Transcriptome Analysis Of Palatal Tissues From Embryonic Mice Treated With AtRA

Objectives:Cleft lip and palate(CLP),regulated by both environmental and genetic factors,is a common congenital maxillofacial malformation in newborns.All-trans retinoic acid(at RA)is an ideal exogenous stimulus to constructing a mouse cleft palate(CP)model,which regulates the transcription of target genes by retinoic acid signaling.To further understand the effect of at RA on gene expression profile during palatal development,our study compared the changes in the palatal transcriptome between CP mice and healthy mice by RNA-seq.Methods: Mouse CP model was established using at RA and the embryonic PS of pregnant mice at E13.5,E14.5,E15.5 and E16.5 were collected respectively for RNA-seq to detect the m RNA expression level of each gene in palatal tissue.Standars screening DEGs are as follow s: log 2(fold change)≥1 and probability≥0.8.GO enrichment analysis and pathway enrichment analysis were used to analyze the functions and pathways of DEGs.m RNA level of selected DEGs were by q PCR and IHC was used to localize the protein expression of candidate genes in mouse palatal shelves.Results: 306 DEGs were screened out bycomparing the gene expression profile beween CP group and control group at E13.5-E16.5.A total of 4.2% were members of crystallin family.There are 14 genes encoding ECM components at E14.5.GO enrichment analysis revealed that DEGs were significantly involved in cellular components,such as macromolecular complex,extracellular matrix,collagen trimer,and intermediate filament,whereas their pathway enrichment analysis showed differences with palatal development.DEGs were mainly enriched in metabolism pathways,such as metabolism of xenobiotics by cytochrome P450,porphyrin and chlorophy II metabolism,retinol metabolism.Ttr and Spp1 were observed in the palatal mesenchyme from E13.5 to E16.5 by IHC,moreover,Spp1 was significantly decreased in E15.5 and E16.5 days in CP group(p<0.05).Conclusion: Our study showed that there were significant differences of gene expression profile between CP group and control group,especially members belonging to crystallin family.Furthermore,GO analysis suggested that both ECM and intermediate filament components might play important role in palatal development and CP.Pathway enrichment analysis indicated that at RA may activate multiple metabolic pathways to regulate CP process during palatalogenesis(E13.5-E16.5).In addition,the m RNA and protein level of Spp1,a gene encoding bone ECM,were down-regulated at different timepoints of palatal development,suggesting that at RA may involve in palatal bone formation by regulating Spp1.This study summerized many pathways and genes that may relate to palatogenesis and CP through RNA-seq,proving a direction for subsequent studies on the mechanism and targeted therapy of CP.