f these nucleotides were labeled with fluorescein dUTP, then this could be a very quick way to perform FISH and could be particularly useful in diagnostic applications when speed is important. Meltzer et al. (1992) have described the rapid (24-hr) generation of region-specific FISH probes and applying them to identify chromosomal rearrangements. The strategy they presented was to microdissect chromosomal regions and amplify them in uitro by PCR. PCR products were then labeled with biotin and used as probes onto metaphase preparations. Using such an approach, it is theoretically possible to generate probes (for FISH or other purposes) for any region of the genome and thus unequivocally identify most cytogenetically visible chromosomal rearrangements.

c. Three-Dimensional FISH A future application which is often mentioned in verbal presentations is the possibility of using FISH to map the physical position of genes in the intact interphase nucleus. If a method of preparing and fixing whole nuclei and hybridizing probes (e.g., cosmids) to them could be devised, then not only could the position of genes at interphase be mapped but also the communication between genes in the nucleus could be investigated. In such an application, a confocal microscope (a instrument which can visualize intact specimens in three dimensions) would be an invaluable tool. Trask et al. (1988) have described methods of preparing whole nuclei and performing FISH on them. They used total genomic probes on somatic cell hybrid nuclei and chromosomespecific satellite probes to reveal specific chromosomal domains in the interphase nucleus. Both groups of researchers are reportedly proceeding to the use of cosmid clones. d. Comparative Genome Hybridization This recent innovation in FISH technology was reported by Kallionemi et al. (1992) and du Manoir et al. (1993). It involves the simultaneous hybridization of normal DNA (labeled

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with one colored fluorochrome) and test DNA (labeled with another colored fluorochrome) on the same metaphase. The objective of the exercise is to compare the relative intensities of each color and thus elucidate gross chromosomal differences between the two DNAs. For instance, if the test DNA was from a patient with trisomy 18, the relative intensity of fluorescence from the test DNA would be brighter on chromosome 18 but of an equal ratio in the remaining chromosomes. This technique is still in its developmental stages but has already been used to analyze prenatal samples and tumor specimens.

e. “Ha1o”Preparations Wiegant et al. (1992) have reported a technique of preparing interphase nuclei which results in extended DNA loops arranged around the nucleus in a halo-like structure. Subsequent FISH with cosmid probes on these nuclei gives signals resembling beads on a string rather than discrete dots. It thus provides a simple, high-resolution approach for visualizing DNA strands. It is principally a method for mapping because it can detect the extent to which clones overlap, but it could feasibly be used to detect subtle DNA rearrangements that lead to clinical disorders.

IV. Sexing of Human Cells

Sexing of a human cell by FISH involves the detection of one or both of the sex chromosomes in an interphase or metaphase nucleus. A. Postnatal Samples Postnatal chromosomal diagnosis involving sex chromosomes has its uses when diagnosing XX males and XY females, when ascertaining the sex ratio of cells of an individual who is an XX/XY dispermic chimera, and also when monitoring the progress of a bone marrow transplant when donor and recipient are of opposite sexes. In each of these cases, FISH can obviate the need to prepare analyzable metaphases. B. Prenatal Samples Sexing is offered in the first or second trimester of pregnancy largely for the screening and selective termination of males in those families at risk of transmitting sex-linked recessive disorders. Selective termination of all

22

DARREN K. GRIFFIN

male pregnancies is only offered in the cases where specific diagnosis of the X-linked disorder is not available. FISH diagnosis of sex can be performed on the interphase nuclei of prenatal material when analyzable metaphases cannot be obtained. Sexing of prenatal samples has been cited by a number of authors using FISH probes specific for the Y chromosome (Kozma and Adinolfi, 1988; Guyot et al., 1988; Griffin et al., 1991). Use of a probe specific for the X chromosome on prenatal material (Griffin et al., 1991)eliminates the risk of failure of hybridization leading to misdiagnosis. Use of probes specific for both chromosomes, each detected in a different color (Griffin et al., 1992) is further advantageous though technically a little more difficult.

C. Sexing of Human Preimplantation Embryos

1. The Need to Research Sexing of Preimplantation Embryos Many families at risk of transmitting X-linked disorders (of which there are over 200) have already undergone one or more stressful abortions of male fetuses. Others disagree with terminations on moral or religious grounds. Hence, an effective means of preventing affected offspring would be to sex embryos fertilized in v i m and selectively implant females-socalled “preimplantation diagnosis.” Preimplantation diagnosis of sex has already been achieved using PCR to sex the embryo (Handyside et al., 1990; Handyside, 1991). This approach is, however, plagued with problems of contamination and amplification failure.

2. Early Work on Other in situ Hybridization Protocols Jones et al. (1987) were the first to report sexing of human embryonic nuclei and used both radioactive and nonradioactive ISH approaches. The nonisotopic technique employed enzymatic detection of a biotinylated probe. They reported good results using radioactivity but more ambiguous ones using biotin. West et af. (1987, 1988) using radioactive means attempted sexing on 14 morphologically normal and 9 apparently abnormal embryos. All 14 apparently normal embryos were sexed with confidence; however, clear diagnosis of sex for the 9 abnormal ones was not always possible. Angel1 et al. (1987) reported polyploidy in IVF embryos using an identical approach. Penketh et al. (1989) used an alkaline phosphatasebased detection system and biotinylated pHY2.1 and claimed a 66% success rate; that is, two-thirds of known male nuclei displayed a Y signal.

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3. FISH Studies

Studies using FISH on preimplantation embryos include those by Grifo et al. (1990), who apparently detected two Y chromosomes in a single human embryo, and Pieters et al. (1990), who detected chromosome 1 in a single human preimplantation embryo. However, Griffin et al. (1991) were the first to perform and evaluate FISH for X and Y chromosomespecific probes on human embryonic material. If one determines sex on the basis of number of hybridization signals in the majority of nuclei, the efficiencies with both probes (pHY2.1 and pBamX7) using FISH appeared to be higher in male embryonic nuclei than previously reported using other ISH methods with probe pHY2.1 (Jones et al., 1987; West et al., 1987, 1988;Penketh et al., 1989). For example, 85% of these nuclei gave positive signals, compared with 66% using an enzyme-based biotin detection system (Penketh et al., 1989). Figure 12 shows two X-chromosome signals on an interphase nucleus of a female human preimplantation embryo. Hybridization with probe pBamX7 had not been previously evaluated using any form of ISH on human embryos. There was a high incidence (18%)of nuclei displaying two (or more) signals in interphase nuclei classified as male and four signals in a nucleus designated as female. All of these embryos had more than 10 nuclei on day 5 postfertilization. In most cases, the nuclei with twice the number of expected signals were appreciably larger than those surrounding cells. This can be easily explained because of the occurrence of tetraploidy. Tetraploid nuclei with two Y chromosome signals have been already observed in human embryos (West et al., 1987, 1988), and it has been suggested that their occurrence in early preimplantation embryos may be a culture-induced phenomenon (West, 1990). Single-color FISH employing either probe had its disadvantages with

FIG. 12 Single nucleus from a human embryo displaying two X-chromosome signals.

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DARREN K. GRIFFIN

regard to diagnostic accuracy. Use of the Y chromosome-specific probe pHY2.1 has an efficiency of around 85%. Thus, if two cells were biopsied (leaving six to be transferred), the probability of misdiagnosis would be more than 1%. Because of the frequent occurrence of twice the expected number of signals using the X probe alone, tetraploid male cells and female cells would be indistinguishable, again leaving the probability of misdiagnosisgreater than 1%. It was clear therefore that use of either probe alone was not an ideal approach to sexing human embryonic material. The problems associated with each of these probes alone were, however, alleviated when both were hybridized and detected simultaneously in different colors on the same nucleus (Griffin et af., 1992). Hybridization rates were high and, more important, the chance of misdiagnosis was reduced to a minimum. Positive diagnosis of sex could be made in 78% of interphase nuclei and 93% of metaphase nuclei (these were unsuitable for classical cytogenetic analysis), and efficiencies were greater than with other ISH techniques (Jones et al., 1987; West et af., 1987, 1988; Penketh et af., 1989; Grifo et al., 1990). This approach was an improvement on single labeling because the possibility of misdiagnosing a male as female is virtually eliminated since two clear X and no Y signals had to be seen before the embryo was identified as female.

4. Prospects for Clinical Application The biggest disadvantage of FISH methods for sexing preimplantation embryos compared with PCR protocols which had already been put into clinical applications (see Section 11) is that FISH methods generally take 24 hr to perform whereas the PCR-based sexing strategy takes only 5 hr. This is a disadvantage because, when performing preimplantation diagnosis, it is desirable to biopsy cells from the embryo, determine their sex, and selectively transfer female embryos all on day 3 postfertilization.This ensures maximum diagnostic efficiency and pregnancy potential. As a result, we developed a method of performing FISH sexing within one working day that involves simultaneous detection of X and Y chromosomes in green and red, respectively. Improvementto cytogeneticpreparation and the use of freshly prepared slides allowed the protocol to be followed with only a mild preproteinase digestion, high-stringency hybridization and washes, 90-120 min hybridization time, and no signal amplification. The whole procedure could be performed in 6-7 hr and high hybridization efficiencies were retained. On the basis of these results, it was decided to proceed with clinical application, treating families who were at risk of transmitting X-linked recessive disorders.

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5. Patient Strategies Thus far, 12 treatment cycles involving preimplantation sexing by FISH have been reported by our group (Griffin et al., 1993; Delhanty et af., 1993) and more are likely to be reported in the near future (Griffin et al., 1994). We can successfully sex over 80% of all our preimplantation embryos, and success rates are constantly increasing because of subtle changes in protocols. More important, our misdiagnosis rates are currently 0%. A number of live births have ensued, thus helping families who would have previously either have had to undergo stressful abortions or abstain from having children.

V. Further Fluorescent in Situ Hybridization Diagnoses A. Autosomal Imbalances

1. Chromosome-Specific Repeat Probes The probes most applicable for the detection of aneuploidy and polyploidy are the centromere-specificrepeat probes. When analyzable metaphases are unobtainable, these probes are visible in the interphase nucleus. There are probes available for all the human chromosomes; however, some detect more than one chromosome simultaneously. Use of these probes on live-born individuals includes confirmatory diagnosis and prenatal diagnosis of trisomy (e.g., trisomy 18), and investigations into the nonrandom nature of ploidy in the cells of certain cancers. Human trisomies that result in live births are those of chromosomes 21, 18, and 13. Cremer et al. (1986) described diagnosis of trisomy 18 in the interphase nuclei of prenatal samples using the probe L1.84 (specificfor the centromere of chromosome 18)and employing various ISH techniques. However, there is no current chromosome satellite-specific probe for chromosomes 21 or 13. As mentioned earlier, the probes available detect 21 and 13 together because the satellite DNAs are very similar (Willard, 1990).Furthermore, our own research has shown that the signal size varies on the acrocentric centromeres from individual to individual. Hence use of the 13/21 probe for the detection of trisomy can be problematic. Chromosome-specific repeat probes can also be of use in detecting the chromosomal nature of spontaneous abortions. Spontaneous abortions can arise as a result of polyploidy or aneuploidy in the fetus. The most common polyploidy in human abortuses is triploidy (Delhanty et al., 1961) and the most common aneuploidy is trisomy 16 (Hassold et al., 1984).

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DARREN K. GRIFFIN

Figure 13 shows the probe for chromosomes 13 and 21 detected on a normal lymphocyte chromosome preparation. Note that the signal on one of the chromosomes 21 is dimmer than the other three. Figure 14 shows the detection of trisomy 18 in interphase nuclei of an uncultured CVS sample. A number of individuals are at risk of transmitting aneuploidy because they carry balanced Robertsonian translocations of the acrocentric chromosomes. It is theoretically feasible to use probes specific for the centromeres of the acrocentric chromosomesto screen trisomy in the preimplantation embryos of these individuals. We are working on this but have run into problems because of the aforementioned differences from individual to individual and because overlying signals could lead to misdiagnosis. Most trisomies, however, do not arise as a result of balanced translocations involving the centromere in one of the parents. Hence the use of centromere-specific probes could be useful for preimplantation diagnosis only if a number of chromosomes were screened for and if the family ran a very high risk of transmitting trisomy for a reason other than a parent carrying a balanced translocation. Furthermore, given that many spontaneous abortions arise as a result of ploidy anomalies, it is theoretically possible to screen the embryos of individuals undergoing fertility treatment in in vitro fertilization clinics with a view to improving the success rate of the treatment. Both the above may be particularly applicable in older mothers who run a high risk of trisomic offspring (Morton et al., 1988).

FIG. 13 Detection of chromosomes 13 and 21 centromeres (alphoid probe).

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FIG. 14 Detection of trisomy 18 in interphase nuclei of an uncultured CVS.

2. Chromosome Painting and Locus-Specific Probes

a. Detection of Chromosome 22 Since trisomy 21 is the most common cause of mental retardation in man, much interest has been generated in using FISH to detect chromosome 21 prenatally, in postnatal samples (for instance to detect the level of mosaicism in live-born individuals), and also for preimplantation diagnosis. As mentioned, the centromere probe specific for chromosome 21 also detects chromosome 13 and its uses are limited. Lichter et al. (1990a) have reviewed the feasibility of various chromosome 21 probes, including 13/21 alphoid probes, chromosome painting libraries, and cosmid clones for detection in interphase nuclei. They conclude that use of a single cosmid clone for the detection of chromosome 21 at interphase is the most applicable. However, we find that single cosmids often give weak signals and can be difficult to detect

28

DARREN K. GRIFFIN

in interphase nuclei, Hence while a world-renowned laboratory such as Lichter’s may find no problem in detecting bright, single cosmid signals in interphase nuclei, ordinary diagnostic labs may not be as successful. It is possible to use a pool of overlapping cosmids to increase signal size. Such “contigs” (as they are referred to) are available commercially and we have looked at these in some detail. We find them to be of some use but find that YAC clones can give larger signals and hence are more applicable for the detection of chromosome 21 in interphase nuclei. Figure 15 shows adjacent metaphase and interphase nuclei: A, probed with the chromosome 21 contig; B, probed with a YAC for chromosome 21. Note that the YAC signals are brighter and more easily visible in the interphase nuclei.

6. Other ChromosomeAnomalies To detect chromosome translocations in the interphase nucleus, locus-specific probes such as cosmids, cosmid contigs, and YACs can be used. This was elegantly demonstrated by Arnoldus et af. (1990) and Tkaachuk et af.(1990), who reported detection of the “Philadelphia chromosome” in the interphases of the bone marrow of patients with chronic myeloid leukemia (CML). The Philadelphia chromosome is the der(22) from a reciprocal translocation t(9;22)(q34:qll) and causes the condition as result of fusion of the cancer genes bcr (on chromosome 22) and abl (on chromosome 9). Both groups used probes specific for these two genes in a dual-color detection strategy. Figure 16 illustrates diagrammaticallythe detection of these probes and the Philadelphia chromosome as reported by the two groups. These probes are currently available commercially and diagnostic laboratories interested in detecting the Philadelphia chromosome should perhaps consider using them when production of analyzable metaphases fails. Recently, Knight et al. (1992) and Joos et af. (1992) have used similar approaches to detect translocations in synovial sarcoma and Burkitt’s lymphoma cells respectively. It is theoretically possible to detect any chromosome translocation by these means in interphase nuclei (provided that the relevant probes are available) in postnatal, prenatal, or preimplantation material (Lichter and Ward, 1990). Chromosome painting is an invaluable technique when working with metaphases on which classic banding and analysis is difficult. Such instances arise when a rearrangement is very complex, when chromosomes are very short and/or clumped together, or when banding techniques fail. This can often be the case in some leukemias and also in some CVS preparations. Chromosome painting gives unequivocal identification of any one chromosome (or more if other colors are used). It is also useful in unequivocally determining the chromosomal origin of ring chromosomes

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FIG. 15 (A). Chromosome 21 cosmid contig (metaphase and interphase). (B). Chromosome

21 YAC (metaphase and interphase).

DARREN K. GRIFFIN

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FIG. 16 Detection of the Philadelphia chromosome in interphase nuclei.

on which banding patterns are not always obvious (e.g., Svennevik and Hastings, 1993). Although about 90% of cells from human preimplantation embryos can be arrested in metaphase following overnight treatment with colchicine, analyzable chromosome spreads are universally very difficult to obtain because chromosomes are often short and clumped together (L. J. Wilton, personal communication). Figure 17 shows a typical chromosome preparation from a human embryo. Since karyotype analysis of CVS involves looking at 20-30 metaphases selected for optimal spreading, it is unlikely that preimplantation diagnosis by karyotyping alone is at present a feasible strategy. Hence chromosome painting approaches have been used to study the interphase nuclei of human preimplantation embryos. Figure 18 shows the detection of chromosome 5 (A) and chromosome 18 (B) on metaphase preparations of human preimplantation embryos.

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FIG. 17 Typical metaphase preparation of a human preimplantation embryo.

B. DNA Deletions

Several conditions arise as a result of DNA deletions. Two notable examples are muscular dystrophy and Angelman and Prader-Willi syndromes. Sixty percent of all males with Duchenne’s muscular dystrophy have a

FIG. 18 Chromosome painting of a human preimplantation metaphase. (A). Chromosome 5 . (B). Chromosome 18.

DARREN K. GRIFFIN 32 large deletion in the dystrophin gene on the X chromosome. In 60% of those individuals, the deletion is in exon 45 (Blonden et al., 1990). It is possible to detect exon 45 using an overlapping set of cosmids (a so-called “contig”). Hence it is possible to detect whether an individual is deleted for exon 45. Such an approach is useful postnatally when screening for female carriers (Reid et al., 1990) and prenatally when identifying affected fetuses. We are investigating the possibility of preimplantation diagnosis in couples at risk of transmittingthe deletion and hence Duchenne’s muscular dystrophy to their offspring. Figure 19 shows detection of the cosmid contig for exon 45 in a normal male. The large signal is the centromeric probe for the X chromosome and the two small dots above it are the cosmid signals. Also arising as a result of large deletions are certain cases of PraderWilli and Angelman syndrome. These are two distinct syndromes, both causing severe mental retardation, which can arise from deletions in the same region of chromosome 15 (15qll-12). If the deletion is in the maternal chromosome, the proband has Angelman syndrome and if the deletion is in the maternal chromosome, Prader-Willi syndrome. We have shown (R. J. Gardner, D. K. Griffin, and J. D. A. Delhanty, unpublished results) that it is possible to detect the deletion using FISH. Such a deletion, if relatively small, may easily be missed by classic cytogenetic analysis. Figure 20 shows a cosmid specific for this region detecting a deletion in a patient with Angelman syndrome in that only one chromosome 15 (top

FIG. 19 Detection of exon 45 of the dystrophin gene in a normal male.

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FIG. 20 Detection of a chromosome 15 deletion in a patient with Angelman syndrome.

left) is lit up. (This cosmid also cross-hybridizes to a region of chromosome 2.) Thus it is theoretically possible to detect any DNA deletion of above 40 kb provided the relevant cosmid is available.

C. DNA Duplications A condition arising from a DNA duplication is Charcot-Marie-Tooth disease type A. Lupski et al. (1991) used FISH to demonstrate this using a single cosmid probe on interphase nuclei of affected patients. This is particularly useful for a confirmatory diagnosis of a live-born individual or for prenatal diagnosis. Since preimplantation diagnosis is at present limited to one cell, detection of DNA duplications for clinical purposes would be unfeasible because overlying signals could lead to misdiagnosis. It is likely to be some time before detection of this disorder and ones like it becomes a possibility using FISH technology.

VI. Concluding Remarks

As mentioned in the introduction, there exists a gap in FISH technology between what is possible and what is practicable in a diagnostic laboratory.

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Also, almost inevitably, there is a time lag (often of 5 years or more) between the publication of a new approach and its use as a routine procedure. In the case of use of the chromosome-specific probes, companies (such as ONCOR) now sell these probes labeled with biotin or digoxigenin for all the human chromosomes. With a little training there is no reason why a moderately skilled technician could not perform a FISH experiment with these. Certain companies (such as CytoCell) even try and make things easier for the operator by sticking the probe to a coverslip. Chromosome painting can also be technically quite easy because companies such as ONCOR and CAMBIO produce paints labeled with biotin, digoxigenin, and/or fluorescein. Furthermore, since most of these companies also market probe detection kits, it is sometimes difficult to envisage how one could go wrong. FISH is certainly a much easier technique than it was 4 years ago. Hybridization and detection of locus-specific probes can, however, be more tricky. Because signals can be tiny and because competitive in situ suppression has to be used, experimental failure can often occur. Also, few such probes are marketed commercially. Exceptions to this include a cosmid contig for detection of chromosome 21 and bcrlabl probes for detection of the Philadelphia chromosome. It seems likely however that as the demand for these types of probes in the diagnostic situation increase, more and more will become commercially available. Whether diagnostic laboratories will keep pace with the rapidly moving FISH technology or simply consolidate the technology they are currently training themselves in remains to be seen. Certainly, techniques such as 12-colorchromosome painting and comparative genome hybridization would be useful tools. One could easily envisage it taking another 5 years at least for these to become routine protocols. In the case of preimplantation diagnosis, I cannot stress enough that it is an approach to be attempted only by labs with considerable experience in both embryo manipulation and research cytogenetics. It is a relatively new strategy, has aroused much attention in both the scientific and lay press, and hence could easily get a bad name if practized by the wrong hands. In terms of FISH technology, preimplantation diagnosis is a largely unexplored area. Only sexing has thus far been put into clinical practice. It may not be overoptimistic to hope that diagnosis of any chromosome abnormality or clinically significant DNA deletion could be possible by the end of the century. As for FISH technology itself, publications are constantly being produced announcing novel approaches to the FISH technique. Comparative genome hybridization is one such approach which looks certain to be exploited greatly. Also, multiple-color detection is very fashionable; one often hears mention of chromosomal bar codes where certain chromo-

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somes are banded in multiple fluorescent colors at will. There are also numerous cameras (cooled, charge-coupled device cameras) which can detect tiny signals invisible to the naked eye. Linked up to image-analysis setups, they can be extremely powerful both in grabbing and manipulating images and also in storing large numbers of them. It would be refreshing to see more research into the detection of DNA specimens in threedimensional specimens such as whole-mount embryos; the use of confocal microscopy (i.e., visualizing whole specimens in three dimensions) would help enormously here. The most interesting challenge to anyone involved in FISH research, I believe, would be the development of a FISH-based protocol which could distinguish between maternally and paternally derived homologs of any given chromosome.

Acknowledgments I express my thanks to Dr. Joy Delhanty for her critical reading of the manuscript, for initially giving me the opportunity to work on FISH, and for her help and encouragement throughout. I am also grateful to my friends and colleagues at the Hammersmith Hospital for providing material on which to work and ultimately making preimplantation diagnosis a reality.

References Adinofi, M., and Polani, P. E. (1989). Prenatal diagnosis of genetic disorders in preimplantation embryos: lnvasive and non-invasive approaches. Hum. Genet. 83, 16-19. Angell, R. R., Sumner, A. T., West, J. D., Thatcher, S. S., Glasier. A. F., and Baird, D. T. (1987). Post-fertilisation polyploidy in human preimplantation embryos fertilised invitro. Hum. Reprod. 2, 721-122. Arnoldus, E. P. J., Wiegant, J., Noordmeer, I. A., Wessels, J. W., Beverstock, G. C., Grosveld, G. C., van der Ploeg, M., and Raap, A. K. (1990). Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet. Cell Genet. 54, 108-1 11. Bentley-Lawrence, J., Singer, R. H., and McNeil, J. A. (1990). Interphase and metaphase resolution of different distances within the human dytrophin gene. Science 249,928-932. Blonden, L. A. J., Grootcholten, P. M., den Dunnen, J. T., Bakker, E., Abbs, S., Bobrow. M., van Broekhoven, C., Blaumbach, L., Chamberlain, J., Caskey, C. T., Denton, M., Felicetti, L., Galluzi, G.. Fishbeck. K. H., Franke, U . , Darras, B., Gilgenkrantz, H., Kaplan, J.-C., Henmann, F. H., Junien, C., Boileau, C., Lietchti-Gallati, S., Lindlof, M., Matsumoto, T., Niikawa. N., Muller, C. R., Poncin, J., Malcolm, S.,Robertson, E., Romeo, G., Covone, A. E., Scheffer, H., Schroder, E., Schwartz, M., Verellen, C., Walker, A., Worton, R., Gillard, E., and van Ommen, G. J. B. (1990). 242 breakpoints in the 200-kb deletion-prone P20 region of the DMD gene are widely spread. Genomics 10, 631-639.

Bolton, V. N., Wren, M.E., and Parsons, J. H. (1991). Pregnancies after in-uitrofertilization and transfer of human blastocysts. Fertil. Steril. 55, 830-832.

36

DARREN K. GRIFFIN

Brambati, B., and Lucia, T. (1990). Prenatal diagnosis before the 8th gestational week. Proc. Int. Con$ Early Fetal Diagn. 5th, Prague. Buster, J. E., Busillo, M., Rodi, I. A., Cohen, S. W., Hamilton, M., Simon, 3. A., Thorneycroft, I. H., and Marshall, S. R. (1985). Biologic and morphological development of donated human ova recovered by non-surgical uterine lavage. Am. J . Obstet. Gynecol. 153,211-217.

Cremer, T., Landegent, J . , Bruckner, A., Scholl, H. P., Shardin, M., Hager, H. D., Devilee, P., Pearson, P., and van der Ploeg, M. (1986). Detection of chromosome aberrations in the human interphase nucleus by visualisation of specific target DNAs with radioactive and non-radioactive in-situ hybridisation techniques. Hum.Genet. 14, 346-352. Cremer, T., Lichter, P., Borden, J . , Ward, D. C., and Mannuelidis. L. (1988a). Detection of chromosome aberrations in metaphase and interphase tumour cells by in-situ hybridisation using chromosome specific library probes. Hum. Genet. 80, 235-246. Cremer, T., Tesin, D., Hopman, A. H. N., and Mannuelidis, L. (1988b). Rapid interphase and metaphase assesement of specific chromosomal changes in neuroectodermal tumor cells by in-situ hybridisation with chemically modified DNA probes. Exp. Cell Res. 176, 199-220.

Dauwerse, I. G., Wiegant, J., Raap, A. K., Breuning, M. H., and van Ommen, G. J. B. (1992). Multiple colors by fluorescence in-situ hybridization using ratio-labelled probes create a molecular karyotype. Hum. Mol. Genet. 1, 593-598. Dawson, K. J., Rutherford, A. J., Winston, N. J., Subak-Sharpe, R., and Winston, R. M. L. (1988). Human blastocyst transfer, is it a feasible proposition? Hum.Reprod. 145(Suppl), 44-45.

Delhanty, J. D. A., Ellis, J. R., and Rowley, P. T. (1961). Triploid cells in a human embryo. Lancet 2, 1286.

Delhanty, J. D. A., Griffin, D. K., Handyside, A. H., Harper, J., Atkinson, G. H. G., Pieters, M. H. E. C., and Winston, R. M. L. (1993). Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplantation sex determination by fluorescent in-situ hybridisation. Hum. Mol. Genet. 2, 1183-1 185. Devilee, P., Cremer, T.. Slagboom, P., Bakker, E., Scholl, H. P., Hager, H. D., Stevenson, A. F. G., Hager, .I. Cornelisse, , C. J., and Pearson, P. L. (1986). Two subsets of human alphoid repetitive DNA show distinct preferential localisation to the pencentric regions of chromosomes 13, 18 and 21. Cytogenet. Cell Genet. 41, 193-201. Devilee, P., Kievits, T., Waye, J. S., Pearson, P. L., and Willard, H. F. (1988). Chromosome specific alpha satellite DNA: Isolation and mapping of a polymorphic alphoid repeat from human chromosome 10. Genomics 3, 1-7. du Manoir, S., Speicher, M. R., Joos, S., Schrock, E., Popp, S., Dohner, H., Kovacs, G., Robert-Nicoud, M.,Lichter, P., and Cremer, T. (1993). Detection of complete and partial gains and losses by comparative genome hybridization. Hum. Genet. 90, 590-610. Fuscoe, J. C., Collins, C. C., Pinkel, D., and Gray, J. W. (1989). An efficient method for selecting unique sequence clones from DNA libraries and its application to fluorescent staining of human chromosome 21 using in-situ hybridisation. Genomics 5, 100-109. Gall, J. G., and Pardue, M. L. (1969). Formation of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. U.S.A.63, 378-383. Gardner, R. J. (1992). Towards a diagnostic test for Angleman syndrome. Final year undergraduate research project. Department of Genetics and Biometry, University College, London. Gardner, R. L., and Edwards, R. G. (1%8). Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts. Nature (London) 218, 346-348. Gosden, J. R., Hanratty, D., Starling, J., Fantes. J., Mitchell, A., and Porteous, D. (1991). Oligonucleotide-primedin situ DNA synthesis (PRINS): A method for chromosome map-

FLUORESCENT IN SITU HYBRIDIZATION FOR DIAGNOSING GENETIC DISEASE

37

ping, banding and investigation of sequence organisation. Cytogenet. Cell Genet. 57, 100-104.

Griffin, D. K. (1992). Fluorescent molecular cytogenetics: Preimplantation diagnosis, colorectal cancer and the mapping of chromosome 9q. Ph.D. Thesis, University College, London. Griffin, D. K.,Handyside, A. H., Penketh, R. J. A.. Winston, R. M. L., and Delhanty, J. D. A. (1991). Fluorescent in-situ hybridisation to interphase nuclei of human preimplantation embryos with X and Y chromosome specific probes. Hum. Reprod. 6 , 101105.

Griffin, D. K., Wilton, L. J., Handyside, A. H., Winston, R. M. L., and Delhanty, J. D. A. (1992). Dual fluorescent in-situ hybridisation for simultaneous detection of X and Y chromosome specific probes for the sexing of human preimplantation embryonic nuclei. Hum. Genet. 89, 18-22. Griffin, D. K., Wilton, L. J., Handyside, A. H., Atkinson, G. H. G., Winston, R. M. L., and Delhanty, J. D. A. (1993). Diagnosis of sex in preimplantation embryos by fluorescent in-situ hybridisation. Br. Med. J . 306, 1382. Griffin, D. K., Handyside, A. H., Harper, J., Wilton, L. J., Atkinson, G. H. G., Wells, D., Kontogianni, E. H., Tarin, J., Geber, S., Ao, A., Winston, R. M. L., and Delhanty, J. D. A. (1984). Results of a clinical trial involving preimplantation diagnosis of sex by dual fluorescent in-situ hybridization. In preparation. Grifo, J. A., Boyle, A., Fischer, E., Lavy, G., DeCherney, A. H., Ward, D. C., and Sanyal, M. K. (1990). Preembryo biopsy and analysis of blastomeres by in-situ hybridisation. A m . J . Obstet. Gynecol. 6 , 2013-2019. Guyot, B., Bezin, A., Sole, Y., Julien, C., Daffos, F., and Forrestier, R. (1988). Prenatal diagnosis with biotinylated chromosome specific probes. Prenaral Diagn. 8, 485-493. Handyside, A. H. (1991). Biospy of human cleavage stage embryos and sexing by DNA amplification. I n “Proceedings of the First Symposium on Preimplantation Genetics” (Y. Verlinksy and J. Strom, eds.), pp. 75-83. Plenum, New York. Handyside, A. H., and Delhanty, J. D. A. (1993). Cleavage stage biopsy of human embryos and diagnosis of X-linked recessive disease. I n “Preimplantation Diagnosis of Human Genetic Disease” (R. G. Edwards, ed.). Cambridge Univ. Press, Cambridge. Handyside, A. H., Kontogianni, E. H., Hardy, K., and Winston, R. M. L . (1990). Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature (London) 344, 768-770. Hardy, K., Martin, K. L., Leese, H. J., Winston, R. M. L., and Handyside, A. H. (1990). Human preimplantation development in-uitro is not adversely affected by biopsy at the 8-cell stage. Hum. Reprod. 5 , 708-714. Hassold, T., Chiu, D., and Yamane, J. A. (1984). Parental origin of autosomal trisomies. A m . Hum. Genet. 48, 129-144. Hopman, A. H. N., Wiegant, J., and R a p , A. K. (1986). Bi-color detection of two target DNAs by non-radioactive in-sifu hybridisation. Histochemistry 85, 1-4. Hopman, A. H. N., Ramaekers, F. C. S.. Raap, A. K.. Beck, J. L. M., Devilee, P., van der Ploeg, M., and Voojs, G. P. (1988). In-situ hybridisation as a tool to study numerical chromosome aberrations in solid bladder tumours. Histochemistry 89, 307-3 16. Jalech, A., Daumer, C., Lichter, P., Murken, J., Schroeder-Kurth, T., and Cremer, T. (1990). Chromosomal in-situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics. Hum. Genet. 85, 145-150. Jones, K. W., Singh, L., and Edwards, R. G. (1987). The use of probes for the Y chromosome in preimplantation embryo cells. Hum. Reprod. 2, 439-445. Joos, S., Falk, M. H., Lichter, P., Hakuska, F. G., Henglein, B., Lenoir, G. M.. and Bornkamm. G. W. (1992). Variable breakpoints in Burkitt lymphoma cells with chromo-

38

DARREN K. GRIFFIN

soma1 t(8;14) translocation separate c-myc and the IgH locus up to several hundred kb. Hum. Mol. Genet. 1, 625-632. Julien, C., Bazin, A., Guyot, B., Forrestier, F., and Daffos, F. (1986). Rapid prenatal diagnosis of Down’s Syndrome with in-situ hybridisation of fluorescent DNA probes. Lancet 2, 863-864. Kallionemi, A., Kallionemi, 0.-P., Sudar, D., Rutovitz, D., Gray, J. W., Waldman, F., and Pinkel, D. (1992). Comparative genome hybridization for molecular cytogenetic analysis of solid tumours. Science 258, 818-821. Khalfan, H., Abuknesha, R., Randweaver, M., Prid, R. G., and Robinson, D. (1986).Aminomethyl coumarin acetic acid: A new fluorescent labelling agent for proteins. Histochern. J. 18,497-499. Knight, J., Reeves, B. R., Kearney, L., Monaco, A. P., Lerach, H., and Cooper, C. S. (1992). Localisation of the synovial sarcoma t(X;l8)(pl1.2;qlI .2) breakpoint by in-situ hybridisation. Hum. Mol. Genet. 1, 633-637. Kozma. R., and Adinolfi, M. (1988). In-situ fluorescence hybridisation of Y translocations: Cytogenetic analysis using probes Y190-Y431. Clin. Genet. 33, 156-161. Landegent, J. E., Jansen, D., Wal, N., Baan, R. A., Hoeijmakers, J. H. J., and van der Ploeg, M. (1984). 2-acetylaminofluorene modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences. Exp. Cell Res. lS3, 61-72. Lichter, P., and Ward, D. C. (1990). Is non-isotopic in-situ hybridisation finally coming of age? Nature (London) 345, 93-95. Lichter. P., Cremer, T., Borden, J., Mannuelidis, L., and Ward, D. C. (1988). Delineation of individual human chromosomes in metaphase and interphase cells by in-situ suppression hybridisation using recombinant DNA libraries. Hum. Genet. 80, 224-234. Lichter, P., Jauch, A., Cremer T., and Ward, D. C. (1990a). Detection of Down Syndrome by in-situ hybridisation with chromosome specific DNA probes. In “Molecular Genetics of Chromosome 21 and Down Syndrome” (D. Patterson and C. J. Epstein, eds.). pp. 69-78. Wiley-Liss, New York. Lichter, P., Chang-Tang, C.-J., Call, K., Hermanson, G . , Evans, G. A., Housrnan, D., and Ward, D. C. (1990b). High resolution mapping of human chromosome 11 by in-situ hybridisation with cosmid clones. Science 247, 64-69. Lilford, R. J. (1991). The rise and fall of chorionic villus sampling. Br. Med. J . 303,936-937. Lupski. J. R., Montes de Oca-Luna, R., Slaugenhaupt, S., Penatao, I., Guzzeta, V.. Trask, B. J., Saucedo-Cardenas, O., Barker, D. F., Killian, J. K.. Garcia. C. A., Chakravarti. A., and Patel, P. I. (1991). DNA duplication associated with Charcot-Marie Tooth Disease Type I A . Cell (Cambridge, Mass.) 66, 219-232. Malcolm, S., Cowell, J. K., and Young, B. D. (1986). Specialist techniques in research and diagnostic clinical cytogenetics. I n “Human Cytogenetics: A Practical Approach” (D. Rooney and B. H. Czepulkowski, eds.), Chapter 7. IRL Press, Oxford. Meltzer, P. S.. Guan, X.-Y., Burgess, A.. and Trent, J. F. (1992). Rapid generation of region specific probes by chromosome microdissection and their application. Nut. Genet. 1, 24-28. Morton, N . E., Jacobs, P. A., Hassold, T.. and Wu, D. (1988). Maternal age in trisomy. Ann. Hum. Genet. 52,227-235. Nederlof, P. M., Robinson, D., Abuknesha, R., Wiegant, J., Hopman, A. H. N., Tanke, H. J., and Raap, A. K. (1989). Three colour fluorescence in-situ hybridisation for the simultaneous detection of multiple nucleic acid sequences. Cytometry 10. 20-27. Penketh, R. J. A., and McLaren, A. (1987). Prospects for prenatal diagnosis duringpreimplantation human development. Ballieres Clin. Obstet. Gynecol. 1, 747-764. Penketh, R.J. A., Delhanty, J. D. A., van den Bergh, J. A., Finklestone, E. M., Handyside, A. H.,Malcolm. S., and Winston, R. M. L. (1989). Rapid sexing of human embryos by

FLUORESCENT I N

siru HYBRIDIZATION

FOR DIAGNOSING GENETIC DISEASE

39

non-radioactive in-situ hybridisation, potential for preimplantation diagnosis of X-linked disorders. Prenatal Diagn. 9, 489-500. Pieters, M. H. E. C.. Geraedts, J. P. M.. Meyer. H., Dumoulin, J. C. M., Evers, J. L. H.. Jongbloed, R. J. E., Nederlof, P. M., amd van der Flier, S. (1990). Human gametes and zygotes studied by non-radioactive in-situ hybridisation. Cytogenet. Cell Genet. 53, 15- 19. Pinkel, D., Straume, T.. and Gray, J. (1986). Cytogenetic analysis using quantitative high sensitivity fluorescence hybridisation. Proc. Natl. Acad. Sci. U . S . A . 83, 2934-2938. Pinkel, D.. Landegent. J., Collins, C., Fuscoe, J., Segraves, R.. Lucas, J.. and Gray. J. (1988). Fluorescence in-situ hybridisation with human chromosome specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Nut/. Acad. Sci. U.S.A. 85,9138-9142. Raap, A. K.. Dirks, R. W., Jiwa, N. M., Nederlof, P. M., and van der Ploeg, M. (1990). In-situ hybridisation with hapten modified DNA probes. I n “Modern Pathology of AIDS and Other Retroviral Infections” (P.Raez. A. T. Haase, and J. C. Glucman. eds.), pp. 17-28. Karger, Basel. Reid, T.. Mahler. V., Vogt, P., Blonden, L., van Ommen, G. J. B.. Cremer, T., and Cremer, M. (1990). Direct carrier detection by in situ suppression hybridization with cosmid clones of the Duchenne/Becker muscular dystrophy locus. Hum. Genet. 85, 581-586. Reid. T.. Landes, G., Dackowski, W., Klinger, K., and Ward, D. C. (1992). Multicolour fluorescence in-situ hybridization for the simultaneous detection of probe sets for chromosomes 13, 18.21, X and Y in uncultured amniotic fluid cells. Hum. Mol. Genet. 1,307-313. Rietman, H. C., Moyzis, R. K., Meyne, J.. Burke, D. T., and Olson. M. V. (1989). Cloning human telomeric DNA fragments into Sacchoromyces cereuisiae using a yeast artificial chromosome vector. Proc. Natl. Acad. Sci. U.S.A. 84, 6240-6244. Rowley, J. D., Diaz, M. O., Espinosa, R., Ill, Patel, Y. D., van Melle, E., Ziemin, S.. Taillon-Miller. P.. Lichter, P., Evans, G. A., Kersey, J. H., Ward, D. C., Domer, P. H.. and le Beau, M. (1990). Mapping chromosome llq23 in human acute leukemia with biotinylated probes: Identification of I lq23 translocation breakpoints with a yeast artificial chromosome. Proc. Natl. Acud. Sci. U.S.A. 87, 9358-9361. Rudkin. G. T., and Stollar, B. D. (1977). High resolution detection of DNA-RNA hybrids in-situ by direct immunofluorescence. Nature (London) 265, 472-473. Smith, J.. Rebello. M.. Gray, C., Rooney, D., Leffler, F.. andColeman, D. (1990). Amniocentesis at 11-14 weeks gestation. Proc. In:. Symp. Early Fetal Diagn. 5th, Prague. Steptoe, P. C., and Edwards, R. G. (1978). Birth after implantation of a human embryo. Lancet 2, 366. Svennevik, E. C., and Hastings, R. J. (1993). A translocation ring chromosome 21 and fragment in a child with Down syndrome. Assoc. Clin. Cytogenet. Bull. Z7), 48. Tchen, P., Fuchs, R. P. P., Sage, E., and Leng. (1984). Chemical modified nucleic acids a s immunodetectable probes in hybridisation experiments. Proc. Natl. Acad. Sci. U . S . A . 81, 3466-3470. Telenius, H.. Pelmear. A. H., Tunnacliffe, A., Carter, N . P., Behmel, A., Ferguson-Smith, M.A.. Nordenskjold, M.. Pfrangner, R., and Ponder, B. A. J. (1992). Cytogeneticanalysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes. Chromosomes, Cancer 4, 257-263. Tkaachuk, D. C., Westbrook, C. A.. Andreelf, M.. Donlon,T. A.,Clearly, M. L.. Suryanarayan, K.. Homge, M., Redner, A., Gray, J., and Pinkel, D. (1990). Detection of bcr-abl fusion in chronic myelogenous leukemia by in-situ hybridisation. Science 250, 559-562. Trask, B. J. (1991). Fluorescence in-situ hybridisation applications in cytogenetics and gene mapping. Trends Genet. 7 , 149-154. Trask, B. J., van den Engh, G., Pinkel, D., Mullikin, J., Waldman, F., van Dekken, H., and Gray, J. (1988). Fluorescence in situ hybridisation to interphase nuclei in suspension

40

DARREN K. GRIFFIN

allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organisation. Hum. Genet. 78, 251-259. Trask, B. J., Pinkel, D., and van den Engh, G. (1990). The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs. Genomics 5, 710-717. Trask, B. J., Massa, H., Kenwrick, S., and Gitschier, J. (1991). Mapping of human chromosome Xq28 by two-colour fluorescence in-situ hybridisation of DNA sequences to interphase cell nuclei. Am. J. Hum. Genet. 48, 1-15. Van Prooijen-Knegt, A. C., van Hoek, J. F. M., Bauman, J. G. J., van Duijn, P., Wool, I. G., and van der Ploeg, M. (1982). In-situ hybridisation of DNA sequences in human metaphase chromosomes by an indirect fluorescent immunocytochemicalprocedure. Exp. Cell Res. 141, 397-407. Wada, M., Little, R. D., Abidi, F., Porta, G., Labella, T., Cooper, T., Della-Valle, G., Diloso, M., and Schlessinger, D. (1990). Human Xq24-q28: Approaches to mapping with yeast artificial chromosomes. Am. J. Hum. Genet. 46,95-106. West, J. D. (1990). Sexing the human conceptus by in-situ hybridisation. I n “In-sifu Hybridisation and the Study of Development and Differentiation” (N. Hams and D. Wilkinson, eds.). West, J. D., Gosden, J. R., Angell, R. R., Hastie, N. D., Thatcher, S. S., Glasier, A. F., and Baird, D. T. (1987). Sexing the human pre-embryo by in-situ hybridisation. Lancet 1, 1344-1347. West, J. D., Gosden, J. R., Angell, R. R., West, K. M., Glasier, A. F., Thatcher, S. S., and Baird, D. T. (1988). Sexing whole human pre-embryos by in-situ hybridisation to a Y chromosome specific DNA probe. Hum. Reprod. 3, 1010-1019. Wiegant, J., Reid, T.. Nederlof, P. M., van der Ploeg, M., Tanke, H. J., and Raap, A. K. (1991).In-situ hybridisation with fluoresceinated DNA. Nucleic Acids Res. 19,3237-3241. Wiegant, J., Kalle, W., Mullenders, L., Brookes, S., Hoovers, J. M. N., Dauwerse, J. G., van Ommen, G. J. B., and Raap, A. K. (1992). High resolution in-situ hybridisation using DNA halo preparations. Hum. Mol. Genet. 1, 587-591. Willard, H.F. (1990). Alpha and beta satellite sequences on chromosome 21: The possible role of centomere and chromosome structure in non-disjunction. I n “Molecular Genetics of Chromosome 21 and Down Syndrome” (D. Patterson and C. J. Epstein, eds.), pp. 39-52. Wiley-Liss, New York. Willard, H. F., and Waye, J. S. (1985). Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet. 3, 192-198. Winston, R. M. L. (1987). Why a ban on embryo research would be such a tragedy. Br. Med. J . 295, 1501-1502.

Isolation and Function of Human Dendritic Cells Lisa A. Williams, William Egner, and Derek N. J. Hart Haematology/Immunology Research Group, Christchurch Hospital, Christchurch, New Zealand

1. Introduction and Overview

A. General Understanding of the cellular costimulatory requirements for primary and secondary T-lymphocyte responses has improved markedly since the identification of potent primary antigen-presentingcells (APCs) in the mouse, collectively termed dendritic cells (DCs) (Steinman and Cohn, 1973). These cells were clearly distinct from typical monocyte-macrophages and B lymphocytes. A similar cell population was subsequently confirmed in human subjects (Hart et al., 1981) and DCs have now been delineated in practically every human organ (Daar et al., 1983; Hancock and Atkins, 1984; Hart et al., 1989; Steinman, 1991) with the exception of the brain and central cornea. These tissue-resident DCs (such as the Langerhans cell, LC) are thought to provide an extensive network of sentinel APCs capable of antigen capture, processing, and subsequent migration to local lymphoid tissue where efficient primary T-lymphocyte stimulation occurs. A DC may be defined primarily by the possession of a more potent ability to initiate primary T-lymphocyte proliferation than monocytes or B cells, and an immunophenotype which distinguishes it from these and other leukocyte populations. Other criteria which are considered to support the identification of DCs are either not restricted to DCs alone-such as high-density major histocompatibility complex (MHC) class I1 expression, the presence of dendritiform morphology in uitro, and lack of phagocytosis in vim-or are relative in comparison with other myeloid cells, for example, the presence of lower levels of cytoplasmic enzymes which may be found in a distinctive perinuclear distribution. The morphological identification of DCs is difficult, and the subdivision of DCs into three subtypes on this basis after exposure to metrizamide during preparation lnternafional Review 11fCylo/ogy. Vol. 153

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Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(Knight et al., 1986) is most controversial. Critical interpretation of all studies on DCs must be made with these points in mind, while recognizing that further work may identify DC subpopulations. It is also important to note that there are considerable differences between human DCs and those from other species with regard to such properties as adherence and the expression of certain leukocyte differentiation antigens. Recent work on human DCs in this and other laboratories has emphasized the close relationship of DCs with other myeloid cells, despite their distinctive morphological and functional properties. The precise relationships between the DCs found in different tissues and their relationship to other myeloid cells remain to be conclusively established. Identification of DC precursors in human bone marrow and understanding the regulation of tissue migration and differentiationis still in its infancy, but important advances are being made and we will outline these later. DCs are phenotypically quite different from, and should not be confused with, follicular dendritic cells (FDC), which are B-lymphocyte-associated cells that are involved in the maintenance of B-cell memory and are probably not bone marrow derived. To present antigen effectively, the DCs must be able to acquire intact antigen, process it into small immunogenic peptide fragments, present this fragment on the surface of an MHC class I1 molecule to a T lymphocyte (signal l), and then provide a number of appropriate “accessory” signals (signal 2) sufficient to costimulate proliferation of naive T cells. This multistep process is being dissected in uitro. Intimate membrane contact between DCs and T lymphocytes in cellular aggregates or clusters is essential. Rapid progress is also being made in defining the cellular attributes (cytokines and adhesion or costimulator molecules) responsible for their extremely potent functional activity in presentation of primary antigen to T lymphocytes (Steinman and Young, 1991) as discussed in Section V1,D. Work on DC in uitro has always been hampered by the low yield of purified cells obtained from a cell population present in very small numbers. It is hoped that this situation will be improved by new isolation methods developed by our laboratory and others. Improved yields will be especially important given the interest in the use of DCs as a potent in uiuo immunizing agent for presentation of vaccines against both malignant cell antigens and infective microorganisms. There is also considerable interest in the possible role of DCs in the dissemination of HIV infection within the human host and the role of impaired DC function in the immunological abnormalities of AIDS. An understanding of the signals which activate DCs may allow their already powerful APC properties to be enhanced or suppressed, both attractive propositions which may have many therapeutic applications.

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6. Dendritic Cells: An Overview

DCs were first identified in 1973 as a weakly and transiently adherent cell population in murine spleen (Steinman and Cohn, 1973) which displayed stellate morphology, a paucity of intracellular organelles, and poor phagocytic capabilities in comparison with monocyte/macrophages (Mo/Mp). Cells displaying similar characteristics were isolated from other rodent tissues (Steinman, 1991) and from other species (Hart and McKenzie, 1990). These DCs lacked most of the lineage-associated surface antigens of Mo/Mp or lymphocytes, but expressed MHC class I and class I1 molecules in high density. Human interstitial DCs were first described in 1981 (Hart et al., 1981) and were isolated from peripheral blood (Van Voorhis et al., 1982) using a modification of the method for purifying murine splenic DCs. Subtle differences between the properties of murine and human DCs, however, made alteration of the techniques desirable to maximize yields. DCs occur in a number of different tissues and can be subdivided on the basis of tissue location as shown in Fig. 1. Minor phenotypic and functional differences among these cell populations have led to the hypothesis that these cells may represent different stages of activation, maturation, or tissuespecific differentiation. Human DCs, in situ,are characterized by long cytoplasmic extensions. Most reports describe an irregularly shaped nucleus and a lack of phagolysosomes evident in classical macrophages. DCs also lack, or express lower levels of many myeloid enzymes such as nonspecific esterases (NSE) and acid phosphatase (AP) when assayed by various techniques. Immunophenotypic analysis typically reveals abundant expression of human leukocyte antigen (HLA) locus products and expression of the leukocyte common (CD45) antigen. Other important surface molecules described on DC include the adhesion or costimulator molecules leukocyte function-associated antigen-1 (LFA-1) (CD18/CD1la), LFA-3 (CD58), intercellular adhesion molecule-1 (ICAM-1) (CD54), ICAM-2; and more recently, ICAM-3 (Prickett et al., 1988; Thomas et al., 1993; Starling et al., 1994), CD40 (Hart and McKenzie, 1988), and B7/BB1 (Hart et al., 1993; Young et al., 1992). High levels of adhesion molecule expression account in part for the ability of these cells to establish strong initial contact with surroundingT lymphocytes. While DCs were initially thought to constitutively express MHC class I1 products, adhesion molecules, and important costimulator ligands in high density, the role of in uiuo and in uitro differentiation or activation in upregulating these surface antigens is only now being appreciated. Molecules associated with myeloid and lymphoid lineages are generally absent from DC, although weak CD4 staining has been observed (Landry

Q

Stemcell

@@ (DCprecursor)

Afferent lymph (veiled cells)

Lymph node (interdigitationgcells)

Interstitial DC (Liver, kidney, pancreas, etc)

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et al., 1988; Hart and McKenzie, 1988) both in situ and in uitro. Weak

CD13, CD14, and CD33 expression has also been observed on freshly isolated human blood DCs (Thomas et al., 1993; Egner et al., 1994). Murine DCs have been shown to express an aminopeptidase in common with macrophages which may represent a murine CD13 analog (Leenen et al., 1992). Mature DCs have historically been characterized by a lack of complement or Fc receptors (FcR) (Hart and McKenzie, 1988; Van Voorhis et al., 1982;Steinman and Cohn, 1973).However, some variations have been observed. LCs express low levels of C3bi receptors (CDI lb), FcRyII (CD32), and show functional complement and FcR activity (Teunissen et al., 1990; Romani et al., 1989; Cohen and Katz, 1992). CD32 (FcRyII); also, lesser amounts of CD64 ( FcRyI) have been detected on fresh blood DCs (Thomas et al., 1993). Low-level CD32 expression has certainly been detected on other in situ DCs using sensitive techniques (Prickett et al., 1988; Buckley et al., 1987). The presence of low-level FcR expression has been inferred from the ability of DCs to function in FcR-dependent assays such as CD3 mitogenesis. The most important identifying feature of DCs is their powerful activity in primary T-cell stimulation. The most commonly used assay for this activity is the allogenic mixed leukocyte reaction (MLR), although primary antigen-specific T-Iymphocyte proliferation and autologous MLR are also used. DCs have been shown to exhibit between 10 and 100 times more potent allostimulatory activity than other leukocytes (Van Voorhis et al., 1982). C. Assessment of Purity of Dendritic Cell Preparations

Purities of >90% have been claimed with many isolation methods. One of the more confusing and difficult aspects of the DC literature is the varied criteria used to assess the purity of the final DC population. 1. Morphology

DCs have traditionally been defined morphologically and indeed murine DCs were named because of their distinctive appearance in situ in the mouse (Steinman and Cohn, 1973). Lately, however, it has become clear that morphological identification of human DCs in uitro is extremely diffi-

FIG. 1 Dendritic cell migration and differentiation. Dendritic cells derived from different tissues are suggested to be related via a pathway such as this. Cytokines such as IL-I, TNFa,and GM-CSF appear to influence DC behavior at the various stages indicated.

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

cult. Dendriform morphology can be demonstrated for numerous cell types in uitro, including B lymphocytes (Corradi e l al., 1987; Hart and McKenzie, 1988).DC preparations obtained by plastic adherence and metrizamide contain contaminating low-density, strongly CD 14-positiveMo/Mp, which also display MHC class 11-positiveveiled morphology (Knight et al., 1986). The majority of these cells express low levels of the ectoenzyme 5'-nucleotidase, despite two-thirds clearly expressing Mo/Mp markers and active phagocytosis. 2. Dendritic CeU-Specific Monoclonal Antibodies

Easy identification of DCs has been hampered by the lack of DC-specific monoclonal antibodies (mAb). In rodents, NLDC 145 (Kraal et al., 1986), M342 (Agger et al., 1992),OX-62 (Brenan and Puklavec, 1992), and 33DI (Nussenzweig et al., 1982) have been used to identify certain subtypes of DCs, but they are not restricted to DCs alone and react with macrophage subpopulations (Kraal et al., 1986)and B cells (Agger et al., 1992). Some also appear to act as activation or differentiation antigens with variable expression. The new antihuman mAbs, CMRF44 (Hock et al., submitted) and HBl5a (Zhou et al., 1992) have shown promise and appear to label activated human blood DCs, but like RFDl (Poulter et al., 1986), are not restricted to DCs. The histiocytosis X (an LC malignancy)-specificmAb, Lagl, may prove an important cytoplasmic marker in the future (Ishikawa et al., 1992), but has yet to be evaluated.

3. Cytochemical Characteristics Cytochemical characteristics have been used to differentiate DCs from other myeloid cells. DCs are reported to lack many of the enzymes associated with classic Mo/Mps in certain staining techniques (e.g., myeloperoxidase, MPO) (Buckley et al., 1987; Steinman and Cohn, 1973; Hart and Fabre, 1981). Other reports demonstrate that they do in fact express 5'nucleotidase, dipeptidyl peptidase-I (DPPl) and cathepsin B activity in low levels (Knight et al., 1986; Thomas et al., 1993; Romani and Schuler, 1992). Other intracellular enzymes or lysosomal antigens (CD68) may be present in an unusual intracellular distribution (NSE, AP, CD68). Staining with AP, for example, displays perinuclear dot cytoplasmic staining in contrast to the diffuse appearance of Mo-Mps (Hart and McKenzie, 1988), while staining and CD68 reveals perinuclear distribution in DCs (Prickett et al., 1988; Betjes et al., 1991; Arkema et al., 1991). Significant phenotypic changes may result from certain isolation techniques. Loss of weak FcR expression (Thomas et al., 1993), or changes in antigen expression or morphology may further complicate the identification of DCs or DC precursors. Identification of DCs on the basis of single

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parameters is thus unreliable and the demonstration of potent primary APC activity should be paramount. Criteria such as distinctive enzyme cytochemistry, dendritiform morphology, and the presence of DCassociated antibodies such as CMRF44 or HBlSa may be regarded as confirmatory, but should not be used alone to define DCs. The interrelationship of lymphoid and nonlymphoid DCs (Fig. 1) has been inferred principally from the behavior of murine Langerhans cells in uitro and in uiuo (Rowden et al., 1992), and from the homing of DCs when injected into recipients. DCs move from the bone marrow (BM) via the blood to become resident in tissues. Following antigen acquisition, they migrate to the lymph nodes as veiled cells (Larsen et al., 1990b). Freshly isolated LCs are initially capable of acquiring antigen and processing it in uitro (or in uiuo during contact sensitization), but are weak stimulators of primary T-cell responses. They rapidly become incapable of antigen acquisition and processing, yet become powerful stimulators of primary antigen-specific responses. During this process many of the characteristic features of LCs are altered (Romani et al., 1989; Stossel et al., 1990), as discussed in Section 111,A,2. Antigen-bearing DCs derived from the tissues subsequently migrate centrally to the draining lymph node to become resident antigen-bearing interdigitating DCs (IDCs) capable of initiating a central T-lymphocyte response (Larsen et al., 1990b). DCs are thus part of a sophisticated mechanism for immunological surveillance. They are probably also capable of initiating and perpetuating a local immune response in situ in the periphery, which might be particularly important in certain autoimmune diseases. DCs are distinctive cells in both morphology and function, but their precise relationship to other myeloid cells is unclear, although it appears likely that these cell types share a common BM progenitor at some early stage. While it has been claimed that DCs derive from classic mature monocytes under certain circumstances, most evidence suggests that DCs are a distinctive cellular population with functional properties different from the majority of Mo/Mps, and diverge at an earlier stage of differentiation in the BM. In the next section we discuss the isolation and properties of individual DC types, and highlight the evidence for the interrelationships among them.

II. Blood and Bone Marrow Dendritic Cells A. Bone Marrow Dendritic Cells

DCs bear the CD45 (leukocyte common) antigen and as such are undoubtedly hemopoietic cells derived from the bone marrow. Observations on

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the repopulation of DCs in bone marrow transplant recipients also demonstrate this clearly in mice (Frelinger et al., 1979), rats (Hart et al., 1981), and humans (Katz et al., 1979; Murphy et al., 1985). DC precursors are also thought to circulate in peripheral blood pending entry into the tissues. The study of both cell types is therefore of great benefit in understanding DC interrelationships and ontogeny. Putative precursors of LC-type DCs have been identified in human BM (Reid et al., 1990; de Frassinette et al., 1988) and derived in uitro from human cord blood cells (Caux et al., 1992).This is controversial, however, since one of the principal identifying features of these LC precursors was CDla expression. This antigen appears to be widely inducible on both immature and mature myeloid cells. Furthermore, studies of fetal tissue have shown that in situ LCs are initially CDla negative (Foster et al., 1986). CDla is also virtually undetectable in human cord blood or adult blood (Gothelf et al., 1988) and is not found in fresh human blood DCs (Thomas et al., 1993) or BM DCs (Egner et al., 1993a). This is discussed in more detail in Section VI.

B. Blood DC and Dendritic Cell Precursors

1. Isolation Protocols for Blood Dendritic Cells Human peripheral blood DCs were first isolated by Van Voorhis et al. in 1982, using methods based on those for the isolation of murine splenic DCs. DCs are present in very low numbers in blood and are difficult to isolate in quantity. Isolation protocols have exploited the DC characteristics of nonadherence to plastic, lack of Fc receptors, low buoyant density and lack of myeloid, T- or B-lymphocyte-specificsurface markers. Various protocols have used different combinations of techniques to maximize yield, as illustrated in Fig. 2. The techniques for producing DCs that are based mainly on adherence depletion, density gradients, or removal of residual contaminating cells from other lineages are considered in the FIG. 2 Preparation of human blood DCs. The various methods which have been developed for (A) conventional DC preparation and (B) fresh DC isolation are shown here in diagrammatic form. AD, adherent cells; NAD, nonadherent cells; LD, low-density cells; HD, highdensity cells; non-T, non-T cells; C’, complement; LLME, L-leucyl L-leucine 0-methyl ester; Hu Ig, human immunoglobulin; RAM Ig, rabbit antimouse immunoglobulin. Superscript numbers indicate references used. I. Van Voorhis ef al., 1982; 2. Kuntz-Crow and Kunkel, 1982; 3. Van Voorhis er al., 1983; 4. Knight er al., 1986; 5. Vakkila el al., 1987; 6. Young and Steinman, 1988; 7. Brooks and Moore, 1988; 8. Freudenthal and Steinman, 1990; 9. Xu ef al., 1992; 10. Karhumaki er al., 1993; I I. Thomas ef al., 1993; 12. Markowicz and Engleman. 1990. 13. Egner er al., 1993c.

A PBMC

PBMC

l 6 h r culture

I

16hr culture

-1 carbonyl

gradimt

panning

I

Dc

(non-adherent)

1

50 following paragraphs. Other methods for obtaining less-activated DC preparations are also discussed. a. Adherence Depletion The original techniques for adherence depletion used differential adherence of peripheral blood mononuclear cells (PBMCs) to plastic surfaces (Van Voorhis et al., 1982; Kuntz-Crow and Kunkel, 1982). DCs were transiently adherent during short incubations (60 min), became nonadherent after overnight culture, and could be enriched by readherence cycles, which depleted firmly adherent Mo/Mps. Subsequent separation into low-density (DCs) and high-density fractions (B cells) over a bovine serum albumin (BSA) gradient and a further cycle of adherence enriched the DCs to 20-60%. Depletion of T and B cells using mAb and complement increased this to 70-78% (Van Voorhis et al., 1983). A similar method was followed by Kuntz-Crow and Kunkel(l982) using repeated adherence cycles, but the density gradient was omitted. Nonadherent Mo/Mps were later depleted from nonphagocytic DCs by ingestion of carbonyl iron and magnetic removal. Subsequent rosetting with immunoglobulin-sensitized ox red blood cells (RBC) removed FcRpositive Mo/Mp and B cells, the latter being further depleted by panning with antihuman immunoglobulin. Purities of greater than 90% were claimed on the basis of class I1 positivity and lack of staining for myeloid intracellular enzymes. All of these initial studies estimated the frequency of DCs in the initial PBMC population to be 0.1-1.0% and consistently monitored potent allostimulatory activity in the transiently adherent, lowdensity, FcR fraction. Human DC adherence differs significantly from that of murine DCs. In man, active allostimulatory cells can be detected in both nonadherent and adherent fractions after short (90-min) incubations, which are sufficient to separate murine DCs (Knight et al., 1986). Early work in our own laboratory led us to similar conclusions (Hart and Calder, 1993). Recent work with purified blood DCs also demonstrates considerable adherence in a subpopulation of human DCs (Thomas et al., 1993).These differences increase the difficulty of obtaining purified human DCs, particularly since transient weak adherence is one of the cornerstones of DC purification in the mouse model. Teflon cultureware (which is assumed to preclude adhesion of all but the most strongly adherent macrophages) has been used by some workers (Markowicz and Engleman, 1990; Xu et al., 1992) in the light of growing uncertainty whether adherence alters the phenotype or activity of DC populations. b. Density-Gradient Separations Most DCs, particularly those isolated from solid organs, are of low buoyant density. Careful use of sophisticated

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density gradients has enabled purification of DC from low-density Mo/Mps. These physical separations may isolate only a portion of the DCs and are heavily dependent on density changes induced by a period of 37°C incubation (Youngand Steinman, 1988;Xu et al., 1992;Markowicz and Engleman, 1990).DC precursors in particular may be of higher density than other DCs and density gradients may be less efficient in their isolation. Culture times have been increased to 36 hr to improve separation (Young and Steinman, 1988; Freudenthal and Steinman, 1990; Markowicz and Engleman, 1990;Xu et al., 1992),or omitted altogether to avoid phenotypic changes which may occur in uitro (Karhumaki et al., 1993; Egner et al., 1993b; Thomas et al., 1993). Various gradient media are used but the original BSA gradient (Van Voorhis et al., 1982) has generally been replaced by metrizamide (Knight et al., 1986; Brooks and Moore, 1988; Freudenthal and Steinman, 1990) or Percoll (Young and Steinman, 1988; Xu et al., 1992; Vakkila et al., 1987; Markowicz and Engleman, 1990). Markowicz and Engleman (1990) substituted a new and complex 4-step discontinuous Percoll gradient system and omitted plastic adherence. We have found a Nycodenz gradient to be useful as a final purification step for removing residual contaminating small lymphoid cells (Hart et al., 1993). The gradient media basically perform similar functions, although there is growing concern about the phenotypic or functional changes which cells may undergo when exposed to these materials. For example, Kabel et al. (1989) have suggested that interaction with metrizamide may alter monocytoid cells so that they resemble DCs. Density-gradient separation may be unreliable as a primary method of isolating human DCs for the reasons outlined earlier. Using metrizamide for example, enrichment varied between 2 and 78% (Knight et al., 1986), prompting attempts by some workers to remove density gradients from their protocols altogether. c. Removal of Contaminating Cells i. Panning Techniques Nonadherent surface immunoglobulin (sIg+) and FcR+ cells can be removed by panning (Brooks and Moore, 1988). This has been extensively used prior to, or following density-gradient separation (Freudenthal and Steinman, 1990, Vakkila et af., 1987; Xu et af., 1992; Young and Steinman, 1988). There is also a potential problem with anti-aFcR panning because some DCs express low amounts of FcRyII (CD32) and FcRyI (CD64). CD45RA panning as used by Freudenthal and Steinman (1990) is not widely applied and is based on the unconfirmed observation that DCs lack a particular CD45 epitope (Wood et al., 1991). ii. Depletion ofPhagocytes This is accomplished by the use of carbony1 iron (Kuntz-Crow and Kunkel, 1982; Vakkila et d., 1987) or L-

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

leucyl L-leucine 0-methyl ester (LLME) (Thomas et al., 1993). LLME is metabolized to a toxic by-product and has been used to deplete esterasepositive natural killer (NK) as well as monocytoid cells (Thomas et al., 1993). While the resulting DCs are clearly similar to those prepared by conventional techniques, the functional effects that LLME will have on DCs remain to be determined, as this compound is clearly toxic to LCs (Simon et al., 1992). iii. Zmmunophenotypic Cell Separations Negative selection using an mAb cocktail to remove unwanted cell populations has latterly become a mainstay of DC preparation protocols, although not all studies have demonstrated that these mAbs do not react with the stimulatory cells, a procedure which is in our view absolutely essential (Egner et al., 1993b; Thomas et al., 1993). Depletion is either accomplished by flow cytometry (Freudenthal and Steinman, 1990) where purity is high but yields often low, or by immunomagneticbead depletion (Karhumakiet al., 1993)where yields may be improved and purity approaches that of flow cytometrybased methods. Both of these methods are routinely used in our laboratory with considerable success (Egner et al., 1993b; Hart et al., 1993). d. Isolution ofFresh Dendritic Cells Conventional methods of DC preparation rely on the use of extended culture periods to allow density and adherence changes which enable DCs to be separated from other myeloid cells (Young and Steinman, 1988; Xu et al., 1992; Markowicz and Engleman, 1990). Phenotypic and functional changes may arise as a result, perhaps inducing DC activation. This may make examination of the unactivated DC or DC precursors impossible by these methods (Egner et al., 1993b; Thomas et al., 1993). We have developed an isolation protocol for human blood and BM DCs which enriches for minimally manipulated DCs (Egner et al., 1993a,b) (Figure 2). Processing the PBMC at 4°C throughout the isolation procedure, we deplete T lymphocytes by rosetting with sheep red blood cells (SRBCs), and negatively select by flow cytometry using a mixture of mAbs. Each mAb has been tested individually to confirm that the allostimulatory activity resides in the mAb-negative fraction. Prior depletion with immunomagnetic beads reduces sorting time and can improve yields. These populations of putative DCs are minimally activated, and provide the best example of a constitutive blood DC phenotype to date. Recent data (Hart et al., 1993; Thomas et al., 1993) confirm that these cells have phenotypic and morphologicaldifferences from their counterparts isolated by conventional methods involving prolonged culture. Fresh DCs can acquire an activated immunophenotype upon culture, including the expression of BBl/B7 (Hart et al., 1993; Thomas et al., 1993), or activation and interdigitating cell markers such as CMRF44 and HB15a (G. Starling,

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personal communication). Adhesion molecule expression may also be altered (Thomas et al., 1993; Young et al., 1992).

2. Properties of Human Blood Dendritic Cells Human blood DCs are assumed to be precursors of tissue DCs (Fig. 1) but direct evidence for this is lacking in man (see Section V for detailed discussion). They are clearly potent allostimulatory cells in contrast to other lineages (Van Voorhis et al., 1982; Kuntz-Crow and Kunkel, 1982; Thomas et al., 1993)and present specific antigen in primary systems (Hart and McKenzie, 1988;Thomas et al., 1993).DC-enriched populations were 10-100-fold more efficient than other cell populations and induced significant T-cell proliferation at stimulator/responder ratios as low as 1 : 100. Unlike murine DCs, human blood DCs appear to display low levels of CD13, CD14, and CD33 as detected by sensitive flow cytometry (Thomas et al., 1993; Egner et al., 1994). Detection of these antigens may be quite epitope dependent and some may not be detected by less sensitive techniques such as immunoperoxidase staining. Blood DCs undergo maturational activation changes similar to other DCs upon in uitro culture. As a result, they phenotypically converge with tissue DCs (Hart et al., 1993; Thomas et al., 1993) (Fig. 2). BBl/B7 expression on human blood DC is induced by cellular activation during isolation rather than being constitutively expressed (Hart et al., 1993). Murine organ allograft experiments demonstrate that some DCs can recirculate from nonlymphoid tissue to lymphoid organs via the bloodstream (Larsen et al., 1990a). It is therefore possible that human blood may also contain recirculating DC subsets. There is no direct evidence for this at present, however, and allograft DC behavior may not parallel that of autologous DCs. Human tonsil DCs appear to express lower levels of CD43 (leukosialin) than most Mo/Mps (Egner et al., 1993c), and blood allostimulatory cells show heterogeneous CD43 expression. However, CD43 detection may be epitope and mAb dependent; therefore any attempts to relate CD43 expression to potential DC subsets or activation or differentiation state requires further study.

111. Nonlymphoid and Interstitial Dendritic Cells

Highly MHC class I1 positive cells with morphological similarities to the DCs have been identified in most human tissues (Daar et al., 1983; Hancock and Atkins, 1984; Hart et d.,1989). These cells probably develop from precursor DCs in the blood and BM. DCs from different tissues

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

display many similarities and are collectively known as interstitial DCs. The most extensively studied tissue DC is the Langerhans cell and relatively few studies have been undertaken on DCs from other tissues. A. Langerhans Cells

Epidermal LCs, initially thought to be part of the nervous system, are an important part of the skin immune system (Katz et al., 1979; Frelinger et al., 1979).Skin transplantation and BM reconstitution experiments in mice have shown that these cells are not a static component of the skin, but derive from BM precursors and bear the surface MHC molecules of the donor animal (Katz et al., 1979; Frelinger et al., 1979). They also seem to be subject to neuroendocrine control and are intimately associated with nerve endings (Hosoi et al., 1993). LCs express high-density MHC class I1 antigens, CDla, and possess typical stellate morphology in situ (Davis et al., 1988; Takezaki et al., 1982; Rowden et af., 1977). CDla is an MHC class I-like molecule which is useful for the immunohistological detection of LCs. CDla may have an MHC class I-like function in human LC antigen presentation (Moulon et al., 1991). It is interesting that no other “mature” member of the DC family has been shown to express this molecule, with the possible exception of thymic DCs. Early mAb studies revealed an extensive suprabasal network of epidermal LCs, with branched cytoplasmic processes extending threedimensionally to make physical contact with large numbers of adjacent LCs. Another distinctive feature of LCs is the presence of intracytoplasmic Birbeck granules of uncertain function but which are believed to be associated with endocytosis and phagocytosis (Romani and Schuler, 1992). LCs have proved invaluable as a model to investigate the phagocytic and antigen-processingcapabilities of DCs at different stages of differentiation. This is discussed further in Section III,A,2. CDla-positive LCs are also found deeper in the dermis (Davis et al., 1988) but have not been extensively studied. They may represent LCs that are moving to the afferent lymphatic system. 1. Isolation of Human Langerhans Cells Isolation of DCs from solid organs is more difficult than from blood. LCs have provided an important model for the study of interstitial DCs, and also provide the bulk of the evidence for the migration and differentiation pathway of resident tissue DCs. Most of this work has been done in the

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mouse but an increasing number of human studies suggest that the two systems are very similar. To prepare LCs (Fig. 31, epidermal sheets are separated from the dermis of skin sections (usually operative specimens in humans) by short (<1 hr) 37°C incubations in trypsin (Cohen and Katz, 1992; Moms et al., 1992; Teunissen et al., 1990;Vedel et al., 1992; Romani et al., 1989). Epidermal cell (EC) suspensions are then produced by mechanical disruption of the tissue. Adherence for 18-24 hr in media supplemented with fetal calf serum (FCS) depletes some keratinocytes, and LCs are enriched in the nonadherent fraction (Cohen and Katz, 1992; Moms et al., 1992; Romani et al., 1989). Alternatively, fresh LCs are isolated via density-gradient centrifugation with the omission of a 37°C culture step (Teunissen et al., 1990). Final purification is generally performed by density-gradient separation using BSA columns (Romani et al., 1989) or Ficoll-metrizoate (Teunissen et al., 1990; Cohen and Katz, 1992) as for blood DCs. More recent methods have utilized positive selection for CDla or class IIpositive cells from fresh or cultured EC suspensions using mAb and immunomagnetic bead selection (Hanau et al., 1988; Morris et al., 1992) or flow cytometric sorting (Inaba et al., 1986);they have produced LC preparations enriched up to 95%. Other variations included mAb panning, including the use of plates coated with sensitized red blood cells (RBC) to bind FcR+ LC (Sting1 et al., 1977; Sauder et al., 1984; Morhenn et al., 1983; Vedel et al., 1992), which can then be recovered by RBC lysis. As with blood DCs, it has become apparent that there are phenotypic and functional differences between “fresh” (fLC) and “cultured” LCs (cLC). Methods have been evolved for the production of fLCs (Fig. 3) (Hanau et al., 1988; Morhenn et al., 1983). Most of these fLCs have, however, been exposed to a period of in uitro culture in the presence of trypsin or have been positively selected using mAb to HLA class I1 or CDla. Early work on LC phenotype and function often involved crude EC suspensions or LCs which were cultured during preparation. One concern regarding functional studies involving mixed epithelial cell preparations only partially enriched for DCs is the potential for activated keratinocytes to produce cytokines, express MHC class I1 products, and to act as facultative antigen-presenting cells (APC) in secondary systems, thereby complicating the interpretation of these studies. It is now clear that 37°C culture periods induce dramatic changes in LC form and function (Romani et al., 1989; Teunissen et al., 1990; Cohen and Katz, 1992), with cLCs, more closely resembling lymphoid DCs. The transition from fLC to cLC has been termed “maturation” by some workers (Romani and Schuler, 1992).

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2. Properties of Langerhans Cells

Freshly isolated human LCs express MHC class 11, CDla, and CD45 antigens, but MHC class I1 is much lower than their cultured counterparts. They also express low levels of complement (CDl 1b) and Fc (CD32) receptors, the myeloid antigens CD13 and CD33, the CD4 antigen, and the enzymes ATPase and NSE (Fig. 4, Table I) (Rowden et al., 1977; Teunissen et al., 1990; Davis et al., 1988; Romani and Schuler, 1992). LCs, are, however, MPO negative (Braathen and Thorsby, 1980). In situ these cells are markedly dendritiform but numerous veils are not visible on fLCs (Teunissenet al., 1990;Romani et al., 1989).Although not capable of obvious phagocytosis, fLCs can ingest and process antigens for presentation to T cells in the context of MHC class I1 molecules (Romani et al., 1989; Cohen and Katz, 1992). Antigen uptake is probably endocytic, and processing occurs via a chloroquine-sensitivepathway, indicating the importance of acidic endocytic vesicles (Cohen and Katz, 1992). Paradoxically, LCs were recognized early as potential antigen “scavengers” in the skin (Shelley and Juhlin, 1977)despite their apparently poor phagocytic capabilities. Short-term culture of LCs (Romani et al., 1989; Teunissen et al., 1990; Cohen and Katz, 1992) induces functional alterations. Veiled processes increase in both number and length. Acidic organelles, FcRs, and Birbeck granules decrease or disappear and ATPase and NSE staining is reduced. The characteristic LC marker CDla also disappears. Expression of stable membrane MHC class I1 products increases markedly in cLCs (Romani et al., 1989; Pure et al., 1990), as does CD25, CD40, and CD58, all of which are found on other DC subtypes (Prickett et al., 1988). The ability to cluster with T cells is an adhesion molecule-dependent phenomenon and appears in cLCs in parallel with increased expression of adhesion molecules (Tang and Udey, 1992b).Other potentially important costimulator molecules increase, including ICAM-1(CD54) (Romani et al., 1989; Vedel et al., 1992) and BBl/B7 (Larsen et al., 1992). Although fLCs are initially capable of acquiring endocytic antigens, cLCs lose this property. Comparison of fLCs with cLCs provides further parallels with other DC types. Fresh LCs are capable of antigen acquisition and processing, and secondary antigen presentation to antigen-specific T-

FIG. 3 Variations in tissue DC preparation. AD, adherent cells; NAD, nonadherent cells; LD, low-density cells; SRBC. sheep red blood cells; ox RBC, ox red blood cells. Superscript numbers represent references used. 1. Hanau et a/., 1988; 2. Romani e t a / . , 1989; 3. McKenzie et a/., 1989; 4. Landry et a / . , 1988; 5. Van Nieuwkerk et a / . , 1992; 6. Cohen and Katz, 1992; 7. Moms et a / . , 1992; 8. Vedel et a / . , 1992; 9. Barrett et a / . , 1993; 10. Nicod et a / . , 1987; 1 1 . Morhenn et a / . , 1983; 12. Brennan et al., 1987.

A

FRESH

ICAY-1+

CAM-1

CD14+/-

cMo+/-

CMRFU-

ICAM-1

ICAMJ++ m/EEl+/CMRFU+ HBlS. + CDla

MHCClASSII ++ CQ16CQS+ cQm+ ~81%CD1.

CD14 +/CD13+ . Cm3+ MHC CLASS II CD16-

36-hr CULTURE

ICAM-lY+ + E7/EE1 + CMo CDl6 CD32 CD64MHCCUSSll+++ CD1.

--++

++ +

-

-

+

++

ICAM-2+

IcAM-2+ CD13+ IcAu3++ cm3+

LFA-l++ LFA-3+ 07/#1-

-

16-hr CULTURE

LFA-1 +/LFAd +

MHC CLASS II + + CD16 CD32 +

CD14 +/CD13 +/CD33 + CD15 CDla + CDllb +

ATPase NSE + Bl-

-

CAM-1

++

MHC CLASS II

-

-

FRESH

+

+

-

48 72 hrs CULTURE

+++

ISOLATION AND FUNCTION OF HUMAN DENDRlTlC CELLS

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cell lines, but are poorly allostimulatory in the MLR (see Section V,c). I n vitro hapten and protein-specific T-cell responses can also be elicited

by human LCs (Braathen and Thorsby, 1980). After 72 hr of culture, however, cLCs are highly allostimulatory but are no longer able to acquire and process antigen efficiently(Romani et al., 1989). Similar changes have been shown to occur in mouse spleen DCs (Girolomoni et al., 1990). The properties of fLCs and cLCs define two separate functions in the generation of an immune response: (1) antigen acquisition and processing for assembly into immunogenic MHC class I1 molecules, sufficient to elicit secondary responses from sensitized T lymphocytes; and (2) antigen presentation to naive T cells in association with appropriate costimulatory signals sufficient to elicit a primary immune response. In the former respect, fLCs resemble the capabilities of B cells and Mo/Mps. No direct comparisons have been done to ascertain whether fLCs are more potent than these cells, although this appears to be so for other DC types. Murine fLCs are thus able to effectively process and present native myoglobin to T-cell clones (Aiba and Katz, 1991; Pure et al., 19901, but cLCs cannot. Only cLCs, in contrast, are powerful allogeneic stimulators in the MLR. This reflects a loss of antigen processing ability in cLCs, since they are perfectly good at acquiring “preprocessed” antigen fragments (Pure et a/., 1990). Loss of acidic organelles upon culture in murine LCs may represent the visible manifestation of this alteration (Stossel et al., 1990). A number of stimuli influence LC numbers and function. Antigen exposure causes an increase in CDla-positive cells in the skin (Friedmann et a[., 1993; Brand et al., 1992). These are presumed to be new immigrants from the blood but the mechanisms of accumulation are not understood. The immunologic responsiveness of the skin is decreased on exposure to ultraviolet B (UVB) irradiation, and this is attributed to the inhibition of LC function (Tang and Udey, 1992a). This may block upregulation of surface accessory and adhesion molecules such as ICAM-1, since cLCs (which already express high levels of costimulator molecules) are resistant to UVB treatment (Tang and Udey, 1992b). The detrimental effects of UVB irradiation may be mediated by release of local cis-urocanic acidinduced tumor necrosis factor-a (TNF-a) (Kurimoto and Streilein, 1992). Indeed, TNF-a-treated and antigen-bearing LCs have been shown to migrate from the skin via lymphaticsto draining lymph nodes of mice (Witmer and Steinman, 1984; Knight et al., 1985).TNF-a may also have inhibitory effects on LC primary APC function (Grabbe et al., 1992) while increasing secondary APC function (see Section VI,C,2).

FIG. 4 Summary of phenotypic changes induced in (A) fresh and cultured human blood DCs and (9) Langerhans cells by periods of in v i m culture.

--

CD36 CD40 cD43 CD44 cD45 CD54( ICAM-I) CD58 (LFA-3) CD64(FcRI) CD686 LFA-I(CD1 la) ICAM-2 ICAM-3 B7IBBI VCAM-I VLA-4 Sloo

- OR + / + I - OR + + - OR +

++ +

+ I - OR + + + OR + + + I - OR + I - OR +

++ + ++

- OR +/+/-

+

+

- OR + / -

ND ND ND

ND ND ND ND

ND ND ND ND

+/-OR++ ND -

ND ND

ND ND - OR ND ND

ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND

-(+)

ND ND

+

+

+

+

-

-

ND

+

+

+

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

-

+ -

++ + ++

++ + +

ND

ND ND

+

ND ND ND

ND ND ND ND

+

(+I ND ND ND

+

ND ND ND ND ND

+

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

A

This table presents data assembled from the references in Sections 11, 111, IV. It presents the range of DC phenotypes examined by a variety of different techniques, both in situ and in uitro, fresh and cultured. All DCs are negative for a variety of T, B, and NK lymphoid markers not shown here. - , Negative; + I - , variable or weak staining; + , positive; + + , strongly positive; + + + , very strongly positive; ( + ), subset positive only. Controversial results, some studies negative, others positive. Unusual perinuclear distribution in DCs. LCs only positive after neuraminidase treatment.

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

B. Other Interstitial Dendritic Cells

Interstitial DCs were first noted as strongly MHC class I1 positive cells with irregular membrane processes in rat tissue sections (Hart and Fabre, 1981;Hart et al., 1983). Morphological similarities and a distinctive immunophenotype, with minor variations, suggested a close relationship to LCs and lymphoid DCs. DCs have been identified in many human organs, including liver, kidney, heart, and other connective tissue (Hart et al., 1981, 1989; Daar et al., 1983; Hancock and Atkins, 1984; Nicod et al., 1987). These cells, like the LCs, are thought to form a sentinal network of antigen-receptive cells with a large membrane surface area and high-density MHC class I1 antigen expression combined with antigen presenting and processing capabilities. DCs can be distinguished from the classic tissue macrophages by surface marker staining, functional activity in uitro, or tisuse location in situ. Human interstitial DCs, however, do share some antigens in common with tissue macrophages (Table I) (Buckley et al., 1987), as do cultured blood DCs (Egner et al., 1992). 1. Liver Dendritic Cells Interstitial DCs have been identified in human liver (Prickett et al., 1988). These cells are clearly distinguished from Kupffer cells and classic tissue macrophages by the lack of a variety of widely distributed myeloid markers [such as CDllb, CD7, CD16, (FcR 111) and CD32 (FcR 11); Hart et al., 19891. They also fail to stain with many CD14 antibodies using in situ immunoperoxidase techniques. As with other DC populations, CD14 detection may be epitope dependent, since weak CD14 and CD32 staining can only be detected with certain antibodies (Prickett et al., 1988). Lowlevel expression may only be detected by sensitive flow cytometry, as appears to be the case with human blood DCs (Thomas et al., 1993). DCs in human liver congregate in the portal triads (Daar et al., 1983; Prickett et al., 1988; Hart et al., 1989) and this may be relevant in the rejection of liver transplants (see Section IX,B). Macrophages and Kupffer cells, in contrast, are located in the sinusoids (Daar et al., 1983; Hart et al., 1989). There are no published methods for the isolation of human liver DCs. 2. Mucosal Dendritic CeUs An interdigitating network of DCs has been described in the mucosa of the human oral cavity (Barrett et al., 1993), human intestinal lamina propria (Pavli et d., 1993), and respiratory tract (Sertl et d . , 1986; Holt et

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al., 1985; Nicod et al., 1987). A method for the isolation of oral mucosal DC has been described (Barrett et al., 1993)but these cells have not been extensively studied in uitro, although they appeared to be LC-like on the basis of CDla expression and the presence of Birbeck granules.

3. Lung Dendritic Cells DCs have also been purified from human lung parenchyma on the basis of weak adherence, lack of FcR, and strong MHC class I1 staining (Nicod et al., 1987; Holt et al., 1985).These cells are distinguished from alveolar macrophages by their surface staining and intracellular structure. Lung DCs could not be obtained by broncheolar lavage, unlike macrophages, but required collagenase digestion of the tissue to isolate them (Nicod et al., 1987), which is consistent with their interstitial location. Methods for isolating lung DCs are similar to other solid organ techniques (Fig. 3). Low-density IDCs are isolated after collagenase digestion ( + I - DNAase).

