Central role of the alpha4beta1 integrin in the coordination of avian truncal neural crest cell adhesion, migration, and survival.

Based on functional and histological studies, the fibronectin receptor of the integrin family alpha4beta1 has been ascribed a critical role during neural crest cell migration in the vertebrate embryo. In the present study, because integrins have been shown to participate in multiple basic cellular processes, including cell adhesion, migration, survival, proliferation, and differentiation, we have reexamined in detail the role of alpha4beta1 during avian truncal neural crest cell migration. RT-PCR and immunocytochemical studies revealed that migrating neural crest cells but not premigratory cells explanted in vitro expressed detectable levels of alpha4 messengers and proteins suggesting that alpha4beta1 expression was induced at the time of the initiation of the migration phase. In agreement with this observation, antibody inhibition of alpha4beta1 activity in vitro resulted in a strong, immediate and sustained reduction of neural crest cell motion on fibronectin, as judged on videomicroscopy analyses, but apparently did not prevent their delamination from the neural tube. However, alpha4beta1 appeared to exhibit a broader role in the control of cell migration on a variety of extracellular matrix molecules, presumably by regulating cellular events downstream from integrins. Moreover, blocking alpha4beta1 function caused a severe increase in apoptotic cell death among the neural crest population without influencing notably cell proliferation. Collectively, these results indicate that, notwithstanding its critical implication in cell motion, alpha4beta1 integrin could play a central role in neural crest cell development by coordinating multiple cellular events, such as cell adhesion, locomotion, and survival.

Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched- Smoothened-Gli signaling pathway.

In the vertebrate embryo, neural cell types are organized spatially along the dorsoventral axis of the neural tube and differ by expression of cell-intrinsic determinants and by their adhesive and locomotory properties. Thus, dorsally, neural crest cells (NCC) show a strong propensity to disperse and migrate, whereas cells situated ventrally are highly cohesive and poorly motile. Members of the bone morphogenetic proteins have been shown to exert a dual role in the specification of dorsal neuroepithelial cells and in the dispersion of NCCs. To test whether Sonic hedgehog (Shh), another signaling molecule involved in the patterning of the ventral neural tube, might also contribute to the control of the adhesive and migratory potential of neuroepithelial cells, we analyzed the effect of ectopic Shh on NCC dispersion from neural tube explants cultured in vitro. The addition of Shh to the migration substrate of NCC caused inhibition of their dispersion. The effect of Shh on cell migration was reversible and was not accounted for by alterations of the specification, delamination, proliferation, and survival of NCCs but could be essentially attributed to a decreased cell-substrate adhesion mediated by integrins. In addition, Shh activity on cell migration was mediated by a specific N-terminal region of the molecule and was independent from the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Our study therefore reveals an unanticipated role for Shh in regulating adhesion and migration of neuroepithelial cells that is discernable from its inductive, mitogenic, and trophic functions.

Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the (beta)1 and (beta)3 families.

Based on genetic, functional and histological studies, the extracellular matrix molecule fibronectin has been proposed to play a key role in the migration of neural crest cells in the vertebrate embryo. In the present study, we have analyzed in vitro the repertoire and function of integrin receptors involved in the adhesive and locomotory responses of avian truncal neural crest cells to fibronectin. Immunoprecipitation experiments showed that neural crest cells express multiple integrins, namely (alpha)3(beta)1, (alpha)4(beta)1, (alpha)5(beta)1, (alpha)8(beta)1, (alpha)v(beta)1, (alpha)v(beta)3 and a (beta)8 integrin, as potential fibronectin receptors, and flow cytometry analyses revealed no major heterogeneity among the cell population for expression of integrin subunits. In addition, the integrin repertoire expressed by neural crest cells was found not to change dramatically during migration. At the cellular level, only (alpha)v(beta)1 and (alpha)v(beta)3 were concentrated in focal adhesion sites in connection with the actin microfilaments, whereas the other integrins were predominantly diffuse over the cell surface. In inhibition assays with function-perturbing antibodies, it appeared that complete abolition of cell spreading and migration could be achieved only by blocking multiple integrins of the (beta)1 and (beta)3 families, suggesting possible functional compensations between different integrins. In addition, these studies provided evidence for functional partitioning of integrins in cell adhesion and migration. While spreading was essentially mediated by (alpha)v(beta)1 and (alpha)8(beta)1, migration involved primarily (alpha)4(beta)1, (alpha)v(beta)3 and (alpha)8(beta)1 and, more indirectly, (alpha)3(beta)1. (alpha)5(beta)1 and the (beta)8 integrin were not found to play any major role in either adhesion or migration. Finally, consistent with the results of inhibition experiments, recruitment of (alpha)4(beta)1 and (alpha)v(beta)3, individually or in combination using antibodies or recombinant VCAM-1 and PECAM-1 molecules as a substratum, was required for migration but was not sufficient to produce migration of the cell population as efficiently as with fibronectin. In conclusion, our study indicates that neural crest cells express a multiplicity of fibronectin-binding integrins and suggests that dispersion of the cell population requires cooperation between distinct integrins regulating different events of cell adhesion, locomotion and, possibly, proliferation and survival.