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Haemocytes control stem cell activity in the Drosophila intestine

Nature Cell Biology volume 17, pages 736–748 (2015)Cite this article

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Abstract

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. Here we show that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. We propose that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans.

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Figure 1: Gut-associated haemocytes are required for ISC proliferation.
Figure 2: Haemocytes dynamically recruit to the gut during stress.
Figure 3: Haemocyte-derived DPP acts on ISCs to promote proliferation.
Figure 4: Haemocyte-derived DPP activates BMP signalling specifically within ISCs during proliferation.
Figure 5: ISC proliferation induced by haemocyte-derived DPP through SAX/SMOX signalling is antagonized by TKV/MAD signalling.
Figure 6: Relative expression of SAX and TKV receptors determines SMOX or MAD activation and proliferative response of ISCs.
Figure 7: Haemocyte-derived DPP promotes resistance to acute infection but leads to intestinal dysplasia and increased gut permeability during ageing.
Figure 8: Model.

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Acknowledgements

This work was financially supported by the National Institute on General Medical Sciences (R01 GM100196) and the National Eye Institute (R01 EY018177). We would like to thank J. Karpac for comments.

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Authors and Affiliations

  1. Buck Institute for Research on Aging, 8001 Redwood Boulevard Novato, California 94945-1400, USA,

    Arshad Ayyaz, Hongjie Li & Heinrich Jasper

  2. Department of Biology, University of Rochester, River Campus Box 270211 Rochester, New York 14627, USA,

    Hongjie Li

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Contributions

A.A. and H.J. designed all experiments. A.A. generated transgenic animals and performed experiments on haemocytes, DPP, SAX and SMOX signalling, interactions of SAX/SMOX signalling with TKV/MAD, EGFR and JAK/STAT pathways and role of TKV expression in mitosis and on ageing, dysplasia and lifespan; H.L. validated specificity of TKV and SAX antibodies, performed experiments on TKV expression and on lineage tracing of MAD, MED and TKV mutant ISCs, and provided additional reagents for other experiments. A.A. and H.J. analysed the data and wrote the manuscript.

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Correspondence to Heinrich Jasper.

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Integrated supplementary information

Supplementary Figure 3 Gut-associated hemocytes are required for ISC proliferation.

(A,B) Hemocyte-ablated larvae (A) and adults (B) are shown, generated by expressing the pro-apoptotic gene hid specifically in hemocytes using hmlΔ::Gal4;UAS::GFP. GFP-positive hemocyte clumps are indicated by red arrowheads. (C) hml promoter expression in the whole adult gut is shown using hmlΔ::Gal4 (i). Variable number of extra-epithelial GFP-positive hemocytes can be detected in the proventriculous (ii; note that hemocytes (yellow arrowheads) are not part of the intestinal epithelium (white arrowheads), sometimes attached to the epithelial surface in midgut (iii), or hindgut regions (iv) but are not part of the epithelium that contains Delta-positive and esg::Gal4 expressing ISCs (v, vi). (D,E) hmlΔ::Gal4;UAS::GFP+ hemocytes attaching the abdominal body wall (D) also express the phagocyte marker NimC1 (E). Ovaries and fat body are hmlΔ::Gal4- and NimC1- (E). (F) Hemocyte-deficient flies rapidly succumb while both HDD and wild-type flies survive systemically disseminated P. entomophila. (G) Intestine in hemoless flies is normal in size and structure, and contain a normal distribution of cells (βCatenin/Armadillo labels cell boundaries and is highly expressed in ISC/EB nests; Prospero in enteroendocrine cells, and Delta in ISCs). (H) Intestinal cell composition. Absence of hemocytes does not change the composition of cells in the midgut of young animals. However, an increased number of Delta-positive ISCs observed in old wild-type animals is reduced in hemoless flies. (I) Representative images showing that intact Delta+ ISCs can be detected in the posterior midguts of hemocless flies at 12 h post-Ecc15 infection. Error bars indicate s.e.m. (H: n = 10 and I: n = 8 flies tested in a single experiment, P values taken from Student Ttest; and experiment was replicated 3 (in panel H) and 2 (in panel I) times. Survival curves shown in panel F were compared using Prism software (n = 20 flies tested in one experiment, p values were taken from LogRank test, and experiment was repeated 3 times. Single representative images from 10 flies used in a single experiment are shown in panels A–E and G while experiment was reproduced 3 (in panels A,B,D–I and G) or 2 (in panel C) times. n.s. = non-significant,∗∗p < 0.01,∗∗∗p < 0.001.

