Matrix interactions
Deposition of stromal and basal laminal matrix by human amnion
epithelial cells
23
J.D. Aplin, S. Campbell, Department of Obstetrics and Gynaecology, St. Mary's Hospital, Whitworth Park,
Manchester, M13 0JH. T.D. Allen, Department of Ultrastructure, Paterson Laboratories, Christie Hospital
and Holt Radium Institute, Manchester, M20 9BX
In placental membranes near gestational term, amnion epithelial cells interact with a highly convoluted
basal lamina beneath which is an acellular 'compact layer' of collagenous stroma rich in type III collagen.
We have examined this extracellular matrix by SEM after fractioning the tissue or removing the epithelial
cells enzymically, by TEM and by immunofluorescence with antibodies to matrix macromolecules. Matrix
deposited by cultures of amnion epithelial cells has also been examined using these techniques as well as by
SDS-PAGE. The principal conclusion is that both in vivo and in vitro, the cells deposit stromal as well as
basal laminal macromolecules: collagen types I, III and IV, fibronectin and laminin. In addition, numerous
other poorly characterised components are present. The matrix is not constricted in vitro as it is in
tissue, suggesting the requirement during deposition for an organising template. In vivo the cells initially
secrete matrix components into basal surface invaginations which occur between hemidesmosomes. Stromal
matrix then appears to be assembled through gaps in the basal lamina.
Cell and matrix interactions in cartilage morphogenesis
Charles W. Archer 1 and Paul Rooney 2.1Clinical Research Unit, Institute of Orthopaedics, Brockley Hill,
Stanmore, Middlesex, HA74LP and2Department of Anatomy and Experimental Pathology,
University of St. Andrews, St. Andrews, Fife
The contribution of major matrix components to cartilage morphogenesis has been studied using
cartilaginous long bone rudiments of the chick. We confirmed an earlier report (Fitton-Jackson, 1970) that
agents which disrupted collagen/collagen synthesis had a profound deleterious effect on the morphogenesis
of growing rudiments in vitro. Conversely, agents which disrupted proteoglycan/proteoglycan synthesis
slightly retarded growth but had little effect on morphogenesis.
Light and electron microscopical examination of the treated rudiments showed that collagen disrupting
agents abolished the distinct chondrogenic zones (round cell, flat cell and hypertrophic cell) characteristic of
cartilaginous long bone rudiments (Fell, 1925). In particular, the zone of flattened cells was replaced by
rounded chondrocytes similar and continuous with those normally found in the terminal regions of the
epiphyses. In addition, the oriented collagen 'mat' associated with the matrix of the flattened zone (Rooney,
Archer and Wolpert, 1984) was replaced with randomly oriented collagen fibrils, again, similar to those
associated with the matrix of the rounded cells of the epiphyses. Proteoglycan disrupting agents had no
effect on the cellular arrangements of the rudiments, nor indeed on the orientation of collagen within the
matrix. These and other experiments strongly suggest a reciprocal relationship between cell shape and
collagen orientation within developing cartilaginous structures. We will also present evidence that a change
in chondrocyte shape provides one mechanism whereby matrix may be secreted in a 'directional' manner.
The morphogenetic implications of these observations will be discussed.
FELL, H. B. (1925). The histogenesis of cartilage and bone in the long bones of the embryonic fowl. /.
Morph. Physiol. 40, 417-459.
FITTON-JACKSON, S. (1970). Morphogenetic influences of intercellular macromolecules in cartilage. In
Chemistry and Molecular Biology of the Intercellular Matrix (ed. E. A. Baolazs), 3, (pp. 1771-1779).
London and New York: Academic Press.
ROONEY, P., ARCHER, C. W. & WOLPERT, L. (1984). Morphogenesis of cartilaginous long bone rudiments. In
The Role of Extracellular Matrix in Development (ed. R. L. Trelstad). New York: Academic Press. (In
press).
24
Matrix interactions
Regulation of laminin gene expression in a differentiating system
DeniseP. Barlow', I.C.R.F. Mill Hill Labs, Burtonhole Lane, Mill Hill, London NW7 IAD
Recombinant DNA techniques are being used to study the mechanisms underlying the twenty-fold
increase in laminin gene expression observed when F9 teratocarcinoma cells are treated with retinoic acid
and cyclic AMP. The aim is to identify the DNA sequences controlling the accumulation of the mRNAs for
all four laminin subunits, as well as other matrix glycoproteins synthesised in increased amounts by the
differentiated cells. I have used cDNA clones derived from laminin Bx and B 2 mRNAs to assess changes in
the amount of laminin message as differentiation is induced. Northern analysis shows that a change in the
amount of hybridisable mRNA occurs within twelve hours of induction and continues to increase
dramatically over a period of five days. The Bi subunit mRNA increases to higher levels than the B2 subunit
mRNA. However, both show the same pattern of increase, which is similar for many other
parietal-endoderm specific cDNA clones that I have analysed, including type IV collagen. Such a
synchronous change in mRNA levels suggests that one mechanism is operating to affect a variety of
developmentally regulated mRNAs in this system.
Requirement of fibronectin for amphibian gastrulation
Jean Claude Boucaut*1, Thierry Darribere l, Michel Delarue 1, Jena Francois Riou 1, Shi-de Li1, Habib
Boulekbache 2, Kenneth Yamada 3 and Jean Paul Thiery 4. lLaboratoire de Biologie Experimental,
University Rene" Descartes, 45, rue des Saints Pires, 75270 Paris Cedex 06 France. 2Equipe de Biologie du
Developpement, University Paris 7, 2 place Jussieu 75221 Paris Cedex 05.3Membrane Biochemistry Section,
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20205, USA. *Institut
d'Embryologie du CNRS et du College de France, 49, bis Avenue de la Belle Gabrielle,
Nogent-sur-Mame 94130 France
In vertebrates, gastrulation, a series of morphogenetic movements, leads to the formation of the three
primary germ layers. Recently, fibrillar structures containing fibronectin (FN) were found to underlie the
inner surface of the blastocoel in amphibian embryo gastrulae. The well known properties of FN (ability to
bind both to cell surfaces and to extracellular matrix (ECM) component) suggest that FN may play a role in
cell migration during early amphibian embryogenesis.
To investigate this role, presumptive ectoderm was inverted locally near the blastoporal lip. Mesodermal
cells avoided the inverted region of the roof lacking an FN-rich matrix. Intrablastocoelic injections of
monovalent antibodies against amphibian FN prevented gastrulation. In controls performed with either
preimmune monovalent antibodies or antibodies preincubated with FN, gastrulation occurred on schedule
while mesodermal cells interacted with FN fibrils through their filopodia. Furthermore, microinjections of a
decapeptide corresponding to the cell binding domain of FN resulted in a complete inhibition of the
migration of mesodermal cells. As with monovalent antibodies to FN, the embryo displayed a highly
circonvulated ectodermal cap with deep furrows associated with a voluminous non-invaginated mesodermal
and endodermal mass. Scanning electron migrography of such blocked embryos show that migrating
mesodermal cells remained spherical. Controls, injected with ACTH or a decapeptide corresponding to the
collagen binding site of FN, developed normally. Prevention of the interaction between mesodermal cell
receptors and FN may result either from competitive inhibition (peptide) or from steric hindrance
(antibodies).
