Drosophila SERTAD domain protein Taranis is required in somatic cells for maintenance of male germline stem cells

Abstract

Background: Polycomb proteins are essential for maintaining stem cell identity across different stem cell niches. However, how they function to maintain stem cell niches is not fully understood.

Results: Here we show that the SERTAD protein Taranis (Tara), which is a Polycomb-trithorax group protein, is expressed in the adult testis niche and plays a role in its maintenance in Drosophila. We found that tara is expressed in early cyst cells, likely including somatic cyst stem cells (CySCs) of Drosophila male testis tip region, which houses both germline and somatic cyst stem cells along with the hub cells, forming the stem cell niche. Consistent with its expression, we found that, while loss of tara in germline cells only had minimal effects, tara knockdown in all cells or only in somatic cells of the niche reduced the number of not only somatic cells, but also germline stem cells (GSCs). We further found that Tara might antagonize Notch signaling in CySCs to maintain the stem cell niche.

Conclusions: Our studies suggest that Tara might function in somatic CySCs for GSC maintenance in the Drosophila testis.

Keywords: epigenetic regulation; germline stem cell; notch; somatic cyst stem cell.

Figure 1.. tara is expressed in the adult Drosophila male testis tip

(A) Wild type Drosophila (OrR) testes were dissected and fixed with papaformadehyde and subjected to in situ hybridization with tara antisense probe (left) and sense probe (right). Positive signals (blue) were found in the apical (tip) region of the testis only with antisense probe (left). Scale bar 300 µm. (B) Testes of the enhancer trap line YB712 were stained for DNA (red) to reveal the nuclei, or were immunostained for Vasa (blue) and GFP (green). Note that cells expressing GFP are interspersed between larger nuclei (red) in tara enhancer trap line YB712 (top), and that the GFP-positive cells are negative for the germline marker Vasa (blue in bottom). Scale bar 75 µm. (C) GFP expressing cells are also positive for the somatic cyst cell marker Eya (red). Arrowheads point to nuclei with Eya and GFP colocalization at the testis tip region at the onset of differentiation. Scale bar 25µm. (D) A representative image of ovaries from female flies carrying the YB172 genetrap (tara) reporter immunostained for GFP (green) and Vasa (blue). Note that GFP signals were detected in the ovarian follicle cells, which are derived from somatic stem cells.

Figure 2.. Escargot-Gal4 driven tara knockdown results in stem cell loss in the testis apex.

(A) Testes from 3–5 day old flies expressing tara RNAi or control were dissected and immunostained for Vasa (magenta), Fusome and Hub (red). The GSCs are identified based on their proximity to the hub (white asterisk) and the dotted fusome morphology. Scale bars are 20 µm (top) and 10 µm (bottom). (B) Quantification of GSC number as mean number for the indicated genotypes, N=10 for each genotype. Error bars are standard deviation. ** indicates p < 0.01, by Mann–Whitney U test. (C) A representative testis from 3–5 day old flies for each indicated genotype stained with DAPI. Note that the DAPI condensed region in apical testis, as shown in wild type, is significantly reduced when tara knocked down. Scale bar 75 µm. N=10 for each genotype. Bar graph shows DAPI intensity ratio between the apical region of 2×105 pixels and the same size area immediately posterior to it. Error bars are standard deviation. *** indicates p < 0.001, by Mann–Whitney U test. (D) Testes from 3–5 day old flies expressing tara RNAi or control were immunostained with anti-STAT92E. Note that the apical region (circled with dashed line) has higher levels of STAT92E in control but not in tara RNAi-expressing testis. N=12 for each genotype. Scale bar 50 µm. Bar graph shows STAT92E fluorescence intensity ratio between the apical region of 2×105 pixels and the same size area immediately posterior to it. Error bars are standard deviation. *** indicates p < 0.001, by Mann–Whitney U test.

