Fibroblast growth factor and bone morphogenetic protein signaling are required for specifying prechondrogenic identity in neural crest-derived mesenchyme and initiating the chondrogenic program.

The pharyngeal endoderm is hypothesized as the source of local signals that specify the identity of neural crest-derived mesenchyme in the arches. Sox9 is induced and maintained in prechondrogenic cells during condensation formation and endochondral ossification. Using explant culture, we determined that pharyngeal endoderm was sufficient, but not necessary for specifying prechondrogenic identity, as surrounding tissues including the otic vesicle can compensate for signals from the pharyngeal endoderm. Multiple Fgf genes are expressed specifically in the pharyngeal endoderm subjacent to the neural crest-derived mesenchyme. Fibroblast growth factor (FGF) signaling is both sufficient and required for specification of Sox9 expression and specification of prechondrogenic identity, as demonstrated by the addition of recombinant FGF protein or the FGF receptor inhibitor (SU5402) to explanted tissue, respectively. However, FGF signaling cannot maintain Sox9 expression or initiate the chondrogenic program as indicated by the absence of Col2a1 transcripts. Bone morphogenetic protein (BMP) 4 signaling can induce and maintain Sox9 expression in isolated mesenchyme, but only in combination with FGF signaling induce Col2a1 expression, and thus, chondrogenesis. Given the spatiotemporal expression patterns of FGFs and BMPs in the pharyngeal arches, we suggest that this may represent a general mechanism of local signals specifying prechondrogenic identity and initiation of the chondrogenic program.