The CL/Fr mouse demonstrates heritable bilateral and unilateral cleft lip and palate (CLP) at a rate of approximately 35%, generally above the background “A” strain mouse. Using classical mouse breeding strategies, it has been suggested that at least two disease loci, clf1 and clf2, are involved in the defect and candidate genes have been identified. Additionally, gene‐targeting analyses strongly suggest that Wnt9b contributes to CLP in the “A” strain mice. The aim of this study was to test the expression of clf1 and clf2 candidate genes in the facial prominences of CL/Fr embryos, utilizing microarray analysis. Medial nasal, lateral nasal, and maxillary prominences of phenotypically normal as well as cleft E11.5 CL/Fr mice were dissected and RNA was extracted using standard techniques for Agilent‐microarray protocol. Results indicate that expression of the clf1 candidate gene, Wnt9b, and the clf2 candidate gene, Ube2ql1, are significantly reduced (−3.11 and −1.83 fold, respectively) in the CL/Fr cleft tissues, suggesting these genes may be involved in the CLP mutation in CL/Fr mice. Future gene expression studies through quantitative RT‐PCR and regional expression analyses through immunohistochemistry and whole mount in situ hybridization will be performed to further test the expression of these clf1 and clf2 candidate genes.Grant Funding Source: NIH/NCRR 5P20RR024206 (S.J.S) and R01‐DK‐064752 (SL)
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.
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