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

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 signals. 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.

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

a-c) Representative images of MT-nanotubes visualized by GFP-αTub (nos > GFP-αtub) after 90min 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 > 3 testes for each data point. e) Representative images of MT-nanotubes in each cell cycle stage visualized by GFP-αTub. Panels e’-e’’’ show magnified images of GSCs from panel e representing various stages of the cell cycle. e’) G1-S phase (prior to the completion of the cytokinesis). e”) G2 phase. e’’’) 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 > 10 testes from 3 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 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=21GSCs) from 3 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. Hub is indicated by the asterisk (*). P values from t-test are provided as *P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001. Bar: 10µm.

Extended Data Fig. 2. IFT proteins localize to MT-nanotubes

(a-c) Examples of MT-nanotubes in wild type (a), oseg2^RNAi (b) and klp10a^RNAi (c) testes. nos-gal4 > GFP-αtub was used. Panels a-c are magnified views of squared area in a’-c’, showing examples of measuring length (L) and diameter (D, the base of the MT-nanotubes). d) An example of 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. (f-i) 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. Hub is indicated by the asterisk (*). Bar: 10µm.

Extended Data Fig. 3. Tkv-mCherry or mCherry colocalize with Tkv-GFP in the hub

a) An apical tip of the testis expressing Tkv-GFP in germ cells (nos-gal4 > tkv-GFP). Hub is indicated by broken lines. 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. Hub is indicated by the asterisk (*). e) mCherry and Tkv-GFP expressed in germ cells (nos-gal4 > UAS-tkv-GFP, UAS-mCherry) colocalize in the hub (arrowheads). f) Tkv-mCherry and Tkv-GFP expressed together in germ cells (nos-gal4 > UAS-tkv-GFP, UAS-tkv-mCherry) colocalize 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 Tkv-GFP’s localization to the GSC cortex upon perturbation of MT-nanotubes. Hub is indicated by dotted hemi or full-circle. Bar: 10µm.

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

a, b) Dad-LacZ staining was undetectable in control GSCs (a) but was enhanced in klp10A^RNAi (b) GSCs. c) Quantification of pMad intensity in the 2 cell- or 4 cell-spermatogonia (SG) of indicated genotypes. Graph shows average value and standard deviations. n=30 GSCs were scored from 10≤ testes from 2≤ independent crosses for each data point. d-j) Cytoplasmic microtubule patterns stained with anti-αTubulin antibody upon RNAi-mediated knockdown of indicated genes (d-h) or colcemid treatment.for 90min (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. klp10A knockdown led to hyper stabilization of cytoplasmic MTs (h). Hub is indicated by the asterisk (*). P values from t-test are provided as n.s.: non-significant (P > 0.05). Bar: 10µm.

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

a) Maintenance of klp10A^RNA GSC clones. b) Maintenance of klp10A^{24 null} clones. The number of control GSC clones (+/+, determined by lack of GFP and the number of klp10A^{24 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 klp10A^{24 null} clones in b). GFP-positive GSC clones did not decrease compared with day3), excluding the possibility that klp10A^{24 null} GSC clones were lost due to unrelated mutation(s) on the histoneGFP, 42DFRT chromosome. (a-c) Indicated numbers of GSCs were scored for each data point from 2≤ independent crosses. d) A representative image of a testis with klp10A^{24 null} clones. klp10A^{24 null} germ cells determined by anti-Klp10A staining were encircled by white broken lines. Hub is indicated by the asterisk (*). Bar: 10μm. Average value and standard deviations are plotted in graphs. P values from t-test are provided as *P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001; n.s.: non-significant (P > 0.05).

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

a-c) Double staining of STAT92E and phospho-Mad (pMad) in control (a), klp10A^RNAi (b) and oseg2^RNAi (c) testes. Hub is indicated by the asterisk (*). GSCs (and GBs that are still connected to GSCs) are circled by dotted line. d) Quantification of STAT92E intensity. n=30 GSCs from > 5 testes from 2 independent crosses were scored for each data point. Average value and standard deviations are shown. P values from t-test are provided as n.s.: non-significant (P > 0.05).

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

dpp^hr56/dpp^hr4 (a) or dpp^hr56/CyO (b) testes 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 SGs upon expression of Dpp in somatic cyst cells. e’) Magnified view of a squared region of e. Arrowheads indicate ectopic MT-nanotubes, Hub is indicated by the asterisk. Bar: 10µm.

