Plastic Surgery Research Council

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Investigating The Role Of R-spondin3 In Craniofacial Morphogenesis And Orofacial Cleft Pathogenesis
Nora Alhazmi, BDS, MS1, Claudio Macias Trevino, BS2, Shannon H. Carroll, PhD3, Shawn Hallet, BA3, Edward Li, BA2, Francesca Gori, PhD1, Eric Chien-Wei Liao, MD, PhD3,4.
1Harvard School of Dental Medicine, Boston, MA, USA, 2Harvard Medical School, Boston, MA, USA, 3Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA, 4Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Boston, MA, USA.

PURPOSE: Cleft lip and/or palate (CL/P) are the most common structural birth anomalies. Mutation of IRF6 transcriptional factor plays a significant role in nonsyndromic cleft pathogenesis and Van der Woude syndrome. Using chromatin immunoprecipitation sequencing, we identified Rspo3 to be a transcriptional target of Irf6. We hypothesize that Rspo3 acts in the IRF6 gene regulatory pathway to regulate craniofacial morphogenesis.
METHODS: CRISPR/Cas9 rspo3 zebrafish alleles were generated for functional analysis. Palate and cartilage were visualized by Alcian blue staining. The genetic and functional interaction between irf6 and rspo3 was determined via rescue of irf6 mutant through over-expression of rspo3. In addition, the interaction between rspo3 expression and Wnt-pathway genes (wls, wnt9a, wnt5b and gpc4) was characterized. Analogous expression studies were also carried out in wildtype and Rspo3 mutant mice.
RESULTS: The rspo3-/-zebrafish Alcian blue staining revealed generalized underdevelopment compared to age matched wild type embryo. In addition, small cranium, cardiac edema, cleft palate and abnormal jaw development were noted. qPCR showed reduced rspo3 expression in irf6-/-zebrafish and injection of irf6 mRNA was able to rescue the rspo3 expression.WISH showed reduced expression of rspo3 in wls-/- and wnt9a-/- zebrafish.
CONCLUSION: Detailed phenotype analysis confirm the importance of rspo3 in craniofacial and palatal development. Genetic interaction and rescue experiments confirm that irf6 regulates rspo3, and this pathwayis required for palate development. Further, we show that rspo3 has an important role in Wnt signaling pathway in regulating palate morphogenesis. Taken together, this work uncovered pathways acting downstream of Irf6, advancing our molecular understanding of palate development.


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