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Injury-stimulated Hedgehog signaling promotes regenerative proliferation of Drosophila intestinal stem cells.

Tian A, Shi Q, Jiang A, Li S, Wang B, Jiang J - J. Cell Biol. (2015)

Bottom Line: Inhibition of Hh signaling in the ISC lineage compromised injury-induced ISC proliferation but had little if any effect on homeostatic proliferation.Furthermore, we show that Hh signaling is stimulated by DSS through the JNK pathway and that inhibition of Hh signaling in EBs prevented DSS-stimulated ISC proliferation.Hence, our study uncovers a JNK-Hh-JAK-STAT signaling axis in the regulation of regenerative stem cell proliferation.

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Affiliation: Department of Developmental Biology and Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390.

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DSS up-regulates Hh signaling through the JNK pathway. (A–B′) Expression of a JNK pathway reporter, puc-LacZ, in mock (Suc; A and A′) or DSS-treated (B and B′) midguts for 1 d. puc-LacZ expression was barely detectable in mock-treated guts but was elevated in Su(H)>GFP-positive cells in DSS-treated guts. Arrows indicate Su(H)>GFP+ cells with small nuclei. (C) Quantification of relative hh mRNA levels by RT-qPCR in control or Su(H)ts>BSKDN midguts treated with Suc or DSS for 1 d after adult flies were raised at 29°C for 10 d. Three independent experiments were performed and error bars are standard deviations. *, P < 0.05. (D) Quantification of PH3+ cells in control or Su(H)ts>BSKDN midguts treated with Suc or DSS for 1 d after adult flies were raised for 10 d at 29°C. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (E) Quantification of hh and ptc mRNA levels by RT-qPCR in control and Su(H)ts>PucRNAi guts grown at 29°C for 6 d. Three independent experiments were performed and error bars are standard deviations. Numbers indicate fold change over control guts. (F–I′) Expression of hh-lacZ (F–G′, red) or ptc-lacZ (H–I′, red) in control (F, F′, H, and H′) or esgts>PucRNAi guts grown at 29°C for 5 d. Arrows indicate elevated hh-lacZ (F-G′) and ptc-lacZ (H-I′) in precursor cells. (J–M) Adult midguts expressing esgts (Con; J), esgts>PucRNAi (K), esgts>HhRNAi (L), or esgts>PucRNAi + HhRNAi (M) for 7 d at 29°C were dissected out and immunostained for PH3 and Hoechst. (N) Quantification of PH3+ cells in adult midguts of the indicated genotypes. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (O) A JNK–Hh–JAK–STAT signaling axis mediates DSS-stimulated ISC proliferation in adult midgut regeneration. BM, basement membrane; VM, visceral muscles. See text for details. Bars, 50 µm.
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fig7: DSS up-regulates Hh signaling through the JNK pathway. (A–B′) Expression of a JNK pathway reporter, puc-LacZ, in mock (Suc; A and A′) or DSS-treated (B and B′) midguts for 1 d. puc-LacZ expression was barely detectable in mock-treated guts but was elevated in Su(H)>GFP-positive cells in DSS-treated guts. Arrows indicate Su(H)>GFP+ cells with small nuclei. (C) Quantification of relative hh mRNA levels by RT-qPCR in control or Su(H)ts>BSKDN midguts treated with Suc or DSS for 1 d after adult flies were raised at 29°C for 10 d. Three independent experiments were performed and error bars are standard deviations. *, P < 0.05. (D) Quantification of PH3+ cells in control or Su(H)ts>BSKDN midguts treated with Suc or DSS for 1 d after adult flies were raised for 10 d at 29°C. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (E) Quantification of hh and ptc mRNA levels by RT-qPCR in control and Su(H)ts>PucRNAi guts grown at 29°C for 6 d. Three independent experiments were performed and error bars are standard deviations. Numbers indicate fold change over control guts. (F–I′) Expression of hh-lacZ (F–G′, red) or ptc-lacZ (H–I′, red) in control (F, F′, H, and H′) or esgts>PucRNAi guts grown at 29°C for 5 d. Arrows indicate elevated hh-lacZ (F-G′) and ptc-lacZ (H-I′) in precursor cells. (J–M) Adult midguts expressing esgts (Con; J), esgts>PucRNAi (K), esgts>HhRNAi (L), or esgts>PucRNAi + HhRNAi (M) for 7 d at 29°C were dissected out and immunostained for PH3 and Hoechst. (N) Quantification of PH3+ cells in adult midguts of the indicated genotypes. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (O) A JNK–Hh–JAK–STAT signaling axis mediates DSS-stimulated ISC proliferation in adult midgut regeneration. BM, basement membrane; VM, visceral muscles. See text for details. Bars, 50 µm.

Mentions: Many types of cellular stresses can activate the JNK pathway (Weston and Davis, 2007). Indeed, we found that midguts fed with DSS activated the JNK pathway reporter puc-lacZ in Su(H)>GFP+ EBs as well as adjacent ECs, whereas mock treatment barely activated puc-lacZ in these cells (Fig. 7, A–B′). To determine whether JNK pathway activation is required for DSS-induced Hh pathway activation, we blocked the JNK pathway by expressing a dominant-negative Basket, BskDN, in EBs (Su(H)ts>BskDN). We found that Su(H)ts>BskDN blocked DSS-induced Hh up-regulation and ISC proliferation (Fig. 7, C and D).


