Germline self-renewal requires cyst stem cells and stat regulates niche adhesion in Drosophila testes - PubMed (original) (raw)

Germline self-renewal requires cyst stem cells and stat regulates niche adhesion in Drosophila testes

Judith L Leatherman et al. Nat Cell Biol. 2010 Aug.

Abstract

Adults maintain tissue-specific stem cells through niche signals. A model for niche function is the Drosophila melanogaster testis, where a small cluster of cells called the hub produce locally available signals that allow only adjacent cells to self-renew. We show here that the principal signalling pathway implicated in this niche, chemokine activation of STAT, does not primarily regulate self-renewal of germline stem cells (GSCs), but rather governs GSC adhesion to hub cells. In fact, GSC renewal does not require hub cell contact, as GSCs can be renewed solely by contact with the second resident stem cell population, somatic cyst stem cells (CySCs), and this involves BMP signalling. These data suggest a modified paradigm whereby the hub cells function as architects of the stem cell environment, drawing into proximity cellular components necessary for niche function. Self-renewal functions are shared by the hub cells and the CySCs. This work also reconciles key differences in GSC renewal between Drosophila testis and ovary niches, and highlights how a niche can coordinate the production of distinct lineages by having one stem cell type rely on a second.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Germline stat depletion does not compromise self-renewal

Germline stat depletion (A, D–G; Tj Gal4/UAS HA-stat; statF/stat85c9) or global stat depletion (B; statF/stat85c9) eight days after stat temperature inactivation. Hub (asterisk and Fas3, white); germ cells (vasa, magenta); DNA (blue). (A) Testes with germline stat depletion maintained CySCs (Zfh-1, green), and germ cells. (B) Testes with global stat depletion exhibited germ cell loss (lack of magenta cells) and CySC loss (Zfh-1, green; note, a few CySCs always persisted near the hub). (C) Global stat depletion led to progressive germ cell loss, first the more undifferentiated spermatogonia (blue; this category includes GSCs/single germ cells) followed by spermatocytes (orange), while most testes with germline stat depletion maintained spermatogonia (blue) indefinitely. Similar results were obtained with three CySC lineage Gal4 drivers and three strong stat alleles with the statts allele (Table 1). (D–G) _Stat-_depleted germ cells continually progressed through differentiation, shown by normal expression of the spermatogonial differentiation marker Bam (D, green), the appearance of branching fusomes through germ cell cysts (E, α-spectrin, green), synchronously cycling germ cell cysts (F, Edu pulse-labeled cells, green), and expression of the spermatocyte marker Ptb (G, green). Bar:10μm.

Figure 2

Figure 2. stat depleted GSCs become displaced from the hub

Germ cells (vasa, magenta), Hub (asterisk or Fas3, white). (A) Wild-type GSCs are tightly associated with the hub. (B) Two days after germline stat depletion (Tj Gal4/UAS HA-stat; statF/stat85c9), GSCs became displaced from the hub. (C–F) 8 days after germline stat depletion, germ cells nearest the hub accumulate M5-4 lacZ (C, white, arrows), have spherical or dumbbell-shaped spectrosomes (D, α-spectrin, green, arrows; D′ shows α-spectrin alone), and undergo mitosis as individual cells (E, phosphohistoneH3, green, arrow; a mitotic CySC is also present), all consistent with GSC identity. M5-4 lacZ (C, white) also accumulates in the hub (arrowheads), but not in CySCs (Tj, green), indicating CySCs do not take on hub fate. (F) _stat-_depleted GSCs fail to reliably orient mitotic spindles away from the hub (centrosomin reveals centrosomes, green). Two mitotic germ cells outlined, one oriented (arrow) and one non-oriented (arrowhead). (G) With germline stat depletion, the hub became surrounded by CySCs (Zfh-1, green). (H) Testes with Gal4-driven stat in the CySC lineage (Tj Gal4/UAS stat; stat85c9/+) undergo modest GSC loss, but still maintain GSCs at the hub (arrows, Zfh-1, green). (I) Germline RNAi knockdown of stat (nos gal4 VP16 VDRC 106980) caused GSCs to be displaced from the hub, and the hub became surrounded by CySCs (Zfh-1, green). Bar:10μm.