Matrix interactions
Immunolocalization of fibronectin in fish embryo; involvement in
gastrulation and epiboly
25
H. Boulekbache 1, T. Darribere 2, C. Joly \ /. C. Boucaut 2 and 3. P. Thiery 3.1Equipe de Biologie du
Diveloppement, Universiti PARIS 7, 2 place Jussieu, F-75251 PARIS, France. 2Laboratoire de Biologie
Experimental, University Rene" Descartes, 45 rue des St Pires, F-75270 PARIS, France. 3Institut
d'Embryologie du CNRS et du College de France, 49bis Avenue de la Belle Gabrielle, Nogent-sur-Marne,
94130 France
Immunolocalization of fibronectin (FN) is carried out using two sources of antibodies: anti-FN directed
against Rainbow trout (Salmo gairdneri) plasma FN purified by affinity chromatography and amphibian
anti-FN (Pleurodeles waltlii).
A high level of FN is shown since late blastula and during gastrulation. However, immunolocalization at
the early blastula stage shows that FN is not yet distributed in a typical network form. It is from late
blastula-early gastrula stage that a starry network of FN-fibrils settles down all over the blastocoele roof
surface. These FN-fibrils seem to be bound to cellular adhesive processes (adherons: lamellipodia and
filipodia) which begin to appear at this stage. In gastrula, when the primordial shield appears the
FN-network becomes more dense and thicker. Moreover, FN seems to settle in front of chordomesoderm
cellular migration and the germ ring. This localization confirms the results obtained in chick and amphibian
embryos. In fishes, FN might be implicated in the mechanisms of morphogenetic movements and
symmetrization of the blastodisc. In trout embryo, two sorts of movements are necessary for embryonic
construction: convergence and epiboly. During epiboly, the germ ring extends over the yolk surface layer
while the ectodermal and mesodermal layers spread out over the yolk sphere to form the yolk sac. A thick
FN-meshwork extends just at the beginning of epiboly. This meshwork acts like a 'purse-string' and
therefore mediates in the closing of the yolk plug.
Spatio-temporal distribution of fibronectin in early fish development suggests that FN plays a major role
not only in epiboly but also in gastrulation and embryonic shield symmetrization.
Early identification oiXenopus laevis primordial germ cells cultured on
different extracellular matrix components
J. J. Brustis, B. Cathalot, D. PeyretandJ. D. Gipouloux, Laboratoire de Biologie Animate, avenue des
Faculty, 33405 Talence Cedex, France
In order to identify in vitro the primordial germ cells (PGC) among endodermal cells dissociated from
early embryo, we use two coated substrate types. On fibronectin coated substrate, most of the cells, after
attachment and spreading, form islets which tend to fuse leading to the formation of a polyedric cell
monolayer. Such a fusing is notably reduced on composite substrate (fibronectin+collagen), so that is
possible to distinguish the special morphological features exhibited by the rest of the cells. Some of them
keep the aspect of endodermal gastrula cells in vitro and are engaged in a creeping locomotion. Others,
elongated or spindle-shaped, possess the characteristics of PGC migrating through the endodermal mass
(Kamimura et al. 1980) and show morphological features similar to those of germ cells, removed from the
mesentery, and cultured in vitro (Wylie, 1980). On fibronectin or composite substrates, the PGC exhibit a
random locomotion. Exposed to a gradient of cAMP, which is supposed to be an attractive factor
(Gipouloux et al. 1979), the velocity and the mode of displacement of these cells depend upon cAMP
concentration.
Such an in vitro system allows the identification of the PGC during the early stages of their migration and
thus could prove to be a valuable tool for the analysis of the mechanisms of this migration.
KAMIMURA, M., KOTANI, M., YAMAGATA, K. (1980). The migration of presumptive primordial germ cells
through the endodermal cell mass in Xenopus laevis: A light and electron microscopic study. /. Embryol.
exp. Morph. 59, 22-37.
WYLIE, C. C. (1980). Primordial germ cells in Anuran embryos: their movement and its guidance.
Bioscience 30, 27-31.
GIPOULOUX, J. D., GIRARD, C , DELBOS, M. (1979). Influence de I'ad6nosine monophosphate cyclique sur la
migration des cellules germinales primordiales des Amphibiens Anoures. Anat. micr. Morph. exp. 68,
61-71.
26
Matrix interactions
The distribution of an endogenous lectin in developing tissues
/. W. CattandF. Lynne Harrison, Department of Cell Biology, AFRC Institute of Animal Physiology,
Babraham, Cambridge CB2 4AT
Galaptins are a class of small )3-galactoside specific lectins found in a number of mammalian adult tissues
and also in differentiating tissues where they are developmentally regulated. In rabbit bone marrow a
galaptin is abundant and localised to erythroid cells. Using an antiserum to the galaptin isolated from bone
marrow we have detected identical lectins in all other rabbit tissues investigated. They are particularly
abundant in lung, heart, intestine, spleen and marrow in the adult. Indirect immunofluorescent staining of
semi-thin sections from resin embedded tissues indicates a predominantly extracellular location for these
galaptins. In neonatal tissues galaptins are distributed differently and are abundant in brain, skeletal
muscle and liver tissue in addition to the heart and lung. We have investigated by indirect immunofluorescence techniques, changes in the distribution of galaptins in heart and skeletal muscle during
development, which are consistent with the hypothesis that galaptins are involved in the organisation of
extracellular components either for secretion or within the pericellular matrix.
Basement membrane proteoglycans
John R. Couchman*, Biosciences Division, Unilever Research, Colworth Laboratory, Sharnbrook,
Bedford MK441LQ
Mammalian basement membranes have consistently been shown to be quite highly conserved in terms of
their biochemical composition, possibly resulting from common functional properties. The glycoprotein
laminin, collagen type IV and heparan sulphate proteoglycan have been demonstrated widely in a number
of basement membrane zones (BMZ). Recently collaborative studies with our Laboratory have shown that
monoclonal antibodies against chondroitin 6-sulphate stain many BMZ indicating that a population of
chondroitin sulphate proteoglycans is also present (Couchman, J. R. et ah 1984). Further studies on the
mouse PYS-2 cell line have shown that in addition to laminin and type IV procollagen, the cells synthesise
both heparan and chondroitin sulphate proteoglycans. Characterisation of the chondroitin sulphate
proteoglycan shows it to be of Mr = 200-300,000, with 10-15 chondroitin sulphate chains of Mr = 1416,000. The core protein (when analysed on SDS-PAGE) under reducing or non-reducing conditions gave
two polypeptides of Mr = 27,000 and 34,000. The proteoglycan has, furthermore, an interesting structure
where most of the chondroitin sulphate chains are clustered at one region of the protein core. This region
has an Mr = 8,000 approximately and therefore represents about one-fourth of the total core. The PYS-2
chondroitin sulphate proteoglycan is much smaller in size than the large cartilage proteoglycan and will not
bind hyaluronic acid, in contrast to the cartilage form. It seems that this proteoglycan has a distinct set of
structural and possibly functional attributes. The PYS-2 chondroitin sulphate proteoglycan may well be
related to that demonstrated immunohistochemically by monoclonal antibody directed to chondroitin
6-sulphate but this remains to be established.
