Regulatory logic underlying neural crest contributions to the head versus the heart.

Neural crest cells exhibit axial-level-specific transcriptional programs, yet the regulatory mechanisms underlying their fate decisions remain unclear. Here, we investigate whether differential transcription factor usage governs cardiac neural crest cell identity. Comparing ATAC-seq profiles of cardiac and cranial neural crest revealed open chromatin regions that were both shared and region-specific. Single-cell RNA sequencing further identified putative target genes, including signaling molecules and receptors. We uncovered two adjacent Cxcr4 enhancers: one specific to the cardiac crest containing a TGIF binding site, and another active in both cranial and cardiac crest containing a TFAP2B binding site. Mutating the TFAP2B site abolished cranial, but not cardiac, crest expression. Finally, we show that the cardiac crest-specific Cxcr4 enhancer also drives expression in zebrafish cardiac crest cells, suggesting evolutionary conservation across vertebrates. These findings suggest that transcription factors enriched in distinct neural crest subpopulations may bias cell fate by modulating enhancer activity, shedding light on the regulatory landscape governing neural crest diversity.