Maintenance of niche architecture requires actomyosin and enables proper stem cell signaling and oriented division in the Drosophila testis

Abstract

Stem cells are essential to repair and regenerate tissues, and often reside in a niche that controls their behavior. Here, we use the Drosophila testis niche, a paradigm for niche-stem cell interactions, to address the cell biological features that maintain niche structure and function during its steady-state operation. We report enrichment of Myosin II (MyoII) and a key regulator of actomyosin contractility (AMC), Rho Kinase (ROK), within the niche cell cortex at the interface with germline stem cells (GSCs). Compromising MyoII and ROK disrupts niche architecture, suggesting that AMC in niche cells is important to maintain its reproducible structure. Furthermore, defects in niche architecture disrupt GSC function. Our data suggest that the niche signals less robustly to adjacent germ cells yet permits increased numbers of cells to respond to the signal. Finally, compromising MyoII in niche cells leads to increased misorientation of centrosomes in GSCs as well as defects in the centrosome orientation checkpoint. Ultimately, this work identifies a crucial role for AMC-dependent maintenance of niche structure to ensure a proper complement of stem cells that correctly execute divisions.

Keywords: Drosophila; Cell division; Signaling; Stem cells.

Fig. 1.

AMC components are enriched in the testis niche. (A) Immunostained young w1118 adult testis showing FasIII (white), E-Cadherin (magenta) and Hoechst (cyan). (A′) FasIII only. (A″) E-Cadherin only. Niche outlined by white dashed line. (B) Young w1118 adult testis niche rendered in 3D showing FasIII (white). (B′) E-Cadherin (magenta). Insets in B,B′ show surface imposed on niche. (C) Quantification of sphericity using FasIII and E-Cadherin as markers; sphericity of niche approaches 1. (D-F) Immunostained young testes showing ROK::GFP (green), FasIII (white), Hoechst (magenta) (D), MyoII HC::GFP (green), nanos-lifeact::tdTom (magenta), FasIII (white), Hoechst (cyan) (E), MyoII RLC::3xGFP (green), nanos-lifeact::tdTom (magenta), FasIII (white), Hoechst (cyan) (F). (D′,E′,F′) FasIII only. (D″) ROK only, (E″) MyoII HC only, (F″) MyoII RLC only. In D″, E″ and F″, yellow arrowhead points to a niche–niche interface, white arrow points to niche–GSC interface. (G-I) Quantification of enrichment at niche–GSC interfaces (D″,E″,F″, white arrow) in comparison to niche–niche interfaces (D″,E″,F″, yellow arrowhead) for ROK::GFP (G), MyoII HC::GFP (H) and (I) MyoIIRLC::GFP (****P<0.0001, Mann–Whitney test). Data are median±s.d. Scale bars: 5 µm.

Fig. 2.

ROK is required for niche shape. (A-B‴) Timelapse of young adult niche. Sqh::GFP (green). White arrowheads represent dynamic accumulations of Sqh at the niche–GSC interface, empty white arrows represent deaccumulations of Sqh at the niche–GSC interface (n=8 testes imaged). (C-E′) Immunostained WT testis (C) and 10 day upd>ROK RNAi testis where niche is misshapen (D) or disassembled (E). (C,D,E) Vasa (magenta), FasIII (white), Hoechst (cyan). (C′,D′,E′) FasIII only. Niche shape outlined by white dashed line. (F) Quantification of the niche phenotype shows that ROK RNAi niches are severely misshapen and occasionally disassembled into more than one aggregate compared to age-matched control niches (WT: 5/51 niches misshapen, ROK RNAi: 34/88 niches misshapen, 4/88 niches disassembled, ***P=0.0002, Chi-squared test). (G) Quantification of sphericity between age-matched control and ROK RNAi niches (****P<0.0001, Mann–Whitney test). (H) Quantification of niche cell number shows no difference between age-matched control and ROK RNAi niches (ns, not significant, Mann–Whitney test). (I) Quantification of the average distance of a niche cell nucleus and its three nearest-neighbor nuclei shows increased distance between ROK RNAi niche cells compared to age-matched control (****P<0.0001, Mann–Whitney test). (J,K) Quantification of the niche area (J) and volume (K) shows an increase in ROK RNAi niches compared to WT (***P=0.0001, **P=0.0091, Mann–Whitney test). Data are median±s.d. Scale bars: 5 µm.

Fig. 3.