COUCHMAN, J.
R., CATERSON, B., CHRISTNER, J. E. & BAKER, J. R. (1984). Mapping by monoclonal antibody
detection of glycosaminoglycans in connective tissues. Nature, Lond. 307, 650-652.
Matrix interactions
27
Synthesis and organisation of fibronectin in early stages of amphibian
development
T. Darribere 1, D. Boucher 2, /. F. Riou \ /. C. Lacroix 2 and 3. C. Boucaut *. 1Laboratoire de Biologie
Expirimentale, University Rent DESCARTES, 45, rue des Saints-Peres, 75270 Paris CedexO6., France.
2
Laboratoire de Ginitique du De'veloppement, University Pierre et Marie CURIE, 4, Place Jussieu, 75230
Paris Cedex 05., France
During embryogenesis, the cell-associated glycoprotein, fibronectin (FN), has been identified in locations
where it may promote cell migration. Using indirect immunofluorescence we showed that prior to
gastrulation a FN-rich extracellular matrix appeared on the entire surface of the blastocoel roof on which
mesodermal cells will migrate. No fibrils were detected on other parts of the embryos. As gastrulation
proceeded, the FN-containing matrix was permanently identified in the interface between the invaginating
mesoderm and the
overlaying ectoderm.
Labelling with 35S-methionine or 3H-leucine injected in vivo has been carried out to study the synthesis of
FN from oogenesis to the end of gastrulation. 2 D-electrophoresis of labelled proteins revealed that
FN-synthesis occurred and increased during oogenesis. FN was produced at a low level during the first stages
of cleavage and then in more abundant amounts at late blastula stage and during gastrulation. Inhibition of
transcription by actinomycin D did not affect the labelling of FN. Thus, it can be postulated that during the
early stages of development, the production of FN is dependant on the translation of stored maternal
messengers RNA.
How release of FN and its organisation in a fibrillar matrix are regulated is an open question.
Extracellular matrix during kidney differentiation
Peter Ekblom*, Department of Pathology, University of Helsinki, Haartmanink3, Finland. (Address after
Junel, 1984: Friedrich-Miescher Lab., Max-Planck Society Spemannstrasse 37-39, D-7400 Tubingen,
West Germany)
Kidney differentiation is driven by interactions between three cell lineages, the nephrogenic mesenchyme, the epithelial ureter bud, and the endothelium. The mesenchyme is induced to differentiate by the
ureter bud, and this in turn stimulates the migration of the endothelium. Immunocytochemistry shows that
the mesenchyme in response to induction switches its matrix composition. Interstitial collagens are lost,
while basement membrane (BM) components appear. At the same time, the cells also become responsive to
the serum mitogen, transferrin. Our recent evidence suggest that the initiation of an endogenous BM
production is crucial for further differentiation. BM components (laminin) influence the polarity of the cells.
BM components are also involved in the migration of the endothelium. This has been shown in
interspecies hybrid kidneys, where the endothelium forms from avian vessels whereas the epithelium is of
mouse origin. Both cells produce an endogenous BM as shown with species-specific antibodies against either
avian or mouse type IV collagen. It is noteworthy that the migrating vessels at all stages produce a rather
linear BM; thus they apparently do not use the matrix of the target organ for migration.
28
Matrix interactions
Role of fibronectin in wound healing of hypoblast in the chicken embryo
F. Harrisson andJ. Van Hoof, The Department of Anatomy and Embryology, State University Centre of
Antwerp, 171 Groenenborgerlaan, B-2020 Antwerp, Belgium
Fibronectin (FN) as an adhesive, high molecular weight glycoprotein bound along cell surfaces and in
basement membranes (Harrisson et al. 1984 for references in the chicken embryo) has been implicated in
migration and spreading of cells on different substrata. There is, however, only scant information available
on the role of this extracellular molecule in mediation of such processes in vivo. In this abstract, a system is
presented that allows investigation of this role of FN during wound healing of hypoblast, a tissue repair that
occurs by migration of this tissue sheet over the basement membrane of the epiblast. The experimental
intervention on chicken embryos during late gastrulation consisted of the unilateral removal of the
hypoblast at the level of the endophyllic crescent. The blastoderms were allowed to grow in culture before
fixation in a glutaraldehyde/formaldehyde solution. The embryos were divided into three series: (1) normal
culture conditions; (2) in the presence of affinity-purified sheep anti-human FN antiserum; and (3) in the
presence of the same antiserum after solid-phase adsorption on FN. Time-lapse photocinemicrography
shows that wound healing does not occur during the first 3 h of incubation in the embryos of our second
experimental series, but occurs completely in a same fashion in the two other series. Immunocytochemical
staining of the embryos using peroxidase-labeled swine anti-sheep IgG on sections of paraffin-embedded
material, or in toto prior to embedding for TEM shows that the anti-FN antibody applied in culture on the
blastoderms of the second series is bound to the basement membrane for about 3 h. TEM and SEM shows
adhesion of the hypoblast tissue layer on the epiblast basement membrane in both control series, but not in
the presence of anti-FN antibodies. Healing hypoblast is characterized by the extension of filopodia and
lamellipodia towards the epiblast basement membrane to which the edge of the wound is firmly attached.
This is not the case in the presence of FN, the hypoblast cells being rounded and smooth. It is concluded that
the basement membrane of the epiblast is used in vivo during wound healing of hypoblast as a substrate for
epithelial sheet movement. This process is mediated by the presence of FN in the basement membrane, the
presence of which is necessary for wound healing to occur.
HARRISSON,
F.,
VANROELEN, CH., FOIDART,
J.-M. & VAKAET, L. (1984). Dev. Biol. 101, 373-381.
Parietal endoderm of the mouse embryo: a model system for studying the
regulation of basement membrane genes
Brigid L. M. Hogan*, Denise P. Barlow, Markku Kurkinen and Ivor Mason, Mammalian Development
Laboratory, Imperial Cancer Research Fund, Burtonhole Lane, Mill Hill, London NW7 IAD
The primitive endoderm of the early post-implantation mouse embryo is a bipotential population that can
differentiate into either visceral or parietal extra-embryonic endoderm, the choice apparently depending
upon interaction of the stem cells with adjacent tissues. Visceral and parietal endoderm have very different
morphologies and patterns of gene expression reflecting their different functions during embryonic
development.