Figure 3

Figure 3. GSCs display hub adhesion defects prior to other signs of differentiation upon global stat depletion

(A) Most control GSCs (statF/stat85c9, 18° permissive temperature) have one centrosome oriented toward the hub interface (arrowheads, γ-tubulin (green) shows centrosomes; vasa (magenta) shows germ cells). (B) Upon stat depletion (sixteen hours after upshift) many GSC centrosomes were misoriented (arrows), while others were not (arrowheads). These GSCs are Bam-negative (A′ and B′, white shows Bam with E-cadherin marking the hub). (C) In control GSCs (Nos Gal4 VP16/UAS E-cadherinGFP; stat85c9/+, 16hrs 29°), E-cadherinGFP (green) localizes to the hub interface (vasa, magenta; fas3, white). (D) Upon sixteen hour stat depletion (Nos Gal4 VP16/UAS E-cadherinGFP; statF/stat85c9) E-cadherinGFP was lost (arrows) or highly reduced (arrowheads) from the hub interface. E-cadherinGFP only (C′, D′). (E) Germline Par3GFP (green) colocalizes with E-cadherin (magenta) at hub-GSC interface in controls (Nos Gal4 VP16/UAS Par3GFP; stat85c9/+, 16hrs 29°). (F) Upon sixteen hour stat depletion (Nos Gal4 VP16/UAS Par3GFP; statF/stat85c9) Par3GFP (green) delocalized, with only modest interface enrichment remaining. Quantified in Table S2. (G) Germline UAS E-cadherin expression in statts testes delayed germ cell loss compared to statts alone or with UAS Par3 expression. Asterisk denotes hub. Bar:10μm.

Figure 4

Figure 4. Rescued GSCs are activated for and require the BMP pathway

Germ cells (vasa, magenta), hub (asterisk). Eight days after germline stat depletion, the germ cells near the hub accumulated phospho-Smad (A, green, arrows; A′ pSmad alone) and Dad-lacZ (B, green, arrows; Zfh-1 in white; B′ Dad-lacZ alone). (C) Sustained zfh-1 expression in the cyst lineage (eyaA3 Gal4/UAS zfh-1; tubGal80ts; twelve days) generated excess GSCs (magenta) and these accumulated Dad-lacZ. DNA (blue). (D) Most testes with germline stat depletion retain spermatogonia (blue), but dampening the BMP pathway by coexpression of UAS sogCR1 (P<0.05 for 8d) or kekkon5 (P<0.0001 for 8d) reduced the proportion of testes with rescued germ cells. (E) Model for self-renewal in the testis niche. Bar:10μm.

References

    1. Tulina N, Matunis E. Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science. 2001;294:2546–9. - PubMed
    1. Kiger AA, Jones DL, Schulz C, Rogers MB, Fuller MT. Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue. Science. 2001;294:2542–5. - PubMed
    1. Leatherman JL, Dinardo S. Zfh-1 controls somatic stem cell self-renewal in the Drosophila testis and nonautonomously influences germline stem cell self-renewal. Cell Stem Cell. 2008;3:44–54. - PMC - PubMed
    1. Singh SR, et al. Competitiveness for the niche and mutual dependence of the germline and somatic stem cells in the Drosophila testis are regulated by the JAK/STAT signaling. J Cell Physiol. 223:500–10. - PMC - PubMed
    1. Hardy RW, Tokuyasu KT, Lindsley DL, Garavito M. The germinal proliferation center in the testis of Drosophila melanogaster. J Ultrastruct Res. 1979;69:180–90. - PubMed

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