To identify molecules involved in early steps of neural crest differentiation, we performed a screen for BMP-induced neural genes and identified rhoB, a gene that encodes a member of the rho family of GTP-binding proteins (Madaule and Axel, 1985). rho proteins have been implicated in the control of neuronal cell morphology, axonal growth and dendritic arborization (Jalink et al., 1994; Kozma et al., 1997; Nishiki et al., 1990; Threadgill et al., 1997; Tigyi et al., 1996). This study shows that rhoB is expressed in premigratory neural crest cells and transiently in migrating neural crest cells. Moreover, the expression of rhoB in neural plate cells is induced by BMPs. The inhibition of rho function in vitro blocks the delamination of neural crest cells from the dorsal neural epithelium but appears not to inhibit their initial specification or their later migration. These findings suggest that rhoB activity has a role in the early differentiation of neural crest cells in response to BMP signaling.
rho proteins appear not to function in the specification of premigratory neural crest cells
rho proteins have been shown to function as intermediates in several cytoplasmic signal transduction pathways that link extracellular mitogenic and inductive signals to nuclear transcriptional responses. rhoA has been implicated in the serum-response pathway that activates transcription of the cfos gene (Hill et al., 1995). rho proteins have also been implicated in the activation of the transcription factor NF-kB (Perona et al., 1997) and in photoreceptor cell differentiation in Drosophila (Hariharan et al., 1995). These observations raised the question of whether the early expression of rhoB by premigratory neural crest cells might have a function in the initial specification of neural crest cell fate in response to BMPmediated signaling. Our results show the blockade of rho activity does not inhibit the BMP-induced generation of slug+ premigratory neural crest cells. Moreover, the induction of expression of slug precedes that of rhoB in response to BMP signaling. These data argue against a role for rhoB at this initial step of neural crest cell differentiation.
A role for rhoB in the delamination of neural crest cells
The delamination of neural crest cells from the dorsal neural epithelium represents a specialized instance of the more general phenomenon of epithelial-mesenchymal transition (Duband et al., 1995; Hay, 1995). During this transition, premigratory neural crest cells undergo changes in cell shape and cell-cell interactions and acquire the ability to interact with extracellular matrix (Duband et al., 1995).
Our studies provide evidence that rho activity is involved in the delamination of neural crest cells from the dorsal neural epithelium. The inactivation of rho function in prospective neural crest cells at the border of neural tube explants results in a disorganization of the actin cytoskeleton and impairs the ability of cells to migrate from the neural tube explant. rho proteins have been shown to regulate assembly of the actin cytoskeleton and focal adhesions: events that are important for cell morphology and adhesion (Nobes and Hall, 1995; Ridley and Hall, 1992). Thus, rho activity might contribute to the changes in cell shape and adhesion that occur during the delamination of neural crest cells by regulating actin polymerization and the formation of focal adhesions and stress fibers. Nevertheless, details of the pathway by which rho proteins control neural crest cell delamination remain unclear. In other cell types, rho proteins have been shown to interact with several downstream effector proteins. In particular, the serine/threonine kinases p160ROCK and ROCKII have been shown to be important for focal adhesions and stress fibers formation (Ishizaki et al., 1997; Leung et al., 1996). In addition, the p140mDia, a formin-related protein, has been shown to bind both rho proteins and profilin and may function in the regulation of actin polymerization by rho proteins (Watanabe et al., 1997).
The use of C3 toxin does not permit us to distinguish the contribution of individual rho proteins to the delamination of neural crest cells. However, the preferential association of rhoB expression with sites of neural crest differentiation in vivo supports the idea that rhoB is the primary target of C3 action in premigratory neural crest cells.
