L. J. Rochard1, S. J. Monica1, Y. Kong1, M. J. Grimaldi1, E. C. Liao1 1Massachusetts General Hospital,Center For Regenerative Medicine,Boston, MA, USA
Introduction: Orofacial clefts, such as cleft lip and palate (CL/P), are the most common congenital anomalies with a frequency of 1 in 700 births. Several genetic causes have been described and one of the main signaling pathways involved is WNT. However the precise role of WNT pathway in morphogenesis of the palate remains poorly understood. WNT proteins are secreted factors where intracellular chaperon and export is regulated by WLS. The WLS gene is located on the short arm of the chromosome 1 and several deletions of this region are associated with craniofacial anomalies.
Methods: We utilize a zebrafish wls mutant to genetically dissect the role of WNT in palate formation. Expression analysis of wls, neural crest and Wnt signaling pathway genes were detected by RNA in situ hybridization during embryogenesis. Craniofacial structures in wls mutants were delineated using Alcian blue staining. Cell lineage, apoptosis and proliferation assays were performed to determine effect of wls mutation on these cellular behaviors.
Results:
wls gene expression was detected in the oropharyngeal epithelium, juxtaposed to the palate chondrocytes, which express extracellular ligand frzb. The epithelium also expresses secreted ligand wnt9a, and receptor gpc4. In this mutant, the wls protein is truncated, abrogating the WNT secretion. Phenotype analysis demonstrated that the palate is malformed, shorter in the anteroposterior 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. We also find that migration and proliferation of chondrocytes are normal. Molecular analysis revealed that expression of wnt9a is unaffected with loss of wls, but expression domain of frzb is expanded proximally, while it should be restricted to the proliferative front. Taken together, the palate malformation is due to failure in convergence and extension mechanisms of morphogenesis, where regulation of frzb expression is disrupted.
Conclusion:Analysis of wls mutant demonstrates that WNT pathway components are coordinated to regulate convergence and extension mechanisms. Taking a genetic approach, wnt9a and frzb mutants have been generated via CRISPR to test genetic interaction between these WNT components. We also generated fluorescent epitope tagged proteins of wls, frzb and wnt9a to directly visualize intracellular and extracellular trafficking of these components during cellular morphogenesis. These studies will gain insight into the molecular basis of craniofacial development and highlight the utility of the zebrafish model in complementary genetic and developmental analyses.