4. Synovial Fluid Dendritic Cells Highly MHC class I1 veiled cells have been isolated from the synovial fluid of patients with rheumatoid arthritis (Knight et al., 1989; Waalen et al., 1987; Stagg et al., 1991; Zvaifler et al., 1985; Harding and Knight, 1986)and are probably derived from interstitial DCs within the rheumatoid pannus. These cells readily form clusters with T lymphocytes. Synovial fluid DCs are purified with established techniques using differential adherence and density-gradient centrifugation following culture (Knight et al., 1989; Stagg et al., 1991), producing 30-50% pure preparations. Further purification by FcR panning and mAb labeling with complement lysis (Waalen et al., 1987; Zvaifler et al., 1985) increases purity to 80-85%. These morphological purities should be interpreted with caution given the frequent high percentage of monocytes in the starting cell populations. Variable staining with the HLA-DQ-associated mAb antibody, RFDl (Waalen et al., 1987; Stagg et al., 1991) may be a consequence of changes induced during isolation. Unlike most DCs but like the LCs, synovial fluid DCs have been reported to produce interleukin-1 (IL-1) spontaneously and in response to lipopolysaccharide (LPS) stimulation (Waalen et al., 1986;Bhardwaj et al., 1989).This may reflect an activated or more differentiated phenotype. Synovial fluid contains cells which are highly potent stimulators of the allo-MLR (Waalen et al., 1987; Stagg et al., 1991; Harding and Knight, 1986; Tsai et al., 1988). This activity was localized to the CD14- RFDl+ population containing synovial fluid DCs (Stagg et al., 1991). These DCs

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

are also active in an oxidative mitogenesis assay (Zvaifler et al., 1985; Waalen et al., 1986; Knight et al., 1989). The inflamed joint contains activated cells and numerous cytokines which will undoubtedly influence cellular form and function. The origin of synovial DCs and the relationship between synovial DCs and other DC types remains to be established. 5. Properties of Interstitial Dendritic Cells

Interstitial DCs have a phenotype and cytochemical profile similar to other human and murine DCs. Human interstitial DCs express CD45 and MHC class I1 loci products (HLA-DR, DP and DQ), and may display minor differences in surface antigen expression (e.g., CD4, CD8, and CD14) in different tissue locations (Table I). They do not stain strongly for NSE (Daar et al., 1983), are weakly adherent, and lack sufficient FcR to stain in situ with mAb to FcR I, 11, and I11 (Nicod et al., 1987). However, isolated interstitial DCs (fLCs)do express FcR I1 (CD32)weakly. CD4 and CD14 staining have been demonstrated with antibodies directed against certain epitopes on in situ IDCs (Hart and McKenzie, 1988; Buckley et al., 1987). Rat interstitial DCs are nonphagocytic (Hart et al., 1981) but similar studies are difficult in human subjects due to lack of available tissue. Most human studies have been performed by in situ phenotyping of tissue biopsies and only pulmonary interstitial DCs have been isolated for functional studies. Pulmonary DCs present antigen to T-cell hybridomas in both rats (Holt et al., 1985)and humans (Nicod et al., 1987). Lung interstitial DCs stimulate strong allo-MLR responses whereas alveolar macrophages do not; indeed, macrophages may inhibit the MLR (Holt et al., 1985).

IV. Lymphoid Dendritic Cells In situ phenotyping reveals the existence of a widespread family of dendritiform cells in the lymphoid system with some regional variation in phenotype (Buckley et al., 1987; Landry et al., 1989). Interdigitating cells are found in the T-dependent areas of lymphoid organs such as spleen and lymph nodes, and also in the thymic corticomedullary region (Agger et al., 1992). They have morphological similarities to other DC populations (Wood et al., 1985) but some subtle differences in immunophenotype (Table I) are apparent which may be tissue specific, and require further study. As with other DC types, isolation methods may induce phenotypic changes. Lymphoid DCs are thought to be derived from migratory DCs known as veiled cells (VC), which are found in afferent lymph, and themselves derive from interstitial DCs.

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A. Afferent Lymphatic Dendritic Cells [Veiled Cells)

Afferent lymph contains large mononuclear cells with dramatic veiled morphology (Steinman, 1991). Peripheral tissue DCs migrate in the lymph as VCs to the central lymph nodes following antigen acquisition in the periphery.

1. Isolation of VCs Limited access to human lymph makes extrapolations from animal studies necessary. DCs are isolated from thoracic duct or peripheral afferent lymph and purified using density gradients such as metrizamide (Bujdoso et al., 1989; Rhodes et al., 1989; MacPherson, 1989). Additional purification steps such as FcR rosetting, or immunomagnetic negative selection with an mAb cocktail (MacPherson, 1989) have also been used and preparations of up to 75-85% purity obtained. Human studies have largely been based on microscopic analysis of peripheral limb afferent lymph without purification (Spry et al., 1980).

2. Properties of Veiled Cells Like all members of the DC family, VCs possess high-density MHC class I1 expression and veiled morphology. Some VCs can be shown to be CDla positive (Hopkins et al., 1989), to possess residual Birbeck granules (Sokolowski et al., 1978), or to stain with NLDC-145 in rodents, which strongly suggests an LC origin in skin-derived lymph. LCs which have been induced to migrate on contact with antigen (Knight et al., 1985)become lymph-borne DCs. Peripheral antigen exposure results in a rapid increase in cellularity of afferent lymph (Knight et al., 1985; Bujdoso et al., 1989). Veiled cells can also take up antigen (Rhodes et al., 1989), spontaneously form clusters in vitro (Bjudoso et al., 1989; Galkowska and Olsewski, 1992), and can stimulate resting T-lymphocyte proliferation after in uiuo priming (Rhodes et al., 1989). VCs increase allostimulatory activity and change surface phenotype upon culture in the rat (MacPherson 1989), with decreased NSE staining. Phenotypic heterogeneity within VC populations has been described (Fossum, 1988), but whether this represents heterogeneity in veiled cells or impure preparations is uncertain.

6. Tonsil and Adenoidal Dendritic Cells Tonsils from routine tonsillectomies are the most readily available source of human lymphoid DCs from otherwise healthy donors. These cells proba-

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

bly represent interdigitating DCs and are similar to those cells isolated from human adenoids (Van Nieuwkerk et al., 1992). 1. Isolation of Tonsil Dendritic Cells The quality of human tonsil tissue is variable, but tonsillar DCs of high purity can be obtained (Brennan et al., 1987; Hart and McKenzie, 1988) for phenotypic (Hart and McKenzie, 1988; King and Katz, 1989; Prickett et al., 1992) and functional studies (McKenzie et al., 1989; Prickett et al., 1992). Protocols similar to those described previously for other tissues (Fig. 3) are used. Tonsil tissue is macerated to produce a cell suspension ( + / - collagenase), then subjected to overnight culture and densitygradient separation. Negative enrichment procedures dominate these protocols, which use SRBC rosette depletion of T cells, or FcR and human Ig panning (Brennan et af., 1987). for example. Final purification can be accomplished using negative selection with an mAb cocktail and fluorescence-activatedcell sorting (FACs) (Hart and McKenzie, 1988). The omission of enzymatic tissue digestion (Hart and McKenzie, 1988) precludes contamination with FDCs, which is a significant advantage of our method of tonsil DCs purification. It is also crucial to distinguish tonsil DCs from contaminating B cells, especially when using polymerase chain reaction (PCR) techniques where minor contamination may significantly affect results (Calder et al., 1992). 2. Properties of Tonsil Dendritic Cells

Tonsil DCs possess a characteristic surface antigen profile with strong expression of HLA locus products, low-level CD4, and a lack of CDl lb (Table I). Weak CD14 expression may be detected with some mAbs (Hart and McKenzie, 1988). In common with other isolated IDCs, tonsil DCs express high levels of adhesion and costimulator molecules such as CD39, CD40 (Hart and McKenzie, 1988), CD44, LFA-1, ICAM-1, LFA-3 (Prickett et al., 1992), and ICAM-3 (T. C. R. Prickett and G. C. Starling, unpublished data). Functional studies on tonsil DCs are limited. These cells are potent allostimulatory APCs in comparison with tonsil B cells (McKenzie et al., 1989; Hart and McKenzie, 1988), which are the major contaminating population. The functional importance of adhesion molecules in T-cell costimulation and clustering has been examined (Prickett et al., 1992; King and Katz, 1989). Tonsil DCs also express B7/BBl antigen (Hart et al., 1993) and CD40, both of which may be important in costimulatory function (see Section VI,D,l). CD45 may have a potential role in transducing regulatory signals to tonsil DCs (Prickett and Hart, 1990).Tonsil DCs

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have also been shown to stimulate T cells in an IL-1-independentmanner (McKenzie et al., 1989) (see Section VI). Tonsil DCs, especially in tonsils removed for recurrent inflammation, are exposed in situ to high levels of cytokines such as IFN-y and IL-1, which may influence their phenotype or function. Inhibition studies have shown that human tonsil DC stimulation is dependent on LFA-3 and to a lesser extent on ICAM-1, both in the oxidative mitogenesis assay (King and Katz, 1989), and in the allo-MLR (Prickett and Hart, 1990). ICAM1 appears less important in the allo-MLR, and a search for alternative LFA-1 ligands has revealed the presence of ICAM-2 on tonsil and blood DCs (Hart and Prickett, 1993),which also apparently contributed little to the DC-T-lymphocyte allo-MLR. C. Splenic Dendritic Cells

As with other human lymphoid tissues, studies on human spleen are limited. Buckley et al. (1987) documented the in situ phenotype of human splenic DC and found significant regional variation within the organ, similar to that seen in the mouse. In murine spleen there are two distinct populations of DCs. The first, situated in the T-cell area in a periarteriolar distribution, does not express the DC marker 33D1, but can be induced to express M342 by culture (Agger et al., 1992). The marginal zone DCs stain for 33D1, but fail to express M342 (Agger et al., 1992). Human periarteriolar DCs are similar to interdigitating DCs located in T-cell areas and lack lysozyme, NSE, FcR, and CDllb but express CD4 and CD14 weakly (Buckley et al., 1987) (Table I). It has been suggested that the more peripheral marginal zone DCs are preferentially isolated from mouse spleen by current methodology (Metlay et al., 1990),although in the human these cells appear to have a more macrophage-like phenotype (Buckley et al., 1987). D. Thymic Dendritic Cells

1. Isolation of Thymic Dendritic Cells Thymic DCs congregate at the corticomedullaryjunction (Pelletier et al., 1986). Although it is attractive to think that this DC subpopulation may be instrumental in the generation of T-lymphocyte deletion and tolerance in mice, there is conflicting evidence that other types of cells may also be effective. In mice, thymic DC have been shown to have an important role in deletion of class I and class 11 restricted early thymocytes (Jenkinson et al., 1992; Carlow et al., 1992).

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

Several investigators have isolated DC from human thymus (Landry et Fontaine et al., 1991) by tissue maceration ( + I - enzyme digestion) followed by density-gradient centrifugation over Percoll (Landry et a/., 1988; La Fontaine et al., 1991). The majority of protocols use selective immunolabeling followed by panning (Landry et al., 1988) or immunomagnetic bead selection (La Fontaine et al., 1991). Recently others have separated DCs using mAb labeling and FACS (La Fontaine et al., 1992) in line with current protocols for other DC types (Fig. 3).

a/., 1988; La

2. Properties of Thymic Dendritic Cells Purified thymic DCs show strong MHC class I1 expression together with CDla, CD45, and low levels of CD4 (Landry et a/., 1988) (Table I). Thymic DCs do not express complement (CDllb) or Fc (CD32) receptors. It is an attractive hypothesis that in the mouse CD8 may deliver a tolerogenic signal to responding T-lymphocyte MHC class I a3 domains (Sambhara and Miller, 1991), but unlike the murine version, in humans they are CD8 negative (Landry et a/., 1988). Typically, thymic DC possess long membrane processes, associate readily with surroundingthymocytes, and are nonphagocytic. No Birbeck granules were identified in thymic DC preparations. Recent studies suggest that murine thymic DCs may be of T-cell origin, although the link with in situ thymic DC is tenuous (Vremec et a/., 1992;Ardavin and Shortman, 1993).All cell lineages at an early precursor stage may have some properties in common with DCs, such as a lack of lineage-associated markers and MHC class I1 positivity. Lymphocytedeficient severe combined immune deficiency (SCID) mice have normal LC numbers, it will be interesting to see if this is also true for thymic DCs.

V. Dendritic Cell Ontogeny: Differentiation and Migration A. Bone Marrow Origin and Differentiation

Identification of late forms of DCs is problematic, but recognition of the precursors of tissue or blood DCs has been virtually impossible. Early precursors of well-defined lineages may bear scant resemblance to the form or function of their late progeny. DCs have been extremely difficult to isolate from murine marrow, although recently descriptions of a murine marrow DC precursor have been made based primarily on the allostimulatory activity of impure preparations and morphological assessment. These DCs probably arise from an MHC class I1 negative cell and could not be

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purified to homogeneity. They appeared to be intimately associated with other myeloid cells (Inaba et al., 1993) and respond to the presence of granulocyte-macrophage-colony-stimulatingfactor (GM-CSF) in culture (Inaba et al., 1992b). Human BM has proven equally difficult for DC studies. Several workers have claimed to isolate precursors from BM thought to be early LC type DCs because of their CDla and CD4 positivity (Reid et al., 1990, 1992; Goordyal and Isaacson, 1985; de Frassinette et al., 1988). LCs-DCs occur in intimate association with myeloid cells, and limiting dilution experiments with partially purified CD34+ cells suggested they may both originate from a single precursor. Morphology and the presence of CDla and APC activity by colonies of uncertain purity provided circumstantial evidence that these represent DCs (Reid et al., 1990). CDla, however, appears not to be restricted to DCs being also inducible on mature monocytes (Kasinrerk et al., 1993). Furthermore, CDla positivity in the DC family is restricted to the fLCs (the thymic DC being controversial), and is lost upon culture or antigen acquisition. Human LCs arise from a CDla negative cell (Rossi et al., 1992; Foster et al., 1986). CDla would not therefore be expected to delineate the progenitors of CDla-negative fresh blood DCs, themselves thought to be precursors of tissue DCs. Similar reservations apply to studies of human CD34+ cord blood leukocytes (Caux et al., 1992). Culture in the presence of exogenous TNF-a and GM-CSF undoubtedly increases CDla and MHC class I1 expression, and at least some surface molecules which are allostimulatory. While CDla-positive cells overall had less CD14 antigen than CDla negative cells, early monocytoid and granulocytic cells from the colony-formingunit-granulocyte macrophage (CFUGM) onward would have a similar phenotype, and cannot be differentiated from early DCs on these criteria. Indeed, it has been demonstrated that GM-CSF specifically induces the expression of CDl molecules on monocytes (Kasinrerk et al., 1993). We have clearly demonstrated that freshly isolated human marrow contains potent allostimulatory cells which have the extended phenotype of CDla negative DCs (Egner et al., 1993a). Culture of these cells confirms that the allostimulatory activity of cultured DCs resides in cells which are predominately CDla negative. Many CDla positive cells appear to be derived from committed monocytoid and granulocyte cells (Egner et al., 1994) and are not potent APCs.

6. Blood Dendritic Cell Precursors Several investigators have isolated precursors of tissue DCs from murine (Jnaba et al., 1992a) and human blood (Thomas et al., 1993). It is not

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

clear if all blood DCs are precursors of tissue DCs or whether the DCs isolated in longer term cultures originate from circulating myeloid precursors which copurify with human blood DCs. Most of these blood DCs may, however, only be precursors in the sense that their phenotype in uitro becomes more closely convergent to the tissue DCs upon culture. Whether this represents differentiation or activation is a matter which requires further study. It is probably more correct to describe most of these circulating DCs as representing an intermediate differentiation stage which is functionally active. Definitive studies demonstrating their ability to migrate to the tissues and become IDCs are awaited in humans, although preliminary experiments suggest this is the case in mice (Section V,C). Blood DCs lack some cardinal features of tissue DCs, namely obvious veils, although they may display dendritic morphology in adherent cell culture. It is assumed that the majority of blood DCs are migrating from the bone marrow to the tissues to become resident DCs. However, work on the recirculation of graft DCs in mice (Larsen et al., 1990a) suggests that some blood-borne DCs may be recirculating cells which have migrated from the tissues directly into circulation rather than via the lymph, and may therefore be destined for the spleen. Investigation of this possibility is still at a preliminary stage. Blood DCs (in common with most DC subtypes) lack CDla, which is not inducible by short-term culture in uitro (Thomas et al., 1993). However Gothelf et al. (1988) reported the presence of large numbers of CDlapositive cells in the peripheral blood of human bum patients. It remains unclear whether these cells represent CDla-positive DCs or result from the induction of CDla on monocytes, but they clearly express CD14. GM-CSF seems to play an important role in the survival, differentiation and activation of human blood DCs (see Section VI,D,2). However, fresh human blood DCs, like fresh murine LCs, initially appear to lack GMCSF receptors which are upregulated by other cytokines (V. Calder, personal communication). We are investigating whether human blood DCs are similarly upregulated.

C. DC Migration Similarities in the physical and functional characteristics of DCs from different tissue sources imply that these cell types are related. Evidence from animal models has established that in uiuo functional maturation of LCs does occur, causing increased allostimulatory potency and migration to the lymph nodes via dermal lymphatics (Hill et al., 1990; Austyn and Larsen, 1990; Cumberbatch et al., 1991). TNF-amay provide at least one

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stimulus for the migration of skin LCs to the lymph nodes (Cumberbatch and Kimber, 1992). Painting the skin of animals with ferritin results in accumulation of LC-bearing ferritin in the draining lymph nodes (Silberberg-Sinakin et al., 1976). Macatonia et al. (1986) applied fluorescein isothiocyanate (FITC) to murine skin and detected increased numbers of antigen-bearingdendritic cells in lymph. Antigen-bearing LC-containing Birbeck granules are also detected in lymph nodes following contact sensitization, indicating LC migration to central lymph nodes. Kripke et ai. (1990) isolated APC from the lymph nodes of skin-grafted mice painted with FITC. These were capable of specific T-cell stimulation and were of donor origin. Murine blood DCs will home to the spleen if injected intravenously or when they originate from an allograft (although, oddly, they appear to localize in the B-cell areas), whereas if they are injected intradermally they can migrate to the skin (Austyn and Larsen, 1990). SCID mouse models are very useful for examining the behavior of human LCs in an in uiuo model. Injected human LCs migrate to human skin grafts and become typical resident LCs (Rowden et al., 1992; Demarchez et al., 1992). LCs which have migrated as VCs have also been isolated from human peripheral lymphatics (Spry et al., 1980; Sokolowski et al., 1978). DC migration is also relevant to the induction of contact dermatitis. Murine LCs take up allergens and mediate sensitization in contact dermatitis (Friedmann et al., 1993; Brand et al., 1992),and transport the sensitizing antigen centrally via lymphatics to the T-dependent areas of draining lymph nodes (Fossum, 1988; Macatonia et al., 1986; Silberberg-Sinakin et al., 1976). The route of antigen administration may be important in determining whether a humoral or cell-mediated immune response is generated (Morikawa et al., 1992). D. Relationship t o Other Myeloid Lineages

Culture of human BM DCs has confirmed that potent allostimulatory cells reside in the BM cell fraction lacking most markers of late and committed lymphoid, granulocyte, or Mo/Mps differentiation (Egner et al., 1993a). DCs thus probably arise at the CFUG, level or earlier. In our view, current knowledge of DC ontogeny permits the conclusion that DCs are of myeloid origin, although it appears likely that they diverge from the monocyte/granulocyte lineage at an early stage (Egner et al., 1993a, 1994) (see Fig. 5 ) . The evidence that DCs arise from mature blood Mo/Mps (Peters et al., 1991; Rossi et al., 1992) is much less convincing. Some data suggest that exogenous cytokines and serum-free culture conditions can modify the

BLOOD

BONE MARROW

TISSUES

W33 +, W 1 5 +/-. CO14- COllb-,

w i a :.

CD14 + +

+'+STEMCELL

EARLV yIIlo(D PRECURSOn CD14 +I-

CFWY

\L QRNUICDl4

CD14 +I-

cmcuunm YYEWD PRECURson CD14 +/-

-

FIG. 5 Two possible differentiation pathways linking bone marrow DC precursors and interstitial DCs which fit with current knowledge of Dc ontogeny.

73 phenotype of Mo/Mps such that class I1 expression and other activation markers increase, while CD14 decreases (Peters et al., 1991). However, it is equally true that no comparisons of activity with isolated DCs have been made. Osteoporotic mice (Op/Op) mutants which lack macrophage (M)-CSF production and mature Mo/Mps show normal numbers of DCs and normal primary immune responses, which suggests early divergence in the mouse as well (Takahashi et al., 1993).

VI. Functional Properties of Human Dendritic Cells A. Antigen Acquisition and Processing by Dendritic Cells

In order to function as powerful antigen-presenting cells, DCs must be able to ( 1 ) acquire antigen, (2) process it into antigenic fragments, (3) present this processed fragment to T lymphocytes, and finally, (4) deliver costimulatory signals to the T cell. It has long been recognized that, unlike classic tissue macrophages or monocytes, the DC does not actively phagocytose particulate antigens. Van Voorhis et al. (1982) showed that human DCs failed to phagocytose zymosan or latex particles and showed no active pinocytosis of horse radish peroxidase. This has been confirmed by many workers (Knight et al., 1986; Hart and McKenzie, 1988) and some DC purification protocols have used macrophage phagocytosis of carbonyl iron particles to allow magnetic depletion (Kuntz-Crow and Kunkel, 1982; Vakkila et al., 1987). Nonetheless, DCs are capable of antigen uptake, which can be visualized in cytoplasmic vesicles (Fossum, 1988). We have observed the uptake of BSA by DCs during preparation, which is clearly visible during gel electrophoresis of cellular proteins (B. Hock, personal communication). The processed antigen is then rendered into small (6-9 amino acids for MHC class I and up to 20 for class 11) peptide fragments which bind to the antigen-receptive groove on MHC molecules and thereby become available for recognition by the T-cell receptor (Chain et al., 1986). This is shown in diagrammatic form in Fig. 6. Much of the evidence for antigen processing by DCs has been obtained from the murine model; fLCs are initially highly active in this respect but lose their processing capacity once cultured (see Section 111,A). Endocytosis of antigen leads to proteolysis in lysosomes. Transportation to MHC class 11-containingendosomes results in stable complexing of peptide and MHC molecules, which are then exported to the surface (Cohen and Katz, 1992).

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PRE-LYSOSOMAL

MHC CLASS II AND INV ASSEMBLY

INVARIANTCHAINDISPUCED

ENDOPLASMIC RETICULUM

FIG. 6 MHC clsss I1 antigen processing. Endocytosed whole antigen is degraded in the

presence of peptidases. Small peptides, which are normally prevented from gaining access to the MHC peptide-binding site by the invariant chain (INV), are acquired by the newly synthesized MHC molecule as the INV is displaced and are exported to the cell surface.

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The processing activity of fLCs may explain the initial abundance of acidic intracellular vacuoles and organelles which are subsequently lost upon culture (Stossel et al., 1990). LC processing is inhibited by chloroquine (Cohen and Katz, 1992), which suggests that antigen degradation occurs in acidic endosomes, as predicted for the class 11-mediatedpathway for presentation of exogenous antigens (Fig. 6). The ability to synthesize new MHC class I1 antigens is critical to fLC antigen-presenting capabilities whereas cLCs (in contrast to fLCs) exhibit stable surface MHC class I1 with minimal turnover (Simon et al., 1992). Both fLCs and cLCs can, however, induce T-cell proliferation following pulsing with antigen fragments which do not require processing and are passively acquired by MHC molecules at the cell surface (Cohen and Katz, 1992). The murine spleen DC has a poorly developed lysosomal system (Steinman, 1991) and it has been hypothesized that some processing events may occur on the plasma membrane. Hen egg lysozyme coupled to beads is presented efficiently by (murine) DCs without ingestion of the entire particle (de Bruijn et al., '1992); however, little is known about possible surface processing of antigen in the human DC system. While antigen processing and presentation in the context of MHC molecules is central to allostimulation,it is clearly not the only signal involved in T-cell stimulation. Other surface membrane-associated costimulatory molecules are discussed in Section VI,D, 1.

6. Dendritic Cells as Potent Stimulators of the Primary Immune Response in Vitro and in Vivo

The most striking feature of the DC is its remarkable ability to stimulate naive or primary T-lymphocyte proliferation. DCs can elicit both CD4+ and CD8+ T-lymphocyte responses (Young and Steinman, 1990), but primary stimulation of naive CD4+ T cells appears almost exclusively restricted to DCs. The most convenient in uitro system for measuring the primary immune response has been the allogeneic MLR, which has the advantage of reliability and simplicity. There are no data on whether DCs are particularly capable of stimulating individual T subsets in man. In uitro work with Thl and Th2 clones essentially represents secondary immune responses in preactivated T cells and may not be relevant to primary in uiuo responses.

1. Allogeneic MLR Stimulation in the allogeneic MLR (allo-MLR) was rapidly established as a feature of human DCs from peripheral blood (Van Voorhis et al., 1982;

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

Kuntz-Crow and Kunkel, 1982; Knight et al., 1986; Freudenthal and Steinman, 1990), tonsils (Hart and McKenzie, 1988), thymus (Landry et al., 1988; La Fontaine et al., 1991), lungs (Nicod et al., 1987), synovial fluid (Knight et al., 1989; de Vere Tyndall et al., 1983), afferent lymph (Spry et al., 1980; Sokolowski et al., 1978), and skin (Peguet-Navarro et al., 1992). Human DCs are the most effective stimulators of an allo-MLR on a cellfor-cell basis (Egner et al., 1993b). The allo-MLR involves proliferation of CD4+ and CD8+ cells and both naive and resting and memory T-cell subsets, and thus may not wholly represent an entirely primary response. Some stimulation of an allo-MLR in resting (class I1 negative) T lymphocytes can be accomplished by highly purified, strongly CD 14-positive human monocytes or to a lesser extent by CDlPpositive B cells (Egner et al., 1993b), but they are much less effective than DCs (Guidos et al., 1987). Primary stimulation of purified CD4+ T lymphocytes in most systems appears to be most exclusively restricted to DCs and not to the other cell types. Activation during the isolation procedures, as is the case with LCs, may increase the level of response but does not appear to affect the relative ability of each cell type. DCs also stimulate the allo-MLR in an IL-1independent manner (McKenzie et al., 1989). 2. Autologous MLR In certain circumstances, autologous T cells (derived from the same donor as the DC) can be stimulated to proliferate, although the response is minimal compared with allostimulation. DCs are again the most effective stimulators in this system, and Mos/Mps are inactive (Van Voorhis et al., 1982; Prickett and Hart, 1990; Kuntz-Crow and Kunkel, 1982). Whether the auto-MLR represents low levels of exogenous antigen presentation acquired in uitro is poorly understood. 3. Primary Antigen-Specific T-Lymphocyte Sensitization

One of the cardinal features of DC function is the ability to stimulate antigen-specificproliferation of resting T lymphocytes (primary response) to nominal antigen. This has been extensively documented in mice both in uiuo and in uitro, and in humans in uitro using a variety of antigens. Murine DC have been shown to deliver processed antigen to naive T cells in uiuo (Knight et al., 1985). Antigen-pulsed DCs injected into footpads of mice stimulate antigen-specific T lymphocytes to draining lymphoid tissue in a donor MHC-restricted fashion (Inaba et al., 1990). Other murine experiments have shown that antigen exposure at the skin

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results in accumulation of antigen-bearing DCs in the lymph node which are capable of specific T-cell stimulation in uitro (Macatonia et al., 1986; Fossum, 1988). Similar experiments are obviously impossible in humans but in uitro experiments are described in Section III,A,2. Priming and initial T-cell proliferation take place in clusters where a DC is surrounded by T lymphocytes (Prickett and Hart, 1990). It has been proposed that DCs are constitutively able to adhere to T cells in an antigen-independent manner via high levels of adhesion molecule expression (Inaba et al., 1989). In this way DCs are thought to survey T cells until an antigenspecific lymphocyte is encountered so that antigen presentation and Tcell activation can occur in stable proliferating clusters (Matsui et al., 1991).Once a T cell has been primed by DCs, it is susceptible to restimulation by a variety of different APCs (secondary response), but DCs are again the most effective. C. Dendritic Cells as Powerful Stimulators of Secondary Immune Responses and Mitogenic Assays

DCs also appear to be potent initiators of secondary immune responses by sensitized T cells. Most functional studies have been performed on cells which may have been activated by isolation procedures, but both murine and human fLCs are active in secondary systems prior to acquiring activity in primary systems (Romani et al., 1989; Cohen and Katz, 1992). The cosiimulatory requirements of T-cell lines, T-lymphocyte clones, TT hybrids, and polyclonal mitogenic responses (Con A, CD3 mitogenesis, oxidative mitogenesis) may be heterogeneous and substantially different from the requirements of resting and naive T lymphocytes. Various other leukocytes (B lymphocytes, Mo/Mps) and nonhemopoietic cells (endothelial cells, fibroblasts)may be active in these systems. It is not clear whether this represents a qualitative difference in costimulator repertoire, or a quantitative requirement for threshold costimulator signals. Study of the in uitro costimulator function of DCs will shed some light on this question in a way that nonphysiological costimulation experiments with plasticimmobilized mAbs or costimulator molecule-Ig constructs cannot. B-cell function may be more dependent on LFA-l/ICAM-l interaction than DCs (McKenzie et al., 1992). Monocytes also seem dependent on ICAM-1 (Neumayer et al., 1990), whereas DCs are not (Prickett et al., 1992). Human DCs are active accessory cells in CD3 mitogenesis. This is curious since DCs appeared to lack FcR expression, an essential attribute of the accessory cell in the CD3 system. The paradox is resolved by the recognition that blood DCs may express both CD64 (FcR I) and CD32 (FcR 11) in low levels and these are downregulated by culture in a manner

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analogous to down regulation in the LCs (Thomas et al., 1993). Some tissue DCs have also been reported to express low levels of CD32 (Davis et al., 1988; Larsen et al., 1990b) and CD64 (Reiger et al., 1992), but CD16 is absent from all DC populations studied, hence its use in mAb mixes for negatively purifying DCs. Human DCs are active in oxidative mitogenesis assays, which might be regarded as a type of secondary assay (Van Voorhis et al., 1982; King and Katz, 1989; Knight et al., 1986). Irradiated DC populations stimulate T cells modified with sodium periodate more efficiently than other cell types. Human DCs are also potent stimulators of responses to superantigens (Bhardwaj et al., 1992). D. Costimulatory Properties of Dendritic Cells

1. Costimulatory Adhesion Molecules T lymphocytes require costimulatory signals in addition to TCR-MHC interaction to initiate proliferation. In the absence of these costimulator signals, T-lymphocyte anergy is induced (Schwartz, 1990).Surface contact between DC and T lymphocytes is a prerequisite for T-cell activation. This DC-T lymphocyte contact is mediated by adhesion molecules. Adhesion not only allows antigen MHC complexes on the APCs to interact with the T-cell receptor/CD4 complex on the T lymphocyte (first signal) but also enables coordinated and regulated interaction of a number of other ligand and receptor surface molecules (second signals, accessory, or costimulatory signals), some of which may have dual adhesion and costimulator functions (Fig. 7). Direct evidence in human DCs for the involvement of additional contactmediated signals comes from the work of Peguet-Navarro et al. (1992), who analyzed accessory functions of fresh and cultured LCs by paraformaldehyde fixation prior to T-cell stimulation. The cLCs transduced the surface membrane-associated, contact-mediated signals which were absent from freshly prepared LCs. These molecules were induced by IFN-7, and are further evidence for a process of activation and maturation in the development of maximal APC activity. Similar fixationresistant contact-mediated costimulatory signals can be demonstrated in murine DCs. Several ligand-receptor interactions appear to be involved in APC-Tcell interaction. Some appear to be adhesion molecules with potential costimulator functions, such as ICAM-1/LFA- 1, ICAM-2/LFA-1,ICAM3/LFA-1 and CD2/LFA-3, very late antigen-4 (VLA-4), VCAM-1 etc. Others appear to have a predominantly costimulator function, such as CD28/B7-BBl. The relative importance of each in different systems is not fully understood.

DC

AUTOCRINET CELL STIMUIATION

CYTOKlNES INFLUENCINQ M:FUNCTION

FIG. 7 DUT-cell interaction: bidirectional signaling. The principal adhesion-costimulator molecule interactions between T cells and DCs are shown, as are cytokines and their receptors. The roles of signals 1 and 2 are well established in T-cell costimulation; the roles of potential signals 3 and 4 have yet to be investigated. p56 and p59 represent the src family of protein kinases associated with T-cell signaling.

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Dendritic cells form clusters with antigen-specific T cells (Scheeren et al., 1991; Prickett et af., 1992) through adhesion molecule interactions. Cluster formation also requires an intact cytoskeleton and protein kinase C activation (Scheeren et al., 1991),suggestingthe necessity for intracellular signaling by surface molecules. If clustering is disrupted, the T-cell response will be inhibited. Using mAb to block this initial contact, clustering of human tonsil DCs has been shown to be mediated by a bidirectional CDl laKD18-CD54 (LFA-1-ICAM-1) interaction and a unidirectional CD58(LFA-3)-CD2 interaction (Prickett et al., 1992). Scheeren et al. (1991) and Prickett et al. (1992) found that human blood DCs required a lesser involvement of LFA-3 or CD2 in early clustering, in contrast to their importance in stimulation. The oxidative mitogenesis response of autologous T cells was inhibited by a similar panel of mAb but anti-CDllc had no effect (King and Katz, 1989). We have demonstrated the importance of LFA-1-ICAM- 1 and LFA-3-CD2 interactions in the context of the DC-mediated allo-MLR (Prickett et al., 1992), and this is confirmed by others who showed inhibition by anti-LFA-1, LFA-3, ICAM-1, and HLA-DR antibodies as well as CDllc (King and Katz, 1989). ICAM-1 dianot, however, appear to be the most important LFA-1 ligand on the DC. Other selectin and p-integrin adhesion molecules are present on DCs (Egner et al., 1992) but their functional significance is unclear, although T cells can be costimulated through these ligands in conjunction with solid-phase anti-CD3 or anti-Tcell receptors (TCRs) mAb (eg, VLA-4, VCAM-1). CD45 is crucial for the control of T-lymphocyteproliferation stimulated by DCs. The intracellulardomains of CD45 have protein tyrosine phosphatase activity (Tonks et al., 1988) which interacts with tyrosine kinases such as p561Ck,but extracellular ligand binding by CD45 molecules may not be essential for their functioning (Desai et al., 1993). Inhibition of Tcell proliferation by CD45 mAb requires extensive cross-linking of the antigen using at least two mAbs at relatively high concentrations (Prickett and Hart,1990). Xu et af. (1992) and Young et al. (1992) demonstrated that single CD45 mAbs have no effect on T-cell proliferation. Identification of ligands for other CD45 isoforms on APCs will allow further investigation of control of CD45 function by APCs. Murine spleen DCs have been shown to express hyposialated surface MHC class I1 molecules (Boog et al., 1989). Neuraminidase treatment of murine nonresponder APC restored the T-cell response to alloantigen (Krieger et al., 1988). We have suggested that human DC surface antigens may also be generally hyposialated, further facilitating cell-cell contact by reducing the electrostatic repulsion between negatively charged cells. Reduced expression of the CD43 molecule on mature DCs may also be important in this regard (Egner et al., 1993~).Freshly isolated blood DCs initially express relatively low levels of adhesion molecules (Thomas et

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al., 1993), and these are upregulated along with the acquisition of other activated DC features by a process of in uitro culture, with or without exogenous cytokines (Thomas et al., 1993; Hart et al., 1993; Egner et al., 1994). Many of these molecules are shared by Mo/Mps and DCs (Egner et al., 1992). Some caution is advisable in extrapolating from in uitro models to the interactions of metabolically active cells. Mouse L-cells transfected with ICAM-1 and MHC class I1 only were shown to be potent stimulatory cells (Altmann et al., 1989). However, on closer examination, L cells were found to express the important costimulator molecule B7/BBl (La Salle et al., 1992),and this considerably changes the interpretation of the experiment. Signalstransduced through one molecule may also alter the response to ligation of another so that T-cell activation can be thought of as a signaling cascade. Recently much interest has focused on the activation antigen B7IBBl (Freedman et al., 1987). B7/BBl is the ligand for T-lymphocyte CD28, expressed on most CD4+ and a proportion of CD8+ T lymphocytes (Linsley et al., 1990). B7/BBI also interacts with CTLA-4 on T lymphocytes, which is upregulated in T-cell activation and has a higher affinity for B7/ BBl (Fig. 7). T-cell signaling via MHC-TCR interaction leads to anergy in the absence of CD28-mediated signals in some systems (Schwartz, 1990) and thus this interaction is thought to mediate a crucial costimulatory signal for T-cell proliferation (Linsley et al., 1991a).Transfectants expressing B7/BBl costimulate T cells in concert with antLCD3 mAb. Perhaps more important, CTLA-4 Ig or anti-B7/BBl mAbs have been shown to be immunosuppressive in uiuo (Linsley et al., 1991b). Recently B7/BBl has been detected on murine splenic DCs (Larsen et al., 1992) and human blood and tonsil DCs (Young et al., 1992; Hart et al., 1993), and is upregulated by GM-CSF and IFN-.)I (Hart et al., 1993). B7/BBl is absent from fresh blood DCs, however, and appears to act as an activation antigen in response to cytokine signaling, as it does on monocytes (Fig. 4). Recently, another potentially important costimulatory molecule, the heat-stable antigen (HSA), has been identified in mice where it labels a subset of NLDC-145-positive splenic and thymic DCs (Crowley et al., 1989) and LCs (Halliday et al., 1992). The anti-HSA mAb 20C9 (or Jlld) inhibits the costimulatory activity of murine accessory cells (Liu et al., 1992a),and complement lysis of J1 Id+ splenic DCs abolishes APC activity (Crowley et al., 1990). HSA has also been shown to cooperate with B7/ BB1 in CD4+ T-cell activation (Liu et al., 1992b). A second ligand for B7/BB1, CTLA-4 (Linsley et al., 1991b) can also inhibit CD4+ T-cell growth in concert with 20C9. Murine spleen DCs espress both B7/BB1 and low-level HSA. A costimulatory human HSA homolog has yet to be identified.

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2. Costimdatory Cytokines and Cytokine Receptors The expression of a number of cytokine receptors on DCs suggests the potential to influence DC function and antigen-presentingactivity. Experiments with murine DCs (Inaba et al., 1989) have shown that they do not activate T lymphocytes if they are not physically associated in the same chamber. This clearly excludes a major role for long-range activity of DCsecreted cytokines in the initial steps of DC-mediated T-cell costimulation, but local cytokine release may still be important. Cytokines from other sources may facilitate T-lymphocyte proliferation and expansion or affect the function of DCs (see Section VI,D,4). Although it was once thought to be an important MLR costimulatory signal; IL-1 is not essential to DC-mediated costimulation (McKenzie et al., 1989). Human tonsil and blood DCs stimulate both primary allogenic and antigen-specificT cells in the absence of IL-1 (McKenzie et al., 1989). IL-1 production has not been detected in human blood DCs (Vakkila et al., 1990). Murine DCs have also been shown to be unable to secrete detectable IL-1 (Koide et al., 1987). We have recently detected small amounts of IL-la and /3 mRNA in tonsil DCs, and IL-la only in blood DCs using PCR (Calder et al., 1992). The significance of this is uncertain, since IL-1 is clearly not vital for allostimulation. The situation may differ among members of the DC family. Murine LCs produce IL-1 more readily (Heufler et al., 1992), a situation paralleled by human synovial DCs (Waalen et al., 1986) and thymic DCs (La Fontaine et al., 1991), although the latter is controversial. Although IL-1 may not be essential, DCs may be IL-1 responsive, and the presence of IL-1 may enhance primary responses. TNF-a is an important candidate for a costimulatory cytokine. It has been shown that TNF-a enhances the proliferative response of CD4+ (Shalaby et al., 1988) and CD8+ T cells (Yokota et al., 1988) in both human and murine MLR. Antibodies to TNF-a will effectively block the MLR response (Shalaby et al., 1988; McKenzie et al., submitted) even when added late in the reaction. TNF-a appears to act directly on the Tcell population, enhancing proliferation. We have detected only low levels of TNF-a mRNA (Hart et al., 1994)in tonsil and blood DCs. This suggests that the TNF is produced principally by T cells and is acting as a critical autocnne T-cell growth factor involved in their expansion following the initial events of costimulation, and does not operate by enhancing APC function (Fig. 7) in functional studies on human blood DCs (Hart et al., 1994). Another candidate signal cytokine is IL-6. The effects of this molecule are pleiotropic, including T-cell activation and differentiation. Monocytes produce IL-6 (Bhardwaj et al., 1989). IL-6 mRNA was readily detected in tonsil DCs by PCR, but only small amounts were present in human

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blood DCs (V. Calder, unpublished). Vakkila et al. (1990) detected no IL-6 protein or mRNA in human blood DC by Northern blotting; however, this technique is somewhat less sensitive than PCR. This cytokine may be particularly relevant to DC-induced cytotoxic T-cell activation (Ming et al., 1992).

3. Dendritic Cell Interactions with Other Cells As discussed, DCs are capable of potent in uitro allostimulation in the absence of other cells, but this function can be altered by cytokines such as IL-1, TNF-a, and GM-CSF (Fig. 7). Any cell which provides these cytokines may therefore affect DC function. IL-1 is particularly intriguing since DCs possess IL-1 receptors, yet produce little IL-1 (Steinman and Young, 1991; Hart and Calder, 1993). We have noted from analysis of FACS fresh DC preparations that contaminating CDlCpositive cells enhance the alloresponse to DCs, although the strong CD14-positive Mo/ Mps alone were ineffective allostimulators (Egner et al., 1992). This phenomenon may be IL-1 based and may explain the remarkable potency of impure preparations such as those prepared using metrizamide (which may be two-thirds Mo/Mp). IL- 1 amplification of T-cell costimulation would make physiological sense, but may also contribute to the pathogenesis of inflammatory autoimmune diseases where interruption of this step (by IL-1Ra) may reduce not only proinflammatory effects, but the effectiveness of autoantigen presentation. It has been suggested that nonphagocytic DCs may require preprocessed antigen released from professional phagocytes into the extracellular fluids. However, two-chamber experiments in murine systems (Inaba et al., 1989) produced no evidence for this passive transfer of antigen. Finally, T lymphocytes themselves may influence DC activity both directly and via cytokines (Hart and Calder, 1993; Hart et al., 1993). The effect of this interaction on DC function may prove crucial to understanding costimulation and DC function (see Section VI,D,4). 4. Regulation of Dendritic Cell Activity DCs are capable of responding to various activation or differentiation signals, resulting in increased costimulatory ability. This is paralleled by increases in adhesion molecule, costimulator molecule, and MHC class I1 expression, as well as other functional and phenotypic changes. These are incompletely understood at present but IL-I , GM-CSF, and TNF-a have already been documented to have potentially important roles (Schuler and Romani, 1993). Other cytokines may also

a. CyrokineRegulution

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be involved, At present the data are incomplete but it seems likely that both upregulation and downregulation may occur. We have detected the presence of both TNF receptor (TNFR) I (p55) and I1 (p75) mRNA (p75 in larger amounts) in both human blood and tonsil DCs by PCR analysis (Calder et al., 1992). Immunostaining with mAb has shown TNFR type I1 expression on DCs of the thymus and spleen, and TNFR type I expression on dendritic reticulum cells of human lymph node germinal centers (Ryffel et al., 1991). These observations are important in view of the potential effects of TNF-a on LC function. It is curious that TNF-a in combination with GM-CSF apparently enhances the growth and differentiation of DC precursors from blood and bone marrow (see Section V,A). In the case of the LC, it appears that TNF-a can induce cell migration and decrease primary APC stimulatory activity, but it is reported to increase secondary responses. This may be important in uiuo in the induction of UV-mediated immunosupression (Kurimoto and Streilein, 1992). Both blood and tonsil-derived DCs express type I IL-1R only (Calder et al., 1992). Schuler and Romani (1993) have demonstrated IL-1 binding to human LCs. These results provide a basis for the observation that IL-1 can enhance DC-mediated MLR stimulation. IL-1 treatment upregulates the expression of GM-CSF receptors in mouse LCs (Schuler and Romani, 1993) and may increase the allostimulatory activity of murine DCs (Koide et al., 1987; Steinman, 1988). In the case of LC, this may depend on IFN-.)I pretreatment of fresh LCs which appear to be IL-1 resistant, in contrast to cultured LCs (Peguet-Navarro et al., 1992). This phenomenon has yet to be investigated in the human system. GM-CSF is traditionally associated with maturation of granulocyte and macrophage precursors but recent studies in mouse and man appear to show that it also induces DC development in blood and bone marrow cultures (see Section V,A and B). Markowicz and Engleman (1990) have shown that human blood DCs were maintained in culture by GM-CSF. The DC did not divide or proliferate, but did maintain allostimulatory activity. DCs cultured in the absence of GM-CSF lost this activity. This effect may depend on the degree of differentiationbecause the same effect is not apparent with rat lymphoid DCs (MacPherson, 1989)where survival but not enhanced stimulation has been reported. GM-CSF also causes upregulation of B7/BB1 on human blood DCs (Hart et al., 1993). We have been unable to detect GM-CSF receptor (CSFR) on freshly isolated blood DCs, which may mean that further differentiation takes place during isolation to allow expression of GM-CSFR. Mature tonsil DCs, in contrast, do express GM-CSFR (V. Calder, personal communication), as do mature LCs. It appears that GM-CSF has pleiotropic activating functions in DCs in addition to effects on survival. IL-2R has been detected

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on human thymic DCs and on a subpopulation of those in the splenic Tcell areas (Ryffel et af., 1991), but the functional effects of IL-2 on DCs have not been reported.

b. Regulation by Adhesion and Costimulator Molecules Only preliminary data are available at present but it appears likely that (as with B cells) DC function will be modified by intimate cell contact and signal transduction via ligand-receptor interactions. Costimulation is likely to be a cascade of sequential and coordinated signaling between stimulator (DC) and responder (T) cells. Interference with any aspect of the cascade may interfere with full activation (Fig. 7). T-cell costimulation can be thought of as a two-stage phenomenon with MHC and antigen-T-cell receptor interactions being central. An activated or “mature” DC charged with processed antigen and with high-level expression of adhesion molecules is likely to engage a T cell via adhesion molecules, and present antigen in the context of MHC class I1 (or present antigen-altered self class 11). Signals transduced by the MHC or adhesion molecules (signal 3) and perhaps cytokines interacting with DC receptors (signal 4),may lead to alteration in APC function, and T cell receptivity may occur, leading to costimulation of cell growth (Fig. 7). Signals 3 and 4 may be multiple. APC-T-cell interaction is thus likely to be bidirectional, and as has been shown for the B-cell-T-cell interaction, this may result in signaling to the APC as well (Nabavi et af., 1992). Either signal may alter the conformation, expression, and signaling function of other ligand-receptor interactions. Alterations in adhesion molecule repertoire may be important in directing recruitment and migration. LCs, for example, express VLA-P integrins, which may be important for extracellular matrix interaction (Staquet et al., 1992).

VII. Dendritic Cell Malignancies A. Histiocytosis X It is now accepted that the rare cases of class I histiocytosis are probably Langerhans cell malignancies (Langerhans cell histiocytosis) but proof of clonality is awaited. Cells in these lesions have Birbeck granules and stain positively for ATPase, S-100 protein, and CDla. CDla positivity has been gaining acceptance as a marker to differentiate class I histiocytosis from other malignant histiocytoses, some of which are of macrophage origin (Chu et al., 1987).

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86 6. Hodgkin’s Disease

Hodgkin’s disease (HD) may well include several malignant lymphomas with common histological and clinical features but different cellular ongins. The malignant cell type found in some cases of Hodgkin’s disease, the Reed-Stemberg cell, and its mononuclear variants, may derive from a DC (McKenzie et al., 1992). Many reports using HD tissue (Kennedy et al., 1989; Ellis et al., 1992; Kadin, 1982) or HD-derived cell lines (McKenzie et al., 1992, Hock and Hart, 1992) have described functional and phenotypic characteristics closely resembling those of resident lymph node DCs. The HD cell line L428 also possesses potent stimulatory activity in the MLR and high-density class I1 expression (Fisher et al., 1985). McKenzie et al. (1992) have shown that the HD cell lines L428, HDLM2, and KM-H2, like the DCs, display IL-1 independent APC activity. L428 also expresses B7/BB1, like tonsil DCs (Hart et al., 1993). Hodgkins cells also lack CD43 (leukosialin) expression both in in situ (Ellis et al., 1992) and in uitro cell lines (McKenzie et al., 1992). CD43 cannot be detected on human tonsil DCs by immunoperoxidase staining (Prickett et al., 1992) and this unusual feature may be further evidence of a common origin. Recently Hock and Hart (1992) have analyzed HD cell lines by SDS-PAGEand have identified some surface molecules which appear to be uniquely expressed on these cells.

VIII. Role of Dendritic Cells in Transplantation, and Infectious and Autoimmune Diseases

A. Transplantation

Passenger leukocytes, of which the most important will be the DCs, are important in the initiation of allograft rejection in rodent models (Hart and Fabre, 1981; McKenzie et al., 1984a,b). Reconstitution of donor lymphoid dendritic cells accelerates graft rejection (Lechler and Batchelor, 1982). Graft rejection and graft versus host disease (GVHD) in liver transplantation tend to locate in areas where DCs are present, such as the portal triads (Prickett et al., 1988), and in some rodent models early clustering of T lymphocytes with graft DCs is seen as suggestive of a peripheral sensitization process (Forbes et al., 1986). DCs also contribute to corneal graft rejection (Williams and Coster, 1989). DC depletion by anti-class I1 mAb infusion has been accomplished in renal allografts with some success (Brewer et al., 1989) although formal proof that removal of donor DC alone is effective requires the use of DC-restricted mAb.

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a7

Some workers have argued that the successful acceptance of a graft requires the establishment of stable chimerism (Starzl et al., 1992) with recipient DCs replacing the donor DC in grafts, and donor DCs establishing residence in the recipient. Although donor leukocytes (perhaps from stem cells) may be established in the recipient, there is no evidence as yet that they are derived from the graft DC. Similar replacement of LCs is reported in human skin (Demarche2 et al., 1992). DCs are clearly able to migrate from heart graft to spleen in murine transplantation models (Larsen et al., 1990a). The hypothesis that recipient DCs may acquire and process graft antigens in an indirect manner and thus stimulate rejection has yet to be tested, although it appears from our own work with blood DCs that indirect presentation to T cells by autologous APCs is insignificant in the in vitro allo-MLR (Egner et al., 1993b). Since freshly isolated human BM (unlike murine marrow) clearly contains active DC, bone marrow transplantation may trigger rejection despite intensive immunosuppressive conditioning of the recipient (Egner et al., 1993a). The presence of donor DC may initiate acute graft versus host disease. Inactivation of APC function without affecting stem cell viability and engraftment may be a useful strategy, if it is achievable. Cyclosporin A and steroids are suggested to affect DC function in addition to T cells (Dupuy et al., 1991) but the relative contributions of this mechanism to their efficacy have not been clearly defined. B. Autoimmunity and Rheumatic Diseases

The role of DCs in autoimmune disease is poorly understood at present. However, since DCs are the most potent APCs for the initiation of primary immune responses, they might be expected to play a major role in the presentation of self-antigens to autoreactive T cells. Failed central tolerance and clonal deletion in the thymus, or failed maintainance of peripheral tolerance, have been shown to be important in the generation of murine autoimmunity in several systems, but there is as yet no direct evidence to implicate DCs in these processes in mice or men. Interestingly, in a manner analogous to transplantation experiments, the administration of anti-MHC class I1 antibodies can abrogate the development of autoimmunity in mice made neonatally tolerant to semiallogeneic F1 spleen cells (Kramar et al., 1993), suggesting a central role for MHC class 11-positive APCs. Many aspects of autoimmune disease, however, can be explained by responder T-cell defects, for example, failure of apoptotic signaling and therefore failure to delete autoreactive T cells, or cross-reactive recognition of processed exogenous and self-peptides.

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Autoimmune diseases are probably as heterogeneous in pathogenesis as is immunodeficiency.There is one report of enhanced cluster formation by DCs from non-obese diabetic (NOD) mice (Clare-Salzer and Mullen, 1992). Autoimmunity can be both transferred and cured by bone marrow transplantation in different systems, suggesting a clonal defect. In some cases autoimmunity is only unmasked by infection with microorganisms. Experimental models of diabetes induction in transgenic mice (Ohashi et al., 1991) suggest that APC may trigger autoimmunity by initiating a local or systemic immune response to the infective agent. This may be particularly pertinent to diseases with a potential infectious trigger such as systemic lupus erythematosis (SLE), rheumatoid arthritis (RhA), and diabetes. DCs may also prolong and exacerbate local immune-based inflammatory reactions such as arthritis. Increased numbers of DC-like cells have been described (Zvaifler et al., 1985) in rheumatoid arthritis joints, and DCs may form an early component of the inflammatory infiltrate (Van DintherJanssen et al., 1990). Synovial fluid DCs have also been observed in patients with seronegative arthritis, and these were more stimulatory than PBMC in the MLR (Stagg et al., 1991). While direct experimental evidence is still lacking, it has been hypothesized that DC in the synovial fluids and membranes of inflamed joints may contribute to the induction and perpetuation of the inappropriate immune responses in these conditions (March, 1987) while joint phagocytes may be primarily responsible for the destruction of tissue. Putative DCs are present in chronic inflammatory infiltrates in increased numbers (Poulter and Janossy, 1985) and may be attracted to inflammatory sites. DCs in rheumatoid infiltrates are localized at the periphery.

C. Immunodeficiency

There have been several reports of deficient APC function in primary immunodeficiencies (Rozynska et al., 1989). The majority of patients with common variable hypogammaglobulinemia have normal APC function, however, and the evidence for a central role for DCs is unconvincing. Immunodeficiency with hyperimmunoglobulinof (IgM) has recently been shown to be a result of abnormal CD40/CD40-ligand interaction between B cells and T cells at the level of the CD40-ligand gp39 (Aruffo et al., 1993). These patients have some limited abnormalitiesof cell-mediated immunity, but investigationsof the role of this ligand-receptor interaction in these patients has just begun.

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D. Infectious Diseases

1. HIV There is considerable controversy about the role of DCs in HIV-mediated immunodeficiency. HIV-infected patients undergo profound loss of their ability to mount primary immune responses and this may be in part due to loss of DC function (Eales et al., 1988; Pimpinelli et al., 1991; Cooper et al., 1992). It has been shown that macrophage secondary APC function is unaffected in HIV-positive subjects, yet DC primary APC function is lost (Macatonia et al., 1992). Some workers (Cameron et al., 1992a,b) maintain that DCs do not support productive HIV infection and are not a reservoir of infection (although they may disseminate the virus). Others support the opposing view (Langhoff et al., 1988; Macatonia er al., 1990). The situation remains to be clarified and may reflect heterogeneity in the purity of the DC isolated by different methods. Macatonia et al. (1991) have also shown that DCs can initiate primary responses to HIV in normal human T cells, thus providing a rationale for an immunization strategy and the use of DCs to isolate protective immunogenic peptides. 2. Leprosy

Mycobacteria have been located within the cutaneous infiltrates of leprosy patients (Poulter and Janossy, 1985) and DCs constitute a minor population of the inflammatory infiltrate. It has been proposed that intracellular infection may cause reduced DC function and MHC class I1 expression, thus contributing to the characteristic anergy of the disease.

E. Allergy, Hypersensitivity, and Psoriasis LCs are intimately involved in contact allergen sensitization (Shelley and Juhlin, 1977; Macatonia et al., 1986) and appear to have an affinity for certain metals and haptens. LCs express IgE receptors and may be capable of initiating the late phases of type I hypersensitivity. They may also be involved in the immunological hyperreactivty of psoriasis (Demidem et al., 1991) and atopic dermatitis (Taylor et al., 1991). DCs appear in the early stages of the inflammatory infiltrate and appear to be located in dermal clusters in psoriasis, but appear in diffuse infiltrates in atopic dermatitis (Poulter and Janossy, 1985).

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IX. Clinical Applications A. Immunization

DCs have been described as “nature’s adjuvant” and are undoubtedly very effective in murine primary immunization schedules (Inaba et al., 1990). Autologous DCs may also be capable of initiating enhanced responses to tumor antigens, for example, mutant oncogenes such as H-ras in solid tumors, or fusion proteins such as bcr-abl in chronic myeloid leukemia, which may provide potential cancer immunotherapy. DCs can also be used to clone antigen-specific human T-cell lines with increased efficiency, and this may have potential applications for passive immunization against a variety of antigens (Langerhoff and Steinman, 1989; Pancholi et af., 1992).

6. Transplantation If DCs are central to graft rejection and GVHD responses, then specific removal may abrogate or ameliorate these problems. These studies await the availabilityof DC-specific antibodies and at present only circumstantial evidence has been obtained in man by removal of class 11-bearing cells (Brewer et al., 1989). Immunization strategies using autologous DCs may be useful in the future in order to reduce post-transplant infectious morbidity in the immunosuppressed. With a clearer understanding of the operation of DCs in T-cell costimulation, it may be possible to provide specific interruption of APC function using mAb or soluble cytokine receptors or antagonists as a novel immunosuppressive or toleration strategy. UV irradiation initiates immunosuppression.UV irradiation of blood products can abrogate MHC allosensitization, presumably also through an effect on DCs (Pamphilon et al., 1991). An understanding of the basis for this functional regulation may enable more precise intervention strategies to be designed.

X. Future Dendritic Cell Research and Applications

The induction of long-term tolerance to allo-MHC may be possible once full details of the mechanisms of DC costimulation of T-cell responses are known. It is already apparent that incomplete costimulation as a result of antigen presentation in the absence of appropriate additional signals

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91 can lead to T-lymphocyte anergy. This is likely to be the most fruitful application of DC knowledge in the future, and DCs are likely to be important for the dissection of the in vivo mechanisms of APC function. Clinical trials using peptides as vaccines are likely. Attempts to increase DC availability by generation of DC-specific antibodies, cell culture of DC precursors, and establishment of immortal DC cell lines will take precedence in the near future. References Agger, R., Witmer-Pack, M., Romani, N., Stossel, H., Swiggard, W. J., Metlay, J. P., Storozynsky, E., Freimuth, P., and Steinman, R. M. (1992). Two populations of splenic dendritic cells detected with M342, a new monoclonal to an intracellularantigen of interdigitating dendritic cells and some B lymphocytes. J . Leukocyte Biol. 52, 34-42. Aiba, S., and Katz, S. I. (1991). The ability of cultured Langerhans cells to process and present protein antigens is MHC-dependent. J . Imrnunol. 46, 2479-2487. Altmann, D. M., Hogg, N., Trowsdale, J., and Wilkinson, D. (1989). Cotransfection of ICAM-1 and HLA-DR reconstitutes human antigen-presenting cell function in mouse L cells. Nature (London) 338, 512-514. Ardavin, C., and Shortman, K. (1993).Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature (London)362,761-763. Arkema, J. M. S., Schadee-Eestermans, I. L., Beelen, R. H. J., and Hoefsmit, E. C. M. (1991). A combined method for both endogenous myeloperoxidase and acid phosphatase cytochemistry as well as immunoperoxidase surface labelling discriminatinghuman peripheral blood-derived dendritic cells and monocytes. Histochemistry 95, 573-578. Aruffo. A., Fanington, M., Hollenbaugh, D., Li, X., Milatovich, A., Nonoyama, S., Bajorath, J., Grosmaire, L. S., Stenkamp, R., Neubauer, M., Roberts, R. L., Noelle, R. J., Ledbetter, J. A., Franckell, U., and Ochs, H. D. (1993). The CMO-ligand is defective in activated T cells from patients with X-linked hyper-IgM. Cell (Cambridge. Mass.) 72, 291-300. Austyn, J. M., and Larsen, C. P. (1990). Migration patterns of dendritic leukocytes. Transplantation 49, 1-7. Barrett, A. W., Ross, D. A.. and Goodacre, J. A. (1993). Purified human oral mucosal Langerhans cells function as accessory cells in vitro. Clin. Exp. Imrnunol. 92, 158-163. Betjes, M. G. H., Haks, M. C., Tuk, C. W., and Beelen, H. J. (1991). Monoclonal antibody EBMl1 (anti-CD68) discriminates between dendritic cells and macrophages after shortterm culture. lmmunobiology 183, 79-87. Bhardwaj, N., Santhanam, U.,Lau, L. L., Tatter, S. B., Ghrayeb, J., Rivelis, M., Steinman, R. M., Sehgal, P. B., and May, L. T. (1989). IL-611FN-P2in synovial effusions of patients with rheumatoid arthritis and other arthritides. J . Immunol. 143,2153-2159. Bhardwaj, N., Friedman, S. M., Cole, B. C., and Nisanian, A. J. (1992). Dendritic cells are potent antigen-presenting cells for microbial superantigens. J . Exp. Med. 175,267-273. Boog, C. J. P., Neefies, J. J., Boes, J., Ploegh, H. L., and Melief, C. J. M.(1989). Specific immune responses restored by alteration in carbohydrate chains of surface molecules on antigen-presenting cells. Eur. J . Immunol. 19, 537-542. Braathen, L. R., and Thorsby, E. (1980). Studies on human epidermal Langerhans cells. Scand. J . Immunol. 11,401-408. Brand, C. U., Hunziker, T., and Braathen, L. R. (1992). Studies on human skin lymph

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containing Langerhans cells from sodium lauryl sulphate contact dermatitis. J. Invest. Dermatol. 99, 109s-110s. Brenan, M., and Puklavec, M. (1992). The MRC OX-62 antigen: A useful marker in the purification of rat veiled cells with the biochemical properties of an integrin. J . Exp. Med. 175, 1457-1465. Brennan, A., Katz, D. R., Nunn, J. D., Barker, S., Hewison, M., Fraher, L. J., and O’Riordan, J. L. H. (1987). Dendritic cells from human tissues express receptors for the immunoregulatory vitamin D, metabolite, dihydroxycholecalciferol. Immunology 61, 457-461. Brewer, Y., Bewick, M., Palmer, A., Severn, A., Welsh, K., and Taube, D. (1989). Prevention of renal allograft rejection by perfusion with anti-leukocyte monoclonal antibody is dependent on good uptake of antibody by interstitial DC. Transplant. Proc. 21,1772-1773. Brooks, C. F., and Moore, M. (1988). Differential MHC class I1 expression on human peripheral blood monocytes and dendritic cells. Immunology 63,303-31 1 . Buckley, P. J., Smith, M. R., Braverman, M. F., and Dickson, S. A. (1987). Human spleen contains phenotypic subsets of macrophages and dendritic cells that occupy discrete microanatomic locations. Am. J . Pathol. 128, 505-519. Bujdoso, R., Hopkins, J., Dutia, B. M., Young, P., and McConnell, I. (1989). Characterization of sheep afferent lymph dendritic cells and their role in antigen carriage. J . Exp. Med. 170, 1285-1302. Calder, V. L., Prickett, T. C. R., McKenzie, J. L., and Hart, D. N. J. (1992). Analysis of cytokine and cytokine receptor production of human dendritic cells. In “Proceedings of the 2nd International Symposium on Dendritic Cells in Fundamental and Clinical Immunology,” Vol. 329, 81-85. Plenum, New York (in press). Cameron, P. U., Freudenthal, P. S., Barker, J. M., Gezelter, S., Inaba, K., and Steinman, R. M. (1992a). Dendritic cells exposed to HIV-1 transmit a vigorous cytopathic infection to CD4’ T cells. Science 257, 383-386. Cameron, P. U . , Forsum, U., Teppler, H., Granelli-Piperno, A., and Steinman, R. M. (1992b). During HIV-I infection most blood dendritic cells are not productively infected and can induce allogeneicCD4+T cells clonal expansion. Clin. Exp. Immunol. 88,226-236. Carlow, D. A., van Oers, N. S. C., Teh, S.-J., and Teh, H . 4 . (1992). Deletion of antigenspecific immature thymocytes by dendritic cells requires LFA-I/ICAM interactions. J. Immunol. 148. 1595-1603. Caux, C. Dezutter-Dambuyant, C., Schmitt, D., and Banchereau, J. (1992). GM-CSF and TNF- cooperate in the generation of dendritic Langerhans cells. Nature (London) 360, 258-261. Chain, B. M., Kay, P. M., and Feldmann, M. (1986). The cellular pathway of antigen presentation: Biochemical and functional analysis of antigen processing in dendritic cells and macrophages. Immunology 58,271-276. Chu, T., D’Angio, G. J., Favara, B., Ladisch, S., Nesbit, M., and Pritchard, J. (1987). Histiocytosis syndromes in children. Lancet 1, 208-209. Clare-Salzler, M., and Mullen, Y. (1992). Marked dendritic cell-T cell cluster formation in the pancreatic lymph node of the non-obese diabetic mouse. Immunology 76,478-484. Cohen, P. J., and Katz, S. 1. (1992). Cultured human Langerhans cells process and present intact protein antigens. J . Invest. Dermatol. 99, 331-336. Cooper, D., Oberhelman, L.,Hamilton, T. A., Baadsgaard, O., Terhune, M., Levee, G., Anderson, T., and Koren, H. (1992). HIV exposure reduces immunization rates and promotes tolerance to epicutaneous antigens in humans: Relationship to dose, CDla-DR+ epidermal macrophage induction and Langerhans cell depletion. Proc. Natl. Acad. Sci. U.S.A. 89, 8497-8501. Corradi, P., Jelinek, D. F., Ramberg, J. E., and Lipsky, P. E. (1987). Development of a

ISOLATION AND FUNCTION OF HUMAN DENDRlTlC CELLS

93

cell with dendritic morphology from a precursor of B lymphocyte lineage. J. Immunol. 138,2075-2081. Crowley, M. T., Inaba, K., Witmer-Pack, M., and Steinman, R. M. (1989). The cell surface of mouse dendritic cells: FACS analyses of dendritic cells from difference tissues including thymus. Cell Immunol. 118, 108-125. Crowley. M. T., Inaba, K., and Steinman, R. M. (1990). Dendritic cells are the principal cells in mouse spleen bearing immunogenic fragments of foreign proteins. J . Exp. Med. 172,383-386. Cumberbatch, M., and Kimber, I. (1992). Dermal TNFa induces dendritic cell migration to draining lymph nodes, and possibly provides one stimulus for Langerhan's cell migration. Immunology 75, 257-263. Cumberbatch, M., Gould, S. J . , Peters, S. W., and Kimber, I. (1991). MHC class I1 expression by Langerhans' cells and lymph node dendritic cells: Possible evidence for maturation of Langerhans' cells following contact sensitization. Immunology 74, 414-419. Daar, S. A., Fuggle, S. V., Hart, D. N. J., Dalchau, R., Abdulaziz, Z., Fabre, J. W., Ting, A., and Moms, P. J. (1983). Demonstration and phenotypic characterization of HLADR-positive interstitial dendritic cells widely distributed in human connective tissues. Transplant. Proc. 1, 31 1-315. Davis, A. L., McKenzie, J. L., and Hart, D. N. J. (1988). HLA-DR-positive leukocyte subpopulations in human skin include dendritic cells, macrophages and CD7-netative T cells. Immunology 65, 573-581. de Bruijn, L. H., Nieland, J. D., Harding, J. V., and Melief, C. J. M. (1992). Processing and presentation of intact hen egg-white lysozyme by dendritic cells. Eur. J . Immunol. 22, 2347-2352. de Frassinette, A,, Schmitt, D., Dezutter-Dambuyant, C., Guyotat, D., Zabot, M. T., and Thivolet, J. (1988). Culture of putative Langerhans cell bone marrow precursors: Characterisation of their phenotype. Exp. Haematol. 16, 764-768. de Vere Tyndall, A., Knight, S. C., Edwards, A. J., and Clarke, J. B. (1983). Veiled (dendritic) cells in synovial fluid. Lancet 1, 472-473. Demarchez, M., Asselineau, D., Regnier, M., and Czernielewski, J. (1992). Migration of Langerhans cells into the epidermis of human skin grafted onto nude mice. J . Invest. Derrnatol. 99, 54s-5%. Demidem, A., Taylor, J. R., Grammer, S. F.. and Streilein, J. W. (1991). T-Lymphocyteactivating properties of epidermal antigen-presenting cells from normal and psoriatic skin: Evidence that psoriatic epidermal antigen-presentingcells resemble cultured normal Langerhans cells. J . Invest. Dermatol. 97, 454-460. Desai, D. M., Sap, J., Schlessinger, J., and Weiss, A. (1993). Ligand-mediated negative regulation of a chimeric trans-membrane receptor tyrosine phosphatase. Cell (Cambridge, Mass.) 73, 541-544. Dupuy, P., Bagot, M., Michel, L., Descourt, B., and Dubertret, L. (1991). Cyclosporin A inhibits the antigen-presenting functions of freshly isolated human Langerhans cells in vitro. J . Invest. Dermatol. 96, 408-413. Eales, L. J . , Farrant, J., Helbert, M., and Pinching, A. J. (1988). Peripheral blood dendritic cells in persons with AIDS and AIDS related complex: Loss of high intensity class I1 antigen expression and function. Clin. Exp. Immunol. 71,423-427. Egner, W., Prickett, T. C. R., and Hart, D. N. J. (1992). Adhesion molecule expression: A comparison of human blood dendritic cells, monocytes and macrophages. Transplant. Proc. 24, 2318. Egner, W., McKenzie, J. L., Smith, S. M., Beard, M. E. J., and Hart, D. N. J. (1993a). Identification of a potent stimulatory cel in human bone marrow: A putative differentiation stage of human blood dendritic cells. J . Immunol. 150, 3043-3052.

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Egner, W., Andreesen, R., and Hart, D. N. J. (1993b). Allostimulatory cells in fresh human blood: Heterogeneity in antigen presenting cell populations. Transplantation 56,945-950. Egner, W . , Hock, B. D., and Hart, D. N. J. (1993~).Dendritic cells have reduced cell surface membrane glycoproteins including CD43 determinants. Adu. Exp. Med. Biol. 329, 71-73. Egner, W., and Hart, D. N. J. (1994). The role of GM-CSF and TNFa in the generation of allostimulatory cells from cultured human bone marrow. J . Invest. (submitted). Ellis, P. A., Hart, D. N. J., Colls, B. M., Nimmo, J. C., MacDonald, J. E., and Angus, H. B. (1992). Hodgkins cells express a novel pattern of adhesion molecules. Clin. Exp. Immunol. 90, 117-123. Fisher, R. I., Cossman, J., Diehl, V., and Volkman, D. J. (1985). Antigen presentation by Hodgkins disease cells. J . Immunol. 135, 3568-3571. Forbes, R. D., Parfrey, N. A., Gromersall, M., Darden, A. G., and Guttmann, R. D. (1986). Dendritic cell-lymphoid cell aggregation and MHC antigen expression during rat cardiac allograft rejection. J. Exp. Med. 164, 1239-1258. Fossum, S. (1988). Lymph-borne dendritic leucocytes do not recirculate but enter the lymph node paracortex to become interdigitating cells. Scand. J . Immunol. 27, 97-105. Foster, C. A., Holbrook, K. A., and Farr, A. G. (1986). Ontogeny of Langerhans cells in human embryonic and fetal skin: Expression of HLA-DR and OKT-6 determinants. J . Invest. Dermatol. 86, 240-243. Freedman, A. S., Freeman, G . . Horowitz, J. C., Daley, J., and Nadler, L. M. (1987). B7, a B-cell restricted antigen that identifies preacrivated B cells. J . Immunol. 139,3260-3267. Frelinger, J. G., Hood, L., Hill, S., and Frelinger, J. A. (1979).Mouse epidermal la molecules have a bone marrow origin. Nature (London) 282, 321-324. Freudenthal, P. S.,and Steinman, R. M. (1990). The distinct surface of human blood dendritic cells, as observed after an improved isolation method. Proc. Natl. Acad. Sci. U.S.A. 87, 7698-7702. Friedmann, P. S., Strickland, I., Memon, A. A., and Johnson, P. M. (1993). Early time course of recruitment of immune surveillance in human skin after chemical provocation. Clin. Exp. Immunol. 91, 351-356. Galkowska, H., and Olsewski, W. L. (1992). Immune events in skin. I. spontaneous cluster formation of dendritic (veiled) cells and lymphocytes from skin lymph. Scand. J . Immunol. 35,727-134. Girolomoni, G., Simon, J. C., Bergstresser, P. R., and Cruz, P. D. (1990). Freshly isolated spleen dendritic cells and epidermal Langerhans cells undergo similar phenotypic and functional changes during short term culture. J . Immunol. 145, 2820-2826. Goordyal, P., and Isaacson, P. G. (1985). Immunocytochemicalcharacterisation of monocyte colonies of human bone marrow: A clue to the origin of Langerhans cells and interdigitating reticulum cells. J . Pathol. 146, 189-195. Gothelf. Y.,Hanau, D., Tsur, H., Sharon, N., Sahar, E., Cazenave, J.-P., and Gazit, E. (1988). T6 positive cells in the peripheral blood of burn patients: Are they Langerhans cells precursors? J . Invest. Dermatol. 90, 142-148. Grabbe, S., Bruvers, S., and Granstein, R. D. (1992). Effects of immunomodulatory cytokines on the presentation of tumor-associated antigens by epidermal langerhans cells. J . Invest. Dermatol. 99, 66s-68s. Guidos, C., Sinha, A. A., and Lee, K. C. (1987). Functional differences and complementation between dendritic cells and macrophages in T-cell activation. Immunology 61, 269-276. Halliday, G. M., Lucas, A. D., and Barnetson, R. St. C. (1992). Control of Langerhans cell density by a skin tumour-derived cytokine. Immunology 77,13-18. Hanau, D., Schmitt, D. A., Fabre, M. F., and Cazenave, J. P. (1988). A method for the rapid isolation of human epidermal Langerhans cells using immunomagneticmicrospheres. J . Invest. Dermatol. 91, 274-279.

ISOLATION AND FUNCTION OF HUMAN DENDRlTlC CELLS

95

Hancock, W. W.. and Atkins, R. C. (1984). Immunohistologic analysis of the cell surface antigens of human dendritic cells using monoclonal antibodies. Transplant. Proc. 16, 963-967. Harding, B., and Knight, S. C. (1986). The distribution of dendritic cells in the synovial fluids of patients with arthritis. Clin. Exp. Immunol. 63, 594-600. Hart, D. N. J., and Calder, V. C. (1993). Human dendritic cells: Function and cytokine production. lmmunopharmacology of macrophages and other antigen presenting cells. In “Handbook of Irnmunopharmacology” (C. A. F. M. Bruijnzeel-Loomen, ed.). Academic Press, San Diego, CA. Hart, D. N. J., and Fabre, J. W. (1981). Demonstration and characterization of Ia-positive dendritic cells in the interstitial connective tissues of rat heart and other tissues, but not brain. J. Exp. Me d. 153, 347-361. Hart, D. N. J., and McKenzie, J. L. (1988). Isolation and characterization of human tonsil dendritic cells. J. Exp. M ed . 168, 157-170. Hart. D. N. J., and McKenzie, J. L. (1990). Interstitial dendritic cells. Int. Re v . Immunol. 6, 127-138. Hart, D. N. J., and Prickett, T. C. R. (1993). ICAM-2 expression on human dendritic cells. Cell. Immunol. 148, 447-454. Hart, D. N. J., Fuggle, S. V., Williams, K. A., Fabre, J. W., Ting, A., and Moms, P. J. (1981). Localization of HLA-ABC and DR antigens in human kidney. Transplantation 31, 428-433. Hart, D. N. J.. Newton, M. R., Reece-Smith. H.. Fabre, J. W.. and Moms, P. J. (1983). Major histocompatibility complex antigens in the rat pancreas, isolated pancreatic islets, thyroid, and adrenal. Transplanrafion 36, 43 1-435. Hart, D. N. J.. Prickett, T. C. R., McKenzie, J. L., Martin, M. L., and Beard, M. E. J. (1989). Characterization of interstitial dendritic cells in human tissues. Transplant. Proc. 21,401-403. Hart, D. N. J., Starling, G. C., Calder, V. C., and Fernando, N. J. (1993). B7IBB-I is a leucocyte differentiation antigen on human dendritic cells induced by activation. Immunology 79, 616-620. Heufler. C.. Topar. G.. Koch, F., Trockenbacher, B., Kampgen, E., Romani, N., and Schuler, G. (1992). Cytokine gene expression in murine epidermal cell suspensions: Interleukin Ip and macrophage inflammatory protein lo! are selective expressed in Langerhans cells but are differentially regulated in culture. J. Exp. M e d . 176, 12211226. Hill, S., Edwards, A. J., Kimber, I., and Knight, S. C. (1990). Systemic migration ofdentritic cells during contact sensitization. Immunology 71, 277-281. Hock, B. D., Starling, G. C., Daniel, P. D., and Hart, D. N. J. (1994). Characterization of CMRF-44. a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood. including dendritic cells. (Submitted). Hock, B. D.. and Hart, D. N. J. (1992). Cellular protein profiles of the Hodgkins disease cell lines L428, KM-H2 and HDLM-2: a comparative study. Leuk. R e s . 16, 253-263. Krska, K.,andPlozza,T. (1985).Tcellactivationby Holt, P. G.,Degebrodt,A.,O’Leary.C., antigen-presenting cells from lung tissue digest: Suppression by endogenous macrophages. Clin. Exp. Immunol. 62, 586-593. Hopkins, J., Dutia, B. M., Bujdoso, R., and McConnell, I. (1989). In vivo modulation of CDI and MHC class I1 expression by sheep afferent lymph dendritic cells. J. Exp. M e d . 170, 1303-1318. Hosoi, J. Murphy, G. F., Egan, C. L., Lerner, E. A., Grabbe, S., Asahina, A., and Granstein, R. D. (1993). Regulation of Langerhans cell function by nerves containing calcitonin generegulated peptide. Nature (London) 363, 159- 163. Inaba, K., Schuler, G., Witmer, M. D., Valinsky, J., Atassi, B., and Steinman, R. M.

LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

96

(1986). Immunologic properties of purified epidermal Langerhans cells. J . Exp. Med. 164, 605-613. Inaba, K., Romani, N., and Steinman, R.M. (1989). An antigen-independent contact mechanism as an early step in T cell-proliferative responses to dendritic cells. J . Exp. Med. 170,527-542. Inaba, K., Metlay, J. P., Crowley, M. T., and Steinman, R. (1990). Dendritic cells pulsed

with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ. J. Exp. Med. 172,631-640. Inaba, K., Steinman, R. M., Witmer-Pack, M., Aya, H., Inaba, M., Sudo, T., Wolpe, S., and Schuler, G. (1992a). Identification of proliferating dendritic cell precursors in mouse blood. J. Exp. Med. 175, 1157-1 167. Inaba, K., Inaba, M., Romani, N., Hideki, A., Deguchi, M., Ikehara, S.,Muramatsu, S., and Steinman, R. M. (1992b). Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with GM-CSF. J. Exp. Med. 176, 1693-1702. Inaba, K., Inaba, M., Deguchi, M., Hagi, K., Yasumizu, R., Ikehara, S., Muramatsu, S., and Steinman, R. M. (1993). Granulocytes, macrophages and dendritic cells arise from a common MHC class 11-negative progenitor in mouse bone marrow. Proc. Natl. Acad. S C ~U.S.A. . 90,3038-3042.

Ishikawa, M., Matsuda, M., and Imai, Y. (1992). Monoclonal antibody against histiocytosis X cells. Histol. Histopathol. 7, 405-413. Jenkinson, E. J., Anderson, G . , and Owen, J. J. T. (1992). Studies on T cell maturation on defined thymic stromal cell populations in vitro. J. Exp. Med. 176, 845-853. Kabel, P. J., de Haan-Meulman, M., Voorbij, A. M., Kleingeld, M., Knol, E. F., and Drexhage, H. A. (1989). Accessory cells with a morphology and marker pattern of dendritic cells can be obtained from elutriator-purified blood monocyte fractions. An enhancing effect of metrizamide in this differentiation. Irnmunobiology 179, 395-41 I . Kadin, M. E. (1982). Possible origin of the Reed-Stemberg cell from an interdigitating reticulum cell. Cancer Treat. Rep. 66, 601-608. Karhumaki, E., Viljanen, M. E., Cottler-Fox, M., Ranki, A., Fox, C. H., and Krohn, K. J. E. (1993). An improved enrichment method for functionally competent, highly purified peripheral blood dendritic cells and its application to HIV-infected blood samples. Clin. Exp. Immunol. 91,482-488. Kasinrerk, W., Baumruker, T.,Majdic, O., Knapp, W., and Stockinger, H. (1993). CDI molecule expression on human monocytes induced by GM-CSF. J. Immunol. 150,579-584. Katz, S . I., Tamaki, K., and Sachs, D. H. (1979). Epidermal Langerhans cells are derived from cells originating in bone marrow. Nature (London) 282, 324-326. Kennedy, I. C. S., Hart, D. N. J., Colls, B. M., Nimmo, J. C., Willis, D. A., and Angus, H. B. (1989). Nodular sclerosing, mixed cellularity and lymphocyte-depleted variants of Hodgkin’s disease are probable dendritic cell malignancies. Clin. Exp. Immunol. 76, 324-33 1.

King, P. D., and Katz, D. R. (1989). Human tonsillar dendritic cell-inducedT cell responses: Analysis of molecular mechanisms using monoclonal antibodies. Eur. J . Immunol. 19, 581-587.

Knight, S. C., Krejci, J., Malkovsky, M., Colizzi, V., Gautum, A., and Asherson, G. L. (1985). The role of dendritic cells in the initiation of immune responses to contact sensitizers. Cell. Immunol. 94,427-434. Knight, S . C., Farrant, J., Bryant, A., Edwards, A. J., Burman, S., Lever, A,, Clarke, J., and Webster, A. D. B. (1986). Nonadherent, low density cells from human peripheral blood contain dendritic cells and monocytes, both with veiled morphology. Immunology 57,595-603.

Knight, S. C., Fryer, P., Griffiths, S., Harding, B., Dixey, J., and Mansell, B. (1989). Class

ISOLATION AND FUNCTION OF -HUMAN DENDRlTlC CELLS

97

11 antigens on dendritic cells from synovial fluids of patients with inflammatory arthritis. Clin. Exp. Immunol. 78, 19-25. Koide, S . L., Inaba, K., and Steinman, R. M. (1987). Interleukin 1 enhances T-dependent immune responses by amplifyingthe function of dendritic cells. J. Exp. Med. 165,515-530. Kraal, G . , Breel, M., Janse, M., and Bruin, G. (1986). Langerhans cells, veiled cells, and interdigitating cells in the mouse recognised by a monoclonal antibody. J . Exp. Med. 163, 981-997.

Kramar, G., Schurmans, S., Aguado, T., Izui, S . , Del Giudice, G., and Lambert, P.-H. (1993). Anti-Ia treatment prevents lupus-like autoimmune syndrome in mice neonatally tolerised to alloantigens. J. Autoimrnunol. 1, 27-37. Krieger, J., Jenis, D. M., Chesnut, R. W., and Grey, H. M. (1988). Studies on the capacity of intact cells and purified Ia from different B cell sources to function in antigen presentation to T cells. J . Immunol. 140, 388-394. Kripke, M. L., Munn, C. G . , Jeevan, A., Tang, J.-M., and Bucana, C. (1990). Evidence that cutaneous APC migrate to regional lymph nodes during contact sensitisation. J . Immunol. 145, 2833-2838. Kuntz-Crow, M., and Kunkel, H. G. (1982). Human dendritic cells: Major stimulators of autologous and allogenic MLR. Clin. Exp. Immunol. 49, 338-346. Kurimoto, I., and Streilein, J. W. (1992). Deleterious effects of cisurocanic acid and UVB radiation on Langerhans cells and on induction of contact hypersensitivity are mediated by TNFa. J. Invest. Dermatol. 99, 69s-70s. La Fontaine, M., Landry, D., Blanc-Brunat, N., Pelletier, M., and Montplaisir, S. (1991). IL-1 production by human dendritic cells: Studies on the interrelation with DC accessory function. Cell. Immunol. 135, 431-444. La Fontaine, M., Landry, D., and Montplaisir, S. (1992). The human thymic dendritic cell phenotype and its modification in culture. Cell. Immunol. 142, 238-251. Landry, D., La Fontaine, M., Cossette, M., Barthelemy, H., and Chartrand. C. (1988). Human thymic dendritic cells: Characterization, isolation and functional assays. Imrnunology 65, 135-142.

Landry, D., La Fontaine, M., Barthelemy, H., Paquette, N.. Chartrand, C., Pelletier, M., and Montplaisir, S. (1989). Human thymic dendritic cell-thymocyte association: Ultrastructural cell phenotype analysis. Eur. J . Immunol.19, 1855-1860. Langhoff, E., and Steinman, R. M. (1989). Clonal expansion of human T lymphocytes initiated by dendritic cells. J . Exp. Med. 169, 315-320. Langhoff, E., Terwilliger, E. F., Bos, H. J.. Kalland, K. H., Poznansky, M. C., Bacon, 0. M. L., and Hazeltine, W. A. (1988). Replication of HIV-I in primary dendritic cell cultures. Proc. Natl. Acad. Sci. U.S.A. 88, 7998-8002. Larsen, C. P., Moms, P. J., and Austyn, J. M. (1990a). Migration of dendritic leukocytes from cardiac allografts into host spleens. J . Exp. Med. 171, 307-314. Larsen, C. P., Steinman, R. M., Witmer-Pack, M., Hankins, D. F., Moms, P. J., and Austyn, J. M. (1990b). Migration and maturation of Langerhans cells in skin transplants. J . Exp. Med. 172, 1483-1493. Larsen, C. P., Ritchie, S. C., Pearson, T. C., Licsley, P. S., and Lowry, R. P. (1992). Functional expression of the costimulatory molecule, B7/BBI, on murine dendritic cell populations. J. Exp. Med. 176, 1215-1220. La Salle, J. M., Tolentino, P. J., Freeman, G. J., Nadler, L. M., and Hafler, D. A. (1992). Early signalling defects in human T cells anergised by T cell presentation of auto-antigen. J. Exp. Med. 176, 177-186. Lechler, R. I., and Batchelor, J. R. (1982). Restoration of immunogenicity to passengercell depleted kidney allografts by the addition of donor strain dendritic cells. J . Exp. Med. 155,31-41.

LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

98

Leenen, P. J. M., Melis, M., Kraal, G., Hoogeveen, A. T., and Van Ewijk, W. (1992). The monoclonal antibody ER-BMDMl recognises a macrophage and dendritic cell differentiation antigen with aminopeptidase activity. Eur. J. Immunol. 22, 1567-1572. Linsley, P. S., Clark, E. A., and Ledbetter, J. A. (1990). T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7IBB-I. Proc. Natl. Acad. Sci. U.S.A.87, 5031-5035. Linsley, P. S., Brady, W., Grosmaire, L., Aruffo, A., Damle, N. K., and Ledbetter, J. A. (1991a).Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 173, 721-730. Linsley, P. S., Brady, W.,Grosmaire, L., Damle, N. K., and Ledbetter, J. A. (1991b). CTLA-4 is a second receptor for the B cell activation antigen B7. J. Exp. Med. 174, 561-569. Liu, Y.,Jones, B., Aruffo, A., Sullivan, K. M., Linsley, P. S., and Janeway, C. A., Jr. (1992a). Heat stable antigen is a costimulatory molecule for T cell growth. J. Exp. Med. 175,437-445. Liu, Y ., Jones, B., Brady, W., Janeway, C. A., Jr., and Linsley, P. S. (1992b).Costimulation of murine CD4 T cell growth: Cooperation between B7 and heat-stable antigen. Eur. J. Immunol. 22,2855-2859. Macatonia, S. E., Edwards, A. J., and Knight, S. C. (1986). Dendritic cells and the inhibition of contact sensitivity to fluorescein isothiocyanate. Immunology 59, 509-5 14. Macatonia, S. E., Lau, R., Patterson, S., Pinching, A. J., and Knight, S. C. (1990).Dendritic cell infection, depletion and dysfunction in HIV-infected individuals. Immunology 71, 38-45. Macatonia, S. E., Patterson, S., and Knight, S. C. (1991). Primary proliferative and cytotoxic T-cell responses to HIV induced in vitro by human dendritic cells. Immunology 74, 399-406.

Macatonia, S. E., Gompels, M., Pinching, A. J., Patterson, S., and Knight, S. C. (1992). Antigen presentation by macrophages but not by dendritic cells in HIV infection. Immunology 75, 576-581. MacPherson, G. (1989). Properties of lymph-borne (veiled) cells in culture I. Modulation of phenotype, survival and function: Partial dependence on GM-CSF. Immunology 68, 102-107. March, L. M. (1987). Dendritic cells in the pathogenesis of rheumatoid arthritis. Rheumatol. I n t . 7, 93-100. Markowicz, S., and Engleman, E. G. (1990). GM-CSF promotes differentiation and survival of human peripheral blood dendritic cells. J. Clin. Inuest. 85,955-961. Matsui, K., Boniface, J. J., Reay, P. A., Schild, H., Fazekas de St. Groth, B., and Davis, M. M. (1991). Low affinity interactions of peptide-MHC complexes with T cell receptors. Science 254, 1788-1791. McKenzie, J. L., Beard, M. E. J., and Hart, D. N. J. (1984a). Depletion of donor kidney dendritic cells prolongs graft survival. Transplant. Proc. 16,948-957. McKenzie, J. L., Beard, M. E. J.,andHart, D. N. J. (1984b). Theeffectofdonor pretreatment on interstitial dendritic cell content and rat cardiac allograft survival. Transplantation 38, 371-376. McKenzie. J. L., Prickett, T. C. R., and Hart, D. N. J. (1989). Human dendritic cells stimulate allogeneic T cells in the absence of IL-1. Immunology 67, 290-297. McKenzie, J. L., Egner, W., Calder, V. L., and Hart, D. N. J. (1992). Hodgkin’s disease cell lines: A model for IL-1-independent accessory cell function. Immunology 77,345-353. McKenzie, J. L., Calder, V. L.. Starling, G. C., and Hart, D. N. J. (1994). Role of TNFa in dendritic cell mediated primary MLR. (Submitted).

ISOLATION AND FUNCTION OF HUMAN DENDRlTlC CELLS

99

Metlay, J. P., Witmer-Pack, M. D., Agger, R., Crowley, M. T., Lawless, D., and Steinman, R. M. (1990). The distinct leucocyte integrins of mouse spleen DC as identified with new hamster mAb. J . Exp. Med. 171, 1753-1771. Ming, J. E., Steinman, R. M., and Granelli-Piperno, A. (1992). IL-6enhances the generation of cytotoxic T lymphocytes in the allogeneic MLR. Clin. Exp. Immunol. 89, 148-153. Morhenn, V. B., Wood, G. S., Engleman, E. G., and Oseroff, A. R. (1983). Selective enrichment of human epidermal cell subpopulations using monoclonal antibodies. J . Invest. Dermatol. 81, 127s-131s. Morikawa, Y., Furotani, M., Kuribayashi, K., Matsura, N., and Kakudo, K. (1992). The role of APC in the regulation of delayed-type hypersensitivity. 1. Spleen dendritic cells. Immunology 77,81-87. Moms, J., Alaibac. M., Jia, M.-H., and Chu, T. (1992). Purification of functional active epidermal Langerhans cells: A simple and efficient new technique. J . Invest. Dermatol. 99, 237-240.

Moulon, C., Peguet-Navarro, J., and Schmitt, D. (1991). A potential role for CDla molecules on human Langerhans cells in allogeneic T-cell activation. J . Invest. Dermatol. 97, 524-528.

Morphy, G. F., Merot, Y., Tong, A. K. F., Smith, B., and Mihm, M. C.. Jr. (1985). Depletion and re-population of epidermal DC after allogeneic bone marrow transplantation in humans. J . Invest. Dermatol. 84, 210-214. Nabavi, N., Freeman, G. J., Gault, A.. Godfrey, D., Nadler, L. M., and Glimcher, L. H. (1992). Signalling through the MHC class I1 cytoplasmic domain is required for antigen presentation and induces B7 expression. Nature (London) 360, 266-267. Neumayer, H. P., Schultz, T. F., Peters, J. H., and Dierich, M. P. (1990). lmportance of ICAM-I for accessory cell function of monocytic cells. Immunology 180, 458-466. Nicod, L. P., Lipscomb, M. F., Weissler, J. C., Lyons, R., Albertson, J., and Toews, G. B. (1987). Mononuclear cells in human lung parenchyma. Am. Rev. Respir. Dis. l36, 818-823.

Nussenzweig, M. C., Steinman, R. M., Witmer, M. D., and Gutchinov, B. (1982). A monoclonal antibody specific for mouse dendritic cells. Proc. Natl. Acad. Sci. U.S.A. 79, 16I - 165. Ohashi, P. S., Oehen, S . , Buerki, K., Pircher, H., Ohashi, C. T., Odermatt, B., Malissen, B.. Zinkernagel, K. M., and Hengartner, H. (1991). Ablation of “tolerance” and induction of diabetes by virus infection in viral antigen transgenic mice. Cell (Cambridge, Mass.) 65, 305-317.

Pamphilon, D. H., Alnagdy, A. A., and Wallington, T. B. (1991). Immunomodulation by UV light: Clinical studies and biological effects. Immunol. Today 12, 119-123. Pancholi, P., Steinman, R. M., and Bhardwaj, N. (1992). Dendritic cells efficiently immunoselect mycobacterial-reactive T cells in human blood, including clonable antigen-reactive precursors. Immunology 76, 217-224. Pavli, P., Hume, D. A., Van De Pol, E., and Doe, W. F. (1993). Dendritic cells, the major antigen-presenting cells of the human colonic lamina propia. Immunology 78, 132-141. Peguet-Navarro, J., Dalbiez-Gauthier, C., and Schmidt, D. (1992). Accessory function of human Langerhans cells in the primary allogeneic T-cell response. J . Invest. Dermatol. 99,87~-88~.

Pelletier, M., Tautu, C., Landry, D., Montplaisir, S., Chertrand, C., and Perreault, C. (1986). Characterisation of human thymic dendritic cells in culture. Immunology 58, 263-270. Peters, J. H., Ruppert, J., Gieseler, R. K. H., Najar, H. M., and Xu, H. (1991). Differentiation of human monocytes into CD14 negative accessory cells: Do dendritic cells derive from the monocytic lineage. Pathobiology 59, 122-126.

100

LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

Pimpinelli, N.. Borgrgnoni, L., Ricardi, R., Ficarra, G., Mori, M., Gaglioti, D., and Romagnoli, P. (1991). CD36 (OKM5)' DC in the oral mucosa of HIV- and HIV+ subjects. J . Invest. Dermatol. 97, 537-542. Poulter, L. W., and Janossy, G. (1985). The involvement of dendritic cells in chronic inflammatory disease. Scand. J . Immunol. 21, 401-407. Poulter, L. W., Campbell, D. A., Munroe, C., and Janossy, G. (1986). Discrimination of human macrophages and dendritic cells by means of monoclonal antibodies. Scand. J . Immunol. 24,351-357. Prickett, T. C. R., and Hart, D. N. J. (1990). Anti-leukocyte common (CD45) antibodies inhibit dendritic cell stimulation of CD4 and CD8 T-lymphocyte proliferation. Immunology 69,250-256. Prickett, T. C. R., McKenzie, J. L., and Hart, D. N. J. (1988). Characterisation of interstitial dendritic cells in the human liver. Transplantation 46, 754-761. Prickett, T. C. R., McKenzie, J. L., and Hart, D. N. J. (1992). Adhesion molecules on human tonsil dendritic cells. Transplantation 53, 483-490. Pure, E., Inaba, K., Crowley, M. T., Tardelli, L., Witmer-Pack, M. D., Ruberti, G., Fathman, G., and Steinman, R. M. (1990). Antigen processing by epidermal Langerhans cells correlates with the level of biosynthesis of MHC class I1 molecules and expression of invariant chain. J . Exp. Med. 172, 1459-1469. Reid, C. D. L., Fryer, P. R., Clifford, C., Kirk, A., Tikerpae, J., and Knight, S. C. (1990). Identificationof haemopoietic progenitors of macrophages and dendritic Langerhans cells (DL-CFU) in human bone marrow and peripheral blood. Blood 76, 1139-1 149. Reid, C. D. L., Stackpoole, A., Meager, A,, and Tikerpae, J. (1992). Interactions of TNF with GM-CSF and other cytokines in the regulation of dendritic cell growth in-vitro from early bipotent CD34+ progenitors in human bone marrow. J . Immunol. 149, 2681-2688. Reiger, A., Wang, B., Kilgus, O., Ochiai, K., Maurer, D., Fodinger, D., Kinet, J.-P., and Stingl, G. (1992). FcsRI mediates IgE binding to human epidermal Langerhans cells. J . Invest. Dermatol. 99, 30s-32s. Rhodes, J. M.. Balfour, B. M., Blom, J., and Agger, R. (1989). Comparison of antigen uptake by peritoneal macrophages and veiled cells from thoracic duct using isotope-, FITC-, or gold-labelled antigen. Immunology 68, 403-409. Romani, N., and Schuler, G. (1992). The immunologic properties of epidermal Langerhans cells as a part of the dendritic cell system. Springer Semin. Immunopathol. W, 265-279. Romani, N., Lenz, A., Glassel, H., Stossel, H., Stanzl, U., Majdic, O., Fritsch, P., and Schuler, G. (1989). Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function. J . Invest. Dermatol. 93, 600-609. Rossi, G., Heveker, N., Thiele, B., Gelderblom, H., and Steinbach, F. (1992). Development of a Langerhans cell phenotype from peripheral blood monocytes. Immunol. Lett. 31, 189- 198. Rowden, G . , Lewis, M. G., and Sullivan, A. K. (1977). Ia expression on human epidermal Langerhans cells. Nature (London) 268, 247-248. Rowden, G., Colp, Dean, S., Auger, F., and Lopes Valle, C. (1992). Comparative epidermal Langerhans cell migration studies in epidermal and epidermal/dermal equivalent grafts. J . Invest. Dermatol. 99, 59s-61s. Rozynska, K. E., Spickett, G. P., Millrain, M., Edwards, A., Bryant, A., Webster, A. D. B., and Farrant, J. (1989). Accessory and T cell defects in acquired and inherited hypogammagloblinaemia. Clin. Exp. Irnmunol. 78, 1-6. Ryffel, B., Brockhaus, M., Greiner, B., Mihatsch, M. J., and Gudat, F. (1991).TNFreceptor distribution in human lymphoid tissue. Immunology 74, 446-452.

ISOLATION AND FUNCTION OF HUMAN DENDRlTlC CELLS

101

Sambhara, S. R., and Miller, R. G . (1991). Programmed cell death of T cells signaled by the T cell receptor and the a]domains of class I MHC. Science 252, 1424-1427. Sauder, D. N., Dinarello, C. A., and Morhenn, V. B. (1984). Langerhans cell production of IL-I. J . Invest. Dermatol. 82, 605-607. Scheeren, R. A., Koopman, G., Van der Baan, S., Meijer, C. J. L. M., and Pals, S. T. (1991). Adhesion receptors involved in clustering of blood dendritic cells and T lymphocytes. Eur. J . Immunol. 21, 1101-1105. Schuler, G., and Romani, N. (1993). Epidermal Langerhans cells as a model for studying the effects of GM-CSF and other cytokines on the biology of dendritic cells. I n “Haemopoietic Growth Factors and Mononuclear Phagocytes” (R. Van Furth, ed.), pp. 197-204. Karger, Basel. Schwartz, R. H. (1990). A cell culture model for T lymphocyte clonal anergy. Science 248, 1349-1 356. Sertl, K., Takemura, T., Tschachler, E., Ferrans, V. J., Kaliner, M. A,, and Shevach, E. M. (1986). Dendritic cells with antigen-presentingcapability reside in airway epithelium, lung parenchyma, and visceral pleura. J . Exp. Med. 163, 436-451. Shalaby, M. R., Espevik, T., Rice, G. C., Ammann, A. S., Figari, I. S . , Ranges, G. E., and Palladino, M. A. (1988).The involvementofTNFaandTNFPinthe MLR. J . Immunol. 141,499-503. Shelley, W. B., and Juhlin, L. (1977). Selective uptake of contact allergens by the Langerhans cell. Arch. Dermatol. 113, 187-192. Silberberg-Sinakin, I., Thorbecke, G. J., Baer, R. L., Rosenthal, S. A., and Berezowsky, V. (1976). Antigen-bearing Langerhans cells in skin, dermal lymphatics and in lymph nodes. Cell. Irnmunol. 25, 137-151. Simon, J. C., Thiele, D. L., Schopf, E., and Sontheimer, R. D. (1992). Effects of the immunosuppressivedipeptide L-leucyl-L-leucine0-methyl ester on epidermal Langerhans cells. J . Inuest. Dermatol. 99, 80s-82s. Sokolowski, J., Jakobsen, E., and Johannessen, J. V. (1978). Cells in peripheral leg lymph of normal men. Lymphology 11,202-207. Spry, C. J. F., Mug, A. J., Janossy, G., and Humphrey, J. H. (1980). Large mononuclear (veiled) cells with “la-like” membrane antigens in human afferent lymph. Clin. Exp. Immunol. 39,750-755. S t a g , A. J., Harding, B., Hughes, R. A., Keet, A., and Knight, S. C. (1991). The distribution and functional properties of dendritic cells in patients with seronegative arthritis. Clin. Exp. Irnmunol. 84,66-71. Staquet, M. J., Levarlet, B., Dezutter-Dambuyant, C., and Schmidt, D. (1992). Human epidermal Langerhans cells express p l integrins that mediate their adhesion to laminin and fibronectin. J . Invest. Derrnatol. 99, 12s-14s. Starling, G. C., Egner, W., McLellan. A. D., Daish, A.. Cordell, J., Mason, D. Y.,Simmons, D. L., and Hart, D. N. J. (1994). ICAM-3, a ligand for LFA-I, is constitutively expressed on blood dendritic cells and costimulates T-lymphocyte proliferation. I n “Leukocyte Typing V: White Cell Differentiation Antigens,” (Schlossman, S., Boumsell, L., Gilks, N., Harlan, J., Kishimoto, T., Morimoto. C., Ritz, J., Shaw, S., Silverstein, R., Springer, T.. Tedder. T., and Todd. R., eds.). Oxford University Press (in press). Starzl, T. E., Demetris, A. J., Murase. N., Ildstad, S., Ricordi, C., and Trucco, M. (1992). Cell migration, chimerism, and graft acceptance. Lancet 339, 1579-1581. Steinman, R. M. (1988). Cytokines amplify the functions of accessory cells. Immunol. Lett. 17, 197-202. Steinman. R. M. (1991). The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9, 271-296.

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Steinman, R. M., and Cohn, Z. A. (1973). Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J . Exp. Med. l37, 1142-1161. Steinman, R. M., and Young, J. W. (1991). Signals arising from antigen-presenting cells. Curr. Opin. Immunol. 3, 361-372. Singl, G., Wolff-Schreiner, E. C., Pichler, W. J., Gschnait, F., Knapp, W., and Wolff, K. (1977). Epidermal Langerhans cells bear Fc and C3 receptors. Nature (London) 268, 245-246. Stossel, H., Koch, F., Kampgen, E., Stoger, P., Lenz, A., Heufler, C., Romani, N., and Schuler, G. (1990). Disappearance of certain organelles (endosomes and Langerhans cell granules) accompanies loss of antigen processing capacity upon culture of epidermal Langerhans cells. J. Exp. Med. 172, 1471-1482. Takahashi, K., Naito, M., Schultz, L. D., Hayashi, S., and Nishikawa, S. (1993). Differentiation of dendritic cell populations in M-CSF-deficient mice homozygous for the osteoporosis (op) mutation. J. Leukocyte Biol. 53, 19-28. Takezaki, S., Morrison, S. L., Berger, C. L., Goldstein, G . , Chu, A. C., and Edelson, R. L. (1982). Biochemical characterisation of a differentiation antigen shared by human epidermal Langerhans cells and cortical thymocytes. J. Clin. Immunol. 2, 128s134s. Tang, A,, and Udey, M. C. (1992a). Doses of UV radiation that modulate accessory cell activity and ICAM-1 expression are ultimately cytotoxic for murine epidermal Langerhans cells. J. Invest. Dermatol. 9, 71s-73s. Tang, A., and Udey, M. C. (1992b). Differential sensitivity of freshly isolated and cultured murine Langerhans cells to UV-B radiation and chemical fixation. Eur. J. Immunol. 22, 581-586. Taylor, S., Baadsgaard, 0.. Hammerberg, C., and Cooper, K. D. (1991). Hyperstimulatory CDla+ CDlb+ CD36+ Langerhans cells are responsible for increased autologous T lymphocyte reactivity to lesional epidermal cells of patients with atopic dermatitis. J. Immunol. 147,3794-3802. Teunissen, M. B. M., Wormmeester, D. J., Kreig, S. R., Peters, P. J., Vogels, I. M. C., Kapsenberg, M. L., and Bos, J. D. (1990). Human epidermal Langerhans cells undergo profound morphologic and phenotypical changes during in-vitro culture. J . Invest. Dermatol. 94, 166-173. Thomas, R., Davis, L. S., and Lipsky, P. E. (1993). Isolation and characterisation of human peripheral blood dendritic cells. J . Immunol. 150, 821-834. Tonks, N. K., Charbonneau, H., Diltz, C. D., Fischer, E. H., and Walsh, K. A. (1988). Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase. Biochemistry 27, 8695-8701. Tasi, V., Bergroth, V., and Zvaifler, N. J. (1988). Synovial dendritic cells in rheumatoid arthritis. Scand. J. Rheumatol. 74, 79-88. Vakkila, J., Lehtonen, E., Koskimies, S., and Hurme, M. (1987). Dendritic cells in human peripheral blood: Effective enrichment from the nonadherent cells. Immunol. Lett. l5, 229-236. Vakkila, J., Sihvola, M., and Hurme, M. (1990). Human peripheral blood-derived dendritic cells do not produce IL-la, IL-lfi or IL-6. Scand. J . Immunol. 31,345-352. Van Dinther-Janssen, A. C. H. M., Pals, S. T.. Scheper, R., Breedveld, F., and Meijer, C. J. L. M. (1990). Dendritic cells and high endothelial venules in the rheumatoid synovial membrane. J . Rheumatol. 17, 11-17. Van Nieuwkerk, E. B. J., Van der Baan, S., Richters, C. D., and Kamperdijk, E. W. A. (1992). Isolation and characterisation of dendritic cells from adenoids of children with otitis media with effusion. Clin. Exp. Immunol. 88, 345-349. Van Voorhis, W. C., Hair, L. S., Steinman, R. M.,and Kaplan, G. (1982). Human dendritic

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cells. Enrichment and characterisation from peripheral blood. J. Exp. Med. 155, 1172-1 187. Van Voorhis, W. C., Valinsky, J., Hoffman, E., Luban, J., Hair, L. S., and Steinman, R. M. (1983). Relative efficacy of human monocytes and dendritic cells as accessory cells for T cell replication. J . Exp. Med. 158, 174-191. Vedel, J., Vincendeau, P., Bezian, J. H., and Taieb, A. (1992). Flow cytometry analysis of adhesion molecules on human Langerhans cells. Clin. Exp. Dermatol. 17, 240-245. Vremec, D., Zorbas, M.,Scollay, R., Saunders, D. J., Ardavin, C. F.. Wu, L., and Shortman, K. (1992). The surface phenotype of dendritic cells purified from mouse thymus and spleen: Investigation of the CD8 expression by a subpopulation of dendritic cells. J . Exp. Med. 176, 47-58. Waalen, K., Duff, G. W.. Forre, O., Dickens. E., Kvarnes, L., and Nuki, G. (1986). IL-I activity produced by human rheumatoid and normal dendritic cells. Scand. J. Immunol. 23, 365-371. Waalen, K., Forre, 0.. Pahle, J., Natvig, J. B., and Burmester, G. R. (1987). Characteristics of human rheumatoid synovial and normal blood dendritic cells. Retention of class 11 MHC antigens and accessory function after short-term culture. Scand. J. Immunol. 26, 525-533. Williams, K. A., and Coster, D. J. (1989).The role of the limbus in corneal allograft rejection. Eye 3, 158-166. Witmer, M. D., and Steinman, R. M. (1984). The anatomy of peripheral light-microscopic immunocytochemical studies of mouse spleen, lymph node and peyers patch. Am. J. Anat. 170,465-481. Wood, G. S., Turner. R. R., Shiurba, R. A., Eng, L., and Warnke, R. A. (1985). Human dendritic cells and macrophages. In-situ immunophenotypic definition of subsets that exhibit specific morphologic and microenvironmental characteristics. Am. J. Pathol. 119, 73-82. Wood, G. S., Freudenthal, P. S., Edinger, A., Steinman, R. M., and Warnke, R. A. (1991). CD45 epitope mapping of human CDla+ dendritic cells and peripheral blood dendritic cells. Am. J. Pathol. l38, 1451-1459. Xu, H., Friedrichs, U., Gieseler. R. K. H., Ruppert, J., Ocklind. G., and Peters, J. H. (1992). Human blood dendritic cells exhibit a distinct T-cell stimulating mechanism and differentiation PATT pattern. Scand. J . Immunol. 36, 689-6%. Yokota, S.. Geppert, T. D., and Lipsky, P. E. (1988). Enhancement of antigen- and mitogeninduced human T lymphocyte proliferation by TNFa. J . Immunol. 140, 531-536. Young, J. W., and Steinman, R. M. (1988). Accessory cell requirements for the MLR and polyclonal mitogens, as studied with a new technique for enriching blood dendritic cells. Cell. Immunol. 111, 167-182. Young, J. W., and Steinman, R. M. (1990). Dendritic cells stimulate primary human cytolytic lymphocyte responses in the absence of CD4' helper T cells. J . Exp. Med. 171,1315-1332. Young, J. W., Koulova, L., Soergel, S. A., Clarke, E. A., Steinman, R. M., and Dupont, B. (1992). The B7IBB-I antigen provides one of several co-stimulatory signals for the activation of CD4+ T lymphocytes by human blood DC in-vitro. J. Clin. Inuest. 90, 229-239. Zhou, L. J., Schwarting. R., Smith, H. M., and Tedder, T. F. (1992). A novel cell-surface molecule expressed by human interdigitating reticulum cells, Langerhans cells, and activated lymphocytes is a new member of the immunoglobulin superfamily. J. Immunol. 149, 135-742. Zvaitler, N. J., Steinman, R. M., Kaplan, G., Lau, L. L., and Rivelis, M. (1985).Identification of immunostimulatory dendritic cells in the synovial effusions of patients with rheumatoid arthritis. J. Clin. Invest. 76, 789-800.

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Granulated Lymphoid Cells of the Pregnant Uterus: Morphological and Functional Features Chau-Ching Liu,'" Earl L. Parr,t and John Ding-E Young* *Laboratory of Molecular Immunology and Cell Biology, The Rockefeller University, New York, New York 10021; and ?Department of Anatomy, School of Medicine, Southern Illinois University, Carbondale, Illinois 62901

1. Introduction

A large population of granulated lymphocytes is present in the uterus of many species during pregnancy. The cells are concentrated in the placental bed or decidua basalis, and have been termed granulated metrial gland cells in rodents or endometrial granulocytes in humans. Considerable evidence now indicates that these cells belong to the natural killer (NK) cell lineage and that in rodents they are highly activated. In addition, several other kinds of granulated lymphoid cells are also present in the decidua basalis. Since granulated lymphoid cells are major constituents of uterine decidual tissue and decidual tissue exerts important regulatory effects on these cells, it is pertinent to begin this chapter with a brief description of decidualization. Viviparous mammals are characterized by the intrauterine development of their embryos following conception. During the gestation period, the uterus undergoes morphological and physiological changes in order to accommodate the conceptus. The most obvious changes occur in the uterine endometrium, where various types of cells actively proliferate and differentiate. These changes are referred to as decidualization or the decidual cell reaction (De Feo, 1967; Bell, 1983; Tarchand, 1986; Parr and Parr, 1989) (Fig. 1). Although decidualization is normally initiated by a stimulus from the blastocyst, this process can also be induced by artificial stimuli in pseudopregnant animals (De Feo, 1963; Finn and Keen, 1963; Finn and Hinchliffe, I

To whom all correspondence should be addressed.

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Copyright 8 1994 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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FIG. 1 (A) Schematic representation of the decidualized mouse uterus. Three regions can be delineated: antimesometrial area (AMA), mesometrial area (MA). and the mesometrial triangle (MT) where the metrial gland eventually develops during normal pregnancy. L, uterine lumen. M, myometrium; and ME, mesometrium. (B) Schematic representation of the mouse uterus during midgestation. The embryo-derived portion of the placenta joints the embryo to the decidua basalis (DB).Both the DB and the adjacent metrial gland (MG) are derived from maternal cells.

1964; Psychoyos, 1973; Buxton and Murdoch, 1982). Several molecules, including histamine (Dey, 1981; Dey and Hubbard, 1981),CAMP(Holmes and Bergstron, 1975;Rankin et al., 1977; Dey and Hubbard, 1981),prostaglandins (Kennedy, 1983, 1985), and proteins synthesized specifically by decidual cells (Finn and Hinchliffe, 1964; Denari et al., 1976; Bell, 1979; Mulholland and W e e , 1984; Leavitt et al., 1985) have been implicated in the decidualization process, but the precise role of such putative biochemical signals remains undefined. Preparation of the uterus for the decidual reaction and the maintenance of decidual tissue require estrogen and progesterone (Yochim and De Feo, 1963; Finn and Martin, 1970; Psychoyos, 1973; Glasser and Clark, 1975;Peel and Stewart, 1986).Endometrial stromal cells isolated and cultured in vitro proliferate spontaneously and differentiate into cells that are morphologically similar to some kinds of decidual cells in the absence of steroid hormones (Vladimirsky et al., 1977; Sananes et a/., 1978; Bell and Searle, 1981). The factors involved in differentiation of decidual cells in uitro are unknown. II. Cells Associated with Decidual Tissue

Once decidualization has been initiated, further differentiation leads to the formation of several kinds of decidual tissue, each characteristic of a

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particular time, species, and location in the pregnant uterus. Cell populations in the decidualized tissue are heterogeneous, and may include lymphoid cells (Table I) as well as cells that have differentiated from uterine stromal fibroblasts. Previous reviews of decidual tissue (Finn, 1980; Bell, 1983; Glasser et al., 1987) and decidual lymphoid cells (Stewart, 1993) may be consulted for detailed information. This chapter focuses on the nature and possible relationships among the granulated lymphoid cells of decidual tissue, including natural killer cells, neutrophils, suppressor cells, and granulated metrial gland cells. A. Natural Killer Cells

Natural killer cells can effectively lyse virus-infected cells and tumor cells without prior sensitization (Trinchieri and Perussia, 1984; Herberman et al., 1986; Perussia, 1991). It has been reported that a significant level of lytic activity against YAC-1 cells (mouse lymphoma cells; NK-sensitive target) was detectable in mouse decidual cell suspensions prepared between day 6.5 and day 9.5 of pregnancy (the morning the vaginal plug is TABLE I Murine and Human Decidual Leukocytes

Cell type

Phenotype

Proposed function(s)

Mouse Granulated metrial Large granulated lymphocyte, gland cell Thy1 +,asialo-GMI +, perforin+, LGL-I Natural killer cell Granulated lymphocyte, asialoGM 1 LGL- I , perforin Macrophage F4/80+, MHC class 11'

+'-

+

Neutrophil Suppressor cell

+

+'-

CD45 , polymorponucleate Small granulated cells, non-T, non-B, FcR +

Cytokine production, cytolytic, immunosuppression? Cytolytic, cytokine production Antigen presentation? immunosuppression, cytokine production Phagocytosis Immunosuppression

+

Human Macrophage Endometrial granulocyte Suppressor cells T lymphocytea

CD14+, HLA DR+ CD3-, CD2+, CD56+, CD16-, HLA DR-, perforin+ ?

CD3 +,CD8+

Antigen presentation? Immunosuppression, cytokine production Cytokine production, cytolytic Immunosuppression Immunosuppression? Cytolytic?

a Small numbers of T cells (approximately 10% of the decidual cell population) were identified in human decidua of early pregnancy (King et al., 1989b).

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found is designated as day O S ) , with the peak activity occurring on day 6.5 (Croy et al., 1985). This NK activity declined steadily as pregnancy proceeded and became undetectable by day 12.5 (Croy et al., 1985).Cells responsible for this lytic activity appeared to be Thy-1 and asialo-GM1' NK cells, since treatment of decidual cell suspensions with a combination of complement and anti-Thy-1 or anti-asialo-GM1 antibody could deplete the YAC-1 lytic activity (Croy etal., 1985;Gambel et al., 1985).Immunolocalization experiments using anti-asialo-GM1 antibody demonstrated that these NK cells were localized in decidua basalis, away from the embryo (Croy et al., 1986). These decidual NK cells were not observed to exert harmful effects on blastocysts or embryo-derived cells in v i m (Croy et al., 1985). Decidual NK cells are also found in humans (Bulmer and Sunderland, 1984; Starkey et al., 1988),and have been reported to display lytic activity against K562 cells (human erythroleukemia cells; targets for NK cells), albeit weaker than the activity of peripheral blood NK cells (King et al., 1989a; Manaseki and Searle, 1989). In pigs, NK cells capable of killing YAC-1 cells have been found in pregnant uterus (Croy et al., 1988). This NK activity was detectable as early as day 9 of gestation (when the embryo has not attached to the uterus-preattachment period) and increased gradually until day 28 (postattachment period). G. J. King (1988) also reported the identification of a population of NK-like cells, referred to as intraepithelial lymphocytes, in the pregnant uterus of pigs. The functions of NK cells during pregnancy are still unclear. NK cells may destroy aberrant trophoblast cells and could contribute to the resorption of aborted fetuses (Gendron and Baines, 1988).Since NK cells are known to produce certain cytokines (Kasahara et al., 1983; Ortaldo and Herberman, 1984; Paya et al., 1988; Perussia, 1991; Croy et al., 1991b), they might participate in regulating the differentiation, activation, and growth of other decidual cells. +

6. Neutrophils A group of small spherical cells stained by anti-CD45 (common leukocyte

antigen) was detected among the decidual leukocyte populations in mouse uterus of day 14 gestation (Redline and Lu, 1989; Parr et al., 1990). These cells were identified as neutrophils by nuclear morphology. Similar cells have been observed in cell suspensions prepared from dissociated mouse decidual tissue during mid to late gestation (Slapsys and Clark, 1982). Decidual neutrophils were localized mainly at the interface between the decidua basalis and trophoblasts, or adhered to the lining of vessels near the trophoblast-deciduajunction (Parr et al., 1990). This special location

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of neutrophils suggests that an acute inflammatory process probably occurs near this important maternal-fetal interface. It is possible that one of the functions of decidual neutrophils is to phagocytose cell debris of decidual cells destroyed by invading trophoblasts, or debris of trophoblasts killed by NK cells. C. Suppressor Cells

Immunosuppressive activity associated with decidual tissue has been reported in both mice and humans (Clark et al., 1984, 1986a).Soluble factors extracted from mouse decidua, though yet to be characterized, were shown to inhibit lymphocyte proliferation in vitro in mixed lymphocyte reaction and upon mitogen stimulation (Kirkwood and Bell, 1981; Badet et al., 1983; Clark et al., 1986b). In addition, small cells isolated from murine decidua have been shown to inhibit the infiltration of cytotoxic T lymphocytes into sponge-matrix (Slapsys and Clark, 1983) and to suppress the mixed lymphocyte reaction in vitro (Slapsys and Clark, 1982). The small decidual cells were non-T, non-B, and non-NK, and contained small acidophilic cytoplasmic granules. It has been proposed that these cells are unusual lymphocytesand that they play a role in suppressinga maternal immune response to the fetal “allograft” (Clark et al., 1984). One appealing observation indicating this role is that the frequency of spontaneous abortion increased in female CBA/J mice mated with male DBA/2 mice, in which combination the maternal decidual suppressor cells were found to be deficient (Clark et al., 1980, 1986c; Chaouat et al., 1990). It has been suggested that the suppressive activity of these cells is mediated by soluble factor(s) which may be related to transforming growth factor-@ (TGF-@)(Clark et al., 1988; Lea et al., 1989). The precise mechanisms of suppression employed by these cells, however, need to be investigated more fully. Other unresolved issues regarding these suppressor cells include their identity, origin, and localization in murine decidual tissues. In humans, explants of first-trimester decidua were shown to exhibit some suppressive activity and this activity was also thought to be mediated by soluble factors, perhaps TGF-/3and prostaglandin E2 (PGE2)(Golander et al., 1981; Parhar et al., 1988). Two types of suppressor cells in human decidua have been reported, but apparently neither of them has been further characterized (Daya et al., 1985a,b). More recently, y / 6 + T cells which displayed downregulated alloreactivity and a/@’ CD8+T cells were observed in human decidua of early pregnancy (Mincheva-Nilsson et al., 1992). It has also been speculated that these cells suppress maternal immune responses that might harm the fetus.

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111. Granulated Metrial Gland Cells of Rodents A. Metrial Gland in the Pregnant Uterus of Rodents

The most remarkable cell population among the granulated decidual leukocytes is the granulated metrial gland (GMG) cells. GMG cells owe their name to both their striking morphology and their unique localization in pregnant uterus. The metrial gland (MG), a term first introduced by Selye and McKeown in 1935, is a region of maternal uterine tissue that differentiates between the longitudinal and circular layers of the myometrium at the conceptus implantation sites in pregnant rodents. It extends from the attenuated circular muscle layer adjacent to the decidua basalis out to the serosal surface of the uterus, an area sometimes referred to as the mesometrial triangle (Bridgman, 1948) (Fig. 1). This pregnancy-associated tissue has been described in mice (Smith, 1966; I. J. Stewart and Peel, 1977), rats (Wislocki et al., 1957; Bulmer, 1968; Larkin and Cardell, 1971; Peel and Bulmer, 1977), hamsters (Bulmer et al., 1983), guinea pigs (Asplund and Holmgren, 1940), and voles (I. J. Stewart and Clark, 1990). The metrial gland consists of three major components: fibroblast-like stroma1 cells, vasculature, and GMG cells, all of which are believed to be maternal in origin. Although it is still controversial, the concept that the maintenance of metrial glands probably depends on certain hormones, particularly progesterone, has been widely accepted (Sharma et al., 1986; Stewart, 1987, 1988). One of the studies suggesting the importance of progesterone showed that explants of rat metrial glands survived in uitro only in the presence of progesterone (Adam and Peel, 1983). Hormone dependency is also suggested by the identification of progesterone receptors in metrial glands (Martel et al., 1984). It was reported that, while the progesterone receptor level gradually decreased in both the endometrium and myometrium during decidualization of the uterus, it increased in metrial glands (Martel et al., 1984). However, in a series of experiments in which ovariectomized mice were given replacement hormones, it was observed that administration of progesterone did not always result in the differentiationand maintenance of GMG cells (Stewart, 1987, 1988). The maintenance of GMG cells in metrial glands appeared to correlate with the activation status of decidual stromal cells. Based on these results, it is tempting to conclude that certain yet-undefined factor(s) produced by activated decidual stromal cells may be responsible for inducing the differentiation of GMG cells and maintaining the metrial glands in uiuo (Stewart, 1987, 1988; Zheng et al., 1991a). Such factor(s) might be provided by direct cell-cell interactions, since a close contact between GMG and decidual stromal cells is obvious.

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The transient existence of metrial glands in the uterus, specifically accompanying pregnancy, and the strategic location of metrial glands at the maternal-fetal interface imply that they may play a role in the maintenance of the fetus or in protecting the mother against the fetus. The recent postulate that metrial glands are immune barriers at the maternal-fetal interface (Zheng et al., 1991d) has mainly evolved from studies on GMG cells. In the next sections, some features of rodent GMG cells and their counterparts in other animal species are discussed.

B. Origin of GMG Cells GMG cells were first described in the mouse as “maternal glycogenic cells” by Jenkinson in 1902. More recently, extensive studies on the origin, distribution, morphology, and functions of GMG cells have been carried out by Stewart and Peel (Peel, 1989; Stewart, 1991), Croy (1990; Croy and Kiso, 1993),and others (Parr et al., 1990;Linnmeyerand Pollack, 1991). As previously mentioned, GMG cells reside mainly in the decidua basalis and metrial glands in the pregnant uterus. They are often observed in the maternal blood vessels of the metrial gland and decidua basalis (I. J. Stewart and Peel, 1978; Parr et al., 1987). Similar to the other components of the metrial gland, GMG cells are of maternal origin. For some time it was believed that, as part of the decidualization process in the pregnant uterus, GMG cells could differentiate from fibroblast-like stromal cells in the endometrium and mesometrial triangle (Selye and McKeown, 1935; Larkin and Schultz. 1968). The possibility that GMG cells may be derived from blood cell precursors was first raised in 1966 without receiving timely attention (Smith, 1966). Subsequently, Peel and Bulmer (1977) and 1. J . Stewart and Peel (1977) performed a series of studies to investigate the structure and morphology of GMG cells in the developing decidua basalis and metrial glands in mice and rats. They observed some agranular cells which were lymphocyte-like, yet bore similarities to GMG cells in terms of nuclear form, cytoplasmic staining, and intracellular organelles (I. J. Stewart and Peel, 1977). These cells were thought to represent GMG cells at the early and intermediate stages of differentiation. These results were taken to suggest a lymphoid origin for GMG cells. Further evidence that GMG cells are derived from precursor cells originating in bone marrow was obtained from bone-marrow chimera studies (Peel et al., 1983; Peel and Stewart, 1984, 1986). Originally, mice were given a lethal dose of whole-body X-ray irradiation followed by an intravenous injection of rat or mouse bone marrow cells after a 24-hr interval. Deciduomata formation in these reconstituted mice was induced by supple-

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ments of ovarian hormones and intrauterine physical stimulus. GMG cells in the deciduomata were then examined. The distinct morphological features of rat and mouse GMG cells (see later discussion)allowed the investigators to demonstrate that mice reconstituted with rat bone marrow contained only GMG cells with the rat phenotype, whereas those reconstituted with mouse bone marrow possessed only mouse GMG cells (Peel et al., 1983). In a subsequent study in which part of the uterine horn of the mouse was shielded during the lethal X-ray irradiation, it was shown that GMG cells of both rat and mouse phenotypes could develop in the shielded uterine horn but,only rat GMG cells were present in the unshielded uterine horn (Peel and Stewart, 1984). These results suggested that GMG cells are derived from bone marrow precursors and that some GMG precursors may already reside in the uterus and readily differentiate in situ during deciduomata formation (or pregnancy). GMG cells differentiate and appear in the pregnant uterus in the following sequence (Peel and Stewart, 1979; Stewart and Peel, 1980, 1981). Shortly after implantation of the embryo, GMG cells appear and reside throughout the developing decidua and uterus. The number of GMG cells increases dramatically by day 6 of gestation, with a preferential localization in the decidua basalis and mesometrial triangle at the implantation sites. Simultaneously, GMG cells disappear from other regions of the decidua and uterus. In the mesometrial triangles, GMG cells continue to proliferate and become a prominent cellular population in those areas now termed metrial glands. GMG cell proliferation appears to peak by days 11-13 of gestation. During midgestation, GMG cells remain nonproliferating but probably are functionally active (Croy, 1990; Croy and Kiso, 1993). Toward the end of gestation (term being 19-21 days in mice), GMG cells degenerate and only a few of them remain by parturition. Since it was clear that GMG cells are derived from hematopoieticprecursors originating in bone marrow, the next obvious question was: To which lineage of hematopoietic cells do GMG cells belong? Some information has been provided by studies which showed that GMG cells are present in mouse strains that exhibit deficiencies in their leukocyte populations (Croy et al., 1991a).Mice with severe combined immunodeficiency (SCID) mutation (Bosma et al., 1983) and nude (nu/nu) mice both have normal development of metrial gland and GMG cells, indicating that GMG cells are unlikely to have T- or B-lymphocyte lineage (Croy et al., 1991a). Beige (bg/bg) mice are defective in NK cell activity but possess GMG cells and breed normally (Roder and Duwe, 1979;Yamashiro et al., 1989;Croy and Chapeau, 1990; Croy et al., 1991a). The finding of GMG cells in bg/bg mice, however, does not preclude NK lineage for GMG cells, since these mice still have nonfunctional NK cells which contain abnormal cyto-

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plasmic granules (Roder and Duwe, 1979). Further information about the identity of GMG cells has come from both morphological and immunohistochemical studies, as discussed later. C. Morphology and Phenotypes of Murine GMG Cells

As indicated by their name, GMG cells are made conspicuous by the presence of numerous granules in the cytoplasm. Mouse GMG cells are large, spherical cells, ranging from 20 to 50 pm in diameter (I. J. Stewart and Peel, 1977); they possess abundant cytoplasm, an eccentric nucleus that is sometimes binucleate and occasionally multinucleate (I. J. Stewart and Peel, 1977; Parr et al., 1990), as well as cytoplasmic granules. The granules have an average diameter of 2-3 pm, but infrequently may be as large as 5 p m (I. J. Stewart and Peel, 1977). These large granules contain glycoproteins and can be stained by the periodic acid-Schiff (PAS) procedure (Fig. 2). In addition to the glycoprotein-containing granules, glycogen deposits can also be found in the cytoplasm of GMG cells. Cells analogous to mouse GMG cells have been found in other rodents, such as rats (Wislocki et al., 1957; Bulmer, 1968; Larkin and Cardell, 1971; Peel and Bulmer, 1977), hamsters (Bulmer et al., 1983), and field voles (Stewart and Clark, 1990). In spite of the many similarities between mouse and rat GMG cells, a striking morphological difference in their granules has been noticed. The granules of mouse GMG cells are bounded by a membranous “cap,” which consists of many vesicular structures lying on one side over a homogeneous core (Fig. 2). In contrast, the membranous component of the granules in rat GMG cells is arranged as myelin figures (Dixon and Bulmer, 1971; Larkin and Cardell, 1971; I. J. Stewart, 1978; KanbourShakir et al., 1990). This difference enabled Peel and Stewart to identify the origin of GMG cells in the bone marrow chimera studies (Peel er al., 1983; Peel and Stewart, 1984, 1986) discussed earlier. In parallel with the extensive investigation of the ultrastructures of GMG cells, phenotyping studies have been performed to discern the lineage relationship between GMG cells and various hematopoietic cells. Both mouse and rat GMG cells were found to express the leukocyte common antigen (CD45)(Mitchell and Peel, 1984; Mukhtar et al., 1989), suggesting that GMG cells belong to the leukocyte lineage. Thy-1 antigens were present on GMG cells (Redline and Lu, 1989; Mukhtar er al., 1989; Parr et al., 1990),indicating a possible T lineage for GMG cells but not excluding an NK lineage. NK cells cultured in vitro in the presence of interleukin-2 (IL-2), like GMG cells, are characterized by abundant cytoplasmic granules which

FIG. 2 Mouse GMG cells in the metrial gland of day 14 pregnant uterus. (A) A section of the metrial gland was examined under a light microscope. Some GMG cells are present in the blood vessel (top o the panel), but most of the GMG cells are found in the metrial gland tissue (X240). (B) Another section of the metrial gland was stained with periodic acid Schiff reagent to reveal the characteristic glycoprotein-containing cytoplasmic granules ( ~ 2 4 0 ) . (continued )

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also contain an electron-dense core surrounded by a vesicular cap (Young et al., 1986a; Ojcius et al., 1991). This morphological similarity led Parr (Parr et af., 1987, 1990) to propose that GMG cells may be uterine NKlike cells. Further studies demonstrated that mouse GMG cells expressed an NK cell marker, asialo-GM1, but were negative for CD3, Lyt-1 (CD5), Lyt-2 (CD8), and L3T4 (CD4) (Mukhtar et al., 1989; Parr et al., 1990; Linnmeyer and Pollack, 1991). Parr et al. (1990) further showed that a few mouse GMG cells expressed another NK cell-surface protein, LGL1 (Mason et al., 1988). The expression of LGL-1 on GMG cells appeared to be inversely correlated with that of Thy-] and asialo-GM1; that is, LGL-lbngh'cells displayed little or no staining of Thy-1 or asialo-GM1 whereas LGL-Idimcells showed more intense staining of Thy-1 and asialoGMl . This inverted expression was also observed for a granular protein, perforin (to be discussed later). These observations have suggested that LGL-1+ NK cells may differentiate in situ to GMG cells (Parr et al., 1990). During the differentiation process, precursor NK cells appear to lose LGL- 1 surface antigens and gain a higher concentration of Thy- 1 and asialo-GM1 antigens (Parr et af., 1990). In rats, GMG cells do not express the Thy-1 antigen and several other T-cell associated antigens, but they do express asialo-GM1 and the 3.2.3 antigen, a triggering molecule expressed on rat NK cells (Mitchell and Peel, 1984; O'Shea et al., 1988; Head, 1990). Taken together, these results provide evidence that strongly supports the NK lineage of GMG cells. A relationship between GMG cells and NK cells is further suggested by the finding that mouse GMG cells express several of the granule proteins of NK cells, lymphokine-activated killer (LAK) cells, and cytolytic T lymphocytes (Young et al., 1986a; Masson and Tschopp, 1987). By employing immunofluorescence techniques, our group has shown that GMG cell granules stained brightly with several polyclonal antisera (Parr et al., 1987, 1990) and a monoclonal antibody (Joag et al., 1991) against mouse perforin, a potent cytolytic mediator (Masson and Tschopp, 1985; Podack et al., 1985; Young et al., 1986a) (Fig. 3). These perforin+ cells coexpressed Thy-1 antigen, asialo-GM1, and CD45, and contained typical PAS+ granules in the cytoplasm. A few LGL-1+ cells also contained perforin, and the staining of these two markers was inversely correlated. The numbers of perforin-expressingcells and the level of perforin production appeared to correlate with the time course of the appearance of GMG cells at implantation sites (Zheng et al., 1991b). The positively stained (C) A portion of the tissue obtained from a mouse under similar conditions was processed for immuno-electron microscopy and used here to show the ultrastructure of GMG cells. The presence of abundant cytoplasmic granules is evident. The inset shows the structure of a granule which characteristically contains an amorphous central matrix bounded by a membrane with a vesicular cap ( ~ 8 0 0 0 )B . reproduced from Zheng er al., 1991; C reproduced from Parr ef a / . , 1990.

FIG. 3 Mouse GMG cells of a day 14 pregnant uterus were stained with a polyclonal antimouse perforin antiserum and visualized by either immunofluorescence microscopy (A and B) or immuno-electron microscopy (C). Intensive staining of perforin in the cytoplasmic granules can be , x294, and ( C ) x 16,800. The subcellular localization of perforin and serine esterase is further seen under a fluorescence microscope. (A) ~ 7 5 . 6 (B) demonstrated by immunogold double labeling. Arrows point to the protein A-conjugated gold particles in the cytoplasmic granules. Small particles demonstrrtie antiperforin staining, while large particles represent antiserine esterase staining. (Reproduced from Parr et a/., 1987.)

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cells became detectable by day 6 of gestation and increased in both number and brightness as gestation proceeded. The peak of perforin expression in pregnant uterus occurred around days 12-15 of gestation. By the time of parturition, perforin-expressingcells were almost absent, which is consistent with the loss of GMG cells at that time (Peel and Stewart, 1979; Stewart and Peel, 1980, 1981). The expression of perforin in GMG cells was further validated by in situ hybridization and Northern blotting analysis using nucleotide probes specific for perforin (Zheng et al., 1991b)(Fig. 4). Similarly, the accumulation of perforin messages in metrial gland showed a good correlation with the time course of GMG cell differentiation and proliferation. The perforin message, however, appeared and then diminished 1-2 days earlier than the protein products (Zheng et al., 1991b),which is in agreement with the sequence of gene transcription and translation. Since the expression of perforin coincides with the development of GMG cells in metrial gland, it is likely that these two processes are controlled by the same gestational endocrine and/or paracrine factors. The origin of these factors is postulated to be maternal rather than fetal, since we have observed that perforinexpressing GMG cells were present in deciduomata of pseudopregnant mice (where no fetus was present) (Zheng et al., 1991a). Considering that decidual cells have the capacity to secrete cytokines (see later discussion), it cannot be excluded that certain factor(s) locally produced is(are) involved in the induction of perforin expression in GMG cells. In addition to perforin, two members of the serine esterase family associated with killer lymphocytes (Masson and Tschopp, 1987), SE 1 and SE 2 (Young et al., 1986b) [also known as granzyme A and granzyme B (Masson et al., 1986)],were found to be expressed by mouse GMG cells (Parr et al., 1990). Both the messages and the protein products of SE 1 and SE 2 were detected, respectively, by Northern and Western blotting techniques, in RNA and cell extracts derived from metrial glands or isolated GMG cells (Parr et al., 1990) (Fig. 5 ) . At the ultrastructural level, perforin and serine esterases were colocalized to the cytoplasmic granules of GMG cells (Zheng ef al., 1991~).The identification of cytolytic cell granule proteins in GMG cells provides important information regarding their lineage and may suggest a cytolytic function (see later discussion).

IV. Human Endometrial Granulocytes Though nonrodents lack an organized tissue analogous to the metrial gland, bone marrow-originated cells that are similar to GMG cells have been found in many other species. In humans and other primates, a popula-

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FIG. 4 Detection of perforin messages in mouse GMG cells by either Northern blotting analysis (A) or in situ hybridization (B and C). (A). A 2.9-kb perforin message was detected in total RNA derived from uteris of pregnant mice on day 6 (lane I), day 8 (lane 2), day 12 (lane 3). and day 16 (lane 4). Lane 5 contained RNA of mouse cytotoxic T cells as a positive control. (B and C) Mouse pregnant uterus of day 1 I gestation was labeled, respectively, with the antisense and sense riboprobe specific for mouse perforin. The metrial gland is delineated by arrows. A specific labeling for perforin message in most cells of the metrial gland was clearly demonstrated (1OOx). (Adapted from Zheng et al., 1991.)

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Actin FIG. 5 Detection of perforin and serine esterase messages in mouse decidual cells by Northem blotting analysis. Total cellular RNA derived from asialo-GMI decidual cells (lane I), asialo-GMI- decidual cells (lane 2), and mouse cytolytic lymphocytes (lane 3) were hybridized with cDNA probes specific for perforin (PFP), serine esterase 1 (SE I), serine esterase 2 (SE2), and actin. Note that the messages for perforin, SE 1, and SE 2 were detected only in asialo-GMI cells, suggesting their expression in GMG cells. (Reproduced from Parr et al., 1990.) +

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tion of granulated lymphoid cells known as endometrial granulocytes (EGs) (Cardell et al., 1969; Bulmer et al., 1980; Bulmer and Sunderland, 1983) has been observed in the endometrium during the secretory phase of the menstrual cycle and early pregnancy (King et al., 1989b). These cells are alternatively termed Kornchenzellen (“K”) cells, endometrial stromal granulocytes (Hamperl, 1955; Hamperl and Hellweg; 1958), or decidual large granulocytes (Bulmer et al., 1987).Human EGs are smaller than rodent GMG cells (a maximum of about 12 p m in diameter), but they are characterized by a morphology similar to that of large granular lymphocytes (LGLs) (Fig. 6). Human EGs are present in the secretory phase of the cycle (King et al., 1989b),and their numbers increase remarkably in the decidua as gestation advances into first trimester (Starkey et al., 1988; Pace et al., 1989);at this stage, EGs may account for up to 70%

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of the decidual leukocytes (Starkey et al., 1988; King et al., 1991). EGs begin to disappear afterward and virtually vanish by parturition (Kazzaz, 1972). These cells are distributed throughout the decidua basalis, but are more noticeable around the spiral arteries and near the degenerative endometrial glands (Bulmer and Sunderland, 1983). Detailed characterization of EGs by immunohistocytochemistry and flow cytometry has recently been carried out. The results obtained from these studies indicate that human EGs possess a distinct surface phenotype. The cells express a number of surface molecules, including CD45 (a common leukocyte antigen), CD2 (an early T-cell marker), and CD7 (an early T-cell marker) (King et al., 1989b, 1991; King and Loke, 1991), but they lack the 55 kDa interleukin-2 receptor (IL-2R; CD25) (Bulmer and Johnson, 1986)and other T-cell markers such as CD3, CD4, and CD8 (Bulmer and Sunderland, 1984; Bulmer and Johnson, 1985; Christmas et al., 1990; King and Loke, 1991). Notably, most human EGs have been shown to possess little or no CD16 (Fc,RIII), but strongly express CD56 (Leu 19)(Ritson and Bulmer, 1987; Starkey et al., 1988; King et al., 1991), two surface molecules previously associated with NK cell phenotype. Flow cytometry analysis of single-cell suspensions prepared from firsttrimester decidua, however, revealed a small group of cells expressing a significant level of CD16 but a low level of CD56 (Starkey et al., 1988). Whether this minor population belongs to the EG family (as precursors?) or are NK cells from the contaminating blood remains unclear. CD56, which was originally identified as the NKHl antigen and has recently been shown to be identical to the neural cell adhesion molecule (NCAM), is present on virtually all NK cells (Lanier et al., 1989). This unique phenotype of EGs, CD45+/CD2+/CD7+/CD3-/CD16-/ CD56+bngh',is reminiscent of, yet distinct from, the NK cells circulating in the peripheral blood. Most peripheral blood NK cells express CD16 and a much lower level of CD56 (CD16+/CD56+dim). A small subset of CD56+bngh'N K cells exists in peripheral blood but lacks cytoplasmic granules (Lanier et al., 1986), in contrast to the high granularity of CD56+bright EGs. These results, taken together, suggest that human EGs

FIG. 6 Histology and perforin expression of human gestational endometrium. (A) and (B) A section of human 6-week gestational endometrium was stained by hematoxylin and eosin.

Note that the decidualized stromal cells are large with oval nuclei; some cells with irregular nuclei and abundant cytoplasm (arrows) appear to be lymphoid cells (x312). (C) A frozen section derived from the same gestational endometrium was stained with anti-NKH 1 antibody ( ~ 1 5 6 ) (D) . An adjacent section was stained with a polyclonal antimouse perforin antiserum that cross-reacts with human perforin ( x 156). Note the similar distribution of NKH 1 + and perforin' cells, which suggests that they are probably the same population of endometrial granulocytes.

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are related to NK cells but may represent a special subtype. It should be mentioned that the expression of CD16 on human EGs increases upon IL-2 stimulation in uitro (King et al., 1992). Another line of evidence indicating the NK lineage of EGs comes from identificationof the cytolytic mediator, perforin, in human EGs. Immunohistochemicalanalysis showed that perforin was expressed in EGs in the decidual tissue of first-trimester gestational endometrium (Lin et al., 1991), as in the case of mouse GMG cells.

V. Possible Functions of Uterine Granulated Cells during Pregnancy A. Cytokine Secretion

Cytokines may play important roles throughout pregnancy in regulating the growth and differentiation of various maternal and embryonic cells, and granulated lymphocytes may contribute to this activity (Pollard, 1991; Guilbert et al., 1993). Croy et al. (1991b) analyzed the cytokines present in culture supernatants which had been conditioned by either metrial gland explants or isolated migratory GMG cells (98- 100% homogeneous population). Three cytokines were detectable: colony stimulating factor1 (CSF-l), interleukin-1, and leukemia inhibitory factor (LIF), along with unidentified factor(s) which were cytotoxic against the macrophage cell line Y10.14 as well as early embryos. The discovery that GMG cells produce CSF-1 is of interest since it lends support for the hypothetical function of CSF-1 in regulating the differentiationof trophoblast cells and other placental components (Pollard et al., 1987; Arceci et al., 1989). Since some trophoblast cells are closely associated with GMG cells in decidual tissue (I. J. Stewart, 1990), a local high concentration of CSF-1 might be delivered to trophoblast cells in a paracrine fashion. It is worthwhile mentioning that receptors for CSF-1 have been found on fetal and spongiotrophoblast cells (Pollard et al., 1987). IL-1 is a pleiotropic polypeptide which acts as a central mediator in host defense mechanisms during inflammation, infection, and tissue injury (Durum et al., 1985).This monocyte-produced cytokine has a broad spectrum of biological effects, including activating lymphocytes, regulating the growth and differentiation of lymphocytes and fibroblasts, and modulating the production of acute-phase proteins. Since GMG cells are morphologically associated with decidual cells, trophoblast cells, small lymphocytes, and endothelial cells (I. J. Stewart, 1990), any or all of these

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cells might represent the physiological targets of IL-1 secreted by GMG cells. LIF is another pleiotropic cytokine that has been shown to regulate a variety of cellular activities in in uitro systems (Gearing et al., 1987; Rathjen et al., 1990; Gough and Williams, 1989). The link of LIF with pregnancy is obvious in view of its ability to suppress the differentiation of embryonic stem cells derived from blastocysts (Williams et al., 1988). Moreover, LIF receptors have been shown to be present on murine decidual macrophages, fetal trophoblasts, and certain embryonic tissues (Gough and Williams, 1989; Croy et al., 1991b; Hilton et al., 1991). A recent report showed that abundant uterine LIF expression occurred on days 4-5 of pregnancy in mouse, which coincided with the implantation event (Bhatt et al., 1991). Although the LIF transcript was found mainly in uterine endometrial glands, a smaller amount of the transcript was also detected in the decidual tissue. In the study performed by Croy et al. (1991b), it was shown that LIF production occurred both during and after implantation. It is conceivable that LIF may be involved in the initiation of blastocyst implantation and the subsequent differentiation and maturation of placental tissues. Considering that LIF also has the capacity to regulate the development of lymphocytes, this cytokine may participate in modulating the differentiation and activation of lymphocytes which accumulate in pregnant uterus. By employing the sensitive reverse transcription-polymerasechain reaction (RT-PCR) technique, Croy et al. (1991b) attempted to detect the transcripts of a number of cytokines in the mRNA of migratory GMG cells. Transcripts for several cytokines which are known to be expressed by cells of T or NK lineage were not detected. These include IL-2, IL3, IL-4, IL-6, IL-7, granulocyte-CSF (G-CSF), granulocyte-macrophageCSF (GM-CSF), interferon-y (IFN-y), tumor necrosis factor-a (TNF-a), TNF-P (lymphotoxin),and erythropoietin (Croy, 1990;Croy et al., 1991b). LIF and CSF-1 transcripts, on the other hand, were readily detectable, in accord with the presence of these cytokine activities in the GMG cellconditioned culture supernatant. Cytokine secretion by human EGs has similarly been investigated. CD16-CD56+bn@" cells isolated from decidual tissue of early pregnancy were shown to contain mRNA coding for G-CSF, GM-CSF, TNF-a, IFNy , LIF, and macrophage-CSF (M-CSF), as detected by RT-PCR (Saito et al., 1993).These cytokines were also detected in the culture supernatant of CD 16-CD56+brightcells using bioassays or enzyme-linked immunosorbent assays. It should be pointed out that transcripts for other cytokines, including IL-lp, IL-2, IL-3, IL-4, IL-5, and IL-6, could be detected by RT-PCR in mRNA of decidual mononuclear cells (Saito et al., 1993). These findings indicate that both EGs and non-EG decidual cells have the

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ability to produce different cytokines which may play a role in successful pregnancies. B. Immune-Effector Function

By virtue of the morphological resemblance of GMG cells to lymphokineactivated killer cells (Ginsburg et al., 1989) and the expression of perforin in GMG cells, a cytolytic function has been postulated for these cells. Previous studies have shown that perforin, following its release from cytolytic lymphocytes, is able to bind to and form transmembrane pores in the plasma membrane of target cells. These water and salt-permeable transmembrane pores lead to lysis of the target cell (Young and Cohn, 1988). The lytic activity of perforin is dependent on calcium ions and exhibits no apparent specificity for target membranes; it lyses a wide variety of targets, ranging from liposomes to erythrocytes to nucleated tumor cells. Expression of perforin is associated with the activation of cytolytic cells, since resting cytolytic cells do not contain this molecule and reagents which activate lymphocytes induce perforin expression (Liu et al., 1989, 1990; Joag et al., 1990; Smyth et al., 1990). The high level of perforin expression in GMG cells implies that these cells could be potent cytolytic cells and raises a question concerning the identity of their target cells. Several lines of evidence have led Stewart and colleagues to postulate that some trophoblast cells may be the targets of GMG cells. First, a small number of rodent GMG cells are present in the labyrinthine placenta. Detailed morphological studies have shown that these GMG cells are bound to dead layer I trophoblast cells (I. J. Stewart, 1984). Second, when GMG cells were cocultured with trophoblast cells and examined by timelapse cinematography, the mouse GMG cells were observed to ignore most trophoblast cells but to bind to and rapidly lyse a small proportion of them (Stewart and Mukhtar, 1988). These observations suggest that GMG cells originating in the maternal decidua basalis evidently migrate to the fetal placenta where they bind to and kill a minor population of layer I trophoblast cells. The target cells may be aberrant in some way that causes their recognition and lysis by GMG cells. Other experiments have shown that most trophoblast cells are resistant to lysis by either cytotoxic T lymphocytes or NK cells (Zuckermann and Head, 1987,1988). Interestingly, trophoblast cells were more susceptible to LAK cell lysis (Drake and Head, 1989; Head, 1989). Mouse GMG cells were not effective in lysing YAC-1 cells, the most commonly used target of NK cells (Croy and Kassouf, 1989), although some lysis was observed when IL-2 was added to the medium (Croy et

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al., 1991~).It is possible that GMG cells, which appear to be highly activated NK cells, no longer recognize target structures on YAC-1 cells but instead recognize determinants on aberrant trophoblast cells. King et al. (1989a, 1990) have reported that human EGs isolated from first trimester decidua were capable of lysing the human NK cell target, K562, but were able to kill trophoblast cells and choriocarcinoma JEG-3 cells only after being activated by IL-2 (King and Loke, 1990). Ferry et al. (1991) obtained similar results, except that only choriocarcinoma cells (BeWo cell line), but not term trophoblast cells, were sensitive to the lysis mediated by IL-2-stimulated EGs. An IL-24ke factor which is probably produced by villous syncytiotrophoblastcells has been identified in human placenta by immunostaining with IL-2 antibody (Soubiran et al., 1987). This finding, together with the report of IL-2 gene expression in human syncytiotrophoblast cells (Boehm et al., 1989),implies that factors capable of activating human EGs are present in the uteroplacental unit. Similar observations have not been reported in rodents. The above-mentioned studies in mice and humans imply that one of the physiological functions of GMG cells and EGs may well be to restrain the growth of trophoblast cells within the uteroplacental unit. It is known that, while a proper extent of invasion by trophoblast cells is required for the development of the placenta and the embryo, an excessive invasion may lead to a catastrophic outcome. For example, choriocarcinoma (caused by malignant trophoblast cells), which is highly invasive and can cause extensive tissue destruction, may develop following pregnancy. GMG cells and EGs thus probably play a critical role in eliminating aberrant and/or overly active trophoblast cells prior to their escape from the decidual tissue. Concerning the immuno-effector role of GMG cells, one speculative function remaining to be verified is whether these cells are involved in preventing vertical transmission of viruses from the mother to the fetus. In view of the role that NK cells play in halting viral infection at other sites, a similar function has been proposed for GMG cells. Investigation in this respect is important since viral infections represent one of the leading causes of birth defects (Hanshaw, 1971; Florman el al., 1973). GMG cells might also be involved in certain pathological conditions. In a murine spontaneous abortion model, asialo-GM1+ cells have been reported to infiltrate the necrotic fetus (Gendron and Baines, 1988, 1989). Treatment of abortion-prone pregnant mice with antiserum against asialoGM1 significantly reduced the frequency of fetal loss (De Fougerolles and Baines, 1987; Gendron and Baines, 1989), whereas injection of polyinosinic/cytidilic acid (poly IC), IL-2, or IFN-y, reagents which are known to enhance NK cell activity, increased the abortion rate (De Fougerolles and Baines, 1987; Chaouat et al., 1990; Lala et al., 1990). These results

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suggest that NK-like cells may be involved in spontaneous abortion. Whether the asialo-GM1 cells were identical to or related to GMG cells was not critically investigated. Results of our studies using the mouse spontaneous abortion model showed that distribution of GMG cells was essentially the same near healthy and aborting implantation sites. Only small numbers of GMG cells could be detected near the dying fetus and very few such cells actually infiltrated the dead fetus (Zheng et al., 1993). These results indicate that GMG cells are not directly involved in cellmediated fetal killing in this model. +

C. Nonirnmunological Functions

An endocrinological function for GMG cells was first suggested by Selye and McKeown in 1935 and gained some support in the 1960s when Dallenbach-Hellweg et al. (1965) proposed that rat GMG cells contained relaxin. Subsequent studies showed that relaxin could not be detected in extracts from pregnant rat uterus (Larkin, 1974; Anderson and Long, 1978). Recently, however, relaxin was again reported to be detected in endometrial granulocytes in the hamster uterus (Renegar et al., 1987). Furthermore, using the in situ hybridization technique, two transcripts: 2ar (osteopontin) and SPARC (osteonectin), whose protein products may act as hormones, were identified in GMG cells (Nomura et al., 1988). Thus, an endocrine (or paracrine) role for GMG cells is reemerging. Moreover, these activated NK cells are likely to secrete a variety of cytokines that may influence neighboring cells.

VI. Concluding Remarks

An extensive scrutiny of maternal-fetal immune interactions during pregnancy has been going on over the past several decades. Several types of lymphoid cells have been identified and characterized in the pregnant uterus, but their development and functions are only partially understood. This chapter discusses the occurrence and possible functions of the granulated lymphoid cells in decidual tissue, in particular the GMG cells. Tht. gestational factors that regulate the differentiation and activation of GMG cells in situ are maternal in origin, since we have observed that perforin+ (differentiated and activated) GMG cells were present in deciduomata of pseudopregnant mice (Zheng et al., 1991a). Such factors may include IL2 (Soubiran et al., 1987; Boehm et al., 1989; Saito et al., 1993), IL-6 (Kameda et al., 1990; Robertson et al., 1992; De et al., 1993), TNF-a (De

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et al., 19931, and probably some yet-unidentified factors that may be secreted by decidual leukocytes or stromal cells. Identification of the endocrine and/or paracrine factors that activate NK cells in decidual tissue is an important step toward elucidating the biological role of GMG cells. Trophoblasts, which form a physical barrier between the mother and the fetus, are also known to have the capacity to produce a number of cytokines. These trophoblast-derived cytokines may be involved in modulating the functions of GMG cells and other maternal immune cells. The complicated interactions between GMG cells and other maternal or fetal cells are probably necessary to achieve the least harmful reactions to the developing fetus but provide sufficient surveillance against infectious pathogens and aggressive invading trophoblasts. Improvements in the isolation and culture of GMG cells will be important for future studies concerning the functions of GMG cells and other uterine granulated cells. In addition, studies using in situ hybridization and genetically mutated animal strains (e.g., mice deficient in perforin and/or other NK cytolytic mediators) may be expected to contribute much valuable information and perhaps verify some of the functions proposed for GMG cells. Acknowledgments We thank Drs. Li Mou Zheng and Paul Y. Lin for performing excellent immunohistochemical and electron microscopic studies for some of the work described here. Part of this work was supported by grants from the National Institutes of Health (CA-47307 to J. D.-E Young and HD-24450 to E. L. Pan-), the Wood Johnson Foundation, and the American Heart Association-New York City Affiliate. C.-C. Liu is supported by a Career Scientist Award from the Irma Hirschl Trust and an Investigatorship from the American Heart Association-New York City Affiliate. J. D.-E Young is a scholar of the Leukemia Society of America.

References Adam, E., and Peel, S. (1983). The maintenance of rat metrial gland explants in vitro, in the presence and absence of progesterone. J . Anat. l36,321-328. Anderson, M. L., and Long, J. A. (1978). Localization of relaxin in the pregnant rat. Bioassay of tissue extracts and cell fractionation studies. Biol. Reprod. 18, 110-1 17. Arceci, R. J.. Shanahan, F., Stanley, E. R., and Pollard, J. W. (1989). Temporal expression and location of colony-stimulatingfactor-1 (CSF-1)and its receptor in the female reproductive tract and consistent with CSF-I-regulated placental development. Proc. Null. Acad. Sci. U.S.A. 86,8818-8820. Asplund, J. Y., and Holmgren, H. (1940). In der uteruswand warend Graviditat auftrende metachromatisch granulierete Zellverbande und ihre Stellung zur "Glandula moymetralis." Z. Mikrosk.-Anot. Forsch. 48, 478-528.

128

CHAU-CHING LIU, EARL L. PARR, AND JOHN DING-E YOUNG

Badet, M.-T., Bell, S. C., and Billington, W. D. (1983). Immunoregulatory activity of supernatants from short-term cultures of mouse decidual tissue. J . Reprod. Fertil. 68, 351-358.

Bell, S . C. (1979). Protein synthesis during decidual morphogenesis in the rat. Biol. Reprod. 20,811-821.

Bell, S . C. (1983). Decidualization: Regional differentiation and associated function. Oxford Rev. Reprod. Biol. 5 , 220-271. Bell, S. C., and Searle, R. F. (1981). Differentiation of decidual cells in mouse endometrial cell culture. J . Reprod. Fertil. 61, 425-433. Bhatt, H., Brunet, L. J., and Stewart, C. L. (1991). Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation. Proc. Natl. Acad. Sci. U.S.A. 88, 11408-11412. Boehm, K. D., Kelley, M. F., and Ilan, J. (1989). The interleukin 2 gene is expressed in the syncytiotrophoblast of the human placenta. Proc. Natl. Acad. Sci. U.S.A. 86,656-660. Bosma, G. C., Custer, R. P., and Bosma, M. J. (1983). A severe combined immunodeficiency mutation in the mouse. Nature (London) 301, 527-530. Bridgman, J. (1948). A morphological study of the development of the placenta of the rat. J . Morphol. 83, 61-85. Bulmer, D. (1968). Further studies on the granulated metrial gland cells of the pregnant rat. J . Anat. 103,479-489. Bulmer, D., Stewart, I., and Peel, S. (1983). Endometrial granulocytes of the pregnant hamster. J . Anat. l36, 329-337. Bulmer, J. N., and Johnson, P. M. (1985). Immunohistologicalcharacterization of the decidual leukocytic infiltrate related to endometrial gland epithelium in early human pregnancy. Immunology 55,35-44. Bulmer, J. N., and Johnson, P. M. (1986). The T-lymphocyte population in first-trimester human decidua does not express the interleukin-2 receptor. Immunology 58, 685-687. Bulmer, J. N., and Sunderland, C. A. (1983). Bone-marrow origin of endometrial granulocytes in the early human placental bed. J . Reprod. Immunol. 5 , 383-387. Bulmer, J. N., and Sunderland, C. A. (1984). Immunohistological characterization of lymphoid cell populations in the early human placental bed. Immunology 52, 349. Bulmer, J. N., Stewart, I. J., Mitchell, B. S., and Peel, S. (1980). Endometrial granulocytes in the human uterus. J . Anat. 131, 754. Bulmer, J. N., Hollings, D., and Riston, A. (1987). Immunocytochemical evidence that endometrial stromal granulocytes are granulated lymphocytes. J. Pathol. 153, 281-288. Buxton, E., and Murdoch, R. N. (1982). Lectins, calcium ionophore A23187 and peanut oil as deciduogenic agents in the uterus of pseudopregnant mice: Effects of tranylcypromine, indomethacin, iproniazid and propanolol. Aust. J . Biol. Sci. 35, 63-72. Cardell, R. R., Hisaw, F. L., and Dawson, A. B. (1%9). The fine structure of granular cells in the uterine endometrium of the rhesus monkey (Macaca mutatta) with a discussion of the possible function of these cells in relaxin secretion. Am. J. Anat. W, 307-339. Chaouat, G., Menu, E., Clark, D. A., Dy, M., Minkowski, M., and Wegmann, T. G. (1990). Control of fetal survival in CBA x DBA12 mice by lymphokine therapy. J. Reprod. Fertil. 89,447-458.

Christmas, S. E., Bulmer, J. N., Meager, A., and Johnson, P. M. (1990). Phenotypic and functional analysis of human CD3- decidual leukocyte clones. Immunology 71, 182-189. Clark, D. A., McDermott, M. R., and Szewczuk, M. R. (1980). Impairment of host-vsgraft reaction in pregnant mice. 11. Selective suppression of cytotoxic T cell generation correlates with soluble suppressor activity and with successful allogeneic pregnancy. Cell. Immunol. 526, 106-118. Clark, D. A., Slapsys, R. M., Croy, B. A., and Rossant, J. (1984). Immunoregulation of host-versus-graft responses in the uterus. Immunol. Today 5 , 1 11-1 15.

GRANULATED LYMPHOID CELLS OF THE PREGNANT UTERUS

129

Clark, D. A., Brierley, J., Slapsys, R., Daya, S., Danji, N., Chaput, A., and Rosenthal, K. (1986a).Trophoblast-dependent and trophoblast-independent suppressor cells of maternal origin in murine and human decidua. I n “Reproductive Immunology” (D. A. Clark and B. A. Croy, eds.), pp. 219-226. Elsevier, Amsterdam. Clark, D. A., Chaput, A., Walker, C., and Rosenthal, K. L. (1986b). Active suppression of host-vs-graft reaction in pregnant mice. VI. Soluble suppressor activity obtained from decidua of allopregnant mice blocks the response to IL-2. J . Immunol. W, 1659-1664. Clark, D. A., Chaput, A., andTutton, D. (1986~).Active suppression ofhost-vs-graft reaction in pregnant mice. VII. Spontaneous abortion of allogeneic DBAR X CBA/J fetuses in the uterus of CBA/J mice correlates with deficient non-T suppressor cell activity. J . Immunol. 136, 1668-1675. Clark, D. A., Falbo, M., Rowley, R. B., Banwatt, D., and Stedronska-Clark, J. (1988). Active suppression of host-vs graft reaction in pregnant mice. IX. Soluble suppressor activity obtained from allopregnant mouse decidua that blocks the cytolytic effector response to IL-2 is related to transforming growth factor-beta. J . Immunol. 141,3833-3840. Croy, B. A. (1990). Granulated metrial gland cells. Interesting cells found in pregnant uterus. Am. J . Reprod. Immunol. 23, 19-21. Croy, B. A., and Chapeau, C. (1990). Evaluation of the pregnancy immunotrophism hypothesis by assessment of the reproductive performance of young adult mice of genotype scidl scid.bg/bg. J . Reprod. Fertil. 88, 231-239. Croy, B. A., and Kassouf, S. A. (1989). Evaluation of the murine metrial gland for immunological function. J . Reprod. Immunol. 15, 51-69. Croy, 8 . A., and Kiso, Y. (1993). Granulated metrial gland cells: A natural killer cell subset of the pregnant murine uterus. Microsc. Res. Tech. 25, 189-200. Croy, B. A., Gambel, P., Rossant, J., and Wegmann, T. G. (1985). Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy. Cell. Immunol. 93, 315-326. Croy, B. A., DeRusha, L., and Rossant, J. (1986). Localization of NK cells in murine decidua. Proc. Int. Congr. Immunol., p. 735. Croy, B. A., Waterfield, A., Wood, W., and King, G. J . (1988). Normal murine and porcine embryos recruit NK cells to the uterus. Cell. Immunol. 115, 471-480. Croy. B. A., Chapman, C., Reed, N., Stewart, I. J., and Peel, S. (1991a). Is there an essential role for bone marrow-derived cells at the fetomaternal interface during successful pregnancy? In “Cellular and Molecular Immunobiology of the Fetomaternal Interface” (T. G. Wegmann, T. J. Gill, and E. Nisbet-Brown, eds.), p. 168. Oxford Univ. Press, New York. Croy, B. A., Guilbert, L. J., Browne, M. A., Gough, N . M., Stinchcomb, D. T., Reed, N., and Wegmann, T. G. (1991b). Characterization of cytokine production by the metrial gland and granulated metrial gland cells. J . Reprod. Immunol. 19, 149-166. Croy, B. A., Reed, N., Malashenko, B. A.. Kim, K., and Kwon, B. S. (1991c).Demonstration of YAC target cell lysis by murine granulated metrial gland cells. Cell. Immunol. U3, 116-126. Dallenbach-Hellweg, G., Battista, J. V., and Dallenbach, R. D. (1965). Immunohistological and histochemical localization of relaxin in the metrial gland of the pregnant rat. Am. J . Anat. 117, 433-450. Daya, S., Clark, D. A., Devlin, C., and Farrell, J. (1985a). Preliminary characterisation of two types of suppressor cell in the human uterus. Fertil. Sreril. 44, 778-785. Daya, S., Clark, D. A., Devlin, C., Jarrell, J., and Chaput, A. (1985b). Suppressor cells in human decidua. Am. J . Obstet. Gynecol. 151, 267-270. De, M., Sanford, T. R., and Wood, G. W. (1993). Expression of IL-1, IL-6, and TNFalpha in mouse uterus during the peri-implantation period of pregnancy. J . Reprod. Fertil. 97, 83-89.

130

CHAU-CHING LIU, EARL L. PARR, AND JOHN DING-E YOUNG

De Feo, V. J. (1%3). Determination of the sensitive period of the induction of deciduamata in the rat by different inducing procedures. Endocrinology (Baltimore) 73, 488-497. De Feo, V. J. (1%7). Decidualization. In “Cellular Biology of the Uterus” (R. M. Wynn, ed.), pp. 191-220. Appleton, New York. De Fougerolles, A. R., and Baines, M. G. (1987). Modulation of the natural killer cell activity in pregnant mice alters the spontaneous abortion rate. J. Reprod. Immunol. 11, 147-153. Denari, J. H., Germino, N. I., and Rosner, J. M. (1976). Early synthesis of uterine proteins after decidual stimulus in pseudopregnant rat. Biol. Reprod. 15, 1-8. Dey, S. K. (1981). Role of histamine in implantation: Inhibition of histidine decarboxylase induces delayed implantation in the rabbit. Biol. Reprod. 24, 867-870. Dey, S . K., and Hubbard, C. (1981). Role of histamine and cyclic nucleotides in implantation in the rabbit. Cell Tissue Res. 22Q, 549-554. Dixon, J. S., and Bulmer, D. (1971). The fine structure of cells in the rat metrial gland. J. Anar. 108, 123-133. Drake, B. L., and Head, J. R. (1989). Murine trophoblast can be killed by lymphokineactivated killer cells. J. Immunol. 143,9-14. Durum, S . K., Schmidt, J. A., and Oppenheim, J. J. (1985). Interleukin 1: An immunological perspective. Annu. Rev. Immunol. 3, 263-287. Ferry, B. L., Sargent, I. L., Starkey, P. M., and Redman, C. W. (1991). Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua. Cell. Immunol. l32, 140-149. Finn, C. A. (1980). The endometrium during implantation. In “The Endometrium” (F. A. Kimball, ed.), pp. 43-56. Spectrum, New York. Finn, C. A., and Hinchliffe, J. R. (1964). Reactions of the mouse uterus during implantation and deciduoma formation as demonstrated by changes in the distribution of alkaline phosphatase. J. Reprod. Fertil. 8, 331-338. Finn, C. A., and Keen, P. M. (1%3). The induction of deciduomata in the rat. J . Embryol. Exp. Morphol. 11, 673-682. Finn, C. A., and Martin, L. (1970). The role of estrogen secreted before oestrus in the preparation of the uterus for implantation in the mouse. J. Endocrinol. 47, 431-438. Florman, A. L., Gershon, A. A,, Blackett, P. R., and Nahmias, A. J. (1973). Intrauterine infection with herpes simplex virus. Resultant congenital malformations. JAMA, J. Am. Med. Assoc. 225, 129-132. Gambel, P., Croy, B. A., Moore, W. D., Hunziker, R. D., Wegmann, T. G., and Rossant, J. (1985). Characterization of immune effector cells present in early murine decidua. Cell. Immunol. 93, 303-314. Gearing, D. P., Gough, N. M., King, J. A., Hilton, D. J., Nicola, N. A., Simpson, R. J., Nice, E. C., Kelso, A., and Metcalf, D. (1987). Molecular cloning and expression of cDNA encodinga murine myeloid leukemia inhibitory factor (LIF). EMBOJ. 6,3995-4002. Gendron, R. L., and Baines, M. G. (1988). Infiltrating decidual natural killer cells are associated with spontaneous abortion in mice. Cell. Immunol. 1l3, 261-267. Gendron, R. L., and Baines, M. G. (1989). Morphometric analysis of the histology of spontaneous foetal resorption in a murine pregnancy. Placenta 10, 309-318. Ginsburg, H., Coleman, R., Davidson, S., Khoury, C., and Mor, R. (1989). Lymphokineactivated killer (LAK) cells are identical to the uterine granulated metrial gland (GMG) cells. Transplant. Proc. 21, 186-189. Glasser, S.R., and Clark, J. H. (1975). A determinant role of progesterone in the development of uterine sensitivity to decidualization and ovo-implantation. In “The Developmental Biology of Reproduction” (C. L. Markert and J. Papaconstantinou, eds.), pp. 311-345. Academic Press, New York. Glasser, S. R., Lampelo, S., Munir, M. I., and Julian, J. (1987). Expression of desmin,

GRANULATED LYMPHOID CELLS

OF THE PREGNANT UTERUS

131

laminin, and fibronectin during in situ differentiation (decidualization)of rat uterine stromal cells. Differentiation (Berlin) 35, 132-142. Golander, A., Zakuth, V., Schechter, Y., and Spirez, Z. (1981). Suppression of lymphocytic activity in vitro by a soluble factor secreted by explants of human decidua. Eur. J . Immunol. 11, 849-851. Gough, N. M.,and Williams, R. L. (1989). The pleotropic effects of leukemia inhibitory factor. Cancer Cells 1, 77-80. Guilbert, L., Robertson, S. A., and Wegmann, T. G. (1993). The trophoblast as an integral component of a macrophage-cytokine network. Immunol. Cell Biol. 71, 49-57. Hamperl, H. (1955). The granular endometrial stromal cells: A new cell type. J . Parhol. Bacreriol. 69, 358. Hamperl, H., and Hellweg, G. (1958). Granular endometrial stroma cells. Obsrer. Gynecol. 11, 379. Hanshaw, J. B. (1971). Congenital cytomegalovirus infection: A I5 year perspective. J . Infect. Dis. 123, 555-561. Head, J. R. (1989). Can trophoblast be killed by cytotoxic cells: In vitro evidence and in vivo possibilities. Am. J. Reprod. Immunol. 20, 100-105. Head, J. R. (1990). Distribution of natural killer (NK) cells in the pregnant rat uterus. J. Reprod. Immunol. 22,14. Herberman, R. B., Reynolds, C. W., and Ortaldo, J. R. (1986). Mechanism of cytotoxicity by natural killer (NK) cells. Annu. Rev. Immunol. 4, 651-680. Hilton. D. J., Nicola, N. A., Waring, P. M., and Metcalf, D. (1991). Clearance and fate of leukemia-inhibitory factor (LIF) after injection into mice. J . Cell. Physiol. 148,430-439. Holmes, P. V., and Bergstron, S. (1975). Induction of blastocyst implantation in mice by cyclic AMP. J. Reprod. Ferril. 43, 329-332. Jenkinson, J. W. (1902). Observations on the histology and physiology of the placenta of the mouse. Tudschr. Ned. Dierkd. Ver. 2, 124-207. Joag, S. V., Liu, C.-C., Kwon, B. S., Clark, W. R., and Young, J. D.-E. (1990). Expression of mRNAs for pore-forming protein and two serine esterases in murine primary and cloned effector lymphocytes. J . Cell. Biochem. 43, 81-88. Joag, S. V., Zheng, L. M., Persechini, P. M., Michl, J., Parr, E. L., and Young, J. D.-E. (1991). The distribution of perforin in the normal tissue. Immunol. Leu. 28, 195-200. Kameda, T., Matsuzaki, N., Sawai, K., Okada, T., Saji, F., Matsuda, T., Hirano, T., Kishimoto, T., and Tanizawa, 0. (1990). Production of interleukin-6 by normal human trophoblast. Placenta 11,205-213. Kanbour-Shakir, A., Kunz, H. W., Gill, T. J., 111, Armstrong, D. T., and Macpherson, T. A. (1990). Morphologic changes in the rat uterus following natural mating and embryo transfer. Am. J . Reprod. Immunol. 23, 78-83. Kasahara, T., Djeu, J. Y., Dougherty, S. F., and Oppenheim, J. J. (1983). Capacity of human large granular lymphocytes (LGL) to produce multiple lymphokines: Interleukin2, interferon, and colony stimulating factor. J . Immunol. l31, 2379-2385. Kazzaz, B. A. (1972). Specific endometrial granular cells. A semiquantitative study. Eur. J . Obsrer. Gynecol. 3, 77-84. Kennedy, T. G. (1983). Embryonic signals and the initiation of blastocyst implantation. Aust. J . Biol. Sci. 36, 531-543. Kennedy, T. G. (1985). Evidence for involvement of prostaglandins throughout the decidual cell reaction in the rat. Biol. Reprod. 33, 140-146. King, A., and Loke, Y. W. (1990). Human trophoblast and JEG choriocarcinoma cells are sensitive to lysis by IL-2-stimulated decidual NK cells. Cell. Immunol. 129, 435-448. King, A., and Loke, Y. W. (1991). On the nature and function of human uterine granular lymphocytes. Immunol. Today 12,432-435.

