80
Cell contacts in development
A molecular approach to the analysis of pre-chondrogenic condensation
in the avian limb
/. A. Bee* and K. von derMark, Max-Planck-InstitutfurBiochemie, D-8033 Martinsried, West Germany
Limb bud chondrogenesis in vivo and in vitro appears to depend upon extensive cell contacts leading to
localized increases in relative cell density. When cultured in suspension, dissociated early limb bud cells
segregate into aggregating and non-aggregating populations. While the non-aggregating cells die, resulting
aggregates differentiate exclusively as cartilage. We are investigating the role of the cell surface in
prechondrogenic condensation employing aggregation in suspension culture. Immediately after their
dissociation limb bud cells readily undergo Ca -independent, temperature-dependent aggregation. This
'early' mechanism is completely and reversibly sensitive to cycloheximide, although this drug does not
inhibit recovery of all cell surface proteins. Addition of type I collagen and/or fibronectin, isolated
fibronectin cell binding fragment, or anti-fibronectin Fab' fragments do not affect this process. Fab'
fragments prepared from antisera directed against the surface of these cells also fail to inhibit their
aggregation. Aggregation is partially inhibited by synthetic lecithin vessicles and stimulated by mixed
ganglioside micelles. In contrast,++after 16 h recovery equivalent cells exhibit a different, 'late', aggregation
mechanism which is totally Ca -dependent and demonstrates a distinct sensitivity to cycloheximide.
Immunohistochemistry with an antiserum prepared against these recovered cells preferentially stains limb
bud pre-chondrogenic core cells. Fab fragments prepared from this same antiserum inhibit the 'late', but not
'early', aggregation mechanism. We conclude that two independent mechanisms of limb bud cell
aggregation exist. The 'early' mechanism is most probably mediated through the lipid portion of the cell
surface and leads to non-specific cell association. This initial phase is subsequently replaced by the 'late',
specific mechanism mediated primarily through cell surface protein(s). We are now attempting to isolate the
surface components involved in the 'late' aggregation mechanism and characterize their relevance to
chondrogenic expression.
Induction of tight junctions in a human adenocarcinoma cell line (HT29)
Y. Ben-Shaul, E. Cohen, O. Faff and A. Backer, Tel Aviv University, RamatAviv, Tel Aviv 69978, Israel
and Technical University of Munich, West Germany
The human adenocarcinoma cell line HT29 is devoid of tight junctions (TJ) when grown in culture.
However, the formation of TJ can proceed rapidly (on a time scale of minutes) subsequent to appropriate
treatment. Thus, several endopeptidases such as trypsin, chymotrypsin, elastase, papoin, and pronase
induced the formation of TJ at 37°C. Pronase showed a marked concentration optimum at 30-100 mg/ml,
whereas higher concentrations were not effective. Under optimum conditions, TJ were seen on 60-70 % of
freeze-fractured membranes. At 0°C, the formation of TJ under the influence of trypsin proceeds only
slowly, but is not completely suppressed. However, when cells were trypsinized for very short periods at 0°C
and subsequently incubated at 37°C without trypsin, TJ formed rapidly and abundantly. The formation of
TJ was also induced by ammonium sulfate (0.32°M) at 37°C, but not at 0°C. Other salt solutions used at the
same osmolarity gave various results which indicate that specific effects of bivalent anions may be involved.
We suggest tentatively that proteases and salt solution effect different stages in the so far hypothetical
sequence of events leading to the formation of TJ.
Cell contacts in development
81
Adhesion of Dictyostelium cells to immobilized glucosides: effects on
cell development and gene expression
Salvatore Bozzaro*, Max-Planck-InstitutfurBiochemie,
8033-Martinsried bei Munchen, West Germany
Dictyostelium amoebae bind to polyacrylamide gels derivatized with glucose, N-acetylglucosamine and
mannose. Binding is mediated by three different receptors and is Ca ++ and energy dependent.
When Dictyostelium cells develop on a solid surface, they form aggregation centers and streams of
adhering cells, by sending chemotactic signals from the centers down the streams; finally, after all cells have
migrated to the centers, tight aggregates are formed. These developmental processes occur normally on all
derivatized gels, except derivatives of glucose. On these gels, aggregation centers and streams are formed
normally, but at a certain point the centers stop emitting cAMP signals and both centers and streams
suddenly dissociate, with the cells migrating away by negative chemotaxis. Thereafter, the cells repeatedly
reaggregate and dissociate, failing to form tight aggregates.
Tight aggregates, formed in shaken culture or on solid surface, immediately disaggregate after transfer to
glucoside gels. However, tight aggregates at late stage or slugs, tough binding to glucose gels, do not
disaggregate. The formation of tight aggregates is correlated with the expression of postaggregative genes.
These genes are not expressed in cells developing on glucoside gels and their expression ceases, when tight
aggregates undergo dispersal on such gels. Expression of genes coding for preaggregative or aggregation
stage specific products are not affected by cell interactions with the.bound glucosides.
The immobilized glucoside, therefore, freeze cell development at a critical switch point between late
aggregation and further development. The possibility that the glucose receptors are involved in phagocytosis
and/or cell adhesion is currently investigated.
BOZZARO, S. & ROSEMAN, S. (1983). Adhesion of D. discoideum cells to carbohydrates immobilized in
polyacrylamide gels. I. /. Biol. Chem. 258, 13882-13889.
BOZZARO, S. & ROSEMAN, S. (1983). Adhesion of D. discoideum cells to carbohydrates immobilized in
polyacrylamide gels. II. J. Biol. Chem. 258, 13890-13899.
BOZZARO, S., PERLO, C , CECCARELLI, A. & MANGIAROTTI, G. (1984). Regulation of gene expression in D.
discoideum cells exposed to immobilized carbohydrates. EMBO J. 3, 193-200.
