Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut
- Harvard Medical School, United States
- University of California Santa Barbara, United States
- University of California, Santa Barbara, United States
- Howard Hughes Medical Institute, Harvard Medical School, United States
Figures
Figure 1 with 3 supplements
The calcium channels TRPA1 and RyR are required for damage-induced ISC proliferation.
(A–C) Midguts overexpressing Luc RNAi, trpA1 RNAi or RyR RNAi in ISCs using EGT (esgGal4 UAS-GFP tubGal80ts) for 6d, were fed the oxidant agent paraquat for the last 2d (A’–C’) or co-expressed with S…
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Figure 1—source data 1
Complete results for Figure 1G–I, Figure 1—figure supplement 1E, Figure 1—figure supplement 3A–B.
- https://doi.org/10.7554/eLife.22441.003
Figure 1—figure supplement 1
Validation of knockdown efficiency for trpA1 RNAi and RyR RNAi lines.
(A) Cartoon depicting the four characterized trpA1 isoforms. The target regions of trpA1 RNAi lines used in this study are shown in dashed rectangles. The regions amplified with isoform-specific …
Figure 1—figure supplement 2
trpA1 RNAi and RyR RNAi do not cause ISC apoptosis.
(A–C) Immunostaining of midguts expressing Luc RNAi, trpA1 RNAi or RyR RNAi in ISCs for apoptosis marker cleaved-caspase 3. The channels of cleaved-caspase 3 signal are shown to the right of the …
Figure 1—figure supplement 3
ISC depletion results in midgut shortening.
(A) Length quantification of midguts with inducible expression of rpr in ISCs. In the ‘rpr off’ group, the flies are kept at 18°C to prevent Gal4 activity and rpr expression; in the ‘rpr on’ group, …
Figure 2 with 1 supplement
TRPA1 and RyR are required for ISC self-renewal but not differentiation.
(A–C, A’–C’) MARCM clones deficient for TRPA1 or RyR are analyzed along with their corresponding control genotypes (A for A’, B for B’, C for C’) 10d after clone induction. Cell numbers per clone …
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Figure 2—source data 1
Complete results for Figure 2J–M.
- https://doi.org/10.7554/eLife.22441.008
Figure 2—figure supplement 1
Lineage-tracing experiments provide evidence that TRPA1 or RyR-deficient ISCs can differentiate.
(A–B) MARCM clones expressing Luc RNAi or trpA1 RNAi for 22d are examined for their survival and stained with anti-Pdm1 antibody to examine EC differentiation. Arrowheads highlight examples of ECs …
Figure 3 with 2 supplements
trpA1 and RyR expression in the midgut.
(A–B) Anti-TRPA1 immunostaining of midguts expressing Luc RNAi or trpA1 RNAi in ISCs. The channel of anti-TRPA1 signal is shown below the merged image. Note that anti-TRPA1 staining has high …
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Figure 3—source data 1
Complete results for Figure 3—figure supplement 2B.
- https://doi.org/10.7554/eLife.22441.011
Figure 3—figure supplement 1
The knock-in design of trpA1Gal4CP2A.
By CRISPR/Cas9-induced homologous recombination, Gal4 is inserted into the shared C terminal of trpA1 isoforms right before the stop codon. The knock-in could result in bi-cistronic transcripts …
Figure 3—figure supplement 2
Additional evidence for trpA1 expression and function in ISCs.
(A) RT-qPCR measurement of midguts expressing the cell death gene rpr in ISCs for 4d for the expression of esg, trpA1 (using primers for CD isoforms), EC marker ε-Trpsin (εTrp), Pdm1, or pros. GAPDH …
Figure 4 with 1 supplement
TRPA1 can respond to oxidant agent and its activation stimulates ISC activity.
(A–B) Oocyte clamp measurement of TRPA1-C or TRPA1-D channel activities in response to paraquat. The same amounts of mRNAs for TRPA1-C or TRPA1-D were injected into Xenopus oocytes. 4 mM paraquat is …
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Figure 4—source data 1
Complete results for Figure 4F–H.
- https://doi.org/10.7554/eLife.22441.015
Figure 4—figure supplement 1
Elevated ROS is a common stress signal in various midgut damage conditions.
(A–E) Dihydro-ethidium (DHE) stainings of live midguts expressing GFP in ISCs for 3d from flies fed with normal food, food containing 2 mM paraquat, 25 µg/ml bleomycin for 6 hr, pathogens Pseudomonas…
Figure 5 with 2 supplements
TRPA1 and RyR are required for ROS-mediated Ca2+ increases in ISCs.
(A–C) Imaging of midguts expressing the GCaMP6s reporter alone or together with trpA1 RNAi or RyR RNAi in ISCs. The same guts are imaged again after exposure to 4 mM paraquat in the imaging buffer …
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Figure 5—source data 1
Results for Figure 5D, Figure 5—figure supplement 1D–E, Figure 5—figure supplement 2F.
- https://doi.org/10.7554/eLife.22441.018
Figure 5—figure supplement 1
The total number of ISCs expressing esgGal4>GCamP6s reporter is not significantly reduced by trpA1 RNAi or RyR RNAi.
(A–C) Anti-GFP staining of midguts expressing GCamP6s alone, or together with trpA1 RNAi or RyR RNAi in ISCs detects GCamP6s expression. The images are acquired on a confocal microscope. The …
Figure 5—figure supplement 2
Additional reporters showing that TRPA1 and RyR are required for ROS-mediated Ca2+ increases in ISCs.
