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Nanotubes mediate niche–stem-cell signalling in the Drosophila testis

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Abstract

Stem cell niches provide resident stem cells with signals that specify their identity. Niche signals act over a short range such that only stem cells but not their differentiating progeny receive the self-renewing signals1. However, the cellular mechanisms that limit niche signalling to stem cells remain poorly understood. Here we show that the Drosophila male germline stem cells form previously unrecognized structures, microtubule-based nanotubes, which extend into the hub, a major niche component. Microtubule-based nanotubes are observed specifically within germline stem cell populations, and require intraflagellar transport proteins for their formation. The bone morphogenetic protein (BMP) receptor Tkv localizes to microtubule-based nanotubes. Perturbation of microtubule-based nanotubes compromises activation of Dpp signalling within germline stem cells, leading to germline stem cell loss. Moreover, Dpp ligand and Tkv receptor interaction is necessary and sufficient for microtubule-based nanotube formation. We propose that microtubule-based nanotubes provide a novel mechanism for selective receptor–ligand interaction, contributing to the short-range nature of niche–stem-cell signalling.

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Figure 1: Characterization of MT-nanotubes in Drosophila male GSC niche.
Figure 2: IFT genes are required for MT-nanotube formation.
Figure 3: Dpp signalling components localize to the MT-nanotubes.
Figure 4: MT-nanotubes are required for Dpp signalling activation and GSC maintenance.
Figure 5: Dpp signalling is necessary and sufficient for MT-nanotube formation.

References

  1. Morrison, S. J. & Spradling, A. C. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell 132, 598–611 (2008)

    CAS  Article  Google Scholar 

  2. Tulina, N. & Matunis, E. Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science 294, 2546–2549 (2001)

    CAS  ADS  Article  Google Scholar 

  3. Kiger, A. A., Jones, D. L., Schulz, C., Rogers, M. B. & Fuller, M. T. Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue. Science 294, 2542–2545 (2001)

    CAS  ADS  Article  Google Scholar 

  4. Shivdasani, A. A. & Ingham, P. W. Regulation of stem cell maintenance and transit amplifying cell proliferation by TGF-β signaling in Drosophila spermatogenesis. Curr. Biol. 13, 2065–2072 (2003)

    CAS  Article  Google Scholar 

  5. Kawase, E., Wong, M. D., Ding, B. C. & Xie, T. Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis. Development 131, 1365–1375 (2004)

    CAS  Article  Google Scholar 

  6. Yamashita, Y. M., Jones, D. L. & Fuller, M. T. Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 301, 1547–1550 (2003)

    CAS  ADS  Article  Google Scholar 

  7. Davis, D. M. & Sowinski, S. Membrane nanotubes: dynamic long-distance connections between animal cells. Nature Rev. Mol. Cell Biol. 9, 431–436 (2008)

    CAS  Article  Google Scholar 

  8. Ramírez-Weber, F. A. & Kornberg, T. B. Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs. Cell 97, 599–607 (1999)

    Article  Google Scholar 

  9. Avasthi, P. & Marshall, W. F. Stages of ciliogenesis and regulation of ciliary length. Differentiation 83, S30–S42 (2012)

    CAS  Article  Google Scholar 

  10. Pedersen, L. B. & Rosenbaum, J. L. Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling. Curr. Top. Dev. Biol. 85, 23–61 (2008)

    CAS  Article  Google Scholar 

  11. Goetz, S. C. & Anderson, K. V. The primary cilium: a signalling centre during vertebrate development. Nature Rev. Genet. 11, 331–344 (2010)

    CAS  Article  Google Scholar 

  12. Perrone, C. A. et al. A novel dynein light intermediate chain colocalizes with the retrograde motor for intraflagellar transport at sites of axoneme assembly in Chlamydomonas and mammalian cells. Mol. Biol. Cell 14, 2041–2056 (2003)

    CAS  Article  Google Scholar 

  13. Kobayashi, T., Tsang, W. Y., Li, J., Lane, W. & Dynlacht, B. D. Centriolar kinesin Kif24 interacts with CP110 to remodel microtubules and regulate ciliogenesis. Cell 145, 914–925 (2011)

    CAS  Article  Google Scholar 

  14. Iomini, C., Li, L., Esparza, J. M. & Dutcher, S. K. Retrograde intraflagellar transport mutants identify complex A proteins with multiple genetic interactions in Chlamydomonas reinhardtii. Genetics 183, 885–896 (2009)

    CAS  Article  Google Scholar 

  15. Tran, P. V. et al. THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia. Nature Genet. 40, 403–410 (2008)

    CAS  Article  Google Scholar 

  16. Qin, J., Lin, Y., Norman, R. X., Ko, H. W. & Eggenschwiler, J. T. Intraflagellar transport protein 122 antagonizes Sonic Hedgehog signaling and controls ciliary localization of pathway components. Proc. Natl Acad. Sci. USA 108, 1456–1461 (2011)