Supplementary Figure 4 Hemocytes recruited to stressed guts represent a post mitotic hematopoietic population.

(A) PQ-induced ISC proliferation when hemocytes were ablated at 2 days of age by expressing hid with hmlΔ::Gal4, UAS::GFP, tub::Gal80ts for 4 days. Hemocyte ablation was monitored visually in intact adults (not shown). Flies were exposed to Paraquat (PQ in 5% sucrose) or Mock (5% sucrose) for 16 h at 25 °C. (B) Hemocytes attached to the external surface of midgut regions a and b (identified by how::Gal4, UAS::GFP; see Fig. 2b), following 2 days of P. entomophila infection. Quantification of eater::DsRed+ cells per gut-associated hemocyte clump 2 days after P. entomophila or 4 h after Ecc15 infection; M: mock. (C) 5966::GS, UAS::GFP flies co-expressing eater::DsRed fed RU846 food for 2 days followed by oral infection with Ecc15 for 4 h. (D) Circulating hemocytes change their size and shape at 4 h post-Ecc15 infection. F-actin detected by Phalloidin (red). (E,F) MARCM competent flies co-expressing eater::DsRed (see Supplementary Table 1 for exact genotype) subjected to Ecc15 challenge and simultaneously heat shocked. No GFP+ hemocytes were observed at 12 h after Ecc15 infection (Analysis I) or at 5 days post heat shock (with or without a second Ecc15 challenge: Analysis II) in any tissue (E; and not shown). GFP+ ISC-derived clones can be observed within the intestinal epithelium (F). (G) Expression pattern of dve::Gal4, UAS::GFP in midgut. PV: proventriculus, AM: anterior midgut, PM (b–d): posterior midgut regions b–d, HG: hindgut; Cut marks Copper Cells in middle midgut (compare18), here designated as region ‘a’ of the midgut. Error bars indicate either s.e.m. (A: n = 12 flies) or range (B: for mock/P.e.: n = 22 flies, for mock/Ecc15: n = 32 flies; where boxes show 25–75% percentile and horizontal bar within each box is population median) calculated from single experiment. P values from Student’s Ttest, reproduced in 3 independent experiments. Single representative image from 15 flies is shown in B–D,F and G, experiment was repeated 3 (in panels B,C and G) and 2 (in panel D–F) times. ∗∗∗P < 0.001.

Supplementary Figure 5 Hemocyte-derive Dpp is required for ISC proliferation.

(A) Hemocyte-specific RNAi screen assessing ISC activity upon PQ treatment. (B) Knockdown of Dpp using hmlΔ::GS. RU486 fed for 2 days followed by 8 h Ecc15 challenge. (C) ISC proliferation at 8 h post-Ecc15 in animals with knockdown of Dpp in hemocytes (hmlΔ::G4 and He::Gal4), hindgut (byn::Gal4) or proventriculus (Cardia::Gal4). (D) ISC proliferation upon Paraquat (PQ) feeding after Dpp knock down in ECs (NP1::Gal4), ISCs and EBs (esg::Gal4), trachea (Btl::Gal4) or visceral muscle (how::Gal4). (E) qRT-PCR to assess effectiveness of DppRNAi lines in 10–15 day old adult carcass (containing hemocytes) using hmlΔ::Gal4, or in whole guts when using how::G4ts (29 °C 48 h). (F) RU486-sensitive expression of UAS::mCD8-GFP in hemocytes and lymph gland of hmlΔ::GS larvae (no expression in adult gut epithelium). (G) GFP-positive, Dpp-deficient hemocytes in larvae and adults. Circulating hemocytes in adult hemolymph collected by Nanoinject II and counted using hemocytometer. (H) Dpp-deficient hemocytes attach to the intestinal epithelium during Ecc15 challenge. (I) Percentage of GFP-positive hemocytes 16 h post-PQ treatment in Dpp::Gal4, UAS::GFP flies. (J) Dpp transcript in hemocytes upon infection with Ecc15 (30 min after challenge). (K) Dpp-GFP from hemocytes (2 day feeding RU486) accumulates on ISCs (4 h Ecc15). (L) Clones generated by FRT-mediated reconstitution of split actin::LacZ in wild-type or hemoless (hml::Gal4, UAS::Rpr) backgrounds. (M) Assessment of apoptosis by Apoliner (UAS::mcd8RFP::nlsGFP) expressed in ISC progeny (5966::GS). No apoptotic cells (nuclear GFP) are observed in hml::DppRNAi flies. Apoptotic ECs can be observed during Ecc15 infection. (N) Number of hemocytes retrieved from hemolymph at indicated ages. (O) PQ-induced recruitment of transplanted hemocytes to the gut of hemoless flies (donor: hmlΔ::Gal4, UAS::GFP). Error bars indicate s.e.m. (A: n’s are indicated, composite of two (first graph) or three (second graph) experiments; B,E: n = 10; G: n = 5 (each sample represents a cohort of 20 flies); C,D: n = 10; I,J: n = 10; N: n = 7) or range (O: n = 9; boxes: 25–75% percentile. horizontal bar median value), Student T-test. All experiments, except A, replicated three times independently. Representative image (n = 9) in F–I and K–M; experiment repeated 3 times. n.s. = non-significant,∗∗∗P < 0.001.

