The neural crest constitutes a transient population of migratory progenitor cells that differentiate into a wide variety of peripheral neurons and non-neuronal cells (Le Douarin, 1982). Many of the cellular and molecular mechanisms that control the diversification and differentiation of neural crest cells during their peripheral migration have been defined (Anderson, 1997). However, the earlier steps that contribute to the specification of premigratory neural crest cells within the neural tube and to the delamination of these cells from the dorsal neural epithelium remain less well understood.
Fate mapping studies have shown that neural crest cells originate from the border of the neural plate and epidermal ectoderm (Rosenquist, 1981). This observation raised the possibility that signals derived from the epidermal ectoderm might be involved in the specification of neural crest cell fate. Support for this idea came from transplantation experiments in which the apposition of epidermal ectoderm and neural plate tissue induced the generation of neural crest cells in amphibian and avian embryos (Moury and Jacobson, 1989; Selleck and Bronner-Fraser, 1995). In vitro studies have indicated further that signals from the epidermal ectoderm are sufficient to induce neural crest differentiation in chick neural plate tissue (Dickinson et al., 1995; Liem et al., 1995). Members of the bone morphogenetic protein (BMP) family are strong candidates as mediators of this epidermal signal. In chick embryos, BMP4 and BMP7 are expressed in the epidermal ectoderm at the time of neural crest induction and both BMP proteins can mimic the neural crest inducing activity of the epidermal ectoderm in vitro (Liem et al., 1995). Furthermore, proteins that inhibit BMP4 and BMP7 activity block the ability of the epidermal ectoderm to induce neural crest cells (Liem et al., 1997), providing evidence for the requirement of BMP signaling in the initiation of neural crest cell differentiation.
The pathway by which cells in the neural plate respond to BMP signaling with the acquisition of a premigratory neural crest cell fate, however, is not well understood. Genes encoding several transcription factors, notably Msx and Zic genes, are expressed in the dorsal neural tube and some of these have been implicated in the specification of neural crest cell fate (Brewster et al., 1998; Foerst-Potts and Sadler, 1997; Nagai et al., 1997; Nakata et al., 1997; Shimeld et al., 1996; Wang et al., 1996). Following their specification, neural crest cells delaminate from the dorsal region of the neural epithelium and acquire mesenchymal properties as they begin their migration into the periphery (Duband et al., 1995). In chick embryos, the zinc finger transcription factor slug is expressed in the dorsal folds of the neural plate, in the dorsal neural tube and later in migrating neural crest cells (Liem et al., 1995; Nieto et al., 1994). Perturbation of slug function in vivo by antisense oligonucleotides has been reported to inhibit the emergence of neural crest cells from the dorsal neural tube of chick embryos (Nieto et al., 1994). The delamination of neural crest cells also appears to involve a reorganization of the cytoskeleton and changes in the expression of cell surface adhesion molecules and receptors (Duband et al., 1995). In particular, the expression of two members of the cadherin family, N-cadherin and cadherin6B, is detected at sites of premigratory neural crest cell generation and these genes are downregulated at the onset of neural crest cell migration (Nakagawa and Takeichi, 1995; Revel and Brown, 1976). The loss of these two cadherins is accompanied by the onset of high level expression of cadherin7 (Nakagawa and Takeichi, 1995). Although there is evidence for the involvement of these transcription factors and cell surface proteins in the differentiation of neural crest cells, the extent to which their pattern of expression is regulated by BMP signaling remains unclear.
In this study, we have attempted to identify additional molecules that might participate in the early steps of neural crest differentiation using a PCR-based screen for genes induced by BMPs in neural plate cells. We describe here the cloning and functional characterization of one gene obtained from this screen, rhoB, a member of the rho gene family. rho proteins are small GTP-binding proteins of the ras superfamily and the three major members of this subfamily, rho, rac and cdc42, appear to control many aspects of cellular functions including adhesion, morphology, motility and cell-cycle progression (Hall, 1998; Van Aelst and D’Souza-Schorey, 1997). The rho proteins have also been implicated in the transduction of extracellular signals that control gene transcription (Hill et al., 1995; Perona et al., 1997). Our results show that rhoB is expressed selectively by cells in the dorsal neural tube, that its expression persists transiently in migrating neural crest cells and that the expression of rhoB in neural tissue is induced by BMPs. Blockade of rho activity prevents the delamination of neural crest cells from the dorsal neural epithelium in vitro. These results suggest that rhoB has a role in the delamination of neural crest cells from the dorsal neural tube.
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