Indian hedgehog in craniofacial neural crest cells links to skeletal malocclusion by regulating associated cartilage formation and gene expression

The frontal craniofacial skeleton derived from neural crest cells is vital for facial structure and masticatory functions. The exact role of Indian hedgehog (Ihh) in facial and masticatory development has not been fully explored. In this study, we generated craniofacial neural crest cells-specific Ihh deletion mice (Wnt1-Cre;Ihh^fl/fl;Tomato^fl/+) and found the gradual dwarfism without perinatal lethality. Morphological and histological analyses revealed unambiguous craniofacial phenotypes in mutants, where we observed skeletal malocclusion accompanied by markedly hypoplastic nasomaxillary complex and reversed incisor occlusion. Both the replacement of nasal concha cartilage by turbinate bones and the endochondral ossification of nasal septum ethmoid bone were substantially delayed. We also observed hypoplastic mandibles in mutants where the mandibular ramus was unexpectedly the most affected. Both the condylar process and mandibular angle cartilages were distorted. However, dental examination showed no significant changes in teeth and dentition. Finally, a comprehensive RNA sequence analysis utilizing condylar cartilage identified Ihh-associated gene network including several cell cycle genes and 16 genes related to the extracellular matrix, sulfate transporters, transcription factors, receptors, a ciliogenesis factor, and an adhesion molecule. Our data provide direct in vivo evidence that Ihh plays crucial roles in midface and masticatory system formation, likely by activating key genes.