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Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis

Nature Cell Biology volume 19, pages 52–59 (2017)Cite this article

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Abstract

Ageing results in loss of tissue homeostasis across taxa1. In the intestine of Drosophila melanogaster, ageing is correlated with an increase in intestinal stem cell (ISC) proliferation, a block in terminal differentiation of progenitor cells, activation of inflammatory pathways, and increased intestinal permeability2. However, causal relationships between these phenotypes remain unclear. Here, we demonstrate that ageing results in altered localization and expression of septate junction proteins in the posterior midgut, which is quite pronounced in differentiated enterocytes (ECs) at tricellular junctions (TCJs). Acute loss of the TCJ protein Gliotactin (Gli) in ECs results in increased ISC proliferation and a block in differentiation in intestines from young flies, demonstrating that compromised TCJ function is sufficient to alter ISC behaviour in a non-autonomous manner. Blocking the Jun N-terminal kinase signalling pathway is sufficient to suppress changes in ISC behaviour, but has no effect on loss of intestinal barrier function, as a consequence of Gli depletion. Our work demonstrates a pivotal link between TCJs, stem cell behaviour, and intestinal homeostasis and provides insights into causes of age-onset and gastrointestinal diseases.

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Figure 1: Age-related changes in SJs in posterior midguts from aged flies.
Figure 2: Gliotactin is located at the TCJ in differentiated cells in the intestine.
Figure 3: Loss of Gliotactin in ECs leads to loss of intestinal barrier integrity.
Figure 4: Loss of Gliotactin in ECs induces ISC proliferation and accumulation of esg+ cells.
Figure 5: JNK signalling is activated downstream of Gli to drive changes in ISC behaviour in a non-autonomous manner.

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Acknowledgements

The authors thank H. Jasper (The Buck Institute for Research on Aging, USA), V. Auld (University of British Columbia, Canada), J. F. de Celis (CBMSO, Spain), M. Furuse (Kobe University, Japan), the Vienna Drosophila RNAi Center (VDRC), and Bloomington Stock Center for reagents, M. Cilluffo from the Brain Research Institute at UCLA EM core facility, and the Jones and Walker laboratories for comments on the manuscript. In addition, we would like to thank Y. Wu and E. Stefani for their sharing their time, expertise, and STED technology. This work was supported by the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at the University of California, Los Angeles and the Rose Hills Foundation (D.L.J.), and the NIH: AG028092 (D.L.J.), AG040288 (D.L.J. and D.W.W.), AG049157 (D.W.W.), and a training grant that supports the UCLA-Caltech Medical Scientist Training Program, GM08042 (S.L.).

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  1. Rebecca I. Clark: Present address: School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, UK.

Authors and Affiliations

  1. Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California 90095, USA

    Martin Resnik-Docampo, Christopher L. Koehler, Vivien Sauer, Daniel M. Wong, Sophia Lewis, Cecilia D’Alterio & D. Leanne Jones

  2. Department of Integrative Biology and Physiology, University of California, Los Angeles, California 90095, USA

    Rebecca I. Clark, Joseph M. Schinaman & David W. Walker

  3. Molecular Biology Institute, University of California, Los Angeles, California 90095, USA

    David W. Walker & D. Leanne Jones

  4. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, California 90095, USA

    D. Leanne Jones

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Contributions

M.R.-D. designed, performed and analysed experiments and wrote the manuscript. C.L.K., R.I.C., J.M.S., D.M.W., V.S., S.L. and C.D’A. designed, performed and analysed experiments. D.W.W. designed and analysed experiments. D.L.J. designed and analysed experiments and wrote the manuscript.

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Correspondence to D. Leanne Jones.

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Integrated supplementary information

Supplementary Figure 1 Localization of septate junction (SJ) proteins in the intestinal tract in young and aged flies.

(A) Schematic representation of a Drosophila melanogaster midgut (B–I) Localization of SJ proteins in posterior midgut and hindgut of young flies. (B) Nervana:GFP, (C) Neurexin IV:GFP, (D) Neuroglian:GFP, (E) Snakeskin, (F) Scribble:GFP, (G) Discs large, (H) Coracle, (I) Mesh. Images are representative of at least 5 midguts. Scale bars, 0.1 mm. (J–S) No changes in SJ protein levels were observed in hindguts from aged flies. STED (J–O) and confocal (P,Q) images comparing SJ proteins localization of 10do hindguts to 45do, scale bars, 1 μm and 5 μm. (R,S) STED images showed the AJ protein Arm localization is not affected by aging, scale bars, 1 μm. More than 14 hindguts per condition (10 ECs were observed per hindgut). Samples were dissected and stained in parallel under same conditions, pictures taken at same laser intensity.

