Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells

The dynamic process of embryonic cell motility was investigated by analyzing the lateral mobility of the fibronectin receptor in various locomotory or stationary avian embryonic cells, using the technique of fluorescence recovery after photobleaching. The lateral mobility of fibronectin receptors, labeled by a monoclonal antibody, was defined by the diffusion coefficient and mobile fraction of these receptors. Even though the lateral diffusion coefficient did not vary appreciably (2 X 10(-10) cm2/S less than or equal to D less than or equal to 4 X 10(-10) cm2/S) with the locomotory state and the cell type, the mobile fraction was highly dependent on the degree of cell motility. In locomoting cells, the population of fibronectin receptors, which was uniformly distributed on the cell surface, displayed a high mobile fraction of 66 +/- 19% at 25 degrees C (82 +/- 14% at 37 degrees C). In contrast, in nonmotile cells, the population of receptors was concentrated in focal contacts and fibrillar streaks associated with microfilament bundles and, in these sites, the mobile fraction was small (16 +/- 8%). When cells were in a stage intermediate between highly motile and stationary, the population of fibronectin receptors was distributed both in focal contacts with a small mobile fraction and in a diffuse pattern with a reduced mobile fraction (33 +/- 9%) relative to the diffuse population in highly locomotory cells. The mobile fraction of the fibronectin receptor was found to be temperature dependent in locomoting but not in stationary cells. The mobile fraction could be modulated by affecting the interaction between the receptor and the substratum. The strength of this interaction could be increased by growing cells on a substratum coated with polyclonal antibodies to the receptor. This caused the mobile fraction to decrease. The interaction could be decreased by using a probe, monoclonal antibodies to the receptor known to perturb the adhesion of certain cell types which caused the mobile fraction to increase. From these results, we conclude that in locomoting embryonic cells, most fibronectin receptors can readily diffuse in the plane of the membrane. This degree of lateral mobility may be correlated to the labile adhesions to the substratum presumably required for high motility. In contrast, fibronectin receptors in stationary cells are immobilized in focal contacts and fibrillar streaks which are in close association with both extracellular and cytoskeletal structures; these stable complexes appear to provide firm anchorage to the substratum

Ets-1 Confers Cranial Features on Neural Crest Delamination

Neural crest cells (NCC) have the particularity to invade the environment where they differentiate after separation from the neuroepithelium. This process, called delamination, is strikingly different between cranial and trunk NCCs. If signalings controlling slow trunk delamination start being deciphered, mechanisms leading to massive and rapid cranial outflow are poorly documented. Here, we show that the chick cranial NCCs delamination is the result of two events: a substantial cell mobilization and an epithelium to mesenchyme transition (EMT). We demonstrate that ets-1, a transcription factor specifically expressed in cranial NCCs, is responsible for the former event by recruiting massively cranial premigratory NCCs independently of the S-phase of the cell cycle and by leading the gathered cells to straddle the basal lamina. However, it does not promote the EMT process alone but can cooperate with snail-2 (previously called slug) to this event. Altogether, these data lead us to propose that ets-1 plays a pivotal role in conferring specific cephalic characteristics on NCC delamination

Ségrégation cellulaire lors de la neurogenèse précose (les cadhérines font Sécession)

