The Mechanism Of Retinoic Acid Affecting Palatal Shelf Elevation In Mice By Regulating Non-Canonical Wnt Signaling Pathway Through Wnt5a

Objective: The development of the mammalian palate includes vertical growth of the palatal process,rapid elevation,and horizontal growth until contact fusion.Abnormal palatal elevation is responsible for 90% of cleft palate cases.Retinoic acid(RA)is A functionally active derivative of vitamin A.Excessive intake of RA by pregnant mothers can lead to the occurrence of cleft palate.The mouse model of cleft palate induced by RA is one of the most classical animal models for studying clef t palate.Previous studies have shown that excessive RA causes cleft palate by delaying the elevation of the palatal process,but the underlying biological mechanism is still unclear.Our previous study found that the contraction of palatal mesenchymal cytoskeleton plays a key role in the palatal lifting phase in lithium Lithium-induced cleft palate mouse model,which may provide a reasonable explanation for the rapid palatal lifting process.However,it remains to be confirmed whether this idea can generally explain this palatal elevation delay in other models.In this study,we aimed to investigate the role of Wnt5 a,a key ligand of the non-canonical Wnt signaling pathway,in the regulation of palatal protrusion in a RA-induced cleft palate mouse model,and to provide a theoretical reference for the prevention and treatment of cleft palate.Methods: In this study,a mouse cleft palate model was established using alltrans retinoic acid.The heads of fetal mice were collected and paraffin sections were taken for HE staining to observe the morphological characteristics of RA-induced cleft palate.Ki67 and TUENL were used to detect the effect of RA on the proliferation and apoptosis of fetal palatal mesenchymal cells.In situ hybridization was used to detect the expression of Wnt signaling pathway and downstream related genes.Scratch wound healing assay and phalloidin staining were used to detect the effect of RA on the migration and cytoskeleton of palatal mesenchymal cells.Foxy5(Wnt5a agonist)was used to disrupt the expression of Wnt5 a and its downstream signaling molecules to further verify the changes in cell migration ability and cytoskeleton.Results: HE staining and microscope observation showed that the incidence of cleft palate in pregnant mice embryos was 100% after continuous low-dose RA stimulation at E11.5,E12.5,and E13.5,and no obvious embryo resorption or other malformations occurred.Ki67 and TUNEL assay showed that there was no significant difference in the proliferation and apoptosis of palatal mesenchyme cells in RA group.The changes of cell proliferation after RA treatment were mainly concentrated in the anterior nasal epithelium and the posterior palatal process.No obvious apoptosis was found in the palatal process,and apoptosis was mainly concentrated in the maxillary region.In situ hybridization showed that RA down-regulated Wnt5a-mediated Wnt signaling.The m RNA levels of β-Catenin,Axin2 and Lef1,the key genes of classical Wnt signaling,did not change significantly in the embryonic palatal mesenchyme of RA group,and β-Catenin was only detected in the concave area of the anterior and middle part of the palate.The expression of Dkk2,a secretory antagonist of Wnt signaling,was only slightly changed in the posterior palatal portion.In control embryos,the expression of Wnt5 a,the major ligand of the atypical Wnt signaling pathway,was distributed in a gradient along the anterior-posterior axis of the palatal process,and the expression of Wnt5 a gene in the palatal process was significantly down-regulated after RA treatment,especially in the anterior and middle parts of the palatal process.m RNA levels of Ror2,the major receptor of the non-canonical Wnt signaling pathway,were also significantly reduced in the mesenchyme of the anterior,middle and posterior parts of the palatal process.Immunofluorescence staining showed that Foxy5 increased the level of Wnt5 a.Consistent with the in vivo results,Wnt5 a levels were decreased by excess RA,while Wnt5 a levels were partially restored by RA+ Foxy5.In addition,Wnt5 a levels were significantly increased in the CASIN group and relatively decreased in the RA+ Foxy5+ CASIN group.Cdc42 expression levels were significantly decreased in RA and CASIN groups,but increased in Foxy5 group.Although Foxy5 partially restored the RA-induced reduction in Cdc42 in the RA+ Foxy5 group,this reduction was reobserved with CASIN stimulation in the RA+ Foxy5+ CASIN group.The results of cell scratch healing assay and phalloidine staining showed that RA stimulation led to the shortening and disorientation of stress fibers in the cytoskeleton of palatal mesenchyme cells,and the cell migration was inhibited.These effects were partially restored by Foxy5 stimulation.Conclusion:(1)Continuous low-dose RA(40mg/kg)could induce 100% cleft palate in the embryos of pregnant mice,and there was no significant change in the proliferation and apoptosis of palatal cells.(2)In the continuous low-dose RA induced cleft palate mouse model,RA mainly inhibited the expression of Wnt5a-Ror2 in the non-canonical Wnt signaling pathway,and also inhibited the migration ability and directional remodeling of the cytoskeleton of palatal mesenchymal cells.(3)Wnt5a,the major ligand of the non-canonical Wnt signaling pathway,can regulate the directional remodeling of Cdc42/F-actin cytoskeleton,change the migration ability of palatal mesenchymal cells,and affect the palatal process elevation in RA induced cleft palate mouse model.