Fgf receptors Fgfr1a and Fgfr2 control the function of pharyngeal endoderm in late cranial cartilage development.

Cranial cartilage derives mainly from cranial neural crest cells and its formation requires fibroblast growth factor (Fgf) signaling for early differentiation and survival of developing chondrocytes as well as patterning of the endodermal pouches. Here, we investigate the role of Fgf receptors in chondrocyte maturation at later stages, beyond 24 hpf. Using inducible expression of a dominant-negative Fgf receptor, we show that Fgf signaling is required around 30 hpf for correct cartilage formation. The receptor genes fgfr1a and fgr2 are expressed in pharyngeal endodermal pouches after 24 hpf or 26 hpf, respectively. Depletion of any of these two receptors by microinjection of antisense morpholinos results in severe defects in cartilage formation at 4 dpf and a decrease in expression of the late chondrocyte markers barx1 and runx2b. Although endodermal pouches are correctly formed and patterned, receptor knock down leads to decreased expression of runx3, egr1 and sox9b in this tissue, while expression of fsta, coding for a secreted BMP/Tgfß inhibitor, is clearly increased. Rescue experiments revealed that each Fgfr1a or Fgfr2 receptor is able to compensate for the loss of the other. Thus, we show that minimal amounts of Fgfr1a or Fgfr2 are required to initiate a regulatory cascade in pharyngeal endoderm reducing expression of fsta, thereby allowing correct BMP signaling to the maturing chondrocytes of the head cartilage.

RUNX3, EGR1 and SOX9B form a regulatory cascade required to modulate BMP-signaling during cranial cartilage development in zebrafish.

The cartilaginous elements forming the pharyngeal arches of the zebrafish derive from cranial neural crest cells. Their proper differentiation and patterning are regulated by reciprocal interactions between neural crest cells and surrounding endodermal, ectodermal and mesodermal tissues. In this study, we show that the endodermal factors Runx3 and Sox9b form a regulatory cascade with Egr1 resulting in transcriptional repression of the fsta gene, encoding a BMP antagonist, in pharyngeal endoderm. Using a transgenic line expressing a dominant negative BMP receptor or a specific BMP inhibitor (dorsomorphin), we show that BMP signaling is indeed required around 30 hpf in the neural crest cells to allow cell differentiation and proper pharyngeal cartilage formation. Runx3, Egr1, Sox9b and BMP signaling are required for expression of runx2b, one of the key regulator of cranial cartilage maturation and bone formation. Finally, we show that egr1 depletion leads to increased expression of fsta and inhibition of BMP signaling in the pharyngeal region. In conclusion, we show that the successive induction of the transcription factors Runx3, Egr1 and Sox9b constitutes a regulatory cascade that controls expression of Follistatin A in pharyngeal endoderm, the latter modulating BMP signaling in developing cranial cartilage in zebrafish.