Characterization of Pathways in the Spontaneous Cleft Lip Strain, A/WySn

OBJECTIVES: Cleft lip with or without cleft palate (CL/P) is a common birth defect with approximately 70% of the patients being nonsyndromic. There is a clear genetic contribution to the etiology. However, significant underlying heterogeneity has limited human genetic studies, though a major locus has been identified at 9q22-q33. The study of mouse CLP models is a complimentary approach to identify genes important in primary palatogenesis. The mouse strain, A/WySn, has spontaneous CLP. The genetic susceptibility for this trait has been mapped to two loci, Clf1 on Chr. 11 and Clf2 on Chr. 13. Clf1 has been fine mapped to a 1.6Mb region that contains 16 genes. Inactivation of one of these genes, Wnt9b, results in CL/P. Recently in collaboration with Dr. Diana Juriloff we have established that Wnt9b is allelic to Clf1. Furthermore, Dr. Juriloff identified a retrotransposon insertion 6.6kb downstream from Wnt9b. We believe this mutation alters the expression of Wnt9b in A/WySn mice. PURPOSE: Characterize the Wnt9b and Wnt3 expression in normal C57BL/6J and A/WySn embryos. Determine if candidate genes at 9q22-q33 are expressed during primary palatogenesis. METHODS: Riboprobes were made by RT-PCR from a 10.5 day whole embryo cDNA library. The sequences were BLASTed to rule out cross reactivity. The product was labeled with digoxygenin and used in whole embryo and section in-situ hybridizations from E9.5 through E11.5, corresponding to the period of primary palatogenesis. RESULTS: Section in-situ hybridization of Wnt9b and Wnt3 revealed ectodermal expression in the nasal and maxillary processes. Wnt9b expression was more intense especially in the area of fusion, while Wnt3 expression was weak. Expression of Wnt9b was absent in some, but not all A/WySn embryos. Candidate genes in Wnt signaling pathways are still currently being investigated. CONCLUSIONS: It appears that lack of Wnt9b expression corresponds with CL/P in A/WySn embryos.