A Twist1-regulated distal enhancer crucial for Alx1 gene expression and function during craniofacial development

Disruption of ALX1 causes frontonasal dysplasia syndrome-3, characterized by extreme microphthalmia, severe midfacial hypoplasia, and orofacial clefting. Recent studies have revealed critical tissue-specific roles of ALX1 in patterning both cranial mesoderm and cranial neural crest-derived facial mesenchyme. However, the molecular mechanisms regulating Alx1 gene expression during craniofacial development are largely unknown. In this study, we have identified a distal enhancer (Alx1-DE1) residing in a large intron of the neighboring Lrriq1 gene and demonstrate that deletion of this enhancer specifically affects Alx1 gene expression in the cranial neural crest-derived frontonasal mesenchyme and causes frontonasal and ocular defects partly phenocopying Alx1-deficient mice. We further functionally analyzed four evolutionary conserved regions, ECR1 - ECR4, in the Alx1-DE1 enhancer. Remarkably, ECR1, whose homologous region in the human genome harbors a lead single nucleotide variation significantly associated with facial and cranial vault shape differences, exhibits high enrichment of Twist1 transcription factor occupancy in mouse embryonic frontonasal tissues and drove Twist1-dependent reporter transgene expression specifically in the developing periocular and frontonasal mesenchyme in transgenic mice. These results reveal Alx1-DE1 as a crucial tissue-specific enhancer through which Twist1 and other major craniofacial developmental regulators control craniofacial patterning and morphogenesis.