The Role And Its Mechanism Of Dhx15 In Early Intestinal Development Of Zebrafish

Objective:To investigate the role of dhx15 in intestinal development,zebrafish is used as a model organism to study the role and mechanism of dhx15 in early intestinal development.Methods:In our previous study,we constructed the heterozygous mutant dhx15+/-zebrafish by Talen technology.Dhx15+/+ and dhx15-/-of embryos were obtained by incrossing dhx15-/-zebrafish.Transposase Tol2 system editing technology were used to generate transgenic zebrafish Tg(hsp70:DHX15-2A-mcherry)by inserting CDS of DHX15 into plasmid Tol2-hsp70-2A-mecherry.This transgenic zebrafish can conditionally overexpress DHX15.The following experiments are carried out based on the previous work:1.Observe and compare the intestinal structure in dhx15+/+ and dhx15-/-embryos under microscope.Get the tissue slides from zebrafish intestine at 5.5 days post fertilization(5.5 dpf),compare the anatomical structure of zebrafish embryos by HE stainning.2.Detect the intestinal development of zebrafish embryos(marker genes of fabp2,apoa4b.1,aqp3,and fabp6 are expressed at 5.5dpf in dhx15+/-embryos by WISH(whole mount insitu hydrization),3.Conditionally overexpress DHX15 on dhx15-/-;Tg(hsp70:DHX15-2A-mcherry)zebrafish by heatshocking the embryos under at 42? for 2 hours,and compare intestinal marker gene expression at 5.5dpf to see whether DHX15 can restore intestine defects in dhx15-/-zebrafish.The marker genes include fabp2,apoa4b.1,aqp3,and fabp6.4.Compare proliferation(PH3)and apoptosis(Caspase-3)of intestine cell in dhx15+/+and dhx15-/-embryos by immunofluorescence.5.Detect differentially expressed genes(DEGs)in dhx15+/+ and dhx15-/-zebrafish embryo by using high-throughput RNA-seq.Screen gene cluster of specific expression in zebrafish intestine with bgee database(https://bgee.org).Analyze and screen intestinal specific DEGs by novomagic platform,which was provided by Novogene Bioinformatics Technology Co.Ltd.6.Through GO(Gene Ontology)enrichment analysis and pathway analysis KEGG,screen enriched pathways in dhx15-/-zebrafish embryo DEGs according to transcriptome sequencing analysis.7.Use qRT-PCR to verify the expression changes of dhx15+/+ and dhx15-/-zebrafish embryo signaling pathway related molecules to further confirm the expression of dhx15-affected signaling pathway related genesResults:1.Dhx15-/-zebrafish embryos show abnormal intestine structure in early development,which include obscure intestinal contour,incomplete foregut expansion,loss of intestinal epithelial plica,and thinning of intestinal wall.2.WISH results suggest that epithelial cell specific marker in dhx15-/-zebrafish embryos is significantly reduced.3.Conditional overexpression DHX15 restores intestine defects in dhx15-/-zebrafi sh.4.The proliferated intestinal cells may decrease,and the apoptotic intestinal cells may accelerate in dhx15-/-zebrafish embryos.5.There are 8986 differentially expressed genes(DEGs)in the dhx15-/-group,in which 4535 genes are up-regulated and 4451 genes are down-regulated in the experimental group compared with the control group.Futhermore,there are 7561 intestine specific DEGs in the dhx15-/-group compared with the control group,in which 3179 genes are up-regulated and 4382 genes are down-regulated.6.The GO(Gene Ontology)enrichment analysis for DEGs in the RNA-seq results showed that dhx15-/-mainly affects biological processes involving development and apoptosis.DEGs are enriched in multiple KEGG pathways such as the p53 signaling pathway,wnt signaling pathway,and apoptosis signaling pathway.Most of these pathways are related to cell energy metabolism,cell proliferation,and apoptosis.Thus we speculated that dhx15 may regulate the development of the intestine by affecting cellular energy metabolism,cell proliferation and apoptosis.7.qRT-PCR results further confirmed that dhx15 affects wnt signaling pathway.Conclusions:1.Dhx15 gene is essential for the development of zebrafish intestine.2.Dhx15-/-zebrafish embryos show abnormal intestine structure in early development,which include obscure intestinal contour,incomplete foregut expansion,loss of intestinal epithelial plica,and thinning of intestinal wall.3.The intestinal developmental defects in dhx15+/-zebrafish may mediated by the wnt signaling pathway.