Supplementary Figure 2 Verification of Gli reagents.

(A–C) GLI:GFP and anti-GLI co-localization (wing discs). GliRNAi efficiency demonstrated by immunofluorescence (IF) microscopy (wing discs) and qPCR (posterior midguts). (A–A′′) Wing disc peripodial membrane showing the co-localization of Gli-GFP (green) with Gli (red), DAPI (blue); Scale bars, 10 μm. (B,C) Gli protein is localized at the TCJ between wing cells (red signal in B;) in wild type discs. Gli is strongly reduced in discs expressing GliRNAi (SalPE-GAL4 UAS-GFP UAS-GliRNAi, C;). Scale bars, 50 μm. (D) RT-qPCR of posterior midguts 5966GSGAL4/UAS-GliRNAi flies RU+ and RU− showing a decrease of Gli expression after 5 and 9 days post GliRNAi expression. n = 75 females posterior midguts per condition. Bars are the mean +/− S.E.M (two tailed, unpaired Student’s t-test). ∗∗∗ = P < 0.001, ∗∗ = P < 0.01 represent a statistically significant difference. (E) Flies fed with EtOH (light blue) (5966GSGAL4/+, RU−, n = 270) show no statistical difference in number of flies that have lost barrier function, when compared to those fed RU (blue) (5966GSGAL4/ + fed, RU+, n = 268). Fisher’s exact test; two tailed. (F,G) EM images of SJ (arrowheads) between adjacent ECs in guts from 23do 5966GSGAL4/UAS-GliRNAi flies (12 RU+) (G) compared to control flies (F) 5966GSGAL4/UAS-GliRNAi (15 RU−). For each midgut 9 EC/EC Septate Junctions were observed per experimental condition. Scale bars, 0.1 μm. (H) Lifespan curves of 5966GSGAL4/UAS-GliRNAi [RU+, red (n = 265) and RU−, grey (n = 240)] female flies. T50 was significantly lower in RU+ d30 compared with RU− d37. Data analyzed with non parametric Log-Rank (Mantel-Cox) test; ∗∗∗ = P < 0.001, represent a statistically significant difference.

Supplementary Figure 3 Effects of Gli depletion in the posterior midgut.

(A–F) 5966GSGAL4; esg-GFP flies (RU+ or RU−) crossed with RNAi lines targeting Gli or Dlg. (A–D) Multiple GliRNAi lines induce an increase of ISC/EB cells, when expressed (RU+) (B;D) compared to RU− controls (A;C), similar to results presented in Fig. 4D. (E,F) Depletion of Dlg (dlgRNAi, RU+) (E) induces an increase in ISC/EBs compared to controls (RU−) (F). (G–K) Changes in ISC/EB number after Gli depletion are reversible (5966GSGAL4; esg-GFP UAS-GliRNAiRU+, RU−). (G) Graphical summary showing the statistical significance in changes in ISC/EB number depicted in H–K. 9do RU− UAS-GliRNAi n = 18, 9do RU+ UAS-GliRNAi n = 15, recovery experiment n = 19, 20do RU− UAS-GliRNAi n = 21, 20do RU+ UAS-GliRNAi n = 20. Each data point is an average proportion calculated from 4 independent images per midgut and bars are the mean +/− S.E.M of those averages (ONE-way ANOVA/Tukey’s multiple comparisons test) ∗∗ = P < 0.01, ∗∗∗ = P < 0.001, ∗∗∗∗ = P < 0.0001 represents a statistically significant difference. (H) Intestine from 5966GSGAL4; esg-GFP UAS-GliRNAi (fed RU+) for 9 days. (I) Flies were shifted onto food containing EtOH (RU−) to re-initiate Gli expression for 11 days. Re-expression of Gli resulted in resumption of normal ISC behavior and morphology compared to guts from 20do RU− 5966GSGAL4; esg-GFP UAS-GliRNAi controls (J) or guts from 20do 5966GSGAL4; esg-GFP GliRNAi flies fed RU+ (K). Scale bars, 10 μm. (L) Graphical summary showing that depletion of Gli from EEs using Rab3-GAL4, tubGAL80TS for 6d at 29 °C (red) did not result in any changes in ISC behavior, compared to control flies (y, w1118; grey). Each data point is an average proportion calculated from 4 independent images per midgut and bars are the median with interquartile range of those averages (Kruskal-Wallis/Dunn multiple comparisons test). Ctrl n = 18; UAS-GliRNAi n = 20. (M,N) No significant differences were detected in AMP expression, after 2, 5, 9 and 21 days post GliRNAi expression. ∗∗ = P < 0.01, represent statistically significant difference. (M) Drosomycin, (N) Diptericin. n = 6 replicates of 5. Boxplots display the first and third quartile, with horizontal bar at the median and whiskers showing the most extreme data point, which is no more than 1.5 times the interquartile range from the box.