MyoII is required for niche shape. (A-C′) Immunostained WT testis (A) and 10 day upd>MyoII RNAi testes where niche is misshapen (B) or disassembled (C). (A,B,C) nanos-lifeact::tdTom (magenta), FasIII (white), Hoechst (cyan). (A′,B′,C′) FasIII only. Niche shape outlined by white dashed line. (D) Quantification of niche disassembly shows that MyoII RNAi niches are severely misshapen and, in some cases, disassembled into more than one niche aggregate compared to WT niches (WT: 0/19 niche misshapen or disassembled; MyoII RNAi: 25/35 niches misshapen, 10/35 niches disassembled; ****P<0.0001, Chi-squared test). (E) Quantification of niche sphericity between WT and MyoII RNAi niches (****P<0.0001, Mann–Whitney test). (F) Quantification of niche cell number shows no difference between WT and MyoII RNAi niches (ns, not significant). (G) Quantification of the average distance of a niche cell nucleus and its three nearest-neighbor nuclei shows increased distance between MyoII RNAi niche cells compared to WT (****P<0.0001, Mann–Whitney test). (H,I) Quantification of the niche area (H) and volume (I) shows an increase in MyoII RNAi niches compared to WT (****P<0.0001, **P=0.001, Mann–Whitney test). (J-K″) Immunostained WT testis (J) and MyoII RNAi testis (K). (J,K) nanos-lifeact::tdTom (magenta), FasIII (green), MyoII HC (white), Hoechst (cyan). (J′,K′) FasIII only. (J″,K″) MyoII HC only. MyoII localizes to niche–niche interfaces (white arrowhead, J″) and also to niche–GSC interfaces (white arrow, J″) in WT testes; this localization is abolished in MyoII RNAi testes (K″). Niche shape outlined by white dashed line. (L) Quantification of the sum of MyoII HC fluorescence in the niche in MyoII RNAi niches compared to WT niches (**P=0.0016, Mann–Whitney test). Data are median±s.d. Scale bars: 5 µm.

Fig. 4.

Niches progressively lose sphericity upon MyoII depletion. (A-C) Immunostained WT testis (A), and upd>MyoII RNAi testis, 3 day knockdown (B) and 10 day knockdown (C). (A,B,C) FasIII (white), Hoechst (magenta). (A′,B′,C′) FasIII only. Niche shape outlined by white dashed line. (D) Quantification of niche sphericity between WT and MyoII RNAi 3 day and 10 day knockdown niches (****P<0.0001, *P=0.0157, Mann–Whitney test). (E-H‴) Timelapse of WT niche (E-F‴) and MyoII RNAi niche (G-H‴). (E,G) upd>GFP (green), nanos-lifeact::tdTom (magenta). (F,H) upd>GFP only. Dashed line outlines niche shape at each time point. Data are median±s.d. Scale bars: 5 µm.

Fig. 5.

Disruption to polarized adhesion complexes when compromised for MyoII. (A-B″) Immunostained WT testis (A) and 10 day upd>MyoII RNAi testis (B). (A,B) nanos-lifeact::tdTom (magenta), FasIII (white), Hoechst (cyan). (A′,B′) FasIII only. (A″,B″) E-Cadherin only. Yellow arrowheads point to niche–niche interface, white arrows point to niche–GSC interface. (C-D″) Immunostained WT testis (C) and MyoII RNAi testis (D). (C,D) nanos-lifeact::tdTom (magenta), FasIII (white), Hoechst (cyan). (C′,D′) FasIII only. (C″,D″) N-Cadherin only. Yellow arrowheads point to niche–niche interface, white arrows point to niche–GSC interface. (E,G) Quantification of WT E-Cadherin (E) and N-Cadherin (G) enrichment at niche–niche interfaces (A″,C″, yellow arrowheads) in comparison to niche–GSC interfaces (A″,C″, white arrows) (****P<0.0001, Mann–Whitney test). (F,H) Quantification of E-Cadherin (F) and N-Cadherin (H) enrichment at niche–niche interfaces (yellow arrowheads) normalized to niche–GSC interfaces (white arrows) in MyoII RNAi niches (B″,D″) compared to WT (A″,C″) (****P<0.0001,*P=0.0118, Mann–Whitney test). Data are median±s.d. Scale bars: 5 µm.

Fig. 6.

