Role Of The Transcription Factor E93 On Wing Development In Drosophila

Transcription factor E93 is one of the primary response factors at the downstream of ecdysone in insects and plays an important role during the pupal-adult metamorphosis.E93 is both involved in the regulation of programmed cell death of larval tissues and the formation of adult tissues and organs during metamorphosis.However,there are few researches on the molecular mechanism of E93 in regulating the formation of adult tissues and organs during metamorphosis so far.E93 was first identified in Drosophila and expressed specifically at the prepupal and pupal phase during the transition from larva to adult.At pupal stage,Drosophila larval organs such as the salivary gland and midgut undergo apoptosis,while the wing discs undergo dramatically morphological changes and eventually develop into adult wings.Based on the dramatical metamorphosis of the wing at pupal stage and the easy observation of adult wings,the wing is often used as an ideal organ model to study the development of organs.In this study,we used the Drosophila as the model organism to study the molecular mechanism of E93 in regulating wing development.By using the Gal4/UAS system,we carried out E93 RNAi specifically in Drosophila wing and observed the phenotypes,then we identified the potential target genes at the downstream of E93 by analyzing ChIP-seq data.Together with the molecular biology experiments at cell and tissue levels and phenotypic analysis at individual level,we revealed the molecular mechanism by which E93 transcription factor regulates the development of Drosophila wings.The main findings were as follows: 1.The effects of wing-specific RNAi of Drosophila E93 gene on wing developmentTo investigate the effect of E93 expression in Drosophila wing on the wing development,we used the Gal4/UAS system to carry out E93 RNAi specifically in the Drosophila wing.In this study,we selected four wing-specific Gal4 lines(nubbin-Gal4,MS1096-Gal4,ptc-Gal4,en-Gal4)and two E93 RNAi lines(V104390,#57868).Firstly,the expression pattern of the four wing-specific Gal4 in the wing disc was detected using Gal4/UAS system.We crossed the virgins of wing-specific Gal4 lines with the males of UAS-Cherry line that expressed red fluorescent protein,then wing discs of the progeny were taken,and the expression of red fluorescent signal was observed.The results showed that nubbin-Gal4 drived expression of the red fluorescent protein in the whole wing pouch region;MS1096-Gal4 drived expression of the red fluorescent protein in the dorsal(D)wing pouch region;en-Gal4 and ptc-Gal4 drived expression of the red fluorescent protein at the posterior(P)region and the anterior/posterior(A / P)boundary in the wing disc,respectively.We selected the most widely expressed nubbin-Gal4 to cross to two E93 RNAi lines(V104390 and #57868)and the corresponding control strains,respectively.Then the progeny wing discs were dissected to analyse the expression of E93 through RT-PCR and qRT-PCR assay.The results showed that compared with the control,wing-specific RNAi of the E93 gene led to significantly downregulated of E93 expression in the wing.Observation of the phenotypes and statistical results showed that the wing-specific RNAi of E93 gene resulted in significant decrease of the adult wings and developmental defects of the wing veins.The wing area in these two groups with E93 RNAi decreased 76.2% and 82%,respectively.In addition,we used three other wing-specific Gal4 lines,MS1096-Gal4,ptc-Gal4,and en-Gal4,to cross with the E93 RNAi line V104390,respectively.Their offsprings also showed smaller wings with wing vein defects.These results above indicated that E93 plays an important regulatory role in the Drosophila wing development.2.Screening of the Drosophila E93 potential target genes based on ChIP-Seq dataTo reveal the molecular mechanism of E93 in regulating Drosophila wing development,we analyzed the E93 ChIP-seq data in Drosophila wing to identify the E93 potential target genes.Genome-wide localization analysis indicated that 28.51% of the E93 ChIP peaks located in the promoter region(It is defined as the region of 2000 bp upstream of the transcription start site),which corresponded to 1,868 genes;43.96% of the E93 ChIP peaks located in the intron region;13.65% of the E93 ChIP peaks located in the intergenic region.Considering that transcription factors usually exert transcriptional regulatory functions by binding to the key DNA motif in the promoter regions of the target genes,we