The parietal endoderm (PE) synthesises large amounts of the complex extracellular matrix glycoproteins
type IV collagen, laminin, entactin, and heparan sulphate proteoglycan, which are assembled into a thick
basement membrane known as Reichert's membrane. Although these glycoproteins are made by a variety
of other embryonic cells, the PE is a particularly powerful system in which to study factors controlling their
biosynthesis and assembly into a three-dimensional extracellular matrix. The strategy we have used to
isolate cDNA clones for proteins specifically made in large amounts by PE cells will be described. The
ultimate aim of this work is to identify the cisacting DNA regulatory sequences co-ordinating the expression
of a set of genes presumably scattered throughout the genome. Analysis of the laminin cDNAs has also
thrown light on the structure of the B chains of this multisubunit protein. Other interesting properties of PE
cells will be briefly discussed, including the synthesis of a Mr = 47,000 plasma membrane protein (colligin)
which binds type IV collagen, and the cellular oncogene, c-fos.
Matrix interactions
29
Distribution of fibronectin, laminin and type IV collagen during the hair
growth cycle of the rat vibrissa follicle
C. Jahoda, A. Mauger and P. Sengel, Laboratoire de Zoologie et Biologie animate, University scientifique et
M6dicale de Grenoble, B. P. 68, 38402 St-Martin-d'Hires Cedex, France
Hair growth in adult animals involves repeated reorganization of lower follicle structure. Using indirect
immunofluorescence we investigated the distribution of fibronectin (FN), laminin, and type IV collagen
(antibodies prepared as previously described, Mauger et al. 1982, 1983) during the growth cycle of the rat
vibrissa follicle.
Through anagen (the active hair growth phase) FN, laminin and type IV collagen marking was continuous
around the inner dermal papilla-epidermal junction. By late catagen (termination of fibre production),
during telogen (no hair growth), and in very early anagen, FN antibody labelling was restricted to a small
region of the lower interface. Laminin and type IV collagen remained uninterrupted and intensified over the
same period. Intrapapillary labelling with FN and laminin antibodies was heaviest around telogen/early
anagen, the time of maximum papilla cell condensation.
For the external dermal-epidermal junction, marking with all three antibodies was relatively feeble
throughout anagen, with one particular patch of very weak labelling at the base of the epidermal 'horn'.
During catagen, marking became uniform and intensified, reaching a maximum at telogen/early anagen.
Transmission electron microscopy revealed that the inner basal lamina changed from a uniform structure
in mid-anagen, to become convoluted and multilayered at telogen/early anagen, while the outer glassy
membrane thickened and extended proximally.
The increased presence and altered distribution of these three extracellular matrix components at the time
of greatest morphogenetic activity suggests that they may be closely involved in the series of
dermal-epidermal interactions which regulate follicle activity.
MAUGER, A., DEMARCHEZ, M., HERBAGE, D., GRIMAUD, J. A., DRUGUET, M., HARTMANN, D. & SENGEL, P.
(1982). Immunofluorescent localization of collagen Types I and III, and of fibronectin during feather
morphogenesis in the chick embryo. Devi Biol. 94, 93-105.
MAUGER, A., DEMARCHEZ, M., HERBAGE, D., GRIMAUD, J. A., DRUGUET, M., HARTMANN, D. J., FOIDART, J.
M. & SENGEL, P. (1983). Immunofluorescent localization of collagen types I, III, IV, fibronectin and
laminin during morphogenesis of scales and scaleless skin in the chick embryo. Wilhelm Roux's Arch, devl
Biol. 192, 205-215.
Distribution and characterization of anionic sites in the basal laminae of
developing human fetal membranes
Barry F. King, Department of Human Anatomy, University of California, Davis, California 95616, U.S.A.
Basement membranes (basal laminae) have been shown to play a role in regulating tissue morphogenesis
in a number of organs, as well as providing a structural scaffolding that supports the organ. In addition,
certain basal laminae have been shown to function as permeability barriers. Anionic sites associated with the
basal laminae are believed to be responsible for establishing this permeability barrier. In this study we have
used the cationic stain ruthenium red to examine the distribution of anionic sites in the basal laminae of
amniotic and trophoblastic epithelia during gestation. Human amnions and placental villi from 8-12 weeks
of gestation and at term were incubated for 45 min at 37°C in the presence or absence of chondroitinase
ABC, Streptomyces hyaluronidase, neuraminidase or heparitinase in appropriate buffer systems, then
exposed to ruthenium red, and prepared for electron microscopic examination. In amnion, control
preparations demonstrated ruthenium red-positive granules at periodic intervals along the laminae rarae of
the basal lamina. These anionic sites were present at 8-12 weeks of gestation and at term. Incubation in the
presence of heparitinase resulted in the disappearance of the ruthenium red-positive granules from the basal
lamina, but other enzymes were without effect. The basal lamina of trophoblast also had ruthenium
red-positive, heparitinase-sensitive granules during early gestation, but these were decreased in number at
term. We conclude that anionic sites associated with these basal laminae, and demonstrable with ruthenium
red, consist of glycosaminoglycans rich in heparan sulfate, probably present as heparan sulfate proteoglycans. Because the amniotic epithelium lacks tight junctions anionic sites in amnion basal lamina may have an
important role in controlling permeability of macromolecules across the amnion. The decrease in basal
lamina anionic sites observed in trophoblast may correlate with the increased permeability of this epithelium
to macromolecules that occurs during later gestation.
30
Matrix interactions
Synthesis and possible role of proteoglycans during Volvox development
Raphael Kopan and Michael Ovadia, Dept. of Zoology, Tel Aviv University, Ramat Aviv 69978, Israel
During the embryonic development of Volvox the cells synthesize proteoglycans of relatively small
molecular size up to 14 h after inversion. In the following stages there is a gradual transition towards larger
forms, which attain the rest of the 72 h life cycle. The smaller embryonic molecules are used by the
spheroids as precursors for the larger ones and the increase in molecular size is due to a higher proportion of
sulfated sugar moieties. The presence of sulfate in the medium was obligatory for the synthesis of the larger
forms. None of these molecules could be dissociated in 4.0M GuHCl, neither could they be digested by
glucosidases. Subtilisin, a bacterial protease, fully digested both proteoglycan populations, whereas pronase
or trypsin only partially digested the molecules.
The possible role of proteoglycans during Volvox morphogenesis has been examined by four developmental characteristics: spheroid motility, size, aggregation and rate of cleavage. Somatic proteoglycans
caused spheroids to start their swimming earner, although they inhibited their expansion. Their growth was
stopped at a diameter of 50 microns in comparison to 250 microns of normal spheroids. Spheroids cultured
in sulfate-free medium start their swimming 24 hrs later than spheroids in the standard medium, probably
because of their inability to synthesize mature proteoglycans. However, the addition of these molecules in
the sulfate-free medium could compensate for their absence in the spheroid and induce motility somewhat
later (4 h) than in the normal forms.
