Experimental Study On The Expression Of The Receptor Of RARα And Dishevelled2 And Vangl2 And β-Catenin Proteins In The Development Of Cleft Palate Induced By Retinoid Acid In KM Mice

Objectives:Cleft palate is a major congenital craniofacial birth defect that has a complex etiology of both genetic and environment factors,and in humans is notable for significant morbidity.Palate development is a most copmplex biological process which is controlled accurately by various correlated genes.If any one of the correlated genes expresses anomaly,it may result in cleft palate.Along with the development of molecular biological techniques,it was proved that retinoic acid is a necessary morphogen during embryonic development.During the sensitive development stage of the mouse palatum,RA can induce cleft palate in the infant.RA plays an important role in the developing process of cleft palate,and it can participate in morphogenesis of cleft palate through regulating the expression of RARαin the second palate.The Wnt protein is a kind of secreted glycoprotein and activate the Wnt signal transduction by binding the receptors of cell membrane and proteins, which is correlated closely with the embryonic development and organofaction.B -catenin is an indispensable effector in the canonical Wnt signaling pathway and participate in a series of bioloy processes such as embyonic development and craniofacial morphogenesis.Its abnormal expression can induce craniofacial development defers and obstacle of facial pattern formation.Dishevelled is an adaptor of many Wnt signaling pathways and vangl2 is also an indispensable effector of wnt signal pathway.They and many other proteins codey by correlated genes constitute a most complex cell signal transduction passway,which control the process of embryonic development,the fate of cells and morphogenesis of organ and tissue..We plan to establish a KM mouse model for cleft palate induced by RA and detect the expression and distribution changes of RARα,Dishevelled2,β-catenin and Vangl2 proteins in the developing palates of this model with the techniques of immunohistochemistry.Methods1.Establishing a mouse model for cleft palate Virginal female KM mice mated with the same strain of males.The day of detection of the vaginal plug was considered gastric day(GD)0.The pregnant mice were randomly divided into different research groups.The experimental groups were orally treated with 60mg/kg,80mg/kg,and 100mg/kg of all-trans RA dissolved in peanut oil on GD10 to decide the best dose of atRA.Then the other two experimental groups were orally treated with 80mg/kg of all-trans RA at a selective time of GD8 and GD12 to decide the best time of administration.The controlled mice were administrated with a volume of peanut oil, equal to that of the experimental group.All animals were killed by cervical dislocation on GD16.The mice embyo were excisedand observed with dissecting microscope and some of the embryos were fixed with 4%paraformaldehyde,then dehydrated,embedded in wax,finally the specimens were sliced into section of 5um in thickness and stained with heamatoxylin and eosin(HE),and observed with microscope.2.The detection of RARα,Dishevelled2,β-catenin and Vangl2 proteins in RA-treated cleft palates:The proteins expression of RARα,Dishevelled2,β-catenin and Vangl2 in the palates of RA-treated mouse embryos were deteced with the techniques of immunohistochemistry and image analysis.Results1.Animal models:The oral administration of 80mg/kg of all-trans RA on GD10 can induced one handred percent of the cleft palate in KM mice and it is an ideal animal cleft palate model. 2.The expressions of RARα,Dishevelled2,Vangl2 andβ-catenin proteins(1)The expressions of RARαControl groups:In the epithelium of palate shelves,the expression of RARαgradully increased firstly during GD13⁸～GD14⁸ and achieve the peak value,then decreased during GD14⁸～GD14²²,latterly the expressive level increased gradually again. The expression trend in the mesenchyme was similar to that of epithelium,but the expressive level was obviously lower than that of epithelium.Experimental groups:The expressive trend of RARαin palatal shelves epithelium was similar to that of the control group,but it decended rapidly at GD15²². Maternal RA treatment resulted in a decreased expression of RA in the fetuses during GD13¹⁴～GD14⁸.All of the experiment groups had a decreased expression except that of the experimental group of the GD14²².(2)The detection of expression of the Dishevelled2 proteinExpression of Dishevelled2 protein displayed a ruled change both in the epithelium and mesenchyme with the embryonic development.The expression of Dishevelled2 protein increased firstly between GD13⁸ and GD13²²and achieved the peak value at GD13²²,then decreased rapidly between the time of GD13⁸ and GD13²²,latterly increased again between the time of GD14¹⁴and D15²²in the control groups.RA treatment groups resulted in a significant increase of Dishevelled2 protein in the fetuses at the stage of GD13⁸ to GD13¹⁴and the time of GD15⁸ in both the epithelium and mesenchyme.(3)Expression of Vangl2 proteinThe expressive level of Vangl2 protein in mesenchyme was a significantly lower than that of in the epithelium in the control groups.The expressions level of Vangl2 protein incended slowly first during the time of GD13⁸ to GD13²²,then decended gradually.There was no obvious regularity in epithelium in the control groups.The RA treatment groups had a significant increase at the time of GD13⁸～GD14⁸ in the mesenchyme.There was a significant decrease at the time of GD14¹⁴and GD15⁸ compared to the corresponding control groups.In the epithelium of the experimental group,the expression of Vangl2 present a wave-like trand,there was no obvious regularity also.(4)Expression ofβ-catenin proteinExpression ofβ-catenin protein displayed a ruled change with the embryonic development,and the expressive level ofβ-catenin protein in mesenchyme had a significant decrease compared with that in the epithelium.The expressions decended slowly at first,then increased rapidly and achieve the peak value at the time of GD14¹⁴. The RA treatment groups displayed a wave-like trend in the epithelium.The expression ofβ-catenin protein in the RA treated groups had a significant increase except the groups of GD14¹⁴and GD15⁸ compared to the control groups.There was no obvious regularity in mesenchyme after the treatment compared to the control groups.Conclusions1 Oral administration with 80mg/kg of all-trans RA on GD10 can induce cleft palate in KM mice and it was an ideal model.2 RARα,Dishevelled2,β-catenin and Vangl2 proteins take part in the regulating process of the normal development of palate in the mice.3 RA can interfere the normal expression of RARα,Dishevelled2,β-catenin and Vangl2 proteins in the developing palates,causing the cleft palate.