(A–B) Confocal calcium imaging of midguts that express bi-cistronic UAS-tdTomato-P2A-GCaMP5G reporter alone or together with trpA1 RNAi in ISCs with exposure to 2 mM paraquat for 5 min (A’–B’). The …
Figure 6 with 2 supplements
High cytosolic Ca2+ is necessary and sufficient to activate Ras/MAPK in ISCs.
(A–D) Midguts expressing Luc RNAi, SERCA RNAi, trpA1 RNAi, or RyR RNAi in ISCs for 2-3d are stained for the Ras/MAPK activity marker dpErk. The channel of dpErk signal is shown below the merged …
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Figure 6—source data 1
Complete results for Figure 6—figure supplement 2H–I.
- https://doi.org/10.7554/eLife.22441.028
Figure 6—figure supplement 1
Prolonged induction of high cytosolic Ca2+ in ISCs results in a nonspecific and variable pattern of Ras/MAPK activation.
(A–C) Midguts expressing SERCA RNAi together with Luc RNAi, Ras1 RNAi, or Yki RNAi in ISCs for 5d are stained for dpErk. The channel of dpErk signal is shown below the merged image. A and A’ are …
Figure 6—figure supplement 2
Src might be a mechanism by which cytosolic Ca2+ can activate Ras/MAPK in ISCs.
(A–D) Midguts expressing the constitutive active form of Src42A (Src42Aca), Src64B, or Ras1A in ISCs for 2d are stained for the Ras/MAPK activity marker dpErk. The channel of dpErk signal is shown …
Figure 7 with 3 supplements
Ras/MAPK activity, not CanA/CRTC/CrebB, is sufficient for ISC proliferation in the absence of TRPA1.
(A–E) Midguts over-expressing calcium responsive signaling molecules such as active Ras1 (Ras1A), constitutively active Calcineurin A1 (CanA1ca), CREB-regulated transcription coactivator (CRTC), or …
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Figure 7—source data 1
Complete results for Figure 7F, Figure 7—figure supplement 1F–G.
- https://doi.org/10.7554/eLife.22441.032
Figure 7—figure supplement 1
Ras/MAPK activity, but not CanA1/CrebB, is required for ISC proliferation induced by calcium influx.
(A–E) Midguts expressing SERCA RNAi together with Luc RNAi, EGFR RNAi, CanA1 RNAi, CrebB RNAi, or CRTC RNAi in ISCs for 3d are stained for mitosis marker pH3. (F) Mitosis quantification of midguts …
Figure 7—figure supplement 2
Ligands for receptor tyrosine kinases (RTKs) are affected by cytosolic Ca2+ signaling.
(A) RT-qPCR measurement of midguts expressing Luc RNAi or trpA1 RNAi in ISCs for 6d, with the last day feeding on normal food or paraquat. rp49 is used for normalization. The data are presented as …
Figure 7—figure supplement 3
Ras/MAPK activity is sufficient for ISC proliferation in the absence of RyR.
(A–B) Midguts, expressing RyR RNAi alone, or together with SERCA RNAi in ISCs for 5d, are stained for mitosis marker pH3.
Figure 8
Connection between ROS, intracellular calcium, and stem cell activity.
(A) Under tissue homeostasis conditions, basal levels of ROS and other unidentified stimuli can activate TRPA1 and RyR channels at low levels to allow low levels of cytosolic Ca2+, autocrine EGFR …
Author response image 1
Videos
Video 1
Calcium imaging of ISCs in response to paraquat.
Midguts expressing GCaMP6s reporter in ISCs are dissected and imaged in adult hemolymph-like (AHL) buffer. Z-stack images were acquired with 10 s interval. A maximal intensity z-projection was shown …
Video 2
Calcium imaging of ISCs expressing trpA1 RNAi in response to paraquat.
Midguts expressing GCaMP6s reporter together with trpA1 RNAi in ISCs were dissected and imaged in AHL buffer. Z-stack images were acquired with 10 s interval. A maximal intensity z-projection was …
Video 3
Calcium imaging of ISCs expressing RyR RNAi in response to paraquat.
Midguts expressing GCaMP6s reporter together with RyR RNAi in ISCs were dissected and imaged in AHL buffer. Z-stack images were acquired with 10 s interval. A maximal intensity z-projection was …
Video 4
Calcium imaging of ISCs in response to AITC.
Midguts expressing GCaMP6s reporter in ISCs are dissected and imaged in AHL buffer. Z-stack images were acquired with 10 s interval. A maximal intensity z-projection was shown in the movie. A final …
Video 5
Calcium imaging of ISCs expressing trpA1 RNAi in response to AITC.
Midguts expressing GCaMP6s reporter together with trpA1 RNAi in ISCs were dissected and imaged in AHL buffer. Z-stack images were acquired with 10 s interval. A maximal intensity z-projection was …
Video 6
Calcium imaging of ISCs expressing RyR RNAi in response to AITC.
Midguts expressing GCaMP6s reporter together with RyR RNAi in ISCs were dissected and imaged in AHL buffer. Z-stack images were acquired with 10 s interval. A maximal intensity z-projection was …
Additional files
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Supplementary file 1
List of Drosophila genotypes used in each figure.
- https://doi.org/10.7554/eLife.22441.037
