TGF-beta signaling and Creb5 cooperatively regulate Fgf18 to control pharyngeal muscle development

The communication between myogenic cells and their surrounding connective tissues is indispensable for muscle morphogenesis. During late embryonic development in mice, myogenic progenitors migrate to discrete sites to form individual muscles. The detailed mechanism of this process remains unclear. Using mouse levator veli palatini (LVP) development as a model, we systematically investigated how a distinct connective tissue subpopulation, perimysial fibroblasts, communicates with myogenic cells to regulate mouse pharyngeal myogenesis. Using single-cell RNAseq data analysis, we identified that TGF-beta signaling is a key regulator for the perimysial fibroblasts. Loss of TGF-beta signaling in the neural crest-derived palatal mesenchyme leads to defects in perimysial fibroblasts and muscle malformation in the soft palate in Osr2(Cre);Tgfbr1(fl/fl) mice. In particular, Creb5, a transcription factor expressed in the perimysial fibroblasts, cooperates with TGF-beta signaling to activate expression of Fgf18. Moreover, Fgf18 supports pharyngeal muscle development in vivo and exogenous Fgf18 can partially rescue myogenic cell numbers in Osr2(Cre);Tgfbr1(fl/fl) samples, illustrating that TGF-beta-regulated Fgf18 signaling is required for LVP development. Collectively, our findings reveal the mechanism by which TGF-beta signaling achieves its functional specificity in defining the perimysial-to-myogenic signals for pharyngeal myogenesis.