Cell Cycle Arrest of a ‘Zippering’ Epithelial Cell Cluster Shapes the Face and is Disrupted in Craniofacial Disorders

Facial features identify individuals, but the mechanisms shaping the human face remain elusive. Orofacial clefting (OFC), the most common craniofacial abnormality, results from failed fusion of the facial prominences that is in part caused by persistence of the cephalic epithelium. Here we uncover the identity, behaviors, and molecular blueprints of a novel craniofacial epithelial population, the Zippering Lambda (ZL), which mediates prominence fusion and is characterized by cell cycle arrest in mouse and human embryos. Remarkably, cell cycle is unleashed in the ZL of Pbx1/2 and p63 mutant mice with OFC. Intersection of ZL-enriched genes with human OFC whole-genome sequencing datasets identifies ZFHX3 variants in affected individuals and cephalic epithelial Zfhx3 deletion causes murine OFC. ZFHX3 and PBX1 genetically interact and synergistically regulate cell cycle inhibitor genes within a complex in embryonic faces. Collectively, we deconstruct new mechanisms that pattern the face, connecting cell cycle arrest to developmental tissue fusion.