Table Of ContentNATO ASI Series
Advanced Science Institutes Series
A series presenting the results of activities sponsored by the NA TO Science
Committee, which aims at the dissemination of advanced scientific and
technological knowledge, with a view to strengthening links between scientific
communities.
The Series is published by an international board of publishers in conjunction with
the NATO Scientific Affairs Division
A Life Sciences Plenum Publishing Corporation
B Physics London and New York
C Mathematical and Kluwer Academic Publishers
Physical Sciences Dordrecht, Boston and London
o
Behavioural and
Social Sciences
E Applied Sciences
F Computer and Springer-Verlag
Systems Sciences Berlin Heidelberg New York
G Ecological Sciences London Paris Tokyo
H Cell Biology
Series H: Cell Biology Vol. 26
The ASI Series Books Published as a Result of
Activities of the Special Programme on
CELL TO CELL SIGNALS IN PLANTS AND ANIMALS
This book contains the proceedings of a NATO Advanced Research Workshop held within the
activities of the NATO Special Programme on Cell to Cell Signals in Plants and Animals, running
from 1984 to 1989 under the auspices of the NATO Science Committee.
The books published as a result of the activities of the Special Programme are:
Vol. 1: Biology and Molecular Biology of Plant-Pathogen Interactions. Edited by J. A. Bailey. 1986.
Vol. 2: Glial-Neuronal Communication in Development and Regeneration.
Edited by H. H. Althaus and W. Seifert. 1987.
Vol. 3: Nicotinic Acetylcholine Receptor: Structure and Function. Edited by A. Maelicke. 1986.
Vol. 4: Recognition in Microbe-Plant Symbiotic and Pathogenic Interactions.
Edited by B. Lugtenberg. 1986.
Vol. 5: Mesenchymal-Epithelial Interactions in Neural Development.
Edited by J. R. Wolff, J. Sievers, and M. Berry. 1987.
Vol. 6: Molecular Mechanisms of Desensitization to Signal Molecules.
Edited by 1 M. Konijn, P. J. M. Van Haastert, H. Van de(Starre, H. Van der Wei,
and M. D. Houslay. 1987.
Vol. 7: Gangliosides and Modulation of Neuronal Functions. Edited by H. Rahmann. 1987.
Vol. 9: Modification of Cell to Cell Signals During Normal and Pathological Aging.
Edited by S. Govoni and F. Battaini. 1987.
Vol. 10: Plant Hormone Receptors. Edited by D. Klambt. 1987.
Vol. 11 : Host-Parasite Cellular and Molecular Interactions in Protozoal Infections.
Edited by K.-P' Chang and D. Snary. 1987.
Vol. 12: The Cell Surface in Signal Transduction.
Edited by E. Wagner, H. Greppin, and B. Millet. 1987.
Vol. 19: Modulation of Synaptic Transmission and Plasticity in Nervous Systems.
Edited by G. Hertting and H.-C. Spatz. 1988.
Vol. 20: Amino Acid Availability and Brain Function in Health and Disease.
Edited by G. Huether. 1988.
Vol. 21: Cellular and Molecular Basis of Synaptic Transmission. Edited by H. Zimmermann. 1988.
Vol. 23: The Semiotics of Cellular Communication in the Immune System.
Edited by E. E. Sercarz, F. Celada, NA Mitchison, and 1 Tada. 1988.
Vol. 24: Bacteria, Complement and the Phagocytic Cell. Edited by F. C. Cabello and C. Pruzzo. 1988.
Vol. 25: Nicotinic Acetylcholine Receptors in the Nervous System.
Edited by F. Clementi, C. Gotti, and E. Sher. 1988.
Vol. 26: Cell to Cell Signals in Mammalian Development.
Edited by S. W. de Laat, J. G. Bluemink, and C. L. Mummery. 1989.
Cell to Cell Signals in
Mammalian Development
Edited by
S.W de Laat
J. G. Bluemink
C.L. Mummery
Hubrecht Laboratory
The Netherlands Institute for Developmental Biology
Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
Springer -Verlag
Berlin Heidelberg New York London Paris Tokyo
Published in cooperation with NATO Scientific Affairs Division
Proceedings of the NATO Advanced Research Workshop on Cell-to-Cell Signalling
in Mammalian Development held in Les Arcs, France, from February 21-26, 1988
Library of Congress Cataloging-in-Publication Data. NATO Advanced Research Workshop on Cell to Cell
Signals in Mammalian Development (1988: Les Arcs, Savoie, France) Cell to Cell signals in mammalian
development! edited by S. W. de Laat, J. G. Bluemink, C L. Mummery. p. cm.-(NATO ASI series. Series H,
Cell biology; vol. 26) "Proceedings of the NATO Advanced Research Workshop on Cell to Cell Signals in
Mammalian Development held in Les Arcs, France, from February 21-26,1988"-Tp. verso. "Published in
cooperation with NATO Scientific Affairs Division."
