Mechanisms Of Cleft Palate Formation In Wnt5a Gene Knockout Mouse

Background: Cleft palate (CP) is one of the most common birth defects in human beings. CP is caused by complicated genetic factors and interaction with environmental factors,therefore its mechanisms is not clearly known. The proliferation and apoptosis of medial edge epithelium (MEE) cells, embryonic palatal mesenchyme (EPM) cells and tongue play important roles in maintain of palatal growth process, and also closely relative with formation of cleft palate.Wnt5a is a unique member of Wnt super family, mainly through the Wnt/Ca2+ pathway involved in the embryonic development. Another interesting character of Wnt5a is its protein distributes in both epithelium and mensenchyme. It has been reported that Wnt5a gene knockout mice showed under growing of all out-growth structures, such as mandible, maxillary, limb, tail and genital turbicle, as well as under-extension of the primary embryonic A-P axis. BrdU results indicate that knocking-out of Wnt5a leads to the reduced proliferation level of progress zone. However, there is no report about the abnormality of orofacial and palatal development in the Wnt5a gene knockout mice. Current experiments observed morphology of Wnt5a knockout mice palate through the crown series sections. In order to study the mechanisms underlying abnormality of palatal development in the Wnt5a knockout mice, Cell proliferation and apoptosis were detected in secondary palate and tongue by performing BrdU labeling and TUNEL methods.Objective: Taking advantage of Wnt5a gene knockout mouse models to observe the changes of proliferation and apoptosis in the palate. Furthermore, discover the roles of Wnt5a gene in the orofacial and palatal development. Methods: Wnt5a gene knockout mice was kindly given by Dr. McMahon and his research group in Harvard University, and then fed in the Animal Center, Keck's School of Medicine, University of Southern California. The female mice were housed overnight with the male mice and then checked for vaginal plugs the next morning, which was designated as day 0 of gestation(GD0). All the pregnant mice were sacrificed at 10 o'clock in morning on GD14, GD15 and GD18. BrdU with 100mg/kg of body weight was injected via abdominal cavity into the mice by 20 minutes before the pregnant mice were killed. Embryos of Wnt5a knockout and control were collected and fixed in 4% PFA. 5μm- thick series sections were prepared for HE staining, BrdU labeling and TUNEL methods.Results: Comparison with control, all Wnt5a knockout mouse showed identical mutant phenotype as following. 1. Morphological detections showed severe apparent defects of the oromaxillo-facial region, such as the under growing of mandible and maxillary, the abnormal shape and position of the tongue which does not drop in the oral cavity and the cleft of palate resulted from the shortage of secondary palates and the failure of elevating into a horizontal position. 2. BrdU staining exhibited no difference in the percentage of BrdU positive cells at the embryonic day 14.5 (E14.5) with the stage of palatal fusion between the mesenchymal compartments of palate, and between the mesenchymal and epithelial compartments of tongue. However, it is abnormal that no BrdU positive cell is detected at the Medium Edge Epithelium (MEE). 3. TUNEL detection showed that abnormally level of TUNEL positive cell was detected in mesenchyme of the palate and epithelium on dorsal surface of the tongue, as well as within the certain portions of the tongue at E14.5. In addition, no TUNEL positive cell located at the MEE.Conclusion:1. Wnt5a knockout mouse show abnormalites during orofacial development, and exhibit cleft palate and abnormal shape and position of the tongue. 2. Knocking out of Wnt5a may lead to cleft palate occurred by regulating both cell proliferation and apoptosis and by affect shape and position of the tongue.