    CAS  ADS  Article  Google Scholar 

  17. Cortellino, S. et al. Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4. Dev. Biol. 325, 225–237 (2009)

    CAS  Article  Google Scholar 

  18. Roy, S., Huang, H., Liu, S. & Kornberg, T. B. Cytoneme-mediated contact-dependent transport of the Drosophila decapentaplegic signaling protein. Science 343, 1244624 (2014)

    Article  Google Scholar 

  19. Michel, M., Raabe, I., Kupinski, A. P., Perez-Palencia, R. & Bokel, C. Local BMP receptor activation at adherens junctions in the Drosophila germline stem cell niche. Nature Commun. 2, 415 (2011)

    ADS  Article  Google Scholar 

  20. Goshima, G. & Vale, R. D. Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells. Mol. Biol. Cell 16, 3896–3907 (2005)

    CAS  Article  Google Scholar 

  21. Schulz, C. et al. A misexpression screen reveals effects of bag-of-marbles and TGF beta class signaling on the Drosophila male germ-line stem cell lineage. Genetics 167, 707–723 (2004)

    CAS  Article  Google Scholar 

  22. Dudu, V. et al. Postsynaptic Mad signaling at the Drosophila neuromuscular junction. Curr. Biol. 16, 625–635 (2006)

    CAS  Article  Google Scholar 

  23. Yagi, R., Mayer, F. & Basler, K. Refined LexA transactivators and their use in combination with the Drosophila Gal4 system. Proc. Natl Acad. Sci. USA 107, 16166–16171 (2010)

    CAS  ADS  Article  Google Scholar 

  24. Wharton, K., Ray, R. P., Findley, S. D., Duncan, H. E. & Gelbart, W. M. Molecular lesions associated with alleles of decapentaplegic identify residues necessary for TGF-β/BMP cell signaling in Drosophila melanogaster. Genetics 142, 493–505 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Xie, T. & Spradling, A. C. Decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary. Cell 94, 251–260 (1998)

    CAS  Article  Google Scholar 

  26. Avidor-Reiss, T. et al. Decoding cilia function. Cell 117, 527–539 (2004)

    CAS  Article  Google Scholar 

  27. Radford, S. J., Harrison, A. M. & McKim, K. S. Microtubule-depolymerizing kinesin KLP10A restricts the length of the acentrosomal meiotic spindle in Drosophila females. Genetics 192, 431–440 (2012)

    CAS  Article  Google Scholar 

  28. Ghiglione, C. The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis. Development 129, 5437–5447 (2002)

    CAS  Article  Google Scholar 

  29. Salzmann, V., Inaba, M., Cheng, J. & Yamashita, Y. M. Lineage tracing quantification reveals symmetric stem cell division in Drosophila male germline stem cells. Cell. Mol. Bioeng. 6, 441–448 (2013)

    Article  Google Scholar 

  30. Bischof, J., Maeda, R. K., Hediger, M., Karch, F. & Basler, K. An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc. Natl Acad. Sci. USA 104, 3312–3317 (2007)

    CAS  ADS  Article  Google Scholar 

  31. Van Doren, M., Williamson, A. L. & Lehmann, R. Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr. Biol. 8, 243–246 (1998)

    CAS  Article  Google Scholar 

  32. Image J. (U.S. National Institutes of Health, 1997–2014)

  33. Rogers, G. C. et al. Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase. Nature 427, 364–370 (2004)

    CAS  ADS  Article  Google Scholar 

  34. Xu, T. & Rubin, G. M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237 (1993)

    CAS  Google Scholar 

  35. Grieder, N. C., de Cuevas, M. & Spradling, A. C. The fusome organizes the microtubule network during oocyte differentiation in Drosophila. Development 127, 4253–4264 (2000)

    CAS  PubMed  Google Scholar 

  36. Kuzhandaivel, A., Schultz, S. W., Alkhori, L. & Alenius, M. Cilia-mediated hedgehog signaling in Drosophila. Cell Rep. 7, 672–680 (2014)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank T. Kornberg, S. Roy, T. Avidor-Reiss, E. Laufer, A. Rodal, D. Sharp, S. Noselli, A. C. Spradling, T. E. Haerry, E. R. Gavis, C.-Y. Lee, T. Xie, B. McCabe, K. S. McKim, Bloomington Drosophila Stock Center, Vienna Drosophila Resource Center and the Developmental Studies Hybridoma Bank for reagents; S. Roy, T. Kornberg, G. Boekhoff-Falk and D. King for comments and advice; K. Luby-Phelps, A. Bugde and M. Acar for advice for imaging/image data processing; and the Yamashita and Buszczak laboratory members for discussion. The research in the Yamashita laboratory is supported by the Howard Hughes Medical Institute. Y.M.Y. is supported by the MacArthur Foundation.