The role of discoidin I on Dictyostelium discoideum aggregation as
ascertained by n-butyrate pretreatment
A. Cano, L. Boto, R. F. Pinilla and A. Pestana, Instituto de Investigaciones Biomidicas del CS1C, Fac.
Medicina de la (JAM, Arzobispo Morcillo, 4. Madrid-34, Spain
Pretreatment of Dictyostelium discoideum (Dd) amoeba, Ax-2 strain, with 10 mM n-butyrate induces
changes in several biochemical parameters and in morphogenetic events during Dd development. The whole
developmental cycle is reduced by 4 h while aggregation competence (contact sites A) and cell streaming is
reached 1-2 h earlier than in controls. Using univalent antibodies against pure discoidin I, we have
previously shown (A. Cano and A. Pestana, /. Cell. Biochem., in press) the direct involvement of these
lectins in the aggregation processes in Dd. Additional evidence is provided by the following observations in
starved amoeba after pretreatment with n-butyrate. 1) Discoidin I synthesis and functional expression (as
measured by a hemagglutination assay) takes place 2 h in advance to control cells. 2) Cell surface discoidins
have been specifically eluted with N-acetyl-D-galactosamine from cells labelled with tritiated leucine during
11 h of development. The specific activity of discoidin I eluted from n-butyrate pretreated cells was at least
twice of the activity detected in controls. A marked difference in the specific activity of discoidin I from both
types of cells was also observed by blotting whole cell extract proteins to nitrocellulose paper followed by
immunoautoradiographic detection. 3) Initial immunofluorescence studies with specific antidiscoidin I
serum indicates that the expression of discoidin I on the cell surface takes place 2-3 h earlier in pretreated
cells than in controls. Serial studies consisting in a combination of specific elution of discoidins from the cell
surface (with N-acetyl-D-galactosamine) and further detection of discoidins by radioimmunoelectrophoresis
are now in progress in order to better define the observed differences in the timing of cell surface expression
as well as m the specific activities of discoidin I between treated and control cells. (With the help of a
research grant from the Fondo National de Investigation Cientifica. L.B. is a fellow from the Fondo de
Investigaciones Sanitarias.)
82
Cell contacts in development
Temperature-sensitive mutants of Dictyostelium discoideum that have
reduced late-stage adhesion
C. M. Chadwick, P. Collodi and M. Sussman, Department of Biology, University of Pittsburgh, PA 15260,
U.S.A.
During development of D. discoideum, late-stage cell adhesion is mediated by a glycoprotein of 95,000
MT. We have studied two temperature-sensitive mutants in late-stage adhesion in our effort to relate
adhesion and morphogenesis.
In a previous study of late development the mutant JC5 was described. This showed a reduced
cohesiveness at 27 °C but was itself a mutant of FR17, which completes development early and forms an
abnormal sorocarp. Using techniques of parasexual genetics it has now been possible to obtain the
temperature-sensitive late-stage mutation in a wild-type background (JC36).
At 27 °C JC36 develops to the finger stage whereupon no further development occurs and the upright
fingers lose cells from the bottom and thereby regress in shape. Under environmental conditions which
produce migrating slugs, cells are sloughed-off the rear of the moving slug but not from those slugs
incubated at 22 °C. From the trail of cells formed at 27 °C, additional slugs are formed if reverted to 22 °C.
EDTA-resistant adhesion assays indicate that the cells are less cohesive at 27 °C than at 22 °C. JC36 fruits
successfully at 22 °C but at 27 °C no spores are formed. The mutant would therefore seem to be direct
evidence that cell cohesion is necessary for late gene expression.
The pattern of developmental regulation of glycoproteins that bind to wheat-germ agglutinin has also
been studied. Silver-stained gels of WGA binding material show that a 95,000 Mt protein is expressed
maximally at 17 h of development in DbB. Nevertheless it is still just visible in vegetative and culmination
stage cells. In JC5 high levels are visible throughout development at 22 °C, probably as a result of the FR17
background. However, there is a decline in the relative level of the molecule during late development at
27 °C.
The detection of micromere-specific cell surface proteins on the
fertilized sea urchin egg surface
Douglas W. DeSimone 1 andMelvin Spiegel, Department of Biological Sciences, Dartmouth College,
Hanover, New Hampshire 03755, U.S.A.xAddress after June 15,1984: P.O. Box 162, Woods Hole,
Mass. 02543, U.S.A.
The unequal 4th division of the sea urchin embryo is marked by the appearance of 8 mesomeres at the
animal pole, 4 large macromeres, and 4 small micromeres
at the
vegetal pole. The cells of early cleavage
++
++
stage embryos can be dissociated by exposure to Ca and Mg -free seawater (CMFSW) and individual
blastomeres of the 16-cell stage separated according to size on sucrose gradients. We have previously
demonstrated in Arbacia punctulata and Strongylocentrotus drobachiensis that micromeres display a
cell-type specific pattern of 125I-labelled cell-surface proteins different from that observed on macromeres
and mesomeres, as resolved by 1 dimensional SDS-polyacrylamide gel electrophoresis. In the present study,
four micromere-specific, iodinated cell-surface proteins are described for Strongylocentrotus purpuratus.
Cell-surface iodination of CMFSW-dissociated early cleavage stage blastomeres revealed that two high
molecular weight micromere-specific proteins are already present at the cell surface following fertilization.
These two proteins were found on fertilized eggs, 2,4 and 8-cell stage blastomeres. By the 16-cell stage, they
were localized on the micromeres but not macromeres or mesomeres. In contrast, two lower molecular
weight micromere-specific cell-surface proteins were first observed at the 16-cell stage.
This is the first demonstration in the sea urchin that micromere-specific proteins, which are present on the
surface of the fertilized egg, become restricted during cleavage to the micromeres.
Supported in part by Research Grant No. PCM-8021631 from the National Science Foundation.
