Foxc1 establishes enhancer accessibility for craniofacial cartilage differentiation

The specification of cartilage requires Sox9, a transcription factor with broad roles for organogenesis outside the skeletal system. How Sox9 gains selective access to cartilage-specific cis-regulatory regions during skeletal development had remained unclear. By analyzing chromatin accessibility during the differentiation of neural crest cells into chondrocytes of the zebrafish head, we find that cartilage-associated chromatin accessibility is dynamically established. Cartilage-associated regions that become accessible after neural crest migration are co-enriched for Sox9 and Fox transcription factor binding motifs. In zebrafish lacking Foxc1 paralogs, we find a global decrease in chromatin accessibility in chondrocytes, consistent with a later loss of dorsal facial cartilages. Zebrafish transgenesis assays confirm that many of these Foxc1-dependent elements function as enhancers with region- and stage-specific activity in facial cartilages. We propose that Foxc1-dependent chromatin accessibility helps directs the versatile Sox9 protein to a chondrogenic program in the face. Wildtype embryos double positive for fli1a:GFP; sox10:DsRed (36 hpf and 48 hpf), col2a1a:GFP; sox10:DsRed (72hpf )and foxc1a; foxc1b mutant embryos and sibling controls double positive for pou3f3b>GFP; sox10:Dsred (36 hpf and 48 hpf) were selected under a fluorescent dissecting microscope, dechorionated, and dissociated. Cells were sorted based on GFP and DsRed expression on a MoFlo Astrios instrument. Approximately 5,000 cells were collected into PBS with 5% FBS and used to construct μATACseq libraries.