ISBN-13: 978-3-642-73 144-0 e-ISBN-13: 978-3-642-73 142-6
DOl: 10.1007/978-3-642-73142-6
1. Mammals-Cytology-Congresses. 2. Mammals-Development-Congresses. 3. Cell interaction-
Congresses. I. Laat, S. W. de (Siegfried W.), 1945- II. Bluemink, J. G. (Johannes Gerardus), 1935-
III. Mummery, C. L. (Christine L.), 1953- IV. North Atlantic Treaty Organization. Scientific Affairs Division.
V. Title. VI. Series. QL739.15.N371988 599'.0333-dc 19 88-36813
This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concern!3d,
specifically the rights oftranslation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on
microfilms or in other ways, and storage in data banks. Duplication of this publication or parts thereof is only
permitted under the provisions of the German Copyright Law of September 9, 1965, in its version of June 24,
1985, and a copyright fee must always be paid. Violations fall under the prosecution act of the German
Copyright Law.
© Springer-Verlag Berlin Heidelberg 1989
Softcover reprint of the hardcover 1s t edition 1989
2131/3140-543210 - Printed on acid-free paper
Preface
Developmental biology is the area of the Life Sciences that deals
with the r~gulatory principles underlying the generation of a new organism.
Virtually all multicellular organisms originate from a single cell, the fertilized
egg cell or zygote, through a complex and integrated process of cell multi
plication, cell differentiation and organisation of cells into tissues and organs
in the right positions and proportions within an embryo.
Until quite recently, our knowledge of the molecular and cellular mechanisms that
guide mammalian development was minimal. The rapid advances of molecular biology
and cell biology have radically changed this situation however. Major contributions
have come from (i) the recognition that regulatory genes identified in invertebrate
morphogenesis have their evolutionarily conserved homologues in vertebrates; these
include certain proto-oncogenes and homeotic genes which can now be readily
characterized; (ii) the identification and molecular characterization of genes and
gene products involved in cell-to-cell signalling such as polypeptide growth fac
tors and extracellular matrices which may in their turn control morphogenesis, cell
proliferation and differentiation; and (iii) the use of cultured equivalents of
early embryonic cells, such as embryo-derived stem cell lines, and the application
of transgenic methodology.
The NATO Advanced Research Workshop on "Cell-to-Cell Signalling in Mammalian
Development" held in Les Arcs, France, from February 21 to 26, 1988, provided an
opportunity to review and discuss intensively the most recent advances in this area
of research. The organizers had in mind to create a platform for discussion among
colleagues who view the problems of mammalian development and, more specifically,
the molecular nature and developmental significance of cell-to-cell signals, from
different perspectives. Presentations and discussions focused on (i) hoxgenes,
'their products and their potential role in mammalian morphogenesis; (ii) growth
factors, their receptors and signal transduction, and their role in guiding cell
multiplication, differentation and transformation; and (iii) cell adhesion and
extracellular matrix molecules, as short-range cell-to-cell signals in mammalian
development.
This book presents the contents of the main lectures and a selection of relevant
poster contributions presented during this workshop. It is intended to make the
proceedings of this meeting accessible to a larger audience. We hope that it will
stimulate further exchange of views and news in this fascinating area of research.
The organizers of this Workshop wish to acknowledge the NATO Scientific Affairs'
Division for their generous support, and the International Socie~ of Developmental
Biologists, the European Developmental Biology Organisation and the Hubrecht
Foundation for their contributions. We also wish to thank Ms. Sabina Rusch for her
excellent secretarial assistance, and Drs. Peter Gruss, Joseph Schlessinger and
Jean Paul Thierry for co-organizing the workshop.
