Unknown,Transcriptomics,Genomics,Proteomics

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JAK/STAT coordinates cell proliferation during disc regeneration with Dilp8-mediated developmental delay in Drosophila melanogaster

ABSTRACT: Regeneration of fragmented Drosophila imaginal discs occurs in an epimorphic manner, involving local cell proliferation at the wound site. Following disc fragmentation, cells at the wound site activate a restoration program through wound healing, regenerative cell proliferation and repatterning of the tissue. However, the interplay of signaling cascades, driving these early reprogramming steps, is not well understood. Here we profiled the transcriptome of regenerating cells in the early phase within twenty-four hours after wounding. We found that JAK/STAT signaling becomes activated at the wound site and promotes regenerative cell proliferation in cooperation with Wingless (Wg) signaling. In addition, we demonstrated that the expression of Drosophila insulin-like peptide 8 (dilp8), which encodes a paracrine peptide to delay the onset of pupariation, is controlled by JAK/STAT signaling in early regenerating discs. Our findings suggest that JAK/STAT signaling plays a pivotal role in coordinating regenerative disc growth with organismal developmental timing. In order to analyze transcriptome change in early regenerating imaginal disc, Drosophila prothorasic leg discs were fragmented (to anterior one-quarter or posterior three-quarters) and cultured ex vivo in adult fly abdomen. Regenerating cells in early regeneration phase (at 12 or 24 hours after wounding) were subjected to transcriptome profiling with Affymetrix microarrays. For control samples, the corresponding regions of uncut-cultured discs and uncut-uncultured discs were used.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Tomonori Katsuyama

PROVIDER: E-GEOD-54868 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Research on the ability of certain organisms to reconstruct missing structures of their bodies is still in its infancy, despite numerous efforts performed in multiple species. Using expression profile analyses on different timepoints that cover wound healing and regeneration processes (0, 24 and 72 hours post injury), we studied the regenerative behaviour of fragmented wing imaginal discs of D. melanogaster implanted into adult flies. First, through the comparison between cut and intact discs, we identified the effect of implantation on the regeneration process. Filtering this information, we then constructed the specific early regeneration gene signature of wing discs in which multiple transcription factors, immune response genes and members of the JNK, Notch and WNT signaling pathways seem to play an important role. We next compared the transcriptomes of discs 24 and 72 hours after fragmentation to characterize the regenerative machinery and observed a temporal decrease in the cellular metabolic processes, RNA processing and gene expression machinery, suggesting the discs normal activity was stopped on this first interval of time in response to injury and implantation offences. Based on the expression patterns, we elaborated a catalogue of genes involved in wing disc regeneration divided into four classes (wound healing, regeneration, quick response, constant response). Imaginal discs from third instar larvae were 3/4 fragmented, implanted into the abdomen of adult flies and cultured. In order to subtract the noise due to implantation, we repeated the same protocol using intact discs that were cultivated for 24h. Therefore, a total of 12 microarrays were hybridized: four microarrays (with total RNA from non cut wing discs at 0 and 24 hours) were used to assess the effect of the implantation procedure in intact wing discs and the rest (8) were used to measure changes in gene expression in the first 24 hours after disc dissection and implantation (total RNA from wing discs at 0 and 24 hours was hybridized) and during the period between 24 and 72 hours after the cut (total RNA from wing discs at 24 and 72 hours was hybridized). At least two independent total RNA extractions were carried out (using the RNeasy Protect Mini Kit from Qiagen Inc.) for each condition. The four microarrays hybridized for each pair of conditions were done in biological replicate pairs with dyes swapped to take dye bias into account. The whole set of microarrays was normalized following the same protocol, extracting in each case the list of significant genes (at least 1.5-fold change, FDR adjusted pvalue < 0.05).

Project description:Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes. FAIRE-Seq in Drosophila wild type eye-antennal imaginal discs (2 wt strains); ATAC-Seq in Drosophila wild type eye-antennal imaginal discs (3 wt strains) ; FAIRE-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila eye discs with Unpaired over-expression (2 biological replicates); CTCF ChIP-seq in Drosophila eye discs; ChIP-seq input in Drosophila eye discs

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JAK/STAT coordinates cell proliferation during disc regeneration with Dilp8-mediated developmental delay in Drosophila melanogaster

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