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Craniofacial development is a complex and tightly regulated process and disruptions can lead to structural birth defects, the most common being nonsyndromic cleft lip and palate (NSCLP). Previously, we identified FOS as a candidate regulator of NSCLP through family-based association studies, yet its specific contributions to oral and palatal formation are poorly understood. This study investigated the role of fos during zebrafish craniofacial development through genetic disruption and knockdown approaches. Fos was expressed in the periderm, olfactory epithelium and other cell populations in the head. Genetic perturbation of fos produced an abnormal craniofacial phenotype with a hypoplastic oral cavity that showed significant changes in midface dimensions by quantitative facial morphometric analysis. Loss and knockdown of fos caused increased cell apoptosis in the head, followed by a significant reduction in cranial neural crest cells (CNCCs) populating the upper and lower jaws. These changes resulted in abnormalities of cartilage, bone and pharyngeal teeth formation. Periderm cells surrounding the oral cavity showed altered morphology and a subset of cells in the upper and lower lip showed disrupted Wnt/β-catenin activation, consistent with modified inductive interactions between mesenchymal and epithelial cells. Taken together, these findings demonstrate that perturbation of fos has detrimental effects on oral epithelial and CNCC-derived tissues suggesting that it plays a critical role in zebrafish craniofacial development and a potential role in NSCLP.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect, affecting approximately 1 in 700 births. NSCL/P has complex etiology including several known genes and environmental factors; however, known genetic risk variants only account for a small fraction of the heritability of NSCL/P. It is commonly suggested that gene-by-environment (G×E) interactions may help explain some of the “missing” heritability of NSCL/P. We conducted a genome-wide G×E interaction study in cases and controls of European ancestry with three common maternal exposures during pregnancy: alcohol, smoking, and vitamin use using a two-stage design. After selecting 127 loci with suggestive 2df tests for gene and G x E effects, 40 loci showed significant G x E effects after correcting for multiple tests. Notable interactions included SNPs of 6q22 near VGLL2 with alcohol and 6p22.3 near PRL with smoking. These interactions could provide new insights into the etiology of CL/P and new opportunities to modify risk through behavioral changes.

Loss of ALX1 function causes the frontonasal dysplasia syndrome FND3, characterized by severe facial clefting and microphthalmia. Whereas the laboratory mouse has been the preeminent animal model for studying developmental mechanisms of human craniofacial birth defects, the roles of ALX1 in mouse frontonasal development have not been well characterized because the only previously reported Alx1 mutant mouse line exhibited acrania due to a genetic background-dependent failure of cranial neural tube closure. Using CRISPR/Cas9-mediated genome editing, we have generated an Alx1-deletion mouse model that recapitulates the FND craniofacial malformations, including median orofacial clefting and disruption of development of the eyes and alae nasi. In situ hybridization analysis showed that Alx1 is strongly expressed in frontonasal neural crest cells that give rise to periocular and frontonasal mesenchyme. Alx1del/del embryos exhibited increased apoptosis of periocular mesenchyme and decreased expression of ocular developmental regulators Pitx2 and Lmxb1 in the periocular mesenchyme, followed by defective optic stalk morphogenesis. Moreover, Alx1del/del embryos exhibited disruption of frontonasal mesenchyme identity, with loss of expression of Pax7 and concomitant ectopic expression of the jaw mesenchyme regulators Lhx6 and Lhx8 in the developing lateral nasal processes. The function of ALX1 in patterning the frontonasal mesenchyme is partly complemented by ALX4, a paralogous ALX family transcription factor whose loss-of-function causes a milder and distinctive FND. Together, these data uncover previously unknown roles of ALX1 in periocular mesenchyme development and frontonasal mesenchyme patterning, providing novel insights into the pathogenic mechanisms of ALX1-related FND.