mainly focused on these 1,868 genes with E93 ChIP peaks located in the promoter region.Gene ontology annotation showed that 513 genes from these 1,868 genes were enriched in the biological process of wing metamorphosis.Further co-expression analysis of these 513 genes were performed based on the transcriptome data of the Drosophila wing at pupal stage,and the co-expression assay at five time points as 6 h,18 h,24 h,36 h and 44 h after puparium formation was mainly focused.These gene were grouped into eight clusters according to the expression pattern.E93 was classified into cluster 2,its expression showed an increase trend at prepupal stage and was highest at 24 h after puparium formation,then the expression level gradually decreased with development.Among the 513 genes,there were 60 genes had the similar expression pattern to E93.Furtherly,KEGG pathway enrichment analysis showed that,four signaling pathways related to wing development were enriched,which were the Dpp,MAPK,Notch,and Hippo signaling pathways.A total of 12 genes involved of Dpp,Tkv,Sog,Nej,Numb,Ser,Wts,Baz,Kibra,Cka,Cic,and Ttk were enriched in these four signaling pathways.3.The molecular mechanism of E93 in regulating Drosophila wing development.In the potential target genes of Drosophila E93 screened based on ChIP-Seq data,we noted that the Dpp gene,encoding the ligand of the Dpp signaling pathway,was enriched and its expression changes at pupal phase were similar to those of E93.Given that the important role of the Dpp signaling pathway in Drosophila wing development has been widely reported,we selected the Dpp gene as a potential target gene of E93 for further investigation.There were four isoforms of the Drosophila Dpp gene according to the Drosophila genome database FlyBase,namely Dpp-RA,Dpp-RB,Dpp-RC,and Dpp-RE,respectively.These four isoforms had different promoter regions but encoded the same protein after processing.The ChIP-Seq data showed that there were six E93 ChIP peaks located in the Dpp promoter region.There were two peaks located in the promoter region of Dpp-RA and Dpp-RB,separately,namely Dpp-RA-D(D,distal)and Dpp-RA-P(P,proximal),Dpp-RB-D and Dpp-RB-P,respectively,based on their distance from the transcription start site.There was one E93 ChIP peak located in the promoter region of Dpp-RC and Dpp-RE,respectively.ChIP-PCR assay in the Drosophila S2 cells showed that E93 can directly bind to these six peaks at the promoter region of the Dpp gene.We investigated the regulation of E93 on the promoters of Dpp through dual luciferase assay.The results indicated that E93 overexpression significantly up-regulated the promoter activities of Dpp-RA and Dpp-RC,but had no significant effect on the promoter activity of Dpp-RB and Dpp-RE.Subsequently,we tested the effects of E93 on the activities of promoters in different truncated forms of Dpp-RA and Dpp-RC isoforms.The results showed that E93 overexpression also induced the activity of the promoter containing only the proximal ChIP peaks(Dpp-RA-P)in Dpp-RA.But E93 overexpression no longer had a significant effect on the promoter activity when all the ChIP peaks on Dpp-RA and Dpp-RC isoforms were cut off.These results above indicated that E93 can bind directly to the Dpp promoter region and activated its promoter activity.We further detected the effects of the change of E93 expression on the transcription of genes involved in Dpp signaling pathway.qRT-PCR results showed that wing-specific E93 RNAi driven by nubbin-Gal4 downregulated the expression of Dpp,Tkv,and Mad,the key genes involved in Dpp signaling pathway.E93 overexpression in Drosophila S2 cells significantly upregulated the expression of Dpp,Tkv,and Mad.Immunofluorescence assay showed that wing-specific E93 RNAi inhibited the expression of Dpp and the phosphorylation and activation of Mad by Dpp in the pupal wing.We also searched the effects of the expression changes of genes in Dpp signaling pathway on Drosophila wing development.We crossed the wing-specific UAS-dicr2;nubbin-Gal4 to the RNAi lines of Dpp,Tkv,and Mad,three key genes involved in the Dpp signaling pathway,respectively,and observed the phenotypes of the offspring.Compared with the wild-type wings,wing-specific RNAi of Dpp,Tkv,and Mad all resulted in significant reduction of the adult wing and loss of wing veins,which phenocopied the defects of wing-specific E93 RNAi.Together,these results above confirmed that Drosophila E93 mediated wing development by directly and positively regulating the key genes in the Dpp signaling pathway.