An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells

Abstract

Stem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed and how morphology impacts function. To address this, we leverage the Drosophila gonadal niche, which affords genetic tractability and live-imaging. We have previously shown mechanisms dictating niche cell migration to their appropriate position within the gonad and the resultant consequences on niche function. Here, we show that once positioned, niche cells robustly polarize filamentous actin (F-actin) and non-muscle myosin II (MyoII) toward neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. We additionally show that germ cells aid in polarizing MyoII within niche cells and that extrinsic input is required for niche morphogenesis and function. Our work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.

Keywords: Drosophila; actomyosin contractility; feedback; morphogenesis; niche; stem cell; testis.

Figure 1:. Niche compaction is characterized by a change in niche shape and size

A-A”‘) Ex vivo gonadal timelapse (5-hour), revealing niche compaction; Six4-Moe::GFP (somatic F-actin, green); His2Av::mRFP1 (nuclei, magenta). A) 5 niche cells in-view reveal an elongated, jagged niche at the anterior. A’) Niche cells rearranged with neighbors, moving closer together. A”) 6 niche cells in-view present a more rounded contour. A”‘) 7 niche cells in-view are yet closer, presenting a circular contour. B-C) Head-on views of niches from fixed ES16 (B) or late ES17 (C) gonads developed in vivo; Six4-Moe::GFP; His2Av::mRFP1. B’-C’) FasIII. D-G) 3D analysis of niches developed in vivo. Niche circularity (D) increases post-compaction. Niche surface area (E), niche cell internuclear distance (F), and niche cell number (G) decrease post-compaction (** p <0.01, ****p<0.0001, Mann-Whitney). In D, E, and G, each dot represents one gonad pre- (n=17) and post- (n=31) compaction; in F, each symbol represents one niche cell pre- (n=277) or post- (n=435) compaction. Unless stated otherwise, for all figures: scale bars = 10 microns; images = single Z slice; yellow dashes outline entire gonad; white dashes outline niche.

Figure 2:. F-actin and MyoII are enriched along the niche-GSC interface during compaction

A) Ex vivo gonadal timelapse of niche compaction; Six4-Moe::GFP; MyoII::mCherry. A’) Somatic F-actin. A”) MyoII. B-C) Quantifications of F-actin (B) and MyoII (C) along niche-GSC interfaces (magenta) relative to non-niche somatic interfaces (blue) at 0h, 2.5h, and 5h post-dissection. Each symbol represents normalized fluorescence along one interface (n=27 for both). D-G) Fixed images of lineage-specific F-actin or MyoII. D) Somatic F-actin (Six4-Gal4 > UAS-F-Tractin::TdTomato.) E) Somatic MyoII (Six4-Gal4::VP16 > MyoII HC::GFP). F) Germline F-actin (Nanos-Moesin::GFP). G) Germline MyoII (Nanos-Gal4::VP16 > MyoII HC::GFP). H-K) Fluorescence along the niche-GSC interface (pink) compared to niche-niche (blue, H-I) or GSC-GSC interfaces (blue, J-K). Equal numbers of niche-GSC interfaces were analyzed compared to respective controls (H: n=57, I: n=52, J: n=57, K: n=79; **p<0.01, ***p<0.001, ****p<0.0001, Mann-Whitney). Arrows and arrowheads = polarization towards or away from niche-GSC interfaces, respectively. Asterisk = niche.

Figure 3:. AMC induces tension along the niche-GSC interface

A-C) Six4-Moe::GFP gonads developing ex vivo. Outlines = interfaces selected for severing pre-cut (A, B, C; red line = interface targeted) and 5s post-cut (A’, B’, C’). A”-C”) Interface pre-cut and montage of 5s intervals post-cut. Dashed lines show displacement of vertices. Scalebars = 1 micron. A) Control niche-niche interface. B) Control niche-GSC interface. C) ROKi-treated niche-GSC interface. D) Initial retraction velocities along niche-niche (blue, n=17) and niche-GSC (magenta, n=15) control interfaces show higher tension along niche-GSC interfaces. E) Initial retraction velocities along niche-GSC interfaces in controls (magenta; same as D) and ROKi-treated gonads show tension is decreased upon AMC inhibition (black, n=11; *p<0.05, *** p<0.001 Mann-Whitney).

Figure 4:. AMC is required for niche morphogenesis

A-B) 5-hour timelapse of control (A) or ROKi-treated (B) Six4-Moe::GFP (somatic F-actin, green); His2Av::mRFP1 (nuclei, Magenta). C-D) Niche circularity measurements at 0h and 5h of untreated (C, n=15 gonads; previously shown in ref.) or ROKi-treated gonads (D, n=15 gonads). Lines pair the same niche at 0 and 5h (**p<0.01, Wilcoxon test). E-H) Six4-moe::GFP, His2Av::mRFP1 control (E, G) or Six4-Gal4 > MyoII RNAi gonads (F = HC RNAi; H = RLC RNAi) developed in vivo. E’-H’) FasIII. I) Niche circularity is decreased when either MyoII is depleted (***p<0.001, ****p<0.0001, Mann-Whitney; See Figure S1). J-K) Niche surface area (J) and niche cell number (K) show no difference between RNAi treatments and respective controls (Mann-Whitney). For graphs I-K, n=17 gonads for all conditions, except n= 21 for MyoII RLC RNAi.

