Zfp750 prevents oral adhesions and promotes temporary epithelial fusions

The differentiation cascade that converts basal keratinocytes into suprabasal layers, including periderm, depends on the activity of transcription factors. Mutations in the genes encoding many of these transcription factors, including <i>TP63, IRF6</i> and <i>GRHL3</i> , disrupt periderm development. Such mutations can also interfere with embryonic fusion and septation events that depend on periderm development, including palatogenesis, digit separation and the formation of temporary epithelial fusions between digits, between eyelids, and between pinnae and the scalp. ZNF750 (Zfp750 in the mouse) is a transcription factor required for keratinocyte differentiation, but whether mutations in <i>ZNF750</i> contribute risk for orofacial cleft, and the role of Zfp750 in periderm development, are unknown. To address these questions we sequenced <i>ZNF750</i> in 5,659 individuals including 2,125 with nonsyndromic OFC. We identify 33 rare missense variants with frequencies less than 0.1% in gnomAD. Of these, about half are predicted to be damaging with <i>in silico</i> tools. Collectively, these missense variants are not overtransmitted from parents to children with OFCs. Two of the variants have lower activity than the reference variant in a zebrafish embryo-based assay but no phenotype in the corresponding murine model. However, in murine embryos homozygous for a frame-shift mutation in <i>Zfp750</i> ( <i>Zfp750</i> ^<i>fs</i> ) that we generated, palatal shelves are fused but intra-oral adhesions are present, a phenotype seen in murine mutants of several <i>bonafide</i> OFC genes. In addition, temporary epithelial fusions are absent in <i>Zfp750</i> ^<i>fs</i> neonates. RNA sequencing of forelimbs from <i>Zfp750</i> ^<i>fs</i> embryos reveals decreased expression of epidermal terminal differentiation genes, and both increased and decreased expression of distinct periderm genes. Immunofluorescence shows the consistent presence of periderm proteins within the oral adhesions in <i>Zfp750</i> ^<i>fs/fs</i> embryos. Together these studies suggest that while mutations in <i>ZNF750</i> are not a major contributor to OFC risk, Zfp750 does contribute to periderm-dependent morphogenic events.