Les transitions de cadhérines sont souvent impliquées dans des phénomènes de ségrégation cellulaire mettant en jeu un phénomène de Transition Epithélium-Mésenchyme (TEM). Cependant, lors de la formation du système nerveux central, la transition E-/N-cadhérine n entraîne pas de TEM et, contrairement au modèle en vigueur, nos résultats montrent que celle-ci n est absolument pas un prérequis nécessaire aux mouvements morphogénétiques de la neurulation. Le point important lors de la formation du système nerveux central semble surtout être le contrôle de la cinétique de cette transition E-/N-Cadhérine. Le système nerveux central d oiseau se forme au cours du développement selon des modes bien distincts : dans la région antérieure de l embryon, la neurulation primaire ; dans la région postérieure, la neurulation dite secondaire conduit à un tube nerveux généré par accrétion cellulaire dont la lumière centrale est créée par cavitation. Dans la région thoracique, le tube neural se forme selon un mode totalement original ayant certaines caractéristiques des deux modes classiques, c est la neurulation intermédiaire. Les précurseurs neuraux du tube neural intermédiaire et secondaire effectuent une TEM puis migrent postérieurement de manière coordonnée et dirigée grâce au dépôt polarisé de fibronectine induit par la protéine de la polarité planaire, Prickle, puis se ré-épithélialisent. Les Cellules de la Crête Neurale (CCN) constituent un tissu à part du tube neural. Nous montrons que ces cellules se distinguent du reste du neuroépithélium par un répertoire d expression de cadhérines spécifiquesCadherin transitions play a part in cell segregation phenomenon, involving an Epithelium Mesenchyme Transition phenomenon (EMT). However, during the development of the central nervous system, the E-/N-cadherin transition doesn t cause an EMT and, unlike the current model, our results show that the E-/N-cadherin switch is not a necessary element in morphogenetic movements of neurulation. The important part in the central nervous system development seems to be mainly the control of the kinetic of this E-/N-Cadherin switch. Avian central nervous system develops according to specific ways: in the anterior part of the embryo, the primary neurulation ; the secondary neurulation which ends to the development of the neural tube by cells accretion which central lumen is created by cavitation. In the thoracic part of the body, the neural tube develops in an original way, with few characteristics from the two classic processes. It is the transition neurulation. The neural precursor of the transition neural tube and secondary neural tube make an EMT, and then migrate in a coordinated and directed way thanks to the polarized deposit of fibronectin, induced by the protein of the planar cell polarity, Prickle, and then re-epithelialized themselves. Neural Crest cells represent a specific population of the neural tube cells. We show that these cells are different from the rest of neuroépithélium thanks to a repertory of expression of specific cadherins: E-Cadherin expressed by non-neural ectodermal cells, Cadherin-6B expressed by neural crest cells and N-cadherin expressed by canonical neural cells. This segregation is orchestrated by a subtle balance between BMP4 and FGFPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

ROLE COORDONNE DES INTEGRINES LORS DE LA MIGRATION DES CELLULES DES CRETES NEURALES IN VITRO ET REGULATION DE LEUR ACTIVATION PAR SONIC HEDGEHOG