Cell contacts in development
83
Modulation and specificity of desmocollins, the adhesion molecules of
desmosomes, during desmosome formation
D. R. Garrod*, D. L. Mattey and A. Suhrbier, CRC Medical Oncology Unit, Southampton General
Hospital, Southampton, Hants S09 4XY
We have identified adhesion molecules of epithelial desmosomes. In bovine nasal epithelium these are
glycoproteins of molecular weight 115,000 and 100,000 which we refer to as desmocollins I and II
respectively (Cowin et al. 1984). We have used antibodies against desmocollins, as well as against
desmosomal plaque constituents, to study desmosomes formation in human keratinocytes and
MDBK cells.
Keratinocytes
can be induced to form desmosomes rapidly in tissue culture by raising Ca2+ concentration.
2+
In low Ca desmocollins
are distributed over the entire cell surface and are not attached to the
cytoskeleton. Raising [Ca2+] causes rapid (15 min) movement of desmocollins to peripheral regions of cell
contact where they become associated with plaque components and the cytoskeleton. With MDBK cells a
much slower modulation of desmocollins occurs, associated with the development of cell polarity. We
propose that desmosome formation involves, (i) a recognition event between desmocollins on adjacent cell
surfaces, (ii) a modulation or redistribution of desmocollins, (iii) a linking of desmocollins to plaque
components and the cytoskeleton.
Our fluorescent antibody studies have suggested that desmosomal components are widely distributed and
highly conserved in the tissues of vertebrate animals (Cowin et al. 1984; Cowin & Garrod, 1983).
Immunoblotting shows that the molecular weights of the various components are similar in man, cow,
chicken and frog. We have been able to demonstrate desmosome formation between all combinations of
HeLa cells (human), MDBK cells (bovine), MDCK cells (canine), chick embryonic corneal epithelial cells,
Rana pipiens (frog) adult corneal epithelial cells and cells from the anus of the fat head minnow (fish). This
suggests that the adhesion recognition sites of desmosomes are conserved between different tissues and
species.
COWIN, P., MATTEY, D. L. & GARROD, D. R. (1984). Identification of desmosomal adhesion molecules
(desmocollins) and inhibition of desmosome formation by specific Fab'. /. Cell Sci. (in press).
COWIN, P. & GARROD, D. R. (1983). Antibodies to epithelial desmosomes show wide tissue and species
cross-reactivity. Nature 302, 148-180.
Elucidation of the mechanism of action of cytochalasin H using invertebrate
and vertebrate developing systems
Surendra Ghaskadbi1 and Leela Mulherkar, Indian Drugs Research Laboratory, 561-B, Shivajinagar,
Pune-411005, India.1Present address: Department of Zoology, University ofPoona, Pune-411007
Present investigation was made to study the mechanism of action of cytochalasin H (CH). Various
systems like hydra, sponge cells, embryonic chick cells and chick embryos cultured in vitro were used. The
major effect of CH was its interference with the phenomenon of cell adhesion. CH brought about
disaggregation of cells in hydra and chick embryos and inhibited the normal phenomenon of reaggregation
of sponge cells. In addition, CH brought about enucleation in hydra cells, inhibition of cytokinesis in sponge
and chick cells and inhibition of primary morphogenesis of heart and neural tube closure in chick embryos.
The effects were found to be dose-dependent and reversible. Reversibility of CH-action was evident when
lost tentacles were regenerated in treated hydra returned to CH-free medium.
Biochemical and histochemical techniques were employed to study the mechanism of action of this drug.
Exogenously added or-D-glucosamine, a precursor of complex carbohydrates brought about reaggregation
of cells in presence of CH. Also, reduction in glycoproteins and glycosaminoglycans was observecfin treated
chick embryos. On the other hand, capping of concanavalin A receptors in sponge cells and isoelectric
points of chick embryonic neural retina cells remained unaffected indicating respectively that CH does not
alter microfilament function and cell surface charge. Moreover, CH had no effect on the cell motility and
cytoplasmic streaming. The results strongly suggest that the interference of CH with the phenomenon of cell
adhesion is primarily due to its effects on cell surface macromolecules and not due to arrested cellular
motility.
84
Cell contacts in development
Cell flattening and junctional coupling in the mouse 8-cell embryo
Harry Goodall*, Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB23DY
Gap junctional coupling first arises in the mouse embryo during the 8-cell stage (Lo & Gilula, 1979;
Goodall & Johnson, 1984) and is superimposed upon the coupling that already exists within quartets of
blastomeres connected by persistent midbodies (Goodall & Johnson, 1984). Whilst gap junctions appear not
to be a prerequisite for one of the major facets of compaction, that of cell polarisation (Goodall & Johnson,
1984) they may themselves be dependent upon the extensive cell flattening that also characterises the onset
of compaction.
Of three treatments that prevented cell flattening, only two, depletion of extracellular calcium and an
antiserum to an embryonal carcinoma cell lines (anti-EC; Johnson et al. 1979) also prevented gap junctional
coupling and reduced the extent of2+ midbody-mediated coupling. The third, a monoclonal antibody
(ECCD-1), recognising a major Ca -dependent cell-cell adhesion system (Yoshida-Noro et al. 1984)
reduced neither mode of coupling. None of these treatments could reverse established coupling between
blastomeres even after several hours culture in the agents and the complete reversal of cell flattening.
It is concluded from these results that cell coupling may arise independently of cell flattening and that the
surface properties permitting
the onset of coupling require extracellular calcium and a moiety recognised by
anti-EC but not by the Ca2+-dependent cell-cell adhesion system recognised more specifically by ECCD-1.
GOODALL, H. & JOHNSON, M. H. (1984). /. Embryol. exp. Morph. 79, 53-76.
JOHNSON, M. H., CHAKRABORTY, J., HANDYSIDE, A. H., WILLISON, K. & STERN, P. (1979). /. Embryol. exp.
Morph. 54, 241-261.
Lo, C. W. & GILULA, N. B. (1979). Cell 18, 399-409.
YOSHIDA-NORO, C , SUZUKI, N. & TAKEICHI, M. (1984). Devi Biol. 101, 19-27.