132

CHAU-CHING LIU, EARL L. PARR, AND JOHN DING-E YOUNG

King, A., Birkby, C., and Loke, Y. W. (1989a). Early human decidual cells exhibit NK activity against the K562 cell line but not against first trimester trophoblast. Cell. Immunol. 118, 337-344. King, A., Wellings, V., Gardner, L., and Loke, Y. W. (1989b). Immunocytochemicalcharacterization of the unusual large granular lymphocytes in human endometrium throughout the menstrual cycle. Hum. Irnmunol. 24, 195-205. King, A., Kalra, P., and Loke, Y.W. (1990). Human trophoblast cell resistance to decidual NK lysis is due to lack of NK target structure. Cell. Immunol. 127, 230-237. King, A., Balendran, N., Wooding, P., Carter, N. P., and Loke, Y.W. (1991). Phenotypicand morphologic characterization of novel CD3-, CD56bright lymphocytes in the pregnant human uterus. Dev. Immunol. 1, 169-190. King, A,, Wheeler, R., Carter, N. P., Francis, D. P., and Loke, Y. W. (1992). The response of human decidual leukocytes to IL-2. Cell. Immunol. 141, 409-421. King, G. J. (1988). Reduction in uterine intra-epithelial lymphocytes during early gestation in pigs. J. Reprod. Irnmunol. 14,41-46. Kirkwood, K. J., and Bell, S . C. (1981). Inhibitory activity of supernatants from murine decidual cell cultures on the mixed lymphocyte reaction. J. Reprod. Immunol. 3, 243252. Lala, P. K., Scordras, J. M., Graham, C. H., Lysiak, J. J., and Parhar, R. S. (1990). Activation of maternal killer cells in pregnant uterus with chronic indomethacin therapy, IL-2 therapy, or a combination therapy is associated with embryonic demise. Cell. Immunol. 127, 368-381. Lanier, L. L., Le, A. M., Civin, C. I., Loken, M. R., and Philips, J. H. (1986). The relationship of CD16 (leu-] 1) and leu-I9 (NKH-I) antigen expression on human peripheral blood NK cells and cytotoxic lymphocytes. J. Immunol. 136, 4480-4486. Lanier, L. L., Testi, R., Binal, J., and Phillips, J. H. (1989). Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule. J. Exp. Med. 169, 2233-2238. Larkin. L. H. (1974). Bioassay of rat metrial gland for relaxin using the mouse interpubic ligment technique. Endocrinology (Baltimore) 94, 567-570. Larkin, L. H., and Cardell, R. R., Jr. (1971). Differentiation of granulated metrial cells in the uterus of the pregnant rat: An electron microscopic study. J. Anat. 132, 241-251. Larkin, L. H., and Schultz, R. L. (1968). Histochemical and autoradiographic studies of the formation of the metrial gland in the pregnant rat. Am. J . Anat. 122, 607-619. Lea, R. G., Clark, D. A., Manuel, J., Banwatt, D. K., and Harley, C. B. (1989). RNA for a pregnancy associated suppression factor, related to TGF-beta2, can be identified in murine decidual tissue by in situ hybridization using the pcD-GIG2 probe. Am. J . Reprod. Immunol. Microbiol. 19, 82. Leavitt, W. W., MacDonald, R. G., and Schwaery, G. T. (1985). Characterizationofdecidua marker protein synthesis during decidualization in the rat. J . Reprod. Ferril. 83,441-449. Soong, Y.-K.,andKuo,T.-T. (1991). Lin, P. Y., Joag, S. V.,Young, J. D.-E.,Chang, Y.-S., In vivo expression of perforin by natural killer cells within first trimester endometrium in humans. Biol. Reprod. 45, 698-703. Linnmeyer, P. A., and Pollack, S. B. (1991). Murine granulated metrial gland cells at uterine implantation sites are natural killer lineage cells. J. Immunol. 147, 2530-2535. Liu, C.-C., Rafii, s.,Granelli-Piperno, A., Trapani, J. A., and Young, J. D.-E. (1989). Perforin and serine esterase gene expression in stimulated human T cells: Kinetics, mitogen requirements, and effects of cyclosporin A. J . Exp. Med. 170, 2105-2118. Liu, C.-C., Joag, S. V., Kwon, B. S., and Young, J. D.-E. (1990). Induction of perforin and serine esterases in a murine cytotoxic T lymphocyte clone. J. Immunol. 144, 11%-1201. Manaseki, S., and Searle, R. (1989). Natural killer (NK) cell activity of first trimester human decidua. Cell. Immunol. 121, 166-173. +

GRANULATED LYMPHOID CELLS OF THE PREGNANT UTERUS

133

Martel, D., Monier, M.,Psychoyos, A., and De Feo, V. J. (1984). Estrogenand progesterone receptors in the endometrium, myometrium, and metrial gland of the rat during the decidualization process. Endocrinology (Baltimore) 114, 1627-1634. Mason, L., Giardina, S. L., Hecht, T., Ortaldo. J., and Mathieson, B. J. (1988). LGL-I: A non-polymorphoic antigen expressed on a major population of mouse natural killer cells. J . Immunol. 140,4403-4412. Masson, D., and Tschopp, J. (1985). Isolation of a lytic, pore-forming protein (perforin) from cytolytic T-lymphocytes. J . Biol. Chem. 260, 9069-9072. Masson, D., and Tschopp, J. (1987). A family of serine esterases in lytic granules of cytolytic T lymphocytes. Cell (Cambridge, Mass.) 49, 679-685. Masson, D., Nabholz, M., Estrade, C., and Tschopp, J. (1986). Granules of cytolytic T lymphocytes contain two serine esterases. EMBO J . 5, 1595-1600. Mincheva-Nilsson, L., Hammarstrom, S., and Hammarstrom, M.-L. (1992). Humandecidual leukocytes from early pregnancy contain high numbers of gamm/delta+ cells and shown selective down-regulation of alloreactivity. J . Immunol. 149, 2203-221 1. Mitchell, B. S., and Peel, S. (1984). Identification of cells bearing leucocyte surface antigens in metrial gland tissue from rats of different gestational ages, strains or parities. Immunology 53,63-68. Mukhtar, D. D. Y., Stewart, I. J., and Croy, B. A. (1989). Leucocyte membrane antigens on mouse granulated metrial gland cells. J . Reprod. Immunol. 15, 269-279. Mulholland, J., and Villee, C. A. (1984). Protein synthesized by the rat endometrium during early pregnancy. J . Reprod. Fertil. 72, 395-449. Nomura, S., Wells, A. J., Edwards, D. R., Heath, J. K., and Horgan, B. L. M. (1988). Developmental expression of 2ar (Osteopontin) and SPARC (Osteonectin) RNA as revealed by in situ hybridization. J . Cell Biol. 106, 441-450. Ojcius, D. M., Zheng, L. M., Sphicas, E. C., Zychlinsky, A., and Young, J. D.-E. (1991). Subcellular localization of perforin and serine esterase in lymphokine-activated killer (LAK) cells and a cytotoxic T cell line by immunogold labelling. J. Immunol. 146, 4427-4432. Ortaldo, J. R., and Herberman, R. B. (1984). Heterogeneity of natural killer cells. Annu. Rev. Immunol. 2, 359-379. O’Shea, J. D., Cerini, M. E., and Ward, H. A. (1988). Expression of leukocyte antigens by cells from the metrial gland of the pregnant rat. Cell Tissue Res. 252, 199-206. Pace, D., Momson, L., and Bulmer, J. N. (1989). Proliferative activity in endometrial stromal granulocytes throughout menstrual cycle and early pregnancy. J . Clin. Pathol. 42, 35-39. Parhar, R. S., Kennedy, T. G., and Lala, P. K. (1988). Suppressionoflymphocytealloreactivity by early gestational human decidua. I. Characterization of suppressor cells and suppressor molecules. Cell. Immunol. 116, 392-410. Parr, E. L., and Parr, M. B. (1989). The implantation reaction. In “Biology of the Uterus” (R. M. Wynn and W. P. Jollie, eds.), pp. 233-278. Plenum Medical Book Co., New York. Parr,E. L., Parr, M. B., and Young, J. D.-E. (1987). Localization of a pore-forming protein (perforin) in granulated metrial gland cells. Biol.Reprod. 37, 1327-1336. Parr, E. L., Young, L. H. Y., Parr, M. B., and Young, J. D.-E. (1990). Granulated metrial gland cells of pregnant mouse uterus are natural killer-like cells that contain perforin and serine esterases. J . Immunol. 145, 2365-2372. Paya, C. V., Kenmotsu, N.. Schoon, R. A., and Leibson, P. J. (1988). Tumor necrosis factor and lymphotoxin secretion by human natural killer cells leads to antiviral cytotoxicity. J . Immunol. 141, 1989-1995. Peel, S. (1989). Granulated metrial gland cells. Adv. Anat., Embryol. Cell Biol. 115, 1-112. Peel, S., and Bulmer, D. (1977). The fine structure of the rat metrial gland in relation to the origin of the granulated cells. J . Anat. 123, 687-697.

134

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Peel, S., and Stewart, I. (1979). Ultrastructural changes in the rat metrial gland in the latter half of pregnancy. Anat. Embryol. 155, 209-219. Peel, S., and Stewart, I. (1984). The differentiation of granulated metrial gland cells in chimeric mice and the effect of uterine shielding during irradiation. J. Anar. 139,593-598. Peel, S . , and Stewart, I. (1986). Oestrogen and the differentiation of granulated metrial gland cells in chimeric mice. J . Anat. 144, 181-187. Peel, S., Stewart, I., and Bulmer, D. (1983). Experimental evidence for the bone marrow origin of granulated metrial gland cells of the mouse uterus. Cell Tissue Res. 223,647-656. Perussia, B. (1991). Lymphokine-activated killer cell, natural killer cells and cytokines. Curr. Opin. Immunol. 3, 49-55. Podack, E. R., Young, J. D.-E., and Cohn, Z. A. (1985). Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules. Proc. Natl. Acad. Sci. U.S.A. 82,8629-8633. Pollard, J. W. (1991). Lymphohematopoietic cytokines in the female reproductive tract. Curr. Opin. Immunol. 3, 772-777. Pollard, J. W., Bartocci, A., Areci, R., Orlogsky, A., Lander, M. B., and Stenley, E. R. (1987). Apparent role of the macrophage growth factor, CSF-1, in placental development. Nature (London) 330,484-487. Y.)31, Psychoyos, C. A. (1973). Hormonal control of ovoimplantation. Vitam. Horm. (N. 201-256. Rankin, J. C.. Ledford, B. E., and Baggett, B. (1977).Early involvement of cyclic nucleotides in the artificially stimulated decidual cell reaction in the mouse uterus. Biol. Reprod. 17, 549-554. Rathjen, P.D., Toth, S.,Willes, A., Heath, J. K., and Smith, A. G. (1990). Differentiation inhibiting activities produced in matrix-associated and difhable forms that are generated by promoter usage alternate. Cell (Cambridge, Mass.)6, 1105-1 114. Redline, R. W., and Lu, C. Y. (1989). Localization of fetal major histocompatibility complex antigens and maternal leukocytes in murine placenta. Lab. Invest. 61, 27-36. Renegar, R. H., Cobb, A. D., and Leavitt, W. W. (1987). Immunocytochemical localization of relaxin in the golden hamster (Mesocricetus auratus) during the last half of gestation. Biol. Reprod. 37, 925. Ritson, A., and Bulmer, J. N. (1987). Endometrial granulocytes in human decidua react with a natural-killer (NK) cell marker, NKHl. Immunology 62, 329-331. Robertson, S. A., Mayrhofer, G., and Seamark, R. F. (1992). Uterine epithelial cells synthesize granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL6) in pregnant and non-pregnant mice. Biol. Reprod. 46, 1069-1079. Roder, J., and Duwe, A. (1979). The beige mutation in the mouse selectively impairs natural killer cell function. Nature (London) 278, 451-453. Saito, S., Nishikawa, K., Morii, T., Enomoto, M., Narita, N., Motoyoshi, K., and Ichijo, M. (1993). Cytokine production by CDlGCD56bright natural killer cells in the human early pregnancy decidua. Znr. Immunol. 5,559-563. Sananes, N., Weiller, S., Baulieu, E. E., and Le Gooascogne, C. (1978).In vitro decidualization of rat endometrial cells. Endocrinology (Baltimore) 103, 86-95. Selye, H., and McKeown, I. (1935). Studies on the physiology of the maternal placenta in the rat. Proc. R. SOC.London, Ser. B 119, 1-31. Sharma, R., Bulmer, D., and Peel, S. (1986). Effects of exogenous progesterone following ovariectomy on the metrial glands of pregnant mice. J . Anat. 144, 189-199. Slapsys, R. M.,and Clark, D. A. (1982). Active suppression of host-vs-graft reaction in pregnant mice. IV. Local suppressor cells in decidua and uterine blood. J. Reprod. Immunol. 4,355-364. Slapsys, R. M.,and Clark, D. A. (1983). Active suppression of host-vs-graft reaction in

GRANULATED LYMPHOID CELLS OF THE PREGNANT UTERUS

135

pregnant mice. V. Kinetic specificity and in vivo activity of non-T suppressor cells. Am. J . Reprod. Immunol. 3, 65-71. Smith, L. J. (1966). Metrial gland and other glycogen containing cells in the mouse uterus following mating and through implantation of the embryo. Am. J . Anat. 119, 15-24. Smyth, M. J., Ortaldo, J. R., Shinkai, Y.-I., Yagita, H., Nakata, M., Okumura, K., and Young, H. (1990). Interleukin-2 induction of pore-forming protein gene expression in human peripheral blood CD8+ T cells. J. Exp. Med. 171, 1269-1281. Soubiran, P., Zapitelli, J.-P., and Schaf€ar, L. (1987). IL 2-like material is present in human placenta and amnion. J . Reprod. Immunol. 12, 225-234. Starkey, P. M., Sargent, I. L., and Redman, C. W. G. (1988). Cell populations in human early pregnancy decidua: Characterization and isolation of large granular lymphocytes by flow cytometry. Immunology 65, 129-134. Stewart, I. (1987). Differentiation of granulated metrial gland cells in ovariectomized mice given ovarian hormones. J . Endocrinol. 1l2,23-26. Stewart, I. (1988). Granulated metrial gland cells in the non-traumatized regions of the uterus of ovariectomized mice with deciduomata maintained on progesterone. J . Endocrinol. 116, 11-15. Stewart, I.. andMukhtar, D. D. Y.(1988).The killingofmouse trophoblastcells by granulated metrial gland cells in vitro. Placenta 9, 417-425. Stewart, I., and Peel, S. (1980). Granulated metrial gland cells at implantation sites of the pregnant mouse uterus. Anat. Embryol. 160, 227-238. Stewart, I., and Peel, S. (1981). Granulated metrial gland cells in the virgin and early pregnant mouse uterus. J . Anat. 133, 535-541. Stewart, I. J. (1978). A comparative study of the mouse and rat metrial gland cells. J . Anat. 126,410. Stewart, I. J. (1984).A morphological study of granulated metrial gland cells and trophoblast cells in the labrinthine placenta of the mouse. J . Anat. 139,627-738. Stewart, I. J. (1990). Granulated metrial gland cells in the mouse placenta. Placenta 11, 263-275. Stewart, I. J. (1991). Granulated metrial gland cells: Pregnancy specific leukocytes? J . Leukocyte Biol. 50, 198-207. Stewart, I. J. (1993). Leukocytic function in the endometrium. In “Local Immunity in Reproductive Tract Tissues” (P. D. Griffin and P. M. Johnson, eds.), pp. 205-228. Oxford Univ. Press, New Delhi. Stewart, I. J., and Clark, J. R. (1990). Granulated metrial gland (GMG) cells in the uterus of virgin and pregnant short-tailed field voles (Microtus agrestid). J . Reprod. Fertil. 6, 21 (abstr.). Stewart, 1. J., and Peel, S. (1977). The structure and differentiation of granulated metrial gland cells of the pregnant mouse uterus. Cell Tissue Res. 184, 517-527. Stewart, 1. J., and Peel, S. (1978). The differentiation of the decidua and the distribution of metrial gland cells in the pregnant mouse uterus. Cell Tissue Res. 187, 167-179. Tarchand, U . (1986). Decidualisation: Origin and role of associated cells. Biol. Cell. 57, 9-16. Trinchieri, G., and Perussia, B. (1984). Human natural killer cells: Biologic and pathologic aspects. Lab. Invest. 50, 489-513. Vladimirsky, F., Chen, L.. Amsterdam, A., Zor, U . , and Lincon, H. R. (1977). Differentiation of decidual cells in cultured rat endometrium. J . Reprod. Fertil. 49, 61-68. Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Nicola, N. A,, and Gough, N. M. (1988). Myeloid leukemia factor maintains the developmental potential of embryonic cells. Nature (London) 336,684-687. Wislocki, G. B., Weiss, L. P., and Burgose, M. H. (1957). The cytology, histochemistry

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and electron microscopy of the granular cells of the metrial gland of the gravid rat. J. Anat. 91, 130-140. Yamashiro, S., Bast, T., and Croy, B. A. (1989). Failure of the beige genotype to alter the granule morphology of uterine granulated metrial gland cells. Proc.-Annu. Meet, Electron. Microsc. SOC.Am. 47, 980-981. Yochim, J. M., and De Feo, V.J. (1963). Hormonal control on onset, magnitude and duration of uterine sensitivity in rat by steroid hormones of the ovary. Endocrinology (Baltimore) 72, 317-326. Young, J. D.-E., and Cohn, Z. A. (1988). How killer cells kill. Sci. Am. 256, 38-44. Young, J. D.-E., Hengartner, H., Podack, E. R., and Cohn, Z. A. (1986a). Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity. Cell (Cambridge, Mass.) 44, 849-859. Young, J. D.-E.. Leong, L. G., Liu, C.-C., Damiano, A., Wall, D. A., and Cohn, Z. A. (1986b). Isolation and characterization of a serine esterase from cytolytic T lymphocytes. Cell (Cambridge, Mass.) 47, 183-194. Zheng, L. M., Joag, S.V., Parr,M. B., Parr, E. L., and Young, J. D.-E. (1991a). Perforinexpressing granulated metrial gland cells in murine deciduomata. J. Exp. Med. 174, 1221-1226. Zheng, L. M., Liu, C.-C., Ojcius, D. M., and Young, J. D.-E. (1991b). Expression of lymphocyte perforin in the mouse uterus during pregnancy. J . Cell Sci. 99, 317-323. Zheng, L. M., Ojcius, D. M., Liu, C.-C., Krarner, M. D., Simon, M. M., Parr, E. L., and Young, J. D.-E. (1991c). Immunogold labeling of perforin and serine esterases in granulated metrial gland cells. FASEB J. 5 , 79-85. Zheng, L. M., Ojcius, D. M., and Young, J. D.-E. (1991d). Role of granulated metrial gland cells in the immunology of pregnancy. Am. J. Reprod. Immunol. 25,72-76. Zheng, L. M.,Ojcius, D. M.. and Young, J. D.-E. (1993). Perforin-expressing cells during spontaneous abortion. B i d . Reprod. 48, 1014-1019. Zuckermann, F. A., and Head, J. R. (1987). Murine trophoblast resists cell-mediated lysis. I. Resistance to allospecific cytotoxic T lymphocytes. J . Immunol. 139,2056-2064. Zuckermann, F. A., and Head, J. R. (1988). Murine trophoblast resist cell-mediated lysis. 11. Resistance to natural cell-mediated cytotoxicity . Cell. Immunol. 116, 274-286.

The Replication Band of Ciliated Protozoa' Donald E. Olins and Ada L. Olins The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and The Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

1. Introduction

"The observations show that the DNA content of the macronucleus doubles during reorganization and that DNA synthesis takes place only at or near reorganization bands. Furthermore, there is reason to believe that a basic protein component, presumably histone, is duplicated simultaneously with the DNA" (Gall, 1959, p. 296). This important conclusion about a substructure within the macronucleus of the ciliated protozoan Euplotes eurystornus was based on a light microscopic autoradiographic analysis of the sites of [3H]thymidineincorporation into live cells, and on a spectrophotometric analysis of specifically stained nuclei. Gall's classic study represents the turning point in a field that had fascinated biologists since the middle of the nineteenth century. The purpose of this chapter is to convey present-day concepts as well as an appreciation of the basis of continued fascination with the replication band (RB). Although RBs are principally confined to a special group of animals (viz., hypotrichous ciliated protozoa), they offer the potential to answer fundamental questions concerning eukaryotic chromatin replication. Ciliated protozoa (Phylum Ciliophora) constitute a remarkably diverse group of unicellular animals (Nanney, 1980). Their complex cortical structure with organized cilia and an oral apparatus has attracted considerable study and constitutes the major morphological criteria for constructing phylogenetic relationships (Small and Lynn, 1985; Lynn and Corliss,

'

This review is dedicated to the memory of Dr. Richard F. Kimball, who died on January 4, 1993. In collaboration with Professor David M. Prescott, Richard Kimball made some of the key early observations on the behavior of replication bands. Inrernurionol ReuLw of Cylology, Vol. 153

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Copyright 0 1994 by Academic Press, Inc. All righls of reproduction in any form reserved.

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1991). Besides the characteristic cortical structures, ciliates possess an additional distinguishing feature, nuclear dimorphism. Two types of nuclei coexist within a single cell body: micronuclei (MIC), constituting the germline; and macronuclei (MAC), the transcriptionally active nuclei of the vegetative cells. Macronucleiare generated from micronucleifollowing the sexual mechanism of conjugation; subsequently the old macronuclei are discarded. Despite these overall similarities, it is becoming clear that the evolutionary divergence among major classes of the ciliates may be quite considerable. A summary of present concepts of the phylogenetic relationships among contemporary ciliates based upon comparison of nucleotide sequences in large rRNA has been published recently (BaroinTourancheau et al., 1992). A similar comparison has been based upon analysis of SS-rRNA sequences (Nanney et al., 1991). From the point of view of this chapter, it is sufficient to note that the hypotrichs are believed to have diverged somewhat late in ciliate evolution during a major diversification of unicellular body plans. The hypotrichous ciliates can be distinguished from other ciliates by their possession of extensive rows of fused cilia in the oral membranelles and by the existence of cirri (also fused cilia) positioned on the ventral surface of cells. The hypotrichs can often be observed “walking” on surfaces in their aquatic world, propelled by the beating cirri. Another major distinction of the hypotrichs, compared with other ciliates, is their unique MAC genomic organization, which consists of highly endoreplicated, short, linear, gene-sized DNA molecules (Kraut et al., 1986; Klobutcher and Prescott, 1986; Raikov, 1989; Prescott, 1992). These macronuclear DNA molecules are highly streamlined single coding regions, flanked by nontranscribed regions and terminated with species-specific, simple, repetitive nucleotide sequence telomeres. Although numbers vary among the different macronuclei of hypotrich species, estimates range within the followingbounds: (1) DNA lengths, 0.5 to 20 kb, most molecules about 2-2.5 kb; (2) number of different types of DNA molecules in a macronucleus, lo4 to 4 X lo4; (3) number of copies of each DNA type, lo3 to lo6 copies per macronucleus; (4) total number of DNA molecules per macronucleus, lo6to lo8molecules. In situ hybridization studies (Pluta and Spear, 1981; Olins and Olins, 1993) indicate that the numerous gene copies are scattered randomly throughout the MAC. It is obvious that the macronucleus does not possess true chromosomes. There is no compelling evidence for kinetochores or an extensive spindle apparatus. Nuclear division appears to occur by a statistical partitioning of DNA molecules into the daughter nuclei (Witt, 1977), an amitotic mechanism. The existence of macronuclear replication bands represents an additional characteristic feature of hypotrichs. RBs can be readily visualized in stained cells by light microscopy (Fig. lA), accounting for their rela-

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FIG. 1 (A) Acetocarmine-stained Euplores eurystomus, revealing the single micronucleus (arrowhead) and the single elongate macronucleus with two RBs (thin arrows). In Euplores the RBs progress from the tips of the macronucleus, eventually fusing about midway. Bar = 10 pm. (B) Probably the earliest drawing of Euplores with RBs (Stein, 1859). The micronucleus is not shown, but the macronucleus (labeled "n") is represented as an elongate structure. The apparent emptiness of the spalffiirmige hohle is clearly indicated.

tively long history of investigation. Their existence is apparently not completely unique to hypotrichs, however. Light and electron microscopic studies have indicated their presence in the macronuclei of Halteria (Order Oligotrichida), and Spirochona and Chlarnydodon (Order Cyrtophorida), among several other species (Raikov, 1978). Macronuclear gene-size DNA molecules have also been identified in Halteria and the cyrtophorid Trithigmostoma (Metenier and Hufschmid, 1988). Direct demonstration (by labeled nucleoside incorporation into RBs) that these are the sole (or major) macronuclear sites of DNA synthesis has only been accomplished with the hypotrichs. Our laboratory has focused attention on the RBs of the hypotrich Euplotes eurystornus (Fig. 1A), the same organism examined by Gall (1959) and most other investigators in this field. RBs have been explored in a number of other hypotrichs, including representatives of Stylonychia, Oxytricha, Urostyla, and Aspidisca (Raikov, 1978).

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140 II. Early History: Prior to 1959

Microscopic observations of the ciliates have a long and venerable history beginning at the end of the seventeenth and early eighteenth centuries with A. van Leeuwenhoek (1632-1723) and L. Joblot (1645-1723). Contributors during the early-to-mid nineteenth century include C. G. Ehrenberg (1795-1876), F. Dujardin, 0. F. Muller, and A. Pritchard, among others. However, the earliest observations of what we now know as RBs can probably be attributed to Stein (1859). He presented a description and drawing of “Euplotes patella” (Fig. 1B) which is remarkably similar to more modern presentations. Der sehr lange stangformige Nucleus liegt auf der linken seite in der Nahe des Mittelfeldes, seine beiden Enden sind rechtwinklig nach innen umgebogen: das hintere umfasst den Hinterrand des Mittelfeldes. Zuweilen zeigte sich kurz vor den beiden Enden eine quere spaltformige Hohle. The very long, pole-shaped nucleus lies on the left side near the centralfield, both ends bend at right angles towards the interior: the posterior surrounds the posterior edge of the central-field. Occasionally, a transverse crevasse-shaped cavity becomes visible a short distance from the ends. (pp. 135-136)

This description was written 100 years before the determination of the true function of RBs (Gall, 1959). It was, of course, impossible for Stein to have guessed the significance of the spaltfiirrnige hohle, since a realistic understanding of the nucleus as the vehicle for heredity did not evolve until the end of the nineteenth century, with the studies of Hertwig, Strasburger, Flemming, and others. That the spaltfiirmige hohle aroused no particular interest until the twentieth century is clear from an examination of the Manual of the Znfusoria (Kent, 1881), in which the earlier illustrations of Stein were redrawn, including the macronuclear substructure; however, they were not otherwise described or commented upon. Griffin’s (1910) investigations on “Euplotes worcesteri” contain beautiful illustrations (Fig. 2A) and display a clear beginning of an understanding of RBs. These nuclear substructures were viewed in the context of preparation of the cell for division: RECONSTRUCTION OF THE MEGANUCLEUS By this is meant that a progressive change occurs in which all the chromatin of the meganucleus is actually dissolved and then reconstructed. The first state of this process is the appearance at each end of the cord-shaped meganucleus of a band in which there is a complete absence of the ordinary chromatin reticulum. It will be convenient to refer to these as the reconstruction bands. They pass rapidly from the ends of the nucleus toward the center, finally meeting, and then disappearing. Each band consists of two planes of about equal thickness, the one on the central side staining darkly and uni-

17

19 20

22

23

24

FIG. 2 Early drawings of Euplores macronuclei with RBs. (A) Grimn (1910) clearly represents the two compartments of RBs, and illustrates a difference in the texture of chromatin in front of and behind RBs. Postmitotic micronuclei are also shown. (B) Yocum (1918) also shows the compartments of RBs. Included is a sketch of a micronucleus in mitosis. ( C ) Turner (1930) presents very detailed drawings of macronuclei with RBs, emphasizing the texture of condensed chromatin granules in front of and behind RBs, the elongate and irregularly shaped nucleoli, and the two parts of RBs.

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formly, while the other is not stained and consequently shows distinctly. (P. 316) It would seem that during the ordinary life and activities of the cell, the chromatin either accumulates a certain amount of inert substance which can play no part in the activities of division, and which it would be useless, perhaps harmful, to carry over to the daughter cells; or else that a portion of the chromatin itself is so modified by its activities that it loses some of the properties essential to its sharing in division, and therefore is eliminated before or during that process. (p. 319) The reconstruction of chromatin by complete solution and reformation, such as occurs in Euplotes, is aprocess of a higher order . . . . the possibility exists that the new chromatin may be entirely composed of new material derived from the cytoplasm. (p. 319)

Griffin clearly noted the two zones of RBs which will be discussed later, and obviously believed that the chromatin reconstruction process was an orderly phenomenon. Several years later, Yocom (1918), in a study of “The Neuromotor Apparatus of Euplotes Patella,” discussed division of the macronucleus in the context of his own and Griffin’s prior observations (1910). Yocom renames the macronuclear substructure and interprets its significance in terms of contemporary observations and writings in the areas of differentiation and senescence.

. . . in place of the terms reconstruction bands as given by Griffin (1910) we would suggest ‘reorganization bands,’ indicating a purely physical reorganization of the nuclear material through a contraction of the chromatin, rather than a solution of the chromatin in the karyolymph. (p. 370) . . . we assume that at the time of division the cytoplasm of Euplotes has undergone certain dedifferentiatingchanges and has assumed a physiological young condition with a greatly increased metabolism. Such being the case it would be necessary for the macronucleus, the important vegetative organelle of the animal, to undergo corresponding changes of dedifferentiation and rejuvenescence in order to properly control the rejuvenated cytoplasm. These changes in the nucleus are undoubtedly of two kinds, physical and chemical. The physical changes we are able to witness, while the only evidence of the chemical changes is the difference in staining reactions, which with the stains we have used tell us practically nothing of the nature of this very important phase of the whole reorganization process. (p. 372) (Yocum, 1918) Yocom’s drawings of RBs do not appreciably improve upon those by Griffin (Fig. 2B). Turner (1930), on the other hand, presents remarkably accurate and detailed camera lucida drawings of macronuclei and RBs in Euplotes patella (Fig. 2C). In addition, he includes some of the earliest photomicrographs of these structures. His descriptions of the two zones of RBs, still using the names suggested by Griffin (i.e., “solution plane,” now referred to as forward zone or FZ; “reconstruction plane,” now rear zone or RZ) are so excellent as to be quite valid today (e.g., see p. 203 of Turner). He

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continues to invoke the vague concept of “rejuvenation.” Turner reviews the earlier literature by Griffin, Yocom, and others. However, his interpretation of the significance of RBs had not evolved beyond that of Yocom (1918). During division a double reorganization band passes through the C-shaped macronucleus from each tip to the middle, where the two bands unite and disappear immediately before macronuclear contraction which precedes fission. It is suggested that these reorganization bands cause a phase reversal of a colloidal system, in which the chromatin changes from the continuous (reticulum) to the dispersed (granular)phase, comparable to the change within the micronucleus. This probably has a rejuvenating influence upon the macronucleus and consequently upon the cytoplasm (Turner, 1930, p. 229)

No further contributions to our understanding of the function of RBs appears between Turner (1930) and Gall (1959). However, in 1957 two groups published the first ultrastructural studies of RBs (Faurk-Fremiet et al., 1957; Roth, 1957); these investigations will be described in a later part of the chapter (see Section IV). The breakthrough observations by Gall were made possible by technical advances in autoradiography (Doniach and Pelc, 1950), Feulgen staining for DNA (Swift, 1955), and the alkaline fast green staining method for histones (Alfert and Geschwind, 1953). Employing these methods, Gall was able to convincingly demonstrate DNA synthesis in the RB, with both the DNA and histone concentration duplicated in its wake. At this time (1959) there was no understanding that the macronucleus of hypotrichs was composed of endoreplicated gene-sized DNA molecules. Gall wisely avoided interpreting the pattern of replication in terms of specific chromosome arrangements. The name “replication band” was suggested by Kimball and Prescott (1962). It was, perhaps, a convenient poetic quirk of history that permits us to employ the abbreviation “RB” for the macronuclear band of “reconstruction,” “rejuvenation, ” “reorganization,’’ or “replication. ”

111. Functional Characteristics of Replication Bands in Cells

Much of our present understanding of RB in uiuo behavior and physiology derives from early investigations by D. M. Prescott and co-workers, including R. F. Kimball (Prescott and Kimball, 1961; Kimball and Prescott, 1962; Prescott et al., 1962; Prescott, 1966; Evenson and Prescott, 1970a,b). Employing techniques (i.e., microscopic autoradiography of incorporated [3H]thymidine,uridine, and amino acids, and cytochemical stains for

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DNA, RNA and protein) extending beyond Gall’s methods (1959), the papers by Prescott and co-workers amved at numerous conclusions, most of which remain valid. In their culture conditions, Euplotes eurystomus exhibits a doubling time of about 14 hr. MAC G I lasts about 3 hr (i.e., cytokinesis being denoted to). The S-phase, with diagnostic RBs, lasts for 8-10 hr; G, plus cell division, about 1 hr. MIC DNA synthesis begins as the MAC S-phase ends; the MIC S-phase terminates before amitosis and cytokinesis (Prescott et al., 1962). An important conclusion of this study is that MACs and MICs appear to be under independent control, in terms of initiating DNA replication. Ringertz and Hoskins (1965)obtained somewhat different cell cycle parameters for Euplotes eurystomus: for a generation time of 20 hr, G, was 5 hr; S, 12.5hr; G,, 2.5 hr. Despite this difference, there was agreement that in growing Euplotes, the S-phase with RBs constitutes the largest proportion of the cell cycle. Staining methods show that the two parts of RBs (the FZ and RZ) have distinctly different properties: DNA and protein could be detected in the FZ, but not in the RZ; RNA could not be detected in either zone of RBs (Prescott and Kimball, 1961). Autoradiographic studies indicated that the leading portion of the RZ was the locale of DNA synthesis. RNA synthesis occurred throughout the MAC, except in RBs. Protein synthesis measured by histidine incorporation was primarily confined to the RZ (Prescott and Kimball, 1961). This basic amino acid is probably indicating deposition of nascent histones into RBs. This conclusion is consistent with later measurements of protein and histone synthesis in biochemical amounts of synchronized Euplotes (Prescott, 1966). Two studies (Prescott, 1966; Evenson and Prescott, 1970a) examined the bulk synthesis of DNA, RNA, and protein during the cell cycle in Euplotes: DNA and histones were not appreciably synthesized during G, , but were synthesized at approximately constant rates throughout the Sphase; total protein was synthesized constantly during the entire cell cycle; a burst of RNA synthesis occurred during GI, declining through the remainder of the cell cycle. The apparent constant rate of DNA and histone synthesis during the S-phase is somewhat contradictory to studies on the rate of RB movement (Ringertz and Hoskins, 1%5), which estimate that the RB of Euplotes eurystomus picks up speed (i.e., ca. 4-fold) during the last third of S. In our laboratory, autoradiographic analysis of [H3]thymidineincorporated into RBs ( O h and Olins, 1993), as well as immunochemical detection of incorporated bromodeoxyuridine (BrdU) (Fig. 3), appear to indicate a wide range of synthesis rates in RBs that are in similar parts of the S-phase. Clearly, the issue of bulk versus microscopic rates of DNA, RNA, and protein synthesis requires further investigation. Concerning the question of RNA synthesis and RBs, ultraviolet microspectrophotometric

FIG. 3 DNA synthesis and gene distribution within the macronucleus of Euplores eurystomus. (A) I n uiuo incorporation of ['Hlthymidine into late S-phase nucleus, detected by autoradiography. (B) I n siru hybridization of an ['Hlthymidine-labeled 5s RNA gene probe

with a macronucleus, illustrating the random distribution of endoreplicated genes (see Olins and Olins, 1993, for details). (C) I n uiuo incorporation of BrdU into RBs, detected with a monoclonal antibody against BrdU. This unusual macronuleus has three RBs. (D) Phase micrograph of permeabilized cell shown in (C). (E) and (F) I n uitro incorporation of biotindUTP into RBs, detected with antibiotin antisera (see Olins and O h , 1987, for details). The thin arrows indicate the positions or RBs. Bars = 10 pm. A and B are at identical magnifications; C and D are alike; E and F are the same magnification.

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measurements of fixed Euplotes MACs with (or without) prior RNAse treatment showed that the FZ contains more protein and RNA than the RZ (Salvano, 1975). This apparent evidence for some RNA in RBs is at odds with the earlier cytochemical and autoradiographic studies and awaits some explanation. Electron microscopic autoradiographic studies have confirmed the localization data from light microscopy and furnished information at somewhat higher resolution. [H3]Thymidineincorporation was observed to be in the RZ, often at the junction between the FZ and RZ (Stevens, 1963; Evenson and Prescott, 1970b; Lin and Prescott, 1985).RNA synthesis, on the other hand, was principally observed over nucleoli and surrounding condensed chromatin bodies (Stevens, 1963). RBs normally begin almost simultaneously in both tips of a MAC (Kimball and Prescott, 1962). Most MACs have two RBs, with abnormal RB patterns seen at a frequency of about (Evenson and Prescott, 1970b). These normal rules of RB behavior could be disrupted by heat shocking cells during the S-phase (Evenson and Prescott, 1970b). Within 15 min of heat shock (i.e., ca. 35-36”C), RBs cease thymidine incorporation. Cells heated for longer periods (e.g., 6.5 hr at 35.3”C) exhibited a plethora of abnormally active RBs during subsequent recovery from the shock. The frequency of abnormal RB patterns increased about lO’-fold. Many of the original RBs regained activity, but new RBs often developed. RB migration was frequently backward and numerous cells revealed more than two RBs. Heat shock clearly produced profound effects on the normal behavior of RBs. A number of other factors have also been shown to affect the activity of RBs. Ringertz and Hoskins (1965) observed inactive RBs (i.e., no [H’lthymidine incorporation into clearly visible RBs) with very slowgrowing cultures of Euplotes. Our laboratory has recently demonstrated (Olins and Olins, 1993) that, in addition to heat shock, inactive RBs were observed following cell crowding (e.g., ca. 5-10 x lo3 celldml), aphidiColin treatment, various CAMP-phosphodiesterase inhibitors (i.e., caffeine, theophylline, and 3-isobutyl-l-methylxanthine,IBMX) (Fig. 4) and the Ca2’ -calmodulin inhibitor, trifluoperazine. Inhibition of RB synthetic activity by these various treatments undoubtedly involves more than one mechanism. For example, aphidicolin is known to have direct inhibitory effects on DNA polymerases a and 6 (Sheaff et al., 1991). Cell crowding, on the other hand, is likely to operate by more indirect effects. It is interesting to note that conditions of high cell concentration and starvation promote secretion of mating-type pheromones (“gamones”; see Kusch and Heckmann, 1988) in the freshwater Euplotes octocarinatus. In contrast to studies demonstrating suppression of RB activity, there has been little reported success in the premature induction of RBs. Removal of the

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Dilutekrowded

Aphidicolin

'001

r-, ++ + + Theoph ylline

lM)1

Heatshock 60 20

IBMX

I

I

60

fli

20

Caffeine

r-1 # I I

t

I

I

1

1

60

I

20

I

3H-dT Incorporation FIG. 4 Influence of culture conditions and added chemicals on in uiuo activity of Euplores RBs, as determined by ['Hlthymidine incorporation and autoradiography. Data are presented as histograms of the percentage of RBs with varying levels of RB replication activity. In all situations, the control RB activity is represented by solid lines, the experimental group by dashed lines. In the case of the crowded versus dilute cells, the dotted line represents a group of cells diluted after inhibition by crowding, illustrating a partial recovery of replication activity. Notice the profound inhibitory effects upon in uiuo activity by the various added chemicals. Conditions for the various treatments were aphidicolin, 1 pg/ml, 2 hr; heat shock, 36.5"C, 90 min; theophylline, 5 m M , 16 hr; IBMX, 2 m M , 16 hr; caffeine, 2.5 mM, 16 hr. Complete details of these and other experiments can be found in Olins and Olins (1993).

micronucleus during G I in Euplotes octocarinatus by micromanipulation (Mikami et al., 1985) does not prevent the subsequent Gl/S transition and the appearance of (presumably active) RBs; but cells were blocked in the following G I . Reimplantation of a micronucleus into the blocked cells permitted a resumption of the Gl/S transition, suggesting an undefined MIC activity in MAC DNA synthesis. One brief report (YOW, 1961) claimed that microsurgical removal of "locomotor organelles" (i.e., cim)

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DONALD E. OLlNS AND ADA L. OLlNS

induced the appearance of active RBs. This abstract was never followed by a detailed publication, nor followed up by other investigators, but it does suggest that chemical deciliation might be attempted to bring this observation to a biochemical level of study. Despite the high degree of organization of DNA replication in RBs, after the S-phase is completed there appears to be considerable mixing of MAC DNA prior to cytokinesis (Kimball and Prescott, 1962). This conclusion was based upon the relatively uniform dispersal throughout MACs of DNA synthesized during short pulses. Thus the MAC appears not to distinguish between DNA synthesized during different times in S, in contrast to higher eukaryotes (see Section VI). However, this conclusion has been challenged by Bonifaz and Plant (1974). These authors followed the fate of pulse-labeled DNA for several generations and concluded that DNA molecules that are replicated simultaneously return to be replicated simultaneously in the alternate generation. This phenomenon has not been pursued further and is especially puzzling in view of present concepts that the endoreplicated MAC DNA molecules of any particular gene are scattered randomly throughout the entire nucleus (see Fig. 3B and Olins and Olins, 1993). The behavior of RBs has been most thoroughly studied in the hypotrich Euplotes eurystomus, which constitutes a paradigm for all ciliates with RBs. During normal rapid vegetative cell growth, cells of a critical size (Ringertz and Hoskins, 1965) initiate RBs at species-specific MAC locations. Once initiated, RBs migrate through the entire MAC, accomplishing a normal duplication of DNA and histones. At the microscopic level, abnormalities of behavior are only rarely observed. Recognition of these properties leads to the formulation of several fundamental questions, none of which can be adequately answered at this stage of knowledge in the hypotrich field: (1) What mechanism determines the reproducible location in MACs for initiation of RB? (2) What is the propelling force for RB movement? (3) What permits RBs to remain organized and localized during the entire length of the S-phase? A full appreciation of this third question, and at least one possible answer, requires a summary of the numerous electron microscopic studies of hypotrich RBs.

IV. Replication Band Ultrastructure

The earliest electron microscopic studies of RBs (FaurC-Fremiet et al., 1957; Roth, 1957) were published prior to Gall’s autoradiographic investigations (Gall, 1959). FaurC-Fremiet et al. (1957) were evidently unaware of Gall’s observations; Roth (1957) clearly knew of the observations before publication. The studies by FaurC-Fremiet et al. provide a more complete

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149

description of the chromatin within RBs than those of Roth. Their primary focus, however, appears to have been in disproving the earlier suggestions by Griffin (1910) that the forward zone of the RB (termed, “solution plane” by Griffin) was not a colloidal solution, but instead was a reticulum of -35 nm filaments. The rear zone (“reconstruction plane”) consisted of even finer filamentous structures of about 8 nm diameter. Roth (1957) also observed up to 40-nm filaments within the FZ, but did not observe structures within the RZ. Roth, aware of Gall’s observations, speculates that “DNA replication cannot take place in the granular state of the macronucleus” (Roth, 1957, p. 996). Since the replication band represents a profound change of state of the MAC chromatin, it is worthwhile detailing the typical ultrastructure of a vegetative MAC. These were accurately described in the earliest electron microscopy studies (FaurC-Fremiet et al., 1957; Roth, 1957; Kluss, 1962). Most of the Eupfotes MAC is composed of three categories of structures: (1) spheroidal condensed chromatin granules (CG) varying in size from about 0.3 to 1 .O pm in diameter, composed of tightly packed fibrils about 5-10 nm and estimated to be about 10-20 x lo3 CG per MAC (Kluss, 1962; Ringertz et u f . , 1967; A. L. Olins et a f . , 1981, 1988); (2) irregularly shaped nucleoli, with diameters similar to CGs, often with multiple cavities and composed of a granular substructure (Kluss, 1962; Ringertz et a f . , 1967; A. L. Olins et al., 1981,1988);and (3) diffuse chromatin, ameshwork of irregularly shaped fibrils and particles in between CGs. Several studies have reported the existence of helical particles within the diffuse chromatin (Kluss, 1962; Murti, 1976; Ruffolo, 1979); these are presumed to represent a unique form of RNP packaging. The ultrastructural perturbations observed in the advancing RB are primarily confined to two categories. The CGs are remodeled and the diffuse chromatin disappears. Nucleoli apparently remain largely unaffected throughout the entire RB. Following passage of the RB and chromatin duplication, the general ultrastructure reverts to its original appearance. In other hypotrichs the condensed chromatin of MACs may be less spheroidal. In Gastrostyla steinii, for example, the condensed chromatin appears more thread-like, with a variable thickness (Walker and Goode, 1976). Similar thick chromatin threads are described in Urosfyfugrundis (Inaba and Suganuma, 1966). Ultrastructural investigations of RBs have now been performed on a variety of hypotrichs, yielding a generally consistent set of descriptions and dimensional measurements of the FZ and RZ (Table I and Fig. 5 ) . Most studies have involved staining sections of embedded cells with uranyl and lead salts, which contrasts nucleic acids and proteins. In one abstract (Phegan and Moses, 1967) and in recent studies from our group (A. L. Olins et al., 1988; Olins and Olins, 1990), RB ultrastructure has also been examined by employing nucleic acid-specific staining.

DONALD E. OLINS AND ADA L. OLINS

150 TABLE I Chromatin Fiber Dimensions in RB Zones

Species Euplotes patella Euplotes eurystomus

Oxytricha platystoma Oxytricha fallax Oxytricha hymenostoma Oxytricha sp. Stylonychia mytilus

Kakliella acrobates Gastrosiyla steinii Urostyla grandis

Number of MACs/cell 1 1

2 2 2 2 2

2 4 40-80

FZ(nm) RZ(nm) 540 -35 11 20-30 14 -40 47 40-50 50-60

-42' -58' 58

-606 43' -60 -55 -45 -70

References

-

Roth (1957) Fad-Fremiet et al. (1957) Kluss (1962) Phegan and Moses (1967) Ringertz et al. (1%7) Ruff010 (1978)" Olins et al. (1981) A. L. Olins et al. (1988) Chakraborty (1%7) Grimes (1973)" Banchetti et al. (1980)" Olins et al. (1981) Walker and Goode (1976)" Olins et al. (1981) Kaul and Sapra (1991)o Fleury et al. (1985)" Walker and Goode (1976)' Inaba and Suganuma ( 196616

-8

-

8-20

-

2-10 6

10

-

-10

" Estimated from the published micrographs; fiber dimensions were not supplied by the authors. Atypical RB. ' Data include normal and atypical RBs. In our view, the authors reversed their designations of FZ and RZ.We estimated FZ fiber dimensions from their published micrograph. Several general conclusions can be derived from the observations and the measurements: (1) The FZ is composed of distinct chromatin fibers. (2) The FZ fibers are thicker and easier to visualize than the thinner filaments of the RZ. (3) The junctions between the pre-RB chromatin and the FZ, and between the FZ and RZ are fairly well defined, and span the entire diameter of the MAC.(4) CGs gradually re-form at the rear portion of the RZ, first forming small granules, then enlarged ones. ( 5 ) The nuclear envelope remains intact throughout the RB. Most measurements have been performed on the RB of Euplotes eurystomus. Emphasizing the most recent determinations of RB fiber diameters (Ruffolo, 1978; A. L. Olins et al., 1981, 1988) yields the following nominal values: FZ fibers, 4050 nm; RZ fibers, about 10 nm. The few measurements of Stylonychiu ~

~~

FIG. 5 Thin-section election microscopy of normal and atypical RBs, visualized after uranyl

and lead staining. (A) Normal RB of Euplotes eurystomus illustrating the FZ (large arrowhead) with thicker chromatin fibers than observed within the RZ (small arrowhead). Notice the irregularly shaped nucleoli within the FZ and the progressive increase in the diameters of condensed chromatin granules within the RZ. (B)Atypical RB from heat-shocked Euplotes

eurystomus, demonstrating the disappearance of the RZ and the persistence of the FZ.

(A) and (B), bar = I pm. (C) Stereo electron microscope images of an atypical RB within a macronucleus of Stylonychia mytilus, reprinted from Olins et al. (1981). Notice the remarkable regularity of the -50 nm FZ chromatin fibers. Bar = 0.2 pm.

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DONALD E. OLlNS AND ADA L. OLlNS

RB fibers fall in the same range of dimensions (Olins et al., 1981). The chromatin fibers within the FZ of Oxytricha RBs appear to be slightly thicker, and might be described as being 50-60 nm in diameter. In our experience, there are also qualitative differences among the RBs of different species; these are especially notable with respect to FZ chromatin fibers. The 40-50-nm fibers of Stylonychia appear as separate units spanning the FZ from front to rear. Those of Euplotes appear to formjunctions, creating a network of 40-50-nm chromatin fibers. The FZ fibers of Oxytrichu seem more irregular and less like continuous fibers. Atypical replication bands have been observed in thin sections of intact, untreated hypotrich cells (Walker and Goode, 1976; Banchetti et al., 1980; Olins et al., 1981), and in cysts of Oxytrichafallax (Grimes, 1973). In such RBs, the RZ is absent and the FZ chromatin fibers connect to condensed chromatin granules on both sides of the RB (Fig. 5B,C). Adverse culture conditions can generate a similar morphologic picture. For example, Stylonychia mytilus grown in 60% D20 reveal clear images of atypical RBs (Walker and Goode, 1976). We now suspect that these atypical RBs are nonfunctional. Ultrastructural studies from our laboratory using Euplotes eurystomus (A. L. Olins et a/., 1988; Olins and Olins, 1993) indicate that when replicational activity is inhibited by heat shock, by cell crowding, and by CAMP phosphodiesterase inhibitors (i.e., caffeine, theophylline, and IBMX), the RZ disappears and the FZ chromatin fibers persist for a somewhat longer time, yielding an image of the atypical RB. Heat-shocked Sfylonychiu mytilus exhibits a similar ultrastructural disappearance of the RZ (Kaul and Sapra, 1991), but experiments were not performed to demonstrate inhibition of DNA synthesis. Thus, it is likely that in those cases observed with untreated cells or after growth in D,O, the atypical RBs are probably not functional in replication. The ultrastructural studies verify a remarkable degree of organization within RBs, with clear stratification between the FZ and RZ, and the preRB chromatin and the FZ. This is all the more amazing when it is recalled that MAC DNA is composed of short gene-size molecules. One possible basis for this architectural stability is the recent electron microscopic demonstration in Euplotes eurystomus MACs of 10-nm nonchromatin filaments (Fig. 6) contained within CGs and the forward zone of RBs, and occasionally attached to the nuclear envelope (Olins and Olins, 1990). Demonstration of the 10-nm filaments involved the use of a cytoskeleton stabilizing buffer and fixation with glutaraldehyde-tannic acid, followed by OsO,. These procedures suggest, but do not prove, a relationship to cytoskeletal elements. That the filaments are not DNA was demonstrated by their absence of staining with the Feulgen electron microscopic stain for DNA, osmium ammine (A. L. Olins et al., 1988; Olins and Olins, 1990). These 10-nm filaments appear either as isolated elements or as

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FIG. 6 Stereo electron microscope images demonstrating 10-nm filaments distributed throughout macronuclear condensed chromatin (A) and in the RB region (B). Permeabilized cells were fixed with glutaraldehyde-tannic acid and postfixed with Os04, conditions necessary for visualization of these filaments; see Olins and Olins (1990) for details. Thin arrows point to a few of the filaments. Notice that the 10-nm filaments appear to be inside and parallel to the 40-50 nm FZ chromatin fibers. Bar 0.2 pm.

154

DONALD E. OLINS AND ADA L. OLINS

parallel bundles within CGs. They reorient in the RB and are observed running down the center of the FZ 40-50 nm chromatin fibers. Unfortunately, they have not been characterized further, nor have they been demonstrated in other hypotrich RBs. Discussion of the possible role of these 10-nm nonchromatin filaments in organizing the MAC genome and stabilizing the RB will be reserved for a later section (i.e., Section VI).

V. Cytochemical and lmmunochemical Studies on Replication Bands

The remarkable stratification of RB structure, involving a lateral amplification of simultaneous events in the chromatin replication process, forms the basis for exploiting its advantages for cytochemical and immunochemical microscopy studies. The dimensions of the RB in Euplotes eurystomus (i.e., ca. 5 p m in length, including FZ and RZ, by ca. 10-15 p m diam.) are convenient for careful analysis with the light microscope. Indeed, the early studies of Gall (1959), Prescott and Kimball(1961),and Kimball and Prescott (1962) employed general cytochemical stains for DNA, RNA, and protein. These studies demonstrated a doubling of DNA and proteins by the RB, a DNA- and protein-rich FZ, and a DNA- and proteinpoor RZ. We initiated cytochemical studies to explore whether proteins of the RB can be demonstrated to be qualitatively different than those of the surrounding chromatin. Two staining techniques (i.e., reaction with AgN03 in the presence of formaldehyde, and reaction with the thiolspecific reagent, coumarin phenylmaleimide, CPM) revealed clear and intense reactions with the RBs of MACs isolated from Euplotes eurystomus (Fig. 7), Stylonychia mytilus, and Oxytricha noua (Allen and Olins, 1984). Strong reactions were also observed with the numerous nucleoli, but not nonRB chromatin. The reactions with RBs and nucleoli were demonstratedto result from trypsin-sensitiveproteins. Although the chemistry of silver staining is extremely complex and not easily interpretable, the staining by CPM clearly demonstrated high concentrations of protein thiol groups within RBs. This latter assay with CPM (or, more recently, iodoacetamidofluorescein,IAF) has proved to be a useful diagnostic tool for developing biochemical methods of RB enrichment (Allen et al., 1985). The silver staining technique has also been of use, but requires care to prevent overstaining. The silver staining procedure was also adapted to electron microscopy, further establishing that specific reactions were with RBs and nucleoli, but not with condensed chromatin granules (A. L. Olins et al., 1988). Analysis of the nuclear proteins in Euplotes eurystomus responsible for

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FIG. 7 Cytochernical staining of RBs in the macronuclei of Euplores eurystomus. (A) Reaction with AgNOl in the presence of formaldehyde. (B)Reaction with coumarin phenylmaleimide. revealing the distribution of reactive thiol groups. Thin arrows point to RBs, probably the FZ regions. Bars = 10 pm. For additional details, see Allen and Olins (1984) and A. L. Olins e f al. (1988).

both types of staining procedures has not received much attention. At present, it is known that the silver staining proteins of the RB are acidextractable and therefore may be basic proteins. The thiol-rich RB proteins, on the other hand, resist acid extraction. SDS-PAGE of total MAC proteins after reaction with CPM reveals numerous labeled peptides. Perhaps a combination of chemical labeling and enrichment of RBs (Allen et al., 1985) will narrow down the candidates of thiol-rich proteins of the RB. Immunochemical methods have the clear potential advantage of localizing specific proteins of known structure or function into regions of the RB, establishing correlations with a stage in the chromatin replication process. Anyone familiar with antibody staining procedures can appreciate that these are often precarious methods fraught with artifacts. Much of the recent effort of our laboratory has been an attempt to map proteins into the RB. At various times, we have employed mono- or polyclonal

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DONALD E. OLINS AND ADA L. OLINS

antibodies directed against Euplotes or heterologous proteins. Immunostaining of Euplotes is technically quite different from analogous experiments on tissue culture cells. For example, Euplotes (and most ciliates) does not attach to glass slides during growth. Consequently, cellular materials must be fastened to subbed slides. In addition, Euplotes eurystornus is a large cell (ca. 150 p m long x ca. 75 p m wide x ca. 20-30 p m thick) covered with cilia and cirri, and often filled with stored food (e.g., the alga Chlorogoniurn elongaturn). This generates considerable endogenous fluorescence, especially if cells are fixed with glutaraldehyde. Our laboratory has settled on two standard procedures of cell preparation prior to the immune reaction. In one procedure ( O h et al., 1989b), Euplotes is permeabilized with 0.5% Triton X-100 in a cytoskeletonstabilizing buffer (PHEM, Schliwa and Van Blerkom, 1981),prior to fixation with formaldehydeand centrifugationonto subbed slides. This method leaches most of the algae and cytoplasm from the cells, which flatten significantly upon centrifugation. The cortex with cilia remains intact; the MAC and MIC remain close to their original cellular positions. This stability of nuclear position is probably due to the extensive array of intracellular microtubules, which form baskets around both MAC and MIC (Olins et al., 1989b). The second procedure employed in our laboratory (Olins et al., 1991) is a rapid lysis of Euplotes eurystornus with 0.5% NP-40 in buffer, liberating MACs and disrupting most of the cell cortex. The liberated MACs are fixed with formaldehyde and centrifuged onto subbed slides. Often both procedures are tried with each antibody. Sometimes the two methods yield comparable results; sometimes not. Frequently, antibodies give strong nonspecific reactions with cilia, cirri, and cortical components, necessitating the use of liberated MACs rather than permeabilized cells. Generally, we dispense with blocking, doing antibody binding or washing steps in phosphate-buffered saline (PBS). When possible, we prepare identical slides after incubation with preimmune sera at a dilution similar to the antisera. Unless the antibody supplier has specific recommendations, we usually perform antibody incubations for 1 hr at 37"C, under a coverslip and within a moistened petri dish. Commercial FITC-affinity purified antirabbit, antimouse, or antihuman y-globulinsare routinely employed. Some of the successful and convincing immunostaining of Euplotes eurystornus RBs is summarized in Table 11. This table represents a small fraction of the antibodies actually tested in our laboratory, most of which yielded negative immunostaining, compared with preimmune sera, unrelated monoclonal antibody (MAb) controls, or PBS controls. One of the most dramatic demonstrations was with human autoimmune sera with documented reactivities against proliferating cell nuclear antigen (PCNA), an accessory factor of DNA polymerase 6. Antigenic determi-

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TABLE II Antigenic Determinants Localized in Euplotes RBs by lmmunostaining

Antigen

Antibody

PCNA (accessory protein for A.K.' DNA polymerase 6) E.B.' Acetylated histone H4 Pentab Tetrab Unacetylated histone H4 Unb Phosphorylation epitope MPM-2d Phosphorylation epitope RT97d Histones H2A/H2B

2Bld

RB chromatin

Clod

Principal nuclear localizations RZ and MICs (during Sphase) RZ and diffuse chromatin, not MICs FZ and RZ(?)', not MICs RZ and diffuse chromatin FZ and RZ(?)', diffuse chromatin FZ and RZ(?)', diffuse chromatin FZ and RZ(?)'

References Olins e t a / . (1989a) Olins et a/. (1991) Olins et a/. (1991) Unpublished' Unpublished Unpublished' Allen et al. (1986)

Human autoimmune sera. Rabbit antipeptide antisera. FZ and RZ(?) indicates that staining of FZ makes it dimcult to determine whether the RZ is also stained. Mouse monoclonal antibodies. MPM-2, a gift of Drs. J. Kuang and P. N. Rao, M. D. Anderson Hospital, Houston, TX (Davis et al., 1983). f RT97, a gift of Dr. J. Wood, Sandoz Inst. Med. Res. Horsforth, Leeds U.K. (Anderton et al., 1982). 8 2B1, a gift of Drs. R. W. Burlingame and R. L. Rubin, Scripps Clinic, La Jolla, CA (Rubin and Theofilopoulos, 1988). a

nants of PCNA could be demonstrated within the RZ of putatively active RBs, whereas negligible staining was observed in RBs from crowded or heat-shocked cells. This observation and the ultrastructural findings described earlier support the conclusion that during such adverse conditions the replicational apparatus at the FZ/RZ junction is dismantled, with a collapse of the RZ. Micronuclei were also stained with the anti-PCNA sera. The timing in the cell cycle of MIC staining included the period in late MAC S-phase, when MIC replication is believed to occur (Prescott et al., 1962). Immunostaining also revealed a high concentration of acetylated histone H4 in the RB (Fig. 8). Anti-histone H4 antisera had been prepared as rabbit antibodies to peptide-keyhole limpet hemocyanin (KLH) conjugates (Lin et al., 1989). The peptide parts of the KLH conjugates were based upon known sequences of the Tetrahymena thermophila histones. The antibodies reactive with acetylated H4 (i.e., penta and tetra) yielded a

FIG. 8 Immunochemical staining of permeabilized Euplotes eurystornus with rabbit antisera directed against Terrahymena macronuclear hyperacetylated and unacetylated histone H4. (A) and (B) Immunofluorescent and phase images after reaction with penta. Note brilliant staining of RZ, negligible staining of FZ, and strong staining of dispersed chromatin. (C) and (D). Moderate reaction with “un” antisera, primarily confined to FZ. Thin arrows point to FZ. Bar = 10 pm. Experimental details published in Olins et 01. (1991).

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characteristically bright staining of the RZ and staining throughout the MAC, probably confined to the diffuse chromatin regions. By contrast, anti-unacetylated H4 (i.e., “un”) was weaker, probably due to dissimilarities between Tetrahymena and Euplotes H4,and principally stained the FZ. No reaction was observed with MICs using any of these antipeptideKLH sera. Since H4 acetylation has been associated with both chromatin transcription and replication, this pattern of staining was not surprising. The intense reaction of penta (and tetra) with RZ might correspond to an accumulation of nascent acetylated H4 at the site of chromatin replication. A number of monoclonal antibodies with specificityto protein phosphorylation sites have also been used to stain RBs. Two examples (i.e., MPM2 and RT97) are cited in Table 11. Immunostaining results with RT97 are shown in Fig. 9. These and other antibodies support the view that a wave of protein phosphorylation occurs during the chromatin restructuring of the FZ and subsequent replication. Unfortunately, the particular protein targets within Euplotes MACs that react with these antibodies are not yet

FIG. 9 lmmunochemical staining of lysed Euplotes eurysfomus with the antiphosphorylation epitope MAb RT97. Top row corresponds to fluorescent images; bottom row, equivalent phase images. Columns, left to right: mid S-phase, RT97; late S, RT97; very late S. RT97; late S. buffer control. Notice that cortical fragments also react with RT97. Thin arrows indicate positions of RBs. Bar = 20 pm.

160

DONALD

E. OLlNS AND ADA L. OLlNS

known. Such studies will require combining selective extraction and RB enrichment procedures, and are in progress. From immunoblottingexperiments (D. E. Olins, A. L. Herrmann and L. H. Cacheiro, unpublished), all that we can conclude is that MPM-2 and RT97 each recognize different sets of proteins and that these proteins are not histones. A particularly interesting antihistone antibody (i.e., 2B 1; Rubin and Theofilopoulos, 1988) has specificity for the H2A/H2B complex. It stains the RB very intensely-slightly stronger than the surrounding MAC chromatin-and is abolished by treatment of MACs with high NaCl. A number of anti-Euplotes antibodies have been generated in our laboratory. A potent rabbit antisoluble MAC chromatin serum (Herrmann et al., 1987) contains antibodies to many MAC proteins, including histone H1, and intensely stains the entire MAC (unpublished). Chromatin from enriched RBs (Allen et al., 1985) was used to generate a panel of MAbs (Allen et al., 1986). Several of these MAbs stained RBs and reacted with proteins (ca. 90-100 kda) by immunoblotting. However, some of these MAbs showed distressing levels of immunostainingof cortical components. Two in particular (i.e., H3 and G l l ) were examined in a study of cortical alveolar plate proteins from Euplotes eurystomusand Euplotes aediculatus (Williams, 1991). With immunoblotting, MAb H3 reacted with all of the alveolar plate proteins; MAb G11 with a more restricted set. Immunofluorescence studies demonstrated strong staining of cortical plates. It is not yet clear whether this reactivity to cortical components in antibodies prepared against RB chromatin represents true cross-reaction by related (or unrelated) proteins, or is due to contamination of RBs by cortical proteins during the preparation procedure. A few apparently negative immunostaining experiments are also worth noting here. No significant immunostaining could be detected with (1) MAbs to yeast DNA polymerase I (L. Chang, Bethesda), (2) rabbit antiyeast DNA polymerase I11 (P. Burgers, St. Louis), and (3) rabbit antiyeast replication factor-A or antihuman replication factor-A (B. Stillman, Cold Spring Harbor). From these and other negative results, we believe that future attempts to map components of the replicational apparatus onto RBs should be conlined to highly cross-reactive antibodies or those generated against purified Euplotes MAC proteins. Immunostainingmethods have also provided techniques for demonstrating replicational activity of RBs in uitro and in uivo, as an alternative to i3H]thymidine incorporation and microautoradiography. In uitro incorporation of biotin-deoxyuridine triphosphate (dUTP) into isolated MACs (Olins and Olins, 1987) or into PHEM-permeabilized cells (Olins et al., 1989a)was readily demonstrated by immunostainingwith rabbit antibiotin antibodies, followed by FITC- or gold-conjugated secondary antibodies. Using this method, we demonstrated that in uitro activity was inhibited

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by N-ethylmaleimide or aphidicolin (inhibitors of DNA polymerase a and 61, or prior in uiuo heat shock. Biotin-dUTP has also been used successfully to monitor in uitro replication with other cell types, including mammalian nuclei (Nakayasu and Berezney, 1989;Banfalvi et al., 1989).More recently we have succeeded in obtaining clear localization of BrdU incorporated in uivo into RBs. Such a method has also been employed successfully in numerous laboratories looking at sites of replication in more typical nuclei (Nakamura et al., 1986; Coltrera and Gown, 1991; O’Keefe et al., 1992; Humbert et al., 1992; Manders et al., 1992). These immunostaining methods for demonstrating RB activity have the advantage of greater spatial resolution and more rapid results than can be obtained with autoradiography. Quantitation of fluorescent reaction intensity, however, may be somewhat more difficult than estimating the density of silver grains. Although not strictly an immunochemical mapping method, the use of fluorescently labeled lectins and neoglycoproteins to study RBs has yielded striking and puzzling findings. (D. E. Olins et al., 1988). The clearest binding was detected with FITC-N-acetyl-0-D-glucosaminebovine serum albumin (BSA) and FITC-cw-~-mannose-6-phosphate-BSA. Other neoglycoproteins and BSA exhibited much weaker (or negligible) binding to RBs. It is a complete mystery why RBs should exhibit specific lectin-like activity. Perhaps components of the replicational apparatus or putative scaffold are glycoproteins.

VI. Relevance t o Current Models of Chromatin Replication

The past decade has witnessed major advances in our understanding of eukaryotic DNA replication, in large measure because of the profitable exploitation of several cellular systems using biochemistry, molecular biology, and genetics (especially employing yeast, mammalian tissue cultures, and SV-40). Some of these areas of advancement include an increased understanding of (1) structure of replication origins, (2) organization of the replication fork, (3) cell-cycle controls on initiation of replication, (4) deposition and stability of histones and nucleosomes at the replication fork, and ( 5 ) spatial and temporal organization of replicons in a eukaryotic nucleus. A number of excellent reviews summarize these advances (So and Downey, 1992; Held and Heintz, 1992; Umek et al., 1989; Stillman et al., 1992; Benbow et al., 1992; Huberman, 1991; Reed, 1992; Gruss and Sogo, 1992) and constitute the framework for discussions in this section. Due to a paucity of information about the organization of ciliate MACs and RBs, much of this analysis represents inquiries

DONALD E. OLlNS AND ADA L. OLINS 162 into how these emerging concepts of eukaryotic replication relate to RBs. A “replicon” may be defined as the length of DNA replicated from a single origin. In typical eukaryotes, the average distance between origins (i.e., replicon size) is approximately 100 kb, ranging from 15 to 300 kb. What constitutes a replicon in a hypotrich MAC, where DNA fragment sizes average about 2.2 kbp (range 0.5-20 kb)? There is no evidence for ligation of gene-sized DNA in RBs. It seems a reasonable guess that, applying this concept to hypotrichs, each gene-sized molecule represents one (or more than one) replicon. This issue is intimately connected to additional questions. What is an “origin of replication” on the gene-sized molecules? How many origins are present on these tiny DNA fragments? Only two published studies relate to this latter question, those by Murti and Prescott (1983)and Allen et al. (1985). Both studies employed electron microscopy to visualize putative replicating MAC DNA molecules. Murti and Prescott (1983) hand isolated MACs with RBs; Allen et al. (1985) performed biochemical enrichment of RBs. The results are summarized in Table 111. Several points emerge from these two studies: (1) The percentage of MAC DNA molecules replicating during the experimental time frame is very small; that is, less than 1.5%. (2) The vast majority of replicating molecules (i.e., 80-98%) exhibit only a single replication fork. (3) Preparations of enriched RBs resulted in about a 3-fold enrichment of replicating DNA molecules. It seems likely that most MAC DNA molecules initiate at a single origin close to one end. In principle, the origin could be at one

TABLE 111 Properties of Replicating MAC DNA Molecules

Species Euplores

Replicating/total molecules. range in dflerent preparations

I-fork

2-fork

Bubbles

References

MAC

0.01-0.1%

80.4

11.8

7.8

MAC

0.08-1.37 0.44-3.77“

97.9

1.6

0.5

Murti and Prescott (1983) Allen er a / . (1985)

0.1-0.5

95.0

1.7

3.3

Preparation

Fraction of replication forms (%)

eurystomus

RB sryx sp’

MAC

Murti and Prescott (1983)

a The average enrichment of replicating DNA molecules, compared with preparations of total MACs, was threefold. * This species has been recently named Onychodromus quadricormufus(Foissner ef al.,

1987).

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telomere or close enough to the telomere such that a replication bubble (i.e., two close, diverging replication forks) possesses only a short lifetime. This apparent proximity of hypotrich MAC DNA replication origins to one telomere stands in marked contrast to evidence that yeast telomeres exert a strong inhibitory effect on the activation of regions that replicate autonomously (ARS) for up to about 20 kb from the telomere (Ferguson and Fangman, 1992). Attempts to define a particular origin nucleotide sequence in MAC DNA molecules by examining the handful of sequenced genes have not been especially enlightening. Current models of eukaryotic replication origins generally postulate the following nucleotide sequence components: ( I ) recognition sequences (e.g., ARS), (2) “unwinding elements,” that is, regions of lowered melting point, (3) transcription factor binding sites, and (4)scaffold attachment sites. Not enough is understood about hypotrich MAC DNA molecules to ascertain which of these components might be present. The general structure of a gene-sized DNA molecule consists of a single coding region, flanked on both sides by more AT-rich nontranscribed regions. Preliminary studies in collaborationwith L. Hauser (Univ. of Tennessee) and R. Wartell (Georgia Inst. Tech., Atlanta) suggest that most sequenced Euplores MAC genes contain one major region of low melting point close to one telomere. Perhaps in hypotrich MACs the essential property of a replication origin is behaving as an unwinding element. Current concepts of the geometry of protein molecules around a replication fork and estimated DNA synthesis rates constitute another puzzling comparison with imagined properties of MAC DNA replication. DNA synthesis rates in uiuo are generally estimated to be about 3 kb/min. This would imply that the average MAC DNA molecule is completely synthesized in about 2/3 min. The complex of proteins at a replication fork (sometimesreferred to as a “replisome”) possesses considerable total protein mass (i.e., ca. 1.2 MDa, adding up helicase, DNA polymerases a and 6 , and accessory protein factors). The average MAC chromatin molecule with about 10 nucleosomes and 2 telosomes would have an estimated mass of about 2.2 MDa, only twice the size of a replisome. Smaller MAC DNA molecules, such as the 5s rRNA (Roberson er al., 1989) and polyubiquitin (Hauser et a / . , 1991) genes, have only four nucleosomes each, making them slightly smaller (i.e., ca. 1 MDa) than a functioning replication complex. If recognition of origin, unwinding, and assembly of the replication complex are the rate-limiting steps of DNA replication compared with nucleotide polymerization, the frequency of these steps must be highly magnified in hypotrichs compared with typical cell nuclei. The integrity of RBs with highly ordered chromatin in the FZ may function in at least two ways to counter this anticipated frequent assembly and

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disassembly: (1) The substrate chromatin of the FZ may be organized so as to expose (and/or destabilize) replication origins. (2) The structure of the FZ may function to bind components of the replicational complex and/or stabilize the complex, once formed. Replication of chromatin also involves deposition of new histones onto the DNA daughter strands. Progress toward achieving an understanding of these events has been slower. Summaries of current concepts and research directions can be found in Van Holde (1989) and Gruss and Sogo (1992). The phenomenon of chromatin maturation refers to the time required for nascent chromatin to demonstrate sensitivity to nucleases equal to bulk chromatin. Initially, nascent chromatin is more sensitive to nuclease digestion than is bulk chromatin. Maturation takes about 20 min. The mechanism of maturation is not known, but may correspond to the compacting of chromatin into a higher order structure. Examination of election micrographs of RBs (such as Fig. 5A) reveals a progressive enlarging of condensed chromatin granules within the RZ. Is this the ultrastructural equivalent of nascent chromatin maturation? Although suggestive, no biochemical evidence exists to verify a correspondence. An issue of some interest is the nature of parental chromatin just prior to disruption by the replication fork. Our immunofluorescent data (Olins et al., 1991) suggest that the prereplicative chromatin contains underacetylated H4. It will be of interest to see whether a similar phenomenon can be detected in prereplicative regions of higher eukaryotic chromatin. Other characteristics of the FZ (e.g., silver staining, high concentration of reactive thiols, and nonhistone phosphorylation)also await comparison with other eukaryotic systems. In most suitably investigated typical cell nuclei, different genes replicate reproducibly at different times during the S-phase (Hatton et al., 1988; Pierron et al., 1989). The general pattern is that tissue-specific, transcriptionally active genes replicate earlier than inactive genes; housekeeping genes appear to replicate throughout the S-phase. This generalization is usually regarded as having the functional significance of exposing the early S nascent chromatin to larger pools of transcription factors which may compete more effectively with histones to maintain transcriptional activity. Unfortunately, this appealing model becomes problematic when applied to hypotrich RBs. Autoradiographic studies, cited earlier, indicate that transcription occurs throughout the MAC; yet, RBs always begin in defined regions. Combined with the knowledge that particular genes are randomly arranged throughout MACs, we are forced to conclude that no particular gene appears to replicate early or late in the S-phase, and that transcriptional activity does not predispose MAC genes toward early replication. The spatial organization of DNA replication in hypotrich MACs is ex-

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tremely localized in comparison with the hundreds of foci observed throughout S-phase nuclei in most cells (Nakamura et al., 1986; Nakayasu and Berezney, 1989; Humbert et al., 1992; O’Keefe et al., 1992; Coltrera and Gown, 1991 ; Manders et al., 1992). The observation that nascent DNA in nonhypotrich nuclei remains attached to a nuclear matrix has led to the suggestion that the matrix is the biochemical basis of nuclear organization (Nakayasu and Berezney, 1989; Neri et al., 1992). Although attractive, the concept of a nuclear matrix remains controversial, principally because of the harsh methods applied to extract nonmatrix nuclear components (especially histones, DNA, and RNA). The goal of much recent work in the matrix field has been to obtain a clear demonstration of its existence without harsh techniques (Hozak et al., 1993). The concept of a matrix might help to explain the high degree of organization of RBs. In this regard, our demonstration of highly regular nonchromatin 10-nmfilaments within Euplotes MACs may have significance. Electron microscopy of the IO-nm filaments suggests that they exist within CGs and rearrange with the chromatin in the FZ. So far we have not succeeded in identifying the protein components of the 10-nm filaments. However, their existence has prompted a line of investigation within our laboratory. Preliminary results have demonstrated that RBs are unusually stable, compared with bulk chromatin, during preparation of nuclear matrix. In addition, as with other nuclei, nascent DNA remains associated with the (RB) matrix. The concept of a nuclear matrix is seductively omnipotent. It has the capability of explaining how the RB is so highly organized and stable. Applying notions developed by advocates of the nuclear matrix, we might suggest that much of the replicational complex remains intact and does not disassemble as it migrates from one MAC gene to the next. Several significant concepts relevant to telomere replication have emerged in recent years. An RNA-containing enzyme, telomerase (Blackburn, 1992) has been described in Tetrahymena and in the hypotrichs, Oxtricha and Euplotes. Telomerase extends the 3’ single-strand DNA tails each round of replication, preventing loss of the telomere nucleotide sequence. Telomeres of hypotrichs and other cell types are normally associated with nonhistone telomere-binding proteins (Zakian et al., 1990; Price, 1992; Biessmann and Mason, 1992) which appear to protect the telomere DNA and may prevent terminal fusion and recombination. It seems reasonable to postulate that telomerase would be localized and functioning at the RB. Unfortunately, no antibody probes are yet available to study the nuclear distribution of telomerase. Antiserum to a telomere binding protein in Euplotes eurystomus has been prepared in our laboratory. It gives a weak immunostaining throughout the MAC, with slightly enhanced reactivity at the RB ( O h er al., 1993). As with nascent histone

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deposition, we anticipate that nascent telomere binding protein might localize in the vicinity of the RB. Advances will be made when reagents or probes become available that are sufficient to distinguish nascent from parental telomere binding proteins.

VII. Conclusions and Speculations The replication band of ciliates is unique in cell biology. The entire complement of nuclear DNA is duplicated by a migrating, highly organized structure which can be conveniently studied using a variety of microscopy techniques. Most other eukaryotic nuclei exhibit hundreds of dispersed foci of replication which activate at different times during the S-phase. RBs initiate at species-distinctive positions within the ciliate macronucleus, signalingthe beginning of the S-phase, and progress as a wave of chromatin modification and replication, which disappears at the termination of the S-phase. Throughout this entire period of migration and DNA replication, the RB maintains a remarkable ultrastructural stratification spanning the diameter of the MAC. The forward zone, consisting of highly regular chromatin fibers about 50 nm in diameter represents an extremely clear instance of prereplicative chromatin organization. These regular fibers disperse at the junction with the rear zone, which is the site of DNA synthesis. Condensed chromatin reappears at the distal end of the RZ. In the RB, the temporal events of DNA synthesis and chromatin assembly are displayed in a vectorial fashion. This vectorial property forms the basis for a significant scientific utility; namely, cytochemical and immunochemical mapping of chromatin replication components and events. RBs are apparently confined to nuclei consisting of gene-sized DNA molecules, a characteristic of a subset of ciliate MACs. Indeed, it may be that these two phenomena (i.e., RBs and gene-sized DNA) are inseparable. The high degree of RB organization could have evolved as a mechanism to ensure ordered replication of the short MAC DNA molecules. In contrast to the unique properties of RBs mentioned above, immunochemical evidence exists (i.e., localization within RBs of PCNA, acetylated histone H4,and numerous phosphorylated proteins) which is in general agreement with observations from typical eukaryotic cells. Clearly, much more biochemical information is required on the chromatin events surrounding replication sites within typical nuclei and RBs before we can adequately discriminate between general and specific mechanisms and modifications.

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A host of unanswered and intriguing questions concerning RB properties still remain. Several were mentioned earlier: (1) Why do RBs always initiate at unique MAC positions? For example, in Euplotes eurystomus, RBs begin at the ends of MACs. Clearly, the origin sites are not simply related to specific gene (or DNA sequence) localization, nor to sites of RNA synthesis, both properties being spread fairly uniformly throughout the MAC. We speculate that the MAC initiation sites have unique nuclear and cytoplasmic properties. Perhaps there is a localized activation of CDC2-type kinase by G,-type cyclins, resulting in confined chromatin phosphorylation. Possibly, transport properties of the nuclear envelope and pores are unique at the ends of MACs, compared with elsewhere along the nucleus. Microinjection studies are feasible in the large hypotrichs and might allow a controlled and localized perturbation, inhibitingor activating RB formation. (2) How is the high degree of RB ultrastructural organization maintained throughout the S-phase as the RB progresses through the MAC? We speculate that there is a nuclear structural framework (i.e., the proteinaceous 10-nm filaments) which tethers the gene-sized minichromatin molecules, directing the chromatin organization within the RB and the rest of the MAC. In an effort to characterize this postulated organizing framework, we have begun studies involving nuclear matrix preparations. (3) What is the motor that promotes RB progression? Is the energy derived from deoxyribonucleoside triphosphates (dNTPs) and DNA polymerases adequate or are there ATP-dependent mechanisms? A profitable direction of research would be to address this type of question through in uitro replication assays involving permeabilized cells and added cell extracts. (4) Do proteins target to the RB? Certainly, nascent histones (and associated transport proteins) might be expected to flood through nuclear pores surrounding an RB. This is another area where microinjection studies in combination with immunochemical analyses might form a convenient assay for transport into a defined nuclear domain of known function. This chapter was written to stimulate interest in a nuclear structure (the replication band) which has received too little attention since its discovery in 1859. Some of the hypotrichous ciliates are easy and inexpensive to maintain in the laboratory. They can be grown in relatively large quantities for biochemical studies. Probably the major perceived drawbacks of the nypotrichs are their obvious differences from most higher eukaryotic cells. In fact, their most serious limitation may be difficulties in utilizing genetic techniques, although several groups are currently exploring mating properties of the hypotrichs. We believe that if more investigators would develop ingenious approaches to explore the properties of RBs, the harvest of fascinating and important discoveries would be considerable.

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Acknowledgments The authors express gratitude to Drs. John Cook and William Harvey for suggestions and criticism of the manuscript. Our studies on the replication band have been funded by a variety of sources since their inception in 1980. These include the National Institutes of Health, the American Cancer Society, the National Science Foundation (NSF), and the Humboldt Foundation. Present funding is provided to D. E. Olins from NSF (DCB 870057) and to A. L. Olins from NSF (DCB 8915715), and by the Office of Health and Environmental Research, U. S. Department of Energy, under Contract DE-AC-05-840R21400 with the Martin-Marietta Energy Systems.

References Alfert, M., and Geschwind, I. (1953). Proc. Natl. Acad. Sci. U.S.A. 39, 991. Allen, R. L., and O h , D. E. (1984). Chromosoma 9 1 , 8 2 4 6 . Allen, R. L., Olins, A. L., Harp, J. M., and Olins, D. E. (1985). Eur. J . Cell Biol. 39, 217-223. Allen, R. L., Kennel, S . J., Cacheiro, L., O h , A. L., and Olins, D. E. (1986). J. Cell Biol. 102, 131-136. Anderton, B. H., Breinburg, D., Downes, M. J., Green, P. J., Tomlinson, B. E., Ulrich, J . , Wood, J. N., and Kahn, J. (1982). Nature (London) 298, 84-86. Banchetti, R., Ricci, N., Cetera, R., and Nobili, R. (1980). MonitoreZool. Ital. 14, 191-198. Banfalvi, G., Wiegant, J., Sarkqr, N., and Van Duijn, P. (1989). Histochemistry 93,81-86. Baroin-Tourancheau, A., Delgado, P., Perasso, R., and Adoutte, A. (1992). Proc. Natl. Acad. Sci. U.S.A. 89,9765-9768. Benbow, R. M., Zhao, J., and Larson, D. D. (1992). BioEssays 14, 661-669. Biessmann, H., and Mason, J. M. (1992). Adu. Genet. 30, 185-249. Blackburn, E. H. (1992). Annu. Reu. Biochem. 61, 113-129. Bonifaz, E. L., and Plant, W. (1974). Chromosoma 46, 261-278. Chakraborty, J. (1%7). J. Protozool. 14, 59-64. Coltrera, M. D., and Gown, A. M. (1991). J . Hisrochem. Cytochem. 39, 23-30. Davis, F. M., Tsao, T. Y.,Fowler, S. K., and Rao, P. N. (1983). Proc. Natl. Acad. Sci. U.S.A. 80,2926-2930. Doniach, I., and Pelc, S. R. (1950). B . J . Radiol. 23, 184-192. Evenson, D. P., and Prescott, D. M. (1970a). Exp. Cell Res. 61,71-78. Evenson, D. P., and Prescott, D. M. (1970b). Exp. Cell Res. 63,245-252. Faurk-Fremiet, E., Rouiller, C., and Gauchery, M. (1957). Exp. Cell Res. 12, 135-144. Ferguson, B. M., and Fangman, W. L. (1992). Cell (Cambridge, Mass.) 68, 333-339. Fleury, A., Iftode, F., Deroux, G., and Fryd-Versavel, G. (1985). Protistologicu21,505-524. Foissner, W., Schlegel, M., and Prescott, D. M. (1987). J . Protozool. 34, 150-159. Gall, J. G. (1959). J . Biophys. Biochem. Cytol. 5 , 295-308. Griffin, L. E. (1910). Philipp. J . Sci. 5, 315-336. Grimes, G. W. (1973). J. Protozool. 20, 92-104. Gruss, C., and Sogo, J. M. (1992). BioEssays 14, 1-8. Hatton, K. S., Dhar, V., Brown, E. H., Iqbal, M. A., Stuart, S., Didamo, V. T., and Schildkraut, C. L. (1988). Mol. Cell. Biol. 8, 2149-2158. Hauser, L. J., Roberson, A. E., and O h , D. E. (1991). Chromosoma 100,386-394. Held, P., and Heintz, N. H. (1992). Biochim. Biophys. Acta 1U0, 235-246.