Fig. 1. Characterization of MT-nanotubes in Drosophila male GSC niche

a) A schematic of the Drosophila male GSC niche. GSCs are attached to the hub cells. The immediate daughters of GSCs, the gonialblasts (GBs) are displaced away from the hub, and become spermatogonia (SGs). b) An apical tip of the testis expressing GFP-αTub in germ cells (nos > GFP-αtub). MT-nanotubes are indicated by arrowheads. Graphic interpretation of b) is shown in b’). c) Orientation of nanotubes toward the hub in GSCs vs. GBs/SGs. The size of each vector represents the frequency of MT-nanotubes oriented toward each direction. Indicated numbers of nanotubes (N > 30 testes) were scored from 3 independent experiments. d-g) 3D rendering images of MT-nanotubes (brackets) in fixed (d) or live tissue (e-g), with indicated cell membrane markers. nos-gal4 > GFP-αtub was used for d-e.

Fig. 2. IFT genes are required for MT-nanotube formation

a) Effect of RNAi-mediated knockdown or overexpression (OE) of indicated genes on MT-nanotube morphology. Boxplot shows 25–75% (box), median (band inside) and min to max (whiskers). Indicated numbers of nanotubes (Extended Data Table 1) from 2≤independent crosses were scored for each data point. P values from t-test are provided as *P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001. b) Examples of MT-nanotubes stained by anti-Klp10A antibody in GFP-αTub-expressing testis. c) Apical testis tip expressing GFP-Oseg3 in germ cells. MT-nanotube is indicated by brackets. GSCs are indicated by blue lines. Hub is indicated by the asterisk. Bar: 10µm.

Fig. 3. Dpp signaling components localize to the MT-nanotubes

a) A GSC clone expressing Tkv-mCherry, GFP-αTub and GFP (hs-flp, nos-FRT-stop-FRT-gal4, UAS-GFP, UAS-GFP-αtub, UAS-tkv-mCherry). b) An apical tip of the testis expressing TIPF and Tkv-mCherry in germ cells. Arrowheads point to a few of colocalizing puncta. c) An apical tip of the testis expressing Dpp in the hub and Tkv in germ cells (dpp-lexA^ts > dpp-GFP, nos-gal4^ts > tkv-mCherry). g-k) Tkv-GFP expressed in control (d), klp10A^RNAi (e), oseg2^RNAi (f) germ cells (nos-gal4^ts > UAS-tkv-GFP, UAS-RNAi). Black and white of micrograph was inverted for better visibility of Tkv localization to the hub and plasma membrane. g) Average number and standard deviations of Tkv-GFP puncta within hub area/testis for indicated genotypes. N=15 testes from 2≤ independent crosses were scored. P values from t-test are provided as *P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001. Bar: 10µm.

Fig. 4. MT-nanotubes are required for Dpp signaling activation and GSC maintenance

a-c) Phosphorylated Mad (pMad) staining in control (a), klp10A^RNAi (b) and oseg2^RNAi (c) testes. pMad signal in somatic cyst cells (arrowheads), which remains unaffected by germ-cell specific modulation of MT-nanotube components, was used to normalize pMad levels in GSCs. d) Quantification of pMad intensity in the GSCs of indicated genotypes. Indicated numbers of GSCs (Extended Data Table 1) from 2≤ independent crosses were scored for each data point. e, f) Maintenance of che-13^RNAi, osm6^RNAi (e) and oseg2⁴⁵² (f) mutant GSC clones. Indicated numbers of GSCs (Supplementary Table 1) from 2≤ independent experiments were scored for each data point. g) A klp10A^RNAi GSC clone (72 hours after clone induction, blue circle) with a higher pMad level, compared to control GSCs (white circle). klp10A^RNAi SG clone (yellow circle) and control SG clone (pink circle) have similar pMad levels. Hub is indicated by asterisks. Bar: 10µm. Average value and standard deviations are shown in each graph. P values from t-test are provided as *P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001; n.s.: non-significant (P > 0.05).

Fig. 5. Dpp signaling is necessary and sufficient for MT-nanotube formation

a, b) Quantification of MT-nanotube thickness and length in GSCs of indicated genotypes. Each scored value is plotted as an dot. Red line indicates average value and standard deviations. Indicated numbers of nanotubes (Extended Data Table 1) from 2≤ independent crosses were scored for each genotypes. P values from t-test are provided as *P ≤ 0.05; **P ≤ 0.01; *** P ≤ 0.001; n.s.: non-significant (P > 0.05). c, d) MT-nanotube formation in absence (c) or presence (d) of Dpp expression in somatic cyst cells. c’, d’) Magnified images of squared regions of c, d. Arrowheads indicate ectopic MT-nanotubes. Hub is indicated by the asterisk. Bar: 10µm. e) Model. Dpp induces MT-nanotube formation, and receptor-ligand interaction occurs at the surface of MT-nanotubes, leading to signaling activation in GSCs.