Patterns of junctional communication in the early amphibian embryo
Sarah C. Guthrie 1, Department of Anatomy, University College, Gower Street, London WC1E6BT
Various lines of evidence suggest that gap junctions may be involved in the control of early pattern
formation. Experiments in different systems using dye transfer to determine the permeability of embryonic
junctions have yielded conflicting results, raising the possibility that transfer of molecules depends on cell
position in the early embryo. The permeability of cell to cell junctions in early stages of Xenopus laevis was
investigated using intracellular injection of the fluorescent dye Lucifer Yellow (Afr 450). At the 32-cell stage,
inspection of injected embryos and sections revealed that dye transfer occurred between some animal pole
blastomeres which were not daughters, as well as between daughter cells. Injection of FTTC Dextrans (Afr
10,000), which can pass between cells via cytoplasmic bridges, but not via gap junctions, showed no transfer
except between daughters, suggesting that movement of Lucifer was via gap junctions. Injection of Lucifer
into individual blastomeres, identified positionally, showed that cell to cell transfer was not uniform within
the animal pole; transfer was maximal near the dorsal (grey crescent) side, and minimal at the ventral (non
grey crescent) side. A similar pattern of communication was present at the 16 and 64-cell stages, though the
incidence of lateral dye transfer was greatly reduced at the 64-cell stage.
This pattern may be due to differences in permeability or numbers of gap junctions across the embryo,
and may explain the lack of consistency among previous results, where transfer was determined without
respect to position. Thesefindingsimply a role for gap junctions during early events in development, which
may be supported by experiments which use gap junctional antibodies to disturb the normal pattern of
communication, producing disruptions in development, (see Warner, Guthrie and Gilula, submitted to
Nature, March 1984; and Warner, this meeting).
1
Supported by an MRC studentship.
Cell contacts in development
Study of the developmental role of the gap junction using metabolic
co-operation-defective embryonal carcinoma variants
85
M. Hooper* and T. A. Smith, Department of Pathology, University Medical School, Teviot Place, Edinburgh
Embryonal carcinoma (e.c.) cells, the pluripotent cells of teratocarcinomas, share with cells of the early
embryo the capacity to differentiate into a variety of differentiated tissues (reviewed in Silver et al. 1983).
Previous work in our laboratory has resulted in the isolation of a metabolic co-operation-defective R5/3,
with a reduced gap junction incidence, from the e.c. cell line PC13TG8 (see Hooper, 1982). We have now
isolated four further metabolic co-operation-defective metabolic co-operation-defective variants from the
feeder-dependent e.c. cell line PSA4TG12, which is capable of differentiation in vitro. The variant cell lines
exhibit differing degrees of communication-deficiency when this is quantified by autoradiographic monitoring of the transfer of uridine-derived nucleotides, with a parallel reduction in the extent of gap
junction-mediated rescue from ouabain toxicity by resistant fibroblasts. The early stages of their in vitro
differentiation have been compared to those of the parent cell line, which forms suspension aggregates
whose surface cells differentiate into endoderm, after which a cavity frequently develops within the residual
core of embryonal carcinoma cells: this cavity appears to be a prerequisite for further differentiation in
suspension. Endoderm formation and cavitation are both defective in all four variant cell lines to differing
extents. There could be a number of reasons for this: 1. Normal levels of metabolic co-operation are
required for differentiation to proceed normally. 2. Reduction in developmental capacity is a consequence
of secondary changes which accumulate in the cells during the selection procedure. 3. Normal embryonal
carcinoma cells undergo differentiation when metabolic co-operation is blocked. Therefore, only a
subpopulation of cells in which this coupling is disturbed is available for the selection of co-operationdefective variants which can be maintained in the undifferentiated state.
Experiments designed to distinguish between these possibilities are in progress.
HOOPER, M. (1982). Interaction of teratocarcinoma-derived cells in culture - a useful model? In The
Functional Integration of Cells in Animal Tissues (ed. I. D. Pitts and M. E. Finbow), Cambridge
University Press, p. 195.
SILVER, L. M., MARTIN, G. R. & STRICKLAND, S. (eds, 1983). Teratocarcinoma Stem Cells. Cold Spring
Harbor Laboratory, New York.
An S.E.M. study of neurulation in normal and lethal gray (Lg) Syrian
hamsters Mesocricetus auratus
Alison J. Holloway, Janet E. Hornby and Louise Muntz, Department of Pure & Applied Zoology, University
of Reading, Whiteknights, Reading, Berkshire RG6 2AJ
Lethal gray is an autosomal mutant of the Syrian hamster: heterozygotes have a gray coat colour and
homozygotes die in utero (Nixon and Connelly, 1967). Embryos homozygous for lethal gray form abnormal
blastocysts and die shortly after implantation. Embryos heterozygous for lethal gray show reduced growth
between 5H5£ dpc followed by a period of compensating growth. By 10 dpc the surviving heterozygotes
closely resemble the control embryos (Holloway and Hornby, 1983).
SEM studies reveal that, in normal embryos at the beginning of neurulation the neuroepithelial cells are
columnar, tightly packed together and have a large number of cytoplasmic projections between them. As
the neural plate begins to fold up to form the neural tube, number of cytoplasmic projections is reduced, and
the cells become flask shaped. When the neural plate folds more, the flask shape of the cells becomes
accentuated, and they become pseudostratified.
Heterozygous lethal gray embryos differ from normal embryos during neurulation. At the beginning of
neurulation they are clearly retarded and smaller than the controls. The neuroepithelial cells become
pseudostratified and the cytoplasmic projections disappear at an earlier stage in the folding than is seen in
the controls, and when formed, the neural tube is irregularly shaped.
A. J. & HORNBY, J. E. (1984). Homozygous lethality and heterozygous recovery in the 'Lethal
gray' hamster (Mesocricetus auratus). Genet. Res. in press.
NIXON, C. & CONNELLY, M. E. (1967). Dark gray and lethal gray - Two new coat colour mutations in Syrian
hamsters. /. Hered. 58, 295-296.
