Twist1 Acts Upstream of the Dlx5-Hand2 Pathway to Pattern the Mammalian Jaw

Both the upper and lower jaws develop from cranial neural crest cells (CNCCs) populating the first pharyngeal arch in all gnathostomes. Previous studies showed that the Edn1/Ednra-Dlx5/Dlx6-Hand2 signaling pathway is necessary for lower jaw formation and that ectopic expression of Edn1 or Hand2 throughout the CNCCs partly transformed the upper jaw to lower jaw structures, but the molecular mechanisms regulating upper jaw development remain unclear. Here we show that the basic helix-loop-helix transcription factor Twist1 is required for upper jaw development. Whereas the Twist1^fl/fl;Wnt1-Cre mouse embryos, with tissue-specific inactivation of Twist1 in premigratory CNCCs, exhibited aberrantly persistent expression of the key neuroglial lineage regulator Sox10 in the postmigratory CNCCs populating the facial primordia, we found that genetic inactivation of Sox10 did not rescue the defects in CNCC survival and patterning in Twist1^fl/fl;Wnt1-Cre embryos. However, analysis of Sox10^fl/+;Twist1^fl/fl;Wnt1-Cre mice revealed duplicated mandibular structures, including ectopic Meckel's cartilage, in place of the maxilla. Both Sox10^fl/+;Twist1^fl/fl;Wnt1-Cre and Sox10^fl/fl;Twist1^fl/fl;Wnt1-Cre embryos exhibited ectopic expression of Dlx5 and Hand2 in the developing maxillary processes at E10.5. Furthermore, we found that Twist1^fl/fl;Wnt1-Cre embryos also expressed Dlx5 and Hand2 ectopically in the maxillary domain at E10.5 and subsequently developed Meckel's cartilage-like cartilage rods bilaterally at the maxillary region. However, the expression of Edn1 was unaltered in the developing Twist1^fl/fl;Wnt1-Cre embryos, indicating that Twist1 functions in the CNCC-derived facial mesenchyme to regulate the Dlx5-Hand2 pathway without affecting Edn1 expression in the epithelial and mesodermal compartments. We further show that Twist1 represses reporter gene activation driven by the Dlx5/Dlx6 intergenic enhancer known to drive Dlx5/Dlx6 expression in the developing mandibular arch. Together, these data identify a new role of Twist1 in patterning the regional identities of the CNCC-derived facial mesenchyme and provide novel insight into the pathogenic mechanisms underlying TWIST1-related craniofacial developmental disorders.