Without children is required for Stat-mediated zfh1 transcription and for germline stem cell differentiation

Abstract

Tissue homeostasis is maintained by balancing stem cell self-renewal and differentiation. How surrounding cells support this process has not been entirely resolved. Here we show that the chromatin and telomere-binding factor Without children (Woc) is required for maintaining the association of escort cells (ECs) with germ cells in adult ovaries. This tight association is essential for germline stem cell (GSC) differentiation into cysts. Woc is also required in larval ovaries for the association of intermingled cells (ICs) with primordial germ cells. Reduction in the levels of two other proteins, Stat92E and its target Zfh1, produce phenotypes similar to woc in both larval and adult ovaries, suggesting a molecular connection between these three proteins. Antibody staining and RT-qPCR demonstrate that Zfh1 levels are increased in somatic cells that contact germ cells, and that Woc is required for a Stat92E-mediated upregulation of zfh1 transcription. Our results further demonstrate that overexpression of Zfh1 in ECs can rescue GSC differentiation in woc-deficient ovaries. Thus, Zfh1 is a major Woc target in ECs. Stat signalling in niche cells has been previously shown to maintain GSCs non-autonomously. We now show that Stat92E also promotes GSC differentiation. Our results highlight the Woc-Stat-Zfh1 module as promoting somatic encapsulation of germ cells throughout their development. Each somatic cell type can then provide the germline with the support it requires at that particular stage. Stat is thus a permissive factor, which explains its apparently opposite roles in GSC maintenance and differentiation.

Keywords: Drosophila; GSC; Niche; Stat92E; Woc; ZMYM; Zfh1.

Fig. 1.

Somatic woc function is required for GSC/CB differentiation. (A) Wild-type germarium. Terminal filament (TF) and cap cells (CC) are at the anterior (left). Germline stem cells (GSCs) and their daughters (cystoblasts, CBs) carry spherical fusomes (yellow). Germline cysts contain branched fusomes and contact escort cells (ECs). (B,C) Germ cells labelled by anti-Vasa (green). Anti-Hts antibody (magenta) labels somatic cell membranes and fusomes. (B) Wild-type germarium: fusomes are spherical in GSCs/CBs (arrowheads) and branched in dividing cysts (arrows). (C) A compression of several z-sections of a woc-RNAi germarium filled with germ cells carrying spherical fusomes (arrowheads). (D,E) Anti-SMAD3 marks pMAD (magenta). (D′,E′) Anti-GFP (detects bamP-GFP, green). (D″,E″) Merged images of D,D′ and E,E′, respectively, including anti-Hts (white). (D-D″) A wild-type germarium, pMAD staining is strong in GSCs (arrowheads). bamP-GFP is undetected in GSCs, low in CBs (outlined) and strong in dividing cysts. (E-E″) A woc-RNAi germarium. pMAD is expressed in GSCs (arrowheads), and is weaker further away from the niche. Cells express low levels of bamP-GFP, similar to wild-type CBs. (F) Comparison of cells with spherical fusomes between wild-type and woc-RNAi germaria. The different categories and t-test P-values are as follows: GSCs (pMAD+ inside the niche, P=0.37, not significant), pMAD+ outside the niche (P=6.98E-6), pre-cystoblasts (double negative, P=1.98E-16) and CBs (bam+, P=5.15E-14). (G,H) Ovaries are labelled using anti-Hts (magenta). (G) hs-bam ovaries following heat shock. Branched fusome close to the niche (arrow) indicates a differentiated GSC. (H) hs-bam expression in woc-RNAi ovaries. Spherical fusomes are observed (arrowheads). Scale bars: 10 μm (bar in B applies to B,C; bar in D applies to D,E; bar in G applies to G,H).

Fig. 2.

Woc is required in ECs to maintain cell protrusions and to allow GSC differentiation. (A-C) A wild-type germarium. Anti-Woc (green in A,B) stains somatic and germ cell nuclei (DAPI, blue in A, greyscale in C). (D-L) Anti-Hts is in magenta. (D-I) GFP marks wild-type cells. (D) Control ovary FRT82B with GFP-deficient somatic cells. GSCs differentiate into normal cysts (arrows). (E-G) When niche cells are WT (arrowheads) and somatic ECs are mutant (no GFP, arrows) for woc²⁵¹ (E), woc⁴⁶⁸ (F) or woc^rgl (G), germ cells fail to differentiate and carry spherical fusomes. (H-I) Germ cells mutant for woc^rgl (H, arrows), or woc²⁵¹ (I, arrow) can develop into cysts. (J) woc-deficient germ cells (anti-Vasa, green) differentiate normally into cysts (arrows). (K,L) Somatic cell membranes are marked by Fax-GFP (anti-GFP, green). Arrows mark somatic cells. Several compressed z-sections are shown. In WT (K), somatic cell protrusions extend between cysts. (L) In woc-RNAi ovaries, ECs fail to send protrusions and GSCs fail to differentiate. Scale bar: 10 μm.

Fig. 3.

Woc is required for soma-germline association during gonadogenesis. Anti-Tj (magenta) stains IC nuclei. (A,B) PGCs are labelled by anti-Vasa (green). (A) Wild-type ovary. ICs are interspersed between PGCs. (B) In woc-RNAi ovaries, ICs are located around the PGC region (solid line), and only a few intermingle. (C-E) GFP labels wild-type cells. (C) Wild-type clones still intermingle with PGCs (outlined). (D,E) Large somatic clones of woc^B11 (D) or woc^rgl (E) mutant ICs (GFP-negative) organise outside the germ cell region (outlined) and very few cells intermingle with PGCs. (F) Overexpression of Woc results in increased IC numbers (compare F with A). Scale bars: 10 μm.