HOLLOWAY,
86
Cell contacts in development
The role of different cell layers in the support of hemopoiesis
N. G. Khrushchov, T. V. Michurina, G. P. Satdykova, A. A. Ivanov and T. V. Vasilieva, Koltzov Institute of
Developmental Biology USSRAcad. ScL, Vavilovst., 26, Moscow, 117334, USSR
The macrophage-fibroblastic layer formed on an acetate cellulose membrane (ACM) in subcutaneous
connective tissue can not support the growth of hemopoietic colonies. On the contrary, colonies are
developed on ACM implanted into the peritoneal cavity and in the mesenterium. Peculiarities of the cell
layer on the ACM from subcutaneous connective tissue are as follows: 1) the predominance of cells
expressing hemopoietic cell-specific antigens, 2) the abundance of intercellular fibronectin. The stroma of
hemopoietic colonies in mesenterium contains macrophages, adipocytes and fibroblasts, sometimes contacts
between hemopoietic cells and mesothelial cells are observed. Erythroid, myeloid and megakaryocytic
differentiation of mesenteric hemopoietic colonies can be detected while the majority of hemopoietic foci on
peritoneal cell-coated ACM are granuloid in nature.
Cell contacts and coated pits in the early eye primordia of the embryos
of three amphibian species
Juhani Kohonen, Department of Biology, University of Turku, SF-20500 Turku, Finland
Studies on the embryos of higher vertebrates have shown that there is a gap between the optic vesicle and
prospective lens ectoderm during the lens induction period, and thus it has been postulated that the
inductive signals are transmitted through matrix interactions, or by a diffusible agent (Piatigorsky, 1981).
Electron microscopical observations on the embryos of smooth newt (Triturus vulgaris), midwife toad
(Alytes obstetricans), and grass frog (Rana temporaria) show that, in the late neurula stage, the optic vesicle
and the prospective lens ectoderm are interconnected with pseudopodia and close cell membrane
appositions with an interspace of 15 to 30 nm. The membranes of interacting heterotypic cells also form
focal contacts, and occasional gap junctions. Homotypic cells are connected with gap junctions and,
infrequently, with desmosomes. In the beginning of the tailbud stage, the optic vesicle and the lens
ectoderm separate leaving a wide cleft between them. Simultaneously, the prospective retinal cells form
large gap junctions, which later are removed from the cell surface and are seen as annular formations in the
cell interior.
The observations suggest that (1) direct cell membrane interactions are possible in the lens induction in
the amphibians, and that (2) gap junctions may be involved in the coordination of determination and
differentiation processes in the homotypic cells.
In the neurulae, and especially in the early tailbud embryos there are numerous coated pits on the cell
membranes. They seem to originate from vesicles produced by the Golgi complex. Because the pits appear
on both ectodermal and optic vesicle cells, they probably are not directly involved in the lens induction.
PIATIGORSKY, J. (1981). Lens differentiation in vertebrates. A review of cellular and molecular features.
Differentiation 19, 134-153.
Cell contacts in development
Changes of plasma membrane antigens during cell differentiation in
Dictyostelium discoideum
87
Akiko Kumagai and Koji Okamoto, Department of Botany, Faculty of Science, Kyoto University,
Kyoto 606, Japan
The cellular slime mould, Dictyostelium discoideum provides a superb model system to the studies of a
molecular basis of cell-cell contact and its role in cell differentiation because of rather simple process of
development. It was previously shown that cells dissociated at the early aggregation stage undergo prespore
differentiation in a snaking culture containing glucose, albumin, EDTA and cAMP (Okamoto, 1981). In
this medium, dissociated cells become adhesive and form agglomerates within a few hours of shaking in a
cAMP-dependent manner. Prespore specific proteins begin to be synthesized only after cells form
agglomerates through newly formed contact sites. Both the cAMP-induced cell contact and the continuous
presence of cAMP were shown to be essential for prespore differentiation (Oyama, Okamoto & Takeuchi,
1982).
To correlate membrane changes with agglomerate formation and prespore differentiation in above
culture system, we have examined changes of plasma membrane antigens under various conditions by the
use of polyacrylamide gel electrophoresis of the antigens prepared by immuno-affinity columns. We found
that three antigens appear specifically in differentiating cells. One of them (10SK protein) appears while
cells become adhesive, but before they synthesize prespore specific proteins. The appearance of the 105K
protein is strictly dependent on the presence of cAMP. Two others (80K and 58K proteins) appear during
prespore differentiation after cells form agglomerates. These results suggest that the 105K protein may be
involved in cell-cell contact and play an important role in differentiation of prespore cells.
OKAMOTO, K. (1981). Differentiation of Dictyostelium discoideum cells in suspension culture. /. Gen.
Microbiol. 127, 301-308.
OYAMA, M., OKAMOTO, K. & TAKEUCHI, I. (1982). Effects of cyclic AMP on contact formation and
differentiation in Dictyostelium discoideum. J. Cell Sci. 56, 223-232.