Embryonic low molecular weight proteoglycans (ELMWPG) caused aggregation of the spheroids, an
effect that appeared also in the sulfate-free medium. The size of the aggregates and their life-span were
concentration-dependent. Cleavage of the spheroid cells was influenced by each of the proteoglycan
populations; the rate of cleavage was decreased by high concentrations.
Normal epithelial morphogenesis in the absence of collagen I
Klaus Kratochwil*1, Ju'rgen Lohler 2 and Rudolf Jaenisch 2.1Institutfiir Molekularbiologie, Osterreichische
Akademie der Wissenschaften, Billrothstr. 11, A-5020 Salzburg, Austria. 2Heinrich-Pette-Institutfur
Experimentelle Virologie und Immunologie an der Universitdt Hamburg, Hamburg 20, West Germany
Interstitial fibrillar collagen has for a long time been implicated as a mediator of mesenchymal control of
epithelial morphogenesis. The distribution of collagen fibres, e.g. in the embryonic lung or salivary gland,
suggested a stabilizing role of this matrix component on epithelial branching. Experimental analysis of the
developmental function of collagen in embryonic organogenesis so far was limited to the application of
collagenase (1, 2), or to the use of inhibitors of collagen synthesis, hydroxylation, or cross-linking (e.g. 3).
Severe 'depatterning' of epithelial morphogenesis was obtained by such (relatively non-selective) interference.
Recently, an elegant system has become available in which the role of type I collagen can be studied most
specifically and without interfering in the cells' metabolism: insertion of a Moloney murine leukemia
provirus into the first intron of the collagen al(I) gene has resulted in a complete block of the gene. This
gene is inherited in Mendelian fashion in a mouse strain ('Moye-13'), but homozygous carriers die as
embryos on day 12 (4, 5). No type I collagen is demonstrable in their tissues by immunohistochemical
methods. Organ rudiments of such embryos were explanted on day 11 and maintained in organ culture for
up to 13 days. In the complete absence of collagen I, all the organs tested (lung, kidney, pancreas, skin with
in vitro formation of hair, vibrissa, and mammary rudiments) developed typically in culture, without any
deviation from the normal morphogenetic pattern.
GROBSTEIN, C. & COHEN, J. (1965). Science 150, 626-628.
WESSELLS, N. K. & COHEN, J. (1968). Dev. Biol 15, 237-270.
SPOONER, B. S. & FAUBION, J. M. (1980). Dev. Biol. 77, 84-102.
SCHNIEKE, A., HARBERS, K. & JAENISCH, R. (1983). Nature 304, 315-320.
HARBERS, K., KUEHN, M., DELIUS, H. & JAENISCH, R. (1984). Proc. Nat. Acad. Sci. U.S.A. 81,
1504-1508.
Matrix interactions
31
Differences in the deposition of a fibronectin matrix by embryonic
corneal epithelial cells cultured on collagenous and non collagenous
substrata
Derek L. Mattey, C.R.C. Medical Oncology Unit, Southampton General Hospital, Southampton,
Hampshire SO9 4XY
Corneal epithelial cells from 15 day chick embryos demonstrate differences in attachment, spreading and
deposition of a fibronectin matrix when cultured on various collagenous and non collagenous substrata in
serum free medium. Dissociated cells quickly attached and spread on different types of collagen dried onto
glass coverslips. No apparent differences were seen on type I, IV or V collagen. In contrast cells attached
poorly and spread slowly on gelatin and heat denatured collagen. A significant degree of attachment and
spreading was obtained on fibronectin and laminin, although less than that on the collagens. Very little
attachment to fibrinogen was seen, although a few cells eventually did attach and spread. After 4 days the
cell cultures were examined for fibronectin using fluorescent antibody staining. An extensive fibrillar matrix
was found beneath those cells that had spread on fibrinogen, gelatin, heat denatured collagen, laminin and
fibronectin. Cells cultured on collagen IV and V had a less extensive matrix but demonstrated more
cytoplasmic staining, while cells an collagen I showed little matrix or intracellular staining. Thus, there are
distinct differences in the deposition of a fibronectin matrix on different substrata. Furthermore
anti-fibronectin antibodies inhibit attachment and spreading on fibronectin and gelatin but not on collagen.
This suggests that the cells are able to attach and spread directly on collagen via specific collagen receptors
or by using some other type of attachment/spreading factor, while on non-collagenous substrata or
denatured collagen the cells need to deposit a fibronectin rich matrix to maintain attachment and promote
cell spreading.
Extracellular matrix fibrils and cell migration in amphibian gastrulae:
correlated in vivo and in vitro studies
Norio Nakatrsuji*, Meiji Institute of Health Science, Narita, Odawara 250, Japan
In amphibian gastrulae, the presumptive mesodermal cells migrate from the blastopore toward the animal
pole by using the inner surface of the ectoderm layer as their substratum for cell movement. An
anastomosing network of extracellular fibrils, which can be observed only at high magnifications by scanning
electron microscopy, is present on this natural substratum, but not on the mesoderm or endoderm cells, in
gastrulae of all the amphibian species examined so far, including three urodeles (1, 2) and three anurans
(2, 3). The fibrils appear shortly before the start of cell migration in normal embryos (2) and in hybrid
embryos that gastrulate normally (3). On the other hand, no fibril or only fewer fibrils appear in hybrid
embryos that are arrested at the initial gastrula stage (3). When the fibrillar network is transferred onto a
cover slip surface, it supports attachment and active movement by mesoderm cells from gastrulae (4). Most
interestingly, filopodia of the migrating cells show preferential attachment to the fibrils both in vivo (1) and
in vitro (4), providing direct evidence that the fibrils serve as the attachment sites of the cell movement.
Furthermore, in urodele gastrulae, in vitro experiments strongly suggest that an alignment in the fibril
network gives orientation for the cell migration by the mechanism of contact guidance (4, 5). Finally, the
latest study using SEM immunocytochemistry (6) has shown that these extracellular fibrils contain
fibronectin, probably as a major component, providing direct evidence for the hypothesis that the
fibronectin-containing extracellular matrix fibrils provide attachment sites for filopodia and other
locomotory organelles and guides the cell migration during morphogenetic movements.
NAKATSUJI,
NAKATSUJI,
NAKATSUJI,
NAKATSUJI,
NAKATSUJI,
NAKATSUJI,
N.,
GOULD, A. C. & JOHNSON, K. E. (1982). /. Cell Sci.
& JOHNSON, K. E. 1983 . /. Cell Sci. 59, 61-70.
& JOHNSON, K. E. 1984 . /. Cell Sci.
i. (In press).
