Shox2/Alx1 Signaling Axis Regulates The Formation Of The Maxilla In Mice

Cleft palate is one of the most common birth defects.The incidence rate of cleft palate is about 1‰-2‰in the world,and about 1.82‰in China.It is characterized by soft tissue deformity of palate,and often accompanied by bone tissue defects and deformities of different degrees.According to the region of the defect,cleft palate can be divided into the hard cleft palate and soft cleft palate.The cleft palate can be divided into complete cleft palate and incomplete cleft palate according to the degree of the cleft.Cleft palate not only brings serious facial deformity to patient children,but also affects the language,swallowing and even hearing of patient children,which brings heavy burden to the patients and their families and health care system.In the process of mammalian embryo development,the genetic factors and environmental factors are the main causes of cleft palate,among which genetic factors play a major role.Many signal pathways have been reported to participate in the development of palate.Among them,Shox2 is a transcription factor,which is expressed specifically in the front of palate of mice.It has been shown that the conditional knockout of Shox2 at the front of palate can cause incomplete cleft palate,and the mechanism is that Shox2 promotes the osteogenesis and differentiation of palate.After analyzing the sequencing data of the anterior palate of Shox2 knockout mice,it was found that the gene expression including Alx1 was significantly down regulated.ALX family plays an important role in the development of human skull and facial bone.The mutation of ALX not only causes a special craniofacial development deformity--frontonasal dysplasia（FND）,in which ALX1 is related to type 3 FND,and the absence of Alx in mice will cause bone development abnormality of limbs.However,there is no relevant study on the effect of this gene on the development of palate.Therefore,this study explores the role of Alx1 in the development of palate in mice.First,based on the analysis of RNA-Seq and Ch IP-Seq of Shox2,it was found that Alx1 was down regulated in Shox2 knockout mice,and a binding signal of Shox2 was found near the Alx1 of chromosome 10.According to the Real-Time PCR results,compared with Shox2^cre/+;Nkx2.5^f/f mice,the expression of alx1 in Shox2^cre/-;Nkx2.5^f/f mice was significantly down regulated.Therefore,Alx1 is considered to be a member of Shox2 signal pathway in palate.Then we constructed Alx1^+/fmice,and by mating Wnt1-Cre and Alx1^+/fmice,we obtained the mice with Alx1 mutation in the mesenchymal cells from the cranial neural crest.The results of histomorphology showed that the mice had incomplete cleft palate phenotype in the middle portion of the palatine process and posterior portion of the palatine process,and died within one day after birth.The results showed that cleft palate was caused by the inability of the middle line after the palatal process was elevated,and the development of adjacent organs of palate was not abnormal,which indirectly affected the shape of palate.In addition,we detected the proliferation,apoptosis and neural crest cell migration of palatal process.It was found that the proliferation and apoptosis level of palatal process cells were not abnormal at E13.5,and the mesenchymal cells transferred from cranial neural crest did not show any decrease in the whole.Focusing on the development of the bone in the hard palate,the development of the maxilla of the mice was abnormal,and the osteoblast markers（SP7）were detected to confirm that the development of the mouse maxilla was abnormal.The results showed that Alx1 affected the formation of maxilla and the integrity of palate during the development of palate in mice.Finally,we named the specific binding peak found by SHOX2 in the Ch IP-Seq analysis in palate as Alx1^En.We constructed Alx1^En-Hsp68-Lac Z reporting mice,and verified the active mode of Alx1^En in palate,and the active only found in the anterior part of palate mesenchymal cell.In Shox2 knockout mice,Alx1^En activity disappeared,which proved that SHOX2 protein was first bound to Alx1^En enhancer region,Then,the chromatin folding was combined with Alx1 promoter region to regulate Alx1 transcription.In this study,we have revealed that Shox2,as an upstream transcription factor,binds to Alx1 promoter by Alx1^En through histomorphology,bioinformatics and genome non coding DNA.Alx1 regulates the expression of osteoblasts related genes in the mesenchymal cells of palate,which ensures the normal morphogenesis of palate in mice.This study is of great significance to understand the fine molecular mechanism of gene regulation in the development of palate and the mechanism of pathological development in cleft palate mice.