Author information

Authors and Affiliations

Authors

Contributions

M.I. conceived the project, and executed experiments. All authors designed experiments, analysed the data, and wrote and edited the manuscript.

Corresponding authors

Correspondence to Michael Buszczak or Yukiko M. Yamashita.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 MT-nanotubes are MT-based structures that form in a cell-cycle-dependent manner.

ac, Representative images of MT-nanotubes visualized by GFP–αTub (nos-gal4>GFP–αtub) after 90 min ex vivo treatment of mock (a, DMSO), colcemid (b) or cytochalasin B (c). d, Thickness and length of MT-nanotubes after mock (DMSO), colcemid or cytochalasin B treatment. Each scored value is plotted as an open circle. Red line indicates average value with standard deviation; n indicates the number of MT-nanotubes scored from more than three testes for each data point. e, Representative images of MT-nanotubes in each cell cycle stage visualized by GFP–αTub. The three smaller panels to the right show magnified images of GSCs from the larger left-hand panel representing various stages of the cell cycle: left, G1–S phase (before the completion of the cytokinesis); middle, G2 phase; right, mitosis. f, g, Frequency of MT-nanotubes/GSC after mock (DMSO), colcemid or cytochalasin B treatment (f) or during cell cycle (g); n indicates the number of GSCs scored from more than ten testes from three independent experiments for each data point. h, Frames from a time-lapse live imaging of a MT-nanotube visualized by GFP–αTub. GSC in anaphase at 0 min is indicated by the red dotted circle, which undergoes cell division and grows MT-nanotubes (arrowheads) at 40 min (see Supplementary Video 1). MT-nanotubes typically formed during telophase to early S phase of the next cell cycle, within an hour after mitotic entry (95.2%, n = 21 GSCs) from three independent experiments. i, An example of a GSC that does not have the centrosome (arrows) at the base of the MT-nanotubes. MT-nanotubes are indicated by arrowheads. Centrosomes are indicated by arrows. Asterisk indicates hub. P values from t-tests are provided as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bar, 10 µm.

Extended Data Figure 2 IFT proteins localize to MT-nanotubes

a–c, Examples of MT-nanotubes in wild type (a), oseg2RNAi (b) and klp10aRNAi (c) testes; nos-gal4>GFP–αtub was used. Upper panels are magnified views of squared areas in lower panels, showing examples of measuring length (L) and diameter (D, the base of the MT-nanotubes). d, An example of a MT-nanotube stained by anti-Klp10A antibody in WT testis. e, Validation of anti-Klp10A antibody, showing that klp10A mutant clones (arrowheads and dotted circles) have completely lost the staining 3 days after clone induction. fi, Examples of testis apical tips expressing Oseg1–GFP (f), Oseg2–GFP (g), Oseg3–GFP (h), GFP–Dlic (i) driven by nos-gal4. Arrowheads indicate MT-nanotubes illuminated by anti-Klp10A staining. GSCs are indicated by blue lines or yellow dotted circles. Asterisk indicates hub. Scale bar, 10 µm.

Extended Data Figure 3 Tkv–mCherry or mCherry co-localize with Tkv–GFP in the hub.

a, An apical tip of the testis expressing Tkv–GFP in germ cells (nos-gal4>tkv–GFP). Broken lines indicate hub. b, An apical tip of the testis expressing GFP in germ cells (nos-gal4>GFP). c, Fully functional Tkv–GFP protein trap shows punctate pattern within the hub area. d, Frames from a time-lapse live observation of Tkv–mCherry puncta (arrowheads) moving along a MT-nanotube. Asterisk indicates hub. e, mCherry and Tkv–GFP expressed in germ cells (nos-gal4>UAS-tkv–GFP, UAS–mCherry) co-localize in the hub (arrowheads). f, Tkv–mCherry and Tkv–GFP expressed together in germ cells (nos-gal4>UAS-tkv–GFP, UAS-tkv–mCherry) co-localize in the hub (arrowheads). g, An apical tip of the testis expressing Dome–GFP in germ cells (nos-gal4>dome–GFP raised at 18 °C to reduce the expression level). h, i, Tkv–GFP localization in control (h, DMSO) or colcemid (i) treatment, revealing the localization of Tkv–GFP to the GSC cortex upon perturbation of MT-nanotubes. Dotted hemi- or full circles indicate hub. Scale bar, 10 µm.