& JOHNSON, K. E. 1983 . /. Cell Sci.
i. 59,
59, 43-60.
& JOHNSON, K. E. 1984 . Nature 307, 453-455.
N., SMOLIRA, M. A. & WYLIE, C. C. (1984). Submitted.
N.
N.
N.
N.
56, 207-222.
32
Matrix interactions
Extracellular matrix components as positive and negative constraints on
neural crest cell migration
D. Newgreen*, Max-Planck-Institutfiir Entwicklungsbiologie, Spemannstrasse 35, D7400 Tubingen,
West Germany
In vertebrate embryos, neural crest (NC) cells originate in the dorsal side of the neural anlage, but they
migrate as mesenchyme away from this position and eventually become widely distributed.
Light and electron microscopy of early embryos indicate that initial NC migration is restricted to regions
of cell-free space, where NC cells contact the extracellular matrix (ECM). However, not all spaces are
occupied to equal degrees. One explanation for this is that positional variation in the distribution of ECM
components may dictate environments that are more or less permissive for NC cell adhesion and
locomotion.
The ECM component fibronectin (FN) occurs in regions of NC migration in vivo, and is highly favorable
for NC cell adhesion and locomotion in vitro, whereas collagen (Type I) can support adhesion but is far
inferior as a locomotory substrate in vitro. The basal lamina component laminin is functionally equal to
fibronectin in vitro, but may be irrelevant in vivo, at least during early migration because NC cells rarely
contact the basal lamina. Thus, FN may be a molecular component which defines an environment permitting
NC cell migration.
Despite the above, some embryonic areas with high amounts of FN are not rapidly occupied by NC cells.
These areas also have chondroitin sulphate proteoglycan (PG) at high concentrations. When assayed in
vitro, nasal cartilage PG can inhibit NC cell adhesion to FN, and chondroitinase-sensitive ECM produced by
the notochord of embryos can prevent NC cells from occupying FN and collagen substrates. Therefore, it is
likely that chondroitin sulphate PG reduces the permissive effects of FN. Important morphogenetic controls
to NC cell migration could be mediated by temporal and positional variations in the balance of these two
ECM components.
Synthesis and possible role of proteoglycans during embryonic
development of sea urchin
Michael Ovadia and Adina Haiim-Matias, Dept. of Zoology, Tel Aviv University, Ramat Aviv 69978, Israel
Proteoglycans or their covalently bound side chains, the glycosaminoglycans, have been assigned a role in
cellular adhesion, migration, proliferation and differentiation in various embryonic systems. During the
embryonic development of the sea urchin, Paracentrotus lividus, proteoglycans of relatively small molecular
size are synthesized up to the morula stage. During the following stages there was a gradual transition
towards larger molecular forms which were apparently formed from the smaller ones by aggregation. The
aggregate molecules could be dissociated by 4.0 M GuHCl and could be digested by nyaluronidase;
proteases digest the molecules only partially and chondroitinase ABC showed no detectable change.
Incubation of the proteoglycan molecules with sea urchin embryonic cell suspensions of various
developmental stages resulted in the aggregation of the cells. Proteoglycans extracted from Volvox carteri
did not cause aggregation and the cells remained dispersed. These results may indicate that proteoglycans
synthesized during the embryonic stages may serve as intercellular adhesive substances which are specific to
a certain extent.
Matrix interactions
33
The basement membrane of mesonephric and paramesonephric ducts of
rat fetuses
Jorma Paranko, Jean-Michel Foidart and LauriJ. Pelliniemi, Department of Anatomy and Laboratory of
Electron Microscopy, University of Turku, SF-20520 Turku 52, Finland, and Clinic ofGynecology and
Obstetrics, University ofLi&ge, B-4020 Liege, Belgium
The changes in the basement membranes were studied to elucidate the mechanisms of the development
and regression of mesonephric and paramesonephric ducts in male and female rat fetuses at the ages of 15 to
21 days. Basement membrane components, type IV and V collagen, laminin, and heparan sulfate
proteoglycan were localized by immunocytochemistry at the light and electron microscope level.
Ultrastructurally all components were localized in the basement membrane and on the surface of fibrillar
and flocculent extracellular material attached to the surface of the basal lamina. At the age of 15 days the
basement membrane of the mesonephric duct (MD) was continuous in both sexes, whereas on the medial
half of the paramesonephric duct (PD) it was discontinuous. The mesenchyme surrounding the PD
contained components of the basement membrane. The developing MD in males and the PD after 16 days in
females were surrounded by a continuous basement membrane. In 16-day-old male fetuses the basement
membrane became discontinuous all around the PD and the condensed mesenchyme around the duct lost its
basement membrane matrix. The regression of the male PD and the female MD was completed at the age of
18 and 19 days, respectively. Concomitantly with the decrease in the diameter of the female MD, cytolysis,
gradual detachment, and large bullous extensions of the basement membrane were frequently seen. This
allowed a gradual protrusion of dying cells out of the duct and simultaneous invasion of the surrounding
mesenchyme. In males basement membrane components began to accumulate in the mesenchyme
surrounding the MD as an early sign of the epididymal smooth muscle differentiation. The results suggest
that the early discontinuities in the basement membrane of the PD and accumulation of basement
membrane matrix around the duct have an important role in the action of the paramesonephric regressor
substance. The later dissolution of the matrix suggests that the paramesonephric regressor also acts through
the mesenchyme by activation of proteolysis. The differences in the regression mechanisms in the male and
female ducts observed here, probably help to elucidate the mode of action of the respective regulatory
factors, the paramesonephric regressor in the male and the lack of testosterone in the female duct.
Embryonic extracellular matrix adsorbed in vivo onto microcarriers
induces phenotypic expression in cultured neural crest cells
Roberto Ferris* and Jan Lofberg, Department of Zoology, Uppsala University, S-75122 Uppsala, Sweden
A microcarrier system has been developed to test the effects of embryonic extracellular matrix (ECM) on
phenotypic expression in cultured neural crest cells. Subepidermal ECM from axolotl embryos was
adsorbed in vivo onto microcarriers prepared from Nuclepore filters. The ECM covered microcarriers were
then transferred to a serum-free culture solution and neural crest cells were deposited on them. To obtain a
crest cell population as pure as possible, the cells had been isolated from the dorsal part of the premigratory
crest cell string.
As a marker for phenotypic expression we used the appearance of pteridines, known to be characteristic
for xanthophores. These pteridine-containing pigment cells can be identified prior to external visibility by
alkali treatment which elicits a specific bright blue fluorescence. Control cultures, consisting of explanted
crest cells grown directly on untreated Nuclepore filters did not exhibit xanthophoric traits. In contrast,
neural crest cells cultured on the microcarrier attached ECM developed into xanthophores.