Injury-stimulated Hedgehog signaling promotes regenerative proliferation of Drosophila intestinal stem cells.

Tian A, Shi Q, Jiang A, Li S, Wang B, Jiang J - J. Cell Biol. (2015)

DSS up-regulates Hh signaling through the JNK pathway. (A–B′) Expression of a JNK pathway reporter, puc-LacZ, in mock (Suc; A and A′) or DSS-treated (B and B′) midguts for 1 d. puc-LacZ expression was barely detectable in mock-treated guts but was elevated in Su(H)>GFP-positive cells in DSS-treated guts. Arrows indicate Su(H)>GFP+ cells with small nuclei. (C) Quantification of relative hh mRNA levels by RT-qPCR in control or Su(H)ts>BSKDN midguts treated with Suc or DSS for 1 d after adult flies were raised at 29°C for 10 d. Three independent experiments were performed and error bars are standard deviations. *, P < 0.05. (D) Quantification of PH3+ cells in control or Su(H)ts>BSKDN midguts treated with Suc or DSS for 1 d after adult flies were raised for 10 d at 29°C. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (E) Quantification of hh and ptc mRNA levels by RT-qPCR in control and Su(H)ts>PucRNAi guts grown at 29°C for 6 d. Three independent experiments were performed and error bars are standard deviations. Numbers indicate fold change over control guts. (F–I′) Expression of hh-lacZ (F–G′, red) or ptc-lacZ (H–I′, red) in control (F, F′, H, and H′) or esgts>PucRNAi guts grown at 29°C for 5 d. Arrows indicate elevated hh-lacZ (F-G′) and ptc-lacZ (H-I′) in precursor cells. (J–M) Adult midguts expressing esgts (Con; J), esgts>PucRNAi (K), esgts>HhRNAi (L), or esgts>PucRNAi + HhRNAi (M) for 7 d at 29°C were dissected out and immunostained for PH3 and Hoechst. (N) Quantification of PH3+ cells in adult midguts of the indicated genotypes. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (O) A JNK–Hh–JAK–STAT signaling axis mediates DSS-stimulated ISC proliferation in adult midgut regeneration. BM, basement membrane; VM, visceral muscles. See text for details. Bars, 50 µm.
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fig7: DSS up-regulates Hh signaling through the JNK pathway. (A–B′) Expression of a JNK pathway reporter, puc-LacZ, in mock (Suc; A and A′) or DSS-treated (B and B′) midguts for 1 d. puc-LacZ expression was barely detectable in mock-treated guts but was elevated in Su(H)>GFP-positive cells in DSS-treated guts. Arrows indicate Su(H)>GFP+ cells with small nuclei. (C) Quantification of relative hh mRNA levels by RT-qPCR in control or Su(H)ts>BSKDN midguts treated with Suc or DSS for 1 d after adult flies were raised at 29°C for 10 d. Three independent experiments were performed and error bars are standard deviations. *, P < 0.05. (D) Quantification of PH3+ cells in control or Su(H)ts>BSKDN midguts treated with Suc or DSS for 1 d after adult flies were raised for 10 d at 29°C. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (E) Quantification of hh and ptc mRNA levels by RT-qPCR in control and Su(H)ts>PucRNAi guts grown at 29°C for 6 d. Three independent experiments were performed and error bars are standard deviations. Numbers indicate fold change over control guts. (F–I′) Expression of hh-lacZ (F–G′, red) or ptc-lacZ (H–I′, red) in control (F, F′, H, and H′) or esgts>PucRNAi guts grown at 29°C for 5 d. Arrows indicate elevated hh-lacZ (F-G′) and ptc-lacZ (H-I′) in precursor cells. (J–M) Adult midguts expressing esgts (Con; J), esgts>PucRNAi (K), esgts>HhRNAi (L), or esgts>PucRNAi + HhRNAi (M) for 7 d at 29°C were dissected out and immunostained for PH3 and Hoechst. (N) Quantification of PH3+ cells in adult midguts of the indicated genotypes. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (O) A JNK–Hh–JAK–STAT signaling axis mediates DSS-stimulated ISC proliferation in adult midgut regeneration. BM, basement membrane; VM, visceral muscles. See text for details. Bars, 50 µm.
Mentions: Many types of cellular stresses can activate the JNK pathway (Weston and Davis, 2007). Indeed, we found that midguts fed with DSS activated the JNK pathway reporter puc-lacZ in Su(H)>GFP+ EBs as well as adjacent ECs, whereas mock treatment barely activated puc-lacZ in these cells (Fig. 7, A–B′). To determine whether JNK pathway activation is required for DSS-induced Hh pathway activation, we blocked the JNK pathway by expressing a dominant-negative Basket, BskDN, in EBs (Su(H)ts>BskDN). We found that Su(H)ts>BskDN blocked DSS-induced Hh up-regulation and ISC proliferation (Fig. 7, C and D).

Bottom Line: Inhibition of Hh signaling in the ISC lineage compromised injury-induced ISC proliferation but had little if any effect on homeostatic proliferation.Furthermore, we show that Hh signaling is stimulated by DSS through the JNK pathway and that inhibition of Hh signaling in EBs prevented DSS-stimulated ISC proliferation.Hence, our study uncovers a JNK-Hh-JAK-STAT signaling axis in the regulation of regenerative stem cell proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Biology and Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390.

Show MeSH
Related in: MedlinePlus