Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche

Abstract

Tissue homeostasis often requires a properly placed niche to support stem cells. Morphogenetic processes that position a niche are just being described. For the Drosophila testis, we recently showed that pro-niche cells, specified at disparate positions during early gonadogenesis, must assemble into one collective at the anterior of the gonad. We now find that Slit and FGF signals emanating from adjacent visceral mesoderm regulate assembly. In response to signaling, niche cells express islet, which we find is also required for niche assembly. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. The function of such niches is severely disrupted, with niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. Our work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.

Keywords: Drosophila; FGF; Slit; biniou; islet; niche; stem cell; testis; visceral mesoderm.

Figure 1.. Visceral mesoderm is required for niche assembly and positioning

(A) Control Stage 17 gonad when niche morphogenesis is complete, immunostained with Vasa (red, germ cells) and Fas3 (white, niche cells, arrow). (B) biniou mutant; Vasa (red) and Fas3 (white) reveal a dispersed niche (arrowheads). (C) Control and (D) biniou mutant gonads immunostained with Vasa (red) and expressing upd-Gal4, UAS-GFP in niche cells (white, arrow). (A′ and B′) Fas3 alone; (C′ and D′) GFP alone. Dotted lines, gonad boundary. (E and F) Quantification using (E) Fas3 or (F) upd > GFP as marker (p < 0.001, p = 0.004, respectively, Fisher’s exact test). (G) Number of niche and non-niche SGPs specified in biniou mutants compared with siblings. (H) Niche assembly is not affected in jeb mutants compared with siblings. (I) Control and (J) jeb mutant embryos (Stage 13, before gonad coalescence); arrows show SGPs (Traffic jam, red) in contact with Vm cells (Fas3, white). Jeb mutants have a different arrangement of Vm precursors. Scale bars, 10 μm.

Figure 2.. Slit and FGF signaling promote anterior niche assembly

(A–D and I) Stage 17 gonads, merge of Vasa (red, germ cells), Hoechst (blue, DNA), and Fas3 (white, niche cells), or single channel (Fas3); dotted line, gonad boundary. Scale bars, 10 μm. Prime panels show Fas3 (niche cells, arrowheads) alone. (A) Sibling controls have a single, anterior niche. (B and C) Df(2R)BSC25 gonads, with a deletion removing pyr and ths genes, exhibit niche defects such as (B) dispersed niche cell aggregates and (C) niches not at the gonad anterior (asterisk, gonad posterior). (D) slit[2] mutant gonads often have dispersed niche cell aggregates. (E–H) Quantification of niche defects (Fisher’s exact test), (E) with pyr and ths removed (fgf) (p = 0.016), (F) FGF htl receptor mutant (p = 0.0003), (G) slit mutant (p < 0.0001), and (H) robo2, robo1 double mutant (p = 0.024). (I and J) Combined mutant with slit, and pyr and ths removed (fgf) exhibit dispersed niche cells (p = 0.003). (K) Niche morphogenesis defects were not significant (n.s.) in Slit overexpression embryos.

Figure 3.. biniou is required for anterior movement of pro-niche cells

(A and C) Stage 17 gonads expressing mcd8GFP in PS11 cells (magenta), and immunostained with Vasa (white, germ cells) and Fas3 (green, all niche cells). (A) A control, with a single anterior niche (left, green) containing cells deriving from both PS 10 (A′, green alone, asterisk) and PS 11 (A′ and A′′, magenta and green, arrow). (C) biniou mutant with dispersed niche cell aggregates (green). Anterior niche cells deriving from PS 10 (C′, green alone, asterisk) do not associate with PS 11-derived niche cells (C′′, magenta and green, arrows). Ectopic PS11 niche cells were distinguishable from PS13 msSGPs, which do not express the niche cell marker Fas3. (B and D) Cartoons illustrating the distribution of PS11 niche cells in (B) control and (D) biniou mutants. (E–G) Quantifications comparing biniou mutants and sibling controls (Fisher’s exact test) by how often (E) PS 11-derived niche cells are located at anterior (p < 0.0001), (F) PS 11 niche cells contact anterior PS 10 niche cells (p < 0.0022), and (G) niche cells are located within 2 μm of the gonad periphery. Scale bars, 10 μm.