These results indicate that the embryonic subepidermal ECM induced the expression of xanthophores in
isolated crest cells. We propose that the ECM in the embryo act as a regulating factor for the establishment
of xanthophoric traits. Studies are now in progress to find out if the embryonic ECM also directs the
development of melanophores and neuronal cell types in the neural crest.
34
Matrix interactions
Study of the fusion of palatal shelves and the closure of eyelids in the rat
A. M. Renard, C. Merveille and E. Baeckeland, Department of Embryology, University of Liege, ruede
Pitteurs20, B-4020 Liege, Belgium
The fusion of mesenchymal buds covered with epithelia is a very frequent morphogenetic process. In this
work we compare the changes taking place during such a process, namely during the fusion of palatal shelves
and the adherence of eyelid buds occurring at the closure of the eyelids.
Palatal shelves taken at day fifteen p.c. and eyelid buds taken at 17 p.c. from the fetus of the albino Wistar
rat were placed in organ culture according to Chen to observe these phenomena in vitro. After 12 and 24 h,
cultures were fixed and slides made to study the distribution of laminin or type I or type III collagen by
immunofluorescence.
We have obtained evidence for the presence of type I and III collagens and laminin, in cultures of palatal
shelves and eyelid buds removed just before the onset of their junction.
In the course of the development of the eyelids, the patterns of distribution of the laminin and of the type
III collagen remained constant during the culture. The type I collagen like the type III collagen is distributed
homogeneously in the mesenchyme from the 12th hour of culture.
The rapid stabilization of these three distributions could be associated with the persistence of an epithelial
seam limited by intact basement membranes between mesenchymal eyelids from the stage of junction, to
the later stage of disjunction, of the eyelids.
Conversely, the secondary palate development is characterized by a disappearance of fluorescence
indicative of the laminin, at the level of the epithelial seam basal lamina, and by a change in the distribution
of type I and III collagens with time in vitro.
Biosynthesis of cell surface glycoproteins during gastrulation of the
amphibian Pleurodeles waltlii
J. F. Riou, T. DarribereandJ. C. Boucaut, Laboratoire de Biologie Expirimentale, University Rene"
DESCARTES, 45, rue des Saints-Peres, 75270 Paris Cedex 06, France
Morphogenetic movements which occur in early development involve important changes in cell
morphology and adhesivity. At the molecular level, much interest has been focused recently on cell surface
glycoproteins.
In early Pleurodeles waltlii development, cell surface proteins have been previously characterized by
specific radiolabelling methods: iodination, metaperiodate - 3 H NaBH4 and galactose oxidase - 3 H NaBH4 .
We have now analysed their biosynthesis during gastrulation.
Using 35S methionine, 3 H mannose, 3H fucose, 2D-electrophoresis and fluorography, we observed
significant biosynthesis both at the3 initiation and the end of gastrulation. Among the hundred of
radiolabelled glycoproteins inserting H mannose, twelve were identified as major cell surface proteins. No
qualitative variation of these glycoproteins could be shown during gastrulation. With 3 H fucose, about
fifteen glycoproteins were detected. Four of them were cell surface glycoproteins which equally
incorporated H mannose.
In an attempt to characterize cell surface glycoproteins synthesized by gastrulating amphibian embryos,
we have used Concanavalin A sepharose. Con A binding glycoproteins were then analysed by 2D-gel
electrophoresis and autoradiography.
Thus ninety biosynthesized Con A binding proteins were separated,
most of them correspond to 3H mannose radiolabelled glycoproteins and ten were major cell surface proteins.
These results indicate that major cell surface glycoproteins are synthesized prior to and during
gastrulation of Pleurodeles waltlii embryos.
Matrix interactions
Long-term cultures of hydromedusa cells spreading on artificial
substrata
35
Walter K. Schlage and Volker Schmid, Zoological Institute, University of Basel, Rheinsprung 9,
CH-4051 Basel, Switzerland
The experimental requirements for the transdifferentiation of striated muscle cells from the hydromedusa Podocoryne carnea finally resulting in the regeneration of an entire manubrium suggest the
involvement of ECM components in controlling the destabilization process (Schmid, V., Weber, C. &
Keller, D. (1984). Factors affecting DNA synthesis in an in vitro system. Roux's Arch. Dev. Biol. 193,
36-41.). These events occur in grafted tissue explants floating in artificial sea water. In order to test the
influence of extracellular matrix components on proliferation and transdifferentiation under continuous
microscopic observation, an effort was undertaken to grow medusa cells spread on coated glass surfaces.
The tissue explants (interradial fragments of the umbrella) were treated with dispase (800 U/ml sea water,
23 °C) for 1 h and cultured in Rose-chambers under a cellophane strip in a sea water medium supplemented
with amino acids, fetal calf serum (5-20 %), HEPES buffer (10 mmoles/1), Pepton from meat (600 mg/1),
and antibiotics. On uncoated glass, the explants flattened within 20 h and lamellipodia-like extensions
became visible, which disappeared a few days later. One type of ameboid cells left the explants being highly
motile on glass as well as on dialysis membrane. On the other hand, on a poly-L-lysine coat the lamellipodia
persisted and, in addition to the ameboid cells, a number of oval-shaped, granulated cells migrated off the
explants as established small colonies. These cells, exhibiting a low proliferative activity, could be cultured
for up to 14 weeks. This system proves to be useful in testing a variety of substrates with respect to cell
behaviour. Furthermore, medium components and compositions can be investigated in order to elaborate
appropriate conditions for the in vitro culture and establishment of medusan cell lines, which have not been
reported so far.
Ontogeny of the peripheral nervous system
1
Jean Paul Thiiry* , Jean-Loup Duband \ Annie Delouve'e 1, Gordon Tucker 1, Hirohiko Aoyama l,
Thomas J. Poole 1 and Kenneth M. Yamada 2. lInstitut d'Embryologie CNRS et College de France, 49bis av.
de la Belle Grabrielle 94130 Nogentsur Marne, France. 2National Cancer Institute, NIH,
Bethesda, MD20205, U.S.A.
All of the peripheral nervous system with the exception of a few cranial sensory ganglia derives from the
neural crest. Cells from the neural crest migrate within transient acellular pathways delineated by the
surrounding epithelial tissues. Adhesion and migration of crest cells in the extracellular matrix (ECM) is
thought to require fibronectin, a multifunctional glycoprotein: crest cells adhere specifically to fibronectin
and not to other ECM components. Movements of crest cells result from an intrisic ability to migrate in
young embryonic environment and weak but specific interactions with fibronectin. Directed cell migration is
apparently a consequence of both population pressure from crest cell proliferation and physical barriers
which are encountered during migration. Gangliogenesis results from the apearance of physical obstacles,
modulation of adhesive properties of the crest cells and modification of the composition of the ECM.
