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Objectives: The CL/Fr mouse displays cleft lip and palate (CLP) at a rate of 35%. The clf1 mutation is associated with CLP in related “A” strain mice and affects the gene Wnt9b. The purpose of this study was to determine tissue specific expression of Wnt9b during facial prominence morphogenesis in CL/Fr mice and provide new details concerning gene variants associated with CLP. Methods: Facial prominences from CLP(-) and CLP(+) CL/Fr and 3H1 wild-type (WT) mice at embryonic day 11.5 (E11.5) were collected for expression assays (DNA microarray analysis, qRT-PCR, immunostaining, and in situ hybridization). A modified  Chi square test was used to analyze microarray data while a student t-test was used to statistically compare qRT-PCR  values (p<0.05). Results: There was a partial and variable loss of Wnt9b in facial prominences of E11.5 CLP susceptible CL/Fr mice, with a greater  loss associated with CLP(+). Two genes in the clf2 locus, Adcy2 and Ube2q11 also showed decreased expression. Two regulators  of palatogenesis, Runx2 and Osr2 were significantly downregulated, while an inhibitor of cell proliferation, somatostatin (Sst), was  elevated in CLP(+) relative to CLP(-) mice. Conclusion: Results indicate a role for Wnt9b in the pathogenesis of CLP and supports previous reports concerning its involvement  with CLP in “A” strain mice. Misexpression of Sst suggests that it may be a downstream target of Wnt9b causing reduced  overall growth possibly hindering fusion of facial prominences and contributing to the development of CLP.

Reduction in Wnt9b and associated gene expression in the embryonic midface of CL/Fr mice with heritable cleft lip and palate