The Mechanisms Of Abnormal Palatal Development In Wnt5a Mutant Mice

Cleft palate is a common congenital birth defect.Wnt signaling pathway is essential for the palate development.Wnt5a as one of Wnt family members, has dual roles in signaling:activating the Wnt canonical signal; and also activating the Wnt noncanonical signal by inhibition canonical signal. It was recently reported that Wnt5a deficiency leads to complete cleft of the secondary palate.In the development of palate, The results will reveal whether Wnt5a regulates Wnt canonical and BMP signals,and screen out the downstream target of Wnt5a in Wnt5a mutant.It demonstrates that the mechanisms of palatal abnormal development in Wnt5a mutant mice.Firstly, Collecting the TOP-gal embryos of 14.5, a transgenic reporter of activation of the canonical Wnt signaling pathway, by LacZ stain, the result shows that the canonical signal failed to be activated. Then collecting E14.5 Wnt5a-/-;TOP-gal mice, by LacZ stain, it fails to find that the positive signal along anterior-posterior axis in palate. The result shows that the loss of Wnt5a cannot upregulate the canonical Wnt signaling pathway.Secondly, It shows that overlapping expression between Wnt signaling pathway and BMP signaling pathway, they are interact with each other in the craniofacial development. Collecting E13.5 BRE-lacZ mice, a transgenic reporter of activation of the canonical BMP signaling pathway, by LacZ stain, the canonical BMP signaling pathway is activated in posterior of palate. Collecting E13.5 Wnt5a-/-;BRE-LacZ embyros by LacZ staining, the result is similar to BRE-LacZ controls. The result shows that the loss of Wnt5a cannot activate the canonical BMP signal.Finally, to explore the differential genes of Wnt5a regulate in palate by microarray analysis. The results show that the expression level of various genes increase significantly, such as API family members:Fos?Fosb?Junb?Jun, meanwhile Cfl1,participating in cytoskeletal regulation, its expression rate increased. RT-qPCR is performed to validate the microarray results. Using Rhodamine Phalloidin staining cytoskeleton, It finds that cytoskeleton is disorder in the palate mesenchyme when Wnt5a is knock out.Above all, Wnt5a cannot regulate Wnt and BMP canonical signaling pathway at early stage of palatal development; The expression level of API family genes is upregulated, then causing cytoskeleton disordered and cleft palate when Wnt5a is knock out.