Improved ER quality control alleviates dysplasia of aging ISCs.

<p>(A) Intestines of wild-type flies stained with anti-bGal antibodies at 5 days or 33 days of age and of old flies over-expressing Hrd1 or Xbp1 (Xbp1^d08698) in ISCs/EBs (esg::Gal4) and carrying the gstD1::lacZ reporter. Arrowheads point to ISCs/EBs (marked by GFP). Quantification of relative bGal staining shown in the right panel. Ratio of fluorescence intensity in ISCs/EBs and nearby ECs is shown. ISCs/EBs identified by GFP. P values from Student's T test. N> = 3. (B) Quantification of mitotic figures in aging wild-type flies and in flies expressing Xbp1 (Xbp1^d08698, Xbp1^EP2112) and Hrd1 in ISCs/EBs (esg::Gal4, UAS::GFP). Averages and SEM are shown. P values from Student's T test, N>160 (from 4–5 guts each). N>20(5 d), N>20 (25 d), N>25 (35 d), N>7 (45 d), n>14 (55d)) (C) Model of UPR^ER/ROS signaling network regulating ISC proliferation, through ER stress is required for the induction of ISC proliferation. Inhibition of CncC activity by JNK in response to ER-induced ROS is further required to permit ISC regenerative responses. (D) Age-related loss of proliferative homeostasis as a consequence of increased ER stress. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004568#pgen.1004568.s005" target="_blank">Figure S5</a>.</p

The UPR^ER as a rheostat for stem cell proliferation.

<p>(A) Quantification of pH3⁺ cells in wild-type flies and in flies over-expressing Xbp1 (<i>xbp1^d08698</i>, <i>xbp1^EP2112</i>), Hrd1, or Hsc3 under the control of esg::Gal4 exposed to mock treatment (5% sucrose) or tunicamycin (TM). Averages and SEM are shown. P values from Student's T test, N = 10. (B) Quantification of pH3⁺ cells in wild-type flies and in flies over-expressing Xbp1 (<i>Xbp1^d08698</i>, <i>Xbp1^EP2112</i>), Hrd1, or Hsc3 under the control of esg::Gal4, UAS::GFP; tub::Gal80^ts exposed to mock treatment (5% sucrose) or tunicamycin (TM). Averages and SEM are shown. P values from Student's T test, N = 10. (C) Quantification of pH3⁺ cells in wild-type flies and in flies over-expressing Xbp1 (<i>Xbp1^d08698</i>, <i>Xbp1^EP2112</i>), or Hsc3 under the control of esg::Gal4, UAS-GFP; tubG80^ts exposed to mock (5% sucrose) and oxidative-stress inducer paraquat (5 mM PQ). Averages and SEM are shown. P values from Student's Test, N = 10. (D) Spliced Xbp1 prevents ISC over-proliferation induced by excessive ER stress. Quantification of mitotic figures of wild-type flies and flies over-expressing spliced Xbp1 (Xbp1^spliced) in ISCs/EBs (esg::Gal4, UAS-GFP; tubG80^ts) exposed to mock treatment (5% sucrose) or paraquat (5 mM) or tunicamycin(50 µM). Averages and SEM are shown. P values from Student's Test, N = 10. (E) Over-expressing spliced Xbp1 in ISCs only (using esg::Gal4, Su(H)-Gbe::G80,tub::Gal80ts) inhibits stress-induced ISC proliferation. Averages and SEM are shown. P values from Student's T test, N>10. (F) Over-expressing spliced Xbp1 in ECs has no effect on ISC proliferation upon stress. Quantification of PH3⁺ cells in intestines of wild-type flies and in flies expressing Xbp1^spliced specifically in ECs (using NP1::Gal4; tub::Gal80^ts). Averages and SEM are shown. P values from Student's T test, N = 10. (G) Quantification of pH3⁺ cells in intestines of wild-type flies and in flies expressing spliced Xbp1 (Xbp1^DSΔ28) or Hrd1 specifically in EBs (using Su(H)Gbe::Gal4,tubG80^ts) exposed to mock treatment (5% sucrose), paraquat (5 mM), or tunicamycin (50 µM). Averages and SEM are shown. P values from Student's Test, N = 10. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004568#pgen.1004568.s001" target="_blank">Figure S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004568#pgen.1004568.s002" target="_blank">S2</a>.</p

