Study On The Mechanism Of Transcription Factor ISL1 Regulating Palatal Development Through SHH Signaling Pathway

Cleft lip and palate（CL/P）is one of the most common congenital maxillofacial defects,which seriously affects the swallowing and pronunciation of patients.The palatal development is precisely regulated,and the molecular mechanism of palatal development has been the focus of research.ISL1 is a Lim family transcription factor,which plays an important role in the development of islets and mandibles.Isl1 gene was first strongly expressed in the epithelial cells of the maxillary process of E11.5-day-old mice,and Isl1 gene was expressed in the palatal plate epithelium of both E13.5 and E14.5 mice.Pitx2^Cremice are widely used in palatal development studies,and X-Gal staining showed that Cre enzyme activity could be detected as early as day E10.5 in maxillary process epithelial cells.In this study,Isl1^f/fmice were mated with Pitx2^Cremice to construct mutant mice(Isl1^Pitx2Cre)with conditional knockout of Isl1 gene by palatal epithelial cells,and the mutant mice developed a complete cleft palate phenotype.The process of palatal plate elevation was not affected by HE staining in the head sections of E13.5-E14.5 mice.We further found that cell proliferation was significantly reduced in the palatal plate of E13.5-day Isl1^Pitx2Cre-mutant mice,whereas apoptosis did not change significantly.We used RNA-Seq technique to screen the differentially expressed genes in the palatal plate of E13.5-day mutant mice.RNA-Seq and in situ hybridization results showed that the gene Sox2 associated with palatal plate development was significantly downregulated in mutant mice(Isl1^Pitx2Cre),The expression of genes related to Hedgehog signaling pathway,such as Shh,Ptch1 and Gli1,was also significantly decreased in the mutant mouse palatal plate.In vitro overexpression results further confirmed that the transcription factor ISL1 can regulate Shh expression,and Ch IP assay experimental results showed that the transcription factor ISL1 can bind to the Shh gene distal enhancer MRCS1 sequence.Our results suggest that Isl1 plays an important regulatory role in the development of palatal plate,and Isl1 gene may regulate the development of palatal plate by regulating the expression of Shh and transcription factor Sox2.