Figure 3.. Effects of tara knock down with germline- or the hub-specific Gal4 and Survival of tara loss-of-function GCSs

(A, B) Testes from 3–5 days old flies of the indicated genotypes were immunostained with indicated antibodies. Hub is marked by asterisk. Scale bar 25 µm. Note that (A) Nos-Gal4 (germline specific) driven tara RNAi in germ line (top) did not reduce GSC number compared with control (Bottom), and that (B) the hub specific Upd-Gal4 driver was used to knockdown tara in the hub, and it did not affect GSC numbers. (C) 0–2 days old male flies of the genotype hs-Flp; FRT^82B, ubi>GFP/FRT^82B, tara⁰³⁸⁸¹ or hs-Flp; FRT^82B, ubi>GFP/FRT^82B, + (control) were heat-shocked to induce tara^–/– clones (marked by loss of GFP). Testes were dissected 10 days after clone induction and immunostained Vasa (magenta) and GFP (green). Representative images of control (+/+) and tara^−/− clones. Arrows point to GSCs homozygous for tara⁰³⁸⁸¹ or control, marked by loss of GFP. Scale bar = 20 μm.

Figure 4.. Tara is required for somatic stem cell maintenance.

(A) Testes from 3–5 day old flies of the indicated genotypes were immunostained with anti-Eya (red). Note that Patched-Gal4 mediated tara knockdown and overexpression reduce and increase, respectively, the number of Eya+ somatic lineage in the same apical region of the testis (defined as the apical region of 2×105 pixels, marked by dashed white line). Scale bar 75 µm. Quantification of Eya+ nuclei in testis apical region of the indicated genotypes (N=12 for each genotype). * indicates p < 0.001 by Kruskal-Wallis test. (B) Testes of Ptc-gal4>mCherry RNAi (control) and Ptc-Gal4>taraRNAi males were immunostained for Vasa (magenta), Fusome and Hub (red), and DNA (blue). GSCs (marked by white dots) were counted. The bar graph shows mean GCSs and standard deviation (N=30 for each genotype). *** indicates p < 0.001 by Mann–Whitney U test. (C) Testes of in Ptc-Gal4>mCherry RNAi (control) and Ptc-Gal4>taraRNAi males were immunostained with anti-phospho (Serine 10) histone 3 (pH3, white). Scale bar 20 µm. Bar graph to the right shows mean numbers of pH3+ cells at the apical (mitotic) region of the testes. Error bars are standard deviations. N=18 for each genotype. *** indicates p < 0.001 (Mann–Whitney U test). (D) “Flip-out” clones (positively marked by GFP) were induced in adult testis (0–2 days) by heat shock (see Methods). Testes were immunostained 15 days post clone induction for Eya and hub (red) and GFP (green). Representative images of the indicated genotypes (N>20 testes observed for each genotype) are shown with magnified view of the tip of the testis (dashed line). Note the colozalization of Eya+ cells (Red) and GFP+ cells (green) in the differentiation zone (arrow). Scale bars are 75 µm.

Figure 5.. Tara antagonizes Notch signaling in stem cell niche

(A) Adult eyes of male flies of the indicated genotypes are shown. Note that the Notch gain-of-function allele N^spl1 exhibits rough-eye phenotype in hemizyous, and that this phenotype is enhanced in trans with loss-of-function alleles of tara (tara¹ and tara⁰³⁸⁸¹). (B) Images of part of the posterior wing of the indicated genotypes are shown. Note that the typical “delta” phenotype (yellow arrow) of Dl⁷ heterozygotes was suppressed in trans with tara¹ or tara⁰³⁸⁸¹. (C) Third instar wing imagical discs expressing the indicated transgenes were immunostained with anti-Cut. Note the “gap” in Cut expressing cells (arrow) presumably due to tara overexpression by Ptc-Gal4, drives expression in the anterior-posterior boundary running perpendicular to cut expressing cells. (D) Adult testes were with immunostaining for Notch intracellular domain (red, Left) or anti-β-gal (for Notch reporter (Gbe-Su(h)LacZ) activity, right). Note that β-gal (green) is enriched in the cytoplasm of elongate d somatic cyst cells (arrows). (E) Testes from 2–3 days old males of the indicated genotypes were immunostained for Vasa (magenta) and Hub (red, white *), as well as DNA (blue). GSCs are marked by white dots. Scale bar 25 µm. (F) Quantification of GSC numbers. Note that the number of GSCs in N^spl−1; tara ⁰³⁸⁸¹/+ testes is significantly reduced, as determined by Kruskal–Wallis test.