Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues

Abstract

Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on loss of access to these signals. Here, we demonstrate that the differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot differentiate properly. We find that an insulin peptide produced by somatic cells immediately outside of the stem cell niche acts locally to promote somatic differentiation through Insulin-like receptor (InR) activation. These results indicate that there is a local 'differentiation' niche that upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, we demonstrate that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, we show that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that the local production of positive and negative InR signals regulates the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation are actively induced by signaling.

Keywords: Drosophila; Insulin; PI3K; Stem cell differentiation; Testis; Tor.

Fig. 1.

PI3K and Tor activity are observed during CySC differentiation. (A) Simplified model of the PI3K/Tor pathway. Here, PI3K is activated by the insulin receptor (InR) tyrosine kinase following binding of a Drosophila insulin-like peptide (Dilp). InR phosphorylates PI3K, which in turn phosphorylates PIP₂ lipids to create PIP₃. Pten is a lipid phosphatase that catalyzes the reverse reaction. Subsequently, PIP₃ activates Akt, which inactivates the Tor inhibitor Tsc1/2. The TORC1 complex is then activated and leads to the phosphorylation of S6K and 4E-BP. The secreted Dilp antagonist ImpL2 blocks Dilps from binding to InR. Phosphorylation events are indicated by the yellow symbols. (B) The PH-GFP reporter of PIP₃ consists of GFP fused to the pleckstrin homology (PH) domain of Grp1, which binds PIP₃. This GFP fusion is cytoplasmic, but when PIP₃ levels are high it translocates to the plasma membrane. (C) The PI3K reporter PH-GFP (green) was predominantly cytoplasmic in CySCs around the hub, indicating low PI3K activity. Arrow in C′,C‴ marks a CySC with low membrane PH-GFP. Membrane-associated PH-GFP was observed as cyst cells begin to differentiate, one further cell diameter away from the hub. Arrowheads in C′,C‴ mark differentiating cyst cells that have upregulated membrane PH-GFP. Tj (blue and C‴) labels CySCs and early cyst cells, Dlg (red and C″) marks somatic cell membranes. (D) The Tor activity reporter p4E-BP (green) was detected at high levels in somatic cells adjacent to stem cells. Arrow in D-D″ marks a Zfh1-positive CySC next to the niche that has low p4E-BP. Arrowhead in D-D″ labels a Zfh1-positive early CySC daughter cell primed for differentiation that is one cell diameter away from the CySCs and that has high levels of p4E-BP. Arrowhead in D‴ marks an Eya-positive differentiating cyst cell. Fas3 (blue and D‴) marks the hub; CySCs are the Zfh1-positive cells (red and D″) that are closest to the hub. Dotted red line outlines the hub.

Fig. 2.

Dp110 and Akt1 are required autonomously for CySC differentiation. MARCM clones labeled by GFP expression. (A,B,D) Control (A) and Dp110 (B) or Akt1 (D) mutant clones marked with membrane-targeted GFP (green and A′,B′,D′). Vasa (red and A″,B″,D″) labels germ cells and Tj (blue and A‴,B‴,D‴) marks somatic cells. At 7 dpci, control clones (A) contained labeled CySCs contacting the hub (A′, arrow) and differentiated cyst cells ensheathing large germline cysts (A′, arrowheads). By contrast, Dp110 (B′,B‴, arrows) and Akt1 (D′,D‴, arrow) mutant clones contained labeled CySCs contacting the hub but lacked labeled differentiated daughter cells. Note that in B only part of the hub is in the plane. (C,E) Dp110 (C) or Akt1 (E) mutant clones marked with nuclear GFP (green and C′,E′, arrows). Mutant cells expressed the CySC marker Zfh1 (red and C″,E″, arrows) and lacked expression of the differentiation marker Eya (blue and C‴,E‴). The hub is outlined by a red dotted line.

Fig. 3.

Tor is required autonomously for CySC differentiation. (A) Control nuclear GFP-labeled clone at 7 dpci showing labeled CySCs adjacent to the hub (A′, arrow) and differentiated cyst cells that have moved away from the hub (A′, arrowhead). (A′) A maximum projection of the confocal stack to display all the labeled GFP-positive cells in this sample. (B) Tor^ΔP mutant clone at 7 dpci. All the mutant cells (B′, arrows) in the clone remained close to the hub, as seen in the maximum projection in B′. (C) Quantification of the distance between marked cells in clones and the hub. Tor^ΔP clones remained significantly closer to the hub than control clones (P<1×10⁻⁴, Mann–Whitney). (D) Preventing cell death in Tor^ΔP clones at 7 dpci by expressing the caspase inhibitor P35 does not lead to the recovery of differentiated cells (D′, arrows). (E) Control clones contained both Zfh1-expressing CySCs (E′,E‴, arrow) and Eya-expressing differentiated cyst cells (E′,E″, arrowhead). (F) By contrast, Tor^ΔP mutant clones contained only Zfh1-expressing cells (F′,F‴, arrow) and no Eya-expressing cells (F″). The hub is outlined with a red dotted line.

