During vertebrate embryonic development, cranial neural crest cells (CNCCs) emerge from the developing neural tube and undergo precisely coordinated migration, proliferation, and differentiation to form the harmonious facial structure, offering a valuable model for studying spatiotemporal gene regulation. We previously demonstrated that Sox9, a key transcription factor for skeletal development and facial morphogenesis, is regulated by distal enhancers located over 1 Mb from its promoter in CNCC-derived facial cartilages, but not in mesoderm-derived limb cartilages. We further showed that in CNCCs, a chromatin loop forms between the Sox9 distal enhancer and promoter, instructing lineage-specific enhancer usage (Sci Adv 2022). Through a systematic analysis of the 3D genome architecture in embryonic stem (ES) cells and craniofacial tissues at different developmental stages, we have now shown that many key genes involved in craniofacial development are regulated by long-range enhancer-promoter loops, in a fashion similar to Sox9. These loops emerge during early CNCC development and precede the expression of craniofacial developmental genes, suggesting they may predetermine the gene expression program for later stages of CNCC differentiation. Furthermore, we observed that the formation of these chromatin loops correlates with increased CTCF occupancy at loop anchors, indicating that developmentally regulated CTCF binding plays a critical role in orchestrating 3D genome topology. Conditional knockout of CTCF in the CNCC lineage disrupts the activation of craniofacial developmental genes, blocks CNCC differentiation, prevents craniofacial structure formation, and ultimately results in embryonic lethality. These findings underscore the importance of 3D genome topology in coordinating CNCC development and shaping craniofacial morphology, providing key insights for interpreting the genetic basis of craniofacial pathologies.

3D genome,CTCF,enhancer-promoter contact,craniofacial development,neural crest