REPLICATION BAND OF CILIATED PROTOZOA

169

Herrmann, A. L., Cadilla, C. L., Cacheiro, L . H., Came, A. F., and Olins, D. E. (1987). Eur. J . Cell Biol. 43, 155-162. Hozak, P., Hassan, A. B., Jackson, D. A., and Cook, P. R. (1993). Cell (Cambridge, Mass.) 73, 361-373.

Huberman, J. A. (1991). Chromosoma 100,419-423. Humbert, C., Santisteban, M. S., Usson, Y., and Robert-Nicoud, M. (1992). J . Cell Sci. 103, 97-103.

Inaba. F., and Suganuma, Y. (1966). J. Protozool. 13, 137-143. Kaul, S. C., and Sapra, G. R. (1991). Cell Struct. Funct. 16, 95-103. Kent, W. S. (1881). “A Manual of the Infusoria.” Vol. I, pp. 797-800 and Plate XLIV. David Bogue, London. Kimball, R. F., and Prescott, D. M. (1962). J . Protozool. 9, 88-92. Klobutcher, L. A., and Prescott, D. M. (1986). I n “The Molecular Biology of Ciliated Protozoa” ( J . G.Gall, ed.), pp. 111-154. Academic Press, Orlando, FL. Kluss, B. C. (1962). J . Cell Biol. 13, 462-465. Kraut, H., Lipps, H. J., and Prescott, D. M. (1986). Int. Rev. Cytol. 99, 1-28. Kusch, J., and Heckmann. K. (1988). J. Protozool. 35, 337-340. Lin, M., and Prescott, D. M. (1985). J . Protozool. 32, 144-149. Lin, R., Leone, J. W., Cook, R. G., and Allis, C. D. (1989). J . Cell Biol. 108, 1577-1588. Lynn, D. H., and Corliss, J. 0. (1991). “Microscopic Anatomy of Invertebrates: Protozoa” (F.Harrison and E. E. Ruppert, eds.), Vol. 1, pp. 333-467. Wiley-Liss, New York. Manders, E. M. M., Stap, J., Brakenhoff, G. J., Van Driel, R., and Aten, J. A. (1992). J . Cell Sci. 103, 857-862. Metenier, G., and Hufschmid, J.-D. (1988). J . Protozool. 35, 71-73. Mikami, K., Kuhlmann, H.-W., and Heckmann, K. (1985). Exp. Cell Res. 161,445-459. Murti, K. G. (1976). Exp. Cell Res. 99, 423-425. Murti, K. G., and Prescott, D. M. (1983). Mol. Cell. Biol. 3, 1562-1566. Nakamura, H., Morita, T., and Sato, C. (1986). Exp. Cell Res. 165, 291-297. Nakayasu, H., and Berezney, R. (1989). J . Cell Biol. 108, 1-11. Nanney, D. L. (1980). “Experimental Ciliatology.” Wiley, New York. Preparata, R. M., Meyer, E. B., and Simon, E. M. (1991). Nanney, D. L., Mobley, D. 0.. J . Mol. Evo~.32, 316-327. Neri, L. M., Mazzotti, G., Capitani, S., Maraldi, N. M., Cinti, C., Baldini, N., Rana, R., and Martelli, A. M. (1992). Histochemistry 98, 19-32. O’Keefe, R. T., Henderson, S. C., and Spector, D. L. (1992). J . Cell Biol. 116, 1095-1 110. Olins, A. L., and Olins, D. E. (1990). Chromosoma 99, 205-211. Olins, A. L., Olins, D. E., Franke, W. W., Lipps, H. J., and Prescott, D. M. (1981). Eur. J . Cell Biol. 25, 120-130. Olins, A. L., O h , D. E., Derenzini, M., Hernandez-Verdun, D., Gounon, P., RobertNicoud, M., and Jovin, T. M. (1988). Biol. Cell. 62, 83-93. Olins, A. L., Cacheiro, L. H., Herrmann, A. L., Dhar, M. S., and Olins, D. E. (1993). Chromosoma 102, 700-71 1. Olins, D. E., and Olins, A. L. (1987). J . Cell Biol. 104, 1125-1132. Olins, D. E., and Olins, A. L. (1993). J. Euk. Microbiol. 40,459-467. O h , D. E., O h , A. L., SCve, A.-P.. Bourgeois, C. A., Hubert, J., and Monsigny, M. (1988). Biol. Cell. 62, 95-98. O h , D. E., O h , A. L., Cacheiro, L. H., and Tan, E. M. (1989a). i. Cell Biol. 109, 1399- 14 10.

O h , D. E., Olins, A. L., Robert-Nicoud, M., Jovin, T . M., Wehland, J. and Weber, K. (1989b). Biol. Cell. 66, 235-246.

170

DONALD E. OLINS AND ADA L. OLINS

Olins, D. E., Olins, A. L.. Herrmann, A. L., Lin, R., Allis, C. D., and Robert-Nicoud, M. (1991). Chromosoma 100, 377-385. Phegan, W. D., and Moses, M. J. (1967). J . Cell Biol. 35, 103A. Pierron, G., Benard, M., Purion, E., Flanagan. R., Sauer, H. W., and Pallota, D. (1989). Nucleic Acids Res. 17, 553-566. Pluta. A. F., and Spear, B. B. (1981). Exp. Cell Res. 135, 387-392. Prescott, D. M. (1966). J. Cell Biol. 31, 1-9. Prescott. D. M. (1992). BioEssays 14, 317-324. Prescott, D. M., and Kimball, R. F. (1961). Proc. Nafl. Acad. Sci. U.S.A. 47, 686-693. Prescott, D. M., Kimball, R. F., and Carrier, R. F. (1962). J. Cell Biol. W, 174-176. Price, C. M. (1992). Curr. Opin. Cell Biol. 4, 379-384. Raikov, 1. B. (1978). “The Protozoan Nucleus,” Cell Biol. Monogr. 9. Springer-Verlag, New York. Raikov. I. B. (1989). Prog. Profisrol. 3, 21-86. Reed, S. (1992). Annu. Rev. Cell Biol. 8, 529-561. Ringertz, N. R., and Hoskins, G. C. (1965). Exp. Cell Res. 38, 160-179. Ringertz, N. R., Ericsson, J: L. E., and Nilsson, 0. (1967). Exp. Cell Res. 48, 97-117. Roberson. A. E., Wolffe, A. P., Hauser, L. J., and O h , D. E. (1989). Nucleic Acids Reu. 17,4699-4712.

Roth, L. E. (1957). J. Biophys. Biochem. Cyfol. 3, 985-999. Rubin, R. L., and Theofilopoulos, A. N. (1988). Int. Rev. Immunol. 3, 71-95. Ruffolo, J. J. (1978). J . Morphol. 157, 211-222. Ruffolo, J . J. (1979). J . Morphol. 159, 81-87. Salvano, P. (1975). J. Profozool. 22, 230-232. Schliwa, M., and Van Blerkom, J. (1981). J. Cell Biol. 90, 222-235. Sheaf€, R., Ilsley, D., and Kuchta, R. (1991). Biochemisrry 30, 8590-8597. Small, E. B., and Lynn, D. H. (1985). In “Illustrated Guide to the Protozoa” (J. J. Lee. S. H. Hutner, and E. C. Bovee, eds.), pp. 393-575. Society of Protozoologists, Lawrence, KS. So, A. G., and Downey, K. M. (1992). Crit. Rev. Biochem. Mol. Biol. 27, 129-155. Stein, F. (1859). “Der Organismus Der Infusionsthiere.” Engelmann, Leipzig. Stevens. A. R. (1963). J . Cell Biol. 19, 67A. Stillman, B., Bell, S. P., Dutta, A., and Marahrens, Y . (1992). Ciba Found. Symp. 170, 147-160.

Swift, H. (1955). Nucleic Acids 2, 51. Turner, J. P. (1930). Univ. Calif., Berkeley, Publ. Zool. 33, 193-258. Umek, R. M., Linskens, M. H. K., Kowalkski, D., and Huberman, J. A. (1989). Biochinr. Biophys. Acfa 1007, 1-14. Van Holde, K. E. (1989). “Chromatin.” Springer-Verlag, New York. Walker, G. K., and Goode, D. (1976). Cyobiologie 14, 18-37. Williams, N. E. (1991). Eur. J . Prorisrol. 27, 21-25. Witt, P. L. (1977). Chromosoma 60, 59-67. Yocom, H. B. (1918). Univ.Calif., Berkeley, Publ. Zool. 18, 338-396. YOW,F. W.11961). J . Profozool. 8, Suppl., 20. Zakian, V. A., Runge, K., and Wange, S.-S. (1990). Trends Genef. 6, 12-16.

Whole-Chromosome Hybridization S. D. Bouffler Biomedical Effects Department National Radiological Protection Board, Chilton, Oxfordshire OX1 1 ORQ, United Kingdom

I. Introduction

The study of chromosomes-cytogenetics-is essentially a visual science. Cytogeneticists depend upon staining methods to aid their understanding of chromosome structure and to identify structural alterations in chromosomes, some of which are involved in the pathology of human diseases. Two of the major goals in cytogenetics are first, to increase the specificity and resolution of chromosome staining methods and second, to increase the speed of chromosome analysis. The development and exploitation of in situ hybridization methods in recent years have contributed to both of these areas. In situ hybridization allows a given nucleic acid sequence to be seen in its normal cellular, nuclear, or chromosomal context. The subject of this chapter, whole-chromosome hybridization, covers a subset of in situ hybridization methods in which the aim is to stain a chromosome or chromosomes by using a collection of nucleic acid fragments specific for the chromosome(s) in question. In order to provide a perspective, the achievements and limitations of some other chromosome techniques require consideration. The history of modern cytogenetics began with the introduction of hypotonic salt solution treatments to aid in spreading metaphase chromosomes (Hsu, 1952). Prior to the use of hypotonic treatments, mammalian cytogeneticists in general used squashed preparations in which the chromosomes did not spread particularly well. Thus, it was not until 1956 that, with the aid of hypotonic treatment, Tijo and Levan (1956) accurately determined the human diploid chromosome complement to be 46. Hypotonic treatment in combination with general chromosome "block" stains allowed the enumeration and partial characterization of chromosomes from many species. Numerical alterations and gross rearrangements could be visualized but it was not possible to identify unequivocally each of the human chromosomes. This latter aim was realized in the 1970s. The lnrernarronal Review' of Cvtology, Vol. 153

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introduction of Q-banding (Casperson et al., 1970) and G-banding (Seabright, 1971; Sumner et al., 1971; Patil et al., 1971; Drets and Shaw, 1971) allowed the identification and classification of each pair of chromosomes in the diploid human cell nucleus. These chromosome banding techniques give rise to a pattern of alternating dark and light bands along the long axis of a chromosome. This pattern is consistent and characteristic for a given chromosome. With chromosome banding, more subtle alterations to the chromosome constitution of cells can be determined. For example, many tumor cells have been shown to carry chromosomal translocations or deletions. These changes were often not noted when chromosome block staining was employed. The high level of resolution that can be achieved with chromosome banding is offset by the high level of skill required to score and interpret preparations. Currently, the technique of in situ hybridization is finding a wide range of applications in cytogenetics. In situ hybridization has advantages over other methods both in terms of resolution and in ease of analysis. While the method was first developed in the late 1960s (Gall and Pardue, 1969; John et al., 1969), more recent refinements to the technique and advances in molecular biology have rendered it of much greater value to the cytogeneticist. As mentioned above, in situ hybridization allows the detection of specific nucleic acid sequences in normal cellular or chromosomal locations. Thus, the technique can be used to study gene expression, RNA processing, developmental biology, and several other disciplines as well as in cytogenetics. Three distinct categories of experiment are in common use in cytogenetics. These are classified on the basis of the type of DNA probe used: 1. Individual genes can be located by the use of unique sequence DNA probes; these experiments are useful in gene mapping. 2. The use of DNA repeat sequence probes allows the visualization of chromosome structural features. For example, satellite DNA probes can stain centromeres, and certain interspersed DNA repeats have a nonuniform distribution throughout chromosomes. 3. The use of chromosome-spec$c DNA probe libraries allows the specific staining of a chromosome or chromosome pair in a metaphase preparation. This method, whole chromosome hybridization (also known as chromosome painting), is the subject of this chapter.

The first section concerns the development of in situ hybridization methods. Essentially, the technique is a specialized form of nucleic acid hybridization and as such involves the selection of nucleic acid probes, labeling of probes, hybridization to the specimen, removal of nonspecifically bound probe, and finally, the detection and visualization of the hybridizing probe (Fig. 1). The ability to perform whole-chromosome

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METAPHASE PREPARATION -RNase digest -proteinase digest denature

173 PROBE -label -denature -supress repeats

-hybridize -remove excess probe -detection reactions -observe under microscope Outline of the in siru hybridization method. For full details, see Section 1I.B. especially Section II,B,S.

FIG. 1

hybridization experiments depends entirely upon the availability of nucleic acid probes which bind to specific chromosomes. These can take the form of genomic DNA, used, for example, to identify human chromosomes in human-rodent hybrid cell lines. A more refined approach is the use of cloned chromosome-specificDNA probe libraries; such libraries are available for each of the 24 human chromosomes. More recently, systems have been developed which allow the rapid production of DNA probe libraries from limited amounts of starting material by a polymerase chain reaction. In Section II,C these reagents, which are central to whole-chromosome hybridization methods, are discussed. The final sections of this chapter cover the applications of whole-chromosomehybridization. These include both fundamental and more applied studies. Questions of chromosome evolution can be addressed by cross-species hybridizationsand interphase cell analysis can help elucidate the structure of the nucleus. In clinical cytogenetics, whole-chromosome hybridization helps the identification of chromosomal rearrangements in cancer cells, and genetic toxicology studies can be aided by the method, facilitating rapid analysis of chromosome translocation. Whole-chromosome hybridization is, therefore, a technique with a wide range of applications in several areas of cytogenetics. Refinements to the technique will, no doubt, continue and the range of applications will widen with time. My aim here is to describe the techniques currently used and discuss major applications.

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II. Development and Principles of Whole-Chromosome Hybridization Techniques A. Landmarks in the Development of Whole-Chromosome Hybridization

The fundamental process underlying all in situ hybridization methods is the reassociation of complementary nucleic acid strands to form a doublestranded molecule. This process, nucleic acid hybridization, was first described in mathematical terms by Marmur and Doty (1961). Initially all nucleic acid hybridization analysis was performed in solution; the first descriptions of in siru hybridization were made by John et al. (1969) and Gall and Pardue (1969). Working independently, these two groups demonstrated the presence of DNA which codes for ribosomal RNA in the nuclear caps of Xenopus laeuis oocytes. The probe used for these experiments was the abundant ribosomal RNA of Xenopus radiolabeled with 3H. At that time, prior to the era of molecular cloning of DNA, a limited range of nucleic acid probes for in situ hybridization was available. However, it proved possible to visualize ribosomal RNA in tissue sections (Buongiorno-Nardelliand Amaldi, 1970),satellite DNA in mouse chromosomes (Pardue and Gall, 1970), globin messenger RNA (Harrison et al., 19731, and viral sequences in tumor sections (Orth et al., 1971) and in chromosome preparations (McDougall et al., 1972). By the beginning of the 1980s, technical advances allowed the detection of single-copy genes by in situ hybridization (Gerhard et al., 1981; Harper et al., 1981; Malcom et al., 1981).Since those early reports, the increasing availability of cloned DNA probes has allowed the detection and mapping of many genes on human chromosomes and the chromosomes of several other species. All the work described above used radioactively labeled probes. 32P, "S, '"I, and 3H have been used, 'H most commonly owing to the good spatial resolution which can be achieved. Although no theoretical reason exists for avoiding the use of radiolabeled probes for whole-chromosome hybridization, few studies have been reported which employ radioactive rather than nonradioactive detection methods. This is probably largely due to the parallel development of nonradioactive labeling and detection systems with whole-chromosome hybridization reagents and methods. Several nonradioactive labeling methods have been described and are discussed in detail later (Section II,B,2). Currently, the most widely used nonradioactive label is biotin; biotin labeling of DNA (Langer et al., 1981) was described at about the same time as the early reports of single-copy gene localization by radioactive in siru hybridization. Initially, nonradioactive in situ hybridization methods were considered inferior to isotopic

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methods largely because of their lack of sensitivity. The advantages of nonradioactive systems include speed of analysis, good spatial resolution, probe stability, and the relatively nonhazardous nature of the procedures. These advantages encouraged the refinement of nonradioactive labeling and detection systems. The sensitivity of nonradioactive methods now rivals that of radioactive methods. It is now possible to detect sequences of -500 bp by nonradioactive methods (Heppell-Parton et al., 1992). The remaining crucial component for whole-chromosome hybridization is the probe. Early studies describing whole-chromosome hybridization employed human and rodent hybrid cell lines. Metaphase preparations of hybrid cell lines carrying one or a few human chromosomes were hybridized with labeled human genomic DNA. After washing and detection, the human chromosomes could be readily distinguished from the rodent material (Durnam et al., 1985; Manuelidis, 1985; Schardin et al., 1985; Pinkel et af., 1986). In the early 1980s, DNA cloning techniques were combined with flow cytometric separation and purification of human chromosomes to produce human chromosome-specificDNA libraries (Davies et al., 1981;Krumlauf et al., 1982). Subsequently, largely as a result of the major efforts of two U.S. national laboratories, Livermore and Los Alamos, a complete set of human chromosome-specific libraries became available (van Dilla et af., 1986). These libraries were soon successfully exploited for wholechromosome hybridization (Lichter et al., 1988a; Pinkel et al., 1988). From this point on, whole-chromosome hybridization techniques and applications have improved and increased; this is also the case with nonradioactive in situ hybridization in general. To appreciate the diversity and versatility of whole chromosome hybridization techniques, a full description of the principles and methods is necessary.

6.In Situ Hybridization Methods and Principles 1. Nucleic Acid Hybridization There are a number of critical factors which affect the rate and stability of nucleic acid hybridization. These factors have been studied widely and are considered in detail by Britten et af. (1974) and in Hames and Higgins (1985). The majority of work in this area concerns solution hybridization and to a lesser extent, filter hybridization. While a detailed study of the physical chemistry of in situ hybridization is not available, the factors influencing solution hybridization are at least qualitatively applicable to in situ hybridization. An understanding of these factors can be of great use in designing and troubleshooting in situ hybridization protocols.

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The reassociation of nucleic acids involves two steps-an initial nucleation reaction involving complementary nucleotides of two single-stranded nucleic acid molecules followed by a “zippering” of the two single strands into a duplex molecule. Mathematical modeling of the reassociation process indicated that the initial nucleation reaction was the rate-limiting step. A hybridization reaction involving complex mixtures of nucleic acids, as found in whole-chromosome hybridization, will involve a random interaction among many differing molecules, with nucleations occurring when two strands with complementary nucleic acid sequence come into close proximity. The subsequent zippering will stabilize exactly complementary duplexes. However, many nucleations will lead to partial duplexes containing both double-stranded areas of complementary base pairing and areas of mismatch between the nucleotides of the two interacting strands. The stability of such imperfectly matched molecules will depend upon the reaction conditions. Thus, in reality a hybridization reaction involves a complex series of interactions between single-stranded molecules, some forming stable duplexes and others being unstable and so dissociating. Before hybridization reaction conditions are described, the stability of reassociated nucleic acids must be considered. The stability of a DNA-DNA duplex or DNA-RNA hybrid is generally measured in terms of melting temperature, T, that is, the temperature (in degrees Celsius) at which one half of the strands are dissociated and one half remain in double-stranded form. The T, for a given duplex is influenced by a number of factors: 1. Base composition. GC base pairs are more stable than AT base pairs (G and C pair with three hydrogen bonds as opposed to 2 in AT). Mathematically T,,, is related to GC content by the formula T,,,= 0.41 (% GC)

+ 69.3.

2. Fragment length. Longer duplexes are more stable than shorter duplexes. An estimate of the effect of fragment length is given by D = SOOIL,

where D is the reduction in T, measured in “C and L is the length of the duplex. 3. Mismatching base pairs. These reduce the T, of a duplex, with an approximate decrease of 1°C in T, for each 1% mismatch. 4. Salt concentration. Higher monovalent cation concentrations stabilize duplexes. T, increases by about 16°C for each 10-fold increase in salt concentration up to 0.1 M.Divalent cations exert an even greater stabilizing influence on duplexes.

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5 . Organic solvent concentration. Formamide is commonly used in hybridization experiments to reduce T,. A 0.72”C drop in T, occurs for each 1% formamide. A general formula that can be used to estimate T, is: T , = 81.5

+

16.6 (log S) + 0.41 (GC) - 0.72 ( F ) - I ( M ) - 500/L,

where S is the molar salt concentration; GC is the percentage GC content of duplex; F is the percentage formamide; M is the percentage mismatch; L is the length of duplex in base pairs. The factors influencing the rate of reassociation of DNA are broadly similar to those affecting the T , of duplexes. The parameters which may be altered or are of importance in experimental design are as follows: 1. Incubation temperature. The maximum rate of reassociation is generally obtained at approximately 25°C below T,. 2. Salt concentrution. This has a profound effect on reassociation rates, higher concentrations increasing the rate. 3. Nucleic acid concentration. Increasing the initial concentration of nucleic acid increases the rate of reaction. 4. Presence of high-molecular-weight polymers. In particular, the presence of dextran sulfate increases the rate of reassociation. The inert polymer is thought to act by effectively concentrating the nucleic acid probe. 5 . Mismatching base pairs. A 10% mismatch reduces the reaction rate about 2-fold. 6. Nucleic acid fragment length. This has a complex effect upon reaction rates. In the case of in situ hybridization, one has little control over the specimen DNA (or RNA) fragment length; probe lengths of 200500 bp are considered optimal, the ability to penetrate the specimen being considered important here. 7. Sequence complexity. Eukaryotic genomes are composed of three distinct classes of DNA defined by the frequency of reiteration within the genome. Thus the reassociation of mammalian genomic DNA occurs in three distinct phases, highly repeated DNA reassociating the fastest, followed by middle repetitive DNA and finally single-copy DNA sequence. These differing reassociation rates are of great importance in wholechromosome hybridization. Any collection of ’‘chromosome-specific” genomic DNA will contain a proportion of DNA fragments which are not in fact chromosome specific but occur repeatedly throughout the genome. These repeats must be removed from the hybridization reaction to allow chromosome-specific hybridization to be achieved. This is usually accomplished by preincubating the labeled “chromosome-specific’’ genomic

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DNA probe with an excess of unlabeled genomic DNA. Hybridization between probe and the unlabeled “competitor” DNA is allowed to proceed for a limited time so that repeat sequences in the labeled probe complex with the competitor but the chromosome-specific sequences remain in single-stranded form. In this way the nonspecific repeats can be effectively removed from the probe, which is apFiied to the specimen. Such competitive, or suppression hybridization strategies were first developed for use with genomic DNA in large cosmid clones (Landegent et al., 1987) and then for whole-chromosome hybridization (Lichter et al., 1988a; Pinkel et al., 1988). A refinement of the suppression technique is to use Cot-1 DNA (i.e., the fastest reassociating component of the mammalian ,genome which contains the most abundant repeats) in place of genomic DNA as a competitor. Cot-1 DNA does not contain unique (chromosome-specific) sequences and therefore the fraction of the probe which is of interest cannot be competed out. The higher concentration of repeats in Cot-I DNA also speeds the competition reaction rate.

The preceding discussion highlights the fact that nucleic acid hybridization is a complex process which is influenced by a number of factors. The conditions employed in whole-chromosomehybridization experiments are well established and fairly standardized: a typical method is given in Section II,B,S. Nonetheless, an appreciation of the factors influencing nucleic acid hybridization can be useful in development of techniques and in designing new methods. 2. Probe Labeling Methods

The objective of probe labeling is to tag the nucleic acid probe so that it can subsequently be detected and then visualized when observed under a microscope. A wide range of tags have been used, both radioactive and nonradioactive. Radioactive labeling methods will not be considered in any detail here. As previously mentioned, the vast majority of wholechromosome hybridization studies have employed nonradioactive labeling systems of one type or another. However, most of the enzymatic labeling systems described later were originally developed for use with isotopically labeled nucleotides and are thus applicable to both types of labeling method. Two broad categories of nonradioactive probe labeling methods exist: (1) enzymatic incorporation of labeled nucleotides and (2) direct chemical derivation. Currently the enzymatic methods are most widely used. A number of nonisotopically labeled nucleotides are available for enzymatic probe labeling. These include nucleotides labeled with biotin (Langer et af., 1981; Brigati et al., 1983; Lo et al., 1988; Weier et af.,

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19901, digoxigenin ((Muhlegger et al., 1989; Kessler et al., 1990), and dinitrophenol(Langer et al., 1981 ;Lichter et al., 1990b).Following hybridization of probes labeled with these reagents, detection reactions are necessary. These include the binding of fluorochromes for observation by fluorescence microscopy or the binding of enzymes such as alkaline phosphatase and subsequent reaction with chromogenic substrates (see Section II.B,3). Such detection reactions can be bypassed by the use of nucleotides carrying fluorescent tags. Wiegant et al. (1991) described the use of directly fluoresceinated nucleotides. A number of nucleotides carrying fluorescent tags such as rhodamine and hydroxycoumarin are now commercially available and have been assessed for in situ hybridization (Wiegant et al., 1993). Various enzyme labeling systems are available for incorporating tagged nucleotides into probes. Practical details for their use can be found in molecular biology technical manuals such as that of Sambrook et al. (1989). Nick translation (Rigby et al., 1977) exploits the properties of two enzymes. DNase 1 and Escherichia coli DNA polymerase 1. DNase 1 creates single-stranded nicks in double-stranded DNA from which E. coli DNA polymerase 1 synthesizes a new DNA strand in a 5' + 3' direction, incorporating labeled nucleotides. The template for the polymerase is exposed by the 5' 3' exonuclease activity of DNA polymerase 1 . By controlling the DNase 1 activity, probes of the desired length can be obtained. A number of primer-directed labeled reactions are available. The random-primed labeling method (Feinberg and Vogelstein, 1984) uses small oligonucleotides (usually hexamers) of random sequence to prime the synthesis of DNA by the Klenow fragment of E. coli polymerase 1 . When fragments to be labeled are cloned into vectors of known sequence, defined primers may be used to produce labeled insert and/or vector in a similar fashion. The polymerase chain reaction (PCR)provides a useful method for producing probes. DNA primers flanking the DNA region to be amplified are extended by a thermostable DNA polymerase [generally that of Thermus aqus (Taq)]. With primers present in excess, multiple cycles of denaturation, primer annealing, and extension yield large quantities of probe. PCR can be used to produce probes from fragments in cloning vectors or directly from genomic DNA if sequence information is available. A further method for generating labeled DNA probes employs terminal deoxynucleotidyl transferase. This enzyme mediates the addition of deoxyribonucleotides to the 3' ends of either single- or double-stranded DNAs. This can be a useful method for labeling small probes such as oligonucleotides of 20-50-base pairs. In situ hybridization initially used RNA probes produced by laborious '

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purification procedures. RNA probes can now be more readily produced by in uitro transcription reactions. There are several DNA cloning vectors which include RNA polymerase promoter sites, such as SP6, T3, and T7. Although not currently in wide use for chromosome hybridization, RNA probes have some advantages over DNA probes, for example, no requirement to denature and high specific activities; however, they have the disadvantage of being less stable than DNA probes. Chemical derivation of probes extends the range of labels which can be employed. Bauman et al. (1980) developed direct fluorescent labeling of RNA, which is perhaps most closely analogous with the enzymatic incorporation of fluorescent nucleotides into probes. A number of directlabeled, fluorescent, whole-chromosome hybridization probes are now commercially available. However, as with the enzymatic labeling methods, most of the described chemical derivation techniques are indirect, requiring further detection steps prior to microscopic examination. Biotin can be directly linked to DNA and RNA in a photochemical reaction (Foster et al., 1985). Acetylaminofluorene is a compound that covalently binds to guanine residues in DNA, a property which may be exploited for probe labeling (Landegent et al., 1984; Tchen et al., 1984). The physically damaging agent, ultraviolet light (UV), can dimerize adjacent thymine residues in DNA. Thymine dimers can be distinguished immunologically, thus UV-irradiated cDNAs have been used for in situ hybridizations (Nakane et al., 1987).Probes can be sulfonated with sodium bisulfite (Sverdlov et al., 1974; Viscidi et al., 1986) or mercury labeled (Hopman et al., 1986a,b). Chemically synthesized oligonucleotides can be prepared with a variety of labels added during synthesis. However, oligonucleotide probes are currently little used for whole-chromosome hybridization owing to problems inherent in visualizing small probes. The methods described thus far allow probes to be labeled with a single tag molecule. The desire to detect multiple DNA sequences on a single metaphase preparation has driven further developments in probe labeling. The hybridization of two probes labeled with different tags allowed the detection of two different DNA sequences simultaneously (Hasse et al., 1985;Cremer et al., 1986;Hopman et al., 1986~).The simultaneous detection of three target sequences was achieved through the use of probes labeled with mercury, acetylaminofluorene, or biotin (Nederlof et al., 1989). The detection of more than three targets demanded the labeling of probes with more than one tag. Following fluorescent detection reactions, probes appear a single color, for example fluorescein isothiocyanate (FITC) green or rhodamine red, when labeled with a single tag. However, when labeled with a combination of two tags, both fluorochromes bind to the probe on detection and so a novel mixed-color fluorescence is observed. Such “combinatorial labeling” or “ratio labeling” first allowed

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the detection of four targets (Nederlof et af., 1990) and more recently, seven targets in a single specimen (Ried et al., 1992). In practice, the more complex combinatorial labeling methods require sophisticated digital imaging systems to resolve the different probes reliably. The multiple tags required on probes can be added by enzymatic methods (Ried et al., 1992) or chemical derivation (Nederlof et af., 1990). It is also possible to synthesize oligonucleotides containing multiple tags (Nelson et af., 1992). A technique which employs two sequential enzyme labelings-nick translation followed by tailing with terminal transferase (Scherthan et af., 1992a)-could be exploited for dual labeling with, for example, biotin and digoxigenin. The extent to which these multiple labeling techniques prove to be of practical use rather than a display of technical bravado remains to be seen.

3. Detection Methods As previously, here I concentrate on nonradioactive methods. The detection of radiolabeled probes requires standard autoradiographictechniques; methods specific for in situ hybridization are given in Pardue (1985). The majority of nonradioactive methods for probe detection involve immunocytochemical procedures. The notable exception here is probes directly labeled with fluorochrome, which require no detection steps but can be directly visualized by fluorescence microscopy. While the direct methods are simple, immunocytochemistry has the advantage of flexibility. The original immunocytochemical detection method applied to in situ hybridization employed antibodies raised against hybrid DNA-RNA molecules; an RNA probe was used to visualize DNA structures (Rudkin and Stollar, 1977). Although this method avoids the need to label the probe, technical problems of antibody specificity prevented its general use. The widely used biotin labeling system is frequently detected with FITCconjugated avidin. Avidin binds to the biotin incorporated into the probe with great specificity and strength. The FITC signal can be amplified by using alternating layers of FITC-avidin and biotinylated antiavidin antibody, as described by Pinkel et af. (1986). While FITC avidin is probably most commonly used to detect a biotinylated probe, other fluorescent conjugates of avidin are available, for example, tetramethylrhodamine isothiocyanate (TRITC) avidin. The biotin-avidin system is also compatible with nonfluorescent detection methods (see later discussion). The detection of probes labeled with agents other than biotin depends upon the availability of antibodies specific to the label. Thus, antidigoxigenin, antidinitrophenol,antithymine dimer, antiacetylaminofluorene,and antisulfonate antibodies have been used to detect probes. The mercury labeling system requires a pretreatment with a mercury-binding sulfhydryl-

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dinitrophenol reagent prior to detection with antidinitrophenol antibodies (Hopman ef al., 1986a,b). In some cases the primary detecting antibody employed is itself fluorescent and so can be visualized by microscopy. More frequently, a secondary antibody detection layer is employed. Further signal amplification is possible through the use of multiple layers of labeled antibody. Signals from probes directly labeled with fluorochrome can also be amplified by the use of, for example, a secondary antifluorescein antibody followed by a tertiary fluorescein-labeled antibody raised against the secondary. Detection through the use of fluorochromes has several advantages, in particular the high sensitivity and resolution achievable. Also, unlike many of the colored reaction products visible under a light microscope (see later discussion), fluorochromes do not mask structural features in the specimen. The most commonly used fluorochromes are FITC (green fluorescence), Texas red and TRITC (red fluorescence),and 7-amino-4-methyl coumarin-3-acetic acid (AMCA, blue fluorescence). New fluorochromes are being introduced at regular intervals; some expand the range of colors available in the visible range, while others, Cy 5 for example, emit far red fluorescence. The use of flurochromes which emit spectra outside the visible range demands sophisticated imaging electronics. Fluorochromes can be distinguished on the basis of emission spectrum, as in the examples above, or on the basis of fading characteristics. Thus, following a pulsed exposure to exciting light, each fluorochrome emits fluorescence for a characteristiclength of time. By using fluorochromes modified to fluoresce for varying lengths of time, multiple targets can be visualized. This principle requires the use of a “time-resolved” fluorescence microscope (see, for example, Seveus et al., 1992; Beverloo et al., 1992). While fluorescence detection has advantages in resolution and sensitivity, a significant drawback is the fading of fluorescence signal. Thus a preparation detected by a fluorescence method is not permanent; where preservation of the specimen and hybridization signal is important, nonfluorescent detection methods should be used. Most such methods are based upon well-established histochemical techniques. The enzymatic methods employing alkaline phosphatase (AP) or horseradish peroxidase (HRP) are widely used. Antibodies or avidin carrying AP or HRP can be used in detection reactions in a manner similar to that used for fluorescence detection and signal amplification, the difference being at the final stages of detection, which depend upon the reaction of the enzyme with a chromogenic substrate. The substrate used with AP is usually nitroblue tetrazolium-bromochloroindoyl phosphate (NBT-BCIP) and, for HRP, diaminobenzidine. The enzyme reaction forms a dense, insoluble product visible in the light microscope. Since the enzyme should be located only at the sites of probe hybridization, signal should be seen only at those points.

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An alternative to enzymatic nonfluorescent detection is the use of antibodies or avidin labeled with gold, which can be used in light or electron microscopy (Narayanswami and Hamkalo, 1991; Thiry et al., 1991; Fetni et al., 1992). Antibodies carrying gold particles of varying size can be used in conjunction with different probe labels such as biotin and digoxigenin to detect multiple targets at the electron microscope level (Narayanswami and Hamkalo, 1991; Narayanswami et al., 1989). In order to make goldlabeled material more readily visible under a light microscope, it is common to perform a reaction depositing silver onto the gold particles. This silver enhancement changes the gold particles to an intense black, rather like the silver grains seen in autoradiography. As more labeling and detection systems are developed, it becomes difficult to categorize methods. A case in point is the combination of enzyme labels with fluorescent substrates. Alkaline phosphatase-labeled detection reagents can be visualized in a reaction employing fast red (Murdoch et al., 1990; Ziomek et al., 1990). The product of this reaction is both colored red and fluorescent and so is visible by light or fluorescence microscopy. Optimization of the method for DNA in situ hybridization (Speel et al., 1992) has demonstrated that the detection of fluorescence is sensitive and long lived. In a similar vein, enzyme-conjugatedantibodies can be detected with labeled antienzyme antibodies; for example, the detection of HRP-conjugated antibodies with gold-tagged anti-HRP antibodies (Roth et al., 1992). Fluorescent-tagged antienzyme antibodies can be used, as can enzyme-tagged anti-FITC antibodies. Despite this diverse and growing range of methods, whole-chromosome hybridization experiments in general use a rather limited range of labeling and detection methods. Enzymatic labeling of DNA probes with biotin or digoxigenin followed by detection with FITC, Texas red, or rhodamine are currently in common use. Table I provides a summary of labeling and detection methods. Several of the newer methods of detection require sophisticated microscopic techniques. Some of the available methods are described in the next section. 4. Microscopy In situ hybridization in general and whole-chromosome hybridization in

particular do not require the use of sophisticated microscopy. Nonetheless, more specialized techniques for visualization of very small, lowsignal-intensity probes, multicolor hybridization, and some fluorescent dyes demand the use of nonstandard microscopes. The basic requirements for viewing whole-chromosome hybridization preparations, along with some of the more recently developed specialized systems, are described in this section. The type of microscope used is determined in a large part by the detection system. Since fluorescence labeling systems are the most

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184 TABLE I Summary of Major DNA Probe Labeling and Detection Systems

Probe labeling method Enzymatic Nick translation Random-primed PCR Other primerdirected Terminal transferase

Label incorporated Biotin

Detection system

Gold-fluorochrome-enzyme-labeled avidin, amplification with biotinylated antiavidin

Fluorochrome Digoxigenin Dinitrophenol

Directly or following amplification with specific antibodies Specific antibodies followed by fluorochrome-gold-enzyme-labeled antibodies

Chemical derivation Biotin Fluorochrome Acetylaminofluorine Sulfonate Thymine dimer Mercury

Avidin-antiavidin, as above Direct or following amplification as above Specific antibodies followed by fluorochrome-gold-enzy me-labeled antibodies Dinitrophenol addition followed by antidinitrophenol antibodies and labeled secondary antibody

highly developed and useful, fluorescence microscopy techniques have been refined to the greatest extent. In contrast, the microscopic techniques and image analysis systems used for nonfluorescent labeling and detection are relatively straightforward. Enzyme precipitates, such as those formed with AP-NBT-BCIP and HRP-diaminobenzidine, can be visualized by simple light microscopy. Either bright-field, phase-contrast, or Nomarski optics can be used. Similarly, silver-enhancedgold labels can be seen with light microscope optics. Detections using gold label can also be visualized by electron microscopy given appropriate preparation of specimens (Narayanswamiand Hamkalo, 1991). In order to visualize probes labeled and detected with fluorochromes, a fluorescence microscope is necessary. An epifluorescence microscope, preferably with a high-power mercury arc lamp as a light source, is ideal. The range of fluorochromes which can be seen is determined mainly by the filter sets available for the microscope. Fluorescence is the light emitted by a fluorochrome following irradiation with light of a shorter wavelength (excitation light). The excitation wavelength spectrum and emission spec-

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trum are characteristic for a given fluorochrome. For example, the excitation maximum for FITC is 490 nm and maximal fluorescence is emitted at 535 nm. To allow the effective observation of fluorescence, the excitation light must be separated from the light emitted. In an epifluorescence microscope, this is achieved by the use of two filters and a dichroic mirror (or beam splitter). The light from the mercury lamp first passes through an exitation filter, which transmits light within a narrow range of wavelengths. The light then passes to the dichroic mirror, which reflects light up to a defined wavelength and transmits light of greater wavelengths. Thus, the exitation light is reflected down through the microscope objective and to the specimen. The interaction of the excitation light with the bound fluorochromes in the specimen leads to the emission of fluorescence of a longer wavelength. This emission light is transmitted through the dichroic mirror and emission filter (or barrier filter), which is a longwavelength pass filter, that is, a filter that transmits only light of a defined wavelength or greater to the eye pieces. These optical characteristics are illustrated in Fig. 2. Filter sets suitable for use with all commonly used fluorochromesare available from major microscope manufacturers. Table I1 gives details of the excitation and emission characteristics of a number of fluorochromes used for probe labeling and general DNA staining of specimens. The standard epifluorescence microscope must be considered the workhorse for whole-chromosomehybridization, although many more complex systems are available. There are three significant limitations to the use of standard epifluorescence microscopy with a human observer: (1) the inability to detect low light intensities, (2) limited color discrimination, (3) poor resolution in three dimensions. Improvements in low-light sensitivity and color discrimination have been made by the use of epifluorescence microscopy in conjunction with digital imaging and analysis systems, while the problems posed by analysis of three-dimensional structures are at present best resolved by using confocal laser scanning microscopy. The most sensitive digital imaging system developed to date is the cooled, charge-coupled device (cooled CCD, Hiraoka et al., 1987). The cooled CCD is an efficient photon-counting device across a broad spectrum. Although cooled CCDs for color are available, the best resolution is possible with noncolor-discriminating CCDs. Digital images captured with a CCD can be readily stored and analyzed by computer. While CCDs are very sensitive, allowing the capture and analysis of weak signals, the lack of color discrimination presents problems for capturing multicolor images. In practice, several images are captured by using various filter sets. The merging of images to form a composite requires the assignment of pseudocolors to each image and accurate register of the images. Image

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186 % transmission 1

wavelength, nm transmission ratio, % 2

I 400 500 700

900

600

, 0

wavelength, nm

% transmission

1

3

wavelength, nm FIG. 2 Optics of the epifluorescence microscope. Lefr-hand side. Path of excitation light from the lamp through the excitation filter (1); reflection by the dichroic mirror (2), through the objective to the specimen. Fluorescence emitted from the specimen travels through the objective, dichroic mirror (2) and emission filter (3) to the observer. Right-handside. Graphs illustrating the optical properties of (1) the excitation filter, (2) the dichroic mirror, and (3) the emission filter. See text for further details. Filters 1 and 3 absorb nontransmitted light; the dichroic mirror reflects nontransmitted light. The transmission ratio is the ratio of transmission to reflection.

alignment can be facilitated by including a fixed marker (Albertson et al., 1988) or the choice of a defined object in the image as a marker (Waggoner et al., 1989; du Manoir et al., 1993; Ried et al., 1992). Digital imaging systems also allow the quantification of fluorescence image signals (Nederlof et al., 1992a,c);however, variation within preparations limit absolute measurement of fluorescence intensity (Nederlof et al., 1992a,c). Fluorescence ratio measurements can be more accurately determined, thus allowing “ratio” or “combinatorial**labeled probes (see

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WHOLE-CHROMOSOME HYBRIDIZATION TABLE II Excitation and Emission Maxima for Commonly Used Fluorochromes

Fluorochrome

FITC Texas red TRITC AMCA

hopidium iodide DAPI Quinacrine

Excitation max (nm)

Emission max (nm)

490 590 560 350 520 365 420

525 615 580 430 610 450 500

Section II,B,2) to be distinguished (Nederlof et al., 1992b; Ried et al., 1992; du Manoir et al., 1993). While digital imaging and analysis allows the simultaneous viewing of multiple targets electronically, multiple targets can be directly seen in a microscope by the use of specialized optics. Dual band-pass filters allow two flurochromes with differing spectral properties [e.g., 6-diaminophenylindoledihydrochloride (DAPI) and FITC] to be viewed simultaneously without the need to change filter sets. Such filter sets are composed of exitation and barrier filters, each with two narrow bands of transmission, and a beam splitter with two regions of reflection and transmission, the properties of the reflection and transmission regions being tailored for the desired pair of fluorochromes. The use of such filter sets overcomes the image register problem noted earlier. More recently, triple and quadruple band-pass filter combinations have become commercially available. Although it is possible to perform three-dimensionalanalysis with conventional epifluorescence optics and sophisticated image processing (see, for example, Agard and Sedat, 1983), the instrument of choice for threedimensional work is the confocal laser scanning microscope. Images obtained by conventional epifluorescence contain much fluorescence from outside the focal plane; thus, images of specimens with any appreciable depth are blurred. Confocal laser scanning microscopes are designed to image precisely a single, thin, optical section at any one time; thus blurring is minimized. Further details of confocal laser scanning microscopy can be found in Brakenhoff et al. (1979) and Schotton (1989). By storing and then reconstructing serial optical sections, high-quality,three-dimensional reconstruction is possible. Thus confocal microscopy is being exploited in studies of nuclear architecture (Kett et al., 1992; Shaw et al., 1992; Ferguson and Ward, 1992).

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As mentioned in Section II,B,3, a number of novel DNA probe detection systems have been developed which demand specialized microscopic techniques. A notable example here is the use of a time-resolved fluorescence microscope to discriminate among fluorochromes with differing fading characteristics (Beverloo et al., 1992; Seveus et al., 1992). Such systems may allow the detection of multiple targets in excess of that which is currently possible. The recently developed technique of atomic force microscopy is applicable to the study of DNA and chromosome structures (Henderson, 1992), and can be used to detect bound probe in situ hybridization preparations (deGrooth and Putman, 1992; Rasch et al., 1993; Putman et al., 1993). There is clearly a desire for researchers to improve the resolution of in situ hybridization and to extend its use. Optics and electronics have contributed significantly in these areas. The automation of fluorescence microscopy is also an area of active research. Automated metaphase location and analysis of in situ hybridization signals could be highly beneficial in a number of areas. Despite the range of sophisticated systems which have been developed, at present the technology only serves to extend the possibilities of in situ hybridization rather than being a requirement. 5. Typical Whole-Chromosome Hybridization Method

This chapter is not intended to be a step-by-step practical guide. However, given the diversity of experimentalparameters which have been discussed, it seems useful to indicate commonly employed conditions for wholechromosome hybridizations. Much useful practical information can be found in Lichter and Cremer (1992). Figure 3 is a diagramatic representation of the process. As with all cytogenetic methods, good-quality chromosome preparations are essential. The most important factor in any whole-chromosome hybridization experiment is the quality of the labeled probe. Irrespective of the method used for labeling, the probe should be checked for size (optimum 100-200 bp) and extent of label incorporation; 1 pg of a goodquality probe should be detectable in a dot blot assay. Almost all wholechromosome hybridization probes require a suppression step to remove common, nonchromosome-specific repeats from the hybridization to the specimen. Thus an excess of genomic or Cot-1 DNA (human-for-human probes, mouse-for-mouse probes, etc.) is denatured with the probe and allowed to reanneal for a limited period. Following suppression, the probe is added to a slide (denatured by immersion in 70% formamide, 2x SSC at 70°C) and allowed to hybridize. Frequently slides are pretreated with RNase and proteinase prior to denaturation. The probe concentration

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l5 FIG. 3 Whole-chromosome hybridization method. (Top left) Probe manipulation ( 1 ) Labeling. (2) Denaturation and suppression of repeats. (Top right) Manipulations of the metaphase preparation (3) Denaturation of chromosomal DNA. (4) Slide and probe are combined and allowed to hybridize. (5) Finally the preparations are washed and the sites of probe hybridization detected.

employed will vary widely, depending upon the probe itself, the presence of vector sequences in cloned probes, and other factors. The hybridization solution contains formamide at 50%, 2x SSC and 10% dextran sulfate. Additional components such as EDTA (0.1-0.5 mM) and sodium phosphate (10-50 mM) are sometimes included. Hybridizations are usually

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allowed to proceed overnight and sometimes longer at 37°C (occasionally 42°C or 45°C). Posthybridization washes are used to remove nonspecifically bound probe; conditions used for washing vary considerably among authors. The wash of highest stringency is of great importance in determining the extent to which nonspecifically bound probe is removed. Usually, a wash of 2x SSC/50% formamide at 45°C is employed; aqueous washing is also possible; 0.1 x SSC at 60°C is an approximately equivalent stringency but chromosome structure can be adversely affected by high-temperature washing. The procedure for probe detection will vary, depending upon the labeling and detection method being used. All procedures share a blocking step to reduce nonspecific binding of detection reagents. Following detection reactions, the chromosomal DNA is usually counterstained with DAPI and/or propidium iodide when fluorescence detection is used. Propidium iodide can be viewed with the same standard filter set as FITC, which can be useful for orienting signals on chromosomes. A number of methods have been devised to allow chromosome identification on in situ hybridization preparations. DAPI fluorescence can sometimes provide sufficient chromosome band information. Alternatively, a replication banding method can be used if bromodeoxyuridine is incorporated in cells during the S-phase prior to making chromosome preparations. An in situ hybridization method for chromosome banding has been developed (Lichter et a/., 1990a; Boyle et al., 1990a; Baldini and Ward, 1991). This method exploits the fact that some interspersed repeat elements are nonuniformly distributed throughout the genome, thus Alu sequences will hybridize to human chromosomes to give an R-band pattern. Similarly, L1 repeats can be used to produce a band pattern on mouse chromosomes. C. Probes for Whole-Chromosome Hybridization

In addition to the technology required for performing in situ hybridization, whole-chromosome hybridization is critically dependent upon the availability of suitable DNA probes. Early studies relied upon the use of whole genomic DNA as a probe applied to interspecific hybrid cell lines. In this way, DNA sequence divergence between species was exploited to allow specific chromosome staining. The development of cloned DNA probe libraries from specific, purified chromosomes, in conjunction with suppression hybridization techniques (see Sections II,B, 1 and II,B,5), enabled specific whole-chromosome hybridization in normal human cells. These libraries are probably the most widely used reagents for wholechromosome hybridization at present. However, developments in technol-

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ogy continue, and so a genomic DNA hybridization method for detecting subtle differences among genomes of the same species-comparative genomic hybridization-promises to be a powerful technique (see later discussion).

1. Use of Genomic DNA The earliest reports of whole-chromosome hybridization were made in 1985 (Schardin et al., 1985; Durnam et al., 1985; Manuelidis, 1985). In an investigation of nuclear organization, Schardin et al. (1985) performed in situ hybridization of human genomic DNA in a hamster x human hybrid cell line carrying a single X chromosome as the only identifiable human material. 'H-labeled human genomic DNA was found to locate almost exclusively to the human X at metaphase and a single domain of hybridization was noted in interphase nuclei. Biotinylated human genomic DNA detected with an avidin-alkaline phosphatase system was seen to behave in the same manner as 3H-labeledprobe. Similarly, Durnham et al. (1985) found human chromosomes in man-mouse hybrids using peroxidase detection. The use of fluorescence detection of biotinylated human genomic DNA in human x hamster hybrids soon followed (Pinkel et al., 1986). Schardin et al. (1985) noted that translocations between human and hamster chromosomes could be detected by this method, a finding exploited by Pinkel et al. (1986) to assess radiation-induced translocation frequencies. The detection of species-specific chromosomes in somatic cell hybrids can also be performed in suspension (Trask et al., 1988), allowing either flow cytometric or microscopic analysis. Genomic DNA hybridizations have proved to be useful in analyzing the genomes of cell hybrids (Giaccia et al., 1990; Boyle et al., 1990b; Kievits et al., 1990). The work of Boyle et al. (1990b) demonstrated the utility of hybridization of mouse DNA to mouse-hamster hybrids and the converse experiment of hybridizing DNA from the mouse-hamster hybrid to a normal mouse metaphase. This reverse experiment allows the chromosomal origin of the mouse component of the hybrid to be analyzed. Kievits et al. (1990)were able to identify the human components in mouse-human hybrids by an analogous method. While many somatic cell hybrids contain identifiable species-specificchromosomes, rearrangements can occur (see, for example, Giaccia et al., 1990).The reverse experiment of hybrid DNA to normal metaphase allows the origin of the rearranged chromosome segments to be identified. Genomic DNAs from human-hamster hybrid cells containing normal or rearranged human chromosomes have been used to analyze human tumor material. Suijkerbuijk et al. (1991) demonstrated that the marker chromosome of testicular germ cell tumors was, as had been suspected, an iso-12p chromosome.

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While increasing numbers of chromosome-specifichybridization probes are being developed (see later discussion), there remain many species for which genomic DNA is the sole probe available. For example, the equine chromosome composition of mouse-horse hybrids can be analyzed by hybridization of equine DNA to the hybrids (Lear et al., 1992). Hybridizations using genomic DNA have probably been more widely exploited in plant than in mammalian systems. These studies have proved of use in identifying chromosomes in man-made hybrid plants (e.g., Le et al., 1989; Schwarzacher et al., 1989;Anamthawat-Jonsson et al., 1990; Leitch et al., 1990). More recently, genomic DNA hybridization has shown the naturally occurring grass Milium montianum to be an allopolyploid (Bennett et al., 1992). Thus genomic DNA in situ hybridization can be used to analyze plant and animal hybrids. In this way, problems of taxonomy, chromosome evolution, and genome organization in hybrids can be addressed. (Further discussion of these areas can be found in Section 111.) Genomic DNA hybridizations are now being used to detect genome differences of a more subtle nature. The technique known as comparative genomic hybridization (CGH) allows a direct visual comparison of the genetic constitution of normal and tumor cell nuclei (Kallioniemi et al., 1992; du Manoir et al., 1993). In CGH, tumor cell DNA is labeled and mixed with control normal cell DNA differentlylabeled and Cot-1-blocking DNA. This mixture is denatured, reassociated for a limited period to allow common repetitive DNA sequences to reanneal, and then applied to denatured normal chromosome preparations. The hybridized labeled DNAs are detected with different fluorochromes, for example, FITC for normal DNA and TRITC for tumor DNA. Chromosomes which are present in the normal DNA and tumor DNA at the same copy number should then fluoresce with the same intensity with both fluorochromes, while chromosomes present at reduced or increased copy numbers in the tumor DNA should fluoresce with reduced or increased intensity signal with the tumor DNA probe relative to the normal cell DNA fluorescence signal. Thus, whole-chromosome duplications or losses can be detected and smaller scale deletion or amplification of chromosome segments can also be visualized. The accurate determination of fluorescence ratio measurements relies upon capture and processing of digital images. One of the major strengths of this technique is that it allows cytogenetic analysis of cells from which it is difficult to obtain good-quality metaphase preparations. Therefore CGH should prove particularly useful for the analysis of solid tumors where the production of quality metaphases can be problematic. Kallioniemi et al. (1992) demonstrated the versatility of CGH by analyzingseveral tumor cell lines and two primary tumors. Losses and gains of chromosome segments in a breast tumor cell line (600PE)

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could be detected; deletions in the 10-20 Mbp range around the retinoblastoma gene locus could be visualized, and amplifications in several tumor types seen. Precise quantitation of the level of DNA amplification was unreliable; however, genomic regions not previously suspected of being involved in tumorigenesis could be seen to be amplified. Du Manoir et al. (1993) were able to demonstrate the presence of excess chromosome 21 in a Down’s syndrome patient and provide a detailed analysis of gains and losses in a renal carcinoma cell line and a primary T-cell leukemia. A relatively simple modification of the CGH technique has also been successfully developed and applied (Joos et al., 1993). In this method, labeled tumor cell DNA is hybridized with Cot-1 DNA suppression to normal metaphases. Amplifications of chromosome segments encompassing the c-myc locus could be readily detected; whole chromosome losses were also detectable. These genomic DNA hybridization strategies have great potential for investigation of a variety of genomic alterations, particularly because they do not require prior knowledge of candidate loci for rearrangement. Rather, CGH can identify genomic regions of potential importance in tumorigenesis.

2. Cloned Chromosome-Specific DNA Probe Libraries The most widely used probes for whole-chromosome hybridization currently are the cloned human chromosome-specific DNA probe libraries. In general, these have been produced by flow cytometric sorting of chromosomes, either from normal diploid cells or somatic cell hybrids, followed by molecular cloning. As mentioned previously, the first set of such libraries was produced by a U.S.national laboratories project (van Dilla et al., 1986) and is now available through the American Type Culture Collection. These libraries were produced by the complete digestion of the various flow-sorted human chromosomes with Hind111 or EcoRl and cloning into the appropriate site in the bacteriophage lambda vector Charon 21A. These probes were used for the first chromosome-specific painting in normal human cells (Lichter et al., 1988a; Pinkel et al., 1988). In order to achieve chromosome-specific hybridization, these libraries require suppression of the nonspecific repeat sequences which occur in all “chromosome-specific’’ libraries. The early studies employed sheared total genomic DNA for suppression; however, the repeat-enriched Cot- 1 fraction of genomic DNA is being increasingly employed. If repetitive DNA is not suppressed, a significant signal is observed on all chromosomes. The principle of suppression (or competitive) in situ hybridization is described in Section II,B,l. In the study by Lichter et al. (1988a1, chromosome-specific libraries in the original phage vectors were used as

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a source of probe, while Pinkel et al. (1988) subcloned the inserts from the phage vector into a plasmid prior to use. A basic problem with the phage libraries is that the ratio of vector to insert is high (approx. 10: 1). Thus, Lichter et al. (1988a) report that superior specificity of hybridization is obtained when the cloned insert is purified from the phage vector prior to labeling with biotin and hybridization. These authors demonstrate that both fluorescence (FITC) and enzymatic (alkaline phosphatase-NBT-BCIP) detections can be successfully employed. An alternative approach was used by Pinkel et al. (1988) to resolve the vector-to-insert ratio problem. Prior to use as probes, the inserts from phage libraries for chromosomes 4 and 21 were subcloned into a smaller plasmid vector. In this way whole plasmid plus insert libraries can be successfully labeled and applied as probes, in contrast to a comment to the contrary by Lichter et al. (1988a). Subsequently, a complete set of chromosome-specific libraries has been subcloned from the original Charon 21A vector into plasmids (Collins et al., 1991). An alternative method for producing probes from phage libraries has been developed by Burde and Leary (1992). The polymerase chain reaction was used to amplify specifically the inserts in a human chromosome 1 phage library. PCR products biotinylated by nick translation were then used as a chromosome 1 hybridization probe (Burde and Leary, 1992). Both the original phage chromosome-specific libraries and the subcloned plasmid libraries are in use in laboratories worldwide. The intensity and specificity of hybridization of the various chromosome-specific libraries is not uniform. Three types of problems can be encountered: (1) lack of hybridization to certain regions of the target chromosome, (2) strong hybridization to nontarget chromosome regions, and (3) relatively high background signal on nontarget chromosomes. Collins et al. (1991) provide a detailed characterization of their plasmid libraries. Ten of the 24 libraries are considered to be top grade, wholechromosome hybridization probes. Lack of uniform staining along the target chromosome, particularly at terminal regions and centromeres, is a problem with some libraries, while others stain nontarget regions, frequently centromeres, to a significant extent. Nonetheless, they represent a very useful resource for whole-chromosome hybridization. In general, the phage libraries have the same problems as the plasmid libraries, as would be expected since one was derived from the other (Lichter et al., 1988a). The greatest part of whole-chromosome hybridization probe development to date has concentrated on human chromosomes. However, wholechromosome hybridization probe sets are available for some other species. A collection of Saccharornyces cereuisiae chromosome V-specific recom-

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binant phage clones which cover a significant proportion of this chromosome has been assembled (Scherthan et al., 1992b). This collection of clones does not produce a uniform hybridization signal over the entire chromosome; rather, the clones cluster into three groups, each group being distinguishable as an area of hybridization. Differential labeling of the clusters of clones allowed chromosome orientation during meiosis to be followed (Scherthan et al., 1992b). The genome of the domestic pig is under close investigation at present and chromosome painting probes are becoming available. Flow sorting of chromosomes followed by PCR amplification and cloning into plasmid vectors has produced a pig chromosome 1-specific library suitable for whole-chromosome hybridization (Miller et al., 1992).Chromosome-specificprobe collectionsfor laboratory animals, such as the mouse and rat, are also under development. A sophisticated method that simultaneously hybridizes whole-chromosome probes and chromosome region-specificyeast artificial chromosome (YAC) probes, known as chromosomal bar coding, has been described recently (Lengauer e? al., 1993). By selecting and hybridizing appropriate YAC clones covering defined chromosome regions and wholechromosome hybridization probes, novel banding patterns could be produced on human chromosomes. Thus, specific sets of reagents tailored to the requirements of specific translocation analysis in, for example, tumor material, could be assembled. Such reagents could revolutionize such areas as clinical cytogenetics. The bar codes produced can be distinguished on the basis of location, color, size, and intensity of signal. This technique has been successfully applied with the combinational labeling method of Ried et al. (1992) (see Section II,B,2) to produce distinctive bar code patterns on at least seven chromosome pairs in a single metaphase (Lengauer et al., 1993). With the increasing availability of YAC clones spanning significant tracts of the human genome, this chromosomal bar coding approach promises to be a powerful analytical tool in cytogenetics. 3. Polymerase Chain Reaction Methods for the Production of Whole-Chromosome Hybridization Probes

The development and application of the polymerase chain reaction has revolutionized many areas of molecular biology, including the development of probes for whole-chromosome hybridization. Three major types of PCR technique have been exploited for probe production: interspersed repetitive sequence (IRS) PCR, linker-adaptor PCR, and randomprimed PCR. IRS-PCR exploits a feature of all mammalian genomes, namely, the existence of frequent and widely dispersed short and long interspersed repeat elements. Broadly similar families of repeats exist in several spe-

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cies, although there is sufficientsequence divergence between human and rodent IRS elements so that species-specific PCR primers can be produced. Thus DNA lying between adjacent IRS elements in the appropriate orientation can be amplified by PCR with species-specific IRS primers. The human DNA component of human-rodent somatic cell hybrids can be isolated with the use of human A h primers (Nelson et al., 1989). This method can also be exploited to isolate and label the cloned insert in yeast artificial chromosomes. The utility of the long interspersed repeat LlH in IRS-PCR has also been demonstrated, (Ledbetter et al., 1990). The use of IRS-PCR in chromosome hybridization studies was demonstrated by Lengauer et al. (1990) and Lichter et al. (1990a). IRS-PCR products were produced from human-rodent somatic cell hybrids, labeled with biotin, and used to probe normal human mitotic preparations. Human-specific IRS-PCR products from hybrids containing either chromosomes 7, 22 (Lichter et al., 1990a), or 7, 11, 20, and X (Lengauer et al., 1990) as the human material hybridized specifically to the respective chromosomes in normal human preparations. Furthermore, the composition of previously identified human marker chromosomes in hybrids could be established. A useful feature of this technique is that the IRS-PCR products used as probes do not always hybridize uniformly along a given chromosome; rather, a band pattern analogous to R-banding can be distinguished (Lichter et al., 1990a). IRS-PCR probes with improved specificity and signal intensity can be produced by using two Alu PCR primer sets (Liu et al., 1993). Combining flow cytometry with IRS-PCR and in situ hybridization extends the application of these methods still further. While the early studies using chromosome hybridization probes generated by IRS-PCR depended upon the availability of suitable somatic cell hybrids, it has now been shown that target chromosomes for IRS-PCR can be purified by flow cytometry (Suijerbuijkeral., 1992).Normal or abnormal (marker) chromosomes can be purified and suitable whole-chromosome hybridization probes prepared. Suijerbuijk et al. (1992) were able to produce probes for several normal human chromosomes and confirm previous cytogenetic analysis of three marker chromosomes. A broadly similar method has been employed to reveal the identity of a marker chromosome from a bladder tumor cell line (Boschman et al., 1993). Random-primed PCR methods promise to be of wide use in molecular cytogenetics. The flexibility of these methods lies in the fact that no DNA sequence information is required. The degenerate oligonucleotide-primed PCR (DOP-PCR) technique of Telenius et al. (1992) employs a single PCR primer containing a run of six random nucleotides. Flow-sorted chromosomes are amplified with this primer, initially with low-temperature annealing cycles to allow random priming, followed by more stringent

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annealing cycles. As with the IRS-PCR methods, both normal and marker chromosomes can be sorted and used as targets, allowing the classification of abnormal chromosomes (Telenius et al., 1992; Carter et al., 1992). Flow cytometry allows the purification of perhaps several hundreds of chromosomes, but not all marker chromosomes are readily purified by flow sorting. The DOP-PCR technique can be combined with chromosome microdissection (Meltzer et al., 1992). Microdissection should allow the purification of any marker chromosome, and furthermore allows the production of chromosome band and region-specific libraries for in situ hybridization (Meltzer et al., 1992; Deng et al., 1992). The random-primed PCR systems are not limited in application to a single species. DOP-PCR has been used to produce pig chromosome painting libraries (Langford et al., 1992) and another method using very low-temperature primer annealing steps has been used to produce pig chromosome 1- and 18-specific libraries (Milan et al., 1993). As an alternative to random-primed PCR, purified chromosome-specific DNA can be amplified by PCR following restriction and ligation of linkeradaptors. The linker-adaptor sequence is used for priming the PCR reactions. This method has been used successfully to microclone chromosome 21 fragments (Kao and Yu, 1991). More recently, a set of human chromosome-specific libraries has been produced from flow-sorted chromosomes by linker-adaptor PCR (Vooijs et al., 1993). It is claimed that these libraries are stable to amplification through more than 200 cycles of PCR. This set of libraries, based on a complete set of newly sorted chromosomes, has improved upon some of the libraries derived from the original U.S.national laboratories project on phage libraries. An example of whole-chromosome hybridization using one of these probe collections is shown in Fig. 4. While the majority of studies have, understandably, concentrated on human material, many of the techniques that have been developed for producing whole-chromosome hybridization probes are not restricted to use with human chromosomes. The current range of methods available for producing whole-chromosome hybridization probes should, at least in principle, allow the production of a probe for any normal or abnormal chromosome from any species. 111. Applications of Whole-Chromosome Hybridization

The preceding sections of this chapter have indicated the range of methods available for whole-chromosome hybridization analysis. These tools have been developed to address a number of issues in the disciplines of cytogenetics and genome analysis. Some applications are of a fundamental na-

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D. B O W L E R

FIG. 4 Whole chromosome hybridization of probe library pcr-2 (originally obtained from

Dr.J. Gray) to a normal human lymphocyte metaphase. The two copies of chromosome 2 stain brightly (white in this picture). The probe was biotin labeled and detected with FITCavidin. Scale, 1 cm = 3 pm.

ture, such as the investigation of evolutionaryrelationships among species, while others have a very practical purpose, for example, biological assessment of radiation doses. The sections which follow consider some of the major applicationsof whole-chromosome hybridization. In this way I hope to highlight the achievements made by applying these techniques and indicate some future uses and applications of the technology.

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A. Evolutionary and Taxonomic Studies Traditional cytogenetic analysis has contributed much to the fields of evolutionary biology and taxonomy. For example, band analysis suggested that human chromosome 2 originated through the fusion of two acrocentric chromosomes-the ancestral chimpanzee chromosomes 12 and 13 (de Grouchy et al., 1972). The human chromosome-specificDNA probe libraries provide a set of useful reagents for studying chromosome evolution among primates and have now been applied to many species. Wienberg et al. (1990) used biotinylated phage libraries for human chromosomes 1 , 2, 3, 4, 5, 7, 9, 17, X, and Y to probe metaphases of many great ape and lower primate species. These authors were able to demonstrate the evolutionary conservation of the X chromosome during primate evolution. While the staining of the X chromosome was uniform in the great apes, a tendency for an R band patterning was seen on the X chromosomes of the lower primates (Maccacafuscata, Cercopithecus talapoin, Papio ursinus, Lagotrix lagorrix, Galago demidouii). However, chromosome hybridization of the human X library to cat (Felis catus) mouse (Mus musculus), or vole (Microtus sauii) chromosomes could not be achieved. Where whole-chromosome hybridizations do not work, the alternative technique of single-copygene in situ hybridization can be useful for studies of chromosomal evolution (e.g., Maccarone et al., 1992). As stated above, the analysis of chromosome band patterns has suggested that the human chromosome 2 had its origins in the fusion of ancestral chromosomes 12 and 13 of the chimpanzee. Application of a human chromosome 2 hybridization probe to chimpanzee metaphases highlighted most of the length of chimpanzee chromosomes 12 and 13 (Wienberget al., 1990; Luke and Verma, 1992), confirming band analyses. While a large interstitial telomere tract has been cloned from the presumptive fusion point on human chromosome 2 (Ijdo et al., 1992), further events in addition to simple fusion, such as centromere inactivation and deletion of heterochromatic regions, must have occurred (Luke and Verma, 1992). The notion that gorilla chromosomes 4 and 19 were derived from a reciprocal translocation between chromosomes homologous to human chromosomes 5 and 17 (Yunis and Prakash, 1982) has been confirmed by using whole-chromosome hybridization probes for human chromosomes 5 and 17 on gorilla metaphases (Wienberg er al., 1990; Stanyon et al., 1992). The chromosomal relationships between humans and the great apes are relatively simple. A detailed whole-chromosome hybridization study confirmed that the fusion origin of human chromosome 2 and the t(5;17) were the only detectable large-scale rearrangements to have occurred in

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the great apes in comparison with humans (Jauch et al., 1992).By contrast, the organization of the gibbon karyotype is significantly different from that in humans. Again, through whole-chromosome hybridization and banding analyses, it was deduced that numerous translocations divided the ancestral human homologous autosomes into 5 1 segments to form the 21 gibbon autosomes (Jauch et al., 1992). These studies on higher mammals illustrate the power of whole-chromosome hybridization methods in comparative cytogenetics. As wholechromosome hybridization probes for a wider range of species become available, further evolutionary questions will be answerable. There are some particularly intriguing problems in chromosome evolution, for example, the chromosomal relationships between the Indian muntjac (2n = 78,69) and the closely related Chinese muntjac (2n = 46). Perhaps it may be possible to take “evolutionary walks” through the karyotypes of various species by selecting and generating appropriate wholechromosome hybridization probes. Thus, large-scale chromosome events associated with speciation may be identified, such as the loss of heterochromatin from chimpanzee chromosomes 12 and 13 being involved in human evolution, as suggested by Luke and Verma (1992). Plant studies do not have the advantage of the availability of chromosome-specificprobe libraries. Nonetheless, genomic DNA probing can be informative in plant genome analysis. Bennett et al. (1992) were able to confirm the close relationship between the large chromosomes of Milium montianum and those of M . vernale by probing M . montianum metaphases with biotinylated M . vernale DNA. Novel hybrids can also be analyzed by genomic DNA probing. Hybrid Nicotiana plumbaginifolia x Nicotiana sylvestris plants generated by protoplast fusion with irradiation of one partner have been analyzed by probing metaphases with genomic DNA (Parokonny et al., 1992b). Similarly, the origin of chromosomes in F, hybrids between two species of Gibasis, which share very similar chromosome morphology, can be verified with genomic DNA hybridization (Parokonny et al., 1992a). Genomic DNA hybridization analysis has also enabled the origin of “alien” chromosomes in certain wheat strains to be ascertained (Murata et al., 1992; Schwarzacher et al., 1992). Whole-chromosomehybridization will aid studies of plant genetics and evolution, just as in studies of primate evolution. Given the widespread occurrence of hybridization among plant species, the lack of chromosomespecific probes has not hindered progress because genomic DNA probing can be successful, although more detailed studies of evolutionary relationships demand such probes. There can be little doubt that genomic DNA probing will prove to be of great use in plant breeding programs (Schwarzacher et al., 1992).

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6. Nuclear Architecture

The structure of the interphase nucleus has been a subject of great interest to biologists since the proposals from Rabl (1885) and Boveri (1909) that chromosomes occupy discrete regions within the interphase nucleus. The availability of whole-chromosomehybridization methods has allowed the detailed study of some aspects of interphase nuclear structure. The early studies by Schardin et al. (1985) and Manuelidis (1985) examined human-rodent somatic cell hybrids. Cells were probed with labeled (tritium or biotin) human genomic DNA. Schardin et af. (1985) observed a discrete area of hybridization in interphase nuclei of a hybrid carrying a single X as the sole human chromosome. The location of this area of hybridization was not consistent, but apparently frequently appeared in the form of a two-lobed structure. The significance of this observation remains obscure. A hybrid carrying the human chromosomes 11, 17, and X was examined by the same authors and again discrete areas of hybridization were noted; these areas could appear separate or fused. These experiments perhaps represent the first direct suggestion of a territorial organization of chromosomes in the interphase nucleus of higher eukaryotes. The study by Pinkel et al. (1986) also noted the presence of discrete chromosome territories in human-hamster hybrid cells. Chromosome territories have also been studied in hybrid hamster egg-human sperm zygotes (Brandriff and Gordon, 1992). As with all studies of interphase nucleus organization, these hybridization experiments are sensitive to fixation techniques, and one has to be aware of possible artifacts when interpreting data. The somatic cell hybrids themselves may be a special case. The extension of these findings to normal diploid cells through the use of chromosome-specific probes was an important step (see later discussion). However, studies with somatic cell hybrids have an advantage in that hybridizations do not have to be performed under suppression conditions. Thus the entire chromosome, unique sequences and repeats, can be visualized. This is not possible in normal cells where the use of chromosome-specific probes demands suppression of repeat-sequence hybridization. The two early papers demonstrating chromosome-specifichybridization in metaphase human cells using cloned DNA probe libraries also noted discrete areas of hybridization in interphase nuclei (Lichter et al., 1988a; Pinkel et al., 1988). Two areas of hybridization were, in general, noted, presumably representing the two homologs of each chromosome. Pinkel et al. (1988) also demonstrated that three areas of hybridization could be detected in a cell line carrying a stable translocation t(4;11), using a chromosome 4 probe library. However, the scoring of chromosomal abnormalities in interphase with whole-chromosome hybridization probes

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is not simple. The most successful and convincing interphase analyses rely upon chromosome-specificcentromere probes which produce intense hybridization signals or large, cloned, unique-sequence probes. Nonetheless, Cremer et al. (1990) have demonstrated that abnormalities in interphase nucleus chromosome domain structure, detected with probes for human chromosomes 1 and 7, correlated with ionizing radiation dose. Thus, a linear relationship was found between the square of a %o gammaray dose and the percentage of nuclei with aberrant chromosome domain structures. The study of interphase nucleus organization demands three-dimensional imaging and image reconstruction systems. Confocal laser scanning microscopy is widely used in studies of nuclear structure. It effectively allows these optical sections to be imaged without interference from scattered light or background, out-of-focus light. A number of recent articles have covered the use of confocal microscopy for in situ hybridization studies (Shaw et al., 1992; Kett et al., 1992; Hulspas and Bauman, 1992). Standard fluorescence microscopy can be used in three-dimensional studies but the reconstruction of images requires the use of complex algorithms. This task can be helped through the use of specimen tilting devices such as that developed by Brad1 et al. (1992). Bischoff et al. (1993) employed both three-dimensional confocal and two-dimensional analysis of active and inactive X-chromosome domains in human amniocytes. Some discrepancy between the techniques was found in the measured sizes of the inactive and active X domains. The two-dimensional analysis suggested that the active X domain was nearly twice the size of the inactive X domain. The three-dimensional analysis did not detect such a large difference. Ferguson and Ward (1992) examined chromosome arrangements in human lymphocytes at various stages of the cell cycle. Human T lymphocytes were stimulated to divide, fixed, stained with propidium iodide, and sorted into G , , S, and G2 populations by flow cytometry. Hybridization of a chromosome 8 library to such nuclei revealed that in G, the centromeric region of this chromosome was commonly found in close association with the periphery of the nucleus, with the chromosome arms oriented toward the interior of the nucleus. By contrast, in G2,the centromeric region was found in a more internal location, with chromosome arms directed toward the outer edge of the nucleus. Through the use of a number of chromosome region-specificprobes, these authors demonstratedthat in G,, centromeres tend to be located in the peripheral regions of the nucleus, sometimes in contact with the nuclear envelope, while telomeric regions tend to lie in the interior nuclear region. Homologous chromosome segments were not in pairs. The pattern for G, nuclei tended to confirm the result with the chromosome 8 probe, that is, that centromeres took a more internal posi-

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tion, with telomeres occupying more peripheral areas. Broadly similar arrangements have been found in mouse T cells, the analysis here depending entirely upon chromosome region-specific probes (Vourc’h er al., 1993).These detailed analyses highlight the dynamic nature of interphase chromatin arrangement. The majority of the detailed studies of nuclear organization in mammals have relied upon chromosome region-specific probes rather than wholechromosome hybridization probes. As pointed out earlier, when using whole-chromosome hybridization probes on normal cells, one needs to be aware of the technical limitations imposed by the requirement to compete out (and so not visualize) common repetitive elements. In this regard, hybrid cell studies may have advantages. However, the artifical nature of hybrid cells also presents problems of interpretation-the foreign chromosomes may not be integrated into the nucleus in the normal fashion. Analysis of nuclear organization in plants has also benefitted from the availability of whole-chromosome hybridization methods. As with the evolutionary and taxonomic studies discussed in the previous section, it is genomic DNA hybridization which has been most extensively employed. The existence of stable, viable, interspecific, and intergeneric hybrids in plants provides ideal material in which to study nuclear organization by genomic DNA hybridization methods. Extensive studies using classic cytogenetic methods suggested that the parental genomes in hybrid plants remain separated in mitosis and furthermore appear to act independently to some degree (reviewed by Heslop-Harrison and Bennett, 1990). Analysis of sectioned material from a barley (Hordeurn uulgare) X Secale africanum hybrid with genomic DNA suggested that the separation of parental genomes is maintained throughout interphase as well as during mitosis (Leitch el al., 1991). Parental genome separation has also been demonstrated in an interspecific hybrid between H . uulgare and H . bulbosum (Anamthawat-Jonsson et al., 1993). Single or small numbers of chromosomes in plant material can be studied using plant strains carrying a limited number of alien chromosomes; in this way Schwarzacher et al. (1992) demonstrated the existence of chromosome territories in interphase nuclei of wheat strains. As is the case with human cells, in diploid strains, pairing of territories was not observed, suggesting that homologs behave independently during interphase. Homologous chromosome pairing does, however, occur during meiosis. The behavior of meiotic chromosomes has been examined by chromosome hybridization techniques in human testicular material (Goldman and Hulten, 1992) and yeast (Scherthan et al., 1992b). The latter authors demonstrate that in Saccharomyces cerevisiae, chromosomes condense and homologous segments align prior to synaptonemal complex formation. Whole-chromosome hybridization has also been used to examine the mei-

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otic behavior of abnormal chromosomes in human testicular biopsy material (Goldman and Hulten, 1993). The understanding of nuclear organization in mitotic and meiotic cells will benefit from the continued application of whole-chromosome hybridization techniques. In particular, the dynamic aspects of nuclear structure are now coming under scrutiny. C. Mechanisms of Formation of Chromosome Aberration

Studies of the type described in the previous section demonstrate the use of whole-chromosome hybridization experiments in elucidating the structure of the interphase nucleus. Some of the consequences of exposure to agents that damage DNA are likely to be influenced by interphase nuclear structure. The physical location of chromosomes within the nucleus must constrain the range of chromosome segments which can potentially interact to form chromosome aberrations. This is particularly important with respect to “exchange-type” aberrations such as dicentrics and translocations, which involve the interaction of (at least) two chromosome segments. Understanding the dynamic nature of nuclear organization, as revealed by the studies of Ferguson and Ward (1992) and Vourc’h et al. (1993), will be of particular importance. The involvement of specific chromosomal translocations and deletions in the origin and development of some human tumors highlights the need to gain an understanding of how such chromosome aberrations are formed. The two predominant types of exchange chromosome aberrations are dicentrics and translocations; both involve an exchange of material between chromosomes. In the case of a dicentric, the exchange includes rejoining the centromere-containing portion of two chromosomes, giving rise to one dicentric and an associated acentric fragment. Translocations, in contrast, involve the exchange of chromosome segments, the event forming two monocentric but abnormal chromosomes (see Fig. 5). Translocations at the chromosome level can appear as reciprocal where there has been precise mutual exchange of material, or terminal where it appears that one chromosome has donated material to the end of another without any being returned. Mechanistically, dicentrics and reciprocal translocations are thought to arise through the same process, with the chromosomal breakpoints and rejoining determining the outcome of the event(s). Biologically, reciprocal translocations and dicentrics differ in stability in transmission through cell division. In general, dicentrics are unstable and so are lost as a result of cell death caused by the difficulties a chromosome with two centromeres presents for the mitotic spindle, while reciprocal translocations are usually

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H

a

1

2

3

b

L

FIG. 5 Stable exchange-type chromosomal aberrations. (a) Two normal chromosomes and possible derivatives; 1, dicentric plus fragment; 2, reciprocal translocation; 3, terminal translocation. (b) Complex translocation among three chromosomes; normal chromosomes are to the left and translocated derivatives to the right. Exchange of material among the chromosomes gives rise to a "three-color triplet."

considered stable, presenting no difficulties at mitosis. In terms of DNA sequence loss, both reciprocal translocations and dicentrics (at least until they are lost at division) are genetically neutral events; loss of material is not normally seen at the light microscopic level. However, fine-scale changes to DNA sequence cannot be excluded.

