FGF 2 promotes skeletogenic differentiation of cranial neural crest cells

Cells of the cranial neural crest make a major contribution to the skeletal tissues of the skull, differentiating into both primary and secondary cartilage, endochondral bone and membrane bone (Hall and Horstadius, 1988; Couly et al., 1993). Whether or not there is an established skeletogenic lineage within the cranial neural crest before migration is unclear. Moreover, the developmental events determining the skeletogenic fate of a sub-population of neural crest and their derivatives have only been identified at the level of epithelialmesenchymal interactions (Bee and Thorogood, 1980) and interactions with the extracellular matrix (Hall, 1982, Thorogood, 1993). A significant clue to the molecular mechanisms that underlie cranial skeletogenesis has come from identification of mutations in the genes encoding fibroblast growth factor receptor (FGFRs) in individuals with craniosynostosis. In this group of diseases, the sutures of the cranial vault lose their proliferative function and display precocious bone differentiation and fusion (Gorlin et al., 1990; Wilkie, 1997; Burke et al., 1998). Several FGFR mutations create constitutive activation of FGF signalling, endowing the receptor with a degree of ligand-binding independence for signal transduction (Webster and Donoghue, 1997). These studies point to a role for FGF signalling in the skeletogenic differentiation of neural crest-derived tissues. The precise role of FGF-mediated signalling in cranial neural crest cell development is, however, unclear. FGF2 is known to stimulate neural crest proliferation in cultures of mouse neural tube (Murphy et al., 1994) and in micromass cultures of chick facial ectomesenchyme, where growth is usually accompanied by an increase in chondrogenesis (Richman and Crosby, 1990). Correspondingly, FGF2and FGF4-soaked beads implanted in vivo into facial tissue promote growth locally and increase the size of cartilage elements at that site (Richman et al., 1997). The facial ectoderm is a local source of FGF2 and is required for expression of FGFR2 in the underlying mesenchyme (Matovinovic and Richman, 1997), which further suggests a role for FGF signalling in the growth and differentiation of facial processes. The possibility that these mechanisms may have a broader and more fundamental role in skull morphogenesis, perhaps underlying the tissue interactions that direct skeletogenic differentiation of cranial neural crest cells, has not been fully explored. We aimed to answer three questions relating to the role of FGF signalling in the skeletogenic differentiation of cranial neural crest cells. First, does neural crest responsiveness to FGF exist before migration? Second, does FGF-mediated signalling play a causal role in determining the skeletogenic fate of neural crest cells? Third, can the relationship between FGF and neural crest development be related to the clinical phenotype of craniosynostosis, in which neural crest-derived c lls of the suture differentiate precociously? We find that FGF2 serves as a survival factor for avian pre-migratory mesencephalic neural crest cells in culture and elicits a concentration-dependent response, with proliferation at low FGF2 concentrations and skeletogenic differentiation at high 2143 Development 128, 2143-2152 (2001) Printed in Great Britain © The Company of Biologists Limited 2001 DEV2720