Infection-induced <i>upd3</i> expression in ECs requires the TFs D-Jun and D-Fos, activated by upstream Src-MAPK pathways.

<p>(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 <i>upd3-lacZ</i> activity under <i>Ecc15</i> infection or UC conditions. (C) RT-qPCR measurements of total midgut <i>upd3</i> expression corroborate <i>upd3-lacZ</i> results. (D) In addition to their requirement for <i>upd3-lacZ</i> activity, activation of the MAPKs and SFKs can also induce <i>upd3-lacZ</i> expression in UC conditions. SAPKs can also induce this activity when stimulated. (E, F) Immunostaining against phosphorylated forms of ERK and Src reveals that these kinases are activated in response to infection in ECs. Scale bar is 100μm. 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).</p

Combination of <i>in vivo</i>, <i>in vitro</i>, and <i>in silico</i> TF screens identifies direct and indirect regulators of <i>upd3</i> transcription.

<p>(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 <i>upd3</i> induction, as well as by their TF category: putative direct regulators of <i>upd3</i> 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 <i>upd3</i>, and epigenetic regulators that may influence <i>upd3</i> expression by modifying genomic DNA structure. The seven genes that were positive hits for all three screens are indicated by red text. (D) Schematic representation of D-Fos and Sd binding motifs present in <i>upd3</i> enhancer regions C (Green), I (Blue), and R (Purple).</p

ISC proliferation and survival following <i>Ecc15</i> infection are compromised by inhibition of the TFs and pathways that are required for <i>upd3</i> induction.

<p>(A) Total pH3+ cell counts in unchallenged and <i>Ecc15</i> infected guts demonstrate that knockdown in ECs of <i>D-Fos</i>, <i>yki</i>, <i>sd</i>, <i>Trl</i>, and <i>sna</i> 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 <i>Ecc15</i> alongside RNAi-induced knockdown of indirect <i>upd3</i> 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).</p

Infection-induced expression of <i>upd3</i> in ECs requires the Hippo and Dpp pathways.

<p>(A-C) Measurements of midgut <i>upd3-lacZ</i> activity under <i>Ecc15</i> infected and UC conditions during EC-specific knockdown or overexpression of Hippo and Dpp pathway components. Depletion of the Hippo TFs <i>sd</i> or <i>yki</i>, or overexpression of an upstream inhibitor (Msn) blocks basal and infection-induced <i>upd3-lacZ</i> expression. Likewise, knockdown of <i>trr</i>, an epigenetic enhancer of Yki/Sd activity, also inhibits infection-induced <i>upd3-lacZ</i>. Alternatively, overexpression of Yki or knockdown of its upstream inhibitors <i>wts</i> and <i>msn</i> is sufficient to induce <i>upd3-lacZ</i>. Knockdown of the Dpp pathway TF <i>Mad</i>, either of the three Dpp pathway receptors, <i>tkv</i>, <i>sax</i>, or <i>put</i>, or overexpression of the <i>Mad</i> inhibitor, Sgg all blocked <i>upd3-lacZ</i> activity. Overexpression of Dpp itself or knockdown of <i>sgg</i> induced <i>upd3-lacZ</i>. (D) RT-qPCR was used to directly measure <i>upd3</i> transcription levels, and confirm that the function of the Hippo and Dpp pathway TFs are required for <i>upd3</i> 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).</p

Bacterial infection, stress and the microbiota induce <i>upd3</i> through distinct enhancers.

<p>(A) RT-qPCR measured <i>upd3</i> expression is significantly induced by <i>Ecc15</i> and <i>Pe</i> 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 (<i>Ecc15</i> and <i>Pe</i>) and chemical stressors (bleo and DSS). (C) RT-qPCR measurements of <i>upd3</i> transcription in the gut of germ-free (GF) flies shows reduced expression compared to their conventionally reared (CR) counterparts. (D, E) Confocal imaging shows that <i>upd3-C-GFP</i> and <i>upd3-R-GFP</i> strongly induce GFP expression in response to all presented stresses, except for DSS treatment. (F) In contrast, enhancer I responds exclusively to <i>Ecc15</i> and marginally to <i>Pe</i> infection by GFP induction. (G) Measuring GFP expression in <i>upd3-C-GFP</i> and <i>upd3-R-GFP</i> flies by RT-qPCR, normalized to the GFP expression in each line under CR conditions, reveals a reduction in basal enhancer C and R activity in GF conditions. Scale bars are 50μm. 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).</p

The <i>upd3</i> gene is regulated by cell-specific, region-specific and infection-responsive enhancers.

<p>(A) Schematic of the <i>upd3</i> gene and the 21 overlapping sequences used to create GFP reporter lines. The <i>upd3</i> 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 <i>esg-lacZ</i> 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 <i>Myo-</i>positive cells (ECs) throughout the midgut. (E) Enhancer region R drives infection-induced GFP expression in <i>esg</i>-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.</p