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Until the advent of whole-chromosomehybridization methods, the scoring of reciprocal translocations largely depended upon G-band analysis, a time-consuming operation requiring great skill. The ability to detect translocations in hybrid cells was demonstrated by Pinkel et al. (1986) and Schardin et al. (1985). Pinkel et al. (1986) were able to demonstrate a dose-dependentinduction of translocationsbetween human and hamster chromosomes in a hybrid cell line exposed to neutron irradiation. A more refined method of applying cloned, whole-chromosome hybridization probes to human cells to detect translocation has also been developed (Pinkel et al., 1988; Cremer ef al., 1988) and is now widely used (see following sections). There are two major types of model for the formation of exchange-type aberrations. In the “classic” models, exchange aberrations are initiated by the formation of double-strand breaks (dsbs; these occur “spontaneously” or as a consequence of damage, such as that caused by ionizing radiation or the processing of altered DNA bases) in DNA. The broken ends are then envisaged to separate and move independently throughout the nucleus and so are potentially capable of independently interacting with other broken ends. The alternative “exchange” model proposes that the broken ends formed by a dsb are held in close association, possibly by specific proteins, until successful repair is achieved or interaction with another broken chromosome occurs and the dsb restitution forms a novel chromosomal structure, that is, a translocation, dicentric, or acentric fragment. Recent experiments by Lucas and Sachs ( 1 9 3 ) directly tested models for the formation of exchange aberrations. Whole-chromosome probes for human chromosomes I and 4 or 2 were simultaneously hybridized to human metaphases and detected with different fluorochromes. Thus exchanges among chromosomes 1, 2, or 4 and other chromosomes could be detected. Lucas and Sachs (1993) observed not only simple reciprocal translocations but more complex events involving at least 3 chromosomes. The existence of “three-color triplets” (mutual exchange of material among three chromosomes, one detected with green fluorescence, one with pink fluorescence, and one with red) in the complex aberrant metaphases, with chromosomes appearing redlgreen, redlpink, and greenlpink (see Fig. 5 ) , has implications for the mechanism of aberration formation. One of the basic assumptions of the exchange model of aberration formation is that pairwise interactions of broken chromosome ends lead to aberrations. Thus the existence of these more complex three-color triplets strongly argues against the exchange model being correct, at least in a strict sense. A comparison of the experimental data obtained (i.e., frequencies of two-color twin exchange events, three-color triplets, and insertions) with mathematical predictions of the two models suggested that the classic

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model more faithfully represents the biological process(es) of formation of aberrations. Whole-chromosome hybridization data from several laboratories indicate that in irradiated cells the frequency of translocations is greater than the frequency of dicentrics (Lucas et al., 1989; Cremer et al., 1990; Natarajan et al., 1992; Schmid et al., 1992). From the data available it appears that the majority of this excess is due to terminal translocation events-that is, a single segment of chromosome is found attached to another chromosome in the absence of the reciprocal event. However, some discrepancy may be due to overscoring of reciprocal translocations relative to dicentrics, especially when scoring is performed within the first few divisions following irradiation. The morphology of chromosomes which have been stained by a hybridization probe can be somewhat masked by the hybridization signal, particularly when the combination of FITC-labeled probe and propidium iodide counterstain is used. These two fluorochromes can be simultaneously viewed, the FITC signal being superimposed upon the background propidium iodide. This masking of chromosome morphology can obscure the centromeres of chromosomes, making it difficult to score dicentrics. Counterstaining with DAPI allows centromeres to be more clearly visualized in the absence of interfering signals. i4 more sophisticated approach, developed by Weier et al. (1991), is to hybridize simultaneously whole-chromosome probes and an a-satellite DNA probe which binds to the centromere of all chromosomes. In this way both centromeres and specific chromosomes can be identified under the fluorescence microscope either by changing filters to observe the two signals separately or by using a multiple-band-pass filter. More detailed analyses of this nature will resolve the issue of relative frequency of stable and unstable aberration types. Studies of the kinetics of chromosome aberration formation following DNA damage can also shed light on the origins of chromosomal aberrations. The phenomenon of premature chromosome condensation (Johnson and Rao, 1970) allows chromosomes to be visualized in phases of the cell cycle other than mitosis. By fusing mitotic cells to GI, S, or G2-phase cells, the chromosomes of the latter are forced to condense by factors provided by the mitotic cells. The cell cycle stage of the prematurely condensed chromosomes can be determined morphologically, G , chromosomes being composed of a single chromatid, G2chromosomes having two chromatids, and S-phase chromosomes having a pulverized appearance. Thus, by inducing premature chromosome condensation (PCC) in a synchronized population of cells at various times after exposure to a DNA damaging agent, the time course of appearance and disappearance of chromosomal aberrations can be determined. By means of this technique

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it was established that ionizing radiation rapidly introduces breaks into chromosomes and that these breaks are rejoined with distinctive biphasic kinetics (Cornforth and Bedford, 1983). More recently, Vyas et al. (1991) applied the PCC technique to investigate the kinetics of rejoining chromosome breaks and formation of dicentric chromsomes in human lymphocytes irradiated in the Go-phase with X-rays. The data presented by these authors suggest that dicentrics are formed very rapidly after irradiation and that dicentric formation is not related to the detectable rejoining of chromosome breaks. This study relied upon conventional Giemsa staining of PCC preparations; results reported by Brown and Evans (1992) were obtained using a combination of PCC with whole-chromosome hybridization. This latter study examined chromosome breakage and exchange formation in chromosome 4 in gammairradiated confluent cultures of the human primary fibroblast, AG 1522. Standard PCC preparations were hybridized with a cloned chromosome 4 probe. By contrast with the results of Vyas et al. (1991), Brown and Evans (1992) report that exchange-typeaberrations involving chromosome 4 form over approximately 10 hr following irradiation. Furthermore, these kinetics are mirrored by a decline in chromosome 4 breaks over a similar time scale. These data therefore suggest that exchange aberrations do form as a consequence of rejoining of chromosome breaks. In addition, the authors report that at times of up to 6 hr postirradiation, the majority of exchanges appeared as nonreciprocal events while at 18-24 hr reciprocal exchange events predominated. This latter observation suggests that the formation of exchange aberrations is a relatively slow, multistep process. In stark contrast, the data of Vyas et al. (1991) suggest that the process is very rapid and cannot be resolved into discrete stages. These two opposing sets of findings could be due, at least partly, to differences in cell types, irradiation conditions, or some other experimental parameters. Without further experimental data, it is difficult to determine the true kinetics of exchange-type aberration formation. The application of wholeichromosome hybridization to investigations of the mechanisms of exchange aberrations is in its infancy, as evidenced by the number of recent papers on the subject. It is clear that wholechromosome hybridization studies can help elucidate the mechanisms, but too few data are yet available to construct with confidence a mechanistic model of the formation of these aberrations. D. Genetic Toxicology Scoring of dicentric chromosomes in human peripheral blood lymphocytes has been used for many years as a “biological dosemeter” of ionizing radiation exposure (e.g., Lloyd, 1984). Dicentrics are readily sc:ored in

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simple Giemsa-stained metaphases; however, the frequency of cells carrying dicentrics decreases with time after exposure, while the frequency of reciprocal translocations is stable with time (Awa et al., 1978; Buckton et al., 1978). On theoretical grounds at least, the frequencies of dicentrics and translocations induced by radiation should be equal at the firstdivision following irradiation. Until recently, estimates of absorbed doses of radiation have relied almost entirely upon the scoringof the readily recognizable dicentrics, fragments, and ring chromosomes. It is possible to score translocations by conventional banding methods but it is very laborious and consequently little used. The advent of whole-chromosome hybridization has provided a much-simplified method for scoring translocations. Such a system, which is simple and rapidly scored, has long been an objective for the discipline of biological radiation dosimetry. The major advantages are that the aberrations scored are stable and so can potentially indicate a cumulative exposure to radiation. As mentioned previously, the early experiments in whole-chromosome hybridization, performed with human-rodent hybrid cell lines, revealed the presence of translocations between human and rodent chromosomes (Schardin et al., 1985; Pinkel el al., 1986). Pinkel et al. (1986) presented a neutron irradiation dose-responsecurve for translocations between human and hamster chromosomes which was scored in a human-hamster hybrid cell line hybridized with genomic human DNA. The simiplicity and speed of scoring translocations in these preparations was noted, as was the potential for the automation of scoring (Pinkel et al., 1986). Translocation scoring in normal human cells by an in situ hybridization method was first achieved by using probes which hybidized to regions at either end of chromosome 1 (van Dekken and Bauman, 1988; Lucas et al., 1989). At about the same time, it became evident that cloned whole-chromosome hybridization probes could be used to detect translocations (Cremer et al., 1988; Lichter et al., 1988b; Pinkel et al., 1988). Before the scoring of stable translocations in lymphocytes can become an established technique in biological dosimetry, it must be extensively verified. Over the past 4 years a number of studies of in uitro irradiation dose-response relationships and investigations of known or suspected radiation overexposure cases have been carried out. Reliable in uiuo dose estimation is based upon in uitro irradiation doseresponse data where aberrations are scored in the first metaphase following irradiation-normally during the Go-stage-in human peripheral blood lymphocytes, A study by Cremer et al. (1990) examined the gammaradiation (60Co)dose-response of lymphocytesfor both unstable and stable translocations of chromosomes 1 and 7 by using the appropriate biotinylated phage library probes. The frequency of induced translocations was found to be directly proportional to the square of the radiation dose. A similar relationship is found for dicentrics, but the number of transloca-

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tions was significantly greater. The results of Natarajan et al. (1991) also indicate that translocations (reciprocal, terminal, and interstitial) occur more frequently than dicentric chromosomes. These authors employed mixtures of phage probes for chromosomes 1, 3, and X or 2,4, and 8 for hybridization. No significant differences in translocation frequency scored with the two probe mixes were noted. Also, the background frequency of translocations was greater than expected from previous dicentric data; donor variability in background translocation frequency was also seen. Lucas et al. (1992a) have presented a detailed analysis of radiationinduced translocations scored in gamma-irradiated lymphocytes hybridized with combinations of plasmid library probes for chromosomes 1, 2, 3, 4, and 15. The scoring of translocations was limited to reciprocal translocations. A formula for converting translocation frequency as determined by whole-chromosomehybridization (which detects translocations of a limited subset of the entire chromosome complement) to overall genomic translocation frequencies was presented and shown to fit experimental data. By means of this equation, the frequency of reciprocal translocations seen in first-division metaphases after irradiation appeared quantitatively very similar to previously established dicentric frequencies. This close correspondence of stable and unstable aberration frequencies, which contrasts with the findings of Cremer et al. (1990) and Natarajan et al. (19921, can probably be accounted for by two factors. First, Lucas et al. (1992a) compared only reciprocal translocation and dicentric frequencies, while the other studies considered all types of translocation. Second, as pointed out in the preceding section, there may be a problem of distinguishing dicentric chromosomes from translocations in wholechromosome hybridization preparations. Lucas et al. (1992a) were able to overcome this by applying an a-satellite DNA probe which hybridized to all centromeres along with the whole-chromosome probes. This technique, developed by Weier et al. (19911, allows dicentrics and translocations to be visualized in the same preparation with in situ hybridization techniques. Chromosome-specific probes and the centromere probe are differentially labeled and detected with different fluorochromes. Thus they can be distinguished under a fluorescencemicroscope, allowing unambiguous discrimination of translocations and dicentrics. Schmid et al. (1992) have presented an extensive set of data comparing X-ray-induced stable and unstable aberrations detected in wholechromosome hybridization preparations. As with the studies of Cremer et al. (1990) and Natarajan et al. (19921, an excess of translocations over dicentrics was detected. Schmid et al. (1992) attribute this to difficulty in distinguishing dicentrics and translocations in their preparations. The investigations outlined go some way to establishing translocation scoring by whole-chromosome hybridization as a reliable biological radia-

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tion dosimeter. The data obtained seem quantitatively sound and, as long as trouble is taken to distinguish dicentrics from translocations, the 1 : 1 relationship between dicentrics and translocations appears to hold. Care needs to be taken in establishing background translocation frequencies. Since these aberrations, unlike dicentrics, are stable, age-related increases in translocations may be significant. The previous exposure history of an individual could confound the assessment of radiation dose from a particular incident. Little detailed information is yet available concerning translocations induced by radiation of differing linear energy transfer (LET) as assessed by whole-chromosome hybridization. The question of stability of translocations over periods of many years has been addressed for a number of cases of known radiation overexposure. In these cases, the problem of confounding of scoring caused by difficulties in distinguishing dicentrics from translocations is much reduced because the majority of cells carrying dicentrics will have been eliminated from the circulation over time. Popp et al. (1990) looked at translocation frequencies in patients treated with Thorotrast, which was used as an Xray contrast medium and contains the a-particle emitter 232thori~m. The half-life of 232This long, 1.4 X 10'O years, and so persons exposed to Thorotrast will have been subjected to continuous a-particle irradiation following the initial injection of the contrast medium. The retained 232Th is found deposited in the liver, spleen, and red bone marrow and therefore hemopoietic tissue is subject to irradiation. Popp et al. (1990) were able to demonstrate that two Thorotrast-exposedpersons (with 42 and 47 years of exposure after treatment) had significantlyelevated frequencies of translocations, deletions, and insertions as assessed by hybridization of phage probes for chromosomes 1-5 individually to metaphases. Two studies of individuals exposed to I3'Cs gamma radiation as a result of the accident in Goiania, Brazil in 1987 have been published. Straume et al. (1991) published data from five exposed persons. Dose estimates were made on the basis of three assays-conventional dicentric scoring, translocation analysis by whole-chromosome hybridization, and mutation frequency at the glycophorin A locus-all employing peripheral blood lymphocytes. Dicentrics showed a significant drop in frequency when samples taken 1 month or less and 1 year following exposure were compared. Translocations were assessed 1-1.4 years following the accident. In one subject there was reasonable agreement between initial dicentric yield and translocation yield at -1 year after exposure, although in the two cases where comparison is possible, translocation analysis gave relatively low dose estimates. Straume et al. (1991) discuss a number of factors which may contribute to this discrepancy, not least among which are our lack of knowledge about lymphocyte growth patterns within the hemopoietic system and the probability of an exposed cell forming a stable and

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an unstable aberration. Overall, the authors conclude that the various dose estimation systems are in reasonable agreement. Natarajan et al. (1991) investigated the frequencies of unstable and stable aberrations and mutations at the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene locus in 14 victims of the Goiania accident 12-15 months after exposure. It is evident from the data presented that translocations are more stable than dicentrics. However, the correspondence between the dose estimate based on dicentric scoring soon after the accident and the detection of translocations and deletions 1 year plus after the accident is poor. Elevated frequencies of lymphocytes with mutant HPRT genes was also observed in 4 cases. One individual included in this study grdup showed frequent chromosome 2 abnormalities in which breakpoints were clustered in the 2pl and 2q2 bands, possibly indicating expansion of an abnormal clone of cells. A case of suspected overexposure to radiation has been assessed by a number of novel biological dosimetric assays (Straume et al., 1992). In this case the individual concerned suspected that physical dosimetry had underestimated his accumulated dose over a 36-year-period. Conventional G-band analyses of translocationsand dicentrics were performed alongside whole-chromosome hybridization analysis with a chromosome 4 probe alone or aprobe mix for chromosomes 1,3, and 4. An estimated cumulative dose of 0.6 Sv was derived from the chromosome hybridization data. When data obtained from conventional and novel cytogenetic end points along with glycophorin A mutation data, a dose estimate of 0.4-2 Sv was indicated. This range includes the physical dosimetric estimate while the dose suspected by the worker (2.5 Sv) was considered unlikely to have been received. While this study in itself was unable to provide a firm cumulative dose estimate, it provided a valuable test of the utility of the assays. Straume et al. (1992) include information gained from studying translocation frequencies in lymphocytes of Hiroshima atomic bomb survivors. A hybridization-based assessment of translocation frequency, measured 45 years after exposure, was compared with the predicted frequency of aberrations that would have been scored in first-division cells after in vitro irradiation with a dose equivalent to that thought to have been received by the individuals concerned. The ratios of these measured to predicted translocation frequencies were not significantly different from 1 in 16 of 19 cases, a strong indicator of the stability of translocations over extended periods of time (Straume et al., 1992). Further verification of the stability of translocations over a number of years is provided by Lucas et al. (1992b). In this case, the subject had incorporated 35 GBq of tritiated water. Dosimetry based upon analysis of urine and conventional dicentric

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scoring performed 39 days following the incident indicated that a dose of 0.4 Gy had been absorbed by the soft tissue of the body (Lloyd et al., 1986).

Comparison of the frequency of dicentrics at 39 days and translocations at 6 years showed very close agreement (a net of 33 dicentrics and 36 translocations; Lucas et al., 1992b).This is a particularly important and encouraging study because it is the only case available to date where accurate biological dosimetry was performed close to the time of the accident for comparison with the retrospective whole-chromosome hybridization translocation analysis. The close quantitative agreement of the assays is impressive and suggests that retrospective translocation analysis will become a valuable assay. The final retrospective study of translocations assessed by wholechromosome hybridization concerns individuals exposed as a result of the atomic bomb explosions in Japan and the Oak Ridge Y-12 criticality accident (Lucas et al., 1992a). A very close correspondence was noted between conventionally (G-band) scored translocations and wholechromosome hybridization-assessed translocations for both sets of individuals. A reasonable correspondence of hybridization-assessed translocations and predicted first-division translocation frequency based on DS86 dose estimates (which carry an uncertainty of ? 30%; Roesch, 1989) was also found for A-bomb survivors. The correlation between measured retrospective translocation frequency and physical dose estimation was reported to be less good for the Y-12 causes, possibly because of errors in the dose estimation at the time of exposure (Lucas et al., 1992a). This report, therefore, also indicates that retrospective translocation assessment has good prospects. The combination of whole-chromosome hybridization and premature chromosome condensation was mentioned in Section III,C. Experiments of this type promise to be of use in studies of aberration formation and repair of radiation-induced damage (Evans et al., 1991; Brown and Evans, 1992),and may extend the range of cells in which biological dose estimation can be performed to include cells that do not normally divide. This could be of use in cases of highly localized suspected overexposure. Furthermore, it may be possible to assess differences in tumor cell radiosensitivity by this technique (Evans et al., 1991). Whole-chromosomehybridization methods hold great promise for biological radiation dosimetry. The reports discussed in this section provide reason to be optimistic about the utility of such assays. Certainly more validation of the methods is required, as is more detailed analysis of translocation frequencies in normal individuals of all ages and both sexes. The field of radiation protection is clearly going to benefit from the application of these techniques.

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E. Clinical Studies

The studies described in the preceding section show that the use of wholechromosome hybridization techniques can have practical benefits. These techniques have found application not only in genetic toxicology but also in clinical cytogenetics. Any methods that increase the speed, resolution, and accuracy of analysis should be welcomed, as should methods that extend the range of disorders that can be detected cytogenetically. Currently there is much interest in the whole range of in situ hybridization techniques capable of providing diagnostic or prognostic information. Not surprisingly for a relatively young technology, much of the work to date has concerned studies of a fundamental nature and developmental work rather than full-scale application in a clinical setting. Clinical cytogenetics can provide information pre- or postnatally on constitutive genetic abnormalities and on chromosomal changes which occur during life, particularly in tumor cells. This information can then be used for diagnosis, prognosis, prediction of response to therapy, and to chart the progress of therapy. Studies applying whole-chromosome hybridization to each of these areas have been performed. DNA in situ hybridization methods in general are also finding use in clinical situations. Gene and/or locus-specific probes can be used to define losses, gains, or rearrangements of genetic material both in metaphase and interphase. For example, differentially labeled probes including the bcr and abl genes have been successfully used to identify the specific translocation between chromosomes 9 and 22 in chronic myelogenous leukemia (e.g., Thachuk et al., 1990). Cosmid contigs spanning 100 kb or so of specific chromosomes have been used to perform prenatal aneuploidy screening in amniocytes in what is probably the most extensive clinical evaluation of an in situ hybridization technique to date (Klinger et al., 1992; Ward et al., 1992,1993).Similarly,chromosome-specific repeat sequences can be used for prenatal analysis (e.g., Griffin et al., 1992). Chromosome-specific repeats, particularly centromeric satellite DNAs, have been widely used to analyze tumors (reviewed by Anastasi, 1991). The strong, discrete signals produced by these probes make them ideal for analyzing chromosome losses in interphase cells. Thus they are often used on specimens in which it is difficult to obtain good quality metaphase spreads. The whole-chromosome hybridization studies of a clinical nature can be broadly categorized into three groups: those using probes specific for normal human chromosomes, those which generate probes for specific abnormal chromosomes, and those employing the comparative genomic hybridization method described in Section II,C,l . The majority of these studies are based on metaphase analysis. Interpretation of the rather diffuse whole-chromosomehybridization signal patterns in interphase nuclei

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is in general too complex to be reliable at present. Each of the three categories of approach has advantages. The wide availability of normal chromosome-specific probes, some in a prelabeled form, makes this method accessible to most laboratories. However, in the absence of prior indications of the chromosome(s) involved in a particular abnormality, some time may be spent in performing relatively noninformative hybridizations until the chromosomes of interest are identified. The generation of probes for specific abnormal chromosomes is technically more involved and usually demands flow sorting, microdissection, or species-specificPCR. Once a probe library for the aberrant chromosome has been produced, the chromosome’s composition can be determined from a single hybridization experiment on a normal metaphase. However, information on the relative position of the various fragments of normal chromosome in an aberrant chromosome may be dimcult to obtain by this method. The strength of the comparative genomic hybridization technique is that it demands no prior knowledge or assumptions about the genetic abnormalities in the cell of interest. Again, this is a technically demanding method which at present requires sophisticated image analysis for interpretation of results. In the following paragraphs I discuss a selection of the ever-increasing number of clinical cases investigated by using each of the three broad approaches. Attempts to apply whole-chromosomehybridization probes to prenatal diagnosis of chromosome imbalances, especially trisomy 21 in interphase cells, have not met with great success (Lichter et af., 1988b; Kuo et af., 1991; Zheng et a / . , 1992). The use of contiguous cosmid clones appears to be the most reliable method for this diagnosis (Klinger et af.,1992;Ward et al., 1993). Whole-chromosome hybridization probes have, however, proven useful in postnatal characterization of constitutive chromosomal imbalances. Potential cases of chromosomal imbalance are often identified on the basis of observation of a greater or lesser degree of mental retardation and/or morphological abnormalities. In these cases of viable individuals with chromosomal imbalance, classical cytogenetic techniques cannot always provide a definitive karyotype analysis. Isolation of the abnormal chromosomes in these cases can be problematic because of the difficulties of identifying the abnormal chromosome(s)and of the availability of cells; thus, a probe specific for the chromosome abnormality can be prepared only rarely. Consequently, the use of normal chromosome probes is often the method of choice. Examples of this approach include analysis of t( 13;15)in patients with craniofacial abnormalities and mental retardation (Mangelschots et af.,1992), characterization of a chromosome 8 deletion associated with mild mental retardation and dysmorphicfeatures (Pettenati et af.,1992),investigation of potential monosomy 21 cases (Viljoen et af., 1992), definition of t(4;18) in a child with growth retardation and delayed

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development (Mewer et al., 1992), identification of marker chromosomes in cat eye syndrome (Liehr et al., 1992),identification of a cryptic translocation in a patient with cri du chat syndrome (Bemstein et al., 19931, characterization of chromosome 14 abnormalities in a patient with a psychological disorder (Magnani et al., 1993),and characterization of chromosome 9 abnormalities (Spinner et al., 1993; Petty et al., 1993). Cremer et al. (1988)applied normal human chromosome hybridization to tumor cells to identify aberrations. Generally it is tumors of the lymphoid system that have been investigated, owing to the (normally) simple preparation of metaphases. Tumors examined include chronic myelogenous leukemia (Amoldus et al., 1990; Seong et al., 1993), hyperplasic myelofibrosis (Trautmann et al., 1992),chronic lymphocytic and prolymphocytic leukemia (Dohner et al., 1993), acute promyelocytic leukemia (Speicher et al., 1993),acute myeloid leukemia/myelodysplasticsyndrome (Zhao et al., 1993b),and acute monocytic leukemia (Cherifet al., 1993). Interphase analysis of leukemias with whole-chromosome probes is possible, but is not as reliable as other methods (Amoldus et al., 1990). Marker chromosomes in metaphases derived from solid tumors of the prostate (Brothman and Patel, 1992) and germ cells (Suijkerbuijk et al., 1991) have been characterized by whole-chromosomehybridization. The interphase analysis of tumor cell chromosomal aberrations has been achieved by applying a combination of whole-chromosomehybridization probes and appropriate gene-specific (Lengauer et al., 1992) or region-specific probes (Poddighe et al., 1992). In these latter cases, the coincidence of signals from the two probes represents the normal situation while the separation of probe signals indicates a chromosomal aberration (see Fig. 6). In addition to defining the chromosomal rearrangements present in tumor cells, whole-chromosome hybridization has been used to predict tumor cell radiosensitivity in standard metaphases or prematurely condensed chromosomes(Brown et al., 1992),to detect residual leukemic cells following treatment (Zhao et al., 1993a), and to follow the damaging effect of tumor treatments on other somatic cells (Smith er al., 1992). It is clear that the use of whole-chromosome hybridization probes for normal chromosomes can be of great benefit in studying chromosome abnormalities in clinical material. The number of clinical cytogenetic studies that include whole-chromosomehybridization techniques is increasing rapidly. These probes, together with locus and region-specific probes, are already coming into use in clinical cytogenetics laboratories. The approachesdescribed in the followingparagraphs cannot yet be considered routine procedures for clinical cytogenetics. At present they are more specialized research techniques. The use of probes for normal chromosomes on material suspected of being abnormal is often referred to as “forward” chromosome painting.

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FIG. 6 Diagrammatic representation of a scheme for detecting chromosomal aberrations in interphase nuclei. (a) Normal nucleus with two domains of whole-chromosome hybridization (gray), each containing a single signal from a locus-specific probe (black dots). (b) Aberrant nucleus. One locus-specific signal lies outside the whole-chromosome hybridizationdomain.

The converse or “reverse” experiment-applying a probe library specific for an abnormal chromosome to a normal metaphase preparation-has also proven useful. These reverse experiments depend upon chromosome purification methods such as flow cytometry, microdissection, and the generation of interspecies hybrid cells followed by DNA cloning, or more commonly PCR amplification of the desired material. The DOP-PCR and IRS-PCR methods used to generate probes have been described in Section II,C,3. A number of tumor-associated chromosome abnormalities have now been characterized by reverse hybridization with probes generated both by IRS-PCR (Suijkerbuijk et al., 1992; Michalski et al., 1992; Boschman et al., 1993) and DOP-PCR (Telenius et al., 1992; Carter et al., 1992; Blennow et al., 1992).These methods are of use in characterizing abnormal marker chromosomes and will continue to play an important part in cancer cytogenetics. However, the methods are technically demanding and there-

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fore unlikely to make a rapid transition into routine use. Nonetheless, useful probes can be generated and if made widely available, some could be adapted for work of a more routine diagnostic nature. DOP-PCR has also been used to investigate three cases of constitutional chromosome 16 abnormalities (Rack et al., 1993). The recently developed technique of comparative genomic hybridization (see Section II,C,l) promises to be of great use in characterizing the genetic constitution of abnormal cells. Again, this is a technically complex method currently used largely for research purposes. The strengths of the method lie in the facts that it allows genetic changes (deletion, amplification, duplication) in all regions of the genome to be detected in a single experiment and prior knowledge of the chromosomal loc?.tion of such changes is not required. Cell lines of tumors with bladder, breast, colon, lung, neural (A. Kallioniemi et al., 1992; 0. P. Kallioniemi et al., 1993) and kidney (du Manoir et al., 1993) origins have been examined using CGH. Primary tumor material can also be investigated by CGH (Kallioniemi et al., 1992; du Manoir et al., 1993). In some cases, the simpler technique of reverse hybridization of tumor cell genomic DNA to normal metaphases can be used to detect amplified DNA segments (Joos et al., 1993). The body of information gained through the application of CGH will undoubtedly increase in coming years. Current approaches to the genetic characterization of tumor cells depend upon the selection of the relevant probes, be they for chromosomal changes or gene mutations. Hence, the genetic profile of a tumor is constructed piecemeal. CGH offers a method for scanning the entire genome of a cell for large-scale alterations. Through CGH it will be possible to gain some insights into the genetic heterogeneity of specific tumor types. Similarly, regions of the genome harboring asyet-unknown oncogenes or tumor suppressor genes may be identified by CGH. While CGH studies published to date all concern human material, the technique is in principle applicable to any species. Thus, laboratory animal studies may also benefit from this method. CGH could also be adapted as a genome-wide screen for large-scale mutations in cells or populations exposed to DNA-damaging agents. The demands of clinical cytogenetics have long been a major driving force in the development of cytogenetic methods. Indeed, some in situ hybridization methods have been developed with clinical problems in mind. Whole-chromosome hybridization techniques are applicable in a range of clinical studies. Routine clinical cytogenetics requires simple, reliable, and rapid methods. The use of whole-chromosome probes to analyze tumor cells or constitutive chromosome abnormalities in metaphase is a candidate for a routine method. However, screening techniques,

219 as in the case of trisomy detection in interphase amniocytes, are more readily performed with high-signal-intensity cosmid probes. The interpretation of whole-chromosome hybridization signals in interphase cells is not reliable or simple enough for these situations. Recently Ledbetter (1992) has written on the current excitement and enthusiasm for applying fluorescence in situ hybridization to clinical problems but also warns of the difficulties and pitfalls which may lay ahead. Whole-chromosome hybridization methods are well suited to clinical research work. The examples quoted demonstrate the use of these methods in tumor cytogenetics and the analysis of inherited chromosomal abnormalities. WHOLE-CHROMOSOME HYBRIDIZATION

IV. Concluding Remarks

Whole-chromosome hybridization is a subgroup of in situ hybridization methods in which the aim is to stain specifically complete chromosomes. While a clear distinction is not always possible (for example, in the case of comparative genomic hybridization)and is perhaps somewhat arbitrary, whole-chromosome hybridization usefully defines a series of methods which have been developed since the observations of Durnam et al. (1985), Manuelidis (1985), and Schardin et al. (1985). Since then, the technology has developed rapidly, with refinements in probe construction, hybridization and detection methods, and microscopy. The technical developments have been rapidly exploited in many of the major areas of cytogenetics. The achievements made through the application of whole-chromosome hybridization-for example, insights into nuclear structure, rapid and reliable analysis of inherited and tumor chromosome abnormalities, the development of a simple cumulative biological dosimetry system for radiation exposure-are impressive, especially when it is considered that the techniques have been in existence less than a decade. A strength of the techniques is that they can be adapted both as sophisticated research tools (in, for example, cell cycle-dependent chromosome positioning) and as routine workhorse methods (radiation-induced translocation analysis, for example). The history of cytogenetics is characterized by periodic introductions of novel techniques which invigorate the field. The developments of in situ and whole-chromosome hybridization certainly rank among these technical advances. There can be little doubt that whole-chromosome hybridization techniques and applications will continue to be refined and extended for some time to come. In this way the capabilities of cytogenetics will continue to expand.

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Acknowlegments I thank Dr. M. Fairman for critical reading of the manuscript, Mr. D. Perry for literature searches, and Mrs. K. Brooks for typing the manuscript.

References Agard, D. A., and Sedat, J. W. (1983). Three-dimensional architecture of a polytene nucleus. Nature (London) 302,676-681. Albertson, D. G., Fishpool, R., Shemngton, P. N., Acheva, E., and Milstein, C. (1988). Sensitive and high resolution in situ hybridization to human chromosomes using biotin labelled probes: Assignment of the human thymocyte CDI antigens to chromosome 1. EMBO J . 7,2801-2805. Anamthawat-Jonsson, K., Schwarzacher, T., Leitch, A. R., Bennett, M. D., and HeslopHarrison, J. S. (1990). Discrimination between closely related Tririceae species using genomic DNA as a probe. Theor. Appl. Genet. 79,721-728. Anamthawat-Jonsson, K.,Schwarzacher, T., and Heslop-Hamson, J. S. (1993). Behaviour of parental genomes in the hybrid Hordeum vulgarae x H . bulbosum. J . Hered. 84,78-82. Anastasi, J. (1991). Interphase cytogenetic analysis in the diagnosis and study of neoplastic disorders. Am. J . Clin. Pathol. 95, Suppl. I , 522-528. Arnoldus, E. P. J., Wiegant, J., Noordermeer, I. A., Wessels, J. W., Beverstock, G. C., Grosveld, G. C., van der Ploeg, M., and Raap, A. K. (1990). Detection of the Philadelphia chromosome in interphase nuclei. Cytogener. Cell Genet. 54, 108-1 I I . Awa, A. A., Sofuni, T., Honda, T., Itoh, M.,Neriishi, S., and Otake, M. (1978). Relationship between the radiation dose and chromosome aberrations in atomic bomb survivors of Hiroshima and Nagasaki. J . Radiar. Res. 19, 126-140. Baldini, A., and Ward, D. C. (1991). In situ hybridization of human chromosomes with Alu PCR products: A simultaneous karyotype for gene mapping studies. Genomics 9,770-774. Bauman, J. G. J., Wiegant, J., Borst, P., and van Duijn, P. (1980). A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome-labelledRNA. Exp. Cell Res. l38,485-490. Bennett, S. T., Kenton, A. Y.,and Bennett, M. D. (1992). Genomic in situ hybridization reveals the allopolyploid nature of Milium montianum (Gramineae). Chromosoma 101, 420-424. Bernstein, R., Bocian, M. E., Cain, M. J., Bengtsson, U., and Wasmuth, J. J. (1993). Identificationof a cryptic t(5-7) reciprocal translocation by fluorescent in situ hybridization. Am. J . Med. Genet. 46, 77. Beverloo, H. B., Vanschadewijk, A., Bonnet, J., Vandergeest, R., Rumia, R.. Verwoerd, N. P., Vrolijk, J., Ploem, J. S., and Tanke, H. J. (1992). Preparation and microscopic visualization of multicolour luminescent immunophosphors. Cytometty W, 561-570. Bischoff, A., Albers, J., Kharboush, I., Stelzer, E., Cremer, T., and Cremer, C. (1993). Differences in size and shape of active and inactive X-chromosome domains in human amniotic fluid cell nuclei. Micros. Res. Tech. 25, 68-77. Blennow, E., Telenius, H., Larsson, C.,De Vos, D., Bajalica, S., Ponder, B. A., and Nordeskjold, M. (1992). Complete characterization of a large marker chromosome by reverse and forward chromosome painting. Hum. Genet. 90,371-374. Boschman, G . A., Buys, C. H. C. M., van der Veen, A. Y.,Kens, W., Osinga, J., Slater, R. M.,and Alten, J. A. (1993). Identification of a tumour marker chromosome by flow sorting, DNA amplification in vitro and in situ hybridization of the amplified product. Genes, Chromosomes, Cancer 6, 10-16.

WHOLE-CHROMOSOME HYBRIDIZATION

221

Boveri, T. (1909). Die blastomerenkerne von Ascaris megalocephala und die theorie de chromosomenindividualitat . Arch. ZeNforsch. 3, 181-268. Boyle, A. L., Ballard. S. G., and Ward, D. C. (1990a). Differential distribution of long and short interspersed element sequences in the mouse genome: Chromosome karyotyping by fluorescence in situ hybridization. Proc. Natl. Acad. Sci. U.S.A. 87, 77577761. Boyle, A. L.. Lichter, P., and Ward, D. C. (1990b).Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization. Genomics 7 , 127-130. Bradl, J., Hausmann, M., Ehemann, V.,Komitowski, D., and Cremer, C. (1992). A tilting device for three dimensional microscopy-application to in situ imaging of interphase cell nuclei. J . Microsc. (Oxford) 168, 47-57. Brakenhoff, G. J., Blom. P., and Barends, P. (1979). Confocal scanning light microscopy with high aperture immersion lenses. J. Microsc. (Oxford) 117, 219-232. Brandriff, B. F., and Gordon, L. A. (1992). Spatial distribution of sperm-derived chromatin in zygotes determined by fluorescence in situ hybridization. Mutar. Res. 296, 33-42. Brigati, D. J., Myerson, D., Leary, J. J., Spalholz, B., Travis, S. Z.. Fong, C. K. Y., Hsiung, G. D., and Ward, D. C. (1983). Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labelled hybridization probes. Virology 126932-50. Britten, R. J.. Graham, D. E., and Neufeld, B. R. (1974). Analysis of repeating DNA sequences by reassociation. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 29E. pp. 363-418. Academic Press, New York. Brothman, A. R., and Patel, A. M. (1992). Characterization of ten marker chromosomes in a prostatic cancer cell line by in situ hybridization. Cytogenet. Cell Genet. 60, 8. Brown, J. M., and Evans, J. W. (1992). Fluorescence in situ hybridization: An improved method for quantitating chromosome damage and repair. Br. J . Radiol., Suppl. 24,61-64. Brown, J. M., Evans, J., and Kovacs, M. S. (1992). The prediction of human tumour radiosensitivity in situ: An approach using chromosome aberrations detected by fluorescence in situ hybridization. Int. J . Radiat. Oncol. Biol. Phys. 24, 279-286. Buckton, K. E., Hamilton, G. E.. Paton, L., and Langlands, A. G. (1978). Chromosome aberrations in irradiated ankylosing spondylitis patients. In “Mutagen-induced Chromosome Damage in Man” (H. Evans and D. Lloyd, eds.), pp. 142-150. Edinburgh Univ. Press, Edinburgh. Buongiorno-Nardelli, M., and Amaldi, F. (1970). Autoradiographic detection of molecular hybrids between RNA and DNA in tissue sections. Nature (London) m,946-948. Burde, S., and Leary, J. F. (1992). Detection of individual human chromosomes by chromosome in siru suppression hybridization using PCR-amplifiedbacteriophage libraries. Genet. Anal. Tech. Appl. 9, 64-67. Carter, N. P., Ferguson-Smith, M. A., Perryman, M. T., Telenius, H., Pelmear, A. H., Leversha, M. A., Glancy, M. T., Wood, S. L., Cook, K., Dyson, H. M., FergusonSmith, M. E., and Willatt, L. R. (1992). Reverse chromosome painting: A method for the rapid analysis of aberrant chromosomes in clinical cytogenetics. J . Med. Genet. 29, 299-307. Casperson. T., Zech, L., and Johnasson, C. (1970). Differential banding of alkylating fluorochromes in human chromosomes. Exp. Cell Res. 60, 315-319. Cherif, D.. Romana, S., Der-Sakissian, H., Jones, C., and Berger, R. (1993). Chromosome painting in acute monocytic leukaemia. Genes, Chromosomes, Cancer 6, 107-1 12. Collins, C., Kuo, W. L., Segaves, R., Fuscoe, J., Pinkel, D., and Gray, J. W. (1991). Construction and characterisation of plasmid libraries enriched in sequences from single human chromosomes. Genomics 11,997-1006. Cornforth, M. N., and Bedford, J. S. (1983).X-ray induced breakage and rejoining of human interphase chromosomes. Science 222, 1141-1 143.

222

S. D. B O W L E R

Cremer, T., Landegent, J., Briickner, A., Scholl, H. P., Schardin, M., Hager, H. D., Devilee, P., Pearson, P., and van der Ploeg, M. (1986). Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: Diagnosis of trisomy 18 with probe LI.84. Hum. Genet. 74, 346-352. Cremer, T., Lichter, P., Borden, J., Ward, D. C., and Manuelidis, L. (1988). Detection of chromosome aberrations in metaphase and interphase tumour cells by in situ suppression hybridization using chromosome-specific library probes. Hum. Genet. 80, 235-246. Cremer, T., Popp, S., Emmerich, P., Lichter, P., and Cremer, C. (1990). Rapid metaphase and interphase detection of radiation-induced chromosome aberrations in human lymphocytes by chromosomal suppression in situ hybridization. Cytometry 11, 110- 118. Davies, K. E., Young, B. D., Elles, R. G., Hill, M., and Williamson, R. (1981). Cloning of a representative X-chromosome library after sorting by flow cytometry . Nature (London) 293,374-376. de Grouchy, J., Truleau, C., Roubin, M., and Klein, M. (1972). Evolution caryotypique de I’homme et du chimpanzk: fitude comparative des topographies de bandes apres dknaturation mbnagke. Ann. Genet. Is, 79-84. deGrooth, B. G., and Putman, C. A. J. (1992). High-resolution imaging of chromosomerelated structures by atomic force microscopy. J. Microsc. (Oxford) 168, 239-247. Deng, H. X., Yoshiura, K., Dirks, R. W., Harada, N., Hirota, T., Tsukamoto, K., Jinno, Y., and Niikawa, N. (1992). Chromosome band specific painting-chromosome in siru suppression hybridization using PCR products from a microdissected chromosome band as a probe pool. Hum. Genet. 89, 13-17. Dohner, H., Pohl, S., Bulgaymorschel, M., Stilgenbauer, S., Bentz, M., and Lichter, P. (1993). Trisomy 21 in chronic lymphoid leukaemias: A metaphase and interphase analysis. Leukemia 7,516-520. Drets, M. E., and Shaw, M. W. (1971). Specific banding patterns of human chromosomes. Proc. Natl. Acad. Sci. U.S.A. 68,2013-2077. du Manoir, S., Speicher, M. R., Joos, S.,Schrock, E.,Popp, S., Dohner, H., Kovacs, G., Robert-Nicoud, M., Lichter, P., and Cremer, T. (1993). Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Hum. Genet. 90,590-610. Durnam, D. M., Gelinas, R. E., and Myerson, D. (1985). Detection of species-specific chromosomes in somatic cell hybrids. Somatic Cell Mol. Genet. 11, 571-577. Evans, J. W., Chang, J. A., Giaccia, A. J., Pinkel, D., and Brown, J. M. (1991). The use of fluorescence in situ hybridization combined with premature chromosome condensation for the identification of chromosome damage. Br. J . Cancer 63,517-521. Feinberg, A. P., and Vogelstein, B. (1984). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Eiochem. 137, 266-267. Ferguson. M., and Ward, D. C. (1992). Cell cycle dependent chromosomal movement in pre-mitotic human T lymphocyte nuclei. Chromosoma 101, 557-565. Fetni, R.. Lemieux, N., Macfoy, B., Dutrillaux, B., Messier, P. E., and Richer, C. L. (1992). Detection of small single-copy genes on protein-G-banded chromosomes by electron microscopy. Cytogenet. Cell Genet. 60, 187-189. Foster, A. C., McInnes, L., Skingle, D. C., and Symons, R. H. (1985). Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res. l3, 745-761. Gall, 1. G., and Pardue, M. L. (1969). Formation and detection of RNA-DNA hybrids in cytological preparations. Proc. Natl. Acad. Sci. U.S.A. 63, 378-383. Gerhard, D. S., Kawasaki, E. S., Bancroft, F. C., and Szabo, P. (1981). Locdisation of a unique gene by direct hybridization in situ. Proc. Natl. Acad. Sci. U.S.A. 78,3755-3759.

WHOLE-CHROMOSOME HYBRIDIZATION

223

Giaccia, A. J., Evans, J. W., and Brown, J. M.(1990). Use offluorescent in situ hybridization to detect chromosomal rearrangements in somatic cell hybrids. Genes. Chromosomes, Cuncer 2, 248-251. Goldman, A. S. H., and Hulten, M.A. (1992). Chromosome in situ suppression hybridization in human male meiosis. J. Med. Genet. 29, 98-102. Goldman, A. S. H., and Hulten, M. A. (1993). Analysis of chiasma frequency in the first meiotic segregation in a human male reciprocal translocation heterozygote t( 1;11) (q36.3;q13.I ) using fluorescence in silu hybridization. Cytogenet. CeN Genet. 63, 16-23. Griffin, D. K., Wilton, L. J., Handyside, A. H., Winston, R. M. L., and Delhanty, J. D. A. (1992). Dual fluorescent in situ hybridization for simultaneous detection of X and Y chromosome-specific probes for the sexing of human pre-implantation embryos. Hum. Genet. 89, 18-22. Hames, B. D., and Higgins, S. J., eds. (1985). “Nucleic Acid Hybridization: A Practical Approach.” IRL Press, Oxford. Harper, M. E., Ullrich, A., and Saunders, G. F. (1981). Localization of the human insulin gene to the distal end of the short arm of chromosome 1 I . Proc. Nut/. Acud. Sci. U.S.A. 78,4458-4460. Harrison, P. R., Conkie, D., Paul, J., and Jones, K. (1973). Localisation of cellular globin messenger RNA by in situ hybridization to complementary DNA. FEBS Lett. 32,109-1 12. Hasse, A. T., Walker, D., Stowring, L., Ventura, P., Geballe, A., Blum, H., Brahic, M., Goldberg, R., and O’Brien, K. (1985). Detection of two viral genomes in a single cell by double-label hybridization in situ and colour microautoradiography. Science 227,189- I9 1. Henderson, E. (1992). Atomic force microscopy of conventional and unconventional nucleic acid structures. J . Microsc. (Oxford) 167, 77-84. Heppell-Parton, A. C., Albertson, D. G . ,and Rabbitts, P. H. (1992). Orderingof six polymorphic DNA markers important in the delineation of 3p deletions in neoplasia. Genes, Chromosomes, Cuncer 4, 228-234, Heslop-Hamson, J. S., and Bennett, M. D. (1990). Nuclear architecture in plants. Trends Genet. 6,401-405. Hiraoka, Y., Sedat, J. W., and Agard, D. A. (1987). The use of a charge-coupled device for quantitative optical microscopy of biological structures. Science 23, 36-41. Hopman, A. H. N., Wiegant, J., and van Duijn, P. (1986a). A new hybridocytochemical method based on mercurated nucleic acid probes and sulphydryl-haptanligands. I. Stability of the mercury-sulphydryl bond and influence of ligand structure on the immunochemical detection of the haptan. Histochemistry 84, 169-178. Hopman, A. H. N.. Wiegant, J., and van Duijn, P. (1986b). A new hybridocytochemical method based on mercurated nucleic acid probes and sulphydryl-haptan ligands. 11. Effects of variations in ligand structure on the in siru detection of mercurated probes. Histochemistv 84, 179-185. Hopman, A. H. N., Wiegant, J., Raap, A. F., Landegent, J. E., van der Ploeg, M.,and van Duijn, P. (1986~).Bicolour detection of two target DNAs by non-radioactive in situ hybridization. Histochemistry 85, 1-4. Hsu, T. C. (1952). Mammalian chromosomes in uitro. I. The karyotype of man. J . Hered. 43, 167-172. Hulspas, R., and Bauman, J. G. J. (1992). The use of fluorescent in situ hybridization for the analysis of nuclear architecture visualized by confocal laser scanning microscopy. Cell Biol. lnt. Rep. 16, 739-747. Ijdo, J. W.. Baldini, A., Ward, D. C., Reeders, S. T., and Wells, R. A. (1991). Origin of human chromosome 2: An ancestral telomere-telomere fusion. Proc. Nutl. Acad. Sci. U.S.A. 88,9051-9055. Jauch. A., Wienberg. J., Stanyon, R., Arnold, N., Tofanelli, S., Ishida, T., and Cremer,

224

S. D. BOUFFLER

T. (1992). Reconstruction of genomic rearrangements in great apes and gibbons by chromosome painting. Proc. Natl. Acad. Sci. U.S.A. 89, 8611-8615. John, H. A., Birnstiel, M. L., and Jones, K. W. (1%9). RNA-DNA hybrids at the cytological level. Nature (London) 223, 582-587. Johnson, R. T., and Rao, P. N. (1970). Mammalian cell fusion induction of premature chromosome condensation in interphase nuclei. Nature (London) 226, 7 17-722. Joos, S.,Scherthan, H., Speicher, M. R.. Schlegel, J., Cremer, T., and Lichter, P. (1993). Detection of amplified DNA sequences by reverse chromosome painting using genomic tumour DNA as probe. Hum. Genet. 90, 584-589. Kallioniemi. A., Kallioniemi, 0. P., Sudar, D., Rutowitz, D., Gray, J. W., Waldman, F. W., and Pinkel, D. (1992). Comparative genomic hybridization for molecular cytogenetic analysis of solid tumours. Science 258, 818-821. Kallioniemi, 0. P., Kallioniemi, A., Sudar, D., Rutowitz, D., Gray, J. W., Waldman, F., and Pinkel, D. (1993). Comparative genomic hybridization: A rapid new method for detecting and mapping DNA amplification in tumours. Semin. Cancer Biol. 4,41-46. Kao, F.-T., and Yu, J.-W. (1991). Chromosome microdissection and cloning in human genome and genetic disease analysis. Proc. Narl. Acad. Sci. U.S.A. 88, 1844-1848. Kessler, C., Holtke, H. J., Seibl, R., Burg, J., and Miihlegger, K. (1990). Non-radioactive labelling and detection of nucleic acids. I. A novel DNA labelling and detection system based on digoxigenin: Antidigoxigenin ELISA principle (digoxigenin system). Biol. Chem. Hoppe-Seyler 371,917-927. Kett, P., Geiger, B., Ehemann, J., and Komitowski, D. (1992). Three-dimensional analysis of cell nucleus structures visualized by confocal laser scanning microscopy. J . Microsc. (Oxford) 167, 169-179. Kievits, T., Devilee, P., Wiegant, J., Wapenaar, M. C., Cornelisse, C. J., van Ommen, G. J. B., and Pearson, P. L. (1990). Direct non-radioactive in situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes. Cytometry 11, 105-109. Klinger, K., Landes, G., Shook, D., Harvery, R., Lopez, L., Locke, P., Lerner, T., Osathanondh, R., Leverone, B., Housed. T., Paulka, K.. and Dackowski. W. (1992). Rapid detection of chromosome aneuploidies in uncultured amniocytes using fluorescence in situ hybridization (FISH). Am. J . Hum. Genet. 51, 55-63. Krumlauf, R., Jean-Pierre, M., and Young, B. (1982). Construction and characterization of genomic libraries of specific human chromosomes. Proc. Natl. Acad. Sci. U.S.A. 79, 2971-2975.

Kuo, W.-L., Tenjin, H., Segraves, R., Pinkel, D., Golbus, M. S.,and Gray, J. W. (1991). Detection of aneuploidy involving chromosomes 13, 18 or 21 by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes. Am. J . Hum. Genet. 49, 112-1 19.

Landegent, J. E., Jansen in deWal, N., Baan, R. A., Hoeijmakers, J. H. J.. and van der Ploeg, M. (1984). Acetyl aminofluorene-modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences. Exp. Cell Res. 153, 61-72. Landegent, J. E., Jansen in deWal, N., Dirks, R. W., Baas, F., and van der Ploeg, M. (1987). Use of whole cosmid cloned genomic sequences for chromosomal localization by non radioactive in situ hybridization. Hum. Genet. 7l,366-370. Langer, P. R., Waldrop, D. A., and Ward, D. C. (1981). Enzymatic synthesis of biotinlabelled polynucleotides: Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. U.S.A. 78,6633-6637.

Langford, C. F., Telenius, H., Carter, N. P., Miller, N. G. A., and Tucker, E. M. (1992). Chromosome painting using chromosome specific probes from flow sorted pig chromosomes. Cytogenet. Cell Genet. 61, 221-223. Le, H. T., Armstrong, K. C., and Miki, B. (1989). Detection of rye DNA in wheat-rye

WHOLE-CHROMOSOME HYBRIDIZATION

225

hybrids and wheat translocation stocks using total genomic DNA as a probe. Plant Mol. Biol. Rep. 7, 150-158. Lear, T. L., Trembicki, K. A., and Ennis, R. B. (1992).Identificationof equine chromosomes in horse x mouse somatic cell hybrids. Cytogenet. Cell Genet. 61, 58-60. Ledbetter, D. H. (1992). The ‘colorizing’of cytogenetics: Is it ready for prime time? Hum. Mol. Genet. 1, 297-299. Ledbetter, S. A., Nelson, D. L., Warren, S. T., and Ledbetter, D. H. (1990). Rapid isolation of DNA probes within specific chromosome regions by interspersed repetitive sequence polymerase chain reaction. Genomics 6, 475-481. Leitch, A. R., Mosgoller, W., Schwarzacher, T., Bennett, M. D., and Heslop-Hamson, J. S. (1990). Genomic in situ hybridization to sectioned nuclei shows chromosome domains in grass hybrids. J . Cell Sci. 95, 335-341. Leitch, A. R., Schwarzacher, T., Mosgoller, W., Bennett, M. D., and Heslop-Harrison, J. S. (1991). Parental genomes are separated throughout the cell cycle in a hybrid plant. Chromosoma 101, 206-213. Lengauer, C., Riethman, H., and Cremer, T. (1990). Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and Li primers. Hum. Genet. 86, 1-6. Lengauer, C., Riethman, H. C., Speicher, M. R., Taniwaki, M., Konecki, D., Green, E. D., Becher, R., Olson, M. V., and Cremer, T. (1992). Metaphase and interphase cytogenetics with alu-pcr amplified yeast artificial chromosome clones containing the bcr gene and the proto oncogenes c-rafl, c-fms and c-erbB2. Cancer Res. 52, 25902596. Lengauer, C., Speicher, M. R., Popp, S., Jauch, A., Taniwaki, M., Nagaraja, R., Riethman, H. C., Doniskeller, H., Durso, M., Schlessinger, D., andcremer, T. (1993). Chromosomal bar codes produced by multicolour fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes. Hum. Mol. Genet. 2, 505-512. Lichter, P., and Cremer, T. (1992). Chromosome analysis by non-isotopic in situ hybridization. I n “Human Cytogenetics: A Practical Approach” (D. E. Rooney and B. H. Czepulkowski, eds.), 2nd ed., Vol. 1, pp. 157-192. IRL Press, Oxford. Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D. C. (1988a). Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80, 224-234. Lichter, P., Cremer, T., Tang, C.-J. C., Watkins, P. C., Manuelidis, L., and Ward, D. C. (1988b). Rapid detection of human chromosome 21 aberrations by in situ hybridization. Proc. Natl. Acad. Sci. U.S.A. 85, 9664-9668. Lichter, P., Ledbetter, S. A., Ledbetter, D. H., and Ward, D. C. (1990a). Fluorescence in situ hybridization with Alu and LI polymerase chain reaction probes for rapid characterization of human chromosomes in hybrid cell lines. Proc. Natl. Acad. Sci. U.S.A. 87, 6634-6638. Lichter. P., Tang, C. C., Call, K., Hermanson, G . , Evans, G. A., Housman, D., and Ward, D. C. (1990b). High resolution mapping of human chromosome 11 by in sifu hybridization with cosmid clones. Science 247, 64-69. Liehr, T., Pfeiffer, R. A., and Trautmann, U. (1992). Typical and partial cat eye syndrome: Identification of the marker chromosome by FISH. Clin. Genet. 42,91-%. Liu, P., Siciliano, J., Seong, D., Craig, J., Zhao, Y.,Dejong, P. I., and Siciliano, M. J. (1993). Dual Alu polymerase chain reaction primers and conditions for isolation of human painting probes from hybrid cells. Cancer Genet. Cytogenet. 65,93-99. Lloyd, D. C. (1984).An overview of radiation dosimetry by conventional cytogenetic methods. I n “Biological Dosimetry” (W. G. Eisert and M. L. Mendelsohn, eds.), pp. 3-14. Springer-Verlag, Berlin.

226

S. D. BOUFFLER

Lloyd, D. C., Edwards, A. A., and Prosser, J. S. (1986). Accidental intake of tritiated water: A report of two cases. Radiat. Prof. Dosim. 15, 191-1%. Lo, Y.-M. D., Mehal, W. Z., and Fleming, K. A. (1988). Rapid production of vector free biotinylated probes using the polymerase chain reaction. Nucleic Acids Res. 16, 8719. Lucas, J. N., and Sachs, R. K. (1993). Using three color chromosome painting to test chromosome aberration models. Proc. Nail. Acad. Sci. U.S.A. 90, 1484-1487. Lucas, J. N., Tenjin, T., Straume, T., Pinkel, D., Moore, D., Litt, M., and Gray, J. W. (1989). Rapid chromosome aberration analysis using fluorescence in situ hybridization. lnt. J . Radial. Biol. 56, 35-44; erratum: p. 201. Lucas, J. N., Awa, A., Straume, T., Poggensee. M., Kodama, Y.,Nakano, M., Ohtaki, K., Weier, H.-U., Pinkel, D., Gray, J. W., and Littlefield, G. (1992a). Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation. lnt. J . Radial. Biol. 64, 53-63. Lucas, J. N., Poggensee, M., and Straume, T. (1992b). The persistence of chromosomal translocations in a radiation worker accidentally exposed to tritium. Cytogenet. Cell Genet. 60,255-256. Luke, S., and Verma, R. S. (1992). Origin of human chromosome 2. Nut. Genet. 2, 11-12. Maccarone, P., Watson, J. M., Francis, D., Selwood, L., Kola, I., and Marshall-Graves, J. A. (1992).The evolution of human chromosome 21: Evidence from in situ hybridization in marsupials and a monotreme. Genomics W , 1119-1 124. Magnani, I., Sacchi, N., Darller, M., Nisson, P. E., Tornaghi, R., and Fuhrmanconti, A. M. (1993). Identificationof the chromosome 14 origin of a c-negative marker associated with a 14q32 deletion by chromosome painting. Clin. Genet. 43, 180-185. Malcolm, S., Barton, P., Murphy, C., and Ferguson-Smith, M. A. (1981). Chromosomal localization of a single copy gene by in situ hybridization-human @-globingenes on the short arm of chromosome 11. Ann. Hum. Genet. 45, 135-141. Mangelschots, K., van Roy, B.. Speleman, F., van Roy, N., Gheuens, J., Benton, J., Buntinx, I., van Thienen, M.-N., Willekens, H.,Dumon, J., Ceulemans, B., and Willems, P. J. (1992). Reciprocal translocation between the proximal region of the long arms of chromosomes 13and I5 resultingin unbalanced offspring: Characterization by fluorescence in situ hybridization and DNA analysis. Hum. Genet. 89, 407-413. Manuelidis, L. (1985). Individual interphase chromosome domains revealed by in situ hybridization. Hum. Genet. 71, 288-293. Mamur, J. G., and Doty, P. (l%l). Thermal renaturation of deoxyribonucleic acids. J. Mol. Biol. 3, 585-594. McDougall, J. K., Dunn. A. R., and Jones, K. W. (1972). I n situ hybridization ofadenovirus RNA and DNA. Nature (London) 236, 346-348. Meltzer, P. S., Guan, X.-Y.,Burgess, A., and Trent, J. M. (1992). Rapid generation of region specific probes by chromosome microdissection and their application. Nut. Genet. 1, 24-28. Mewer, R., Kline, A. D., Jackson, L., and Overhauser, J. (1992). Confirmation of a cryptic unbalanced translocation using whole chromosome fluorescence in situ hybridization. A m . J. Med. Genet. 44, 471-481. Michalski, A. J., Cotter, F. E., and Cowell, J. K. (1992). Isolation of chromosome-specific DNA sequences from an Alu polymerase chain reaction library to define the breakpoint in a patient with a constitutional translocation t( 1;13)(q22;q12)and ganglioneuroblastoma. Oncogene 7, 1595-1602. Milan, D., Yerle, M.,Schmitz, A., Chaput, B., Vaiman, M., Frelat, G., and Gellin, J. (1993). A PCR-based method to amplify DNA with random primers-determining the chromosomal content of porcine flowkaryotype peaks by chromosome painting. Cyrogenet. Cell Genet. 62, 139-141.

WHOLE-CHROMOSOME HYBRIDIZATION

227

Miller, J. R.. Dixon, S. C., Miller, N. G. A., Tucker, E. M.. Hindkjaer. J., and Thompson, P. D. (1992). A chromosomal specific DNA library from the domestic pig (Sus scrofa dornestica). Cyrogener. Cell Genet. 61, 128. Muhlegger, K., Batz, H. G., van der Bohm, S.. Eltz, H., Holtke, H. J., and Kessler, C. (1989). Synthesis and use of new digoxigenin labelled nucleotides in non-radioactive labelling and detection of nucleic acids. Nucleosides Nucleotides 8, 1160-1 163. Murata, M., Nakata, N., and Yasumuro, Y. (1992). Origin and molecular structure of a midget chromosome in a common wheat carrying rye cytoplasm. Chrornosoma 102,27-31. Murdoch. A.. Jenkinson, E. J., Johnson, G. D., and Owen, J. J. T. (1990). Alkaline phosphatase-fast red-a new fluorescent label. Application in double labelling for cell surface antigen and cell cycle analysis. J. Imrnunol. Merhods 132, 45-49. Nakane, P. K.. Morinchi. T., Koji, T., Tanno, M., and Abe, K. (1987). In siru localization of mRNA using thymine-thymine dimerized cDNA. Acra Hisrochem. Cyrochern. 20, 220-243. Narayanswami, S.. and Hamkalo, B. A. (1991). DNA sequence mapping using electron microscopy. Genet. Anal. Tech. Appl. 8, 14-23. Narayanswami, S.. Lundgren, K., and Hamkalo, B. A. (1989). Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy. Scanning Microsc. 3, Suppl., 65-76. Natarajan. A. T., Vyas, R. C., Wiegant, J., and Curado, M. P. (1991). A cytogenetic followup study of the victims of a radiation accident in Goiania (Brazil). Murur. Res. 247, 103-1 1 1 . Natarajan, A. T., Vyas, R. C., Darroudi, F., and Vermeulen, S. (1992). Frequencies of xray induced chromosome translocations in human peripheral lymphocytes as detected by in sifu hybridization using chromosome specific libraries. Int. J. Radiut. Biol. 61,199-203. Nederlof, P. M., Robinson, D., Abuknesha. R.. Wiegant, J., Hopman, A. H. N., Tanke, H. J.. and Raap. A. K. (1989). Three colour fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences. Cyrornerry 10, 20-27. Nederlof. P. M., van der Flier, S., Weigant, J., Raap, A. K., Tanke, H. J.. Ploem, J. S., and van der Ploeg, M. (1990). Multiple fluorescence in situ hybridization. Cytornefry 11, 126-13 1. Nederlof, P. M.. van der Flier, S., Raap, A. K., and Tanke. H. (1992a). Quantification of internuclear and intranuclear variation of fluorescence in situ hybridization signals. Cytornerry 13, 831-839. Nederlof, P. M., van der Flier, S., Vorlijk, J., Tanke, H., and Raap, A. K. (1992b). Fluorescence ratio measurements of double-labelled probes for multiple in siru hybridization by digital imaging microscopy. Cyrornerry W, 839-845. Nederlof. P. M.. van der Flier, S., Venvoerd, N. P., Vorlijk, J., Raap, A. K., and Tanke, H. ( 1992~). Quantification of fluorescence in situ hybridization signals by image cytometry. C.vtornerry 13, 846-852. Nelson, D. L., Ledbetter, S. A,, Corbo, L.. Victoria, M. F., Ramifez-Solis, R., Webster, T. D.. Ledbetter. D. H., and Caskey, C. T. (1989). Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources. Proc. Nut/. AcUd. Sci. U . S . A . 86, 6686-6690. Nelson, P. S., Kent, M., and Muthini, S. (1992). Oligonucleotide labelling methods. 3. Direct labelling of oligonucleotides employing a novel non-nucleosidic 2-aminobutyl1,3-propanediol backbone. Nucleic Acids Res. 20, 6253-6259. Orth, G., Jeanteur, P., and Croissant, 0. (1971). Evidence for and localization of vegetal viral DNA replication by autoradiographic detection of RNA-DNA hybrids in sections of tumours induced by shope papilloma virus. Proc. Natl. Acad. Sci. U.S.A. 68, 18761880.

228

S. 0.B O W L E R

Pardue, M. L. (1985). In situ hybridization. In “Nucleic Acid Hybridization: A Practical Approach” (B. D. Hames and S. J. Higgins, eds.), pp. 179-202. IRL Press, Oxford. Pardue, M. L., and Gall, J. G. (1970). Chromosomal localization of mouse satellite DNA. Science 168, 1356-1358. Parokonny, A. S., Kenton, A. Y., Meredith, L., Owens, S. J., and Bennett, M. D. (1992a). Genomic divergence of allopatric sibling species studied by molecular cytogenetics of their FI hybrids. Plant J . 2,695-704. Parokonny, A. S., Kenton, A. T., Gleba, Y. Y., and Bennett, M. D. (1992b). Genome reorganization in Nicotiana asymetric somatic hybrids analysed by in situ hybridization. Plant J . 2, 863-874. Patil, S. R., Memck, S., and Lubs, H. A. (1971). Identification of each human chromosome with a modified Giemsa stain. Science 173, 821-822. Pettenati, M. J., Rao, N., Johnson, C., Hayworth, R., Crandall, K., Huff, O., and Thomas, 1. T. (1992). Molecular cytogenetic analysis of a familial 8p23.3 deletion associated with minimal dysmorphic features, seizures and mild mental retardation. Hum. Genet. 89, 602-606. Petty, E. M., Gibson, L. H., Breg, W. R., Bums, J. P., and Yang Feng, T. L. (1993). Brief clinical report-mosaic dup-(9p) diagnosis by fluorescence in situ hybridization (FISH). Am. J . Med. Genet. 45,770-173. Pinkel, D., Straume, T.,and Gray, J. W. (1986). Cytogenetic analysis using quantitative high sensitivity fluorescence hybridization. Proc. Natl. Acad. Sci. U.S.A. 83,2934-2938. Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J., and Gray, J. (1988). Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. U.S.A. 85,9138-9142. Poddighe, P. J., Ramaekers, F. C. S.,Smeets, A. W. G. B., Vooijs, G. P., and Hopman, A. H. N. (1992). Structural chromosome I aberrations in transitional cell carcinoma of the bladder-interphase cytogenetics combininga centromeric, telomeric and library DNA probe. Cancer Res. 52,4929-4934. Popp, S., Remm, B.. Hausmann, M., Liihrs, H., van Kaick, G., Cremer, T., and Cremer, C. (1990). Towards a cumulative biological dosimeter based on chromosome painting and digital image analysis. Kerntechnik 55, 204-210. Putman, C. A. J., Degrooth, B. G., Wiegant, J., Raap, A. K., van der We$ K. O., van Hulst, N. F., and Greve, J. (1993). Detection ofin situ hybridization to human chromosomes with the atomic force microscope. Cytometry 14, 356-361. Rabl, C. (1885). Uber zelltheillung. Morphol. Jahrb. 10,214-330. Rack, K. A., Hams, P. C., MacCarthy, A. B., Boone, R., Raynham, H., McKinley, M., Fitchett, M., Towe, C. M., Rudd, P., Arrnour, A. C., Lindenbaum, R. H.. and Buckle, V. J. (1993). Characterization of three de novo derivative chromosomes-l6 by reverse chromosome painting and molecular analysis. Am. J . Hum. Genet. 52,987-997. Rasch, P., Wiedemann, U., Weinberg, J., and Heckl, W. M. (1993). Analysis of banded human chromosomes and in situ hybridization patterns by scanning force microscopy. Proc. Natl. Acad. Sci. U.S.A. 90, 2509-2511. Ried, T., Baldini, A., Rand, T. C., and Ward, D. C. (1992). Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorid fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. U.S.A. 89, 1388-1392. Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. (1977). Labellingdeoxyribonucleic acid to high specific activity in uitro by nick translation with DNA polymerase I. J. Mol. Biol. 1l3, 237-251. Roesch, W. C., ed. (1989). “US-Japan Joint Reassessment of Atomic Bomb Radiation Dosimetry in Hiroshima and Nagasaki,” DS86 Final Report. RERF, Hiroshima.

WHOLE-CHROMOSOME HYBRIDIZATION

229

Roth, J., Saremaslani. P., and Zuber, C. (1992). Versatility of anti-horseradish peroxidase antibody-gold complexes for cytochemistry and in siru hybridization-prepation and application of soluble complexes with streptavidin-peroxidase conjugates and biotinylated antibodies. Histochemistry 98, 229-236. Rudkin, G. T., and Stollar, B. D. (1977). High resolution detection of DNA-RNA hybrids by indirect immunofluorescence. Nature (London) 265, 472-473. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd ed. Cold Spring Harbor Lab., Cold Spring Harbor, NY. Schardin, M., Cremer, T., Hager, H. D., and Lang, M. (1985). Specific staining of human chromosomes in Chinese hamster x man hybrid cell lines demonstrates interphase chromosome territories. Hum. Gener. 71, 281-287. Scherthan, H., Kohler, M., Vogt, P., von Malsch, K.. and Schweizer, D. (1992a). Chromosomal in sifu hybridization with double labelled DNA-signal amplification at the probe level. Cyrogener. Cell Gener. 60,4. Scherthan, H . . Loidl, J., Schuster, T., and Schweizer, D. (1992b). Meiotic chromosome condensation and pairing in Saccharomyces cereuisiae studied by chromosome painting. Chromosoma 101, 590-595. Schmid, E., Zitzelsberger, H., Braselmann, M., Gray, J. W., and Bauchinger, M. (1992). Radiation-inducedchromosome aberrations analysed by fluorescence in siru hybridization with a triple combination of composite whole chromosome-specificprobes. Int. J. Radiar. Biol. 62, 673-678. Schotton, D. (1989). Confocal microscopy: The way ahead. Microsc. Anal. May, pp. 6-13. Schwarzacher. T., Leitch, A. R., Bennett, M. D., and Heslop-Hanison, J. S. (1989). In siru localization of parental genomes in a wide hybrid. Ann. Bor. (London) [N.S.] 64, 315-324. Schwarzacher, T., Anamthwat-Jonsson, K., Harrison, G. E., Islam, A. K. M. R., Jia, J. Z., King, I. P., Leitch. A. R., Miller, T. E.. Reader, S. M., Rogers, W. J., Shi, M.. and Heslop-Hamson, J. S. (1992). Genomic in siru hybridization to identify alien chromosomes and chromosome segments in wheat. Theor. Appl. Genef.84,778-786. Seabright, M. (1971). A rapid banding technique for humanchromosomes. Lancer 2,971-972. Seong, D. C., Liu, P., Siciliano, P., Zhao, Y.,Cork, A., Henske, E., Warburton, D., Yu, M. T., Champlin, R., Trujillo, J. M., Deisseroth, A., and Siciliano, M. J. (1993). Detection of variant Ph-positive chronic myelogenous leukaemia involving chromosome I , chromosome 9 and chromosome 22 by fluorescence in siru hybridization. Cancer Gener. Cyfogenef. 65, 100-103. Seveus, L., Vaisala, M., Syjanen, S., Sandberg, M., Kuusisto, A., Ha~ju,R., Salo, J., Hemmila, I., Kojola, H., and Soini, E. (1992). Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in siru hybridization. Cyrometry W, 329-338. Shaw, P., Highett, M., and Rawlins, D. (1992). Confocal microscopy and image processing in the study of plant nuclear structure. J. Microsc. (Oxford) 166, 87-97. Smith, L. M., Evans, J. W., Moki, M., and Brown, J. M. (1992). The frequency of translocations after treatment for Hodgkins disease. I n f .J. Radiar. Oncol. Biol. Phys. 24,737-742. Speel, E. J. M., Schutte, B., Wiegant, J., Ramaekers, F. C. S., and Hopman, A. H. N. (1992). A novel fluorescence detection method for in siru hybridization based on the alkaline phosphatase-fast red reaction. J . Hisrochem. Cyrochem. 40, 1299-1308. Speicher, M. R., Jauch, A., Pam, A., and Becher, R. (1993). Delineation of translocation t( 15; 17) in acute prornyelocytic leukaemia by chromosomal in siru suppression hybridization. Leuk. Res. 17, 359. Spinner, N. B., Lucas, J. N., Poggensee, M., Jacquette, M., and Schneider, A. (1993). Duplication 9q34-qteridentifiedby chromosome painting. Am. J . Med. Gener. 45,609-613.

230

S. D. B O W L E R

Stanyon, R., Wienberg, J., Romagno, D., Bigoni, F., Jauch, A., and Cremer, T. (1992). Molecular and classical cytogenetic analyses demonstrate an apomorphic reciprocal translocation in Gorilla gorilla. Am. J . Phys. Anthropol. 88, 245-250. Straume, T., Langlois, R. G., Lucas, J., Jensen, R. H., Bigbee, W. L., Ramalho, A. T., and Brandao-Mello, C. E. (1991). Novel biodosimetry methods applied to victims of the Goiania accident. Health Phys. 60, 71-76. Straume, T., Lucas, J. N., Tucker, J. D., Bigbee, W. L., and Langlois, R. G. (1992). Biodosimetry for a radiation worker using multiple assays. Health Phys. 62, 122-130. Suijkerbuijk, R. F., van de Veen, A. Y., van Echten, J., Buys, C. H. C. M., deJong, B.. Oosterhuis, J. W., Warburton, D. A., Cassiman, W., Schonk, D., and Geurts van Kessel, A. (1991). Demonstration of the genuine is0 12p character of the standard marker chromosome of testicular germ cell tumours and identification of further chromosome 12 aberrations by competitive in situ hybridization. Am. J . Hum. Genet. 48, 269-273. Suijkerbuijk, R. F., Matthopolos, O., Kearney, L., Monard, S., Dhut, S., Cotter, F. E.. Herbergs, J., Geurts van Kessel, D., and Young, B. D. (1992). Fluorescent in situ identification of human marker chromosomes using flow sorting and alu element mediated PCR. Genomics W, 355-362. Sumner, A. T., Evans, H. J., and Buckland, R. A. (1971). A new technique for distinguishing between human chromosomes. Nature (London) 232, 31-32. Sverdlov, E. D., Monastyrskaya. G. S.. Guskova, L. I., Levitan, T. L.. Sheichenko, V. I., and Budowski, E. I. (1974). Modification of cytidine residues with a bisulphite-omethyl hydroxylamine mixture. Biochim. Biophys. Acta 340, 153-165. Tchen, P., Fuchs, R. P. P., Sage, E., and Leng, M. (1984). Chemically modified nucleic acids as immunodetectable probes in hybridization experiments. Proc. Nutl. Acad. Sci. U.S.A. 81,3466-3470. Telenius, H., Pelmear, A. H., Tunnacliffe, A., Carter, N. P., Behmel, A., Ferguson-Smith, M. A.. Nordenskjold, M., Pfragner, R., and Ponder, B. A. J. (1992). Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow sorted chromosomes. Genes, Chromosomes, Cancer 4, 257-263. Thiry. M., Scheer, U., and Goessens, G. (1991). Localization of nucleolar chromatin by immunocytochemistryand in situ hybridization at the electron microscopic level. Electron Microsc. Rev. 4, 85-1 10. Tijo. J. H., and Levan, A. (1956). The chromosome number of man. Hereditus 42, 1-6. Tkachuk, D. C., Westbrook, C. A., Andreefe, M., Donlon, T. A., Cleary, M. L., Saryanarayan. K., Homge, M., Redner, A., Gray, J., and Pinkel, D. (1990). Detection of bcr-abl fusion in chronic myelogenous leukaemia by in situ hybridization. Science 250,559-562. Trask, B., van den Engh, G., Pinkel, D., Mullikin, J., Waldman, F., van Dekken, H., and Gray, J. (1988). Fluorescence in situ hybridization to interphase nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization. Hum. Genet. 78, 251-259. Trautmann, U., Rubberts, A., Gramatzk, M., Henschke, F., and Gebhart, T. (1992). Multiple chromosomal changes and karyotypic evolution in a patient with myelofibrosis. Cancer Genet. Cytogenet. 61,6-10. van Dekken, H., and Bauman, J. (1988). A new application of in situ hybridization: Detection of numerical and structural chromosome aberrations with a combination centromerictelomeric probe. Cytogenet. Cell Genet. 48, 188-189. van Dilla, M. A., Deavan, L. L., Albright, K. L.. Allen, N. A.. Aubuchon, M. R., Bartholdi, M. F., Brown, N. C., Campbell, E. W., Carrano, A. V., Clark, L. M., Cram, L. S., Crawford, B. D., Fuscoe, J. C., Gray, J. W., Hildebrand, C. E., Jackson, P. J.. Jett, J. H., Longmire, J. L.. Lozes, C. R., Luedemann, M. L., Martin, J. C., McNinch, J. S., Meincke, L. J.. Mendelsohn, M. L.. Meyne, J., Moyzis, R. K., Munk, A. C., Perlman,

WHOLE-CHROMOSOME HYBRIDIZATION

231

J.. Peters, D. C., Silva, A. J., and Trask, B. J. (1986). Human chromosome specific libraries: Construction and availability. BiolTechnology 4, 537-552. Viljoen. D. C., Speleman, F., Smart. R., van Roy, N., Dutoit, J., and Leroy, J. (1992). Putative monosomy 21 in two patients-clinical findings and investigation using fluorescence in situ hybridization. Clin. Genet. 42, 105-109. Viscidi, R. P., Connelly, C. J., and Yolken, R. H. (1986). Novel chemical method for the preparation of nucleic acids for non-isotopic hybridization. J. Clin. Mircobiol. 23,311-3 17. Vooijs, M., Yu, L. C., Tkachuk, D., Pinkel, D., Johnson, D.. and Gray, J. W. (1993). Libraries for each human chromosome constructed from sorted enriched chromosomes by linker-adaptor PCR. Am. J . Hum. Genet. 52,586-597. Vourc’h, C.. Taruscio, D., Boyle, A. C., and Ward, D. C. (1993). Cell cycle dependent distribution of telomeres, centromeres and chromosome-specific sub satellite domains in the interphase nucleus of mouse lymphocytes. Exp. Cell Res. Ms, 142-151. Vyas, R. C., Darroudi, F., and Natarajan, A. T. (1991). Radiation-induced chromosomal breakage and rejoining in interphase-metaphase chromosomes of human lymphocytes. Mutat. Res. 249, 29-35. Waggoner, A., Debasio, R., Conrad, P., Bright, G. R.. Ernst, L., Ryan, K., Nederlof, P. M., and Taylor, D. (1989). Multiple spectral parameter imaging. Methods Cell Biol. 30,449-478. Ward, B. E.. Gersen, S. L..and Klinger, K. W. (1992). Reply to letter-A rapid (but wrong) prenatal diagnosis. N . EngI. J . Med. 326, 1639-1640. Ward, B. E., Gersen, S. L., Carelli. M. P., McGuire, N. M.. Dackowski, W. R., Weinstein, M.. Sandlin, C.. Warren, R.. and Klinger, K. W. (1993). Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization-clinical experience with 4,500 specimens. Am. J. Hum. Genet. 52, 854-865. Weier, H.-U. G . . Segraves, R., Pinkel, D., and Gray, J. W. (1990). Synthesis of Y chromosome specific labelled DNA probes by in uifro DNA amplification. J . Hisrochem. Cyfochem. 38,421-426. Weier. H.-U. G., Lucas, J. N., Poggensee, M., Segraves, R., Pinkel, D., and Gray, J. W. (1991). Two colour hybridization with high complexity chromosome specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations. Chromosoma 100, 371-376. Wetmur. J. G . . and Davidson, N. (1968). Kinetics of renaturation of DNA. J . Mol. Biol. 31,349-370. Weigant. J., Ried. T., Nederlof, P. M.,van der Ploeg, M., Tanke, H., and Raap, A. K. (1991). In situ hybridization with fluoresceinated DNA. Nucleic Acids Res. 19,3237-3241. Wiegant. J.. Wiesmeijer, C. C.. Hoovers, J. M. N., Schuuring, E., Dazzo, A.. Vrolijk. J., Tanke, H. J.. and Raap. A. K. (1993). Multiple and sensitive fluorescence in situ hybridization with rhodamine labelled, fluorescein-labelled and coumarin labelled DNAs. Cyfogenet. Cell Genet. 63, 73. Wienberg, J., Jauch, A., Stanyon, R.. and Cremer, T. (1990). Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. Genomics 8, 347-350. Yunis. J. J.. and Prakash, 0. (1982). The origin of man: A chromosome pictorial legacy. Science 215, 1525-1530. Zhao, L., Kantajian, H. M., van Oort, J., Cork, A,. Trujillo, J. M.. and Liang, J. C. (1993a). Detection of residual proliferating leukaemic cells by fluorescence in situ hybridization in CML patients in complete remission after interferon treatment. Leukemia 7 , 168-171. Zhao, L.. Van Oort, J.. Cork. A.. and Liang, J. C. (1993b). Comparison between interphase and metaphase cytogenetics in detecting chromosome-7 defects in hematological neoplasias. Am. J . Hematol. 43, 205-21 1. Zheng, Y. L.. Ferguson-Smith, M. A., Warner, J. P., Ferguson-Smith. M. E., Sargent,

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C. A., and Carter, N. P. (1992). Analysis of chromosome 21 copy number in uncultured amniocytes by fluorescence in situ hybridization using a cosmid contig. Prenatal Diagn. 129931-943. Ziomek, C. A., Lepire, M. L., and Torres, I. (1990). A highly fluorescent simultaneous azo dye technique for demonstration of non-specific alkaline phosphatase activity. J . Histochem. Cytochem. 38,437-442.

Neuronal Modulation and Plasticity in Vitro’ Robert A. Smith and Zhi-Gang Jiang Department of Anatomy, University of Glasgow, Glasgow, G12 8QQ Scotland, United Kingdom

1. Introduction

Neuronal plasticity may be defined as the ability of neuronal populations to adapt and make long-term changes in their phenotypic expression and to their synaptic connections in response to local environmental changes and interactions. This has fundamental consequences for the nervous system both during development, if its neurons are to seek and form intercommunicationswhich will become functionally established yet retain an inherent propensity to change, and if it is to possess the potential to undergo regeneration following trauma. Plasticity is considered to be a sequential process involving a number of phases, each of which may be influenced by the local environment, including the initiation of fiber growth (sprouting),elongation of processes, and the capacity to form functional connections with the correct target cells to permit the establishment of aprecise postsynaptic map. The importance of environmental cues, such as the response to the extracellular matrix and trophic factors (Walicke, 1989; Thoenen, 1991; Loughlin and Fallon, 1993), and the role played by growth cones in effecting fiber outgrowth, guidance, and successful targeting to form synapses (GordonWeeks, 1991; Kater and Mills, 1991; Letourneau et al., 1991) have been, and continue to be, realized. The level at which any of these act, the transducing signals involved, and whether mediators such as inducible intermediate early genes, for example,fos andjun (Sheng and Greenberg, 1990; Morgan and Curran, 1991), play a part in regulating neuronal differentiation and plasticity are all exciting questions still requiring detailed answers. Options for plasticity can involve modulation of neuronal function and the potential for subtle regulatory changes at either pre- (or even preI We have pleasure in dedicating this article to Dr. Muriel J. Ord as a “thank you” for her guidance and friendship over the years, and we wish her many happy years of retirement.

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pre-) synaptic or postsynaptic sites. Certainly the importance of control mechanisms for these changes becomes increasingly apparent as more is known of neural circuitry, yet the precision and complexity of developing and mature nervous systems impose obstacles to analysis of plasticity, even with the rapidly expanding technology now available for wholeanimal studies. Capability for change is retained in uiuo into adulthood and underlies the imprinting of memory circuitry as typified by cognitive functioning and hippocampal synaptic plasticity (Lisman and Hams. 1993). It also forms the basis of much-studied behavioral preferences, such as the complex learning patterning in many species of songbirds (Nottebohm, 1989; Marler, 1991). The dichotomy between the stability needed to retain memory and the plasticity needed to allow new learning, or to respond to change, including trauma, is a finely balanced dilemma which the nervous system has to solve (Carpenter and Grossberg, 1993), and which has perplexed the neuroscientist for many years. The problems in deciphering such intricacies of the nervous system continue. The dawn of cell culture methodology, in the opening decade of the century (Harrison, 1907, 1910), however, generated much excitement since it promised means by which the factors influencing and guiding neuronal differentiation could be studied. Hamson’s hanging-drop preparations of frog, and later chick, neural tube and primordia of cranial nerve ganglia cultured in clotted lymph, opened a Pandora’s box of experimental manipulation, the full potential of which is now only being realized with the aid of modem culture and molecular technologies. These early studies predicted that one of the necessary conditions for neurite outgrowth was a medium which afforded some solid support to the fibers (Hamson, 1910); simple saline and dextrose solutions were sufficientfor fiber outgrowth from cultured sympathetic nerves, although again stereotropism by the coverslip surface was implicated in the extension processes (Lewis and Lewis, 1912). Since these early studies, many workers have turned their attention to the advantages of in uitro methods that permit neurons to be cultured in defined and carefully controlled environments in which the influence of specific factors or conditions can be monitored, thus helping to unravel the mysteries of what in part determines polarity, plasticity, and modulation of neuronal function. A number of good and recent treatises have focused on the strategies available for nerve cell culture and are worth detailed scrutiny by those addressing specific questions of nerve plasticity (Beadle et al., 1988; Shahar et al., 1989; Banker and Goslin, 1991; Chad and Wheal, 1991). A briefdescription of the various types of possible neural cultures is given in Section 11. This chapter aims to emphasize the contribution that in uitro studies have made in determining the influence of trophic factors and physical

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environment on neuronal survival and phenotypic plasticity. It also considers the control of neurite extension and growth cone activity in finding pathways and establishing synaptic connections. The part played by culture systems in elucidating the pivotal position of these processes in uiuo in the developing nervous system and in the adult, and in aging and in repair mechanisms following injury, are explored.

II. Neuronal Cell Culwres

Model systems range from cultures of invertebrate neurons to those from vertebrates, primarily of avian and mammalian origin, and are derived from all elements of the nervous system. Neuronal cell lines have also proved beneficial in studies of neuronal plasticity and modulation in precisely controlled in uitro environments. Each system has its advantages for certain types of studies and in addressing specific problems, but also has its own set of limitations which should be realized when interpreting the findings. Cultures of brain slices have also been of use (Andersen, 1981), both in developmental studies of how the cortex establishes functional connections (Bolz et af., 1993) and in toxicopharmacological studies where the value of slices in investigations of hippocampal long-term potentiation have been particularly realized (Fountain et al., 1992). Such systems by and large, however, though of interest and having generated data of significance, fall outside the scope of this chapter, which concentrates on the use of actual cell cultures in studies of plasticity and neuronal modulation. A. Transformed Cell Lines The use of established neuronal cell lines has been advocated by many as having the advantage of generating homogeneous populations that can be cloned and maintained indefinitely (Wood, 1992). Cell lines derived from a mouse neuroblastoma (Augusti-Toccoand Sato, 1969), such as the popular C 1300, and also PC 12 cells, derived from a rat pheochromocytoma (Greene and Tischler, 1976), which can be induced to differentiate into sympathetic-likeneurons, have been instrumental in analyses of the mechanisms which underlie neurite sprouting in response to neurotrophins such as nerve growth factor (NGF) (Pollock et al., 1990). Cell lines continue to have a fundamental role in elucidating signal transduction in molecular studies of NGF receptor expression, including studies of the trk (tyrosine kinase) family (Jing et al., 1992). It is now possible to actually custom

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build cell lines by immortalizing primary neurons by introducing oncogenecontaining retroviruses, although in general only short lengths of DNA can be introduced by this strategy at present (Lendahl and McKay, 1990; Rabizadeh et al., 1993). In the next decade, as molecular biology and gene technologies advance, these lines, together with the culture of neurons from the nervous systems of transgenic animals, will contribute significantly to our understanding of plasticity, particularly in studies of neuronal aging and cell death (Garcia et ul., 1992). The major disadvantage of neuronal lines still remains however: their very nature of being transformed cells, often with unstable karyotypes. They therefore frequently express properties quite different from typical neurons in situ. Biochemical, metabolic, and physiological changes in cloned cells have led us to conclude that primary neuronal cultures are by and large still preferable in in uitro studies of nervous tissue whenever possible (Smith, 1991). B. Invertebrate Neuronal Cells Invertebrate neurons are recommended by their large size, which permits intracellular recording, and also by the fact that individual characterization is possible because of the small number of cells within invertebrate nervous systems. They have phylogenetic and evolutionary interest. The main disadvantage, however, is perhaps this simplicity, which makes extrapolation to higher animals, and especially man, difficult for many neuroscientists, particularly clinicians. Even so, such cultures have generated much information on neuronal behavior, especially with respect to neurite outgrowth, growth cones, the establishment of functional synaptic connections, and reconstruction of neural networks (Bulloch and Syed, 1992). Neurons routinely used include those isolated from annelids such as the leech, Hirudo (Fuchs et al., 1981), and from molluscs such as Aplysiu (Schachter, 1985; Lukowiak and Colebrook, 1988-1989; Bailey and Chen, 19911, and Helisoma (Haydon et al., 1985; Mattson and Kater, 1987). Neuronal cultures from arthropods, such as Drosophila, are increasingly being used in developmental and genetic studies (Wu et al., 1983), and have relevance to neuronal plasticity studies with respect to axonal elongation and growth cone behavior. C. Vertebrate Neurons

Many early studies of vertebrate neurons focused on the use of avian neurons, either in explant or dissociated cultures (Letourneau, 1975), which were of embryonic and peripheral nervous system (PNS) tissue.

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Such cultures demonstrated the effects of extracellular matrix (ECM) components on the elongation and guidance of neurites, dramatically showing how these processes may be regulated (Rogers et al., 1983; Bray et al., 1987; Gundersen, 1987; Hammarback et al., 1988; On-and Smith, 1988). Cultures of retinal tissue from goldfish have been used as a model for the study of central nervous system (CNS) regeneration (Landreth and Arganoff, 1976; Smalheiser et al., 1984; Ford-Holevinski et al., 1986). Mammalian neurons have often been restricted to those of fetal or neonatal origin (Hawrot, 1980), especially in culturing neurons from human sources, that is, fetal sensory ganglia (Baron-Van Evercooren et al., 1982; Kim et al., 1984; Yong et al., 1988). Embryonic or neonatal cultures are now possible from many CNS areas, for example, spinal cord (Mayer et al., 1987), cerebellum (Messer, 1977), striatum (Sebben et al., 1990), cerebral cortex (Frandsen et al., 1993), hippocampus (Mattson et al., 1988a), and mesencephalon (Nagata er al., 1993). Adult mammalian postmitotic cells have traditionally proved more difficult to maintain in culture; however, for the PNS at least, adult neurons can be successfully cultured for several weeks (Scott, 1982; Smith, 1991). As more is learned about the individual growth requirements of each specialized adult neuronal type, it is envisaged that the success rate will continue to increase and will contribute to a clearer understanding of neuronal plasticity and modulation.

111. Neurite Initiation and Elongation

Fiber initiation and elongation, both in development and in response to injury, is considered a prerequisite for neuronal plasticity. Indeed, Ram6n y Cajal (1919) appreciated that the local environment and the influence of target-derived agents and electrical activity might all contribute to the regulation of axonal sprouting. In uitro studies have enabled these processes to be studied in detail and have generated data which have been vital in substantiating hypotheses difficult to prove from in uivo work. Neuronal culture experiments demonstrated that fiber outgrowth can follow time courses similar to those that occur in uivo (Role and Fishbach, 1987), which provides a reassuring baseline for other experimental manipulations performed in uitro in order to investigate the factors and mechanisms involved in the process. In uitro neurons become polarized as they differentiate (Dotti et al., 1988), with dendritic processes containing a high-molecular-weight, microtubule-associated protein, MAP-2, and with a balance of microtubules with (+) and (-) ends directed distally; while axons are usually

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enriched in lower molecular-weight, microtubule-associated tau proteins, and with all microtubules orientated with their (+) end directed away from the perikaryon (Black and Baas, 1989; Sargent, 1989). Nerve process formation, neuritogenesis, and the distal growth cones are affected by a number of extrinsic signals; these range from the physical substratum, and particularly extracellular matrix components (Letourneau, 1975; Orr and Smith, 1988; Sanes, 1989), to an increasing list of known trophic agents (Thoenen, 1991; Lindsay, 1992) and neurite inhibitors (Schwab et al., 1993). Many studies indicate that delicate interactions of these influences would be more likely to generate complex interlinked control mechanisms of neurite outgrowth. Extracellular signaling molecules may be coupled to intracellular metabolism and second messenger systems by a group of guanine nucleotide proteins (G-proteins) whose significance in neuronal cells is now being realized (Jordan, 1992). The intrinsic cues stimulated in response to external influences play an integral role in neuritogenesis, and include specific molecules known as growth-associated proteins, for example, GAP-43 (also known by other names such as BSO) (Skene, 1989; Coggins and Zwiers, 1991; Pfenninger et al., 1992), and also intracellular mechanisms regulated by neurotransmitters (Mattson, 1988; Lipton and Kater, 1989), calcium (Kater and Mills, 1991), and other ionic species. These influences are considered in the following subsections.

