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 <i>Edn1</i> or <i>Hand2</i> 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 <i>Twist1^fl/fl</i>;<i>Wnt1-Cre</i> mouse embryos, with tissue-specific inactivation of <i>Twist1</i> 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 <i>Sox10</i> did not rescue the defects in CNCC survival and patterning in <i>Twist1^fl/fl</i>;<i>Wnt1-Cre</i> embryos. However, analysis of <i>Sox10^fl/+</i>;<i>Twist1^fl/fl</i>;<i>Wnt1-Cre</i> mice revealed duplicated mandibular structures, including ectopic Meckel's cartilage, in place of the maxilla. Both <i>Sox10^fl/+</i>;<i>Twist1^fl/fl</i>;<i>Wnt1-Cre</i> and <i>Sox10^fl/fl</i>;<i>Twist1^fl/fl</i>;<i>Wnt1-Cre</i> embryos exhibited ectopic expression of <i>Dlx5</i> and <i>Hand2</i> in the developing maxillary processes at E10.5. Furthermore, we found that <i>Twist1^fl/fl</i>;<i>Wnt1-Cre</i> embryos also expressed <i>Dlx5</i> and <i>Hand2</i> 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 <i>Edn1</i> was unaltered in the developing <i>Twist1^fl/fl</i>;<i>Wnt1-Cre</i> embryos, indicating that Twist1 functions in the CNCC-derived facial mesenchyme to regulate the Dlx5-Hand2 pathway without affecting <i>Edn1</i> expression in the epithelial and mesodermal compartments. We further show that Twist1 represses reporter gene activation driven by the <i>Dlx5/Dlx6</i> intergenic enhancer known to drive <i>Dlx5/Dlx6</i> 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 <i>TWIST1</i>-related craniofacial developmental disorders.