Extended Data Figure 4 Effect of RNAi-mediated knockdown of IFT components on Dpp signalling and cytoplasmic microtubules.

a, b, Dad-LacZ staining was undetectable in control GSCs (a) but was enhanced in klp10ARNAi GSCs (b). c, Quantification of pMad intensity in the two- or four-cell spermatogonia (SG) of indicated genotypes. Graph shows average values ± s.d.; n = 30 GSCs were scored from at least ten testes from at least two independent crosses for each data point. di, Cytoplasmic microtubule patterns stained with anti-α-tubulin antibody upon RNAi-mediated knockdown of indicated genes (dh) or colcemid treatment for 90 min (i). In control as well as upon knockdown of IFT-B components, cytoplasmic MTs, visible as fibrous cytoplasmic patterns, were not visibly affected, whereas colcemid treatment disrupted cytoplasmic MTs; h, klp10A knockdown led to hyper stabilization of cytoplasmic MTs. Asterisk indicates hub. P values from t-tests are provided as NS, non-significant (P > 0.05). Scale bar, 10 µm.

Extended Data Figure 5 The klp10A mutant clones do not show a competitive advantage in GSC maintenance.

a, Maintenance of klp10ARNAi GSC clones. b, Maintenance of klp10A24 null clones. The number of control GSC clones (+/+, determined by lack of GFP) and the number of klp10A24 null GSC clones (−/−, determined by anti-Klp10A staining) were scored. c, Maintenance of GFP-positive GSC clones from the cross of 42DFRT X histoneGFP, 42DFRT; hs-flp-MKRS/TM2 as a control for klp10A24 null clones in b. GFP-positive GSC clones did not decrease compared with day 3, excluding the possibility that klp10A24 null GSC clones were lost because of unrelated mutation(s) on the histoneGFP, 42DFRT chromosome. ac, Indicated numbers of GSCs were scored for each data point from at least two independent crosses. d, A representative image of a testis with klp10A24 null clones. The klp10A24 null germ cells determined by anti-Klp10A staining are encircled by white dotted lines. Asterisk indicates hub. Scale bar, 10 μm. Average values ± s.d. are plotted in graphs. P values from t-tests are provided as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; NS, non-significant (P > 0.05).

Extended Data Figure 6 STAT92E level is not affected by modulation of MT-nanotube formation.

ac, Double staining of STAT92E and pMad in control (a), klp10ARNAi (b) and oseg2RNAi (c) testes. Asterisk indicates hub. GSCs (and gonialblasts that are still connected to GSCs) are circled by the dotted line. d, Quantification of STAT92E intensity. GSCs (n = 30) from more than five testes from two independent crosses were scored for each data point. Average values ± s.d. are shown. P values from t-test are provided as NS, non-significant (P > 0.05).

Extended Data Figure 7 Dpp pathway is required for the MT-nanotube formation.

a, b, Testes (a, dpphr56/dpphr4) or (b, dpphr56/CyO) expressing GFP–αTub in germ cells (nos-gal4>GFP–αtub) at restrictive temperature. c, d, MT-nanotube formation upon knockdown (c) or overexpression (d) of Tkv visualized by GFP–αTub. e, Ectopic MT-nanotube formation in spermatogonia upon expression of Dpp in somatic cyst cells. The right-hand panel is a magnified view of the squared region in the left panel. Arrowheads indicate ectopic MT-nanotubes. Asterisk indicates hub. Scale bar, 10 µm.

Extended Data Table 1 Effects of primary cilium or cytoneme genes on MT-nanotube formation

Supplementary information

Supplementary Information

This file contains Supplementary Notes 1-2 and Supplementary Table 1. (PDF 231 kb)

A representative video of GSC division visualized by GFP-αTub.

GSC in anaphase at 0 min undergoes cell division and grows a MT-nanotube from GSC-hub interface into the hub area (see Extended Data Fig. 1e). (MOV 531 kb)

A representative video of interphase MT-nanotubes visualized by GFP-αTub.

A representative video of interphase MT-nanotubes visualized by GFP-αTub. (MOV 1012 kb)

Spatial relationships between MT-nanotubes and hub cell junctions.

3d rendering shows that the MT-nanotubes invaginate into a hub cell. A GSC with an MT-nanotube (GFP-αTub, green) Hub-GSC junction and hub cell coltex (Arm staining, red). (MOV 6500 kb)

Membrane lipids around a MT-nanotube.

GFP-αTub (green). FM4-64 Lipophilic Styryl Dye (magenta) shows that the MT-nanotubes are surrounded by membrane. (MOV 3057 kb)

A video of oblique sectioning of an MT-nanotube.

Membrane (magenta) components ensheath a MT-nanotube (green). (MOV 1918 kb)

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Inaba, M., Buszczak, M. & Yamashita, Y. Nanotubes mediate niche–stem-cell signalling in the Drosophila testis. Nature 523, 329–332 (2015). https://doi.org/10.1038/nature14602

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