Figure 4.. islet is expressed in niche cells in response to Vm signals

(A and B) (A) Control and (B) islet mutant Stage 17 gonads immunostained with Vasa (red, germ cells), Fas3 (white, niche cells), and Hoechst (blue, nuclei). (A′ and B′) Fas3 alone (arrows). (C) Quantification of niche assembly in islet versus sibling controls (p = 0.024, Mann-Whitney test). (D) St 17 gonad expressing GFP driven by the islet AMEr enhancer stained with Vasa (germ cells, red) and Fas3 (niche cells, white). (D′) islet AMEr-driven GFP alone. (E–G) Stage 17 gonads immunostained for Islet (white), Fas3 (red, niche cells), and Vasa (not shown, germ cells). Gonad boundaries, dotted lines. Arrowheads, niche cells. (E′–G′) Islet alone. (H and I) Islet accumulation in niche cells from (H) pyr and ths removed (fgf) and (I) slit mutants, compared with sibling controls (p < 0.0001, Mann-Whitney test). Scale bars, 10 μm.

Figure 5.. Niche cells are polarized during assembly

(A) Stills from a time course of a gonad expressing six4-eGFP::moesin to label F-actin in all SGPs. F-actin accumulates at niche-niche interfaces when niche cells begin to associate (arrow), and later repolarizes to niche-stem cell interfaces (asterisk). (B and C) Quantification of F-actin accumulation at niche-niche or niche-germ cell interfaces (B) during and (C) after completion of niche assembly in fixed tissue (p < 0.0001, Wilcoxon test). (D) Niche cells in fixed tissue expressing a somatic cell F-actin label, six4-eGFP::moesin in islet mutants in which niche cells have begun to associate but have not completed assembly. (E) F-actin accumulation at niche cell interfaces in islet mutants. (F) A model illustrating how Vm signals influence niche assembly. (G and I) Niche cells from fixed Stage 17 control gonads (G) expressing six4-eGFP::moesin or (I) immunostained for E-cadherin. (H) F-actin accumulation at niche-GSC interfaces versus niche-niche interfaces (p < 0.0001, Mann-Whitney test). (J) E-cadherin accumulates at niche-niche interfaces compared with niche-GSC interfaces (p < 0.0001, Mann-Whitney test). (K–N) Quantification of polarity loss in Stage 17 niches for (K) F-actin in biniou, (L) E-cadherin in biniou, (M) Ecad with pyr and ths removed (fgf), or (N) Ecad in slit mutants (Mann-Whitney tests). Asterisks, niche-GSC interfaces; arrows, niche-niche interfaces. Scale bars, 5 μm.

Figure 6.. Niche assembly is required for niche function

(A and B) Stage 17 gonad, Stat (white), Fas3 (red, niche cells), and Vasa (germ cells; not shown). Niche cell, asterisk; GSCs, blue dotted line; neighboring germ cells, yellow-dotted line. (A′ and B′) Stat alone. (A″) inverted Stat. (C) Stat accumulation in GSCs relative to neighboring germ cells in control, biniou or combined signaling mutant (slit, pyr, and ths). (D) Quantification of control versus biniou mutants for percentage of GSCs with a centrosome at a GSC-niche interface (p = 0.004, Fisher’s exact test). (E and F) Stage 17 gonad, γ-tubulin (green, centrosomes), Fas3 (white, niche cells), and Vasa (magenta, germ cells). Arrowheads, GSC centrosomes; asterisk, adjacent niche cell. (G and H) Stage 17 gonads pulsed with EdU, fixed and immunostained. Merge shows EdU (white), Fas3 (magenta, niche), and Vasa (green, germ cell), along with single channel Fas3 (G′ and H′), and EdU (G″ and H″). (G′) Control niche cell with no EdU incorporation (blue arrow), and (H′) a biniou mutant niche cell incorporating EdU (yellow arrow). Asterisk, S phase GSC. (I and J) Quantification of control versus biniou mutants for: (I) percentage of gonads with quiescent niches (p = 0.0001, Fisher’s exact test); (J) S-phase index of GSCs. Scale bars, 10 μm.