The hedgehog co-receptors cdon and boc function synergistically to regulate zebrafish craniofacial development

Craniofacial development is a complex developmental process that involves formation of the skeleton, muscle, and tendons. Defects in craniofacial development lead to common hereditary disorders such as orofacial clefting or holoprosencephaly. Among genetic factors regulating craniofacial development, dysregulated hedgehog signaling is commonly associated with craniofacial skeletal defects. Here, we characterize craniofacial phenotypes associated with two hedgehog signaling co-receptors, cdon and boc, in zebrafish. Pedigree analyses have previously linked both CDON and BOC to microform holoprosencephaly skeletal defects, and mutations of Cdon and Boc in mouse result in midline craniofacial phenotypes. However, a detailed analysis of craniofacial phenotypes associated with cdon and boc in zebrafish has not been completed. Our studies show that cdon and boc operate synergistically to promote craniofacial cartilage, tendon, and muscle development in zebrafish. Using RNA-seq and HCR in situ hybridization we show that mutations of cdon and boc result in misregulation of chondrogenesis gene expression including Indian hedgehog ligand, tendon associated thrombospondin genes, and FOX transcription factors. Our data are consistent with a model whereby cdon and boc together modify hedgehog activity in the head, and establishes a foundation for using zebrafish to further understand the role of cdon and boc in craniofacial hereditary disorders such as holoprosencephaly. Overall design: RNA-seq profiling of heads of wildtype and double homozygous mutant cdon/boc embryos at 30 hours post fertillization and at 4 days post fertilization.