Molecular Regulation of Palate Development

Cleft palate is a common major birth defect, of which surgical repair often fail to fully restore the structure and function of palatal muscles, leaving many patients suffering velopharyngeal dysfunction and/or impairment of speech and hearing. To better understand the mechanisms regulating palatal muscle morphogenesis and integration, we performed single-cell multi-omic analysis to jointly profile the transcriptome and chromatin accessibility at the onset of palatal muscle formation. Direct comparison of patterns of transcription factor expression and ATAC-seq profiles of their DNA binding sites in the developing palatal mesenchyme suggest that multiple transcription factors act in concert to regulate connective tissue differentiation and palatal muscle morphogenesis. In particular, the Forkhead-box family transcription factors Foxf2 and Foxd1 exhibit complimentary patterns of expression to pre-pattern the soft palate mesenchyme for palatal myogenesis, with Foxf2 expression enriched in medial mesenchyme progenitors and Foxd1 strongly expressed in perimysial progenitor cells. A missense mutation in FOXF2 has been associated with shortened soft palate with misoriented palatal muscles. We show that mice with specific disruption of Foxf2 display cleft posterior soft palate with failure of palatine aponeurosis formation and malpositioning of palatal muscles. Single cell RNA-seq analysis using E13.5 palatal tissue from Foxf2 null mutant mice comparing with wiltdype control embryos revealed that the expression of Foxd1 and several perimysial mesenchyme marker genes were ectopically up-regulated in the mesenchymal progenitor cells in Foxf2 mutant palate. Together, these results uncover a key role of Foxf2 and Foxd1 in controlling palatal connective tissue differentiation and palatal myogenesis.