Cellular and Molecular Regulation of Upper Lip Fusion

Cleft lip with or without cleft palate occurs in thousands of births every year in the United States and is a leading birth defect worldwide. Upper lip development is a multistep process that includes nasal pit invagination, outgrowth of nasal processes, and fusion of the medial nasal, lateral nasal and maxillary processes. The failure of these processes to join results in cleft lip, but the cellular mechanisms that drive this morphogenesis are largely unclear. Based on the abundance of apoptosis at the fusion junction, it has been hypothesized that cell death is crucial for epithelia removal during lip fusion. To test this, we generated mouse models disrupting the apoptotic regulators Bax and Bak. Surprisingly, we found that complete loss of apoptosis in the epithelium does not result in a discernable lip phenotype. Based on reports of cleft lip‐associated mutations in CTNND1 , CDH1 , and MYH9 , cell‐cell adhesion and actomyosin contractility are indispensable for normal lip development. Indeed, based on recent mouse genetic studies, Ctnnd1 loss of function in the lip epithelium results in a cleft lip, yet the cellular and molecular basis of this phenotype remains unknown. We interrogated lip epithelial‐specific Ctnnd1 and Cdh1 mouse mutants and found that loss of Ctnnd1 , but notably not Cdh1 , resulted in a failure of the medial and lateral nasal processes to contact and fuse. Our parallel analyses of epithelial‐specific Myh9; Myh10 compound mouse mutants with disrupted actomyosin contractility revealed early nasal pit invagination defects and disorganized epithelia. To better understand the cellular functions of these regulators during fusion occurring at a later stage, we have established an ex‐utero time‐lapse imaging protocol to achieve a 4D view of the fusing medial and lateral nasal processes. In this setup, we can now track individual cells and examine the force generation components such as actomyosin contractility. Collectively, our live imaging approach along with mutant mouse models provide a new understanding of the contributions of apoptosis and cell adhesion and new insights into unexplored roles of actomyosin contractility.