Integration of UPR^ER and Oxidative Stress Signaling in the Control of Intestinal Stem Cell Proliferation

<div><p>The Unfolded Protein Response of the endoplasmic reticulum (UPR^ER) controls proteostasis by adjusting the protein folding capacity of the ER to environmental and cell-intrinsic conditions. In metazoans, loss of proteostasis results in degenerative and proliferative diseases and cancers. The cellular and molecular mechanisms causing these phenotypes remain poorly understood. Here we show that the UPR^ER is a critical regulator of intestinal stem cell (ISC) quiescence in <i>Drosophila</i><i>melanogaster.</i> We find that ISCs require activation of the UPR^ER for regenerative responses, but that a tissue-wide increase in ER stress triggers ISC hyperproliferation and epithelial dysplasia in aging animals. These effects are mediated by ISC-specific redox signaling through Jun-N-terminal Kinase (JNK) and the transcription factor CncC. Our results identify a signaling network of proteostatic and oxidative stress responses that regulates ISC function and regenerative homeostasis in the intestinal epithelium.</p></div

Xbp1 coordinates with Keap-CncC in ISC proliferation.

<p>(A) UPR^ER influences CncC activity in ISCs/EBs (esgts). CncC activity assessed by bGal expression from the CncC reporter gstd1::lacZ. DAPI, blue; GFP, green; bGal and Hsc3 are shown as separate channels in white. Arrowheads point to individual ISCs/EBs. Quantification of relative bGal and Hsc3 staining shown in the right panel. Ratio of fluorescence intensities in ISCs/EBs and nearby ECs is shown. ISCs/EBs identified by GFP. P values from Student's T test. N = 3. (B) Control of CncC activity by the UPR^ER in response to tunicamycin treatment. CncC activity assessed by bGal expression from the CncC reporter gstd1::lacZ. DAPI, blue; GFP, green; bGal white, and shown as separate channel in white. Arrowheads point to individual ISCs/EBs. Quantification of bGal staining shown in the lower panel. Ratio of fluorescence intensities in ISCs/EBs and nearby ECs is shown. ISCs/EBs identified by GFP. P values from Student's T test. N = 3. P values from Student's T test. N = 5. (C) GFP-marked MARCM clones for Keap1^EY5, Xbp1RNAi¹⁰⁹³¹², Keap1^EY5; Xbp1RNAi¹⁰⁹³¹² and wild-type control (Frt82B) at 7 days after heat shock. (D) Quantification of MARCM clone sizes at 7 days after heat shock. Number of clones examined: n = 89 (Frt82B); n = 56 (Xbp1RNAi¹⁰⁹³¹²); n = 32 (Keap1^EY5); n = 88 (Keap1^EY5, Xbp1^RNAi109312). Averages and SEM are shown. P values from Student's T test. (E) Increased CncC (by loss of Keap1 or over-expressing CncC) inhibits ISC overproliferation induced by loss of Xbp1 in ISCs/EBs (esg^ts). Averages and SEM are shown. P values from Student's T test. N = 10. (F) CncC in ISCs/EBs (esg::Gal4,tub::Gal80ts) inhibits tunicamycin-induced ISC proliferation. Averages and SEM are shown. P values from Student's T test. N>10. (G) Quantification of MARCM clone sizes for at 7 days after heat shock. Number of clones examined: n = 102 (Frt82B); n = 99 (Xbp1^spliced); n = 23 (Cnc^VL110, Xbp1^spliced). Averages and SEM are shown. P values from Student's T test. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004568#pgen.1004568.s004" target="_blank">Figure S4</a>.</p

The UPR^ER is activated in aging intestines.