Fig. 4.

PI3K or TORC1 pathway knockdown in the somatic lineage prevents CySC differentiation. (A) A Control (Tj>+) testis showing Zfh1 (green and A′) in CySCs and early cyst cells near the hub and Eya (blue) in differentiated cyst cells (A‴, arrowhead). Vasa (red and A″) labels the germ line. Germ cells grow larger as they differentiate and move away from the hub. (B) Akt1 knockdown in the somatic lineage led to expansion of the Zfh1-expression domain (green and B′) away from the hub, loss of Eya expression (blue and B‴) and prevented normal germ cell development, as only small germ cells were seen (red and B″). (C) Tor knockdown in somatic cells by RNAi. Many Zfh1-expressing cells (green and C′) were seen far from the hub, whereas Eya expression was absent (blue and C‴). Germ cell differentiation was also disrupted (C″). (D) Knockdown of the TORC1 component Raptor caused a similar phenotype to Akt1 and Tor knockdown. Zfh1 expression (D′) was maintained in cells distant from the hub, whereas Eya was absent (D‴) and germ cell differentiation was blocked (D″). (E) Feeding flies the TORC1 inhibitor rapamycin prevented differentiation, as Eya expression was absent (E‴) whereas Zfh1 (E′) expanded and germ cells (E″) did not differentiate properly. (F) Hyperactivating Tor by knockdown of Tsc1 rescued the loss of differentiation observed when Akt1 is knocked down by RNAi. Note that in contrast to Akt1 knockdown alone (B), concurrent knockdown of Tsc1 leads to robust Eya expression (F‴) and rescue of germ cell differentiation. The hub is outlined with a red dotted line.

Fig. 5.

PI3K/Tor pathway knockdown in the somatic lineage blocks quiescence. (A) In a control (Tj>+) testis, only CySCs near the hub underwent S-phase, as revealed by EdU incorporation (A-A‴, arrows). (B,C) Like control testes, EdU incorporation was observed in CySCs next to the niche in testes with somatic knockdown of Tor or Raptor (B-C‴, arrows), but unlike control testes EdU incorporation was also observed in somatic cells far from the hub (B-C‴, arrowheads). Tj is green, EdU is red and Vasa is blue in A-C. The hub is outlined with a red dotted line.

Fig. 6.

Localized Dilp6 expression promotes CySC differentiation. (A) Dilp6-Gal4 driving expression of UAS-GFP in a control testis. GFP (green and A′) is expressed in differentiating cyst cells. Vasa (red and A″) marks germ cells and Tj (blue and A‴) labels somatic cells. (B) Dilp6>GFP (green and B′) expression is partially overlapping with p4E-BP (red and B″), suggesting that Dilp6 could act in a juxtacrine or autocrine manner. Tj (blue and B‴) labels somatic cells. Arrowhead in B-B‴ indicates a Tj-positive somatic cell that expresses the Dilp6 driver and exhibits p4E-BP staining. (C) A control (Tj>+) testis stained with Zfh1 (green and C′). (D) Knockdown of Dilp6 in somatic cells results in an accumulation of Zfh1-expressing cells (green and D′). In C,D, Vasa is red and Eya and Fas3 are blue. More Zfh1-positive cells are seen in a Tj>Dilp6 RNAi testis (D) than in a control (C). (E,F) p4E-BP staining (green and E′,F′) in control (E) or dilp6 mutant (F) testes. Note the reduction in both the width of the p4E-BP domain and the levels of staining (brackets in E′,F′). Tj (red and E″,F″) labels somatic cells and Fas3 (blue and E‴,F‴) marks the hub. The hub is outlined with a dotted line.

Fig. 7.

CySCs produce ImpL2 to antagonize local Dilp signaling in stem cells. (A) ImpL2-GFP expression (green) is detected in CySCs contacting the hub (A′,A‴, arrows), in a pattern complementary to p4E-BP (red and A″). Tj (blue and A‴) marks somatic cells. (B,C) p4E-BP levels (green and B′,C′) are altered in testes with somatic depletion of ImpL2. Tj (red) marks somatic cells and Fas3 (blue) labels the hub. p4E-BP is detected in differentiating cyst cells, two cell diameters away from the hub in a control testis (B). However, high levels of p4E-BP are detected in CySCs immediately adjacent to the hub when ImpL2 is knocked down (C′, arrow). (D) Somatic overexpression of ImpL2 results in an accumulation of Zfh1-expressing cells (green and D′) and a delay in the onset of Eya expression (blue and D‴). Vasa labels germ cells (red). The hub is outlined with a dotted line.