Figure 5:. Proper niche shape is required to regulate GSC behavior

A-B) Control (A) or Six4-Gal4 > MyoII HC RNAi (B) stained gonads; Vasa (germline, magenta), FasIII (white). A’-B’) STAT antibody. Asterisk = niche. Solid outlines = niche-adjacent germ cells (‘GSC’) enriched for STAT. Dotted outlines = posterior germ cells (‘GC’) exhibiting lower STAT. C) STAT enrichment in GSCs under control (n= 87 GCs and 145 GSCs) and RNAi (n=108 GCs and 180 GSCs) conditions. D) Number of STAT+ germ cells contacting the niche is increased in MyoII HC RNAi gonads (n= 27 gonads) compared to controls (n=23 gonads). E) Percentage of niche area that contacts the germ line is increased in MyoII HC RNAi gonads. (*p<0.05, ***p<0.001, ****p<0.0001, Mann-Whitney; See Figure S2). F-G) Control (F) and MyoII HC RNAi (G) stained gonads; centrosomes (gamma tubulin, green), Vasa (germline, magenta), and FasIII (white). F) A GSC (solid outline) with one centrosome oriented at the niche (arrow), and one opposite (arrowhead). G) A GSC (solid outline) with two centrosomes (arrowheads) both oriented away from the niche. H) Percentage of cells with mispositioned centrosomes doubled from 9.4 to 20.5% comparing control and MyoII HC RNAi (**p<0.01, Chi-Square; See Figure S1 and S3).

Figure 6:. Germ cells protrude into the niche during early compaction

A, C) Gonads ex vivo expressing Nanos-Moe::GFP (germline F-actin); early (A) or late compaction (C). Asterisk = niche. Niche-GSC interface contour was monitored before laser cut (pre-cut), during cut (red line, 0s), and 10 and 20s post-cut. A’, C’) Inset of analyzed cell. Yellow dashed line = contour pre-cut; magenta = extent of protrusion post-cut, if any. A, A’) Severing the niche-GSC interface during early compaction led to germ cell protrusion into the niche (10s and 20s). C, C’) Severing the niche-GSC interface during late compaction revealed little to no protrusion. B, D) Quantifications of protrusion into the niche, where each line represents the same interface 0s and 20s after severing. Germ cells significantly protrude into the niche during early (B) but not late compaction (D; n=8 cells each; **p<0.01, Wilcoxon Test).

Figure 7:. GSC divisions are required to shape their niche

A-B) Six4-Moe::GFP control (A) or Nanos-Gal4::VP16 > hid.Z gonad (B) developed in vivo; F-actin (green), Vasa (magenta), and FasIII (A’-B’). C-D) Six4-Moe::GFP; His2AV::mRFP1 control gonad (C) or Nanos-Gal4::VP16 > Cdc25 RNAi gonad (D) developed in vivo; F-actin (green), DNA (magenta) and FasIII (C’-D’). A, C, and D are maximum projections of ~10 slices (0.5-micron intervals). E) Total germ cell number is reduced by expressing hid.Z (n=19 gonads) or Cdc25 RNAi (n= 11 gonads) in the germline compared to respective controls (n=21 and 11, respectively). F) Niche circularity is decreased upon germline expression of hid.Z or Cdc25 RNAi (*p<0.05, **p<0.01, Mann-Whitney). G-H) No difference in niche surface area (G) nor niche cell number (H) between conditions (n=18 hid.Z and 21 Cdc25RNAi gonads) and respective controls (n= 20 and 22, respectively). I-J) Control (I) and Cdc25 RNAi (J) gonads expressing MyoII RLC::GFP; Vasa (magenta), FasIII (white). I’-J’) MyoII RLC::GFP shows enrichment in controls towards niche-GSC interfaces (I’, arrow), and decreased but residual polarity towards the niche-GSC interface in Cdc25 RNAi gonads (J’, arrow). MyoII is sometimes mis-polarized away from the niche-GSC interface (J’, arrowhead). K) Quantifications show germline Cdc25 RNAi leads to decreased MyoII enrichment at the niche-GSC interface (n=54 control = interfaces, n=72 Cdc25 RNAi interfaces, ***p<0.001, Mann-Whitney; see Figure S4). L) Percentage of cells with mispositioned centrosomes tripled from 8 to 26% comparing control and Cdc25 RNAi gonads (**p<0.01, Chi-Square). M-N) Control (M) and Cdc25 RNAi (N) gonads; gamma tubulin (green), Vasa (magenta), FasIII (white). M) A GSC (solid outline) with one centrosome anchored at the niche (arrow), and one located opposite (arrowhead). N) A GSC (solid outline) with two centrosomes (arrowheads) oriented away from the niche.