Niches compromised for MyoII are functionally defective. (A-B‴) Immunostained WT testis (A) and 10 day upd>MyoII RNAi testis (B). (A,B) nanos-lifeact::tdTom (magenta), FasIII (green), STAT (white). (A′,B′) FasIII only. (A″,B″) STAT only. (A‴,B‴) nanos-lifeacttdTom only. Dashed white line indicates niche shape, dashed yellow circle indicates GSC, dashed blue circle indicates germ cell. (C-D‴) Timelapse of relatively rare WT GSC loss (C) and more frequent MyoII RNAi GSC loss (D), upd>GFP (green), nanos-lifeact::tdTom (magenta). Yellow dot indicates a GSC, blue dot indicates newly formed gonialblast (Gb); in both cases, GSC has oriented division (C′,D′), GSC-Gb is eventually lost from the niche (C‴,D‴). (E) Quantification of STAT fluorescence intensity levels of GSCs in WT testes and MyoII RNAi testes (****P<0.0001, Mann–Whitney test). (F) Quantification of STAT⁺ GSCs in WT testes and MyoII RNAi testes (****P<0.0001, Mann–Whitney test). (G) Quantification of GSC loss in WT testes and MyoII RNAi testes (WT: 10/70 GSCs; MyoII RNAi: 40/114 GSCs; **P=0.0020, Fisher's exact test). Data are median±s.d. Scale bars: 5 µm.

Fig. 7.

Germ cells repopulate the niche in MyoII knockdown niches. (A-C‴) Timelapse of GSC divisions showing upd>GFP (green) and nanos-lifeact::tdTom (magenta). Yellow dot indicates a GSC, blue dot indicates newly formed gonialblast (Gb). (A) WT niche, GSC has oriented division; (B) WT niche, GSC has oriented division, but the newly generated GSC-Gb pair exhibits relatively rare symmetric renewal, and the Gb makes contact with the niche; (C) 10 day upd>MyoII RNAi niche, GSC has oriented division; white arrowhead indicates niche extending outward to ‘capture’ Gb for symmetric renewal (C‴). (D) The 10 day upd>MyoII RNAi niche, GSC (yellow dot) divides symmetrically to niche (D′, white asterisk indicates niche) and produces a 2 GSC-like daughters, adherent to niche (D″, yellow dots). (E) Quantification of symmetric renewal events of GSC-Gb pairs in WT testes and MyoII RNAi testes; WT: 6/82 GSC-Gb pairs, MyoII RNAi: 29/93 GSC-Gb pairs exhibited symmetric renewal (****P<0.0001, Fisher's exact test). (F) Quantification of division orientation of GSCs in WT and MyoII RNAi testes; WT: 0/82 GSCs, MyoII RNAi: 33/126 GSCs exhibited misoriented divisions (****P<0.0001, Fisher's exact test). Scale bars: 5 µm.

Fig. 8.

Niche MyoII required for centrosome orientation checkpoint in GSCs. (A-C″) Immunostained WT (A) and 10 day upd>MyoII RNAi testes (B,C). (A,B,C) nanos-lifeactd::Tom (magenta), FasIII (green), γ-tubulin (white). (A′,B′,C′) FasIII only. (A″,B″,C″) γ- tubulin only. White dashed lines outline GSC, asterisk indicates niche. (D) Quantification of centrosome anchoring in GSCs in WT testes and MyoII RNAi testes. WT: 15/114 GSCs with misanchored centrosomes; MyoII RNAi: 113/476 GSCs with misanchored centrosomes, 25/476 GSCs with double anchored centrosomes; ***P=0.0010, Chi-squared test). (E-H″) DMSO (E-E″,G-G″) and colcemid-treated (F-F″,H-H″) testes in WT (E-F″) and MyoII RNAi (G-H″) conditions. (E,F,G,H) nanos-lifeact::tdTom (magenta), FasIII (green), PH3 (white). (E′,F′,G′,H′) FasIII only. (E″,F″,G″,H″) PH3 only. White dashed circle outlines GSC, asterisk indicates niche, yellow dashed circle indicates PH3⁺ germ cell. (I) Quantification of M-phase GSCs in WT and MyoII RNAi testes treated with DMSO or colcemid. WT DMSO (n=26); WT DMSO+colcemid (n=23); MyoII RNAi DMSO (n=45); MyoII RNAi DMSO+colcemid (n=42) (****P<0.0001,*P=0.0436, Kruskal–Wallis test). Data are mean±s.d. ns, not significant. Scale bars: 5 µm.