Colloque SBCF-AT’IF’E/CNRS Biologie du Dtveloppement, mars 1995 99

CONTROL OF INTERCELLULAR ADHESION MEDIATED BY N-CADHERIN IN NEURAL CREST CELLS: CROSS TALK

BETWEEN CADHERINS AND INTEGRINS

MONIER Fr6dderiaue and DUBAND Jean-Loup

Laboratoire de Biologie Cellulaire du Dheloppement, Institut Jacques Mouod, Universit6 Paris 7, 2 place Jussieu, 75251 Paris Cedex OS, France

The development of the neural crest (NC) is associated with successive changes in the adhesion properties of cells. In particular, cells lose the cell adhesion molecule, N-cadherin, at the onset of migration and reexpress it during aggregation at the cessation of migration. We have analyzed in vitro the mechanisms of control of N-cadherin expression in migrating NC cells. NC cells moved without establishing permanent intercellular contacts with their neighbors. Although cells presented a weak staining for N-cadherin, they synthesized it at levels comparable to those found in cells exhibiting stable intercellular contacts. These observations suggest that migrating NC cells express N-cadherin but cannot retain permanent cell-cell interactions. We have determined which events are responsible for the instability of N- cadherin junctions using different agents known to affect signal transduction processes. Drugs that inhibit serine-threonine kinases. tyrosine kinases, or phosphatases all restored intercellular associations accompanied by the concentration of N-cadherin to the regions of cell-cell contacts. These results show that N-cadherin is regulated by kinase and phosphatase activities, but drug effects were not found to involve modifications in the phosphorylation state of N-cadherin. Based on the hypothesis that N-cadhetin instability may be linked to the mimatorv urocess, we demonstrated that inhibition of NC cell migration with agints ihat interfere with fibronectin-integrin interactions induced cell aggregation. This aggregation could be inhibited by calcium ionophores. Fu%e&ore, cell agGegaGon was obtained in the presence of an inhibitor of voltage-independent calcium channels, including the integrin- associated protein responsible for the calcium influx regulated by integrins. These different observations suggest that N-cadherin function in NC cells is directly controlled by integrins through signal transduction pathways implicating variation of intracellular calcium.

REPERTOIRE AND FUNCTION OF THE FIBRONECTIN- BINDING INTEGRINS IN

AVIAN NEURAL CREST CELL ADHESION AND MIGRATION

DELANNET Muriel, DESBAN Nathalie et DUBAND Jean-Loup

Laboratoire de Biologie Cellufaire du Dkveloppement, Institut Jacques Monod, Universitk Paris 7,2, place Jussieu, 75251 Paris Ceder OS, France

Fibronectin is an extracellular matrix adhesive molecule which was described as a substratum for neural crest cell (NCC) migration in vitro. Fibronectin can bind numerous cellular receptors belonging the integrin family. In the present study, we have studied the repertoire of the fibmnectii- binding integrins expressed by migrating NCC and defined their roles in cell adhesion and migration on fibronectin.

NCC expressed at least five fibronectin-binding integrins, i.e. a3p1, a4pl. a5B1, avpl and avp3. Inhibition assays of cellular adhesion and migration with function-perturbing antibodies allowed us to define the respective functions of these receptors. a3p1, a@1 and ~$1 integrins were each involved in cell adhesion, but a$1 played a predominant role in this process. avb3 and cx4pl were essentially implicated in cell locomotion. Moreover, immunofluorescence labelling of cultured motile NCC revealed that avpl is practically the sole integrin to be distributed in focal adhesion sites in association with the actin microfilaments, the other integtin receptors being diffuse on the cell surface. Thus, adhesion function of integrins and their cell surface distribution seem to be correlated.

We have previously shown that avpl mediates adhesion of NCC to vitronectin and that avp3 is implicated in locomotion on this substratum @PLANNET M. et at. (1994) Development 120:2687-2702 ). These two parallel works demonstrate that 1) avpl and avp3 mediate the same function on two different substrata and 2) neural crest cell migration requires the cooperation of at least two different integrins, one for cell adhesion and an other for cell locomotion.

We thank D.A. Chcrcsh (Scripps Res. Inst., La Jolla. USA) and L.F. Reichardt (Univ. California. San Francisco. USA) for providing antibodies lo inlegrins so generously. This work wils supported by CNRS. ARC and INSERM.