September 1988 Siegfried W. de Laat
John J. Bluemink
Christine L. Mummmery
Utrecht, The Netherlands
VI
I. HOMEOOOMAIN CONTAINING GENES:
STRUCTIJRE AND ROLE IN DEVEWPMENT
The Murine Homeobox Protein Hox 1.1 is Differentially
Expressed in Embryonal Mesoderm and Ectoderm...... 1
F. Schulze, M. Kessel and P. Gruss
Structure, Expression and Evolutionary Relationships of
Murine Homeobox Genes in the Hox 2 Cluster........ 9
N. Papa1opu1u, A. Graham, J. LDrimer, R. Kenny,
J. McVey and R. Krurn1auf
Regulation of Expression of the Hox 2.3 Gene............ 23
F. Meij1ink, L. de Laaf, P. Verrijzer, W. de Graaff
and J. Deschamps
The Mouse En-1 Gene Encodes a Nuclear Protein Expressed
in the Nervous System of the Developing Embryo.... 43
G.R. Martin and M.A. Frohman
The Hox 1.3 Homeo Box Gene Encodes a Sequence Specific
DNA Binding Phosphoprotein........................ 53
W.F. Odenwa1d, J. Garbern, H. Arnheiter, E. Tournier
Lasserve, H.A. Overton and R.A. Lazzarini
AnalYSis of Transcription of the Murine Homeobox Gene
Hox 1.3........................................... 63
J. Garbern, W. Odenwa1d, E. Tournier-Lasserve and
R. Lazzarini
The Human Cognate of the Murine Hox 1. 3 Homeo Box Gene
is Almost Identical to its Murine Counterpart..... 75
E. Tournier-Lasserve, W. F. Odenwa1d, J. Garbern and
R.A. Lazzarini
Temporal and Cellular Specificity of Expression of the
Murine Homeobox-containing Gene Hox 1.4. . . . . . . .. . . 85
D.J. Wolgemuth, M. Mostoller, C.M. Viviano,
Z.F. Zakeri, Y. Iwaoki and G.L. Mutter
Murine Homeo-Genes: Some Aspects of their Organisation
and Structure. . . . . . . . . . . . . .. .. . . . . . . . . .. .. . . . . . . . . 97
D. Dubou1e, B. Ga11iot, A. Baron and
M.S. Featherstone
VII
II. EXTRACElLULAR MATRICES AND CElL ADHESION MOLECULES
Cell Adhesion in Morphogenesis .......................... 109
J.P. Thiery
Generation of Diversity in the NCAM Adhesion Protein
of Rodents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
C. Goridis, M.-J. Santoni, M.-R. Hirsch, J. Barbas,
D. Barthels and W. Wille
Ultrastructural Localization of the Cell Adhesion
Molecule Uvomorulin using Site-directed
Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
R. Kemler and H. Schwarz
III. GROWTH FACTORS, RECEPTORS AND ONCOGENES:
STRUCTURE AND FUNCTION
Mutational Analysis of the EGF Receptor Proto-Oncogene .. 153
J. Schlessinger, A.M. Honegger and A. Ullrich
Activation of the human epidermal growth factor
receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
L.H.K. Defize, J. Boonstra,
P.M.P. van Bergen en Henegouwen, A.J. Verkleyand
S.W. de Laat .
Insulin Receptor Tyrosine Kinase and Insulin Action ..... 183
E. van Obberghen, S. Garnmeltoft, Y. Le Marchand
Brustel and R. -Ballotti
The Macrophage Colony Stimulating Factor, CSF-l, and its
Receptor (c-fms).................................. 193
Ch.J. Sherr
Signal Transduction by Growth Factor Receptors .......... 209
W.H. Moolenaar
VIII
IV. GROWTH FACTORS, RECEPTORS AND ONCOGENES:
ROLE IN DIFFERENTIATION AND DEVElDPMENT
Growth and Differentiation Factors of Embryonic
Stern Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
J.K. Heath, A.G. Smith, A.J. Wills and D.R. Edwards
Regulation of Growth Factors and their Receptors in
Early Murine Embryogenesis ........................ 231
C. L. Mumnery, J. van den Eijnden-van Raaij ,
A. Feijen, H-C. Tsung and W. Kruijer
Mesodermal Differentiation of Embryonal Carcinoma
Cells in Coculture with Visceral Endoderm Cell
Lines........................... .................. 247
A.H. Piersma, A. Willernse, C.E. van den Brink,
S . W. de laat and C. L. Munmery
Platelet-derived Growth Factor and its role in
Mitogenesis and Transformation .................... 261
B. Westermark and C.-H. Heldin
The Role of Polypeptide Growth Factors in Phenotypic
Transformation of Normal Rat Kidney Cells ......... 271
E.J.J. van Zoelen
Functions of the erbA and erbB Oncogenes in Avian
Erythroblastosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 289
B. Vennstrom, H. Beug, D. Forrest, A. Johnsson,
K. Khazaie, A. Munoz, J. Sap, A. Ullrich and
M. Zenke
c-fos and Polyoma Middle T Oncogene Expression in
Transgenic Mice and Embryonal Stern Cell
Chimaeras. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 301
E.F. Wagner, R. Lindsay and U. Ruther
Expression of Proto-oncogene int-1 is Restricted to
Specific Regions of the Developing Mouse Neural.
