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. 2012 Dec 1;372(1):5-16.
doi: 10.1016/j.ydbio.2012.09.009. Epub 2012 Sep 23.

Jak-STAT regulation of cyst stem cell development in the Drosophila testis

D Sinden  1 , M Badgett, J Fry, T Jones, R Palmen, X Sheng, A Simmons, E Matunis, M Wawersik
Affiliations

Jak-STAT regulation of cyst stem cell development in the Drosophila testis

D Sinden et al. Dev Biol. .

Abstract

Establishment and maintenance of functional stem cells is critical for organ development and tissue homeostasis. Little is known about the mechanisms underlying stem establishment during organogenesis. Drosophila testes are among the most thoroughly characterized systems for studying stem cell behavior, with germline stem cells (GSCs) and somatic cyst stem cells (CySCs) cohabiting a discrete stem cell niche at the testis apex. GSCs and CySCs are arrayed around hub cells that also comprise the niche and communication between hub cells, GSCs, and CySCs regulates the balance between stem cell maintenance and differentiation. Recent data has shown that functional, asymmetrically dividing GSCs are first established at ∼23 h after egg laying during Drosophila testis morphogenesis (Sheng et al., 2009). This process correlates with coalescence of the hub, but development of CySCs from somatic gonadal precursors (SGPs) was not examined. Here, we show that functional CySCs are present at the time of GSC establishment, and that Jak-STAT signaling is necessary and sufficient for CySC maintenance shortly thereafter. Furthermore, hyper-activation of Jak in CySCs promotes expansion of the GSC population, while ectopic Jak activation in the germline induces GSC gene expression in GSC daughter cells but does not prevent spermatogenic differentiation. Together, these observations indicate that, similar to adult testes, Jak-STAT signaling from the hub acts on both GSCs and CySC to regulate their development and differentiation, and that additional signaling from CySCs to the GSCs play a dominant role in controlling GSC maintenance during niche formation.