Fig. 4.

Phenotypic similarity of stat, zfh1 and woc. (A-D,G) Anti-Tj (magenta) labels ICs. (A,B,F-H) Anti-Vasa marks PGCs. Somatic overexpression of Upd (A) or Hop^Tum-l (B) results in additional ICs and PGCs (compare with WT, Fig. 3A). (C-E) GFP (green) marks wild-type cells. Large mutant clones of stat92E alleles result in separation of ICs from PGCs (C,D, outlined). When ECs are stat92E deficient (E, large stat³⁹⁷ mutant clone; F, RNAi, compression of several z-sections), additional single cells (anti-Hts, magenta, arrowheads) are present. (G) In zfh1-RNAi ovaries, ICs (magenta) do not intermingle with PGCs (green, outlined). (H) More single germ cells carrying spherical fusomes (anti-Hts, magenta) are present in zfh1-RNAi ovaries (arrowheads). (I-L) Anti-Coracle (magenta) labels EC extensions and anti-Tj (green) labels EC nuclei. EC extensions protrude into a wild-type germarium (I). stat-RNAi germaria, extensions in region 1 are either missing (J) or reduced (K). Arrowhead in K indicates an EC nucleus close to the niche, which retains a protrusion. (L) ECs in zfh1-RNAi germaria also lack extensions. Scale bars: 10 μm.

Fig. 5.

Woc is required for proper Zfh1 expression. (A,B) Images were taken together with the same confocal settings. Zfh1 (greyscale) stains all somatic nuclei in larval ovaries. (A) Wild-type somatic cells in proximity to germ cells exhibit stronger Zfh1 labelling (compare arrowheads with arrows). (B) In woc-RNAi ovaries, Zfh1 levels in somatic nuclei abutting PGCs are not as elevated as in WT (compare arrowheads in A,B). (C,C′) Wild-type larval ovaries stained with anti-Stat (green in C, greyscale in C′). Higher Stat levels are present in ICs (PGCs are outlined in C). (D,E) Anti-Tj (green) and anti-Zfh1 (D′,E′, greyscale) co-stain EC nuclei. Zfh1 staining is reduced in woc-RNAi ECs (arrowheads E′, compare with D′). woc-RNAi sheath cells outside the outlined germarium still express high Zfh1 levels. Tj levels are unaffected (compare D with E). (F) GFP (green) marks wild-type cells. In woc mutant cells (arrowhead), Zfh1 (magenta in F, greyscale in F′) staining is reduced compared with a neighbouring wild-type cell (arrow). The nuclei of the marked cells are at the same confocal plane and can therefore be compared. (G) Quantification of Zfh1 protein expression in woc- and stat-deficient cells. P-values of Student's t-test and s.e.m. bars are indicated. (H) Real-time qPCR of zfh1 transcripts comparing bam mutant ovaries with bam mutants that were also woc deficient. Two different recombinant lines produced similar results in two independent experiments (shown combined). (I) Real-time qPCR of zfh1 transcripts comparing control cells (lacZ dsRNA) to woc dsRNA, exposed to control or Upd-containing media. Student's t-test P-values of five independent experiments are shown. Scale bars: 10 μm (bar in A applies to A,B; bar in D applies to D-F).

Fig. 6.

Haplo-insufficiency of Zfh1 and rescue of woc-deficient ovaries. (A-D) Compression of several z-sections of WT (A), and of flies heterozygous for three zfh1 alleles (B-D) labelled by anti-Hts (magenta) to highlight fusomes within germ cells (anti-Vasa, Green). Arrowheads mark accumulated single germ cells in heterozygotes. Cysts are also present in the heterozygous flies (arrows). (E-H) Heterozygous zfh1 flies express lower levels of Zfh1 in ECs (marked by arrowheads in E-H and by anti-Tj in E′-H′) compared with WT. Scale bar: 10 μm.

Fig. 7.

Zfh1 overexpression can rescue woc-deficient ovaries. (A-C,G-I) Anti-Hts labels somatic cells and fusomes; anti-Vasa (green) marks germ cells. (A,B) Developing cysts are marked by arrows. Overexpression of either a wild-type (A) or a mutated form of Zfh1 (B, Zfh1*) does not impair germ cell differentiation. (C) Overexpression of Zfh1 in woc-RNAi ovaries rescues GSC differentiation. Elongated fusomes and germline cysts are present (arrows). (D-F) Anti-Coracle stains EC extensions (greyscale), which encapsulate germline cysts in WT (D). (E,F) Compression of several z-sections, taken with the same confocal settings. (E) A woc-RNAi germarium, ECs (arrowhead) are present, but lack cell extensions. (F) Rescue of woc-RNAi ovaries by Zfh1; extensions into the germarium are observed. (G-I) An entire woc-RNAi ovary. Six ovarioles are shown in this confocal section, all filled with single germ cells. Very few cysts and no egg chambers are observed. (H) Upon overexpression of Zfh1, cysts and egg chambers are readily observed in woc-RNAi ovaries. Four ovarioles are shown in this section. (I) No rescue of woc-RNAi ovaries by expressing a mutated form of Zfh1. Single germ cells are observed (arrowheads). (J) A model showing phenotypic and molecular aspects of Woc function in larval and adult ovaries. Woc is required for an increase in Zfh1 expression within ICs and ECs, respectively. Elevated Zfh1 levels are required for soma-germline association and for GSC differentiation. Scale bars: 10 μm (bar in A applies to A-F).