Tube.............................................. 311
D.G. Wilkinson, J.A. Bailes and A.P. McMahon
The Murine Homeobox Protein Hox 1.1 Is Differentially Expressed in Embryonal Mesoderm
and Ectoderm
Frank Schulze, Michael Kessel and Peter Gruss
Dept. Molecular Cell Biology
Max Planck Institute of Biophysical Chemistry
Am Fassberg
3400 Gottingen
West Germany
Introduction
Structural conservation is usually indicative of functional conservation. If this were also true
of developmental control genes then the isolation of sequences homologous to developmental
control genes of Drosophila could lead to the identification of genes with a related function in
the mammalian genome. Indeed, based on this hypothesis, several candidates for mammalian
developmental control genes have been identified. Undoubtedly most effort has gone into
. delineating the possible role of murine homeobox genes. At least 18 members of the Hox
multigene family have been found, most of which are located on mouse chromosomes 6 and
11 (see Martin et al. 1987 for references and nomenclature). Another class of putative control
gene is characterized by a common fmger motif, a structure first suggested for the Xenopus
transcription factor TFIIIA (Miller et aI. 1985; for review see Klug and Rhodes 1987). Murine
finger containing genes have been identified and individual members of this family have been
shown to be specifically active in developing and adult neurons (Chowdhury et aI. 1987; K.
Chowdhury et al., submitted). Finally, a candidate for a third class of murine developmental
control gene (Pax) bears a high degree of homology to Drosophila paired box (U. Deutsch et
aI., submitted). This conserved element is present in Drosophila segmentation genes such as
paired, gooseberry proximal and gooseberry distal (Bopp et al. 1986; Cdt6 et al. 1987).
The best studied example of conserved sequences with a possible role in murine
developmental control is however the homeobox (see Gehring and Hiromi 1986 ~d Scott and
Carroll 1987 for review). Most if not all of the mouse Hox genes are expressed during
embryogenesis in a region specific manner (Dony and Gruss 1987; Gaunt 1987; Gaunt et al.
1986; Utset et al. 1987; Toth et al. 1987; Holland and Hogan 1988; Breier et al. 1988). These
studies were conducted using 35S-labelled RNA as a probe in in situ experiments and thus
only in exceptional cases do they allow resolution down to the single cell level. Furthermore,
NATO ASI Series, Vol. H26
Cen to Cen Signals in Mammalian Development
Edited by S. W. de Laat et al.
© Springer-Verlag Berlin Heidelberg 1989
2
as recently shown for Drosophila caudal and hunchback (Mlodzik and Gehring 1985; Tautz
1988), the level of RNA expressed does not always reflect the amount of protein translated.
Therefore, immunohistochemistry using antibodies directed against a Hox protein should be
employed in order to reach an unambigous answer.
To this end we studied the expression of Hox 1.1 in developing mouse embryos. The
Hox 1.1 protein sequence, as deduced from a cDNA sequence, consists of 229 amino acids
with a molecular weight of 25,740 Dalton (Kessel et al. 1987). Antibodies against synthetic
peptides were shown to bind to a nuclear protein which is associated with chromatin during
meiosis (Kessel et al. 1987; Schulze et al. 1988). We employed these antibodies in
immunohistoc:hemical analyses of mouse embryos at different stages and detected the antigen
in the neural tube, spinal ganglia and also in sclerotomic structures of 12.5-day-old embryos.
However, expression was restricted to sclerotomes of the thoracic region. Moreover, the
ectodermal activity was not in register with the mesoderm expression and thus the Hox 1.1
expression must be spatially and temporally tightly regulated.
Results
Experimental Strategy
The fIrst experimental evidence demonstrating differential expression of murine homeobox
RNA was provided for Hox 1.1 (m6) in differentiating F9 cells (Colberg-Poley et al. 1985a,
b). Monoclonal antibodies directed against the Hox1.1 protein were subsequently used to
show that, in the F9 cell system, the Hox 1.1 protein was expressed with similar kinetics
(Kessel et al. 1987; Schulze et al. 1988). In order to decipher the possible function of murine
homeobox genes during development the spatial and temporal expression pattern has to be
established. Initially "Northern analyses" helped to determine the temporal expression profIle
(Colberg-Poley et al. 1985a, b). The spatial pattern, however, cannot be adequately
determined with this method and in situ techniques are required in order to identify the
respective signal at the single cell level. In particular, immunofluorescence analyses using the
monoclonal antibodies available should allow a precise localization of the protein in the
developing embryo.
Expression of the Hox 1.1 Protein in Mesoderm
Northern analyses revealed the highest amount of stable Hox 1.1 RNA in 12.5 day old
embryos (Colberg-Poley et al. 1985a, b). Therefore, frozen parasagittal sections of similarly
staged mouse embryos were prepared and, in order to localize the Hox 1.1 protein,
