Functional analysis of human orofacial clefting genes in Xenopus laevis

Orofacial clefting (OFC) is one of the most common congenital anomalies and is accompanied by medical, economic, and social burdens on the affected individuals and their families. A significant proportion of OFC cases have a genetic basis, but the majority of genetic variation underlying orofacial clefting remains unknown. Our lab previously conducted a large copy-number variation study of OFC patients aimed at identifying previously unknown OFC loci. Three of the candidate genes identified in this study were ARHGEF38, COBLL1, and RIC1, all members of the Rho/Rab GTPase signaling pathway. Other members of this pathway have been implicated in OFC, suggesting that these candidates are likely to be important in proper facial development. To test this hypothesis, we knocked down these genes in zebrafish (Danio rerio) and frogs (Xenopus laevis) and assessed how their facial tissue developed. In this work, we show that CRISPR-Cas9 and morpholino knockdown of arhgef38, cobll1, and ric1 in zebrafish results in defects in cranial cartilage development. We also show that arhgef38, cobll1, and ric1 are expressed in the developing head in frogs, and that knockdown of these genes also leads to reduced or absent cartilage development. Together, this data supports the validation of ARHGEF38, COBLL1, and RIC1 as human OFC genes, expanding our knowledge of the genetic regulation of mouth development.