The assembly of cell junctions in developing arthropod tissues
Nancy J. Lane*, A.F.R.C. Unit, Dept. of Zoology, Downing Street, Cambridge CB32EJ
During organogenesis in both insects and arachnids, glial cell-cell junctions in the nervous system tend to
differentiate during the last hah* of embryonic development. The contacts that form include septate, tight
and gap junctions as well as desmosomes. The changes in intramembranous particle (IMP) organization that
characterize each of these can be studied in freeze-fracture replicas except for desmosomes, which fail to
exhibit a recognizable profile. These junctions all appear concurrently, often along the same membrane
face, but their specific modifications are quite distinct. Pleated septate junctions initially appear as discrete
8 nm IMPs, lying on the Pface of the presumptive junctional membrane; these become aligned into
imprecise rows which meander over the membrane face. Such IMP-rows in time become more orderly and
oriented in parallel with other, like rows, which ultimately achieve a high degree of organization. These
intramembranous events parallel the appearance of the intercellular septa which characterise these
junctions. (The smooth septate junctions, primarily restricted to the digestive tract, develop in a comparable
way, but their component IMPs become aligned into more closely-packed rows, so that they form bead-like
ridges. These ridges also become arrayed in parallel stacks which undulate over the junctional surface; their
intercellular components, septal ribbons and columns, also become inserted at this time, although the
manner in which these events occur is still obscure.) Tight junctions, restricted to systems like the CNS
which exhibit permeability barriers, develop by the translateral migration over the P face of individual
8-10 nm IMPs. These at first become aligned into short, bead-like ridges which then become fused end on
with one another into the interconnecting network which characterizes the mature occluding junctions. Gap
junctions, in arthropods recognizable as 13 nm IMPs fracturing onto the E face, are initially found as
isolated particles. These connexons appear to be translaterally mobile and aggregate into small clusters or
alignments, which ultimately appear to stream together into the macular plaques of IMPs which are typical
of mature gap junctions. TTiese events are paralleled in thin-sections by the 'zippering' together of the
junctional membranes. Antibodies raised against isolated gap junctions from adult tissues are currently
being used to assess junctional biosynthesis in developing systems. Although the mode of IMP assembly of
all these junctions can be followed, the factors that stimulate individual IMP insertion and migration, as well
as those which determine the final precise patterns of IMP distribution, have yet to be ascertained.
88
Cell contacts in development
Gap junctional communication compartments and the regulation of
development
Cecilia W. Lo*, Biology Department, University of Pennsylvania, Philadelphia, Pa., U.S.A.
The specialized cell-cell contacts known as gap junctions are found amongst almost all cell types
throughout the animal kingdom. Gap junctions are thought to contain membrane channels that permit the
direct passive diffusion of small molecules between cells. We have been interested in the possibility that this
type of cell-cell communication might mediate the intercellular exchange of molecules that may play a
critical role in regulating pattern formation in development. To examine this question, we have directly
analyzed the pattern of cell-cell communication in three developmental systems, the early mouse embryo,
the Drosophila wing imaginal disk, and the early sea urchin embryo. In all of these studies, we utilized
microelectrode impalements to monitor cell-cell communication by measuring the extent that ions or
injected fluorescent molecules are exchanged between cells (ionic or dye coupling). In the mouse embryo,
we observed that gap junctional communication which is turned on at the 8 cell stage is initially very
extensive-basically connecting all the cells of the embryo. However, as development progresses, this
communication begins to break down so that only small groups of cells in the embryo are connected. We
refer to these groups of cells as communication compartments. As the formation of these compartments are
temporally and geographically coincident with specific differentiation events, these results suggest that gap
junctions may play a role in regulating the development of the embryo. This possibility is strongly confirmed
by the further examination of gap junctional communication in Drosophila wing disks. We again observed
the presence of communication compartments and moreover, the boundaries of these compartments exactly
coincide with the known lineage compartment boundaries. Thus given that lineage compartments are
thought to play an important role in organizing the development of the adult cuticle, our results would
suggest that gap junctional communication may play a role in this patterning process. Finally, the
examination of sea urchin, a developmental system which is highly mosaic and would not be expected to
utilize gap junctional communication for modulating its development, we observed very little if any gap
junctional exchange. In summary, all of our results are completely consistent with the notion that gap
junctional communication may mediate the exchange of molecules that may modulate patterning in
development.
Induction of the 16K gap junction protein in mammalian uterus
Caroline M. MacDonald 1 and Richard M. Elliott2. ^Division of Biochemistry, The Todd Centre, University
of Strathclyde, Glasgow G4 ONR. 2MRC Virology Unit, Institute of Virology, University of Glasgow,
Glasgow Gil 5JS
Gap junctions are hydrophilic membrane channels which permit the direct transfer of small molecules
between adjacent cells in culture and in multicellular animals. There has been disagreement in the literature
about the size of the major gap junction protein, but recent evidence (Finbow et al. 1983) indicates that it
has a molecular weight of 16,000 (16K). Gap junctions are virtually absent in the smooth muscle cells of the
myometrium, except in pregnant tissue immediately prior to parturition, when a dramatic increase has been
observed by quantitative thin section and freeze-fracture electron microscopy. We have observed an
increase in the amount of 16K junctional protein which can be purified from mouse uterus immediately prior
to parturition. In vitro translation of mRNA extracted from mouse uterus shows that a processed 16,000
molecular weight protein, with properties similar to those of the mouse liver 16K junctional protein, is
induced in pregnant tissue. These results suggest that the regulation of gap junction protein synthesis is at
the level of transcription.
FINBOW, M. E., SHUTTLEWORTH, J., HAMILTON, A. E. & PITTS, J. D. (1983). Analysis of vertebrate gap
junction protein. EMBO. J. 2, 1479-1486.
Cell contacts in development
Monoclonal antibodies raised against cell surface components of sea
urchin embryos show the expression of different antigenic patterns
89
Valeria Matranga and Melchiorre Cervello, Istituto di Biologia dello Sviluppo del CNR, Via Archirafi 20,
90123 Palermo, Italy
Monoclonal antibodies were raised against surface components extracted with butanol from cells
dissociated from blastulae of the sea urchin Paracentrotus lividus (Noll et al. 1979). Indirect immunofluorescence labeling on sectioned material showed location of antigens expressed at the blastula
stage. The antigens examined in this study can be subdivided into several classes with regard to their
location in the embryo. Cell-cell surface: to this class belong antigens that are localized at the contact area
between cells. External surface: these antigens can be seen at the external surface of the blastula cell
monolayer and on the ciliary band. Blastocoel wall: antigens confined to the cell surface that opposes to the
blastocoelic cavity. Primary mesenchyme: these antigens appear on the primary mesenchyme cells and are
not observed at earlier developmental stages. Blastopore: a particular interest was given to this antigen
because of its different locations at various developmental stages. It is found as a diffuse cytoplasmic
staining in oocytes and unfertilized eggs. After fertilization there is a redistribution of the antigen that
appears only at the plasma membrane of the egg. At the blastula stage it is only seen on the external surface
of the monolayer of cells. As soon as gastrulation begins there is a quantitative loss in the fluorescent
staining at the animal pole while the dorso-ventral region displays an intense labeling and ultimately the
antigen is confined within the invagination area showing an immunofluorescent ring in the region at which
the primitive intestine is formed. In longitudinal sections of gastrula embryos the antigen is localized along
the primitive intestine tract and proceeds together with migration towards the tip of the archenteron. At the
pluteus stage the antigen is found on the endothelium of the intestine tract.