INTECRIN aY SUBUNIT: DISTRIBUTION DURING OOCENESIS AND EARLY AMPHIBIAN DEVELOPMENT SKALSKI Mvleng ALFANDARI Dominique, et DARRJBBRB Thieny. UniversitC P. et M. Curie, URA CNRS 1135, Laboratoire de Biologie Mol6culaire et Cellulaire du Dtveloppement, 9 Quai Saint Bernard, 75005 Paris, France. During development, cell interactions play essential roles in cell differentiation, tissue-specific gene expression and cell migration. During amphibian gastrulation, mesodermal cells migrate on a fibronectin rich extracellular matrix secreted by cells of the blastocel roof. These interactions are mediated by cell surface receptors of the integrin gl family. Integrins consist of transmembrane glycoproteins non-covalently associated in a8 heterodimers. One of the putative a subunit associated to 81 is av. It has been established that the integrin av subunit can associate with several different g subunits. Given the functional diversity of av integrins and the role of the extracellular matrix in early develonment, it seems imoortanr to . . . . . ”

analyse the distribution of the av subunit during.oogenesis and early development of the amphibian Plewodeles waltl. Using antibodies which recorrnize the exnacellu]ar domain of av. we”pr” ,“.-... b]ot inafysis show that the protein is maternal and expressed throughout the deveIopment. By indirect immunofluorescence, we have shown that integrin av subunit is present on the plasma membrane of vitellogenic oocytes, since it is detected only in ovarian tissues surrounding previtellogenic oocytes. At the time of fertilization, the nlasma membrane in the animal hemisohere ic 1.w I . ,

stained. In the vegetal hemisphere, while cytoplasmic staining is apparent there is no significant fluorescence on the egg surface. During early development, a general expression of av in the cytoplasm and a dynamic expression of the protein at the cell surface is observed. During cleavage, the staining remains in the cytoplasm, disappears progressively from the egg surface and appears on the plasma membrane of blastomeres of the marginal zone. During gastrulation, plasma membrane of both blastocoel floor and mesodermal cells are labelled. In particular, the fluorescence appears concentrated in the leading edge of migrating cells. At the end of gastrulation, integtin a, subunit expression at the cell surface disappears progressively. It remains mainly confined to cell-cell contacts in the mesoderm until neurulation. During organogenesis, the protein is detected in head mesenchyme, branchial archs. liver, and pronephros. Isolated cells are labelled in a region where cell migration and recruitment occur for Wolff duct formation. The results show that a, integtins may be present at the egg surface before fertilization and at the surface of migrating cells. They suggest a possible role for av integrins in mesodermal cell migration and organogenesis of the primary ureter.

ORIGIN OF BASEMENT MEMBRANE COLLAGEN GENES: CHARACTERIZATION OF THE MOST PRIMITIVE GENE IN A MARINE SPONGE

BOUTE Nicolas, EXPOSITO Jean-Yves, GEOlJRJON Christophr, GARRONE Robert.

Type IV collagen is one of the major prorein component of basement membranes. To d:tte, six genetically distinct a chains have been identified in manimaliatl basement membt-aces. Those chains can be divided into two parts : al-like chains (~1, a3 and ~5) and a2-like chains (~2, a4 and 06). The mammali~~n collapcn genes have :I unique arrangement in th;tt they arc loc;~trtl pairwisc in a hratl-to- head position on three different chromosomes, sharing bidirection;tl promoters. This arntngemcnt and the highly conservation of the sequences suggest that the genes have evoIvcd by duplications of ~111

ancestor gene. In order to get information on the origin of those type IV collagen genes, we decided to isolate :I b;rsemen~ nlembranr collagen gene in one of the most primitivr Metazoa11 : the martne sponge Pseud~~curricilrrnjtrrrei.

The high conservation of the c-tcrmtn:tt part of type IV collagen chains allowed us to make a specific probe using degenerared primers with RT-PCR. This probe was used to screen a Psell~locorricil~m jurrri cDNA library. Isolation of positive clones enabled us to derive a part of the primary sequence of 21 type IV collagen chain. The analysis with sequences of others type IV coilagen genes from different species help us to understand the first steps leading to the diversity and the organization of the genes coding for type IV collagen chains.