The CCAAT Enhancer Binding Protein Beta (Cebpb)is Essential For The Development Of Enveloping Layer (EVL) In Zebrafish

Epiboly is necessary for blastopore closure and the successful completion of gastrulation in zebrafish,is the coordinated movement of the enveloping layer(EVL),the extra-embryonic yolk syncytial layer(YSL)and the deep cells(DCs).Epiboly begins at the end of the blastocyst stage,and blastocyst cells migrate along yolk cells,leading to large-scale morphogenesis,which is the first morphogenesis event during the development of zebrafish.Previous studies have shown that interference with the differentiation of EVL during the epiboly will lead to abnormal embryo development and even death.However,the developmental mechanism of EVL remains unclear.In order to explore the related functions and marker genes of EVL,we mined the single-cell data from the public database,namely the single-cell RNA sequencing expression matrix at 4 hours after zebrafish fertilization.A total of 105 reliable EVL marker genes were identified,and their specific expression in EVL was verified by in situ hybridization.To better understand the function of the genes that showed significant EVL specificity,Gene Ontology(GO)and KEGG enrichment analysis were performed.We found genes associated with cell junction(cx43.4,cldnb,cldn7 b,f11r.1,cldn23 a,cldne,marveld2 b,jupa),keratin filament(krt4,krt8,krt5),apical junction complex(cldnb,cldn7 b,f11r.1,cldn23 a,cldne),supramolecular fiber(nrap,krt4,krt18,mid1ip1 a,krt8,krt5)and microtubule organizing center(lrwd1,lrmp,nin,ccnf)were significantly enriched,suggesting genes for cell adhesion were highly specific for EVL.In addition,protein-protein interaction(PPI)network was constructed according to the String database and visualized by Gephi software.Then,key genes and important modules were identified by RT-PCR,and the relative m RNA expression levels between 1.75 h and 4.5h after fertilization were obtained.By comparing with rhesus monkey 16-cell,morula and blastocyst embryonic single-cell RNA sequencing analysis,we found that the homologous genes corresponding to EVL marker genes were not specifically expressed in a rhesus monkey tissue,and EVL may have evolved specifically in fish.At the same time,we also found that zebrafish EVL marker genes cldnb,krt8,grhl3,krt4 and krt18 had positive selection in fish lineage compared with other vertebrate developmental tree lineages such as rhesus monkey,mouse,bird,reptile,etc.,which also indicated the evolutionary specificity of EVL and its adaptability to the environment.And most importantly,RT-q PCR and in situ hybridization showed that cebpb gene was expressed preferentially to other marker genes at 2.25 h after fertilization during the zygotic genomic activation(ZGA)phase.To explore the role of cebpb in EVL differentiation,we knocked down its expression by morpholino-labeled antisense RNA.The morphant embryos showed disrupted EVL and developmental termination prior to epiboly.In accordance,expression of the EVL specific cell-adhesion molecule genes were significantly reduced while the expression levels of deep cell marker gene znf995-203 almost remained unchanged.Thus,cebpb is the earliest marker and essential for EVL differentiation in zebrafish.The elucidation of the specific EVL gene set provide us new insights into the molecular mechanisms underlying EVL differentiation and the molecular interaction with other cell layers during gastrulation.