Importance of the bHLH Transcription Factor, Hand2, in Craniofacial Development

Proper development of craniofacial tissues depends largely upon the formation, migration, differentiation, and survival of cranial neural crest (CNC) cells during embryogenesis. These pluripotent cells migrate away from the hindbrain neuroepithelium around the time of neural tube closure, coming to rest in the pharyngeal arches, transient structures on the embryo ventral surface. Within the first mandibular pharyngeal arch, their communication with surrounding tissues (i.e. epithelial ectoderm, paraxial mesoderm, and pouch endoderm) set up hierarchical signaling cascades, mediated by transcription factors, that direct correct spatiotemporal formation of the adult facial skeleton. Objective: To understand the mechanism by which the basic helix-loop-helix (bHLH) transcription factor Hand2, expressed in the distal mesenchyme of the mandibular arch, regulates facial development. While loss of Hand2 in zebrafish leads to craniofacial defects, loss of Hand2 in mice leads to early embryonic lethality from vascular defects, precluding further analysis. Methods: To address the function of Hand2 in mammalian development, we have used Cre-loxP technology to create Hand2fl/fl;Wnt1-Cre embryos in which the Hand2 gene is inactivated in CNC cells, permitting further analysis of its function. We sought to characterize craniofacial deformities in Hand2fl/fl;Wnt1-Cre embryos using hematoxylin and eosin-stained sagittal sections of E18.5 embryos. Furthermore, we examined the expression of two potential Hand2 effectors, Goosecoid and Msx1, via whole mount in situ hybridization using E10.5 embryos. Results: We find that Hand2 mutants exhibit many lower jaw defects including mandibular hypoplasia, absence of the submandibular gland and tongue and the presence of maxillary soft tissue features in the mandible. Furthermore, Goosecoid expression in Hand2 mutants is downregulated while Msx1 is unaffected. Conclusions: These findings suggest that Hand2 promotes distal lower jaw formation through maintenance of distal gene expression, with loss of function resulting in a spread of maxillary fates into the distal domain. Support: NIH/NIDCR DE14181.