Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Craniofacial Anomalies Result from Morphogenetic Defects in Cellular Positional Cues As Revealed By Genetic Analysis Of Wnt Signaling
Lucie J. Rochard, PhD, Stefanie Monica, B.S., Yawei Kong, PhD, Michael Grimaldi, B.S., Eric C. Liao, MD,PhD.
Massachussets General Hospital, Boston, MA, USA.

PURPOSE:
Craniofacial malformations such as orofacial clefts and mandibular hypoplasia are among the most common congenital anomalies. Several genetic causes have been described including derangements in the WNT pathway, which regulates fundamental cellular movements termed convergence extension (CE). We and others have defined several key Wnt components during palate development, extending from the initiation of the Wnt signal from the epithelial cells to the recipient chondrocyte. We reported expression of wnt9a (ligand), wls (trans-Golgi chaperon required for Wnt secretion), and gpc4 (extra-cellular matrix) in the signal sending epithelial cells; and frzb (extra-cellular chaperon), fzd7a (receptor) in the juxtaposed signal recipient chondrocytes. However the molecular and cellular basis of how disruption of Wnt signaling results in craniofacial anomalies remain unclear.
METHODS:
We generated mutants in different components of the Wnt pathway via CRISPR targeting to genetically dissect the role of WNT signaling in palate formation and craniofacial malformation. We analyzed the craniofacial phenotypes of developing embryos with mutations in wls, gpc4 and wnt9a, and compound mutants. Advanced analysis including clonal labeling to track each chondrocyte and its progeny is carried out by multi-spectral lineage analysis via lineage restricted and inducible zebrabow transgenic line.
RESULTS:
Gene expression of wls, gpc4 and wnt9a 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. Proliferation assay show that directional proliferation was intact in the wls mutant. However, multispectral clonal analysis demonstrates 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 novel advances using zebrabow transgenic technology to carry out multispectral clonal analysis. We uncovered that it is cell intercalation defects due to oss of Wnt mediated positional cues for developing chondrocytes that underlie orofacial cleft and mandibular hypoplasia. This work elucidates a fundamental principle of morphogenesis mediated by CE cell movements, which is generalizable to other developmental contexts.


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