Inhibition of rho activity does not significantly alter the migration of neural crest cells in vitro The inhibition of rho function has been shown to inhibit the motility of a wide variety of cell types (Hinsch et al., 1993; Stasia et al., 1991; Takaishi et al., 1993). However, inhibition of rho activity appears to have little effect on the migration of neural crest cells, at least on a fibronectin substratum in vitro. One possible explanation for the absence of a requirement for rho activity is that neural crest cells employ other C3- insensitive GTPases, rac or cdc42, for their later migration. Distinct cell types have been shown to rely on different members of this GTPase superfamily for similar migratory behaviors. For example, in mouse keratinocytes, HGF-induced cell motility appears to be mediated by rho, but not by rac or ras (Takaishi et al., 1994), whereas in MDCK cells HGFinduced cell motility is mediated by ras or rac but not by rho (Ridley et al., 1995). Similarly, rac and cdc42 but not rho have been shown to induce cell motility in mammary epithelial cells (Keely et al., 1997). Thus, rac- or cdc42-dependent activities may be sufficient to promote the later migration of neural crest cells, independent of rho proteins.
BMP signaling and the induction of neural crest differentiation
Our studies on the expression of markers characteristic of sequential stages of neural crest cell development provide evidence that BMP signaling is sufficient to activate a molecular program of neural crest differentiation (Fig. 9). The induction of slug expression by BMPs in vitro precedes that of other markers examined, placing slug upstream of rhoB in a temporal hierarchy of neural crest cell gene expression in the chick embryo. The precise role of slug in the specification of neural crest cells, however, remains unclear. In chick embryos, the antisense oligonucleotide-mediated ablation of slug activity has been reported to perturb neural crest cell differentiation (Nieto et al., 1994). However in mouse embryos, slug expression is initiated only after neural crest cells have emerged from the neural tube and the targeted inactivation of the slug gene does not obviously affect early stages of neural crest cell differentiation (Jiang et al., 1998). It is possible that, in mouse, the closely related zinc finger gene snail serves a function in neural crest development similar to that proposed for the chick slug gene (Sefton et al., 1998; Smith et al., 1992). The sequential expression of cadherin6B and cadherin7 has been reported to control the delamination and later migration of neural crest cells (Nakagawa and Takeichi, 1995, 1998). Our results indicate that the pattern of expression of cadherin6B and cadherin7 in the neural tube is controlled, at least in part, by BMP signaling. In contrast to the initial restriction in slug and rhoB expression to the dorsal neural folds, the expression of cadherin6B is initially detected over a wide domain of the neural plate, absent only from ventral midline cells. It is possible therefore that the restriction of cadherin6B expression to the site of generation of premigratory neural crest cells involves both the BMP-mediated upregulation of cadherin6B dorsally and the Shh-mediated repression of gene expression ventrally. Alternatively, neural plate cells may extinguish cadherin6B expression over time unless exposed to BMP signals. Cadherin7 expression is excluded from the domain of premigratory neural crest cells, appearing only after cells have migrated away from the dorsal neural tube (Nakagawa and Takeichi, 1995). Thus, the induction of cadherin7 expression in neural plate explants by BMPs is likely to be a secondary consequence of the generation of premigratory neural crest cells.
Finally, our studies showing that the level of expression of rhoB is markedly elevated by BMP signaling parallel studies of other cell types in which rhoB, but not rhoA or rhoC, is induced rapidly by growth factors (Jahner and Hunter, 1991). It is possible therefore that rhoB is used generally as an inducible source of rho proteins under conditions in which a high level of rho activity is demanded. In the context of neural crest differentiation, the high level of rhoB activity appears to reflect a function for rho proteins during the delamination of neural crest cells from the dorsal neural tube.
We thank Alan Hall and Catherine Nobes for advice on rho function, for reagents and for their generous hospitality, A. Feig for the C3 plasmid and S. Morton for help with antibody generation. We are grateful to S. Arber, J. Briscoe, K. Lee and S. Price for discussions and comments on the manuscript, and K. MacArthur and I. Schieren for help in its preparation. We also thank K. Liem for providing BMP4 in situ images and for helpful discussions. J-P. L. is supported by a fellowship from the Burroughs Wellcome Fund. T. M. J. is an Investigator of the Howard Hughes Medical Institute.