Orofacial Clefts Result from Defects in Cellular Positional Cues As Revealed By Genetic Analysis Of Wnt Signaling
Lucie Rochard, Ph.D., Yawei Kong, Ph.D., Stefanie Monica, B.S., Christina Nguyen, B.S., Michael Grimaldi, B.S., Eric C. Liao, M.D., Ph.D..
Harvard Medical School, Boston, MA, USA.
PURPOSE: Orofacial clefts such as cleft lip and palate, are the most common congenital anomalies. Several genetic causes have been described including derangements in the WNT pathway. However the cellular basis of cleft pathogenesis is still unclear, which we hypothesize to result from cellular derangements in positional cues that is imparted by WNT signaling.
METHODS: We utilize a wls mutant to genetically dissect the role of WNT signaling in palate formation. Genetic epistasis analysis is carried out with the glypican 4 mutant and CRISPR generated wnt9a mutant.
RESULTS: Expression of wls, gpc4 and wnt9a genes were detected in the oropharyngeal epithelium, juxtaposed to the palate chondrocytes, which express extracellular ligand frzb. Phenotype analysis demonstrated that the palate is malformed, shorter in the anteroposterior (AP) axis and wider in the transverse dimension. On the cellular level, the palate chondrocytes are rounded instead of disc-like, and the cells fail to intercalate and organize in a single layer as in wildtype. Multispectral clonal analysis demonstrate that chondrocytes fail to intercalate and proliferate without axis polarity cues.
CONCLUSION: This work genetically dissects the Wnt pathway in palate development revealing precise coordination of Wnt gradient is necessary to impart positional cue to differentiating chondrocytes. Cellular demonstration of chondrocyte intercalation and directional migration are advances previously unfeasible without application of zebrabow transgenic technology to carry out multispectral clonal analysis. This work elucidates a fundamental principle of morphogenesis generalizable to other developmental contexts, and contributes a cellular understanding to palate development and human cleft pathogenesis.
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