<p>(A) Expression of Gp93, Hsc70-3 and Xbp1 in guts from flies of different ages. Expression was determined using RNAseq in experiments described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004568#pgen.1004568-Guo1" target="_blank">[32]</a>. Expression values are shown normalized to the 2 d timepoint in the graph. Raw RPKM values are shown in the table. (B) Major branches of UPR^ER signaling pathways in <i>Drosophila</i>. (C) Young (5 day old) and old (30 day old) guts of esg::Gal4, UAS::GFP flies immunostained with anti-Hsc3 antibody (DNA: DAPI, blue; ISCs/EBs: GFP, green; Hsc3, red). The Hsc3 channel is shown separately on the right. (D) Reporter line <i>Xbp1_p</i>::<i>DsRed</i> visualizes expression of Xbp1 in young (3 day) and old (42 day) guts (DNA: DAPI, blue; ISCs/EBs: GFP, green; DsRed: red). The DsRed channel is shown separately on the right. (E) Enlarged images for intestines of young (5 day) and old (30 day) flies (esg::Gal4, UAS:: GFP/+) immunostained with anti-Hsc3 antibody (DNA: DAPI, blue; ISCs/EBs: GFP, green; Hsc3, red). White arrowheads indicate ISCs/EBs. The Hsc3 channel is shown separately on the right. (F) Xbp1 splicing reporter (UAS::Xbp1-EGFP) expressed in ISCs/EBs using esg::Gal4. Expression of GFP in young (5 day) and old (62 day) guts is shown. (DNA: DAPI, blue; GFP, green). The GFP channel is shown separately on the right. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004568#pgen.1004568.s001" target="_blank">Figure S1</a>.</p

The UPR^ER is sufficient and required in ISCs to promote proliferation.

<p>(A) Knockdown of Xbp1 (using Xbp1RNAi¹⁵³⁴⁷) in ISCs/EBs (using esg::Gal4, tubG80^ts) leads to ISC over-proliferation in intestines, quantified by counting the number of pH3+ cells/gut. (B) Representative images of wild-type flies and flies with ISC/EB-specific knockdown of Xbp1 (DNA: DAPI, blue; ISCs/EBs marker: GFP, green; ISC marker: DI, red/white). Dl channel is shown separately in lower panels. (C) Loss of Xbp1 or Hrd1 in ISCs/EBs promotes eIF2α phosphorylation in ISCs/EBs, compared to wild-type control. peIF2α channel is shown in white in lower panels. Arrowheads for orientation. DNA: DAPI, blue; ISCs/EBs: GFP, green; peIF2α, grey. (D) ISC-specific knockdown of Xbp1 (using esg::Gal4, Su(H)-Gbe::G80,tub::Gal80ts) induces ISC proliferation. 3 different fly lines expressing dsRNA against Xbp1 in a Gal4-sensitive manner (Xbp1RNAi¹⁵³⁴⁷, Xbp1RNAi¹⁰⁹³¹², and Xbp1RNAi^HMS03015) were used. Averages and SEM are shown. P values from Student's T test, N>20. (E) Knockdown of Hrd1 in ISCs/EBs (using esg::Gal4, tubG80ts), or specifically in ISCs (esgts/Su(H)Gbe::G80) induces ISC proliferation. Averages and SEM are shown. P values from Student's T test, N>10. (F) Quantification of pH3⁺ cells in wild-type flies and in flies expressing RNAi constructs against Xbp1 or Hrd1 in EBs (using Su(H)Gbe::Gal4, tub::Gal80ts) or ECs (using NP1::Gal4, tub::Gal80ts). Averages and SEM are shown. P values from Student's T test, N = 10. (G) Quantification of MARCM clone sizes at 3 days and 7 days after heat shock for <i>Xbp1</i> and <i>hrd1</i> loss-of-function (Xbp1^k13803, Xbp1^RNAi, Hrd1^Delta, Hrd1^RNAi) or gain-of-function (Xbp1^spliced, Xbp1^d08698 and UAS::Hrd1) conditions. Averages and SEM are shown. P values from Student's T test. Number of clones examined: n = 447 (3d FRT82B); n = 42 (3d FRT42D); n = 165 (3d FRT40); n = 95(3d Xbp1^k13803); n = 268 (3d Xbp1RNAi); n = 515 (3d Hrd1^Delta); n = 262 (3d Hrd1RNAi); n = 178 (7d FRT42D); n = 215 (7d FRT40); n = 394 (7d FRT82B); n = 123 (7d Xbp1^k13803); n = 270 (7d Xbp1RNAi); n = 424 (7dHrd1^Delta); n = 119 (7d Hrd1RNAi); n = 99 (Xbp1^spliced); n = 138 (Xbp1^d08698); n = 162 (UAS::Hrd1). (H) Representative images for Xbp1 loss-of-function at 3 days after heat shock and spliced Xbp1 at 7 days after heat shock are shown on the right. (DAPI, blue; GFP, green). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004568#pgen.1004568.s001" target="_blank">Figure S1</a>.</p

