The alx3 gene shapes the zebrafish neurocranium by regulating frontonasal neural crest cell differentiation timing.

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 and differentiate with distinct developmental timing and cellular morphologies. The mechanisms that divide neural crest cells into discrete 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 gene family encoding the Alx transcription factors is enriched in the frontonasal population of neural crest cells. Genetic mutant analyses indicate that alx3 functions to regulate the distinct differentiation timing and cellular morphologies among frontonasal neural crest cell subpopulations. This study furthers our understanding of how genes controlling developmental timing shape craniofacial skeletal elements.