The Regulatory Mechanism Of Msx1 In The Development Of The Secondary Palate

Palatogenesis is a very complicated biological event,which involves the processes of vertical growth,elevation,horizontal growth and fusion.These processes involve a huge network composed of various signal pathways and multiple transcription factors.The deletion or mutation of critical genes in this network system often leads to cleft palate.Cleft lip or palate is the most common birth defect in human.Investigation of the mechanism of cleft lip and palate may shed light on the prevention of cleft lip or palate and the improvement of population quality.The Msx1 protein encoded by Msx1?muscle segment homeobox gene?is a transcriptional factorthat is expressed in epithelial and mesenchymal interaction sites.Msx1 gene has been demonstrated to be a susceptible gene for cleft lip or palate.Mutations in the human MSX1 gene cause cleft palate,while knockout of the Msx1 gene in mice also leads to cleft palate.Msx1knockout mouse model is currently available,providing conditions for the study of Msx1 gene function in the development of the palate.However,as a transcription factor,relatively little is known about the target genes of Msx1 during palatal development..Therefore,to investigate the target genes for Msx1 during the development of the palate,our laboratory constructed a conditional overexpression mouse model(RosaMsx1^fx/fx)for Msx1,and combined with the Msx1 knockout mouse model to study the function of Msx1.Thus far,we have achieved a series of results.First,the successful construction of the Msx1mouse model was confirmed by immunofluorescence and in situ hybridization.We then used Wnt1-Cre mice to mate with Msx1 conditional over-expression mice to express Msx1 in mouse mesenchymal cells.We found that Msx1-overexpression mice have a cleft palate phenotype with an open eyelid phenotype.Next,we used RNAseq to find genes that were differentially expressed in the palate of Msx1-overexpression and wild type miceat E12.5.We found that overexpression of Msx1 leads to changes in the expression of multiple genes in the palate.Among them,the expression of several genes crucial for the development of palate,such as Satb2,Barx1,and Alx1,has significantly changed.Given that the deletion of these genes leads to cleft lip and palate,as a key research subject,we further examined the expression of these genes in the palates of Msx1 overexpression,Msx1 knockout,and wild type mice using in situ hybridization.We found that the in situ hybridization results supported the RNAseq results,and that each gene had an opposite expression pattern in overexpressing mice and knockout mice.We further found that the promoter regions of the Satb2 and Alx1 genes had several Msx1binding sites,indicating that they are target genes for Msx1.The dual luciferase reporter assay results suggest that Msx1 regulated the expression of Alx1 and Satb2.In the promoter region of Barx1,no binding site was found for Msx1,suggesting that Barx1 is indirectly regulated by Msx1,or that Msx1 binds to the enhancer region to regulate Barx1 expression.Taken together,in this project,we found several possible target genes of Msx1,which provided the data basis for molecular function research of Msx1 in the development of palate.