Coordinated control of ISC proliferation by the UPR^ER and ROS signaling.

<p>(A) Increased ROS in ISCs deficient in Xbp1 or Hrd1. Relative DHE fluorescence intensity is calculated by the ratio of fluorescence intensity in ISCs/EBs and nearby ECs. ISCs/EBs identified by GFP. Arrowheads point to selected ISCs/EBs. GFP, green; DHE red; DHE is shown as separate channel in white. Averages and SEM for relative DHE intensity are shown. P values from Student's T test, N>200 (from 6–7 WT, Xbp1^RNAi guts), N>100 (from 3–4 WT, Hrd1^RNAi guts). (B) Over-expression of spliced Xbp1in ISCs/EBs (using esg::Gal4, tubG80^ts) resulted in decreased DHE fluorescence in ISCs under Paraquat treatment. Relative DHE fluorescence intensity is quantified for wild-type flies and flies over-expressing spliced Xbp1. Arrowheads point to selected ISCs/EBs. GFP: green. DHE is shown as separate channel in white. Averages and SEM are shown. P values from Student's Test. (C) Frequency of pH3+ cells are quantification for wild-type fly and flies expressing spliced Xbp1 only, Jafrac1 loss-of-function only, and for flies coexpressing spliced Xbp1 and Jafrac1 loss-of-function. Averages and SEM are shown. P values from Student's Test. N = 10. (D) Over-expression of anti-oxidant enzyme GTPx-1 or Cat in ISCs/EBs (esg::Gal4,tubG80ts) inhibits tunicamycin-induced ISC proliferation. Averages and SEM are shown. P values from Student's T test. N>10. (E) Quantification of pH3⁺ cells in wild-type flies, in flies over-expressing spliced Xbp1 only, and in flies expressing both Jafrac1 loss-of-function and spliced Xbp1 under the control of esg::Gal4, UAS-GFP; tubG80^ts exposed to mock (5% sucrose) and tunicamycin. Averages and SEM are shown. P values from Student's Test. N = 10.</p

JNK is activated by UPR^ER-induced ISC proliferation.

<p>(A) JNK is phosphorylated in ISCs when Xbp1 or Hrd1 is knocked down specifically in ISCs. DAPI, blue; GFP, green; pJNK or Dl shown as separate channels in white. (B) JNK activation when Xbp1 or Hrd1 is knocked down in ISCs/EBs. Anti bGal antibody staining to detect lacZ expression from the puc^E69::lacZ reporter. DAPI, blue; GFP, green; bGal or DI is shown as separate channel in white. (C) JNK activation by tunicamycin exposure. Anti bGal antibody was used to detect expression of lacZ from a puc::lacZ reporter (esg::Gal4, UAS::GFP, tubG80ts/pucE69::lacZ). Flies were exposed to mock treatment (5% sucrose) or tunicamycin (50 µM in 5% sucrose). DNA: DAPI, blue; ISCs/EBs: GFP, green; bGal, white. (D) Over-expression of spliced Xbp1 or of GTPx-1 or Cat in ISCs/EBs (esg::Gal4,tubG80ts) represses tunicamycin-induced JNK activation. bGal antibody staining detecting lacZ expression from the puc^E69::lacZ reporter. DNA: DAPI, blue; ISCs/EBs: GFP, green; bGal, white; DI,white). (E) Repressing JNK activity (using Bsk^RNAi or Bsk^DN) in ISC/EBs (using esg^ts) inhibits ISC over-proliferation induced by loss of Xbp1 or by tunicamycin treatment. Averages and SEM are shown. P values from Student's T test, N = 10. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004568#pgen.1004568.s003" target="_blank">Figure S3</a>.</p

Mtor regulates GSC maintenance.