A. Role of Substratum

1. Synthetic and Patterned Substrates

The importance of in v i m approaches to studying problems of plasticity was shown by the pioneering studies of Letourneau (1975) with embryonic chick dorsal root ganglion (DRG) neurons. This work demonstrated that neuronal behavior was substantially influenced by the adhesiveness of neurons to the substratum. His use of prepared, patterned, substrata to unravel the part played by substrate in enhancing neuronal survival, neurite extension, and growth cone migration has been emulated by many researchers in numerous subsequent studies. Patterns were formed by shadow deposition with pallidium over an electron microscope grid and included collagen or the synthetic cationic coating agent polyomithine (PORN)on uncoated surfaces. Preferential neurite elongation occurred on substrata to which the neurites adhered most strongly, even in cases where the less adhesive substratum was quite capable of supporting neurite elongation if this was the only choice for the regenerating neurites (Letourneau, 1975). The extracellular environment in uiuo may furnish neurons with the regulatory options essential for events leading to plasticity. Syn-

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thetic agents have since continued to be popular as coating agents (Fig. 1). Collagen, polylysine, and PORN have been widely used both in developmental studies (Hawrot, 1980), and in monitoring regenerative capability following injury (Cannella and Ross, 1987). Other workers have attempted to produce more natural threedimensional matrices from natural sources, that is, from secretions by non-neural cells, to demonstrate how subtle changes in composition and organization of substrate-attached material can have profound effects on neuronal survival and neurite elongation (Fridman et al., 1985; Om and Smith, 1988; Halfter and von Boxberg, 1992) (Fig. 2c). Patterned substrates incorporating adsorbed laminin and fibronectin were instrumental in work examining the importance of substrates in guiding neurites and growth cones (Hammarback et al., 1985; Gundersen, 1987; Pixley et al., 1987; Heaton and Swanson, 1988; Clark et al., 1993). High-resolution patterning techniques have now been developed. These include elegant silicon or quartz surfaces photolithographically defined, with the adhesivity controlled by covalently binding amine derivatives to the surfaces using the coupling agent aminotrihydroxysilane(Kleinfeld et al., 1988). Substrates of ethylenediaminepropylsilane (EDA-P) regions to promote adhesion, and n-tetradecane regions to inhibit adhesion were employed to maintain mouse spinal cord or perinatal rat cerebellar neurons for up to 12 days in uitro. Neurons adhered faithfully to the EDA-P patterned regions, providing that serum was present in the medium (Figs. lA,B). The periodicity of the pattern was also important, with granule cells remaining faithful to the pattern if lines were spaced 50 p m apart, while Purkinje cells, which developed electrical activity and extended fibers, bridged nonadhesive regions, even when separated by 60 p m (Kleinfeld et al., 1988). The importance of neurite surface charge properties has also been strikingly shown by use of patterned substrata (Torimitsu and Kawana, 1990). DRG neurons plated on metal oxide patterns deposited on silica grew neurites selectively, guided by the electronegativity of the aluminum or indium (Figs. 3a and 3b), even if the lines were only I pm apart (Torimitsu and Kawana, 1990). Recent studies using microgrooved plastic dishes have shown that the hydrophilic properties and particularly the water contact angle of the plastic can help in elucidating the mechanisms of neurite elongation and orientation (Morikawa et al., 1991). Clearly such in uitro methods are now sufficiently advanced to offer means for studying in detail the physical factors that guide neurites and which may be fundamental in establishing highly ordered neural networks and in regulating plasticity changes. Cell-cell interactions with glial components may also be studied by in uitro experiments and their role in regeneration investigated (Carbonetto et al., 1987; Bixby et al., 1988; Bedi et al., 1992).

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FIG. 1 (A)Postnatal rat cerebellar neurons maintained for 1 div (days in vitro) in serumfree medium and plated on a patterned silicon grid substrate, with n-tetradecane bound to the background. Note that the cells are randomly attached. (B)1-day culture from the same preparation as (A), but with 10% serum added-the cells now faithfully reflect the underlying pattern. Bar = 100 pm. (Reproduced with permission from Kleinfeld et al., 1988.)

FIG. 2 (a) Scanning electron micrograph (SEM) of extensive neurite outgrowth from adult mouse sensory neurons maintained in a muscle-conditioned medium. Note process branching (arrow) and distal growth cones. Bar = 25 pm. (b) Adult sensory neurons in nonneuronal-cell reduced culture maintained for 8 div on a 10 pg/ml fibronectin substratum. Bar = 10 pm. (c) Sensory neurons extending neurites following 4 days in culture and maintained on substratum-attached material prepared from 3T3 fibroblasts. Bar = 10 pm.

FIG. 3 (a) Neurite outgrowth by a DRG neuron maintained in serum-free medium and with 20 ng/ml NGF on an unpatterned silica glass plate. Bar = 40 pm, (b) Directional growth of neurites along a patterned surface: bright lines are 7-pm-wide S O z tracks, and dark lines 1-pm aluminum oxide tracks. Bar = 40 pm. [(a) and (b) reproduced by permission from Torimitsu and Kawana, 1990.1 (c) Phase-contrast micrograph of adult DRG neurons with bipolar appearance following maintenance in medium supplemented with 10 pM retinoic acid for 6 days. Bar = 25 pm. (d) SEM of the distal end of neunte of an adult neuron

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2. Fibronectin

Early candidates of interest in the search for substances which might be involved in regulating initiation of axonal sprouting included components of the ECM because these were suspected of modulating cellular differentiation in vivo and could easily be applied to in vitro regimens for detailed study. The glycoprotein, fibronectin (FN) [initially termed LETS or large external transformation-sensitive protein by Hynes in 1973 owing to its presence on normal but not on transformed lines, and renamed later to signify its association with fibroblasts (Kuusela et al., 1976)] proved of interest. FN-coated culture dishes were shown to support and enhance neurite outgrowth from embryonic chick retinal cells compared with coatings of collagen or poly-L-lysine (Akers et al., 1981). Using defined substrata prepared by embedding different proteins in a hydrophilic gel (2hydroxyethylmethacrylate) of known pore size, Carbonetto and colleagues (1982)demonstrated that fibronectin was effective in supporting outgrowth from embryonic chick DRG neurons. This effect was abolished if the gels were treated with antiserum against fibronectin. In a subsequent study, these workers demonstrated that proteolytic fragments of the larger FN molecule retained the potential to induce fiber extension, providing they contained the portion of the molecule which mediated myoblast attachment (Carbonetto et al., 1983). Since these early studies, others have shown that specific regions of the FN molecule interact with neurites and that different neuronal types might recognize different binding domains (Rogers et al., 1985, 1987; Lewandowska er al., 1990). Cooperation with other molecules, such as some glycosaminoglycans(Akeson and Warren, 1986),gangliosides (Mugnai and Culp, 1987), and heparin-binding peptides (Haugen et al., 1992) are important in promoting outgrowth. The FN-receptor complexes have been localized both in vivo and in v i m by immunocytochemical studies in chick sensory and sympathetic ganglia (Duband et al., 1986); in v i m , the complexes localized on perikarya, axons, and growth cones of neurons (Bozyczko and Horwitz, 1986). A synergistic action of FN and NGF has also been shown for DRG neurons obtained from chick embryos of different stages (E8-10, E16), with the requirements changing at different times in development (Millaruelo et al., 1988). Neurons isolated from the CNS responded differently from those of PNS origin in an elegant set of experiments by Rogers and co-workers (1983) using embryonic chick neurons dissociated from DRG, sympathetic ganglia, the spinal cord, and the retina. While neurites were initiated from peripheral neurons, central neurons cultured in the presence of 10 p M retinoic acid to show branching and growth cones. Bar = 10 pm. [(c) and (d) courtesy of P. S. Chong] (e) SEM of flattened growth cone of DRG neuron maintained on 10 pglml laminin. Note the ruffled leading lamella. Bar = 10 pm. (f) SEM of extensive neurite outgrowth (arrows) of a group of adult neurons maintained on a laminin substratum. Bar = 10 pm.

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cultured under identical conditions aggregated into clusters but did not extend processes in the presence of FN. Many studies of FN effects primarily investigated the response of chick embryonic neurons. Neurite outgrowth was also demonstrated from rat sympathetic neurons of both embryonic (El5 and E20) and young adult (P35) rats, although neurite fasciculation differed with age, partly owing to the response of the non-neuronal cells present in the cultures (Roufa et al., 1983). DRG neurons from newborn and young adult (45-60 days) mice were seen to extend neurites in cultures where the substratum had been coated with 30 pg/ml FN (Horie and Kim, 1984), although survival was limited to a few days in these studies. We found that DRG neurons from 6-month-old mice extended neurites and survived in coculture with a meshwork of non-neuronal cells (Smith and McInnes, 1986), but could also be maintained in cultures where non-neuronal cells were reduced by adding cytosine arabinoside to the culture medium, providing that 10 pg/ ml FN coatings were applied to the culture dishes (Smith and Orr, 1987) (Fig. 2b). If FN was applied together with laminin (LN), the effect was more striking (Om and Smith, 1988). Human fetal sensory neurons have also been shown to be responsive to the effects of FN for neurite initiation and elongation. Baron-Van Evercooren and co-workers (1982) showed that if nerve growth factor was included in the cultures, FN enhanced neurite outgrowth. The influence of FN was not in this case as great as that of laminin, while in a later study the response to FN was reported as comparable to that of laminin (Yong et al., 1988).

3. Lnminin On the whole, neuritogenesis by cultured neurons is greater on substrata containing the larger glycoprotein, laminin, which is present in all basal laminae, than on those with FN (Sanes, 1989).Of particular significancefor neuronal regulation is the well-documented observation that for embryonic nervous tissue at least, both peripheral and central neurons commonly respond to LN (Manthorpe et al., 1983; Rogers et al., 1983; Carri et al., 1988). In this case, longer neurites were extended from DRG, retinal, and spinal cord neurons of embryonic chick than with cells maintained on polylysine, although the actual numbers of nerve fibers were smaller. Similar conclusions with respect to centrally derived neurons resulted from studies of neonatal rat brain cultures where LN, but not FN, was found to be effective for enhanced neurite outgrowth and attachment (Liesi et al., 1984). In addition to stimulating neurite extension, culture experiments of both sympathetic and telenecephalic chick neurons demonstrated that LN also promoted actual neuronal migration (Liang and Crutcher, 1992). With

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human fetal DRGs, more pronounced neuritogenesis in synergy with NGF occurred with LN than with FN (Baron-Van Evercooren et al., 1982), although others considered the two glycoproteins comparable in their efficacy (Yong et al., 1988). Longer neurites were elicited from chick retinal neurons in response to LN than with FN (Cam et al., 1988), and from embryonic chick (E5-9) DRG neurons (Bray et al., 1987) maintained on patterned substrata which had adsorbed LN (Hammarback et al., 1985; Gundersen, 1987). Laminin also advanced the onset of neurite initiation from ciliary ganglion neurons, with an increase in the proportion of multipolar neurons (Davis et al., 1985).A selective role has been reported in initial nerve fiber outgrowth from very early embryonic neurons prepared from neural tube fragments at 40 hr incubation, with six times as many neurons extending neurites on LN-patterned substrata than on collagen after 24 hr in culture (Heaton and Swanson, 1988). With embryonic rat neurons from the cerebral cortex, septal basal forebrain, or spinal cord, Schinstine and Cornbrooks (1988) showed a response to LN on neurite extension by cells isolated at El8 but not E15. Pixley and coworkers (1987), however, in studying El8 rat septal neurons, did not observe a preference for LN by neurites; rather, the glycoprotein caused cocultured astrocytes to migrate, and these in turn guided neuritic outgrowth and elongation. LN proved no better than polylysine in enhancing neuritogenesis for teleost spinal cord cultures prepared from adult Apteronotus albfrons (Anderson, 1993). Work on embryonic rat sympathetic neurons has shown that laminin causes the growth of axons rather than dendritic processes (Lein and Higgins, 1989),and also enhances axonal outgrowth by hippocampal neurons, thereby accelerating the development of neuronal polarity in culture (Lein et al., 1992). A response to LN was seen for adult rat retina following optic nerve lesions (Ford-Holevinski et al., 1986), although David (1988) suggested that CNS outgrowth may be mediated via an astrocyte surface molecule distinct from LN. Beneficial support of adult mouse sensory neuronal survival and neuritogenesis by LN has been demonstrated (Unsicker et al., 1985; Orr and Smith, 1988), LN being a potent modulator of neurite geometry by affecting elongation rather than neurite branching (Itoh et al., 1991) (Fig. 3f). Laminin was found to be an active component in medium conditioned by bovine endothelial cells (Lander et al., 1983, and we also reported a similar result in medium conditioned by muscle (Fig. 2a), which was shown to stimulate neurite outgrowth by adult DRG neurons (Simpson and Smith, 1989). The mechanisms by which laminin regulates neuronal survival and neuritogenesis involve interactions with other molecules, including both integrin and nonintegrin receptors (Sanes, 1989; Hunter et al., 1991). An understanding of the complete molecular mechanisms of LN receptors awaits characterization (Edgar, 1989), although studies using neural cell

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lines, such as PC12 (Akeson and Warren, 1986), and chick sympathetic (Edgar et al., 1984) and spinal cord neurons (Dow and Riopelle, 1992), have all emphasized an association of heparan sulfate proteoglycans and glycosaminoglycans in promoting laminin binding and effecting neurite formation. Studies on the neuroblastoma-gliomahybrid NGlO8-15 cell line have demonstrated that laminin-induced neuritogenesis may be mediated either through cytoskeletal mechanisms (Luckenbill-Edds and Kleinman, 1988), or by dephosphorylation of proteins following laminin binding (Weeks, et al., 1990). LN-mediated effects therefore, have a role in development, and may have a regulatory function in mature neuronal plasticity responses by guiding limited regeneration following injury. They may also be relevant to neurodegenerative disorders such as Alzheimer’s disease and Down’s syndrome (Murtomaki et al., 1992). Further in uitro experimental designs should continue to clarify laminin’sprecise role in such neuronal plasticity. 4. Other Cell Adhesion Molecules A number of other cellular adhesion molecules (CAMS)have been studied in uitro which are of interest to studies of plasticity and neuronal behavior, and which may act by modulating the activity of other ECM components. Tenascin (also known as cytotactin), provided substrates had been precoated with polylysine, stimulated neurite outgrowth from chick sympathetic and sensory neurons, and produced long neurites from spinal cord neurons, with growth rates for the latter similar to those for fibronectin and laminin substrata, although the amount of branching $peared to be inhibited (Wehrle and Chiquet, 1990). Glycoproteins such as neural CAM (NCAM) (Rutishauser et al., 1988; Docherty et al., 1989), L1 (Kobayashi et al., 1992), F11 and G4 (Chang et al., 1987), and the calcium-dependent cadherins (Rutishauser, 1989; Bixby and Zhang, 1990)have all been shown to function in the control of neuron-neuron adhesion and neurite outgrowth and fasciculation. Indeed, for chick ciliary ganglion neurons, Ncadherin promoted more rapid outgrowth than LN (Bixby and Zhang, 1990). Lafont and colleagues (1992) recently demonstrated that addition of heparan chondroitin sulfate to embryonic rat mesencephalic neurons stimulated axonal growth, whereas dermatan sulfate promoted dendritic elongation, thus regulating neuronal polarity. These, and a growing list of matrix molecules which can be identified in uiuo at different stages of development, apparently play an important and interrelated role in the morphogenesis and maintenance of neuronal tissues; their full significance remains to be realized but in uitro manipulation will undoubtedly contribute to a inore complete understanding.

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5. Concanavalin A

Plant lectins, and particularly concanavalin A (ConA), have been used in studying regional localization of membrane receptors and effects on neuronal plasticity, including enhancement of neurite outgrowth and modulation of synaptic activity (Lin and Levitan, 1991). Invertebrate neuronal cultures especially have benefitted from use of ConA (Chiquet and Nicholls, 19871, although neuronal responses can differ from those observed on ECM substrates (Neely, 1993). An understanding of how these differences are generated may be useful in deciphering the complexities of neuronal plasticity.

6. Role of Trophic Factors

Neurotrophic factors have been shown to be essential for the selective survival of neurons and in mediating target-guided axonal growth in the developing vertebrate PNS and parts of the central nervous system (Davies, 1988).Normally for mature neurons, neurotrophic factors are considered more important in regulating differentiation than in ensuring survival, as evidenced by axonal arborization, synaptic connections, and neurotransmitter plasticity (Lindsay, 1992), but nevertheless the realization is growing that the presence of peptide growth factors and subtle regulation by them is essential in the development, maintenance, and repair of nervous system functioning. A number of succinct and recent reviews of neurotrophic factors can be recommended (Barde, 1989; Walicke, 1989; Thoenen, 1991; Loughlin and Fallon, 1993). Nerve growth factor, a polypeptide, has come to represent the prototypical neurotrophic factor, since much could be discovered about its bioactivity because it could be purified in large quantities from the adult male mouse submandibular gland, and its effects in promoting neuritic outgrowth, particularly from peripheral tissue, assayed (as reviewed by Edgar, 1985; Levi-Montalcini, 1987). Other related molecules of the NGF gene family have since been discovered: Brain-derived neurotrophic factor (BDNF) (Lindsay et al., 1985; Davies et al., 1986b), and the neurotrophins (NT-3, NT-4, and NT-5) (Maisonpierre et al., 1990; Berkemeier et al., 1991). The actions of these factors are mediated by structurally distinct receptors, the low-affinity p75NGFR and the high-affinity receptors p140trkA(both binding NGF), p14StrkB(binding BDNF, NT-4, and NT-5), and ~145"~'(which binds NT-3) (see Meakin and Shooter, 1992). Other trophic factors, primarily peptides, which can affect neuritic outgrowth as well as neuronal survival, are also considered briefly here, including ciliary neurotrophic factor

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(CNTF) (Barbin et al., 1984;Manthorpe er al., 1989),and fibroblast growth factors (FGF) (Walicke, 1989).The extent to which these different trophic agents interact and regulate glial cells and each other in the vital specialized functions of different parts of the nervous system is of current interest in neuronal plasticity and an area in which in uitro methodology should help in furnishing explanations.

1. NGF, BDNF, and the Neurotrophms In addition to being a prerequisite for neuronal survival, a role for NGF in initiating neurite elongation in embryonic chick sympathetic and sensory neurons was demonstrated in early investigations (Levi-Montalcini, 1987). This dependence on the trophic factor may be inversely related to the levels of intracellular calcium (Eichler et al., 1992). Mammalian sensory and sympathetic neurons are also responsive during development, as are central cholinergic neurons (Barde, 1989), although rat septa1 neurons studied in very low-density microcultures may survive without NGF (Nonner et al., 1992). NGF, either alone or with glutamate or aspartate, promoted neurite elaboration in cultures of Purkinje cells prepared from rat (E18) cerebella (Cohen-Cory et al.. 1991). Cochleo-vestibular neurons maintained in a medium conditioned by the developing otic vesicle were stimulated to extend neurites; this effect was destroyed if the medium was blocked with anti-NGF (Lefebvre et al., 1990). Neurites from chick (E7 and E12) DRG cultures were shown to undergo rapid reorientation and fast turning toward elevated NGF concentrations if the NGF was applied in local concentration gradients from micropipettes (Gundersen and Barrett, 1980). Such target guidance was considered consistent with an increase in intracellular levels of CAMPand calcium since in parallel experiments, neurites would also grow toward these sources (Gundersen and Barrett, 1980). Antisera to fibronectin or laminin blocked neuronal survival and neuritogenesis from E8-ElO chick DRG neurons even if NGF was present, and similarly, despite the presence of FN and LN substrates, treatments with anti-NGF prevented outgrowth (Millaruelo et al., 1988). Such findings are indicative of a cooperative action between the glycoproteins and the trophic factor in controlling neurite elongation. Using PC 12 cells, Keshmirian and co-workers (1989) found that if cells were on two-dimensional substrata of LN or a basement membrane extract (which included LN, collagen, and heparan sulfate proteoglycan), they responded to NGF by extending neurites; whereas if the same substrata were three-dimensional gels, the neural cells aggregated in response to NGF rather than producing neurites. PC12 cells have also been used to demonstrate increased sodium

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channel expression associated with NGF-induced neurite outgrowth, although this is not the only regulatory mechanism since basic fibroblast growth factor (bFGF) caused a larger sodium current density but less neurite outgrowth (Pollock et al., 1990). With adult rat DRG neurons, 50 ng/ml NGF did not enhance the onset of neurite formation on LN substrata, but did on PORN coatings (Grothe and Unsicker, 1987), although other studies using low-density and single neuronal culture systems led to the conclusion that NGF (and also BDNF) could stimulate axonal regeneration (Lindsay, 1988), and that it particularly promoted neurite arborization or axonal sprouting (Yasuda et af., 1990). These studies (Grothe and Unsicker, 1987; Lindsay, 1988; Yasuda et al., 1990) and those of others where anti-NGF controls were employed (Aguayo and White, 1992; Bedi et al., 1992) all suggested that NGF was not required as a survival factor for the vast majority of adult neurons, although it has to be said that in these particular culture systems, large numbers of cells may be lost in the isolation procedures. A significant role for NGF in controlling both morphological and functional phenotypes continues to be stressed (Lindsay, 1992). Other recent ir: uitro studies have indicated that for sympathetic neurons (Ruit et al., 1990),and for some DRG neuronal subgroups at least, not only in cultures prepared from young adults but also in those from aged rodents, NGF retains a role as a survival factor as well as affecting neurite regeneration (Fukuda et al., 1991; Jiang and Smith, 1992; Manfridi et al., 1992). Our current work is in agreement with these findings, although the effect is reduced for neurons dissociated from 2-year old CBA mice compared with 6-month-old adults (Jiang and Smith, 1993a). Neurite outgrowth was increased by addition of 50-100 ng/ml exogenous NGF to our enriched and nonneuronal, cell-reduced cultures for both adult and aged neurons (Fig. 4a-d), with elongation, rather than arborization, being more obviously enhanced for processes from aged animals (Fig. 5A,B). The complexities of probable synergistic and combined actions of a number of different factors may underlie some of the variation in the conflicting results and interpretations from different laboratories, and as with the contrasting in uiuo reports (Rich et al., 1987; Diamond et al., 1992), further experimentation is needed to clarify the situation with respect to neuritogenesis. Novel molecular manipulation of neural cells may assist in fully untangling this important question of neuronal responsiveness to NGF. This is demonstrated by the use of immortalized cells in correlating apoptotic loss and attenuation by NGF in reacting with the low-affinity NGF receptor, ~ 7 . (Rabizadeh 5 ~ ~ et ~al.,~1993). Such findings may have relevance to plasticity in giving a clearer understanding of certain neurodegenerative disorders.

FIG. 4 (a) Phase-contrast micrograph of adult DRG neurons maintained in non-neuronal cell-reduced cultures without addition of exogenous NGF for 7 days in uitro. (b) Adult neurons supplemented with 100 nglml NGF. Neurite fasciculation is extensive by 7 div. (c) Aged neurons supplemented in the absence of exogenous NGF for 7 div. (d) Enhancement of neurite outgrowth from aged neurons compared with (c), although not as extensive as for adult neurons. Bar on (a) = 25 pm; b-d at same magnification.

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A

- NGF

+ NGF

FIG. 5 (A) Camera lucida drawings of typical neurite extension from adult (6 months) DRG neurons maintained for I , 3,6, and 9 days in culture without exogenous NGF (-NGF), or with 100 ng/ml NGF added to culture from onset ( + NGF). Notice the increase in total neurite length and fasciculation in + NGF cultures. (B) Typical neurite extension from aged (2 years) DRG neurons maintained as for (A). Again the addition of NGF ( + NGF) has an enhancing effect, but this is less pronounced than for adult neurons.

BDNF, which belongs to the same family of proteins as NGF because of the large number of identical amino acids, was shown to support survival and neurite outgrowth from neurons of peripheral sensory ganglia and those derived from ectodermal placodes, including neurons nonresponsive to NGF (Davies et al., 1986a), but had little effect on sympathetic or parasympathetic cells (Lindsay et al., 1985; Davies et al., 1986b). As its name suggests, BDNF is predominantly, though not exclusively, synthesized in the CNS target fields, with high levels in the hippocampus (Hofer et al., 1990). Recent evidence is accumulating that BDNF is required for the survival of certain central cholinergic and dopaminergic neurons (Hyman et al., 1991; Knusel et al., 1991). NGF and BDNF have additive effects on

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embryonic neurons, with substantially more neurons responding when both trophic factors were administered together than when either was applied separately, indicating that different classes of neurons may have different requirements for support (Lindsay et al., 1985). Such hypotheses have been substantiated from in uiuo studies where anti-NGF injections in utero caused a loss of smaller neurons projecting to laminae I and 11, while larger neurons projecting to more ventral spinal laminae were NGFindependent and probably require BDNF or one of the NTs for support (Ruit et al., 1992). In uitro findings demonstrate that BDNF continues to play a role in neuritogenesis by adult neurons (Lindsay, 1988). The third member of the NGF family, NT-3, was identified by molecular cloning techniques (Maisonpierre et al., 1990), and was shown to share about 50% amino acid sequence identity with BDNF. NT-3 has been shown to have a widespread distribution and may influence neuronal function in adult as well as developing nervous tissue. Neurite outgrowth was promoted not only from chick sympathetic ganglionic neurons (i.e., similar to NGF), but also from nodose ganglionic neurons (similar to the effect of BDNF), in addition to DRG neurons. This suggests that this neurotrophic factor has a broader specificity than the other two factors (Maisonpierre et al., 1990), although ciliary ganglionic neurons were nonresponsive. Neurotrophin-4 (NT-4) stimulated neurite outgrowth of cultured embryonic chick sensory, but not sympathetic, neurons and has a somewhat restricted occurrence, being reported only in Xenopus and the snake, Vipera lebetina (Hallbook et al., 1991). NT-4 therefore has a response similar to BDNF, with which it has structural similarities. Bioassays of chick (E10) neurons with the latest neurotrophin to be identified, NT-5, demonstrated a survival effect and neurite outgrowth by 70% DRG neurons and 30% sympathetic ganglionic neurons, whereas NT5 was not effective with parasympathetic or ciliary ganglionic neurons, or with spinal motor neurons (Berkemeier et al., 1991). Clearly, the full range of neurotrophins and their precise role in plasticity of the nervous system, both in development and in differentiated tissue, with respect to neurite elongation will continue to interest neuroscientists for the rest of this century. 2. CNTF

CNTF, an acidic protein, represents a class of neurotrophic factors which was originally shown to be capable of supporting in vitro survival and growth of chick ciliary ganglion neurons (for review, see Manthorpe et al., 1989, 1993). By using single-cell cultures Unsicker and co-workers (1992) have shown that CNTF (10 ng/ml) can have a direct effect on chick

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ciliary ganglion neurons and need not be mediated through Schwann cells or astrocytes which proliferate in response to bFGF. However, the findings do not rule out the possibility of more complex mediation in vivo. Peripheral nerve proved to be a rich source of CNTF, with less in the central nervous system, although levels increase following injury. The full extent of CNTF’s bioactivity is clearly much broader however, and includes survival enhancement of embryonic rat sympathetic (Saadat et al., 1989) and spinal cord (Magal et al., 1991) neurons. With rat embryonic hippocampal neurons, CNTF-treated cultures (maximal effect at 0.1 ng/ ml) had approximately twofold higher numbers than untreated controls, and, by measuring neurofilament protein levels as an index of neurite outgrowth, an extensive increase in neuritogenesis could be observed, particularly between 6 and 8 days in vitro (Ip et al., 1991).

3. Fibroblast Growth Factors Two fibroblast growth factors, one basic (bFGF) and the other an acidic (aFGF) peptide, are known which support the survival of many diverse types of neurons (Walicke, 1989) of both central and peripheral origins (Morrison et al., 1986; Janet et al., 1988). bFGF promoted the survival and differentiation of cultured fetal rat hippocampal (Walicke et al., 1986) and cerebellar granular (Hatten et al., 1988) neurons. Postnatal (d15) neurons from several areas of rat brain (septum, striatum, midbrain, hippocampus) survived significantly better than control cultures when 0.1-1 ng/ ml bFGF was added and exhibited a process-bearingmorphology similar to that achieved with 10-100 ng/ml NGF, indicating that bFGF can act as a neurotrophic factor for postnatal central neurons (Matsuda et al., 1990). Only cerebellar neurons in these postnatal cultures failed to respond to bFGF, although as with the findings of Hatten and co-workers, embryonic cerebellar neurons were responsive (Matsuda et al., 1990). As with CNTF, bFGF can act directly on ciliary ganglion neurons maintained in single culture systems (Unsicker et al., 1992). The distribution of both acidic and basic FGFs in cultured (E15) DRG neurons and in Schwann cells has recently been monitored as a function of time over a 30-day period in vitro (Neuberger and De Vries, 1993a). Initially the neuronal perikarya, but not neurite cytoplasm, were immunoreactive for both FGFs, while at 20 days in vitro, positive intracellular labeling was also seen in neurites, and by 30 div, colocalization of both a- and bFGF was also detected on the surface of neurites. This surface appearance is of interest because it could reflect a role in modulating neuron-glial cell interactions and hence be important in plasticity adaptations (Neuberger and De Vries, 1993a). In a subsequent study, these workers induced neuritic degeneration by compressing the processes at 20 and 30 div and monitored redistributions

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of the FGFs (Neuberger and De Vries, 1993b). They found in this case that after injury, neurite membrane vesiculations containing FGF at 30 div, occurred extensively distal to the crush. FGF was also found on the neuronal perikarya following injury, presumably the result of an upregulated synthesis, and also in increased amounts along the neurite. Such increases may act in signaling glial cells to proliferate and initiate myelin formation, which may have consequences for neuronal survival and axonal elongation (Neubergerand De Vries, 1993b).If this proves to be the case in viuo, such studies represent a significant and exciting advance in elucidating the role of trophic factors in neuronal signaling that has consequences for plasticity after traumatic events. 4. Transforming Growth Factors and Epidermal Growth Factor Evidence is accumulating that peptide factors of the transforming growth factor-p (TGF-P) and epidermal growth factor (EGF) families also have an essential role in neuronal systems. TGF-P2 and 4 3 have been localized in uiuo in large, central, multipolar neurons in the spinal cord, brain stem motor nuclei, hypothalamus, amygdaloid complex, hippocampus, and cerebral cortex, and peripherally in DRG neurons of adult rats (Unsicker et al., 1991). Using dissociated cultures of olfactory epithelium, recent work has demonstrated that TGF-p2 is the probable neurogenic factor that controls neurogenesis in this site of continual neuronal production, although neither TGF-p1 or 2, nor EGF were able to maintain long-term survival in vitro (Mahanthappa and Schwarting, 1993). TGF-pl , the best-characterized TGF-@isoform, has not been localized in viuo in developing or adult mammalian nervous tissue (Millan et al., 19911, but it is tentatively identified as the isoform that is synthesized and secreted by neurons after peripheral nerve injury, and it may modulate neuronal-glial cell signaling(Rogister et al., 1993).Schwann cell proliferation was activated, their behavior modulated, and an environment favorable for peripheral regeneration generated. A similar response to injury also occurred in other neuronal populations (Lefebvre et al., 1991) and warrants further study. Unsicker and colleagues (1991) report that TGF/3l mRNA and protein can be induced in FC12 cells by NGF, while in cultured DRG neurons, NGF induces TGF-p3 and suggests functional relationships among these different peptide growth factors. Recent reports also indicate that TGF-a promoted neurite outgrowth in PC12 cells and survival of embryonic brain neurons (Zhang et al., 1W), and stimulated dopamine uptake by fetal rat ventral mesencephalic neurons (Alexi and Hefti, 1993). Ten nanograms per milliliter of TGF-(Y has an optimal effect on the survival and neuritic outgrowth of neonatal

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rat sensory neurons maintained for up to 2 weeks in uitro, whereas EGF, which has structural homologies with TGF-a, within a treatment range of 0.1-500 ng/ml did not (Chalazonitis et al., 1992). Ten nanograms per milliliter of EGF had previously been shown to promote survival and neuritogenesis of cerebellar neurons, although concentrations as low as 100 pg/ml were also effective (Momson et al., 19881, and although EGF did not affect general neuronal survival of cultured rat septa1 neurons, a neuromodulatory role for cholinergic expression was observed (Kenigsberg et al., 1992). EGFalso affects neurogenesis in avian species (Erickson and Turley, 1987). 5. Other Neurotrophic Factors

The list of trophic factors which have limited but important effects on the survival and differentiation of specific groups of neurons during development and in the mature animal continues to expand rapidly and highlights the enormous task involved in deciphering the full intricacies of neuronal plasticity, particularly its role in axonal elongation and guidance. Insulin and insulin-like growth factors I and I1 have been shown to promote neuritogenesis in cultured embryonic sensory and sympathetic neurons (Recio-Pintoet al., 1986), and can also directly modulate the regeneration of adult sensory neurons in culture (Fernyhough et al., 1993). Classic neurotrophic actions have been reported for a soluble neurite-promoting factor (SN) which is produced in response to sciatic nerve transection (Windebank and Blexrud, 1989). Acetyl-L-carnitine (ALCAR), an acetylated derivative of carnitine, has been shown to have a substantial neuroprotective influence on aged (2 years) rat sensory neurons in culture, although it had little effect on axonal elongation (Manfridi et al., 1992). Interest has also focused on the effects of retinoic acid (RA), a naturally occurring metabolite of vitamin A, since this produced neurite outgrowth from human neuroblastoma cells and chick DRG neurons (Haskell et al., 1987), amphibian spinal cord (Hunter et al., 1991), and neonatal mouse DRG and human spinal cord explants (Quinn and De Boni, 1991). We have recently shown similar responses to 1-100 p M retinoic acid by adult mouse sensory neurons in dispersed cultures (Chong et al., 1994) (Fig. 3c), with the rate of elongation being affected and growth cone morphology modified (Fig. 3d). Haskell and co-workers (1987) reported that one of the effects of RA was to increase the number of both high- and low-affinity nerve growth factor receptors in LA-N-I neuroblastoma cells and also in modifying the cell surface glycoproteins which act as surface receptors for EGF and insulin. Midkine, a retinoic acid-responsive gene product and member of a new family of heparin-binding growth factors, caused a 2-fold increase in neurite extension in spinal cord neurons after 7 days in

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culture and in similarly promoted DRG neuritogenesis 1.7-fold in 13-day fetal rat neurons, although it was ineffective in neonatal DRG neurons (Michikawa et al., 1993). Clearly, retinoic acid and its action are likely to continue to stimulate interest in further neuronal plasticity studies. The complexity of the interplay of different neurotrophic factors, with the potential to modulate regenerative events, including neurite outgrowth, in both embryonic and adult systems, continues to unfold, and its significance is elucidated by in uitro studies. Clearly, our understanding of the role of neurotrophic factors has come a long way since the pioneering days when only NGF was known to be involved. 6. Neurite Inhibitors

Much attention has been given to factors that stimulate neurite outgrowth. However, factors that can inhibit neuritogenesis and which might have an equal function in the negative regulation of neuronal plasticity should also be addressed to fully appreciate the potential interactions leading to neuronal plasticity (Schwab et al., 1993). In an elegant series of experiments, Kapfhammer and Raper (1987)showed that compatability between growth cones and neurites varied according to the source of the embryonic chick neural tissue explants. Inhibition, in the form of retraction of growth cones away from neurites, occurred as a rule for combinations of PNS and CNS neurons but not for homonymous pairings (for example, sympathetic growth cones retracted from diencephalic, but not DRG, neurites), which is indicative of inhibitory neuronal surface molecules (Kapfhammer and Raper, 1987). In the mammalian nervous system, specific neurite growth inhibitors, IN-35 and IN-250, have been identified in rat CNS white matter, but not in CNS gray matter or peripheral nerve (Caroni and Schwab, 1988; Savio and Schwab, 1989). IN-35 and IN-250 prevented cell adhesion and axonal elongation: localized effects causing growth cone arrest and collapse. Further studies by Brandtlow and colleagues (1990) revealed that the growth cones of postnatal (d6-12) DRG neurons responded to different CNS glial cell types in culture: growth velocity was maintained when astrocytes were encountered but was dramatically arrested when oligodendrocytes were contacted. This contact inhibition could be prevented by treatment with the antibody IN-1 directed against IN-35 and IN-250 (Brandtlow et al., 1990). Recent work by Bedi and cclleagues (1992) failed to demonstrate axonal outgrowth by adult DRG neurons on adult nonlesioned peripheral nerve sections, whereas neurite outgrowth did occur if prelesioned or predegenerated sciatic nerve sections were prepared, suggesting that myelin components may indeed inhibit axonal elongation in the normal adult. The negative trophic factors therefore would

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not seem to be acting either in isolation of positive factors or of a downregulation of the inhibitory proteins in peripheral nerve injury mechanisms and plasticity, and more work is needed to fully determine the cellular mechanisms involved. Neurotransmitters, with their vital role in neural communication in mature nervous system functioning, represent a further set of environmental cues which can inhibit neuritic outgrowth, thereby regulating neuronal development, maintenance, and plasticity (Mattson, 1988; Lipton and Kater, 1989). Responses to neurotransmitters were first demonstrated in studies of the pond snail Helisoma where serotonin was shown to act as a potent inhibitor of regenerative neurite extension by specific identified neurons (Haydon et al., 1984).Further studies showed that dopamine also selectively inhibited neurite elongation, and at least one identified neuron responded to both transmitters (McCobb et al., 1988). Mattson and coworkers ( 1988a) showed that the excitatory neurotransmitter, glutamate, had a differential effect on dendritic growth cones of cultured embryonic hippocampal pyramidal neurons at low concentrations (<10 p M ) while the axonal process continued to grow. At high levels (
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substrate (Neely, 1993). Lipton and Kater (1989) point out that, teleologically, the incorporation of fundamental molecules (such as neurotransmitters and substrate) and/or electrical activity as mechanisms for creating and stabilizing connections in the nervous system, which provides further ways of reshaping and eliminating superthous and outdated connections, produces an attractive model for further elucidation of neuronal plasticity.

C. Growth Cones The “decisions” concerning neurite extension or retraction are ultimately regulated by the structure and function of the growth cones, which are specialized distal endings of the extending neurites, and it is these, therefore, which play a fundamental role in effecting neuronal modulation and plasticity. They function by exploring their local environment and responding to external cues and thus direct and guide neurites to establish correct connections both during development and in response to injury. Much work has been carried out on growth cones, and the most salient findings have recently been comprehensively surveyed (Letourneau et al., 1991), updating the previous definitive treatise (Kater and Letourneau, 198% so that only a brief review is given here. There is great variety in growth cone morphology, depending on species differences and on their interactions and adhesiveness with the extracellular environment, for example, the presence of neurotrophic factors (Fig. 3d) and substrate effects (Fig. 3e), which can effect rapid changes in gross form (Bray, 1987; Gundersen, 1987; Kapfhammer and Raper, 1987; Rivas et al., 1992; Neely, 1993). The dynamic nature of growth cones makes their analysis potentially difficult, but since in uitro studies have permitted visualization of living as well as fixed material, investigations using video and digital processing and imaging techniques (Goldberg and Burmeister, 1989; Parpura et al., 1993), modem fluorescent dye techniques (Dailey and Bridgman, 1989; Davenport et al., 1993), and electron microscopy (Lankford and Letourneau, 1991) have yielded much valuable data. In general, growth cones consist of a central cytoplasmic domain rich in organelles, particularly endoplasmic reticulum (Dailey and Bridgman, 1989). This is surrounded by a peripheral cortical domain, veiled by a broad lamellipodium and numerous filopodia, which are abundantly rich in actin filaments (Goldberg and Burmeister, 1989). Filopodia (microspikes) are known to undergo cycles of mobility: contractile activity and the flow of actin filaments produced at the leading edge move laterally, resulting in the growth cone moving forward (Bray and Chapman, 1985). Experiments with isolated filopodia have recently demonstrated that filopodia have a sensory role in growth cones, as well as a motor function,

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since they contain signal transduction mechanisms that allow an autonomous response to environmental cues, and hence act as a fundamental unit of organization in neuronal pathfinding (Davenport et al., 1993). Growth cones have indeed been shown to be involved in neuritic branching, with inhibition of branching being correlated to changes in filopodialorientation. These latter are the result of changes in the number and diameter of microtubules, as can be demonstrated by treatments with drugs, such as taxol, which interfere with microtubule assembly (Letourneau et al., 1986). Microtubule-associated proteins (MAP) such as MAP lB, and in particular the phosphorylated isoform, have been shown to be present in growth cones of embryonic rat cerebral cortex neurons and may play an important role in growth cone microtubule assembly and neurite elongation (Mansfield et al., 1992). The stability of the growth cone cytoskeleton, particularly the actin filaments, may be regulated by calcium, which has a significant role in controlling growth cone behavior. In a series of experiments with cultured chick DRG neurons, Lankford and Letourneau (1989) showed that intracellular calcium increases induced by addition of the ionophore A23 187 caused neurite retraction within 10min, with the growth cone areadecreasing by 62% of the control value. This effect could be blocked or reversed by the actin-stabilizing drug, phalloidin (Lankford and Letourneau, 1989). In a subsequent detailed study, they showed that alterations in intracellular calcium concentration levels, measured by fura 2-AM dye-loading, as low as 50 nM were sufficient to cause detectable changes in growth cones, while the use of actin-filament disrupting agents, cytochalasin B or D, resulted in growth cone collapse indistinguishable from that produced by increasing intracellular calcium (Lankford and Letourneau, 1991). The involvement of intracellular calcium levels in controlling growth cone activity has been well appreciated by many workers (see Kater and Mills, 1991). Focal application of serotonin to specific growth cones of identified Helisoma neurons was shown to cause only localized retraction of the particular growth cone involved, while others on the same neuron retained normal morphology (Haydon et al., 1985). With the buccal ganglion neuron, B19, treatment with 1 p M dopamine for 20 min also caused retraction of the growth cone's thin membranous lamellipodium and filopodia (McCobb et a / . , 1988). Dopamine also regulated chick retinal neurons by inhibiting growth cone motility (Lankford et al., 1988). Other studies using the calcium indicator fura-2 demonstrated that serotonin and also the generation of action potentials caused changes in intracellular calcium levels that inhibited growth cone mobility (Cohan et al., 1987). Mattson and Kater (1987) subsequently used multivalent cation blockers and obtained evidence that plasma membrane Ca2+channels are the primary sites at which serotonin acts to induce calcium influx, and that

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growth cone motility was optimal in a narrow range of intracellular calcium concentrations although lower concentrations can still permit neurite elongation. Results from recent studies using young adult rat DRG neurons and varying cytosolic free calcium concentrations in the range 0-60 nM are consistent with the conclusions drawn from the snail work: neurite outgrowth was maximal at 35 nM free intracellular calcium and reduced at higher or lower levels (Al-Mohanna et al., 1992). As was seen in the study of Helisoma neurons by Cohan and co-workers (1987), electrical activity can also act as an arrest signal for growth cones. More refined techniques using focally applied electric fields now permit detailed study of local filopodial changes in Helisoma neurons which can be linked to increases in intracellular calcium (Davenportand Kater, 1992). A striking orientation of filopodia toward the cathode source was evoked within minutes of applying sustained or brief electric fields in this elegant study (Figs. 6A-6B), with a significant increase in filopodial number following a sustained electric field. Interestingly, Davenport and McCaig (1993) have recently shown in fetal rat (d18) hippocampal neurons that only growth cones on processes characteristic of dendrites oriented to focally applied electric fields, while axons showed no such response to the stimulus. Electrical activity can selectively raise calcium levels in the growth cone, leaving neurite levels low due to channel clustering of calcium “hotspots” in both proximal and distal regions of growth cones and thus modulate behavior; this was shown by patch clamp experiments on N1E-115, neuroblastoma cells (Silver et al., 1990). Other second-messenger mechanisms exist which regulate growth cone behavior in addition to levels of intracellular calcium (Mattson et al., 1988b). In an early study of chick embryo DRG neurites, the turning response toward sources of NGF was linked to elevated cAMP andlor calcium levels in growth cones (Gundersen and Barrett, 1980). Other workers have implicated a direct activation of protein kinase C in stimulating GAP-43phosphorylation in intact growth cones isolated from rat septa1 and hippocampal neurons maintained in low NGF concentrations (Meiri and Burdick, 1991). Lankford and Letourneau (1991) investigated the effects of cAMP elevation and protein kinase C activation on neurite retraction, growth cone structure, and intracellular levels of calcium in their studies of chick DRG neurons. They concluded that the effects of cAMP manipulations, using either forskolin or dB (dibutyryl) CAMP, could be explained, partially at least, by a lowering of calcium levels and a concomitant increased stabilization of actin filaments. Protein kinase C stimulation, produced by the addition of 1 pM phorbol lf-myristate-13acetate, caused neurite retraction but altered neither growth cone shape or motility, with no loss of actin filaments, nor calcium concentrations (Lankford and Letourneau, 1991). Studies of hippocampal neurons also

FIG. 6 (A, A’) Effect of a sustained electric field on Helisoma growth cones. (A) Control phenotype 1 min prior to stimulation. (A’) Taken after an 1 I-min application of a sustained electric field of 1.8 mV/pm from a pipette tip on the right-hand side. Note filopodial elongation and increased number on the cathode side. Bar = 20 pm. (B, B’) Effect of brief electric field upon similar growth cone. (B) Control morphology 1 min before stimulation. (B‘) Recorded 10 min later, following a 5-min pulse of field strength of 4.6 mV/pm from a pipette on the right and a subsequent lag of 4 min post-stimulation. Again, filopodial elongation is obvious. Bar = 20 pm. (Reproduced with permission from Davenport and Kater, 1992.)

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implicate protein kinase C systems in inhibiting, and CAMPin stimulating, neurite growth (Mattson et al., 1988b). In uiuo it is almost certain that growth cones integrate information from many different cues simultaneously, such as from neurotransmitters (Lipton and Kater, 1989) as discussed earlier. In addition, their interaction with the substratum is considered paramount in successful neurotropic functioning(Letourneau, 1975; Gundersen and Barrett, 1980; Sanes, 1989). Factors that promote substratum-bound growth are widely thought to act by providing an adhesion force against which the growth cone protrusions can pull (Bray, 1987). The involvement of receptor molecules for fibronectin and laminin has been reported for DRG neuronal growth cones (Bozyczko and Horwitz, 1986). In embryonic mouse cerebral cortex neurons, a surface glycoprotein has been identified-edge-membrane antigen (EMA)-which is prominent on growth cones and may mediate their interaction with external substratum cues (Baumrind et al., 1992). Recent studies in which 25 pg/ml laminin was applied to growth cones of neonatal rat sympathetic neurons grown in the absence of laminin resulted in changes within 12 min, with the lamellipodium becoming engorged with membranous organelles, vesicles, microtubules, and mitochondria from the central domain, and filopodia lengthening considerably (Rivas et al., 1992). Such observations are similar to earlier studies which demonstrated that growth cones preferentially interacted with laminin substrates compared with adjacent, less conducive substrates (Gundersen, 1987; Hammarback et al., 1988; Clark et al., 1993). Certainly substrate can affect growth cone morphology of leech neurons, with small growth cones on ECM substrates and broad, flat growth cones on concanavalin A surfaces (Neely, 1993). The pioneering aspect of growth cones ensures their prime position in mechanisms of neuronal modulation and plasticity, and especially in the formation of potential synapticjunctions during development and regeneration. The appearance in growth cones of specific integral proteins such as synaptophysin in neurotransmitter-containing vesicles prior to synaptogenesis suggests that growth cones may indeed act in establishing functional connections (Phelan and Gordon-Weeks, 1992). This and other aspects of growth cone structure and behavior will be areas where much work will be focused in the years ahead, and which should help to clarify many fundamental questions concerning plasticity. D. Growth-Associated Proteins

By and large, neurite initiation and elongation during normal development in uiuo are restricted temporally to a brief developmental window, or else become reexpressed as a response to neuronal injury (Skene, 1989). During

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263 these processes, growth-related proteins (GRPs) are synthesized at increased rates and are particularly abundant in growth cones. Once synaptogenesis begins, however, their levels start to decline. As such, there has been growing interest in GRPs, giving rise to many proposals for their role in neuronal plasticity and modulation since some are also prevalent in adult brain regions such as the hippocampus, and undergo phosphorylation during long-term potentiation at synapses (Benowitz and PerroneBizzozero, 1991; Pfenninger et al., 1992). The most researched GRP is the acidic membrane protein designated neuronal growth-associated protein-43 (GAP-43),also known by different research groups as B50, F1, pp46, y5, P-57, and neuromodulin (Coggins and Zwiers, 1991). GAP-43 immunoreactivity can be detected in adult DRG neurons in uitro within 2-3 hr after axotomy, although in uiuo its appearance takes up to 3 days after injury (Woolf et al., 1990), while in autonomic nerves extensive expression occurs in uninjured adult rats in uiuo at levels comparable to those present during development (Stewart et al., 1992). By culturing fetal rat hippocampal cells, Goslin and coworkers (1988) showed that as neuronal maturation occurred and neuronal polarity was established, GAP-43 was found in axonal growth cones but was absent in dendrites, although ultrastructural studies of a small number of selected, isolated neurons failed to fully confirm this finding (Van Lookeran Campagne et al., 1992). Other studies of NGF-stimulated phosphorylation of GAP-43 in growth cones of cultured hippocampal neur,onssupport the proposal that the growth-related protein has a role in the onset of axonogenesis(Meiri and Burdick, 1991), and regulation of transmembrane calcium channel conductance by GAP-43 phosphorylation could well be significant in this respect (Norden et al., 1991). The precise part played by growth-related proteins in neurite initiation, elongation, and interaction with other factors in the developing nervous system and after injury remains to be fully elucidated but represents an intriguing current problem for those studying neuronal plasticity.

IV. Synaptic Connections between Cultured Neurons

Following sproutingand elongation, it is essential that growth cones participate in the formation of synaptic connections iffunctional circuitry is to be established in the developing nervous system, or if axons are to regenerate (although this is usually the least successful phase, particularly for central neurons). Synaptic plasticity plays a vital role throughout normal adult life also in producing the modifications necessary for learning, memory, and routine motor and sensory functioning. The molecular events concerned are thought to involve both presynaptic modifications (e.g., regula-

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tion of neurotransmitter release), and modulation of transmitter receptors at the postsynaptic membrane of chemical synapses (Bliss and Collingridge, 1993; Raymond et al., 1993). Signal transduction from the postsynaptic to presynaptic elements through the generation of retrograde secondary messengers, such as nitric oxide (Garthwaite, 1991), or arachidonic acid (Fazeli, 1992), occurs to coordinate changes at both sites. The difficulties encountered in using intact living animals to investigate the specific cellular and molecular basis of formation (synaptogenesis), maturation, and plasticity of the interactions among functionally linked neurons (and/ or glial cells) have necessitated the incorporation of in uitro methodologies in order to elucidate precisely the whole spectrum of morphological, physiological, and pharmacological mechanisms. The sheer multitude of neurons interconnected within the mature vertebrate nervous system, and the added level of complexity imposed by the formation of multiple synapses, highlights the attractions of studying dispersed vertebrate cell cultures since even with sophisticated brain slice preparations, which permit pharmacological recordings, it is still difficult to fully observe the individual functional connections involved (Gahwiler and Brown, 1985). Significant investigations of synaptic plasticity have also been made using the simplified nervous systems of invertebrates, for example, leeches (Chiquet and Nicholls, 1987) and molluscs (Hayden et al., 1984; Haydon, 1988; Lukowiak and Colebrook, 1988-1989). Data from both vertebrate and invertebrate work, therefore, are briefly reviewed. A. Vertebrate Neurons

The development of synapses between cells has been reported for a wide range of neuronal types from both avian and mammalian sources: neonatal rat nodose ganglia (Cooper, 1984), spinal cord-peripheral neurons of chicks (Choi et al., 1981) and fetal rodents (Bunge et al., 1974; KO et al., 1976; Jahr and Jessell, 1985; Nelson et af., 1989; Wang et al., 1990), chick retinal neurons (Gleason and Wilson, 1989), rat hippocampal neurons (Bartlett and Banker, 1984; Laiwand and Brown, 1992; Craig et al., 1993), striatum (Weiss et al., 1986; Dubinsky, 1989; Sebben et al., 1990), cerebellum (Hirano et al., 1986), and cerebral cortical neurons from chicks (Tokioka et af., 1993) and rats (Dichter, 1978; Huettner and Baughman, 1988; Harris and Rosenberg, 1993). Although far from exhaustive, this list illustrates the diversity of neuronal types which can be induced to form functional synapses in culture, and on which data can now be obtained with reference to factors affecting synaptogenesisand the mechanisms underlying plasticity and modulation of function. Neurons in culture can form synapses between cells which would nor-

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mally be functionally linked, for example, monosynaptic connections between cerebellar granule cells and Purkinje neurons (Hirano et d . , 1986), and between basal forebrain neurons and hippocampal cells (Laiwand and Brown, 1992). Dissociated cells in culture also possess plasticity, however, in displaying an inherent potential for synaptic connections which would not normally be formed in situ. Even these “abnormal” synapses can be useful in shedding light on the regulatory control of synaptogenesis. Cooper ( 1984) demonstrated that synapse formation could occur between neonatal rat nodose ganglion neurons; the ability to do so depended on whether satellite cells were present in the cultures. This led to the conclusion that glial cells have a role in regulating synaptogenesis in uiuo. Approximately 60% of neurons formed synapses in the absence of satellite cells, with acetylcholine (ACh) identified as the neurotransmitter and with nicotinic postsynaptic receptors, whereas those cocultured with satellite cells rarely formed functional synapses and were insensitive to ACh (Cooper, 1984). Early studies with fetal spinal cord explants and dissociated superior cervical ganglion neurons, in coculture for up to 10 weeks, showed the presence of nicotinic cholinergic synapses by spinal cord neurites on principal ganglionic neurons (KOet af., 1976). The synaptic potentials recorded were fast excitatory postsynaptic potentials (EPSPs) with no sign of slow responses. Some evidence of synapses between the sympathetic neurons themselves was also reported (KOet af., 1976). Choi and his colleagues (198 1) investigated inhibitory postsynaptic potentials (IPSPs) in their cocultures of chick spinal cord and DRG neurons. They recorded potentials which were sensitive to bicuculline, which is indicative of mediation by GABA, thus supporting the hypothesis that dorsal root fibers can receive GABAergic innervation in uiuo. In a later study, intracellular recordings were made to identify the excitatory sensory transmitter at synapses between fetal (day 15) rat DRG and dorsal horn neurons (Jahr and Jessell, 1985). Over 80% of the dorsal horn neurons within 1 mm of the DRG explants received at least one fast monosynaptic input which could be excited by L-glutamate, kainate, or quisqualate, whereas aspartate or N-methyl-D-aspartate (NMDA) had little effect on them. This gave good in uitro evidence that glutamate, but not aspartate, is the excitatory transmitter which routinely mediates EPSPs at DRG-dorsal horn synapses (Jahr and Jessell, 1985). Stimulation by glutamate was also demonstrated in excitatory synaptic transmission between motoneurons of chick spinal cord which developed after 4 days in uitro (O’Brien and Fishbach, 1986). Synaptic plasticity was seen to be modulated by electrical activity in an elegant series of experiments using fetal mouse neurons cultured in a three-compartment system (with DRG neurons in the side compartments and with ventral horn spinal cord neurons in the central chamber) (Nelson

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et al., 1989, 1990). DRG axons formed synapses on the spinal neurons within a week in culture, after which a phasic electrical stimulation was applied to the axons extending from one of the side compartments while the other remained unstimulated. Three to five days later, intracellular recordings were made of the spinal cord neurons to determine the number of functional inputs and amplitudes of the EPSPs. In the stimulated axons, the amplitude of EPSPs (synaptic efficacy) in the ventral horn neurons was significantly higher than in culture controls, or in the nonstimulated chamber, indicating that electrical activity induces competitive processes favoring activated rather than inactive pathways (Nelson et al., 1989). A more general increase in the number of axons forming functional connections also resulted from electrical stimulation, although there was little evidence for any actual reduction in synaptic contacts from the unstimulated side where efficacy had been reduced (possibly by a decreased synaptic bouton density). Postsynaptic as well as presynaptic activity was presumed to play a role in the mechanisms involved. Nelson and colleagues (19%) investigated these phenomena further by manipulating calcium levels, reducing postsynaptic activity by use of tetrodotoxin (TTX) and by blocking NMDA receptors. They concluded that changes in calcium levels, linked to influx through NMDA channels but also from other sources, were involved in the processes of activity-dependent synaptic plasticity, and may have a role in the mechanisms which regulate synapse elimination or augmentation caused by electrical activity. Such experimental design typifies the type of question in uitro studies can address in monitoring plasticity of the spinal cord. An understanding of synaptic organization within the striatum has also been enhanced by cell culture methodology, adding to the data obtained from standard neuroanatomical techniques. After 9 days in uitro, axodendritic contacts were observed in mouse striatal neurons maintained in serum-free medium to eliminate contamination from glial cells ( Weiss et al., 1986). On noncoated substrates. these contacts were, however, characterized by the absence of synaptic vesicles, whereas fully differentiated synapses, as revealed by transmission electron microscopy, were seen in cultures on precoated surfaces. Both symmetric and asymmetric synapses with the associated synaptic vesicles were observed by 12 days in uitro, and were shown to contain synapsin I, a protein which may function in the docking and fusion of vesicles to the release sites at the presynaptic membrane (Weiss et al., 1986). Such synaptic contacts remained even after 50 days in uifroin longer term striatal cultures where a low astrocyte density was permitted (Sebben et al., 1990). Striatal cultures from postnatal rat anterior caudate and putamen were used to demonstrate the development of not only inhibitory, GABAergic, synaptic interactions, but also provided the first report of glutamatergic fast excitatory activity

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within the striatum (Dubinsky, 1989).Electrophysiological recordings (of excitatory activity) and immunocytochemical demonstrations of glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, and of synapsin I were employed in this study-techniques which can be accurately monitored in uitro. Both excitatory and inhibitory simple and complex chemical synapses were reported in early studies of fetal rat cortical neurons (Dichter, 1978). Huettner and Baughman (1988)prepared primary cultures of postnatal rat visual cortex maintained in miniature island cultures of 50-100 cells which contained neurons that were synaptically connected, whereas in mass cultures (8000-10,000 cells), neurons exhibited abundant EPSPs and IPSPs, but very few were functionally connected. Pharmacological experiments showed that corticocollicular neurons in culture used an excitatory amino acid neurotransmitter, and that with physiological levels of magnesium the EPSPs were largely due to non-NMDA receptors, whereas in Mg2+-freemedium, most EPSPs were attributable to the NMDA receptor (Huettner and Baughman, 1988). Much attention has been given to hippocampal synaptic plasticity, and particularly long-term potentiation (LTP), which has been recently reviewed in depth (Bliss and Collingridge, 1993).considerations of neuronal modulation and changes of synaptic efficacy in hippocampal cells are most pertinent owing to the fundamental part the hippocampus plays in memory and learning circuitry. Much of the data on the mechanisms involved with LTP have come from use of in uitro hippocampal slice preparations. (Madison et al., 1991). Both NMDA and non-NMDA glutamate (AMPA, a-amino-3-hydroxyl-5-methyl-4-isoxazoleproprionic acid, and kainate) receptors and a resulting influx of calcium are involved during the enhancement of synaptic transmission which forms the basis of LTP. A critical role for protein kinases has been suggested; certainly LTP can be blocked by tyrosine kinase inhibitors (O'Dell et al., 1991),which strongly points toward protein phosphorylation of ionotropic glutamate subunits as having a role in the resulting synaptic changes. Molecular cloning techniques and in situ hybridization of several glutamate receptor subunits have recently been applied to rat hippocampal neurons, predominantly pyramidal cells, developing in culture (Craig et al., 1993). Hotspots of functional non-NMDA ionotropic glutamate receptor subunits were shown to be clustered at specific postsynaptic sites and localized on enriched dendritic spines; these and NMDA receptors developed after presynaptic specializations had developed, which suggested that presynaptic contact is a requisite for postsynaptic spine formation and retention (Craig et al., 1993). Conversely, the presynaptic cell specializations observed in hippocampal neuronal cultures when axonal contact has been established with the appropriate postsynaptic target cells

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could also be investigated (Fletcher et al., 1991). Focal redistribution and accumulation of synapsin and synaptophysin were evident in large clustered vesicles and used as a presynaptic marker in this study. Investigation and characterization of possible intercellular retrograde secondary messengers that functionally integrate and modulate pre- and postsynaptic neurons is another aspect of synaptic plasticity which has been studied in hippocampal cell cultures. Recent studies demonstrating stimulation of arachidonicacid release in a dose-dependent manner following activation of NMDA receptors suggest that it is an example of a likely second messenger candidate (Sanfeliu et al., 1990), and have added new insights to the rapidly expanding comprehension of synaptic plasticity. The mechanisms which direct the development of the synaptic interactions between vertebrate neurons are complex and the interplay of many factors which induce change or modification, for example, adhesion mechanisms (Schubert, 1991) or cytokines (Patterson and Nawa, 1993), are likely to be equally complex. Of interest in this respect is the finding that synaptic neurotransmission mediated by glutamate in cultured cerebellar granular cells can upregulate the levels of mBDNF, highlighting a possible overlapping of plasticity regulatory factors (Bessho et al., 1993). The continued use of cell cultures, linked with cloning techniques and receptor and signal transduction research, promises to yield further data which should eventually make it possible to determine precisely the mechanisms involved in synaptic plasticity. 6. Invertebrate Neurons

Even in invertebrate ganglia, the number of complex interactions makes it difficult to investigate the intricacies of individual connections in the whole organism. Many of the advances in elucidating the mechanisms involved in both electrical and chemical synapses of invertebrate neurons have therefore been made using cell culture systems. Synapses have been reported between neurites (Hadley et al., 1983), but also between neuronal somata in close apposition (Arechiga et al., 1986; Haydon, 1988). A particular attraction of large-sized invertebrate neurons is that they can be used to establish simple neuronal networks in culture for analysis of simple learning and behavioral circuitry that involves sensory and motor neurons (Bulloch and Syed, 1992). Rayport and Schacher (1986) studied neurons which mediated habituation and sensitization in the gill withdrawal reflex of Aplysia by reconstructing the elementary circuitry in culture. After 5 days, the mechanosensory neuron, LE, was monosynaptically connected to a motor cell, L7, with a facilitatory or modulatory neuron (MCC-metacerebral cell) forming synapses onto the presynaptic

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terminals of the sensory neuron. Short-term habituation of the reflex was mediated by homosynaptic depression, which caused a decrease in the transmitter substance, serotonin; whereas presynaptic facilitation caused increased calcium levels and an increase in transmitter release (Rayport and Schacher, 1986). Further studies of these sensorimotor connections showed that application of sertonin produced long-term facilitation and also caused structural changes, with an increase in sensory neuron varicosities in the presynaptic sensory cell (Glanzman et al., 1990). The inhibitory neuropeptide, FMRFamide (Phe-Met-Arg-Phe-amide), produced functional changes that were opposite and structural changes that were parallel to those caused by serotonin in the presynaptic cell if the proper target motor cell, L7, was present, highlighting in this system the integration of pre- with postsynaptic events (Schacher and Montarolo, 1991). Investigations to determine whether secondary messengers were involved in these plasticity events showed that raised CAMPenhanced the strength of the connections between sensory and motor neurons with concurrent modulation of neuritic arborization, while arachidonic acid decreased the synaptic efficacy and decreased the numbers of varicosities (Schacher et al., 1993). Schacher and his colleagues have therefore demonstrated, and continue to do so, many interesting features of synaptic plasticity in using this simple network of gill withdrawal circuitry. Other workers have pointed out, however, that the situation is undoubtedly complex, with learning probably dependent on a number of mechanisms that act at several sites to effect behavioral change (Lukowiak and Colebrook, 1988-1989),including modulation of transmitter release (Edmonds et al., 1990). Early studies with Helisoma demonstrated that isolated neurons extended neurites and formed synaptic contacts (Hadley et al., 1983). Synaptic plasticity was shown by the modulatory action on salivary gland cells, with an increase in synaptic efficacy occurring if B4 (buccal ganglion) motoneurons were depolarized by application of snail cardioactive peptide B (SCPB)(Coates and Bulloch, 1985). By culture manipulation which prevented neurite outgrowth, Haydon ( 1988) demonstrated that isolated buccal ganglia neurons could be induced to form chemical synapses directly between their somata, with an action potential in B5 evoking an inhibitory postsynaptic potential in B 19. The synaptic connection between the somata was shown to be Ca2+-dependentand cholinergic, indicative of the same neurotransmitter as in the synapses between neuritic contacts by these two cells (Haydon, 1988). Chemical synapses without excessive neurite outgrowth have also been observed between perikarya of Retzius and pressure-sensitive (P) neurons of leeches (Chiquet and Nicholls, 1987). The selectivity of synapse formation could be followed in this culture system since Retzius cells were

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always presynaptic in this pairing, forming an inhibitory serotoninergic synapse with the P cell. P cells, on the other hand, were presynaptic to anterior pagoda cells (Arechiga et al., 1986). Such studies, and particularly culture manipulation that causes the formation of different synaptic responses, were instrumental in illustrating synaptic plasticity in culture. Recent work with the cultured Helisorna buccal motoneuron, B19, which normally forms chemical synapses with supralateral radular tensor (SLT) muscle fibers, has shown that the target cells retrogradely regulate calcium influxes in the presynaptic sites of contact in B19 neurites by a mechanism mediated by CAMP-dependent protein kinase (Funte and Haydon, 1993). Such mechanistic approaches to synaptogenesisin invertebrate neurons in many ways parallel similar studies of mammalian synapse formation and plasticity. They reemphasize a type of experimental design offered by cell culture which is not possible in whole-animal studies, and which can greatly advance an understanding of complex functional processes.

V. Phenotypic Expression

One of the most fascinating areas related to neuronal plasticity and which underlies the great diversity in nervous tissues concerns the mechanisms which regulate the development of the neurotransmitter and neuropeptide phenotype in neurons, together with the factors which maintain and/or modulate this once it has been established, both in embryonic and in mature adult cells (Black ef al., 1984).In uitro methods have greatly aided the elucidation of the factors involved since the effects of, for example, growth factors (Lindsay, 1992), extracellular matrix (Gupta and Bigbee, 1992), cell-cell contacts (Dal Toso et al., 1988; Freidin et al., 1993), and target retrograde signaling (Landis, 1990)can all be monitored in a defined environment, which is not possible in the whole animal. Examples of transmitter and neuropeptide plasticity can be found from all regions of the nervous system. Peripheral neurons in which plasticity and modulation have been demonstrated, for example, include both sympathetic (Potter et al., 1986; Moms and Gibbins, 1989; Hart et al., 1991; Smith er al., 1993), and sensory (Lindsay and Harmar, 1989; Schoenen et al., 1989; Barakat-Walker et al., 1991; Jiang and Smith, 1993b) cells. Examples of central plasticity have been reported for spinal cord (Magal et al., 1991), pontine (Kniisel and Hefti, 1988), septa1 (Kenigsberg et al., 1992; Nonner et al., 1992), hippocampal (Ip et al., 1991), mesencephalic (Nagata et al., 1993; Alexi and Hefti, 19931, and cerebral cortex (Poulakos et al., 1993) neurons. The importance of these studies in determining the

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mechanisms involved and the regulation of activity during development and in mature and regenerating neurons will be considered. A. Developing and Neonatal Neurons

Early, detailed studies which highlighted neuronal phenotypic plasticity were carried out on rat neonatal sympathetic neurons dissociated from superior cervical ganglia (SCG) where, in the absence of nonneuronal cells, or if the neurons were chronically depolarized, a noradrenergic phenotype persisted (see Landis, 1990). This was similar to the pattern of maturation expected in uiuo where the nonneuronal cells form only a small proportion of the ganglionic population and where the noradrenergic phenotype typically predominates. In the presence of non-neuronal cells, or even with conditioned medium, which is indicative of diffusible factors, neurons could be induced to switch their differentiation pathway to a cholinergic phenotype (Patterson and Chun, 1974, 1977). In microculture systems, some neurons were even detected which demonstrated a dual status-synthesizing and releasing both noradrenaline and acetylcholine (Potter et al., 1986). Immunocytochemical determination of tyrosine hydroxylase (TH, an adrenergic enzyme), and choline acetyl transferase, ChAT (the biosynthetic enzyme for ACh), revealed cells capable of simultaneous dual expression in a considerable number of quail (E 10) paravertebral sympathetic neurons maintained in defined medium (Barbu et al., 1992). Recent work from the same laboratory, however, indicates that the differentiation of avian sympathetic neurons follows different pathways than in rodents: cholinergic phenotypes were present from the earliest stages of ganglionic development, and depolarization selectively enhanced a cholinergic phenotype, not the catecholaminergic phenotype that occurs with mammals (Smith et al., 1993). Growth factors, such as CNTF, induced ChAT in cultured rat sympathetic neurons, and hence regulated phenotypic plasticity with a S f o l d increase after 7 days in cultures treated with 1 ng/ml (Saadat et al., 1989). In addition to affecting the “classic” neurotransmitter phenotype of sympathetic neurons, external influences in cell culture systems were also seen to control the expression of several neuropeptides in the heterogeneous neuronal populations containing distinctive colocalization combinations (Morris and Gibbins, 1989). Neuropeptide Y (NPY), normally present in approximately 60% of SCG neurons, was found to be highly influenced by cell contact and density in culture: NPY expression decreased as the number of cells per culture was raised from 8000 to 12,000 (Freidin et al., 1993). Treatment with leukemia inhibitory factor (LIF) further reduced NPY expression (Freidin et al., 1993), whereas LIF and

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CNTF increased substance P (SP) synthesis in addition to upregulating ChAT activity and decreasing catecholaminergic markers (Kessler and Freidin, 1991). Interleukin-lp (IL-I@)caused an increase in SP also, its effects being at the level of transcription with an upregulation of precursor preprotachykinin (PPT) mRNA (Hart et al., 1991).The modulatory effects of different peptides, for example, SP and somatostatin (SOM) on SCG neurons, are known to act upon calcium channels via different G-protein pathways (Shapiro and Hille, 1993); this allows the complex phenotypic diversity to be amplified if there are plasticity changes caused by subtle local environmental changes. Thus, neural components may have a vast pharmacological diversity available for either developing autonomic ganglia or promoting recovery following neuronal injury. Neurons cultured from embryonic sensory ganglia can also be used to investigate regulation of phenotypic expression. One example of interest is the restricted localization of a subset of catecholaminergic neurons not found in most adult cranial ganglia and rarely in DRG, although these neurons have more widespread tyrosine hydoxylase activity transiently during development. In a study using cultures of nodose, petrosal, and dorsal root ganglia from rat embryos, Katz (1991) showed that this phenotype could persist in culture until at least the first postnatal week. NGF and cell aggregation may play a role in the regulation of TH cells, with a significant decline in high-density cultures; after E 16.5, TH activity was only detectable if neurons were maintained singly (Katz, 1991). Such cell density interactions may be significant for phenotypic regulation in uiuo. Similarly, control mechanisms involved in the development of SP-containing neurons in chick DRGs have been investigated (Barakat-Walker et al., 1991). In culture, as in uiuo, the majority of DRG neurons are positive for SP at E6, whereas by El0 the proportion falls to 60%. BarakatWalker and colleagues (1991) demonstrated that the proportion of E6 neurons immunoreactive for SP decreased following addition of extracts of El0 muscle, skin, brain, or spinal cord (i.e., peripheral and central targets) to these cultures from earlier stages of embryogenesis. Soluble factors from targets may therefore be responsible for regulating SP expression in these DRG cells, and in development may do so by acting on the transcriptional control of preprotachykinin (Barakat-Walker et al., 1991). This type of experimental design typifies how cell culture systems can begin to answer neuronal plasticity questions which would be extremely dimcult to monitor in uiuo. With rat (E15) DRG neurons, the influence of the substratum in modulation of AChE activity has been demonstrated by observing maintenance on LN, collagen, and Matrigel, a reconstituted basal lamina (Gupta and Bigbee, 1992). Vernadakis and her co-workers have employed cultures of chick CNS neurons prepared from early embryos, as young as 3 days old, to study

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the development of neurotransmitter phenotypes and their plasticity (Mangoura and Vernadakis, 1988; Mangoura et al., 1988, 1990). ChAT activity in neurons prepared from 3-, 6-, or 8-day-old embryos differed in expression. In cultures from the early embryos, it reached a high level after 7 days; in cultures from 6-day embryos, it increased up to 6 days in vitro and then declined; whereas for neurons from 8-day-old embryos, activity was low until 9 days in uitro and then rose after this time. One suggestion was that these differences reflected a loss of plasticity of the cholinergic population with time which might be related to increased cell-cell contacts (Mangoura et al., 1988). GAD activity was low in cultures from 3-dayold chicks but increased with time in uitro for cultures from both 6and 8-day embryos, indicating a pattern of activity for maturation of GABAergic neurons different than that of cholinergic neurons (Mangoura and Vernadakis, 1988). Further studies indicated that in cultures from 3day-old embryos, 10 ng/ml NGF and substratum effects, causing the presence of flat non-neuronal cells, prolonged survival of cholinergic and GABAergic neurons, with both markedly stimulating GABAergic expression, whereas only NGF enhanced cholinergic expression (Mangoura et al., 1990). More recent reports with this early chick embryo CNS system have considered the effect of other trophic factors and shown that acetylL-carnitine has a neuromodulatory role in phenotypic plasticity in that the neuronal balance was shifted to cholinergic neurons at the expense of those with a GABAergic phenotype (Kentroti et al., 1992). There are many examples of trophic factors triggering phenotypic plasticity and modulation in cultured mammalian fetal and neonatal central neurons, and a fuller understanding of the mechanisms involved in regulating these changes is now emerging. NGF has been clearly shown to affect the development and survival of cholinergic neurons in embryonic and adult basal forebrain, and septal and striatal neurons. At 100 ng/ml concentrations it caused severalfold elevations in ChAT activity in septal cultures (Hartikka and Hefti, 1988), although with very low-density (1-7 cells/ well) cultures there is evidence that at least some septal cells can develop cholinergic properties in the absence of NGF or other cells (Nonner et al., 1992). BDNF was shown to increase both survival and differentiation of rat septal cholinergic neurons (Alderson et al., 1990), with recombinant human BDNF exerting a more pronounced effect than NGF on cholinergic forebrain neurons in young cultures. Unlike NGF, however, which is effective when applied late in uitro, BDNFfailed to increase ChAT activity if it was added after 9 days in culture (Knusel et al., 1991). NGF failed to enhance ChAT activity in pontine cell cultures; neither 0.2-2 ng/ml CNTF nor 10-8M-10-5M retinoic acid supported cholinergic pontine or septal neurons. This type of negative finding suggests that neurotrophic

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factor activity may be highly specific in regulating phenotypic expression of distinct neuronal populations, and may require interaction with other signals to induce plasticity (Knusel and Hefti, 1988). With EGF applications to fetal rat septa1 neurons, a lag of 5 days in culture was necessary before significant decreases in ChAT activity or numbers of AChE neurons could be observed; this was attributed to the effect on cholinergic phenotypes being an indirect one mediated by the glial cell which proliferated under the influence of EGF (Kenigsberg et al., 1992). Ip and co-workers (1991) observed increases of 2-, 28-, and 3-fold for GABAergic, cholinergic, and calbindin-immunopositive cells respectively which were elicited directly in rat (E18) hippocampal neurons, plated on polyornithine-laminin substrata, and treated with 100 pg/ml CNTF; the presence of CNTF from the beginning of culture, also prevented a dramatic loss in GABAergic neurons by 8 days in culture. Interestingly, no effect was evident if addition of CNTF was delayed until day 3 of culture, which indicates plasticity in neuronal responsiveness (Ip et al., 1991). Cholinergic phenotypic expression was also seen to be regulated by CNTF in spinal cord motoneurons (Magal et al., 1991). Dal Toso and colleagues (1988) showed that cell density affected the development of dopaminergic and GABAergic neurons in cultures of rat (E13) mesencephalic cells in which non-neuronal cell proliferation was restricted by omitting serum in the medium. Extracts of a 16kDa neurotrophic protein (Lea,clearly distinct from NGF) were also purified from adult rat striatum (SDNF) and these were capable of increasing both dopamine and GABA uptake and attenuating survival of these mesencephalic neurons (Dal Toso et al., 1988). It is possible that SDNF may have been similar or identical to BDNF, since this neurotrophic factor has been clearly shown to enhance expression of dopaminergic neurons in the developing substantia nigra (Hyman et al., 1991; Knusel er al., 1991). bFGF was another possible candidate of similar molecular weight, although this agent’s action, as with EGF, was considered to be indirect, requiring the proliferation of glial components (Knusel et al., 1990),which was not the case for SDNF. A medium conditioned by microglial cells can stimulate TH activity and increase dopamine content of rat (E16) mesencephalic neurons, suggesting that action on these by some neurotrophic factors might influence local regulatory mechanisms for phenotypic expression (Nagata et al., 1993). Developing mesencephalic dopaminergic neurons were also shown to be responsive to administration of TGF-a which, if applied for 2 hr at the time of plating, caused increases in dopamine uptake after 5 days in uitro. With increasing culture age, the responsiveness of the neurons to TGFQ declined so that longer exposure was necessary and even so the response was weaker (Alexi and Hefti, 1993). Such results demonstrate that neu-

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ronal phenotypic plasticity is undoubtedly most complex, with temporal factors also being important in governing the magnitude of the changed expression and likely to be responsible for inducing subtle developmental differences in uiuo. In view of its importance as a mediator of the acute phase of reaction to infection and to endotoxins, the effects of interleukin-1 (IL-1) upon cultured rat El7 diencephalic neurons are of interest: treatments for 24 hours with IL-1 concentrations as low as lO-'OM stimulated somatostatin biosynthesis and release providing evidence that a neuroendocrine response may be significant following infection (Scarborough e f al., 1989). In uitro studies of plasticity and modulation of transmitter and neuropeptide phenotypes in developing neurons, as described, illustrate how the factors involved and the temporal aspects of regulation may be monitored using cell cultures. The complexity of the mechanisms underlying such plasticity and the diversity of neuronal functional modulation it generates are areas which will receive much attention in future developmental investigations. B. Adult Neurons

Examples of phenotypic plasticity are well documented in adult neurons and represent a field of growing interest in view of their relevance to nerve regeneration following traumatic and toxic insult. One of the best examples is seen in the various studies of adult DRG neuronal plasticity and modulation, as revealed by the pioneering work of Lindsay and his colleagues (for review, see Lindsay, 1992),and others (Schoenen et al., 1989; Delree et al., 1992). We have recently demonstrated that DRG neuronal phenotypic expression continues to be influenced by external factors, even in cultures prepared from aged animals (Jiang and Smith, 1993b). The heterogeneity of adult DRG neuronal phenotypes for neurotransmitters and neuropeptides, with colocalization in different subpopulations, has been well studied in uiuo (Price, 1985; Hokfelt e l al., 1987; Ju et al., 1987; Kai-Kai, 1989). Modulatory effects in adult rat DRG neurons were reported in uitro for NGF regulation of substance P and calcitonin-generelated peptide (CGRP): 50 ng/ml doses retained both neuropeptides for 18 days in culture at levels 10- to 15-fold higher than in cultures without exogenous NGF (Lindsay et al., 1989). The expression of the neuropeptides was still upregulated, even if NGF treatments were delayed for up to 10 days after cultures had been initiated. Northern blotting analysis showed that this upregulation by NGF acted at the level of transcription, with a 15-fold increase in the biosynthesis of the mRNAs encoding for preprotachykinin (the precursor of substance P) and for CGRP, in adult

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DRG neurons cultured for 5 days with 25 ng/ml NGF compared with controls without exogenous NGF (Lindsay and Harmar, 1989). Grothe and Unsicker (1987) had previously failed to find any significant differences in the proportions of SP, SOM,or cholecholecystokinin-8 (CCK) immunoreactive neurons in 4.5-day cultures of their preparations from adult rat DRGs supplemented with either 50 ng/ml NGF or 6.4 ng/ml CNTF when they were compared with controls without the neurotrophic factors; nor did the proportions differ from those estimated at 12 hr in culture. On PORN/LN surfaces, the proportionsof SP-positive cells actually fell after 7 days in culture, while on PORN alone, SP-containing neurons were maintained at control levels without the neurotrophins (Grothe and Unsicker, 1987). On the whole, however, this report is at variance with the findings of other groups where potentiation of neuropeptide expression has been observed (Lindsay, 1992; Schoenen et al., 1989; Delree et al., 1992; Jiang and Smith, 1993b). The study by Schoenen and co-workers (1989) was of great interest from a plasticity viewpoint. They undertook a detailed study of possible phenotypic plasticity by using antigens against 22 neurotransmittedneuropeptides and comparing the proportions of immunoreactive neurons after 3 days with those from freshly dissociated cells (Schoenen et al., 1989). An increase in expression was observed for 20 of the 22 neurochemicals tested (including SOM and CCK), and in the case of SP [together with others including GABA, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP)], significantly higher proportions were positive following culture than at dissociation. For some compounds, for example, 5hydroxytryptamine (5-HT), the change in proportions increased by over 30% in culture (Schoenen et al., 1989). These findings indicated that the culture environment can act to stimulate synthesis and storage of neurotransmitters, some of which are not even detectable in uiuo; such a culture system is ideal for investigating the factors responsible for this positive modification of adult neuronal phenotype. Further work by this group has focused on the influence of feeder layers of other cells, and the effects of NGF, CNTF, and bFGF on these phenotypic modificationsfor 5-HT, CGRP, and thyrotropin-releasinghormone (TRH) (Delree et al., 1992). Glial cells and fibroblasts inhibited the expression of 5-HT but not the other two neurochemicals; expression of CGRP and 5-HT was repressed by CNTF or bFGF, but not NGF, which was little changed after 3 days in culture. This finding showed that the regulation of expression of different phenotypes may depend on different instructive factors (Delree et al., 1992). We have recently investigated the neuropeptide phenotypes of aged, in addition to adult, mouse DRG neurons and also considered their responsiveness to 100 ng/ml NGF (Jiang and Smith, 1993b)(Fig. 7a-f). For SP,

FIG. 7 Nine-day cultures of sensory neurons stained by the avidin-biotin complex ICC method for CGRP, NPY, and SOM immunoreactivity using primary antibodies at 1 :20K dilutions. (a), (c),and (e) are representative fields of cultures maintained without exogenous NGF. (b), (d), and (f) are representative fields of cultures supplemented with 100 n g / d NGF. *, IR (positive-stained) neurons; arrows, non-IR (negative) neurons. Bar (for all) = 25 urn.

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immunoreactivity neurons were monitored for up to 19 days in culture. Cultures were routinely screened, however, at 9 days in uitro: all four neuropeptides monitored (SP, SOM,CGRP, and NPY) were retained in neurons from aged animals, although the proportions were less than in adult cells (Fig. 8). For cultures of both aged and adult neurons, supplementation with NGF enhanced the proportions of neurons expressing SP, NPY, and CGRP, while the proportions of SOM-containing neurons were nonresponsive (Figs. 7 and 8). These findings are consistent with, and also extend, those of others with respect to the effects of NGF on maintaining

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FIG. 8 Proportions of CGRP, NPY, SP, and SOM IR-neurons in cultures of (A) adult and (B) aged neurons in the absence (white) or presence (black) of 100 ng/ml NGF. Each bar representscounts of at least 7000neurons from five separate experiments;standard errors are included. Note that NGF enhances neuropeptide expression for calcitonin-gene-regulatorypeptide, neuropeptide Y, and substance P, but not for somatostatin.

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phenotypic differentiation in postmitotic neurons (Lindsay et al., 1989; Vedder and Otten, 1991). It will be pertinent to investigate the effects of the other neurotrophins, and also use in situ hybridization techniques, to determine whether the upregulation is indeed at the level of transcriptional expression. Certainly such studies may be significant in the future in investigations of the longevity of inherent plasticity in aging neurons. Trophic factor regulation of other phenotypic characteristics of adult neurons has been reported from in uitro studies and includes sensitivity to nociceptive stimuli such as capsaicin, at doses of 2 pM (Winter et al., 1988) or 500 nM (Aguayo and White, 1992), 1 pM tetrodotoxin (Aguayo and White, 1992) and 1-10 nM resiniferatoxin (Winter et al., 1990). When cultured without exogenous NGF, neurons were not responsive to the excitotoxins either morphologically, biochemically, or physiologically; treatment with NGF however, restored sensitivity, even after a delay of up to 6 days before a 200 ng/ml dose was administered (Winter et al., 1988). Such phenotypic changes in culture may be useful when assessing the effects of toxic agents in uitro, and are under investigation (Smith, 1991; R. A. Smith and Z. G. Jiang, unpublished observations). The more that is known about the factors that can regulate phenotypic expression and plasticity, the greater will be the understanding of mechanisms governing toxic insult and disease processes.

VI. Concluding Remarks

We have attempted in this chapter to show the types of questions which the use of cell culture methodology can begin to answer in studies of neuronal modulation and plasticity, and thereby augment data on neuronal functional complexity collected through in uiuo experiments. It would be naive to suggest that in uitro models on their own could ever fully elucidate the intricacies and mysteries of neuronal plasticity, and we do not wish to convey such a simplistic viewpoint. Where we believe cell culture does offer a unique and priceless lifeline, however, in the sea of confusion which all too often exists in neurobiological whole-organism research, is in isolating the phases leading to plasticity within developing and adult nervous tissue, thereby enabling either isolated or integrated investigation of the multifactorial environmental cues which are germane to the processes. Cell culture technology is now at an advanced state for both vertebrate (Banker and G o s h , 1991) and invertebrate neurons (Beadle et al., 1988), so that sophisticated and precise manipulation of environment may be achieved and the response of the neurons examined in many different neuronal types.