LES CELLULES DES CRETES NEURALES (CCN) FONT PARTIES DES TYPES CELLULAIRES DE LA MOITIE DORSALE DU TUBE NEURAL QUI SONT INDUITS PAR LES MEMBRES DE LA FAMILLE DES TGF NOTAMMENT BMP4. SOUS LE CONTROLE DE BMP4, LES CCN ACQUIERENT PAR LA SUITE DES PROPRIETES LOCOMOTRICES TRANSITOIRES QUI LEUR PERMETTENT D'ENVAHIR DIFFERENTES ZONES EMBRYONNAIRES AFIN DE S'Y DIFFERENCIER. LEUR MIGRATION S'EFFECTUE GRACE A L'ACQUISITION D'INTEGRINES, RECEPTEURS DE LA MATRICE EXTRACELLULAIRE ENVIRONNANTE ET DONT LA FIBRONECTINE EST L'UN DES CONSTITUANTS MAJEURS. NOUS AVONS TOUT D'ABORD ETABLIT LE REPERTOIRE DES RECEPTEURS INTEGRINES DE LA FIBRONECTINE EXPRIMES PAR LES CCN. CELLES-CI NE PRESENTENT PAS MOINS DE SEPT INTEGRINES A LEUR SURFACE MAIS SEULES QUATRE D'ENTRE ELLES SEMBLENT DIRECTEMENT IMPLIQUEES DANS LA LOCOMOTION DES CCN SUR LA FIBRONECTINE. LES INTEGRINES V 1 ET 8 1 REGULENT L'ADHERENCE TANDIS QUE LES INTEGRINES 4 1, 8 1 ET V 3 CONTROLENT LA MIGRATION. IL SEMBLE QUE L'INTEGRINE 4 1 COORDONNE L'ACTIVITE DES AUTRES INTEGRINES FONCTIONNELLES, CECI QUEL QUE SOIT LE TYPE DE SUBSTRAT UTILISE ET PROBABLEMENT PAR L'INTERMEDIAIRE DE MOLECULES TRANSMEMBRANAIRES ASSOCIEES AUX INTEGRINES, LES TETRASPANINES CD81, CD82 ET CD151. LA POLARITE DORSOVENTRALE DU TUBE NEURAL EST LE RESULTAT DES ACTIVITES ANTAGONISTES DES BMP ET DE SONIC HEDGEHOG (SHH), L'INDUCTEUR DES TYPES CELLULAIRES VENTRAUX. NOUS AVONS EMIS L'HYPOTHESE QUE SHH CONSTITUAIT AUSSI UN SIGNAL ANTAGONISTE DE BMP4 LORS DE L'EMIGRATION DES CCN. EN EFFET, LES CCN EN PRESENCE DE SHH SONT INCAPABLES DE SE DEPLACER. CETTE INHIBITION DE LA MIGRATION PAR SHH N'EST PAS LE FAIT D'UNE MODIFICATION DE LA SPECIFICATION DES CCN NI D'UN CHANGEMENT DE LA POLARITE DU TUBE NEURAL. DE PLUS, LA PROLIFERATION ET LA SURVIE DES CCN NE SONT PAS ALTEREES. SHH N'AFFECTE PAR NON PLUS L'EXPRESSION DES CADHERINES, DE RHOB OU DES INTEGRINES INTERVENANT LORS DE L'EMIGRATION DES CCN. EN FAIT, SHH INACTIVE LES INTEGRINES ET CONTROLE AINSI L'ADHERENCE ET LA MIGRATION DES CCN VIA UNE SEQUENCE SPECIFIQUE ET DISTINCTE DE CELLE IMPLIQUEE DANS L'INDUCTION DES NEURONES. NOUS PROPOSONS QUE SHH N'EST PAS SEULEMENT IMPLIQUEE DANS LA SPECIFICATION DES NEURONES MAIS REGULE AUSSI LE CARACTERE MIGRATOIRE DES CELLULES DU TUBE NEURAL, REVELANT UN NOUVEAU ROLE POUR CETTE MOLECULE DANS LE CONTROLE DE LA MORPHOGENESE.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Rôle du proto-oncogène Ets1 au cours du développement des cellules des crêtes neurales aviaires

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Buckling along boundaries of elastic contrast as a mechanism for early vertebrate morphogenesis

International audienceWe have investigated the mechanism of formation of the body of a typical vertebrate, the chicken. We find that the body forms initially by folding at boundaries of stiffness contrast. These boundaries are dynamic lines, separating domains of different cell sizes, that are advected in a deterministic thin-film visco-elastic flow. While initially roughly circular, the lines of elastic contrast form large ``peanut'' shapes evoking a slender figure-8 at the moment of formation of the animal body, due to deformation and flow in a quadrupolar stretch caused by mesoderm migration. Folding of these ``peanut'' or ``figure-8'' motives along the lines of stiffness contrast creates the global pattern of the animal, and segregates several important territories. The main result is that the pattern of cell texture in the embryo serves simultaneously two seemingly different purposes: it regionalizes territories that will differentiate to different cell types and it also locks the folds that physically segregate these territories. This explains how the different cellular types segregate in physically separated domains

Trunk NCC Delamination Occurs Prematurely is Amplified and Prolonged by <i>h-ets-1</i> Misexpression.