H., MATRANGA, V., CASCINO, D. & VITTORELU, M. L. (1979). Reconsitution of membranes and
embryonic development in dissociated blastula cells of the sea urchin by reinsertion of aggregationpromoting membrane proteins extracted with butanol. Proc. Natl. Acad. Sci. U.S.A. 76, 288-292.
NOLL,
Alterations in the cell surface coat during closure of the neural tube, as
revealed by concanavalin A
R. E. Poelmann*, A. E. Smits-Van Prooije and Chr. Vermeij-Keers, Dept. of Anatomy, University of
Leiden, 2333 AL Leiden, The Netherlands
The neural plate of the mouse embryo is transformed via the neural groove into the neural tube. The cell
coat of the tops of the opposed neural folds is thought to be involved in cell recognition during closure of the
neural tube.
A quantitative method was used to investigate the sugar moieties in the cell coat of the surface ectoderm,
the top cells (i.e. the fusion zone) and the neurectoderm. Aldehyde-fixed embryos of the CPB-S mouse
strain were incubated with concanavalin A (Con A).
After incubation the embryos were treated with a colloidal gold complex (Au) with a diameter of 12 nm.
The number of gold particles on the cell membrane is linearly related to the number of lectin receptors on
that membrane. With Con A-Au, the top of the neural folds is labeled twice as heavy as the neurectoderm
and H times as heavy as the surface ectoderm.
The topographical concurrence of strong Con A-binding and the top cells of the neural folds indicates a
specific participation of ar-D-mannose- and/or ar-D-glucose-containing carbohydrates (both Con Areceptors) during closure of the neural tube. In order to evaluate the role of glucose and mannose,
glucosidase and mannosidase were injected into the amniotic cavity of mouse embryos, cultured in vitro.
After culture with these enzymes no difference was found in the Con A-Au pattern. Experiments with
papain to digest the entire cell coat are in progress.
90
Cell contacts in development
Molecular biology of gap junctions
J.-P. Revel*, S. B. Yancey, J. Cline, M. GoninandJ. Honvitz, Division of Biology, California Institute of
Technology and the Jules Stein Institute, UCLA, Los Angeles, U.S.A.
With the availability of N-terminal and other sequences of the gap junction proteins found in liver, heart
and also in the vertebrate lens, it has been theoretically possible to clone gap junction genes. We have
succeeded in isolating two DNA clones genetically engineered from the mRNA which directs the synthesis
of the lens junction protein. A complete amino acid sequence for lens protein has been deduced. The lens
junction protein 263 amino acids in length and of molecular weight about 28,000, is somewhat larger than
the figure usually given for protein sized by SDS-PAGE. The deduced composition is very close to that
determined by amino acid analysis. Determination of the hydrophobicity of various domains of the
sequence and Fourier transform analysis of the distribution of hydrophobic groups as developed by
Finer-Moore and Stroud has allowed us to draw up a model of the lens junction protein. It appears to consist
of six transmembrane segments connected by linkers. A short amino terminal region and a longer carboxy
terminal region are found in the cytoplasm. One of the transmembrane segments is amphiphiuc and thus
fulfils one of the criteria necessary for the establishment of a pore in conjunction with other protein
subunits. A detailed analysis of the position of various residues fits very well with previously known
experimental data. The model may permit the identification of those regions of the molecule involved in
cell-cell interaction at the level of gap junctions and the availability of the cDNA clone will permit
exploration of a number of developmentally interesting features of gap junctions, particularly as they apply
to the lens of the eye.
Prestalk/prespore differentiation tendency of Dictyostelium cells as
detected by a monoclonal antibody
Ikuo Takeuchi* and Toshiaki Noce, Department of Botany, Faculty of Science, Kyoto University,
Kyoto 606, Japan
Deprivation of food supply from growing amoebae of Dictyostelium discoideum leads to aggregation of
cells into a mass which eventually forms a fruiting body consisting of spores and stalk cells. By the use of
monoclonal antibodies specifically reactive against either cell type, we have previously shown that the
majority of cells of the strain NC-4 first synthesizes prestalk-specific antigens during the early period of
development, although they show considerable heterogeneity in the cellular content of the antigens. After
cell aggregation, however, some of the cells lose the prestalk antigens and instead begin to synthesize
prespore-specific antigens, while others continue to synthesize the prestalk antigens. In consequence, the
normal proportion between prestalk and prespore cells is established within cell aggregates (Tasaka, Noce
& Takeuchi, 1983).
It was conjectured that cells initially containing less prestalk antigen are converted to prespore cells after
aggregation, while those containing more remain prestalk cells. To prove this possibility, we have examined
the production of prestalk (Cl) antigen in glucose- [G(+)] and non-glucose-grown [G(-)] cells of the strain
Ax-2 which are known to have tendencies to become prespore and prestalk cells respectively when mixed
(Leach, Ashworth & Garrod, 1973; Tasaka & Takeuchi, 1981). Unlike NC-4 cells, some Ax-2 cells produce
Cl antigen even during the growth phase, although its production increases during early development. The
addition of glucose or metabolizable sugars during the growth phase or the preaggregation period greatly
reduces the amount of Cl antigen. When G(+) and G ( - ) cells are mixed, they aggregate randomly, but
G ( - ) cells which contain more Cl antigen are sorted out to the prestalk region of cell aggregates and G(+)
cells containing less antigen to the prespore region. These results indicate that the extent of Cl antigen
production during the preaggregation period closely correlates to prestalk/prespore differentiation tendency
of the cells after aggregation.