Supplementary Figure 6 Hemocyte-derived Dpp requires Jak/Stat and EGFR pathways to induce proliferation.

(A–C) Transcription of Jak/Stat pathway activating ligand upd3 (A), expression of Jak/Stat activity reporter STAT::GFP (B), and EGFR ligand reporter Vein::LacZ (C: arrowheads) are normally induced in the intestine of HDD (A,C) and hemoless (B) flies during Ecc15 infection. (D) ISC proliferation induced upon upd2 overexpression in ECs is significantly reduced when Dpp expression is simultaneously blocked in hemocytes using an hmlΔ-DppRNAi lines that does not use Gal4/UAS system (see Methods). (E,F) Expression of UAS::Dpp was induced in adult hemocytes under the control of hmlΔ::Gal4 driver (which co-expressed tub-Gal80ts) by shifting 3 days old adult flies from 18 °C to 29 °C for indicated time intervals. Short-term expression of hemocyte-derived Dpp (that is, for 8 or 48 h) did not induce mitotic response in ISCs (E), while higher ISC proliferation was observed upon a long-term expression of Dpp in hemocytes for 13 days period. Over-expression of Dpp in visceral muscle (How::Gal4) for 8 or 24 h does also not induce ISC proliferation. Error bars indicate s.e.m. (n > 7), P values from Student T test. (G,H) HDD flies exhibit normal feeding in the absence of stress (assessed by the CAFÉ assay; G) or when fed on Ecc15 infection solution mixed with a blue dye for 90 min, following 2 h of starvation period (H; note that digestive tracts are full of blue dye). Error bars indicate s.e.m. (A: n = 4; D–F: n = 10; and G: n = 9 flies were used in one experiment), P values from Student T test, while results were reproduced in 2 independent experiments. Panels B (n > 9), C (n > 8) and H (n > 15) show single representative images from 9 (panel B), 8 (panel C) and 15 (H) flies, and each experiment was replicated twice. n.s. = non-significant,∗P < 0.05,∗∗P < 0.01,∗∗∗P < 0.001.

Supplementary Figure 7 Hemocyte-derived Dpp induces ISC proliferation in all gut regions, independent of Mad.

(A) Reduced ISC proliferative response in HDD flies upon Ecc15 infection is observed in the entire length of the Drosophila adult midgut: anterior midgut (AM), Copper Cell Region (region ‘a’) and posterior midgut (regions ‘b–d’). (B) Induction of local Dpp expression in the visceral muscle at region ‘c’ is observed only after 16 h post-Ecc15 infection. (C) Mad is not phosphorylated in ISCs of region ‘c’ of the PM 4 h post Ecc15 infection, but phosphorylation can be detected at 16 h after challenge (AC): arrowheads. Delta as well as YFP-positive cells are ISCs. (D) Ubiquitous knockdown of Dpp, but not of dActivin or dawdle, using Mifepristone (RU486) drug sensitive ubiquitously expressed driver tub-GS decreases ISC proliferative response 4 h post-Ecc15 challenge. (E) Knock down of Smox using ISC-specific 5961::GS significantly reduces the expression of dad::nGFP specifically in ISCs, but not in other intestinal cells, in region c of the posterior midgut following 4 h of Ecc15 challenge (compare to Fig. 4e). Error bars indicate s.e.m. (A: n = 10 and D: n = 7 flies from one experiment), P values taken from Student T test, while each experiment was repeated 3 times. One representative image is shown from 13 (in panel B), 7 (in panel C) and 10 (panel F) flies used in a single experiment, while each experiment was repeated twice (B) or three times (C,F). n.s. = non-significant.

Supplementary Figure 8 Hemocyte-derived Dpp signals through Sax/Smox to induce ISC proliferation.