<p>(A–a) Analysis of the effects of <i>h-ets-1</i> misexpression in trunk dorsal neural tube assayed at 15hpe (A–E, O–R), 24hpe (F–N) and 48hpe (S–a). (A–J, O–R) Whole mount in situ hybridization using <i>sox-10</i> (A–J, dark blue), <i>cadherin-6B</i> (O–R, dark blue) and <i>h-ets-1</i> (A–E, O–R, light blue) probes. (K–N) Wholemount immunostaining using anti HNK1 antibody. (C–E, H–J, M–N, P, R) Vibratome sections (30 µm) of embryos presented in (B), (G), (L), (O') and (Q') respectively. (V–Y) In situ hybridization on transversal cryosections (20 µm) using <i>sox-9</i> (V–W) and <i>sox-10</i> (X–Y) probes. (Z–a) Transversal cryosections (10 µm) immunolabeled using anti-N-Cadherin antibody. Electroporated cells are detected by GFP expression (S–T, W, Y, a) or DAPI staining (U). At 15hpe in <i>h-ets-1</i> caudally transfected embryos, <i>sox-10</i> trunk NCCs delaminate precociously (A–B, E, arrow heads). Besides, more rostrally, the outflow is increased (C–D) compared to contralateral side and is associated with a loss of <i>cadherin-6B</i> expression (O–P). At 24hpe, at level where delamination is already completed on the control side (H–J, asterisks), <i>h-ets-1</i> expression prolongs delamination of a massive amount of <i>sox-10</i> (H–J, arrow heads) and HNK-1 (M–N) positive NCCs. At 48hpe, <i>h-ets-1</i> transfected cells are still able to leave the dorsal neural tube as a multilayered wave (S–U) but they fail to express NCCs markers such as <i>sox-9</i> (V–W), <i>sox-10</i> (X–Y) and keep a strong expression of N-Cadherin (Z–a). ot, otic vesicle.</p

Ets-1 confers cranial features on neural crest delamination.

<p>(A) Normal delamination of cranial NCCs. Premigratory and migratory NCCs expressing <i>ets-1</i> are in purple. (B) Normal delamination of trunk NCCs. Premigratory and migratory NCCs are in yellow. (C) Consequences of <i>ets-1</i> electroporation in trunk neural tube at dorsal and at intermediate to ventral levels. <i>Ets-1</i> electroporated cells are coloured in green. (D) Cell movements induced by <i>ets-1</i> expression. Proliferating cells are in grey, non-proliferating cells are in blue. <i>Ets-1</i> electroporated cells are dotted in green. Cell-cell junctions involving N-cadherin are represented by black centers. Nuclei in S-phase are colored in black. Basal lamina is represented by twisted red line. Cranial NCCs express <i>ets-1</i> and massively delaminate independently of G1/S transition (A) whereas trunk NCCs do not express <i>ets-1</i> and delaminate progressively as a cell population subjected to successful G1/S transition (B). When <i>ets-1</i> expression is forced in the dorsal part of trunk neural tube, trunk NCCs delamination is greatly enhanced and cells emigrate as multilayered streams (C, green cells). Moreover, they lose their subjection to cell cycle progression indicating that <i>ets-1</i> converts trunk delamination into cranial-like emigration (C). Ectopic <i>ets-1</i> expression in ventral part of the neuroepithelium leads to massive cell movements without affecting cell proliferation or differentiation. Electroporated cells are accumulated close to the basal side of the neural tube and the basal lamina is degraded (C, D). These events are sufficient to initiate delamination. However, other factors such as <i>snail-2</i> are required to perform full delamination and promote EMT and cell migration. M, cell in mitosis.</p

Junctional Neurulation: A Unique Developmental Program Shaping a Discrete Region of the Spinal Cord Highly Susceptible to Neural Tube Defects

International audienceIn higher vertebrates, the primordium of the nervous system, the neural tube, is shaped along the rostrocaudal axis through two consecutive, radically different processes referred to as primary and secondary neurulation. Failures in neurulation lead to severe anomalies of the nervous system, called neural tube defects (NTDs), which are among the most common congenital malformations in humans. Mechanisms causing NTDs in humans remain ill-defined. Of particular interest, the thoracolumbar region, which encompasses many NTD cases in the spine, corresponds to the junction between primary and secondary neurulations. Elucidating which developmental processes operate during neurulation in this region is therefore pivotal to unraveling the etiology of NTDs. Here, using the chick embryo as a model, we show that, at the junction, the neural tube is elaborated by a unique developmental program involving concerted movements of elevation and folding combined with local cell ingression and accretion. This process ensures the topological continuity between the primary and secondary neural tubes while supplying all neural progenitors of both the junctional and secondary neural tubes. Because it is distinct from the other neurulation events, we term this phenomenon junctional neurulation. Moreover, the planar-cell-polarity member, Prickle-1, is recruited specifically during junctional neurulation and its misexpression within a limited time period suffices to cause anomalies that phenocopy lower spine NTDs in human. Our study thus provides a molecular and cellular basis for understanding the causality of NTD prevalence in humans and ascribes to Prickle-1 a critical role in lower spinal cord formation