C. K., ASHWORTH, J. M. & GARROD, D. R. (1973). Cell sorting out during the differentiation of
mixture of metabolically distinct population of Dictyostelium discoideum. J. Embryol. exp. Morph. 29,
647-661.
TASAKA, M. & TAKEUCHI, I. (1981). Role of cell sorting in pattern formation in Dictyostelium discoideum.
Differentiation 18, 191-196.
TASAKA, M., NOCE, T. & TAKEUCHI, I. (1983). Prestalk and prespore differentiation in Dictyostelium as
detected by cell-type specific monoclonal antibodies. Proc. Nail. Acad. Sci. USA 80, 5340-5344.
LEACH,
Cell contacts in development
Phagocytosis in Dictyostelium caveatum
91
David Waddell, Bergische Universitdt GHS Wuppertal, FB9 Biochemie, 5600 Wuppertall, West Germany
D. caveatum is a predatory cellular slime mold which can feed upon both bacteria and other cellular slime
molds by phagocytosis (1). D. caveatum can feed either upon free amoebae or amoebae of other species that
are engaged in multicellular morphogenesis after infiltrating their aggregates when they are formed. This
capacity to feed upon other amoebae implies that the D. caveatum amoebae can distinguish self from
non-self.
I have begun to study how the D. caveatum amoebae make this distinction. For this purpose I have
developed a phagocytic assay based upon a mutant which is resistant to a lytic drug, phallolysin (2) which is
produced by Amanita phalloides mushrooms. In this assay the amount of phagocytosis can be quantitated by
lysing the prey cells and separating the lysate from the D. caveatum amoebae. By using this assay and
observing the phagocytosis offluorescently-labelledcells in the microscope, it is apparent that D. caveatum
amoebae begin to feed upon other amoebae by pieces, a process I have called 'nibbling'. This appears to be
necessary for the smaller D. caveatum amoebae to feed upon larger prey amoebae. Cell nibbling seems to
require that the engulfing pseudopods in some way sense size and close upon bite-size pieces.
WADDELL, D. R. (1982). Nature 298, 464^66.
SEITZ, J., ADLER, G., STOFFT, E. & FAULSTICH, H. (1981). Eur. J. Cell Biol. 55, 46-53.
VOGEL, G., THILO, L., SCHWARZ, H. & STEINHARDT, R. (1980). /. Cell Biol. 86, 456-465.
The role of gap junctions in early development
Anne Warner*, Department of Anatomy, University College London, Gower Street, London WC1E6BT
Cells in the embryos of all species so far studied are interconnected by gap junctions. Since Potter,
Furshpan & Lennox (1966) suggested that this pathway might allow transfer of information during
development, much circumstantial evidence in support of this role has accumulated, although direct
evidence for a developmental role is still lacking.
In adult systems gap junctions allow the transfer of both small ions and molecules up to a limit of about
1000 MT. In embryonic systems both success and failure to transfer dyes such as fluorescein (Mr 332) and
Lucifer Yellow (Mr 450) from cell to cell have been reported. The permeability of gap junctions may depend
on the location of the cell within the developing system. Thus transfer of Lucifer Yellow, but not electrical
coupling, fails at the border between developmentally autonomous segments in insect epidermis (Warner &
Lawrence, 1982), despite transfer between cells lying in the same segment. Also transfer of Lucifer Yellow
is not uniform among animal pole cells of the 32 (xuXenopus embryo (Guthrie, 1984 & this meeting), the
ease of intercellular transfer depending on the position of the cell with respect to the dorso-ventral axis.
Recent experiments (Warner, Guthrie & Gilula, 1984) show that injection of an antibody raised against
the major 27K gap junctional protein extracted from rat liver into one cell of the 8 cell Xenopus embryo
completely blocks electrical coupling and Lucifer transfer. Cell division continues normally and the cells
maintain high resting potentials. Embryos injected with the antibody develop pronounced patterning
defects in the region which arises from the injected cell. These results provide the first direct evidence that
gap junctions play a part in patterning embryonic development.
POTTER, D. D., FURSHPAN, E. J. & LENNOX, E. (1966). Connections between cells of the squid as revealed
by electrophysiological methods. Proc. natn. Acad. Sci. U.S.A. 55, 328-336.
WARNER, A. E. & LAWRENCE, P. A. (1982). Permeability of gap junctions at the segmental border in insect
epidermis. Cell 28, 243-252.
GUTHRIE, S. C. (1984). Patterns of junctional communication in the early amphibian embryo submitted to
Nature.
WARNER, A. E., GUTHRIE, S. C. & GILULA, N. B. (1984). Antibodies to gap junctional proteins selectively
disrupt junctional communication in the early amphibian embryo. Submitted to Nature.
92
Cell contacts in development
Differences in hypostome regeneration of sectioned and treated hydra
Danica Znidarit and Ante Lui, Institute of Zoology, University of Zagreb, 4100 Zagreb, Yugoslavia
Hydra whose hypostome was cut off with a small knife will heal much more regularly and more rapidly
and will regenerate a new hypostome than the hydra of which this part of the body is destroyed by means of
chemicals such as, for instance, some of the insecticides, cytostatics or when it is destroyed by ultraviolet
rays. What causes the delay and irregularities in the regeneration of the thus destroyed hypostome? In the
region of the sectioned hypostome there are much fewer injured cells than in the hydras whose hypostome
was destroyed by a chemical or a physical means respectively. From the thus wounded part, the injured cells
gradually peel. But, when there is a greater quantity of these cells, they can, before their recovery or death,
act chemically or, with their presence, even mechanically upon their normal association and differentiation
of newly arrived healthy cells. Groups of injured cells can be localized in one or more places and so only
these places will then have irregular morphogenesis, and hence the new hypostome will be consequently
malformed.