(A) ISC proliferation at 4 h of Ecc15 challenge. Gbb is knocked in adults in ECs (NP1::Gal4ts) or hemocytes (hmlΔ::Gal4ts). (B) Mad phosphorylation is absent in Tkv-, but not Sax-, mutant ISCs in MARCM clones in region ‘c’ of PM 24 h post-Ecc15 challenge (arrowheads). (C) Over-expression of a constitutively active TkvQD in ISCs at region ‘c’ is sufficient to phosphorylate Mad in the absence of injury. (D) Representative images showing MARCM clones generated under normal conditions by mad12, mad1−2, tkv04415, Tkva12, med13, Sax4 and SmoxMB388 mutant ISCs, and following Ecc15 oral challenge by tkv04415 and mad12 mutant ISCs alone combined with ISC-specific Sax or Smox knockdown. (E) Smox knock down in ISCs suppresses the growth of ISC tumours in Notch loss-of-function conditions, which are formed by the accumulation of symmetrically dividing Delta-positive ISCs, a phenotype observed when NotchRNAi is expressed under the control of ISC/EB specific driver esg::Gal4. (F) ISC over-proliferation induced by expressing EGFR-gain-of-function (EGFR-GOF) or constitutively active Hop (HopTumL) is rescued by simultaneous Smox knockdown. (G) Number of gut-associated eater::DsRed+ hemocytes does not increase upon EGFR-GOF over-expression. Error bars indicate either s.e.m. (A: n = 14 and F: n = 10 flies) or range (G: n = 7 flies; where boxes show 25–75% percentile and horizontal bar within each box is population median) from one experiment, P values from Student T test; while each experiment was repeated twice. One representative image from 7 (panels B,C and G) and 10 (panel E) flies used in a single experiment is shown, while experiment was repeated 3 (panels B,C,E) or 2 (panel G) times. Panel D shows one representative images for each data set quantified in Fig. 5d, e (see legends of Fig. 5d, e for n, P values and repetitions).

Supplementary Figure 9 Sax does not induce Tkv expression.

(A) Sax detection in ISCs is lost upon RNAi knockdown using ISC/EB specific driver esg-Gal4, while an excessive staining is observed upon Sax overexpression within these cells (arrowheads), thus validating the specificity of the antibody used in this study. (B) Low expression of Tkv is detected in ISCs during basal conditions. TkvQD, when over-expressed in ISCs and EBs under the control of esg-Gal4, can be readily detected in these cells (arrows), confirming the specificity of the antibody used. (C) Sax knockdown in ISCs does not prevent late Tkv induction in ISCs following Ecc15 challenge (arrowheads). One representative image from 7 flies used in single experiment is shown in all panels, while each experiment was repeated twice.

Supplementary Figure 10 Hemocyte-derived Dpp promotes intestinal dysplasia by inducing Smox nuclear localization.

(A) Large numbers of hmlΔ::Gal4, UAS::GFP+ hemocytes (both as single cells or clumps) can be found attached to the intestine of young flies 8 h after Ecc15 challenge as well as in 30 day old untreated flies. (B–D) Images show hemocyte-derived Dpp dependent activation of BMP signalling pathway activation (note dad::nGFP reporter activity) in region c of posterior midgut (B), Smox nuclear localization (C) and development of intestinal dysplasia (D; note accumulation of Delta-positive cells). (E) Aged related overactivation of ISC proliferation, identified by the frequency of phospho histone H3+ (pH3+) cells per gut, is significantly reduced when 24 days old 5961::GS flies co-expressing UAS::Tkv transgene were shifted to RU846 drug containing food before observation at 30 days of age, thus, indicating that a relatively acute overexpression (for 6 days) of wild-type Tkv construct in ISCs can inhibit over activation of ISCs in aging animals. (F) Results from lifespan experiments are showing that where hemoless are significantly short-live, HDD flies live as long as their wild-type counter parts. Each survival curve represents a composite of total number of animals of a particular genotype tested (also see methods: lifespan experiments). Error bars indicate s.e.m. (E: n = 14 flies used in one experiment), p values taken from Student T test, while results were reproduced twice. One representative image from 15 (panel A) or 7 (panels B–D) flies used in one experiment is shown, and each experiment was repeated two times.

Supplementary Table 1 List of genotypes.
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Supplementary Table 2 Statistics for lifespan experiments.
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Ayyaz, A., Li, H. & Jasper, H. Haemocytes control stem cell activity in the Drosophila intestine. Nat Cell Biol 17, 736–748 (2015). https://doi.org/10.1038/ncb3174

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