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All possible phases of plasticity, ranging from the regulation of neurite initiation and outgrowth (Lipton and Kater, 1989) to the formation of synaptic connections (Harris and Rosenberg, 1993), integration of successful circuitry (Bulloch and Syed, 1992), and the expression of gross phenotype (Landis, 1990) have been carefully monitored in uitro, and will continue to be investigated in cultured neurons. In our opinion, the interplay of the physical substratum (Sanes, 1989) with trophins (Walicke, 1989; Thoenen, 1991; Loughlin and Fallon, 1993) and with inhibitory factors (Schwab et al., 1993), and its pole position in modulating neuronal choices is of paramount importance in understanding the regulation of plasticity in the nervous system. The means by which neurons perceive these cues and mediate a response underlies developmental decisions throughout differentiation, and continues to be fundamental to plasticity in the mature nervous system. The events at the presynaptic (Fletcher et al., 1991) and postsynaptic (Bliss and Collingridge, 1993) terminals, the role played by signaling molecules (Mattson et al., 1988b; Jordan, 1992), and the intracelMar responses which involve inducible genes (Morgan and Curran, 1991), are all aspects in which in uitro studies have played their part in advancing our understanding. The demonstration that NGF can upregulate its own receptor genes in adult neurons and cause a rapid induction of c-fos (Lindsay et al., 1990) serves as an intriguing example of the kind of mechanistic approach neuronal culture strategies can elucidate. In v i m data can also compliment in uiuo work in developing neuronal tissues, as illustrated recently by monitoring c-fos expression and its role in embryonic remodeling and programmed cell death (Smeyne et al., 1993). Cell culture affords models conducive for such molecular studies of early transcriptional responses and may play an increasingly significantrole in future studies. The events at the growth cone which lead to changes that can direct plasticity have been well studied in uitro for vertebrate and invertebrate neurons (Letourneau et al., 1991). Growth cone regulation by intracellular calcium in response to external signals (Kater and Mills, 1991) can be successfully demonstrated by cell culture observations, as seen in elegant work using electrical fields (Davenport and Kater, 1992; Davenport and McCaig, 1993). Such approaches will continue to be instrumental in further directing future investigations. The factors directing growth cone activity and the establishment of the correct synaptic connections are certainly fundamental to basic mechanisms of modulation and plasticity, and will continue to fascinate as the neuroscientist strives to unravel the processes underlying development, memory and learning, and the changes involved in neuronal aging. A plethora of unanswered questions remain concerning plasticity; we believe, however, that dispersed cell culture methods will generate further pioneering studies to answer these questions and to determine the mechanisms involved.

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Acknowlegments We sincerely thank Drs. Roger Davenport, David Kleinfeld, and Keiichi Torimitsu for so enthusiastically agreeing to our including the micrographs from their excellent studies. The help and advice of Dr. Safa Shehab and Mrs. Christine Smith in the preparation of this manuscript is much appreciated. Thanks are due to Ms. Caroline Moms and Margaret Hughes for their help with artwork and photography. One of us (Z-G. Jiang) acknowledges support from a University of Glasgow Postgraduate Scholarship.

References Aguayo, L. G., and White, G. (1992). Effects of nerve growth factor on ‘ITX- and capsaicinsensitivity in adult rat sensory neurons. Brain Res. 570, 61-67. Akers, R. M., Mosher, D. F., and Lilien, J. E. (1981). Promotion ofretinal neurite outgrowth by substrate-bound fibronectin. Dev. Biol. 96, 179-188. Akeson, R., and Warren, S. L. (1986). PC12 adhesion and neurite formation on selected substrates are inhibited by some glycosaminoglycans and a fibronectin-derived tetrapeptide. Exp. Cell Res. 162, 347-362. Alderson, R. F., Alterman, A. L., Barde, Y.-A., and Lindsay, R. M. (1990). Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture. Neuron 5 , 297-306. Alexi, T., and Hefti, F. (1993). Trophic actions of transforming factor a on mesencephalic dopaminergic neurons developing in culture. Neuroscience 55,903-918. Al-Mohanna, F. A., Cave, J., and Bolsover, S. R. (1992). A narrow window on intracellular calcium concentration is optimal for neurite outgrowth in rat sensory neurones. Dev. Brain Res. 70, 287-290. Andersen, P. (198 1). Brain slices-a neurobiological tool of increasing usefulness. Trends Neurosci. 4, 53-56. Anderson, M. (1993). Differences in growth of neurons from normal and regenerated teleost spinal cord in vitro. In Vitro Cell. Dev. Biol. 29A, 145-152. Arechiga, H., Chiquet, M., Kuftler, D. P., and Nicholls, J. G. (1986). Formation of specific connections in culture by identified leech neurons containing serotonin, acetylcholine and peptide transmitters. J . Exp. Biol. U6, 15-31. Augusti-Tocco, G., and Sato, G. (1%9). Establishment of functional clonal lines of neurons from mouse neuroblastoma. Proc. Narl. Acad. Sci. U.S.A. 64, 311-315. Bailey, C. H., and Chen, M. (1991). Morphological aspects of synaptic plasticity in Aplysia: An anatomical substrate for long-term memory. Ann. N . Y . Acad. Sci. 627, 181-1%.

Banker, G., and Goslin, K. (1991). “Culturing Nerve Cells.” MIT Press, Cambridge, MA. Barakat-Walker, I., Molter, H. U.,and Droz, B. (1991). Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: Regulation by factors present in peripheral and central target tissues. Mol. Brain Res. 10, 107-1 14. Barbin, G., Manthorpe, M., and Varon, S. (1984). Purification of the chick eye ciliary neuronotropic factor. J. Neurochem. 43, 1468-1478. Barbu, M.,PourquiC, 0..Vaigot, P., Gateau, G.. and Smith, J. (1992). Phenotypic plasticity of embryonic sympathetic neurons grown in a chemically defined medium: Direct evidence for noradrenergic and cholinergic properties in the same neurons. J. Neurosci. Res. 32, 350-362.

Barde. Y.-A. (1989). Trophic factors and neuronal survival. Neuron 2, 1525-1534. Baron-Van Evercooren, A., Klienman, H. K., Ohno, S., Marangos, P.,Schwartz, J. P.,

282

ROBERT A. SMITH AND ZHI-GANG JIANG

and Dubois-Dalcq, M. E. (1982). Nerve growth factor, laminin and fibronectin promote neurite growth in human fetal sensory ganglia cultures. J. Neurosci. Res. 8, 179-193. Bartlett, W., and Banker, G. (1984). An electronmicroscopic study of the development of axons and dendrites by hippocampal neurons in cultures. 11. Synaptic relationships. J. Neurosci. 4, 1934-1965. Baumrind, N. L., Parkinson, D., Wayne, D. B., Heuser, J. E., and Pearlman, A. L. (1992). EMA: A developmentally regulated cell-surface glycoprotein of CNS neurons that is concentrated at the leading edge of growth cones. Deu. Dyn. 194, 311-325. Beadle, D. J., Lees, G., and Kater, S. B. (1988). "Cell Culture Approaches to Invertebrate Neuroscience." Academic Press, San Diego. Bedi, K. S.,Winter, J., Berry, M.,and Cohen, J. (1992). Adult rat dorsal root ganglion neurons extend neurites on predegenerated but not on normal peripheral nerves in vitro. Eur. J. Neurosci. 4, 193-200. Benowitz, L. I., and Perrone-Bizzero, N. I. (1991). The relationship of GAP-43 to the development and plasticity of synaptic connections. Ann. N . Y. Acud. Sci. 627, 58-74. Berkemeier, L. R., Winslow, J. W., Kaplan, D. R., Nokolics, K.. Goeddel, D. V., and Rosenthal, A. (1991). Neurotrophin-5: A novel neurotrophic factor that activates trk and trkB. Neuron 7,857-866. Bessho, Y.,Nakanishi, S.,and Nawa, H. (1993). Glutamate receptor agonists enhance the expression of BDNF mRNA in cultured cerebellar granule cells. Mol. Bruin Res. 18, 20 1-208.

Bixby, J. L., and Zhang, R. (1990). Purified N-cadherin is a potent substrate for the rapid induction of neurite outgrowth. J . Cell Biol. 110, 1253-1260. Bixby, J. L., Lilien, J., and Reichardt, L. F. (1988). Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro. J. Cell Biol. 107, 353-361.

Black, I. B., Adler, J. E., Dreyfus, C. F., Jonakait, G. M.,Katz, D., LaGamma, E. F., and Markey, K. M. (1984). Neurotransmitter plasticity at the molecular level. Science 225, 1266-1270.

Black, M. M.,and Baas, P.

W.(1989). The basis of polarity in neurons. Trends Neurosci.

U,211-214. Bliss, T. V. P., and Collingridge, G. L. (1993). A synaptic model of memory: Long-term potentiation in the hippocampus. Nature (London) 361, 31-39. Bolz, J., Gstz, M.,Hlibener, M.,and Novak, N. (1993). Reconstructing cortical connections in a dish. Trends Neurosci. 16, 310-316. Bozyczko, D., and Horwitz, A. F. (1986). The participation of a putative cell receptor for laminin and fibronectin in peripheral nerve extension. J. Neurosci. 6, 1241-1251. Brandtlow, C., Zachleder, T.,and Schwab, M. E. (1990). Oligodendrites arrest neurite growth by contact inhibition. J. Neurosci. 10, 3837-3848. Bray, D. (1987). Growth cones: Do they pull or are they pushed? Trends Neurosci. 10, 431-434.

Bray, D., and Chapman, K. (1985). Analysis of microspike movements on the neuronal growth cone. J . Neurosci. 5, 3204-3213. Bray, D., Bunge, M. B., and Chapman, K. (1987). Geometry of isolated sensory neurons in culture. Exp. Cell Res. 168, 127-137. Bulloch, A. G. M.,and Syed, N. I. (1992). Reconstruction of neuronal networks in culture. Trends Neurosci. Is, 422-427. Bunge, R. P., Rees, R., Wood, P., Burton, H.,and KO, C.-P. (1974). Anatomical and physiological observations on synapses formed on isolated autonomic neurons in tissue culture. Bruin Res. 66,401-412. Cannella, M.S., and Ross, R. A. (1987). Influence of substratum on the retrograde response of the rat superior cervical ganglion in vitro. Exp. Neurol. 95,652-660.

NEURONAL MODULATION AND PLASTICITY IN VlTRO

283

Carbonetto, S. T., Gruver, M.M.,and Turner, D. C. (1982). Nerve fiber growth on defined hydrogel substrates. Science 216,897-899. Carbonetto, S.T., Gruver, M.M.,and Turner, D. C. (1983). Nerve fiber growth in culture on fibronectin, collagen and glycosaminoglycan substrates. J . Neurosci. 3,2324-2335. Carbonetto, S . , Evans, D., and Cochard, P. (1987). Nerve fiber growth in culture on tissue substrates from central and peripheral nervous system. J . Neurosci. 7 , 610-620. Caroni, P., and Schwab, M. E. (1988). Two membrane fractions from rat central myelin with inhibitory properties of neurite growth and fibroblast spreading. J . Cell Eiol. 106, 128 1 - 1288. Carpenter G . A., and Grossberg, S. (1993). Normal and amnesic learning, recognition and memory by a neural model of cortico-hippocampal interactions. Trends Neurosci. 16, 131-137. Cam, N. G., Penis, R., Johansson, S.,and Ebendal, T. (1988). Differential outgrowth of retinal neurites on purified extracellular matrix molecules. J . Neurosci. Res. 19,428-439. Chad, J., and Wheal, H. (1991). "Cellular Neurobiology: A Practical Approach." IRC Press, Oxford. Chalazonitis, A., Kessler, J. A., Twardzik, D. R., and Morrison, R. S. (1992). Transforming growth factor a,but not epidermal growth factor, promotes the survival of sensory neurons in vitro. J. Neurosci. U,583-594. Chang, S., Rathjen, F. G., and Raper, J. A. (1987). Antibodies to F l l and G4 glycoproteins prevent axon fasciculation. J . Cell Biol. 104, 335-362. Chiquet, M., and Nicholls, J. G. (1987). Neurite outgrowth and synapse formation by identified leech neurones in culture. J . Exp. Eiol. 132, 191-206. Choi, D. W., Farb, D. H., and Fishbach, G. D. (1981). GABA-mediated synaptic potentials in chick spinal cord and sensory neurons. J . Neurophysiol. 45, 632-643. Chong, P. S., Smith, R. A., and Jiang, Z.-G. (1994). The effects of retinoic acid on primary cultured adult mouse sensory neurons. J . Anat. (in press). Clark, P., Britland, S., and Connelly, P. (1993). Growth cone guidance and neuron morphology on micropatterned laminin surfaces. J . Cell Sci. 105,203-212. Coates, C. J., and Bulloch, A. G. M. (1985). Synaptic plasticity in the molluscan PNS: Physiology and role for peptides. J . Neurosci. 5, 2677-2684. Coggins, P. J., and Zwiers, H. (1991). B-50 (GAP-43): Biochemistry and functional neurochemistry of a neuron-specific phosphoprotein. J . Neurochem. 56, 1095-1 106. Cohan, C. S.,Conner, J. A., and Kater, S. B. (1987). Electrically and chemically mediated increases in intracellular calcium in neuronal growth cones. J . Neurosci. 7 , 3588-3599. Cohen-Cory, S., Dreyfus, C. F., and Black, I. B. (1991). NGF and excitatory neurotransmitters regulate survival and morphogenesis of cultured cerebellar Purkinje cells. J . Neurosci. 11,462-471. Cooper, E. (1984). Synapse formation among developing sensory neurons from rat nodose ganglia grown in tissue culture. J . Physiol. (London) 351, 263-274. Craig, A. M., Blackstone, C. D., Huganir, R. L.,and Banker, G. (1993). The distribution of glutamate receptors in cultured rat hippocampal neurons: Postsynaptic clustering of AMPA-selective subunits. Neuron 10, 1055-1068. Dailey, M.E., and Bridgman, P. C. (1989). Dynamics of the endoplasmic reticulum and other membranous organelles in growth cones of cultured neurons. J . Neurosci. 9, 1897-1909. Dal Toso, R., Giorgi, O., Soranzo, C., Kirschner, G., Ferrari, G., Favaron. M.,Benvegnh, D., Presti, D., Vicini, S.,Toffano. G., Azzone, G. F., and Leon, A. (1988). Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: 1 . Effects of cell density and of an adult mammalian striatal-derived neuronotrophic factor (BDNF). J . Neurosci. 8,733-745. Davenport, R. W., and Kater, S. B. (1992). Local increases in intracellular calcium elicit local filopodial responses in Helisoma neuronal growth cones. Neuron 9,405-416.

284

ROBERT A. SMITH AND ZHI-GANG JIANG

Davenport, R. W., and McCaig, C. D. (1993). Hippocampal growth cone responses to focally applied electric fields. J . Neurobiol. 24, 89-100. Davenport, R. W.,Dou, P., Rehder, V., and Kater, S. B. (1993). A sensory role for neuronal growth cone filopodia. Nature (London)361,721-724. David, S . (1988). Neurite outgrowth from mammalian CNS neurons on astrocytes in vitro may not be mediated by laminin. J . Neurocytol. 17, 131-144. Davies, A. M. (1988). The emerging generality of the neurotrophic hypothesis. Trends Neurosci. 11,243-244. Davies, A. M., Thoenen, H.,and Barde, Y.-A. (1986a). Different factors from the central nervous system and periphery regulate survival of sensory neurons. Nature (London) 319,497-499.

Davies, A. M., Thoenen, H., and Barde, Y.-A. (1986b). The response of chick sensory neurons to brain-derived neurotrophic factor. J. Neurosci. 6, 1897-1904. Davis, G. E., Manthorpe, M., and Varon, S. (1985). Parameters of neuritic growth from ciliary ganglion neurons in vitro: Influence of laminin, Schwannoma polyornithine-binding neurite promoting factor and ciliary neuronotrophic factor. Deu. Brain Res. 17, 75-84. Delree, P., Martin, D., Sadzot-Delvaux, C., Rogister, B., Leprince, P., Robe, P., Rigo, J.-M., Lefbvre, P. P., Malgrange, B., Schoenen, J., and Moonen, G . (1992). In vitro and in vivo modulation of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and calcitonin-gene related peptide-like immunoreactivities in adult sensory neurons. Neuroscience 51,401-410. Diamond, J., Forester, A., Holmes, M., and Coughlin, M. (1992). Sensory nerves in adult rats regenerate and restore sensory function to the skin independently of endogenous NGF. J . Neurosci. 12, 1467-1476. Dichter, M. A. (1978). Rat cortical neurons in cell culture: Culture methods, cell morphology, electrophysiology, and synapse formation. Brain Res. 149, 279-293. Docherty, P., Barton, C. H., Dickson, G., Seaton, P., Rowlett, L. H., Moore, S. E., Gower, H. J., and Walsh, F. S. (1989). Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms. J. Cell Biol. 109,789-798. Dotti, C. G., Sullivan, C. A., and Banker, G. A. (1988). The establishment of polarity by hippocampal neurons in culture. J . Neurosci. 8, 1454-1468. Dow, K. E., and Riopelle, R. J. (1992). Influence of N-linked oligosaccharides on the processing and neurite-promoting activity of proteoglycans released by neurons in vitro. Cell Tissue Res. 268, 553-558. Duband, J.-L., Rocher, S., Chen, W.-T., and Yamada, K. M. (1986). Cell adhesion and migration in the early vertebrate embryo: Location and possible role of the putative fibronectin receptor complex. J . Cell Biol. 102, 160-178. Dubinsky, J. M. (1989). Development of inhibitory synapses among striatal neurons in vitro. J . Neurosci. 9, 3955-3965. Edgar, D. (1985). Nerve growth factors and molecules of the extracellular matrix in neuronal development. J . Cell Sci., Suppl. 3, 107-113. Edgar, D. (1989). Neuronal laminin receptors. Trends Neurosci. U,248-251. Edgar, D., Timpl, R., and Thoenen, H.(1984). The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival. EMBO J . 3, 1463-1468.

Edmonds, B., Klein, M., Dale, N., and Kandel, E. R. (1990). Contributions of two types of CaZ+channels to synaptic transmission and plasticity. Science 250, 1142-1 147. Eichler, M. E., Dubinsky, J. M., and Rich, K. M. (1992). Relationship of intracellular calcium to dependence on nerve growth factor in dorsal root ganglion neurons in cell culture. J . Neurochem. 58, 263-269.

NEURONAL MODULATION AND PLASTICITY IN VITRO

285

Erickson, C. A., and Turley. E. A. (1987). The effects of epidermal growth factor on neural crest cells in tissue culture. Exp. Cell Res. 169, 267-279. Fazeli, M. S . (1992). Synaptic plasticity: On the trail of the retrograde messenger. Trends Neurosci. 15, 115-1 17. Fernyhough, P., Willars, G. B., Lindsay, R. M., and Tomlinson, D. R. (1993). Insulin and insulin-like growth factor I enhance regeneration in cultured adult rat sensory neurones. Bruin Res. 607,117-124. Fletcher, T. L., Cameron, P., De Camilli, P., and Banker, G. (1991). The distribution of synapsin and synaptophysin in hippocampal neurons developing in culture. J. Neurosci. 11, 1617-1626.

Ford-Holevinski, T. S., Hopkins, J. M., McCoy, J. P., and Agranoff, B. W. (1986). Laminin supports neurite outgrowth from explants of axotomized adult rat retinal neurons. Dew Bruin Res. 28, 121-126. Fountain, S. B., Ting, Y.-L. T., and Teyler, T. J. (1992). The in vitro hippocampal slice preparation as a screen for neurotoxicity. Toxicol. In Vitro 6, 77-87. Frandsen, A., Schousboe, A., and Griffiths, R. (1993). Cytotoxic actions and effects on intracellular CaZ and cGMP concentrations of sulphur-containingexcitatory amino acids in cultured cerebral cortical neurons. J. Neurosci. Res. 34, 331-339. Freidin, M., Dougherty, M., and Kessler, J. A. (1993). Cell density regulates neuropeptide Y expression in cultured sympathetic neurons. Bruin Res. 615, 135-140. Fridman, R., Alon, Y.,Doljanski, F., Fuks, Z., and Vlodavsky, I. (1985). Cell interaction with extracellular matrices produced by endothelial cells and fibroblasts. Exp. Cell Res. +

158,461-476.

Fuchs. P. A., Nicholls, J. G., and Ready, D. F. (1981). Membrane properties and selective connexions of identified leech neurons in culture. J . Physiol. (London) 316,203-223. Fukuda, J., Aosaki, T., Keino, K., and Yamaguchi, T. (1991). Age-associated and cell-type specific changes in NGF requirement for neurite regeneration from trigeminal ganglion cells of the shrew (Suncus murinus). J. Geronrol. 46, B3-Bl6. Funte, L. R., and Haydon, P. G. (1993). Synaptic target contact enhances presynaptic calcium influx by activating CAMP-dependent protein kinase during synaptogenesis. Neuron 10, 1069-1078. Gahwiler, B. H., and Brown, D. A. (1985). Functional innervation of cultured hippocampal neurons by cholinergic afferents from co-cultured septal explants. Nature (London) 3l3, 577-579.

Garcia, I., Martinou. I., Tsujimoto, Y.,and Martinou, J.-C. (1992). Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene. Science 258, 302-304.

Garthwaite, J. (1991). Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci. 14, 60-67. Glanzman, D. L., Kandel, E. R., and Schacher, S. (1990). Target-dependent structural changes accompanying long-term synaptic facilitation in Aplysiu neurons. Science 249, 799-802.

Gleason, E., and Wilson, M. (1989). Development of synapses between chick retinal neurons in dispersed culture. J . Comp. Neurol. Un,213-224. Goldberg, D. J., and Burmeister, D. W. (1989). Looking intogrowth cones. Trends Neurosci. l2,503-506. Gordon-Weeks, P. R . (1991). Growth cones: The mechanism of neurite advance. BioEssays 13, 235-239. G o s h , K., Schreyer, D. J., Skene, J. H., and Banker, G. (1988). Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones. Nature (London)336, 672-674.

286

ROBERT A. SMITH AND ZHI-GANG JIANG

Greene, L. A., and Tischler, A. S. (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromccytoma cells which respond to nerve growth factor. Proc. Nutl. Acad. Sci. U.S.A. 73,2424-2428. Grothe, C., and Unsicker, K. (1987). Neuron-enriched cultures of adult rat dorsal root ganglia: Establishment, characterisation, survival, and neuropeptide expression in response to trophic factors. J. Neurosci. Res. 18, 539-550. Gundersen. R. W. (1987). Response of sensory neurites and growth cones to patterned substrata of laminin and fibronectin in vitro. Dev. Biol. 121,423-431. Gundersen, R. W., and Barrett, J. N. (1980). Characterisation of the turning response of dorsal root neurites toward nerve growth factor. J. Cell Biol. 87, 546-554. Gupta, J. K., and Bigbee, J. W. (1992). Substratum-induced modulation of acetylcholinasterase activity in cultured dorsal root ganglion neurons. J. Neurosci. Res. 31, 454-461. Hadley, R. D., Kater, S. B., and Cohan, C. S. (1983). Electrical synapse formation depends on interaction of mutually growing neurites. Science 221, 466-468. Halfter, W., and von Boxberg (1992). Axonal growth on solubilised and reconstituted matrix from the embryonic chick retina inner limiting membrane. Eur. J. Neurosci. 4, 840-852. Hallbdok, F., Ibiifiez, C. F., and Pearson, H. (1991). Evolutionary studies of the growth factor family reveal a novel member abundantly expressed in Xenopus ovary. Neuron 6, 845-858.

Hammarback, J. A., Palm, S. L., Furcht, L. T., and Letourneau, P. C. (1985). Guidance of neurite outgrowth by pathways of substratum-bound laminin. J. Neurosci. Res. 13, 213-220.

Hammarback, J. A., McCarthy, J. B., Palm, S. L., Furcht, L. T., and Letourneau, P. C. (1988). Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity. Dev. Biol. W, 29-39. Hams, K. M., and Rosenberg, P. A. (1993). Localisation of synapses in rat cortical cultures. Neuroscience 53, 495-508. Harrison, R. G. (1907). Observations on the living developing nerve fibre. Anut. Rec. 1, 116-1 18.

Harrison, R. G. (1910). The outgrowth of the nerve fibre as a mode of protoplasmic movement. J. Exp. Zool. 9, 787-846. Hart, R. P., Shadiack, A. M., and Jonakait, G. M. (1991). Substance Pexpression is regulated by Interleukin-1 in cultured sympathetic ganglia. J. Neurosci. Res. 29, 282-291. Hartikka, J., and Hefti, F. (1988). Comparison of nerve growth factor’s effects on development of septum, striatum, and nucleus basalis cholinergic neurons in vitro. J. Neurosci. Res. 21, 352-364. Haskell, B. E., Stach, R. W., Werrbach-Perez, K., and Perez-Polo, J. R. (1987). Effect of retinoic acid on nerve growth factor receptors. Cell Tissue Res. 247, 67-73. Hatten, M. E., Lynch, M., Rydel, R. E., Sanchez, J., Joseph-Silverstein, J., Moscatelli, D., and Rifkin, D. B. (1988). In vitro neurite extension by granule neurons is dependent on astroglial-derived fibroblast growth factor. Deu. Biol. US,280-289. Haugen, P. K., McCarthy, J. B., Roche. K. F., Furcht, L. T., and Letourneau, P. C. (1992). Central and peripheral neurite outgrowth differs in preference for heparin-binding versus integrin-binding sequences. J. Neurosci. 12, 2034-2042. Hawrot, E. (1980). Cultured sympathetic neurons: Effects of cell derived and synthetic substrata on survival and development. Dev. Biol. 74, 136-151. Haydon, P. G. (1988). The formation of chemical synapses between cell-cultured neuronal somata. J. Neurosci. 8, 1032-1038. Haydon, P. G., McCobb, D. P., and Kater, S. B.(1984). Serotinin selectively inhibits growth cone motility and synaptogenesis of specific identified neurons. Science 226, 561-564. Haydon, P. G., Cohan, C. S., McCobb, D. P., Miller, H. R., and Kater, S. B. (1985).

NEURONAL MODULATION AND PLASTICITY IN VlTRO

287

Neuron-specific growth cone properties as seen in identilied neurons of Helisoma. J. Neurosci. Res. W, 135-147. Heaton, M. B., and Swanson, D. J. (1988). The influence of laminin on the initial differentiation of cultured neural tube neurons. J . Neurosci. Res. 19,212-218.. Hirano, T., Kubo, Y., and Wu, M. M. (1986). Cerebellar granule cells in culture: Monosynaptic connections with Purkinje cells and ionic currents. Proc. Nafl. Acad. Sci. U . S . A . 83, 4957-4961.

Hofer, M.,Paguliusi, S. R., Hohn, A. L.. Eibrock, J., and Barde, Y.-A. (1990). Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain. EMBO J . 9, 2459-2464. Hokfelt, T., Millhorn, D., Seroogy. K., Tsuruo, Y.,Ceccatelli, S., Lindh, B., Meister, B., Melander, T., Schalling, M., Bartfai, T., and Terenius, L. (1987). Coexistence of peptides with classical neurotransmitters. Experienria 43, 768-780. Horie, H., and Kim, S. U. (1984). Improved survival and differentiation of newborn and adult mouse neurons in F12 defined medium by fibronectin. Brain Res. 294, 178-181. Huettner, J. E., and Baughman, R. W. (1988). The pharmacology of synapses formed by identified corticocollicular neurons in primary cultures of rat visual cortex. J . Neurosci. 8, 160-175. Hunter, D. D., Cashman, N., Morris-Valero, R., Bulock, J. W., Adams, S. P., and Sanes, mechanism for adhesion J. R. (1991). An LRE (Leucine-Arginine-Glutamate)-dependent of neurons to S-laminin. J . Neurosci. 11, 3960-3971. Hunter, K., Maden, M., Summerbell, D., Eriksson, U., and Holder, N. (1991). Retinoic acid stimulates neurite outgrowth in the amphibian spinal cord. Proc. Narl. Acad. Sci. U . S . A . 88, 3666-3670. Hyman, C., Hofer, M., Barde, Y.-A., Juhasz, M., Yancopoulos, G. D., Squinto, S. P., and Lindsay, R. M. (1991). BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Narure (London) 350, 230-332. Hynes, R. 0. (1973). Alteration of cell surface proteins by viral transformation and by proteolysis. Proc. Narl. Acad. Sci. U.S.A. 70, 3170-3174. Ip, N. Y., Li, Y., van de Stadt, I., Panayotatos, N., Alderson, R. F.. and Lindsay, R. M. (1991). Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures. J . Neurosci. 11, 3124-3134. Itoh, T.. Sobue, G., Yasuda, T., Mitsuma, T., Takahashi, A., and Kimata, K. (1991). Geometry of adult rat sensory neurons in culture; its modulation by laminin. Neurosci. Left.

U3,212-216.

Jahr, C. E., and Jessell, T. M. (1985). Synaptic transmission between DRG and dorsal horn neurons in culture: Antagonism of monosynaptic excitatory postsynaptic potentials and glutamate excitation by kynurenate. J . Neurosci. 5 , 2281-2289. Janet, T., Grothe, C., Pettmann, B.. Unsicker, K., and Sensenbrenner, M.(1988). Immunocytochemical demonstration of fibroblast growth factor in cultured chick and rat neurons. J. Neurosci. Res. 19, 195-201. Jiang, Z.-G., and Smith, R. A. (1992). The effects of NGF on the survival in vitro of adult and aged mouse sensory neurons. J . Anar. 180, 364. Jiang. Z.-G., and Smith, R. A. (1993a). Effects of nerve growth factor on the survival of primary cultured adult and aged mouse sensory neurons. J. Neurosci. Res. 35,29-37. Jiang, Z.-G., and Smith, R. A. (1993b). The effects of nerve growth factor on neuropeptide expression by aged mouse sensory neurons in vitro. J . Anat. 183, 192. Jing, S.. Tapley, P., and Baracid, M. (1992). Nerve growth factor mediates signal transduction through trk homodimer receptors. Neuron 9, 1067-1079. Jordan, F. L. (1992). Distribution and expression of G-protein in rat cerebral cortical cells. 11. Primary tissue culture. Deu. Brain Res. 67, 11-18.

288

ROBERT A. SMITH AND ZHI-GANG JIANG

Ju, G., Hokfelt T., Brodin E., Fahrenkmg, J., Fischer J. A., Frey, P., Elde R. P., and Brown J. C. (1987). Primary sensory neurons of 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. Kai-Kai, M. A. (1989). Cytochemistry of the trigeminal and dorsal root ganglia and spinal cord of the rat. Comp. Biochem. Physiol. A 93A, 183-193. Kapfhammer, J. P., and Rqper, J. A. (1987). Interactions between growth cones and neurites growing from different neural tissues in culture. J. Neurosci. 7, 1595-1600. Kater, S., and Letourneau, P. C. (1985). Biology of the nerve growth cone. J . Neurosci. Res. 13, 1-335. Kater, S., and Mills, L. R. (1991). Regulation of growth cone behaviour by calcium. J. Neurosci. 11,891-899. Katz, D. M. (1991). A catecholinergic sensory phenotype in cranial derivatives of the neural crest: Regulation by cell aggregation and NGF. J . Neurosci. 11, 3991-4002. Kenigsburg, R. L., Mazzoni, I. E., Collier, B., and Cuello, A. C. (1992). EGF affects both glia and cholinergic neurons in septal cell cultures. Neuroscience 50,85-97. Kentroti, S.,Ramacci, M. T., and Vernadakis, A. (1992). Acetyl-L-carnitine has a neuromodulatory influence on neuronal phenotypes during early embryogenesis in the chick embryo. Dev. Brain Res. 70, 259-266. Keshmirian, J., Bray, G., and Carbonetto, S. (1989). The extracellular matrix modulates the response of PC12 cells to nerve growth factor: Cell aggregationversus neurite outgrowth on 3-dimensional laminin substrata. J. Neurocyrol. 18, 491-504. Kessler, J. A., and Freidin, M. (1991). Regulation of substance P expression in sympathetic neurons. Ann. N . Y. Acad. Sci. 632, 10-18. Kim. J. H., Kim, S. U., and Kito, S. (1984). Immunocytochemical demonstration of pendorphin and 8-lipotropin in cultured human spinal ganglion neurons. Brain Res. 304, 192-196. Kleinfeld, D., Kahler, K. H., and Hockberger, P. E. (1988). Controlled outgrowth of dissociated neurons on patterned substrates. J. Neurosci. 8,4098-4120. Knusel, B., and Hefti, F. (1988). Development of cholinergic pedunculopontine neurons in vitro: Comparison with cholinergic septal neurons and response to nerve growth factor, ciliary neurotrophic factor, and retinoic acid. J. Neurosci. Res. 21, 365-375. Kniisel, B., Michel, P.P., Schwaber, J. S.,and Hefti, F. (1990).Selective and non-selective stimulation of central cholinergic and dopaminergic development in vitro by NGF, bFGF, EGF, insulin and insulin-like growth factors I and 11. J. Neurosci. 10, 558-570. Knusel, B., Winslow, J. W.,Rosenthal, A., Burton, L. E., Seid, D. P., Nikolics, K.. and Hefti, F. (1991). Promotion of central cholinergic and dopaminergic neuron differentiation by brain-derived neurotrophic factor but not NT-3. Proc. Narl. Acad. Sci. U.S.A. 88, %1-%5. KO, C.-P., Burton, H., and Bunge, R. P. (1976). Synaptic transmission between rat spinal cord explants and dissociated superior cervical ganglion neurons in tissue culture. Bruin Res. 117,437-460. Kobayashi, H., Mizuki, T., Wada, A., and Izumi, F. (1992). Cell-cell contact modulates expression of cell adhesion molecule L1 in PC12 cells. Neuroscience 49, 437-441. Kuusela, P., Ruoslahti, E., Engvall, E., and Vaheri, A. (1976). Immunological interspecies cross-reactions of fibroblast surface antigen (fibronectin). Immunochemistry W, 639-642. Lafont, F.. Rouget, M., Triller, A., Prochiantz, A., and Rousselet, A. (1992). In vitro control of neuronal polarity by glycosaminoglycans. Development (Cambridge, U K ) 114, 17-29. Laiwand, R., and Brown, D. A. (1992). Synapse formation between dissociated basal forebrain neurones and hippocampal cells in culture. Neurosci. Lerr. l38,221-224.

NEUAONAL MODULATION AND PLASTICITY IN vim0

289

Lander, A. D., Fujii, D. K., and Reichardt, L. F. (1985). Laminin is associated with the “neurite outgrowth-promoting factors” found in conditioned media. Proc. Nutl. Acad. Sci. U.S.A. 82, 2183-2187. Landis, S. C. (1990). Target regulation of neurotransmitter phenotype. Trends Neurosci. 13, 344-350. Landreth, G . E., and Agranoff, B. W. (1976). Explant culture of adult goldfish retina: Effect of prior optic nerve crush. Bruin Res. 118, 299-303. Lankford, K. L., and Letourneau, P. C. (1989). Evidence that calcium may control neurite outgrowth by regulating the stability of actin filaments. J. Cell Biol. 109, 1229-1243. Lankford, K. L., and Letourneau, P. C. (1991). Roles of actin filaments and three secondmessenger systems in short-term regulation of chick dorsal root ganglion neurite outgrowth. Cell Motil. Cytoskel. 20, 7-29. Lankford, K. L., DeMello, F. G., and Klein, W. L. (1988). DI-type dopamine receptors inhibit growth cone mobility in cultured retina neurons: Evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system. Proc. Natl. Acud. Sci. U.S.A. 85, 2839-2843. Lefebvre, P. P., Leprince, P., Weber, T., Rigo, J.-M., Delree, P., and Moonen, G. (1990). Neuronotrophic effect of developing otic vesicle on cochleo-vestibular neurons: Evidence for nerve growth factor involvement. Bruin Res. 507, 254-260. Lefebvre, P. P., Staeker, H., Weber, T., Van de Water, T. R., Rogister, B., and Moonen, G. (1991). TGFpl modulates bFGF receptor message expression in cultured adult auditory neurons. NeuroReport 2,305-308. Lein, D. J., and Higgins, D. (1989). Laminin and a basement membrane extract have different effects on axonal and dendritic outgrowth from embryonic rat sympathetic neurons in vitro. Dev. Biol. w6, 330-345. Lein, D. J., Banker, G. A., and Higgins, D. (1992). Laminin selectively enhances axonal growth and accelerates the development of polarity by hippocampal neurons in culture. Dev. Bruin Res. 69, 191-197. Lendahl, U., and McKay, R. D. G. (1990). The use of cell lines in neurobiology. Trends Neurosci. W, 132-136. Letourneau, P. C. ( 1975). Possible role for cell-to-cell-substratum adhesion in neuronal rnorphogenesis. Dev. Biol. 44, 77-91. Letourneau, P. C., Shattuck, T. A., and Ressler, A. H. (1986). Branching of sensory and sympathetic neurites in vitro is inhibited by treatment with taxol. J . Neurosci. 6, 1912-1917. Letoumeau, P. C., Kater, S., and Macagno, E. (1991). “The Nerve Growth Cone.’’ Raven Press, New York. Levi-Montalcini, R. (1987). Nerve growth factor 35 years later. Science 237, 1154-1 162. Lewandowska, K., Balza, E., Zardi, L., and Culp, L. A. (1990). Requirement for two different cell-binding domains in fibronectin for neurite extension of neuronal derivative cells. J. Cell Sci. 95, 75-83. Lewis, W. H., and Lewis, M. R. (1912). The cultivation of sympathetic nerves from the intestine of chick embryos in saline solutions. Anut. Rec. 6, 7-31. Liang. S . , and Crutcher, K. A. (1992). Neuronal migration on laminin in vitro. Dev. Brain Res. 66, 127-132. Liesi, P., Dahl, D., and Vaheri, A. (1984). Neurons cultured from developing rat brain attach and spread preferentially to laminin. J . Neurosci. Res. 11, 241-251. Lin, S. S.,and Levitan, I. B. (1991). Concanavalin A: A tool to investigate neuronal plasticity. Trends Neurosci. 14, 273-277. Lindsay, R. M. (1988). Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons. J. Neurosci. 8, 2394-2405.

290

ROBERT A. SMITH AND ZHI-GANG JIANG

Lindsay, R. M. (1992). The role of neurotrophic factors in functional maintenance of mature sensory neurons. In ‘Sensory Neurons: Diversity, Development and Plasticity” ( S . A. Scott, ed.), pp. 404-420. Oxford Univ. Press, New York. Lindsay, R. M., and Harmar, A. J. (1989). Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons. Nature (London) 337, 362-364. Lindsay, R. M., Thoenen, H., and Barde, Y.-A. (1985). Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor. Dev. Biol. llt, 319-328. Lindsay, R. M., Lockett, C., Sternberg, J., and Winter, J. (1989). Neuropeptide expression in cultures of adult sensory neurons: Modulation of substance P and CGRP levels by nerve growth factor. Neuroscience 33, 53-65. Lindsay, R. M.. Shooter. E. M., Radeke, M. J., Misko, T. P., Dechant, G., Thoenen, H., and Lindholm, D. (1990). Nerve growth factor regulates expression of the nerve growth factor receptor gene in adult sensory neurons. Eur. J . Neurosci. 2, 389-3%. Lipton, S. A,, and Kater, S. B. (1989). Neurotransmitter regulation of neuronal outgrowth, plasticity and survival. Trends Neurosci. 12, 265-270. Lisman, J. E., and Harris, K. M. (1993). Quanta1analysis and synaptic activity-integrating two views of hippocampal plasticity. Trends Neurosci. 16, 141-147. Loughlin, S. E., and Fallon, J. H. (1993). “Neurotrophic Factors.” Academic Press, San Diego. Luckenbill-Edds, L.,and Kleinman, H. K . (1988). Effect of laminin and cytoskeletal agents on neurite formation by NGlO8-15 cells. J . Neurosci. Res. 19, 219-229. Lukowiak, K., and Colebrook, E. (1988-1989). Neuronal mechanisms of learning in an in vitro Aplysia preparation: Sites other than the sensory-motor neuron synapse are involved. J . Physiol. (Paris) 83, 198-206. Madison, D. V., Malekenka, R. C., and Nicoll, R. A. (1991). Mechanisms underlying longterm potentiation of synaptic transmission. Annu. Rev. Neurosci. 14, 379-397. Magal, E., Burnham, P., and Varon, S . (1991). Effects of ciliary neuronotrophic factor on rat spinal cord neurons in vitro: Survival and expression of choline acetyltransferase and low-affinity nerve growth factor receptors. Dev. Brain Res. 63, 141-150. Mahanthappa, N. K., and Schwarting, G. A. (1993). Peptide growth factor control ofolfactory neurogenesis and neuron survival in vitro: Roles of EGF and TGF-ps. Neuron 10,293-305. Maisonpierre, P. C., Belluscio, L., Squinto, S., Ip, N. Y., Furth, M. E., Lindsay, R. M., and Yancopoulos, G. D. (1990). Neurotrophin-3: A neurotrophic factor related to NGF and BDNF. Science 247, 1446-1451. Manfridi, A., Forloni, G. L., Anigoni-Martelli, E., and Mancia, M. (1992). Cultures of dorsal root ganglion neurons from aged rats: Effects of acetyl-L-carnitine and NGF. Int. J . Dev. Neurosci. 10, 321-329. Mangoura, D., and Venadakis, A. (1988). GABAergic neurons in cultures derived from three-, six-, or eight-day-old chick embryo: A biochemical and immunocytochemicalstudy. Dev. Brain Res. 40, 25-35. Mangoura, D., Sakellaridis, N., and Venadakis, A. (1988). Cholinergic neurons in cultures derived from three-, six-, or eight-day-old chick embryo: A biochemical and immunocytochemical study. Dev. Brain Res. 40, 37-46. Mangoura, D., Sakellaridis, N., and Venadakis, A. (1990). Evidence for plasticity in neurotransmitter expression in neuronal cultures derived from 3-day-old chick embryo. Deu. Brain Res. 51, 93-101. Mansfield, S. G., Diaz-Nido, J., Gordon-Weeks, P. R., and Avila, J. (1992). The distribution and phosphorylation of the microtubule-associated protein MAP IB in growth cones. J. Neurocytol. 21, 1007-1022. Manthorpe. M., Engvall, E., Ruoslahti, E., Longo, F. M., Davis, G. E., andVaron, S. (1983).

NEURONAL MODULATION AND PLASTICITY IN vim0

291

Laminin promotes neuritic regeneration from cultured peripheral and central neurons. J . Cell Biol. 97, 1882-1890. Manthorpe, M., Ray, J., Pettmann, B., and Varon, S. (1989). Ciliary neuronotrophicfactors. In “Nerve Growth Factors” (R. A. Rush, ed.), pp. 31-56. Wiley, Chichester. Manthorpe, M., Louis, J-C., Hagg, T., and Varon, S . (1993). Ciliary neurotrophic factor. In “Neurotrophic Factors,” ( S . E. Loughlin and J. H. Fallon, eds). Academic Press, San Diego. Marler, P. (1991). Song-learning behavior: the interface with neuroethology. Trends Neurosci. 14, 199-206. Matsuda, S . , Saito, H., and Nishiyama, N. (1990). Effect of basic fibroblast growth factor on neurons cultured from various regions of postnatal rat brain. Bruin Res. 524 310-316. Mattson, M. P. (1988). Neurotransmitters in the regulation of neuronal cytoarchitecture. Bruin Res. Rev. 13, 179-212. Mattson, M. P., and Kater, S. B. (1987). Calcium regulation of neurite elongation and growth cone motility. J . Neurosci. 7,4034-4043. Mattson, M. P., and Kater, S. B. (1989). Excitatory and inhibitory neurotransmitters in the generation and degeneration of hippocampal architecture. Bruin Res. 478, 337-348. Mattson, M. P., Dou, P., and Kater, S. B. (1988a).Outgrowth-regulatingactions ofglutamate in isolated hippocampal pyramidal neurons. J. Neurosci. 8, 2087-2100. Mattson, M. P., Guthrie, P. B., and Kater, S. B. (1988b). Intracellular messengers in the generation and degeneration of hippocampal architecture. J. Neurosci. Res. 21, 447464. Mayer, M. L., MacDermott, A. B., Westbrook, G. L., Smith, S. J., and Barker, J. L. (1987). Agonist and voltage gated Ca entry in cultured mouse spinal cord neurons under voltage clamp measured using Arsenazo Ill. J. Neurosci. 7, 3230-3244. McCobb, D. P., Haydon, P. G., and Kater, S . B. (1988). Dopamine and serotonin inhibition of neurite elongation of different identified neurons. J. Neurosci. Res. 19, 19-26. Meakin, S. 0.. and Shooter, E. M. (1992). The nerve growth factor family of receptors. Trends Neurosci. 15, 323-33 1. Meiri, K. F.. and Burdick, D. (1991). Nerve growth factor stimulation of GAP43 phosphorylation in intact growth cones. J. Neurosci. 11, 3155-3164. Messer, A. (1977). The maintenance and identification of mouse cerebellar granular cells in monolayer culture. Bruin Res. UO, 1-12. Michikawa, M., Kikuchi, S . , Muramatsu, H., Muramatsu, T., and Kim, S. U. (1993). Retinoic acid responsive gene product, midkine, has neurotrophic functions for mouse spinal cord and DRG neurons in culture. J . Neurosci. Res. 35,530-539. Michler, A. (1990). Involvement of GABA receptors in the regulation of neurite growth in cultured embryonic chick tectum. Int. J . Deu. Neurosci. 8, 463-472. Millan, F. A.. Denhez, F., Kondaiah, P., and Ackhurst, R. J. (1991). Embryonic gene expression patterns of TGF p l , p2 and p3 suggest different development functions in vivo. Development (Cambridge, UK)111, 131-141. Millaruelo, A. I . , Nieto-Sampedro, M., and Cotman, C. W. (1988). Cooperation between nerve growth factor and laminin or fibronectin in promoting sensory neuron survival and neurite outgrowth. Deu. Bruin Res. 38, 219-228. Morgan. J. I . , and Curran, T. (1991). Stimulus-transcription coupling in the nervous system: Involvement of the inducible proto-oncogenes fos and jun. Annu. Reu. Neurosci. 14, 421 -45 I . Morikawa, T., Odagawa, Y.,Keino, K., and Fukuda, J. (1991). New plastic plates which enhance neurite extension in culture: Roles of bisphenol-A and tricyclodecanyl units for growth and orientation of neurites on plastic plates with microstructures. Neurosci. Lett. U7. 16-20.

292

ROBERT A. SMITH AND ZHI-GANG JIANG

Moms, J. L., and Gibbins, I. L. (1989). Co-localisation and plasticity of transmitters in peripheral autonomic and sensory neurons. I n t . J . Dev. Neurosci. 7, 521-531. Morrison, R. S., Sharma, A., de Vellis, J., and Bradshaw, R. A. (1986). Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture. Proc. Natl. Acad. Sci. U.S.A. 83, 7537-7541. Morrison, R. S . , Keating, R. F., and Moskal, J. P.(1988). Basic fibroblast growth factor and epidermal growth factor exert differential trophic effects on CNS neurons. J . Neurosci. Res. 21, 71-79. Mugnai, G., and Culp, L. A. (1987). Cooperativity of ganglioside-dependent with proteindependent substratum adhesion and neurite extension of human neuroblastoma cells. Exp. Cell Res. 169,328-344. Murtomaki, S . , Risteli, J., Koivisto, U.-M., Johansson, S., and Liesi, P. (1992). Laminin and its neurite-outgrowth-promoting domain in the brain of Alzheimer's Disease and Down's Syndrome patients. J . Neurosci. Res. 32, 261-273. Nagata, K., Takei, N., Nakajima, K., Saito, H., and Kohsaka, S. (1993). Microglial conditioned medium promotes survival and development of cultured mesencephalic neurons from embryonic rat brain. J . Neurosci. Res. 34, 357-363. Neely, M. D. (1993). Role of substrate and calcium in neurite retraction of leech neurons following depolarisation. J . Neurosci. W , 1292-1301. Nelson, P. G., Yu. C., Fields, R. D., and Neale, E. A. (1989). Synaptic connections in vitro. Modulation of number and efficacy by electrical activity. Science 244, 585-587. Nelson, P. G., Fields, R. D., Yu, C., and Neale, E. A. (1990). Mechanisms involved in activity-dependent synapse formation in mammalian central nervous system cell cultures. J . Neurobiol. 21, 138-156. Neuberger, T. J., and De Vries, G. H. (1993a). Distribution of fibroblast growth factor in cultured DRG neurons and Schwann cells. I. Localisation during maturation in vitro. J . Neurocytol. 22,436-448. Neuberger, T. J., and De Vries! G. H. (1993b). Distribution of fibroblast growth factor in cultured DRG neurons and Schwann cells. 11. Redistribution after neural injury. J . Neurocytol. 22,449-460. Nonner. D., Temple, S., and Barrett, J. N. (1992). Rat embryonic septal neurons survive and express cholinergic properties in isolation and without growth factor. Dev. Brain Res. 70, 197-205. Norden, J. J., Lettes, A., Costello, B., Lin, L.-H., Wouters, B., Bock, S., and Freeman, J. A. (1991). Possible role of GAP43 in calcium regulationlneurotransmitterrelease. Ann. N . Y.Acad. Sci. 627, 75-93. Nottebohm, F. (1989). From bird song to neurogenesis. Sci. Am. 260(2), 56-61. O'Brien, R. J., and Fishbach, G.D. (1986). Excitatory synaptic transmission in chick spinal cord cell cultures. J . Neurosci. 6, 3284-3289. O'Dell, T. J., Kandel, E. R., and Grant, S . G. N. (1991). Long-term potentiation in the hippocampus is blocked by tyrosine hydroxylase kinase inhibitors. Nature (London)353, 558-560. Orr, D. J., and Smith, R. A. (1988). Neuronal maintenance and neurite extension of adult mouse neurons in non-neuronal cell-reduced cultures is dependent on substratum coating. J. Cell Sci. 91, 555-561. Owen, A., and Bird, M. (1993). GABA as a regulator of neurite growth in mouse spinal cord cultures. J. Anat. 182, 140-141. Parpura, V.,Haydon, P. G.,and Henderson, E. (1993). Three-dimensional imaging of living neurons and glia with the atomic force microscope. J . Cell Sci. 104,427-432. Patterson, P. H.,and Chun, L. L. Y.(1974). The influence of non-neuronal cells on catecholamine synthesis and accumulation in cultures of dissociated sympathetic neurons. Proc. Natl. Acad. Sci. U.S.A. 71, 3607-3610.

NEURONAL MODULATION AND PLASTICITY IN VITRO

293

Patterson, P. H., and Chun, L. L. Y. (1977). The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons. I. Effects of conditioned medium. Dev. Biol. 56, 263-280. Patterson, P. H., and Nawa. H. (1993). Neuronal differentiation factorslcytokines and synaptic plasticity. Cell (Cambridge, Mass.) 72INeuron 10, Suppl., 123-137. Pfenninger, K. H., de la Houssaye, B. A., Helmke, S. M., and Quiroga, S. (1992). Growthregulated proteins and neuronal plasticity. Mol. Neurobiol. 5 , 143-151. Phelan, P., and Gordon-Weeks, P. R. (1992). Widespread distribution of synaptophysin, a synaptic vesicle glycoprotein, in growing neurites and growth cones. Eur. J. Neurosci. 4, 1180-1190. Pixley, S . K. R., Nieto-Sampedro, M.. and Cotman, C. W. (1987). Preferential adhesion of brain astrocytes to laminin and central neurites to astrocytes. J . Neurosci. Res. 18, 402-406. Pollock, J. D., Krempin, M., and Rudy, B. (1990). Differential effects of NGF, FGF, EGF, CAMPand dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells. J. Neurosci. 10, 2626-2637. Potter, D. D., Landis, S. C., Matsumoto, S . G., and Furshpan, E. J. (1986). Synaptic functions in rat sympathetic neurons in microcultures. 11. Adrenergiclcholinergic dual status. J . Neurosci. 6, 1080-1098. Poulakos, J. J., Millard, W. J., and Meyer, E. M. (1993). Modulation of neuropeptide Y expression in rat brain neuronal cultures. Deu. Brain Res. 74, 25-29. Price, J. (1985). An immunohistochemical and quantitative examination of dorsal root ganglion neuronal subpopulations. J . Neurosci. 5, 205 1-2059. Quinn, S. D. P., and De Boni, U. (1991). Enhanced neuronal regeneration by retinoic acid of murine dorsal root ganglia and of fetal murine and human spinal cord in vitro. In Vitro Cell. Deu. Biol. 27A, 55-62. Rabizadeh, S . , Oh, J., Zhong, L-T., Yang, J., Bitler, C. M., Butcher, L. L., and Bredesen, D. E. (1993). Induction of apoptosis by the low-affinity NGF receptor. Science ( N Y )261, 345-348. Ram6n y Cajd, S. (1919). Acci6n neurotropica de las epitelios (in English [L. Guth, trans.]). In "Studies on Vertebrate Neurogenesis" (C. C. Thomas, ed.), pp. 149-200. Thomas, Springfield, IL, 1960. Raymond, L. A., Blackstone, C. D., and Huganir, R. L. (1993). Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity. Trends Neurosci. 16, 147-153. Rayport, S. G., and Schacher, S. (1986). Synaptic plasticity in vitro: Cell culture of identified Apl ysia neurons mediating short-term habituation and sensitization. J. Neurosci. 6, 759-763. Recio-Pinto, E., Rechler, M. M., and Ishii, D. N. (1986). Effects of insulin, insulin-like growth factor I1 and nerve growth factor on neurite formation and survival in cultured sympathetic and sensory neurons. J. Neurosci. 6, 121 1-1219. Rich, K. M., Luszczynski, J. R., Osborne, P. A., and Johnson, E. M., Jr. (1987). NGF protects adult sensory neurons from cell death and atrophy caused by nerve injury. J . Neurocytol. 16, 261-268. Rivas, R. J . , Burmeister, D. W., and Goldberg, D. J. (1992). Rapid effects of laminin on the growth cone. Neuron 8, 107-115. Rogers, S. L., Letourneau, P. C.. Palm, S . L., McCarthy, J., and Furcht. L. T. (1983). Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin. Dev. Biol. 98, 212-220. Rogers, S. L., McCarthy, J., Palm, S. L., Furcht, L. T., and Letourneau, P. C. (1985). Neuron-specific interactions with two neurite-promotingfragments of fibronectin. J. Neurosci. 5 , 369-378. Rogers, S . L., Letourneau, P. C., Peterson, P. C., Furcht, L. T.,and McCarthy, J. B.

294

ROBERT A. SMITH AND ZHI-GANG JIANG

(1987). Selective interaction of peripheral and central nervous system cells with two distinct cell-binding domains of fibronectin. J. Cell Biol. 105, 1435-1442. Rogister, B., Delrbe, P., Leprince, P., Martin, D., Sadzot, C., Malgrange, B., Munaut, C., Rigo, J. M., Lefebvre, P. P., Octave, J.-N.. Schoenen, J., and Moonen, G. (1993). Transforming growth factor as a neuronoglial signal during peripheral nervous system response to injury. J . Neurosci. Res. 34, 32-43. Role, L. W., and Fishbach, G. D. (1987). Changes in the number of chick ciliary ganglion neuron processes with time in cell culture. J. Cell Biol. 104, 363-370. Roufa, D. G., Johnson, M. I., and Bunge, M. B. (1983). Influence of gangLon age, nonneuronal cells and substratum on neurite outgrowth in culture. Dev. Eiol. 99, 229-239. Ruit, K. G., Osborne, P. A., Schmidt, R. E., Johnson, E. M., Jr., and Snider, W. D. (1990). Nerve growth factor regulates sympathetic ganglion cell morphology and survival in the adult mouse. J . Neurosci. 10, 2412-2419. Ruit, K. G., Elliot, J. L., Osborne, P. A., Yan, Q., and Snider, W. D. (1992). Selective dependence of mammalian dorsal root ganglion neurons on nerve growth factor during development. Neuron 8,573-587. Rutishauser, U. (1989). N-cadherin: A cell adhesion molecule in neural development. Trends Neurosci. U,275-276. Rutishauser, U., Acheson, A., Hall, A. K., Mann, D. M.,and Sunshine, J. (1988). The neural cell adhesion molecule (N-CAM) as a regulator of cell-cell interactions. Science 240,53-57. Saadat, S., Sendtner, M., and Rohrer, H. (1989).Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture. J. Cell Eiol. 108, 1807-1816. Sanes, J. R. (1989). Extracellular matrix molecules that influence neural development. Annu. Rev. Neurosci. 12,491-516. Sanfeliu, C., Hunt, A., and Patel, A. J. (1990). Exposure to N-methyl-D-aspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and not in astrocytes. Brain Res. 526, 241-248. Sargent, P. B. (1989). What distinguishes axons from dendrites? Neurons know more than we do. Trends Neurosci. U , 203-205. Savio, T., and Schwab, M. E. (1989). Rat CNS white matter, but not gray matter, is nonpermissive for neuronal cell adhesion and fiber outgrowth. J. Neurosci. 9,1126-1 133. Scarborough, D. E., Lee, S. L., Dinarello, C. A., and Reichlin, S. (1989). Interleukin-la stimulates somatostatin biosynthesis in primary cultures of fetal rat brain. Endocrinology (Baltimore) W, 549-55 1. Schacher, S. (1985). Differential synapse formation and neurite outgrowth at two branches of the metacerebral giant cell ofAplysia in dissociated cell culture. J . Neurosci. 5,2028-2034. Schacher, S . , and Montarolo, P. G. (1991). Target-dependent structural changes in sensory neurons of Aplysia accompany long-term heterosynaptic inhibition. Neuron 6, 679-690. Schacher, S., Kandel, E. R., and Montarolo, P. G. (1993). CAMP and arachidonic acid stimulate long-term structural and functional changes produced by neurotransmitters in Aplysia sensory neurons. Neuron 10, 1079-1088. Schinstine, M., and Cornbrooks, C. J. (1988). Age-dependent patterns and rates of neurite outgrowth from CNS neurons on Schwann cell-derived basal lamina and laminin substrata. Dev. Brain Res. 43, 23-27. Schoenen, J., Delree, P., Leprince, P., and Moonen, G. (1989). Neurotransmitter phenotype plasticity in cultured dissociated adult rat dorsal root ganglia: An immunocytochemical study. J . Neurosci. Res. 22, 473-487. Schubert, D. (1991). The possible role of adhesion in synaptic modification. Trends Neurosci. 14, 127-130. Schwab, M. E., Kapfhammer, J. P., and Bandtlow, C. E. (1993). Inhibitors of neurite outgrowth. Annu. Rev. Neurosci. 16,565-595.

NEURONAL MODULATION AND PLASTICITY IN VlTRO

295

Scott, B. S. (1982). Adult neurons in cell culture: Electrophysiological characterisation and use in neurobiological research. Prog. Neurobiol. 19, 187-21 1. Sebben, M., Gabrion, J., Manzoni, O., Sladeczek, F., Gril, C., Bockaert, J., and Dumuis, A. (1990). Establishment of long-term primary cultures of strial neurons. Deu. Brain Res. 52, 229-239. Shahar, A., de Vellis, J., Vernadakis, A., and Haber, B. (1989). "A Dissection and Tissue Culture Manual of the Nervous System." Alan R. Liss, New York. Shapiro, M. S., and Hille, B. (1993). Substance P and somatostatin inhibit calcium channels in rat sympathetic neurons via different G protein pathways. Neuron 10, 11-20. Sheng, M., and Greenberg, M. E. (1990). The regulation and function of c-fos and other intermediate early genes in the nervous system. Neuron 4, 477-485. Silver, R. A., Lamb, A. G., and Bolsover, S. R. (1990). Calcium hotspots caused by Lchannel clustering promote morphological changes in neuronal growth cones. Nature (London)343,75 1-754. Simpson. R. B., and Smith, R. A. (1989). Survival of adult mouse neurons in vitro is improved by the use of conditioned medium. J. Anat. 164, 265. Skene, J. H. P. (1989). Axonal growth-associated proteins. Annu. Reu. Neurosci. 12, 127- 156.

Smalheiser, N. R., Crain, S. M., and Reid, L. M. (1984). Laminin as a substrate for retinal axons in vitro. Deu. Brain Res. U,136-140. Smeyne, R. J.. Vendrell, M., Hayward, M.. Barker, S. J., Miao, G. G., Schilling, K., Robertson, L. M., Curran, T., and Morgan, J. I. (1993). Continuous c-fos expression precedes programmed cell death in vivo. Nature (London)363, 166-169. Smith, J., Vyas, S., and Garcia-Arraras, J. E. (1993). Selective modulation of cholinergic properties in cultures of avian embryonic sympathetic ganglia. J. Neurosci. Res. 34, 346-356.

Smith, R. A. (1991). Primary cultures of adult mammalian sensory neurons and other in vitro systems of use in neurotoxicological studies. Arch. Toxicol., Suppl. 14, 8-14. Smith, R. A., and McInnes, 1. B. (1986). Phase contrast and electron microscopical observations of adult mouse dorsal root ganglion cells maintained in primary culture. J. Anat. 145, 1-12.

Smith, R. A.. and Om,D. J. (1987). The survival of adult mouse sensory neurons in vitro is enhanced by natural and synthetic substrata, particularly fibronectin. J . Neurosci. Res. 17, 265-270.

Stewart, H. J. S., Cowen, T., Curtis, R., Wilkin, G. P., Mirsky, R., and Jessen, K. R. (1992). Gap43 immunoreactivity is widespread in the autonomic neurons and sensory neurons of the rat. Neuroscience 47, 673-684. Thoenen, H. (1991). The changing scene of neurotrophic factors. Trends Neurosci. 14, 165-170.

Tokioka. R., Matsuo. A.. Kiyosue, K., Kasai, M., andTaguchi, T. (1993). Synapse formation in dissociated cell cultures of embryonic chick cerebral neurons. Deu. Brain Res. 74, 146- 150.

Torimitsu, K., and Kawana, A. (1990). Selective growth of sensory nerve fibres on metal oxide pattern in culture. Deu. Brain Res. 51, 128-131. Unsicker, K., Skaper, S. D., Davis, G. E., Manthorpe, M.,and Varon, S. (1985). Comparison of the effects of laminin and polyornithine-binding neurite promoting factor from RN Schwannoma cells on neurite regeneration from cultured newborn and adult rat dorsal root ganglion neurons. Deu. Brain Res. 17, 304-308. Unsicker. K., Flanders, K. C., Cissel, D. S., Lafyatis, R., and Sporn, M.B. (1991). Transforming growth factor beta isoforms in the adult rat central and peripheral nervous system. Neuroscience 44, 613-625. Unsicker, K.. Reichert-Preibsch, H., and Wewetzer, K. (1992). Stimulation of neuron sur-

296

ROBERT A. SMITH AND ZHI-GANG JIANG

vival by basic FGF and CNTF is a direct effect and not mediated by non-neuronal cells: evidence from single cell cultures. Deu. Brain Res. 65, 285-288. Van Lookeren Campagne, M., Dotti, C. G., Jap Tjoen San, E. R. A., Verkleij, A. J., Gispen, W. H., and Oestreicher, A. B. (1992). B-50/GAP43 localisation in polarised hippocampal neurons in vitro: An ultrastructural quantitative study. Neuroscience 50, 35-52. Vedder, H., and Otten, U. (1991). Biosynthesis and release of tachykinins from rat sensory neurons in culture. J. Neurosci. Res. 30,288-299. Walicke, P. A. (1989). Novel neurotrophic factors, receptors and oncogenes. Annu. Rev. Neurosci. 12, 103-126. Walicke, P. A., Cowan, W. M., Ueno, N., Baird, A., and Guillemin, R. (1986). Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. Natl. Acad. Sci. U.S.A. 83,3012-3016. Wang, F. Z., Nelson, P. G., Fitgerald, S. C., Hersh, L. B., and Neale, E. A. (1990). Cholinergic function in cultures of mouse spinal cord neurons. J . Neurosci. Res. 25, 3 12-323. Weeks, B. S.,DiSalvo, J., and Kleinman, H.K. (1990). Laminin-mediatedprocess formation in neuronal cells involves protein dephosphorylation. J. Neurosci. Res. 27, 418-426. Wehrle, B., and Chiquet, M. (1990). Tenascin is accumulated along developing peripheral nerves and allows neurite outgrowth in vitro. Development (Cambridge, U K )110,401-415. Weiss, S., Pin, J. P., Sebben, M., Kemp, D. E..Sladeczek, F., Gabrion, J., and Bockaert, J. (1986). Synaptogenesis of cultured striatal neurons in serum-free medium: a morphological and biochemical study. Proc. Natl. Acad. Sci. U.S.A. 83, 2238-2242. Windebank, A. J., and Blexrud, M. D. (1989). Biological activity of a new neuronal growth factor from injured peripheral nerve. Dev. Bruin Res. 49, 243-25 1. Winter, J., Forbes, C. A., Sternberg, J., and Lindsay, R. M. (1988). Nerve growth factor (NGF) regulates adult rat cultured dorsal root ganglion neuron responses to the excitotoxin capsaicin. Neuron 1, 973-981. Winter. J., Dray, A., Wood, J. N., Yeats, J. C., and Bevan, S.(1990). Cellular mechanisms of action of resiniferatoxin: A potent sensory neurotoxin. Brain Res. 520, 131-140. Wood, J. N. (1992). “Neuronal Cell Lines: A Practical Approach.” Oxford Univ. Press, Oxford. Woolf, C. J., Reynolds, M. L., Molander, C., O’Brien, C., Lindsay, R. M., and Benowitz, L. I. (1990). The growth-associated protein GAP43 appears in DRG cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury. Neuroscience 34,465-478. Wu, C.-F., Suzuki, N., and Poo, M.-M. (1983). Dissociated neurons from normal and mutant Drosophila larval central nervous system. J. Neurosci. 3, 1888-1899. Yasuda, T., Sobue, G., Ito, T.,Mitsuma, T., and Takahashi, A. (1990). Nerve growth factor enhances neurite arborisation of adult sensory neurons; a study in single-cell culture. Brain Res. 524, 54-63. Yong, V. W., Hone, H., and Kim, S. U. (1988). Comparison of six different substrata on the plating efficiency, differentiation and survival of human dorsal root ganglion neurons in culture. Dev. Neurosci. 10, 222-230. Zhang, M., Woo, D. D. L., and Howard, B. D. (1990). Transforming growth factor a and a PC12-derived growth factor induce neurites in PC12 cells and enhance survival of embryonic brain neurons. Cell Regul. 1, 511-521.

Index

A

Biopsy polar body, 4 cleavage-stage, 5 Blood, dendritic cells adherence depletion, 49 contaminating cells, removal, 51-52 density-gradient separations, 49,51 fresh cell isolation, 52-53 immunophenotypic cell separations, 52 isolation protocols, 48-53 panning techniques, 51-52 phagocyte depletion, 52 properties, 53 Bone marrow, dendritic cells ontogeny, 69-70 studies, 48 Brain-derived neurotrophic factor, neurite initiation and elongation role, 247, 251-252

Acetyl-L-carnitine, neurite initiation and elongation role, 255 Acidic fibroblast growth factors, neurite initiation and elongation role, 253 Adhesion molecules, see Cell adhesion molecules Allergy, dendritic cell role in, 90 y-Aminobutyric acid, as neurite inhibitor, 257 Amniocentesis, 2-4 Angelman syndrome, prenatal screening with FISH, 30,32-33 Antibodies, monoclonal, dendritic cell-specific, 46 Antigens, and dendritic cells processing, 72, 74-75 stimulation of primary immune response, 77 Autoimmune disease, dendritic cell role in, 88-89 Autosomal imbalances, FISH diagnosis chromosomes anomalies, 28, 30-31 painting, 28, 30-31 Philadelphia chromosome, 30 specific repeat probes, 25-27 trisomy 21 detection, 27-29 DNA deletions, 30-33 duplications, 33

C Calcitonin-gene-related peptide, phenotypic expression affected by, 275 Calcium, neurite outgrowth regulatory role, 257 Cancer cytogenetics clinical studies, 216-217 comparative genomic hybridization use in examination, 218-219 Cell adhesion molecules and dendritic cells, costimulation of T cells, 78-82 neurite initiation and elongation role, 246 Central nervous system, vertebrate neuronal cell models, 236-237

B Basic fibroblast growth factors, neurite initiation and elongation role, 253

297

INDEX

298 Charged-coupled device, in whole-chromosome hybridization microscopy, 181 Choline acetyltransferase, trophic factor-induced neuronal activity, 27 1-274 Chorionic villus sampling, 2-4 Chromatin, ciliated protozoa, replication models, 161-166 Chromatin granules, in replication band of ciliated protozoa macronuclei, 149-150, 152 models, 164-165 Chromosomes aberrations FISH diagnosis, 25-33 formation mechanisms, 204-208 hybridization, see Hybridization, whole-chromosome locus-specific probes, 27-28 painting, 16-17, 28, 30-31 Philadelphia, 28-30 preimplantation embryo analysis, 4, 22-25 premature condensation, 207-208 specific repeat probes, 25-27 trisomy 21 detection, 27-29 yeast artificial, probes, 195 Ciliary neurotrophic factor, and neuronal plasticity neurite initiation and elongation, 252253 phenotypic expression, 274 Cleavage-stage biopsy, for preimplantation embryo analysis, 5 Clones, chromosome-specificDNA, in whole-chromosome hybridization, 193- 195 Colony-stimulatingfactor- I , secretion by granulated metrial gland cells, 122 Concanavalin A, neurite initiation and elongation role, 247 Confocal laser scanning microscopy, in whole-chromosome hybridization, 187 Cyclic AMP,growth cone regulation, 260, 262 Cytogenetics cancer, 216-217 clinical, 214-219 Cytokine receptors, costimulation by dendritic cells, 82-83

Cytokines, see also specific cytokines dendritic cells affected by costimulation, 82-83 regulation, 84-85 secretion by granulated lymphoid cells of pregnant uterus, 121-123

D Decidualization, uterine, 105-106 Degenerate oligonucleotide-primed polymerase chain reaction, in whole-chromosome hybridization clinical studies, 217-218 method, 1%-197 Dendritic cells, human adenoidal, 66-67 adhesion molecules costimulation, 78-82 effects, 85-86 allergy, 90 autoimmune disease, 88-89 blood adherence depletion, 49 contaminating cells, removal, 5 1-52 density-gradient separations, 49, 5 1 fresh cell isolation, 52-53 immunophenotypic cell separations, 52 isolation protocols, 48-53 panning techniques, 51-52 phagocyte depletion, 52 properties, 53 bone marrow, 48,69-70 characterization, 43.45 clinical applications immunization, 90 transplantation, 90-91 costimulator molecules, effects 85-86 cytochemical characteristics, 46-47 cytokine receptors, costimulation, 82-83 cytokines, costimulation, 82-83 differentiation, 43-44 functional activities, 42-43 functional properties allogeneic mixed leukocyte reaction, 76 antigen processing. 72,74-75

INDEX antigen-specificT-lymphocyte sensitization, 77 autologous mixed leukocyte reaction, 76-77 cellular interactions, 83-84 costimulation, 78-86 cytokine regulation, 84-85 mitogenic assay stimulation, 77-78 regulation of activity, 84-86 hypersensitivity, 90 identification, 41-43,45 immune response, stimulation of primary, 76-77 secondary, 77-78 immunodeficiency, 89 infectious diseases human immunodeficiency virus, 89 leprosy, 89-90 interstitial, 60-64 Langerhans cells isolation, 55-57 properties, 54.57-62 liver, 62-63 lung, 63 lymphatic, afferent, 65-66 lymphoid, 64-68 malignancies histiocytosis X, 86 Hodgkin’s disease, 86 monoclonal antibodies, 46 morphology, 46 mucosal, 63 ontogeny blood cell precursors, 70-71 bone marrow, 69-70 migration, 71-72 myeloid lineages, relationship, 72-73 phenotypic comparisons of subtypes, 60-61 preparations, purity of, 45-47 psoriasis, 90 rheumatic disease, 88-89 splenic, 67 subtypes, 42 synovial fluid, 63-64 thymic, 68 tonsil, 66-67 transplantation, 87.90-91 veiled, 65-66 Dioxigenin, probes for genetic disease diagnosis by FISH, 10

DNA autosomal imbalance diagnosis

deletions, 30-33 duplications, 33 preimplantation embryo analysis, 4 probes for genetic disease diagnosis by FISH, 7, 10-1 1, 13-14 in replication band of ciliated protozoa chromatin replication models, 162-165 cytochemical studies, 154 function, 144- 145, 147- I48 macronuclear models, 137-138, 142 ultrastructure, 152 in whole-chromosome hybridization chromosomal aberration analysis, 204-207 cloned chromosome-specific DNA probe libraries, 193-195 detection methods, 181, 183 genomic probes, 191-193 polymerase chain reaction, 1%-I97 probe labeling methods, 179-180 probe techniques, 190-191 in situ hybridization technique, 172-178 tumor, as whole chromosome hybridization probe, 192 typical methods, 188 DNA synthesis, primed in situ, 20 Dorsal root ganglion neurons, plasticity fibronectin role, 243-244 growth cones, 260 laminin role, 244-245 nerve growth factor role, 248-252 neurite inhibitors, 256 neurotrophic factor role, 255-256 phenotypic expression, 272, 275-276 substrate role, 238-239 synaptic connections, 265

E Embryos preimplantation diagnostic procedure, 2 sexing of, 22-25 in uirro fertilized, sampling, 4 Endometrial granulocytes, human, of pregnant uterus immune-effector function, 124-125

300

INDEX

nonimmunological functions, 126 properties, 105, 117, 119-121 Epidermal growth factor, neurite initiation and elongation, 254-255 Epifluorescence microscopy, in whole-chromosome hybridization, 184-187 Euplotes, replication band chromatin replication models, 163, 165 early studies, 141-142 function, 144, 146 immunochemical studies, 156-157, 159-160 ultrastructure, 149, 152 Euplotes eurystomus, replication band characterization, 137, 139 chromatin replication models, 165 cytochemical studies, 154 function, 144-145, 148 immunochemical studies, 156, 158-160 ultrastructure, 150- 152 Euplotes octocarinarus, replication band, 146- I47 Evolution, in whole-chromosome hybridization studies, 199-200

F Fibroblast growth factors, neurite initiation and elongation role, 253-254 Fibronectin, neurite initiation and elongation role, 243-244 FISH, see Fluorescent in situ hybridization Fluorescein isothiocyanate, in FISH diagnosis of genetic diseases, 11-12 Fluorescence microscopy, in whole-chromosome hybridization, 183- 187 Fluorescent in situ hybridization, for diagnosis of genetic diseases applications, 6-14 autosomal imbalances, 25-33 chromosomes anomalies, 28.30-31 painting, 16-17,28, 30-31 Philadelphia, 30 specific repeat probes, 25-27 trisomy detection, 27-29 comparative genome hybridization, 21

DNA deletions, 30-33 duplications, 33 halo preparations, 21 isotopic, 7 multicolor FISH, 19-20 polymerase chain reaction technology, 20 procedure, 1-2 samples postnatal, 3 preimplantation embryo, 4-6 prenatal, 3-4 sexing of human cells clinical application, 24-25 early studies, 22-23 patient strategies, 25 postnatal samples, 21 preimplantation embryos, 22-25 prenatal samples, 22 research needs, 22 single-color FISH, 24 studies, 23-24 single locus detection, 17-19 tandem repetitive probes, 14-16 technological advances, 33-34 three-dimensional, 20-21 total genomic probes, 18 types, 6-14 Fluorochromes, in whole-chromosome hybridization chromosomal aberration analysis, 206 techniques, 184-188

G Genetic diseases, fluorescent in situ hybridization in diagnosis, see Fluorescent in situ hybridization Genetic studies, whole-chromosome hybridization in, 2 14-219 Genetic toxicology, whole-chromosome hybridization applications, 208-2 13 Genomic hybridization, comparative, 192-193,218 Granulated lymphoid cells, of pregnant uterus, see Lymphoid cells, granulated, of pregnant uterus

301

INDEX

Granulated metrial gland cells, rodent colony-stimulating factor- I secretion by, 122 cytokine secretion, 122 immune-effector function, 123-125 morphology, 113-1 19 nonimmunological functions, 125- I26 origins, 111-113 phenotype, 113-119 properties, 105 Granulocytes, endometrial, of pregnant uterus immune-effector function, 123-125 nonimmunological function, 125- 126 properties, 117, 119-121 Growth-associated proteins, neurite initiation and elongation role, 262-263 Growth cones, neurite initiation and elongation role, 258-262

H Halreria, replication band, 139 Helisoma, neuronal modulation growth cone activity, 259-261 neurotransmitter effects, 257 synaptic connections, 269-270 Histiocytosis X,as dendritic cell malignancy, 86 Hodgkin's disease, dendritic cell association, 86 Human immunodeficiency virus, dendritic cell role in, 89 Hybridization comparative genomic clinical studies, 218 technique, 21, 192-193 fluorescent in sifu, see Fluorescent in siru hybridization Hybridization, whole-chromosome applications characteristics, 197-198 chromosomal aberrations, 204-208 clinical studies, 214-219 evolutionary studies, 199-200 genetic toxicology, 208-2 13 mammals, 199-200 nuclear architecture, 201-204 plants, 200 primates, 199-200

reverse experiments, 217 taxonomic studies, 199-200 Comparative genomic hybridization, 192- I93 development of techniques, 174- 175 experimental uses, 172-173 methods, 171-173,219 probes cloned chromosome-specific DNA probe libraries, 193-195 genomic DNA, 191-193 labeling methods, 178-181 methods, 190-191 polymerase chain reaction methods, 195- I98 in sifu hybridization techniques detection methods, 181-184 microscopy, 183-188 nucleic acid, 175-178 probe labeling methods, 178-181 typical method, 188-190 yeast artificial chromosome probes, 195 Hypersensitivity, dendritic cell role in, 90 Hypotrichs, replication band characterization, 138-139 chromatin replication models, 162-165 early studies, 143 function, 148

I Immunization, dendritic cell role in, 90 Immunochemical studies, replication band of ciliated protozoa, 155-161 Immunodeficiency, dendritic cell role in, 88-89 Immunophenotypic separations, dendritic cell, 52 Infectious diseases, dendritic cell role in human immunodeficiency virus, 89 leprosy, 89-90 Interleukin-I dendritic cell interactions, 82-84 rat diencephalic neurons affected by, 275 secretion by granulated metrial gland cells, 122 Interleukin-2, endometrial granulocyte-stimulated, 124 Interleukin-6, costimulation with dendritic cells, 83

302

INDEX

L Laminin, neurite initiation and elongation role, 244-246 Langerhans cells isolation, 55-57 properties, 54,57-62 Leprosy, dendritic cell role in, 89-90 Leukemia inhibitory factor neuropeptide Y expression affected by, 27 1 secretion by granulated metrial gland cells, 122-123 Leukocyte reaction, mixed, dendritic cell role dogeneic, 76 autologous, 76-77 LIF, see Leukemia inhibitory factor Liver, dendritic cells, 62-63 Long-term potentiation, mechanism, 267 Lung, dendritic cells, 63 Lymphatic dendritic cells, human, 65-66 Lymphocytes, T, see T lymphocytes Lymphoid cells, granulated, of pregnant uterus characterization, 105, 126 decidual tissue-associated cells, 107- 109 natural killer cells, 107-108 neutrophils, 108-109 suppressor cells, 109 endometrium, human, 117, 119-121 functions cytokine secretion, 121-123 immunological, 123-125 nonimmunological, 125- 126 metrial gland cells, rodent characteristics, 110-1 1 I morphology, 113-1 19 origin, 111-113 phenotypes, 113-1 19 Lymphoid dendritic cells, human, 64-68 Lymphoid tumors, whole-chromosome hybridization use in clinical studies, 216

M Mammals, in whole-chromosome hybridization studies, 199-200 Mental retardation, FISH diagnosis, 26-29

Metrial gland, 110-1 11 granulated cells, see Granulated metrial gland cells Microscopy confocal laser scanning, in whole-chromosome hybridization, 187 epifluorescence, in whole-chromosome hybridization, 184-187 fluorescence, in whole-chromosome hybridization, 183-187 in whole-chromosome hybridization, 183- 188 Midkine, neurite initiation and elongation role, 255 Mitogenic assay, dendritic cell-stimulated, 77-78 MLR, see Leukocyte reaction, mixed Monoclonal antibodies, dendritic cell-specific, 46 Mucosal dendritic cells, human, 63 Muscular dystrophy, prenatal screening with FISH, 30-32

N Natural killer cells, decidual, of pregnant uterus, 107-108 Nerve growth factor, and neuronal plasticity neurite initiation and elongation, 247-252 phenotypic expression, 273, 275-279 studies, 235 Neuronal plasticity cell cultures invertebrate neuronal cells, 236 model systems, 235 transformed cell lines, 235-236 vertebrate neuronal cells, 236-237 definition, 233 future investigations, 280 neurite initiation and elongation acetyl-L-carnitine, 255 brain-derived neurotrophic factor, 247, 25 1-252 cellular adhesion molecules, 246 ciliary neurotrophic factor, 252-253 concanavalin A, 247 epidermal growth factor, 254-255 fibroblast growth factors, 253-254

303

INDEX

fibronectin, 243-244 growth-associated proteins, 262-263 growth cones, 258-262 laminin, 244-246 mechanisms, 237-238 midkine, 255 nerve growth factor, 247-252 neurite inhibitors, 256-258 neurotrophins, 248-252 patterned substrate role, 238-242 retinoic acid, 255 soluble neurite-promoting factor, 255 substratum role, 238-247 synthetic substrate role, 238-242 transforming growth factors, 254-255 trophic factors, 247-258 phenotypic expression adult neurons, 275-279 developing neurons, 27 1-275 mechanism, 270-271 neonatal neurons, 271-275 studies, 234-235 synaptic connections excitatory postsynaptic potentials, 265-267 invertebrate neurons, 268-270 mechanism of formation, 263-264 vertebrate neurons, 264-268 technological advances, 279-280 Neurons, see Dorsal root ganglion neurons; Superior cervical ganglion neurons Neuropeptide Y, phenotypic expression, 27 1 Neurotransmitters, role in nervous system function, 257 Neurotrophic factors brain-derived, 247.25 1-252 ciliary, 252-253,274 Neurotrophins, neurite initiation and elongation, 248-252 Neutrophils, decidual, of pregnant uterus, 108-109

0 Oxytricha, replication band chromatin replication models, 165 cytochemical studies, 154 ultrastructure, 150, 152

P Peptide, calcitonin-gene-related, phenotypic expression affected by, 275 Perforin, expression in granulated metrial gland cells, 123-124 Peripheral nervous system, vertebrate neuronal cell models, 236-237 Phagocytes, depletion, dendritic cell, 52 Phenotypes dendritic cells, human, comparisons of subtypes, 60-61 granulated metrial gland cells, rodent, 113-119 Phenotypic expression, and neuronal plasticity adult, 275-279 developing, 271-275 mechanism, 270-27 I neonatal, 271-275 Plants nuclear organization, 203 whole-chromosome hybridization studies, 200 Plasticity, neuronal, see Neuronal plasticity Polar body biop,sy,preimplantation embryo analysis, 4 Polymerase chain reaction in fluorescent in siru hybridization, 20 whole-chromosome hybridization clinical studies, 217 degenerate oligonucleotide-primed, 1%-197,217-218 interspersed repetitive sequence, 195-1%, 217 linker-adaptor, 197 method, 195-198 random primed, 1%-I97 Pregnancy, and granulated lymphoid cells of uterus, see Lymphoid cells, granulated, of pregnant uterus Primates, whole-chromosome hybridization studies, 199-200 Proteins growth-associated, neurite initiation and elongation role, 262-263 neurite elongation and initiation role, 237-238,243 Protozoa, see also specific protozoa replication band, 137-167 Psoriasis, dendritic cell role in, 90

304

INDEX

R Replication band, ciliated protozoa characterization, 137-138, 166 chromatin replication models, 161-166 cytochemical studies, 154- 155 early studies, 140-143 functional characteristics, 143-148 hypotrichs characterization, 138-139 chromatin replication models, 162-165 early studies, 143 function, 148 immunochemical studies, 155-161 macronuclei characterization, 137-139 chromatin replication models, 162-165 cytochemical studies, 155 function, 144, 146, 148 immunochemical studies, 156-157,

single-color FISH, 24 studies, 23-24 prenatal samples, 22 Somatostatin, neuronal phenotypic expression affected by, 272,276-278 Spleen, dendritic cells, 67 Stylonchia, replication band cytochemical studies, 154 ultrastructure, 150, 152 Superior cervical ganglion neurons, phenotypic plasticity, 271 Suppressor cells, decidual, of pregnant uterus, 109 Synapses, and neuronal plasticity formation mechanism, 263-264 invertebrate neurons, 268-270 postsynaptic potentials, 265-267 vertebrate neurons, 264-268 Synovial fluid dendritic cells, human, 63-64

159-160

ultrastructure, 149-150, 152 micronuclei characterization, 138 function, 144 immunochemical studies, 156-157, 159 microscopy, 140. 151 properties, 167 structure, 137-138, 166 ultrastructure, 148- 154 Retinoic acid, neurite initiation and elongation role, 255 Rheumatic disease, dendritic cell role in, 88-89

RNA, in whole-chromosome hybridization detection methods, 181 development of technique, 174 probe labeling methods, 179-180 in situ hybridization technique, 176

S Sexing, of human cells postnatal samples, 21 preimplantation embryos, 22-25 clinical application, 24-25 early studies, 22-23 patient strategies, 25 research needs, 22

T Taxonomy, in whole-chromosome hybridization studies, 200 Tetrahymena, replication band chromatin replication models, 165 immunochemical studies, 157- 159 Tetramethylrhodamine isothiocyanate, in FISH diagnosis of genetic diseases, 11-12

Thymus, dendritic cells isolation, 68 properties, 68 T lymphocytes, dendritic cell interactions antigen-specific sensitization, 77 costimulation, 78-82.84-86 Tonsil, dendritic cells isolation, 66-67 properties, 66-67 Transforming growth factors, and neuronal plasticity neurite initiation and elongation role, 254-255

phenotypic expression, 274 Transplantation, dendritic cell role in clinical applications, 90-91 process, 87 Trisomy, FISH diagnosis, 26-29

305

INDEX

Trophic factors, neurite initiation and elongation role, 247-258 acetyl-L-carnitine, 255 brain-derived neurotrophic factor, 247, 251-252 ciliary neurotrophic factor, 252-253 epidermal growth factor, 254-255 fibroblast growth factors, 253-254 midkine, 255 nerve growth factor, 247-252 neurotrophins, 248-252 retinoic acid, 255 soluble neurite-promoting factor, 255 transforming growth factors, 254-255 Trophoblasts, granulated metrial gland cell modification by, 124-126 Trophoectoderm material, diagnosis, 4-5 Tumor DNA, as whole-chromosome hybridization probe, 192 Tumor necrosis factor, dendritic cell interactions, 83-84 Tumors, whole-chromosome hybridization analysis comparative genomic hybridization, 2 18 lymphoid tumors, 216 methods, 2 18-2 I9

U Uterus, pregnant decidualization, 105-106 granulated lymphoid cells, see Lymphoid cells, granulated, of pregnant uterus

v Veiled dendritic cells, human, 65-66

W Whole-chromosome hybridization, see Hybridization, whole-chromosome

Y Yeast artificial chromosome probes, in whole-chromosome hybridization, 195

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