Study On The Mechanism Of Wnt5a Protein In The Development Of Palate And RA-induced Cleft Palate In Mouse

Cleft lip and/or cleft palate (CL/P) is one of the most common malformations among live births, occurring with an incidence of 1‰～2‰in the world and 1.82‰in China. CL/P is caused by genetic factors and interaction with environmental factors. It is so complicated that until now little is known about its causation. Wnt family is one of the molecular signals regulating the epithelium-mesenchyme interaction. Wnt5a is a unique member in the family since it is the only Wnt expressed in progress zone of growing bone, and is required for extension of the primary embryonic anterior-posterior axis and outgrowth of the limb proximal-distal axis. There is no report about the role of Wnt5a in regulating the development of facial primordial, especially in the secondary palate development. Objective: To study the temporal and spatial distribution of Wnt5a as well as β-catenin the crucial factor of Wnt canonical pathway in order to indicate Wnt5a signaling pathway involving in secondary palate development. Furthermore, analysis of Wnt5a and β-catenin in retinoic acid(RA)-induced cleft palate mouse to discover the mechanism and relationship of Wnt5a signaling molecule and retinoic acid(RA)pathway. Propose to offer experiment guidance and academic support to the prevention, diagnosis and therapy to CL/P. Methods: Using the well established mouse model of RA-induced cleft palate, we detected the expression and distribution of Wnt5a and β-catenin at E13d, E14d, E14.5d, E15d and E16d in control group and RA treated group by immunohistochemistry (Ed: Embryonic day). Results: 1. Higher level expression of Wnt5a was detected in mesenchymal cells of secondary palate at E13d when palatal shelves were in vertically position, and the expression decreased from E14d when palatal shelves went up to the horizontal position. The expression of Wnt5a was detectable evenly in the developing MEE cells until the palatal shelves fused following apoptosis happened in the MEE cells. 2. In RA-induced cleft palate, the expression of Wnt5a decreased faster, and was lower at E15d also disappeared earlier than control group. No significant difference of Wnt5a expression was observed in mesenchymal cells of secondary palate between the control group and RA treated group. 3. β-catenin was detectable both in control group and RA induced cleft palate. There was no significant change in the expression and distribution of β-catenin, and also no relative of β-catenin and Wnt5a distribution pattern and level was observed during palate development. Further detection of β-catenin protein in the control group and RA-induced cleft palate did not show any significant changes. Conclusions: 1.Wnt5a was expressed both in epithelial and mesenchymal components during palate development, the data proved that Wnt5a signaling molecule may involve in the growth and development of secondary palate. Higher level expression of Wnt5a expressed at rapid proliferating stage of E13d and reduced level at the following stage of epithelial differentiation suggested that Wnt5a take part in the proliferation and rarely join the differentiation. 2. RA possibly disabled MEE cells to proliferate and differentiate atthe pivotal stage when palate shelves were to contact and fuse, MEE cells were dysplastic, and then both side of palatal shelves could not contact and fuse. The decreased expression of Wnt5a at this pivotal stage proved that RA may influence the proliferation and differentiation of MEE cells and then induce cleft palate by regulating Wnt5a and other signaling molecules. 3. Wnt5a may not function through the canonical Wnt/β-catenin pathway in regulating the development of palate.