Supplementary Figure 4 Neither changes in bacteria nor death of ECs induces changes in ISC behavior downstream of Gli.

(A–F) Epistatic analysis between Gli and apoptosis inhibitors. (A) ISC/EB counts in flies of indicated genotypes. RU− GliRNAi n = 19, RU+ GliRNAi n = 25, GliRNAi dIAP n = 25, dIAP n = 25, GliRNAi P35 n = 17, P35 n = 15. Each data point is an average proportion calculated from 4 independent images per midgut and bars are the mean +/− S.E.M (ONE-way ANOVA/Tukey’s multiple comparisons test). (B) Mitosis counts in midguts from flies of indicated genotypes. RU− GliRNAi n = 19, RU+ GliRNAi n = 25, GliRNAi dIAP n = 25, dIAP n = 25, GliRNAi P35 n = 17, P35 n = 15. Each data point is an average proportion calculated from 4 independent images per midgut and bars are the median with interquartile range (Kruskal-Wallis/Dunn multiple comparisons test) ∗∗∗∗ = P < 0.0001, represent statistically significant difference. (C–F) IF images of midguts from flies of indicated genotypes stained with DAPI (nuclei, blue), GFP (esg + cells, green) and PH3 (mitotic cells, red, arrowheads) following 9 days of incubation in RU+ or RU−. Co-expression of GliRNAi with dIAP (5966GSGAL4/UAS-GliRNAi; UAS-dIAP, RU+) or P35 (5966GSGAL4/UAS-GliRNAi; UAS-P35, RU+ (A,B;E,F) does not rescue the effect on ISCs produced by depletion of Gli (5966GSGAL4/UAS-GliRNAiUAS-LacZ, RU+) (A,B,D). Compare to control flies fed EtOH (RU−) (5966GSGAL4/UAS-GliRNAiUAS-LacZ, RU−) (A–C). (C–F) Images are representative of at least 15 midguts, 4 images taken per midgut. (G–H′) Under axenic conditions, reduction of Gli expression in ECs still initiates JNK pathway activation, as reported by puc-lacZ expression (G,H: red or G’,H’: grey) in ECs 2 days after reducing Gli expression (H–H’), compared to RU− controls (G–G’). ISC/EBs marked by esg-GFP (green), cell nuclei marked by DAPI (blue). (I) MRS plates showing absence of bacterial colonies from cultured fly homogenates of axenic flies at 7do. (J) RT-qPCR of posterior midguts from 5966GSGAL4/UAS-GliRNAi flies (RU+ and RU−) showing an increase of puc expression after 2, 5, 9 days post GliRNAi expression. n = 75 females posterior midguts per condition. Bars are the mean +/− S.E.M (two tailed, unpaired Student’s t-test). ∗∗∗ = P < 0.001, ∗∗ = P < 0.01, ∗ = P < 0.05 represent statistically significant difference. (K) Lifespan curves of 5966GSGAL4RU+ female flies crossed with yw118 (grey, n = 290), UAS-BskDN (green, n = 277), UAS-GliRNAi flies (red, n = 259) and GliRNAi UAS-BskDN (blue, n = 264). ∗∗∗ = P < 0.001, non parametric Log-rank (Mantel-Cox) test. Scale bars, 10 μm.

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Resnik-Docampo, M., Koehler, C., Clark, R. et al. Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis. Nat Cell Biol 19, 52–59 (2017). https://doi.org/10.1038/ncb3454

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