36
Matrix interactions
Role of the extracellular matrix during epimorphic regeneration in
Owenia fusiformis (Polychaete annelid)
Y. Thouveny, J. Coulon, J. P. Arsanto and M. H. Delgrossi, Laboratoire de Biologie de la Differentiation
Cellulaire, L.A. 179, University d'Aix-Marseille II, Faculti des Sciences de Luminy, F. 13288 Marseille Cedex
9, France
Near the amputation level of the first abdominal segment of the body, early regenerating processes
involve modifications of the cell morphology, of cell to cell adhesion and cell to extracellular matrix
adhesion. At this level, the extracellular matrix (ECM) undergoes a complete disorganization. Healing
epidermal cells migrate on the wound surface. Then, the blastema is formed by cell proliferation (1). In spite
of this ECM disorganization, the first step of regeneration appears to be dependant of some ECM
components. Inhibition of glycosylation by tunicamycin stops the blastema formation and decreases the rate
of DNA synthesis. A high molecular weight glycoprotein, immunologically related to fibronectin, was
localized by immunofluorescence and its biosynthesis and accumulation studied. There is a good correlation
between the deposition of this material, the sprouting of the nerve endings in the blastema and the
proliferative activity of the regeneration cells. A similar sprouting of nerve endings was obtained, in vitro,
on nerve cord explants grown on substrates containing ECM components.
These results indicate that the synthesis and deposition of new extracellular matrix would be the first
major event allowing: 1. the extent of the nerve fibres that were thought to be involved in the regulation of
cell proliferation (2); 2. the organization and the growth of the blastema.
(1) MARILLEY, M. & THOUVENY, Y. (1978). DNA synthesis during the first stage of anterior regeneration in
the polychaete annelid Owenia fusiformis. J. Embryol. exp. Morph. 44, 81-92.
(2) COULON, J. & THOUVENY, Y. (1984). Relation between innervation of cicatricial tissues and proliferative
activity of blastema cells during regeneration in Owenia fusiformis. Arch. Anat. microsc. morph. exp. (in
press).
Immunofluorescent localization of hyaluronate binding proteins on
motile cells
E. A. Turley* andJ. Torrance, Department Pharmacology, University of Calgary, Alberta, Canada T2N4N1
Polyclonal antibodies to hyaluronate binding proteins (HABP) were prepared in rabbits and used to
localize cell associated HABP on 3T3 cell lines and embryonic chick heart fibroblasts. HABP co-localized
with RITC-hyaluronate, but differed from the localization of fibronectin, laminin and heparan sulfate
proteoglycan even though HABP have been shown to bind to the nectin proteins in solid surface binding
assays and could therefore, theoretically, act as 'receptors' for such proteins. The localization of HABP and
RITC-hyaluronate changed with progressive age of the cell cultures and the rate of cell motility. HABP and
hyaluronate were localized in the leading lamellae and, often in the retraction of fibres of freshly seeded
cells (12 h old) that were actively motile. This distribution changed with age and declining rate of motility so
that after 4 days in culture and a motility rate one-half that of newly seeded cells, HABP and hyaluronate
appeared to be uniformly spread over cell body and processes. Interference reflection microscopy indicated
that hyaluronate and HABP coated areas of rapidly motile cells contained both close and focal contracts
although neither junction type was restricted or particularly localized to hyaluronate-rich areas. The
distribution of hyaluronate and HABP is discussed in context with the hypotheses regarding the role of
hyaluronate in the cell locomotory cycle.
Matrix interactions
37
An in vivo study of the relation between the presence of hyaluronate and
cell morphology in the chick blastoderm
/. Van Hoof1, F. Harrisson 1 andL. Vakaet 2. lState University of Antwerp, 171 Groenerborgerlaan, B-2020
Antwerp. 2State University of Ghent, Belgium
From in vitro experiments, it has been suggested that, in addition to its space-creating properties,
hyaluronate may also be important in the modulation of cell behaviour (Toole, 1982). Studies involving the
use of living tissues on this problem are rare. Therefore, blastoderms were presently studied at the
ultrastructural level after microinjection of glycosaminoglycan-degrading enzyme preparations. In these
blastoderms, the extracellular space was drastically decreased, thus confirming previous results (Fisher &
Solursh, 1977). In agreement with Sanders (1979), we observed a continuous remnant of the basal lamina all
over the ventral side of the epiblast. Immunocytochemical staining of these blastoderms showed that, at
least fibronectin, is still present in the basal lamina. Hyaluronidase (testicular and streptococcal) treatment
had also a profound effect on the behaviour of mesoblast cells. Most of these cells had a round or cuboidal
shape and had no cellular extensions. Large areas of close apposition between their cell membranes were
present. Cell junctions between these cells were abundant. While paraffin-embedded blastoderms showed
no contacts between meso- and epiblast, in agreement with previous reports (Fisher & Solursh, 1977), all
Epon-embedded blastoderms did, suggesting the former to be an artifact. In the latter, most mesoblast cells
tended to have large areas of their cell membranes close to the basal lamina instead of focal contacts
normally seen. Controls included Ringer, chondroitinase, and trypsine injections, as well as protease assays
of the enzymes used. Though protease activity was detectable in all preparations, we excluded the possibility
that these contaminants interfered with the experimental results for several reasons, which will be discussed
in detail. The present study provides additional arguments that hyaluronate may indeed be involved in the
modulation of cell behaviour. Particularly the abundant presence of intercellular junctions between
mesoblast cells of hyaluronidase-treated blastoderms, their altered contact pattern, and the change in their
relation to the basal lamina suggest that hyaluronate, by its presence, may prevent cellular communication
and facilitate focal detachment.
TOOLE, B. P. (1982). Connect. Tissue Res. 10, 93-100.
FISHER, M. & SOLURSH, M. (1977). /. Embryol. exp. Morph. 42, 195-207.
SANDERS, E. J. (1979). Cell Tissue Res. 198, 527-537.
The influence of extracellular matrix on reversible
gap junction formation in vitro
Christian Weber, Volker Schmid and Walter K. Schlage, Zoological Institute, University of Basel,
Rheinsprung 9, CH-4051 Basel, Switzerland
In vivo gap junctions are common in the subumbrellar plate endoderm of anthomedusa. When isolated
and cultivated in artificial sea water the tissue consisting of one cell type only forms a spheroid in which all
gap junctions disappear. Gap junction formation, however, can be induced by attachment and consecutive
spreading of the endodermal tissue (spheroid) on stretched extracellular matrix (ECM) material isolated of
the polyp stage (with CaMg-free sea water). Formation, and total loss of gap junctions is reversible and
strictly corresponds with the alteration from the monolayer 'spread' (on stretched ECM) to 'spheroid'
arrangement (no ECM) of the endodermal cells. The functional competence of induced gap junctions is
ascertained by injection of Lucifer Yellow, and the transfer of the dye is used to map the pattern of
communication. The experimental conditions that result in gap junction formation simulate the in vivo
situation of the endoderm. The influence of the ECM on gap junction formation, and the structural
organization of the isolated endodermal tissue in this well defined in vitro system are discussed.