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Figures

Figure 1
Figure 1. Cyst stem cells are present at mid- larval first instar
Adult and mid-first instar larval testes immunostained with anti-Vasa (red) to detect germ cells, anti-Fasciclin 3 (Fas 3; A, B, E, F; blue) to detect the hub, and either anti-ZFH-1 (A, B, green; A', B' alone), anti-β-galactosidase (C, D, green; C', D' alone) to reveal Socs36E-PZ expression, or anti-GFP (G, H, green; E', F' alone). Testis apex/anterior oriented to the left in all images. Hub (yellow dashed lines) and testes (white dotted lines) outlined. [A, B] Adult (A) and mid-L1 (B) testes with high-level ZFH-1 expression (yellow arrows) restricted to nuclei of somatic cells immediately adjacent to the hub in adult, and lower-level ZFH-1 (yellow arrowheads) observed in somatic cells 2–3 cell layers away from the hub. ZFH-1 is also detected in somatic cells surrounding the testis (white arrowheads) at mid-L1. [C, D] Adult (C) and mid-L1 (D) testes with Socs36E-PZ expression detected strongly in the hub, at lower levels in CySCs (yellow arrows), and in somatic cells surrounding the testes (white arrowheads). Location of the hub was confirmed by Fas 3 expression (not shown). [E, F] Adult (E) and mid-L1 (F) testes with expression of a plasma membrane-tethered GFP transgene (UAS-mCD8∷GFP) driven by a somatic driver (c587-Gal4) observed in cells that interdigitate GSCs (yellow arrows). Scale bars at 10 μm.
Figure 2
Figure 2. CySC reporters are detected at the embryo-larval transition
Late-stage embryonic and first instar larval testes immunostained with anti-Vasa (A–H; red), either anti-ZFH-1 (A–D, green; A'–D' alone) and anti-Traffic Jam (TJ; A–D, blue; A”–D” alone) to detect somatic gonadal cells, or anti-β-galactosidase (E–H, green; E'–H' alone) to reveal Socs36E-PZ expression. Testes are from early-mid stage 17 embryos (~19 hrs AEL; A, E), stage 17-late/early L1 (~23 hrs AEL; B, F), early- 1st instar larvae (~36 hrs AEL; D, H). All images oriented with testis anterior to the left. Hub (yellow dashed lines) and testes (while dotted line) are outlined. [A–D] Early-mid stage 17 testis (A) with uniform ZFH-1 expression in SGPs that co-express TJ, and ZFH-1 expressed in likely msSGPs that are TJ negative (yellow arrow). Testis at the embryo-larval transition (B) with ZFH-1 expression enriched in somatic cells located adjacent to the hub (yellow arrowheads). Early- L1 (C) and mid-L1 (D) testes with high-level ZFH-1 restricted to hub-adjacent somatic cells (yellow arrowheads), and lower-level ZFH-1 detected in hub cells and also in somatic cells 2–3 cell diameters away from the hub (white arrows). [E–H] Early-mid stage 17 testis (E) with low-level Socs36E-PZ detected in somatic cells clustered at the testes anterior (yellow arrow). Testes at the embryo-larval transition (F), early-L1 (G) and mid-L1 (H) with low-level Socs36E-PZ (yellow arrowheads) in somatic cells immediately adjacent to hub cells that show high-level Socs36E-PZ expression.
Figure 3
Figure 3. CySC division and cyst differentiation are observed by mid-L1
Late-stage embryonic and 1st instar larval testes immunostained with anti-Vasa (A–D, red; E, F, blue), either anti-Eyes Absent (EYA; A–D, green) to detect cyst cells and msSGPs or anti-phosphorylated-Histone-H3 (pHH3; E, F, green; E', F' alone) to detect mitotic chromatin, and either anti-N-Cadherin (N-Cad; A–D, blue) to detect the hub or anti-TJ (TJ; E, F, red; E”, F”, alone) to detect somatic gonadal cells. Images oriented with anterior to the left. Hub (yellow dashed lines) and testes (white dotted lines) outlined. [A–D] Testes at the embryo-larval transition (A) and early-L1 (B) with EYA expressed in male-specific somatic gonadal precursors (msSGPs; white arrows). Mid-L1 (C) and late-L1 (D) testes with EYA detected in somatic nuclei in the posterior half of the gonad (white arrowheads) where spermatogonia are known to form. [E, F] Testes at the embryo-larval transition (E) and mid-L1 (F) with pHH3 detected in somatic cells adjacent to the hub (yellow arrows). [G] Graph showing the percentage of pHH3/TJ double positive testes over time with pHH3 detected in somatic cells adjacent to the developing hub (in the anterior half of the testes in early-mid stage 17 embryos) or in cells elsewhere in the testes (Non-Adjacent). P-values from Chi-squared analyses are shown.
Figure 4
Figure 4. Jak-STAT signaling is necessary and sufficient for CySC maintenance in larval testes
Late-stage embryos and 1st instar larval testes immunostained with anti-Vasa (A–F, red; E'”& F'” alone), anti-ZFH-1 (A–F, green; A'–D', in black and white with GFP; E' & F' alone), and either anti-GFP (A–D, green; A' & B' in black and white with ZFH-1) to determine genotype of Stat92E06346 mutants (see methods) or anti-EYA (E & F, blue; E” & F” alone) or. All images with testis anterior oriented left. Hub (yellow dashed lines) and testes (white dotted lines) outlined. [A, B] mid-L1 testes from (A) a Stat92E06346 homozygous mutant lacking high-level ZFH-1 expression in somatic cells around the hub and from (B) a sibling controls with ZFH-1 enriched in hub-adjacent cells (yellow arrowheads). [C, D] stage 17-late/L1 early testes from (A) a Stat92E06346 homozygous mutant and (B) a sibling control both showing high-level ZFH-1 in somatic cells adjacent to the hub (yellow arrowheads). [E, F] Late-L1 testes with Jak hyper-activated in the somatic gonad (E; c587-Gal4/+;UAS-hopTumL/+) show expanded expression of high-level ZFH-1 and no EYA expression in cyst cells, while age-matched sibling controls (F; c587-Gal4/+;CyO/+) show high-level ZFH-1 restricted to hub-adjacent CySCs and EYA expression in somatic nuclei interspersed throughout the testis posterior.
Figure 5
Figure 5. Impact of germline vs. somatic Jak-STAT activation on GSC maintenance and gene expression
Late- 1st instar and early 2nd instar larval testes immunostained with anti-Vasa (A–F, red; A”–F” alone), and either anti-1B1 E' alone) to reveal fusomes, or anti-(β-galactosidase (C, D, & F, green; C', D' & F' alone) to reveal expression of the enhancer trap Mgml. All images with testis anterior oriented left. Hub (yellow dashed lines) and testes (white dotted lines) outlined. [A, B] Early-L2 testis with Jak hyper-activated in somatic cells (A; c587-Gal4/+;UAS-hopTumL/+) showing germ cells with rounded fusomes (yellow arrows) and aberrant branching (yellow arrowheads) in the testis posterior, whereas elongation and branching of fusomes (yellow double-arrows) is observed in posterior germ cells of an age-matched sibling control (B; c587-Gal4/+; CyO/+). [C, D] Late-Ll testis after somatic Jak hyper-activation (C; c587-Gal4/+; Mgm1/UAS-hopTumL) showing Mgm1 expression in a small subset of germ cells at the testes posterior (yellow arrowheads), while Mgml expression is restricted to GSCs and early gonialblasts in an age-matched sibling control (D; c587-Gal4/+; Mgm1/+). [E, F] Early-L1 testis with Jak hyper-activated specifically in the germline (E, F; UAS-hopTumL/Mgml; nanos-Gal4/+) showing (E) normal germ cell differentiation with spherical fusomes in GSCs and gonialblasts adjacent to the hub, and branched fusomes (yellow double-arrows) in distally-localized spermatogonia, as well as (F) expression of Mgml in germ cells throughout the testis, including differentiating spermatogonia at the testis posterior (yellow arrowheads).
Figure 6
Figure 6. Model of testis stem cell development
(left gonad) Jak-STAT activation represses SGP differentiation and specifies male GSC fate after gonad formation. (middle gonad) Hub morphogenesis results in restricted expression of UPD to the testes anterior and Jak-STAT activation only in germ cells and somatic cells adjacent the hub. Restriction of Jak-STAT activation in germ cells promotes formation of adherens junctions between GSCs and hub cells that are required for orienting germ cell divisions away from the hub, while restricted Jak-STAT activation in somatic cells promotes CySC behavior and represses cyst differentiation. (right gonad) Somatic cells away from the hub that lack Jak-STAT activation differentiate into cyst cells that act in pairs to promote spermatogonial differentiation, while CySCs adjacent to the hub repress differentiation of neighboring GSCs and divide asymmetrically to produce more cyst cells. Fusomes and spectrosomes are shown in dark green, cell adhesions shown in red, and the mitotic spindle of diving cells shown in maroon.
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