The alx3 gene functions to regulate skeletal cell differentiation timing during zebrafish neurocranium development.

During craniofacial development, different populations of cartilage and bone forming cells develop in precise locations in the head. Most of these cells are derived from pluripotent cranial neural crest cells. The mechanisms that divide neural crest cells into distinct populations are not fully understood. Here we use single-cell RNA sequencing to transcriptomically define different populations of cranial neural crest cells. We discovered that the transcription factor encoding alx gene family is restricted to the frontonasal population of neural crest cells. Furthermore, genetic mutant analyses indicate that alx3 functions to subdivide the frontonasal population into medial versus lateral subpopulations. Our results support a mechanism in which the alx gene family functions as an identity code, subdividing frontonasal neural crest cells into distinct subpopulations. This study furthers our understanding of how different skeletal cell fates are established during craniofacial development and how these mechanisms can go awry in genetic diseases. Overall design: neural crest cell profiling of 24 hpf WT D. rerio