TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway [RNA-Seq]
Cranial neural crest development is governed by positional gene regulatory networks (GRNs). Fine-tuning of the GRN components underly facial shape variation, yet how those in the midface are connected and activated remain poorly understood. Here, we show that concerted inactivation of Tfap2a and Tfap2b in the murine neural crest, even during the late migratory phase, results in a midfacial cleft and skeletal abnormalities. Bulk and single-cell RNA-seq profiling reveal that loss of both Tfap2 members dysregulates numerous midface GRN components involved in midface morphogenesis, patterning, and differentiation. Notably, Alx1/3/4 (Alx) transcript levels are reduced, while ChIP-seq analyses suggest TFAP2 directly and positively regulates Alx gene expression. TFAP2 and ALX co-expression in midfacial neural crest cells of both mouse and zebrafish further implies conservation of this regulatory axis across vertebrates. Consistent with this notion in zebrafish, tfap2a mutants present abnormal alx3 expression patterns, Tfap2a binds alx loci, and tfap2a-alx3 genetic interactions are observed. Together, these data demonstrate TFAP2 paralogs regulate vertebrate midfacial development by activating expression of ALX transcription factors. Overall design: To investigate the role of TFAP2 (Tfap2a and Tfap2b) in midface neural crest cell development, we isolated E10.5 frontonasal and maxillary prominence tissue from 3 Tfap2a;Tfap2b;Wnt1CRE mutants, and 3 sibling matched controls, and conducted bulk RNA sequencing of the cDNA generated.