<p>(A) A schematic drawing of the asymmetric division of a <i>Drosophila</i> male GSC. Both GSCs (green) and CySCs (blue) are anchored around the hub (orange) through adherens junctions (brown). Asymmetric division of a GSC results in a new GSC (green) and a gonialblast (GB) (light green). In the GSC (green), the mother centrosome (dark blue) nucleates more microtubules (dark blue) than the daughter centrosome (pink), which may help to asymmetrically deliver Apc2 (beige) to the cortex, where GSCs contact the hub. At the cortex, Apc2 concentrates at the cell–cell junction (brown) to anchor the mitotic spindles from the mother centrosome perpendicular to the hub, but the daughter centrosome remains on the opposite side (pink). As a result, GSCs divide asymmetrically. (B) Quantification of the number of GSCs associated with the hub (7 days old flies were dissected and stained for each genotype). <i>Nos-Gal4</i>-driven <i>Mtor</i>^RNAi-1 (n = 35), <i>Mtor</i>^RNAi-2 (n = 39), or <i>Mtor</i>^RNAi-3 (n = 41) caused a significant decrease in the number of GSCs associated with the hub compared to the control <i>Nos</i>-<i>Gal4</i>-driven <i>LacZ</i>^RNAi (n = 27). ***p<0.0001. Error bars represent SD. (C-F) GSCs in testes of wild-type control (<i>Nos>lacZ</i>^RNAi) (C), <i>Nos>Mtor</i>^RNAi-1 (D), <i>Nos>Mtor</i>^RNAi-2 (E), and <i>Nos>Mtor</i>^RNAi-3 flies (F) were examined by staining with anti-vasa [red, marks all germ cells including GSCs in white dotted circle (one depicted in a white arrowhead); yellow dotted circle marks the GB (one depicted in yellow arrow)], anti-Fas3 (green, marks the hub cells, asterisks), anti-1B1 (green in dot and branched marks the spectrosomes and fusomes respectively), and DAPI (blue). Some CySCs positioned adjacent to the hub cells (pink dotted circle) after some GSCs were depleted in <i>Nos>Mtor</i>^RNAi testes (D-F). All flies were cultured for 7 days at 29°C before dissection. Scale bars represent 10 μm.</p

<i>Mtor</i> cell-autonomously regulates CySC differentiation.

<p>GFP⁺ clones were generated in the testes of wild-type control (<i>FRT</i>^42D<i>-piM</i>; A-A’ and F-F’) or FRT^42D<i>-Mtor</i>^k03905 (B-E’ and G-H’) flies using the MARCM technique and stained at 1, 2, and 4 days ACI with the anti-GFP (green), anti-Vasa (red) and DAPI (blue). In FRT^42D control testes, we were able to find many GFP-positive CySCs and their differentiated progenies (A-A’, F-F’, I) at 1, 2, and 4 days ACI. In <i>FRT</i>^42D<i>-Mtor</i>^k03905 testes, we were rarely able to find GFP-positive CySCs at 1 and 2 days ACI (B-E’, I). At 4 days ACI, we were not able to find a single GFP-positive CySC in 60 <i>Mtor-</i>mutant testes (G-H’, I). However, we could find many GFP-positive differentiated cyst cells (B-E’ and G-H’) in <i>Mtor</i>-mutant testes at 2 and 4 days ACI. CySCs are highlighted by yellow dotted lines and yellow arrows. White dotted lines arrows highlight GSCs and red arrows point to differentiated cyst cells. Red asterisks mark hubs. Scale bars represent 10 μm. (I) Quantitative data of GFP-positive CySC clones in wild-type control (<i>FRT</i>^42D<i>-piM</i>) or <i>FRT</i>^42D<i>-Mtor</i>^k03905 fly testes at 1, 2, and 4 days ACI.</p