The ALX transcription factors determine frontonasal identity of the neural crest-derived craniofacial mesenchyme

Biallelic disruptions of any of the three Aristaless-like homeobox (ALX) genes, ALX1, ALX3, and ALX4, cause frontonasal dysplasia, a class of congenital craniofacial disorders characterized by hypertelorism and median facial clefting. Studies in mice showed that Alx1, Alx3, and Alx4 exhibit partially overlapping patterns of expression during craniofacial development. Whereas Alx3 mutant mice do not have obvious craniofacial defects and Alx4 mutant mice exhibit parietal foramina, Alx1 mutant mice show frontonasal defects including short snout, flat nasal bridge, upper lip notching, and hypoplastic premaxilla, and die soon after birth. The Alx3/Alx4 and Alx1/Alx4 compound mutant mice show severe nasal clefting and neonatal lethality. In this study, we show that Alx1/Alx3 compound mutant mice have severe nasal clefting with missing upper and lower incisors. We show that Alx1/Alx3 mutants exhibit disruption of frontonasal mesenchyme identity with ectopic expression of jaw mesenchyme markers Dlx1, Dlx2, Lhx6, and Lhx8 during embryonic development. Furthermore, transgenic overexpression of Alx1 in the post migratory cranial neural crest cells (CNCC) resulted in duplication of the premaxilla accompanied by ectopic expression of Alx4, Pax7, and Shh genes and reduction in expression of Dlx1, Dlx2, Dlx3, and Dlx5 genes in the developing maxillary/mandibular mesenchyme. Together these data indicate that ALX family transcription factors are necessary and sufficient to determine the frontonasal identity of the neural crest-derived craniofacial mesenchyme.