TFAP2 Paralogs Regulate Alx Genes During Midface Cranial Neural Crest Development.

Craniofacial anomalies are the 2 leading structural malformation present at birth. Those in the midface, such as orofacial clefts and hypertelorism, can arise from mutations in the Transcription Factor Activating Protein 2 genes TFAP2A and TFAP2B (abbreviated as TFAP2). These mutations do not reduce their gene expression, but instead generate mutant proteins that disrupt the activity of other wild-type and functionally redundant paralogs. Previous studies showed that both paralogs are co-expressed in the embryonic midface, hinting at their cooperative role during face development. Yet, how TFAP2 regulate midface development at the cellular and molecular level is poorly understood. To this end, we leveraged CRE-based approaches in mice to delete both Tfap2 members in the neural crest (NC), a cell lineage that gives rise to craniofacial bone and cartilage. We show that NC-specific knockout of Tfap2 (Tfap2 ) results in a fully penetrant midface cleft compared to controls and single paralog mutants. Lineage tracing analyses in Tfap2 embryos suggest that cranial NC migration into the embryonic face is not severely impaired. Corroborating this result, we find that Tfap2 deletion in either pre-migratory or late-migratory NC cells still results in a midface cleft. This suggests this phenotype stems from disrupted TFAP2 activity in post-migratory NC cells. Moreover, when examining Tfap2 craniofacial skeletons, we observe severe bone malformations and ectopic structures in the frontonasal regions when compared to controls. Single-cell RNA sequencing analysis reveals that loss of Tfap2 results in reduced cranial NC cell numbers and dysregulated Alx1/3/4 (Alx) gene expression. This is supported by the literature, as the Tfap2 midface clefts phenocopy those with loss of various Alxgene combinations. Our ongoing efforts aim to test how TFAP2 regulates Alx genes, if loss of Tfap2 compromises midface NC survival or proliferation, and whether Tfap2 and Alx genetically interact. In sum, we propose the model that TFAP2 regulates Alx gene expression in NC cells during midface development.