Molecular Determination of the Frontonasal Identity of Neural Crest Cells

The entire vertebrate facial skeleton is derived from Hox-negative cranial neural crest cells (CNCCs) that populate the embryonic frontonasal prominence and the first pharyngeal arches. Previous studies have shown that the Edn1/Ednra-Dlx5/Dlx6-Hand2 regulatory network specifies the maxillomandibular identity of CNCCs in the first pharyngeal arches in mice and that the combined application of retinoic acid and noggin to the presumptive maxillary domain in avian embryos transformed maxillary tissues to frontonasal structures, but the molecular mechanisms regulating frontonasal CNCC identity remain unresolved. Biallelic loss of function of any of the aristaless-like homeobox genes, including ALX1, ALX3, and ALX4, which exhibit partly overlapping patterns of expression in the frontonasal CNCCs, causes frontonasal dysplasia. In this study, we show that mice with combined inactivation of Alx1 and Alx3 throughout CNCCs exhibit disruption of frontonasal CNCC identity with ectopic activation of maxillary CNCC developmental profiles. We show that transgenic Alx1 expression throughout the CNCCs, starting from when they were migrating toward the facial primordia, caused ectopic activation of frontonasal CNCC marker genes and suppression of the maxillomandibular marker genes in the developing maxillary and mandibular processes and resulted in the formation of duplicated premaxilla at the expense of maxillary structures. These data identify the ALX transcription factors as key regulators specifying frontonasal CNCC identity and fill a long-standing gap in the molecular mechanism patterning the regional identities of the CNCC-derived craniofacial mesenchyme.