Hippo, TGF-β, and Src-MAPK pathways regulate transcription of the upd3 cytokine in Drosophila enterocytes upon bacterial infection

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Abstract

Cytokine signaling is responsible for coordinating conserved epithelial regeneration and immune responses in the digestive tract. In the Drosophila midgut, Upd3 is a major cytokine, which is induced in enterocytes (EC) and enteroblasts (EB) upon oral infection, and initiates intestinal stem cell (ISC) dependent tissue repair. To date, the genetic network directing upd3 transcription remains largely uncharacterized. Here, we have identified the key infection-responsive enhancers of the upd3 gene and show that distinct enhancers respond to various stresses. Furthermore, through functional genetic screening, bioinformatic analyses and yeast one-hybrid screening, we determined that the transcription factors Scalloped (Sd), Mothers against dpp (Mad), and D-Fos are principal regulators of upd3 expression. Our study demonstrates that upd3 transcription in the gut is regulated by the activation of multiple pathways, including the Hippo, TGF-β/Dpp, and Src, as well as p38-dependent MAPK pathways. Thus, these essential pathways, which are known to control ISC proliferation cell-autonomously, are also activated in ECs to promote tissue turnover the regulation of upd3 transcription.

Figures

  • Fig 1. The upd3 gene is regulated by cell-specific, region-specific and infection-responsive enhancers. (A) Schematic of the upd3 gene and the 21 overlapping sequences used to create GFP reporter lines. The upd3 exons are represented by orange blocks and the introns are light blue. Putative enhancer regions have been color coded by their ability to drive GFP expression as follows: Solid Grey–no midgut signal, Dashed Grey–infection induced signal in scattered cells, Green–infection-induced signal throughout the gut, Blue–constant signal throughout the gut, Pink–infection induced signal in a specific midgut region, Purple–constant signal confined to a specific midgut region. (B) Enhancer region M drives an unvarying GFP signal in esg-lacZ expressing cells (ISCs and EBs) in all regions. (C, D) Both the C and I enhancer region sequences drive GFP in an infection-inducible manner, specifically in Myo-positive cells (ECs) throughout the midgut. (E) Enhancer region R drives infection-induced GFP expression in esg-positive cells (ISCs and EBs). (B, C, D, E) Confocal microscopy images taken at 40x magnification with four color channels. DAPI stained nuclei in Blue, GFP in green and antibody stained β-Gal in red. Scale bars are 50μm.
  • Fig 2. Bacterial infection, stress and the microbiota induce upd3 through distinct enhancers. (A) RT-qPCR measured upd3 expression is significantly induced by Ecc15 and Pe infection, as well as bleomycin (bleo) treatment and DSS. (B) ISC proliferation, measured by phospho-Histone H3 (pH3) immunostaining, is triggered in response to ingestion of harmful bacteria (Ecc15 and Pe) and chemical stressors (bleo and DSS). (C) RT-qPCR measurements of upd3 transcription in the gut of germ-free (GF) flies shows reduced expression compared to their conventionally reared (CR)
  • Fig 3. Combination of in vivo, in vitro, and in silico TF screens identifies direct and indirect regulators of upd3 transcription. (A) Basic schematics of the RNAi (A) and yeast one-hybrid screens (A’) along with the number of positive TF hits for each. (B) Venn diagram displaying the number of positive hit TFs identified by each screen and identified by multiple approaches. (C) Summary table of important TF hits organized by whether they induced or suppressed upd3 induction, as well as by their TF category: putative direct regulators of upd3 that likely bind to enhancer regions of the gene, indirect regulators that lack evidence for direct binding potential but have strong phenotypes and probable cause for controlling upd3, and epigenetic
  • Fig 4. Infection-induced upd3 expression in ECs requires the indirect functions of Snail and its transcriptional co-repressors, as well as homeodomain TFs and epigenetic regulators. (A) Induction of upd3-lacZ by Ecc15 infection is impeded by RNAi-mediated knockdown of Snail (Sna), its corepressors Ebi and CtBP, the epigenetic regulator Trl, and the homeodomain TFs, Rx and Ubx. (B) RT-qPCR measurements of total midgut upd3 expression corroborate upd3-lacZ results. (C) RT-qPCR measurements of sna expression reveal that the gene is transcriptionally upregulated in the midgut following Ecc15 infection. (D) Cell-specific midgut RNA-Seq data reveals that sna is transcriptionally induced specifically in ECs during oral infections by Pe. Statistical significance: mean values of at least 3 repeats are represented ± SEM. *p<0.05, **p<0.01, ***p<0.001 (student’s t-test).
  • Fig 5. Infection-induced expression of upd3 in ECs requires the Hippo and Dpp pathways. (A-C) Measurements of midgut upd3-lacZ activity under Ecc15 infected and UC conditions during EC-specific knockdown or overexpression of Hippo and Dpp pathway components. Depletion of the Hippo TFs sd or yki, or overexpression of an upstream inhibitor (Msn) blocks basal and infection-induced upd3-lacZ expression. Likewise, knockdown of trr, an epigenetic enhancer of Yki/Sd activity, also inhibits infection-induced upd3-lacZ. Alternatively, overexpression of Yki or knockdown of its upstream inhibitors wts and msn is sufficient to induce upd3-lacZ. Knockdown of the Dpp pathway TF Mad, either of the three Dpp pathway receptors, tkv, sax, or put, or overexpression of the Mad inhibitor, Sgg all blocked upd3-lacZ activity. Overexpression of Dpp itself or knockdown of sgg induced upd3-lacZ. (D) RTqPCR was used to directly measure upd3 transcription levels, and confirm that the function of the Hippo and Dpp pathway TFs are required for upd3 induction. Statistical significance: mean values of at least 3 repeats are represented ± SEM. *p<0.05, **p<0.01, ***p<0.001 (student’s t-test).
  • Fig 6. Infection-induced upd3 expression in ECs requires the TFs D-Jun and D-Fos, activated by upstream Src-MAPK pathways. (A-B) Knockdown by RNAi of multiple constituents of MAPK pathways, as well as Src kinases or the TFs D-Jun (Jra) and D-Fos (Kay) inhibits upd3-lacZ activity under Ecc15 infection or UC conditions. (C) RT-qPCR measurements of total midgut upd3 expression corroborate upd3-lacZ results. (D) In addition to their
  • Fig 7. ISC proliferation and survival following Ecc15 infection are compromised by inhibition of the TFs and pathways that are required for upd3 induction. (A) Total pH3+ cell counts in unchallenged and Ecc15 infected guts demonstrate that knockdown in ECs of D-Fos, yki, sd, Trl, and sna as well as upstream components of the MAPK and Dpp pathways is accompanied by a decrease in ISC mitotic activity. Statistical significance: mean values of at least 3 repeats are represented ± SEM. *p<0.05, **p<0.01, ***p<0.001 (student’s t test). (B-D) Survival curves of flies orally infected with Ecc15 alongside RNAi-induced knockdown of indirect upd3 regulators (B), Hippo and Dpp pathways components (C), or MAPK pathway factors (D). Curves represent averaged survival ± SE. Statistical significance: *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001 (Log-rank test).

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Houtz, P., Bonfini, A., Liu, X., Revah, J., Guillou, A., Poidevin, M., … Buchon, N. (2017). Hippo, TGF-β, and Src-MAPK pathways regulate transcription of the upd3 cytokine in Drosophila enterocytes upon bacterial infection. PLoS Genetics, 13(11). https://doi.org/10.1371/journal.pgen.1007091

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