Insulin signaling acts in adult adipocytes via GSK-3β and independently of FOXO to control Drosophila female germline stem cell numbers

Abstract

Tissue-specific stem cells are tied to the nutritional and physiological environment of adult organisms. Adipocytes have key endocrine and nutrient-sensing roles and have emerged as major players in relaying dietary information to regulate other organs. For example, previous studies in Drosophila melanogaster revealed that amino acid sensing as well as diet-dependent metabolic pathways function in adipocytes to influence the maintenance of female germline stem cells (GSCs). How nutrient-sensing pathways acting within adipocytes influence adult stem cell lineages, however, is just beginning to be elucidated. Here, we report that insulin/insulin-like growth factor signaling in adipocytes promotes GSC maintenance, early germline cyst survival, and vitellogenesis. Further, adipocytes use distinct mechanisms downstream of insulin receptor activation to control these aspects of oogenesis, all of which are independent of FOXO. We find that GSC maintenance is modulated by Akt1 through GSK-3β, early germline cyst survival is downstream of adipocyte Akt1 but independent of GSK-3β, and vitellogenesis is regulated through an Akt1-independent pathway in adipocytes. These results indicate that, in addition to employing different types of nutrient sensing, adipocytes can use distinct axes of a single nutrient-sensing pathway to regulate multiple stages of the GSC lineage in the ovary.

Keywords: Adipocytes; Drosophila; GSK-3β; Germline stem cells; Insulin signaling; Oogenesis.

Fig. 1

Insulin signaling in adult adipocytes cell autonomously controls cell size. (A) Diagram illustrating that adipocytes and oenocytes constitute the Drosophila fat body, which underlies the cuticle and surrounds the ovaries (composed of ovarioles). (B) Diagram of ovariole showing the anterior germarium (g) and developing egg chambers (or follicles), each composed of a germline cyst (one oocyte and 15 nurse cells) surrounded by follicle cells. “Pre-vitellogenic” and “vitellogenic” refer to stages prior to or after the onset of yolk uptake by the oocyte, respectively. (C) Diagram of germarium showing GSCs housed in direct contact with cap cells. GSCs generate cystoblasts, which in turn incompletely divide four times to form germline cysts containing 16 interconnected cells. The position and morphology of a special organelle, the fusome (orange), allows for the identification of GSCs, cystoblasts, two-, four-, eight-, and 16-cell germline cysts. (D–F) Adipocytes from females subjected to adipocyte-specific manipulation of insulin signaling. Gal80^ts; Lsp2-Gal4 drove expression of a UAS-nuclear GFP reporter in combination with other UAS transgenes (see Table S1), and females were raised at 18°C and switched to 29°C four days after eclosion for 10 days to induce transgene expression. Nuclear GFP intensity varies between adipocytes indicative of variable driver expression. Adipocytes with relatively robust nuclear GFP expression (arrowheads indicate some examples) were used for quantification shown in (G). GFP (green); E-cadherin (red), cell membranes. Scale bar, 25 μm. (G) Average cell area of adipocytes upon adult adipocyte-specific expression of various transgenes at 29°C for 10 days. GFP^dsRNA was used as RNAi control. Mean + s.e.m. Number of adipocytes quantified are shown inside bars. ****P < 0.0001; ordinary one-way ANOVA. See supplementary material Fig. S1 for additional transgenes tested.

Fig. 2

Inhibition of insulin signaling in adult adipocytes leads to increased rates of GSC loss. (A) Average number of GSCs per germarium at 0 or 10 days of adult adipocyte-specific RNAi against InR or Akt1 compared to GFP RNAi control or overexpression of Pten compared to Gal80^ts; Lsp2-Gal4 only control. Mean + s.e.m. *P < 0.05, **P < 0.01; two-way ANOVA with interaction. (B) Graph showing frequencies of germaria containing zero-or-one, two, or three-or-four GSCs representing the same data used to calculate average GSC number in (A). Number of germaria analyzed indicated inside bars. (C–F) Scatter plot of total E-cadherin intensity at the cap cell-GSC junction (C) with representative images for adult adipocyte-specific RNAi against GFP control (D), InR (E) or Akt1 (F). Mean + s.e.m. Scale bar, 2.5 μm. ****P < 0.0001, no statistically significant difference between Akt1 and control GFP knockdown; ordinary one-way ANOVA. (G–J) Mean nuclear phosphorylated Mad (pMad) intensity in GSCs (G) with representative images for adult adipocyte-specific RNAi against GFP control (H), InR (I) or Akt1 (J). Mean + s.e.m. No statistically significant differences; ordinary one-way ANOVA. Number of GSCs analyzed indicated below each data set (C and G). 1B1 (red), fusome; Lamin C (red), cap cell nuclear envelopes; E-cadherin (green, D–F); pMad (green, H–J). Scale bar, 5 μm. Asterisks indicate cap cells and visible portions of GSCs are outlined (D–F); GSC nuclei are outlined in (H–J).

Fig. 3

Reduced insulin signaling in adult adipocytes causes increased death of early germline cysts and degeneration of vitellogenic follicles. (A–C) Germaria from females at 10 days of adult adipocyte-specific knockdown of GFP control (A), InR (B) or Akt1 (C). 1B1 (red), fusome; Lamin C (red), cap cell nuclear envelopes; Cleaved Dcp-1 (green), dying germline cysts. Scale bar, 10 μm. (D) Percentage of germaria with Dcp-1-positive germline. Hatched and solid regions indicate the percentage of germaria with two or more Dcp-1-positive germline cysts, or just one dying cyst, respectively. Data combine three replicate experiments each for control GFP and InR knockdown and two replicate experiments for Akt1 knockdown. Mean + s.e.m. Total number of germaria analyzed are shown inside bars. **P < 0.01, ***P < 0.001; ordinary one-way ANOVA. (E–G) DAPI-stained ovarioles from GFP control (E), InR (F), and Akt1 (G) knockdown as described in (A–C). Arrow indicates pyknotic nuclei in degenerating follicle. Scale bar, 100 μm. (H) Percentage of ovarioles with degenerating vitellogenic follicles. Mean + s.e.m. Data combine nine replicate experiments each for control GFP and InR knockdown and four replicate experiments for Akt1 knockdown. Total number of ovarioles analyzed are shown inside bars. ****P < 0.0001; ordinary one-way ANOVA.

Fig. 4

Insulin signaling in adult adipocytes does not regulate insulin-like peptide secretion or systemic insulin signaling. (A–C) Clusters of median neurosecretory cells from females carrying ilp5-lacZ (a marker for insulin-producing cells) at 10 days of adult adipocyte-specific knockdown of GFP control (A), InR (B) or Akt1 (C). ILP2 (green); βgal (red), encoded by the ilp5-lacZ transgene. ILP2 shown in grayscale in (A′-C′). Scale bar, 20 μm. (D) Quantification of total ILP2 fluorescence intensity in insulin-producing cells. Mean + s.e.m. Number of adult brains quantified are shown inside bars. No statistically significant differences; ordinary one-way ANOVA. (E–G) Nurse cells of stage 10 follicles from females as described in (A–C) but carrying the tGPH reporter instead of ilp5-lacZ. In the tGPH reporter, GFP is fused to a pleckstrin homology domain such that under conditions of high insulin signaling, PI3K activity recruits GFP to the plasma membrane (Britton et al., 2002). GFP shown in grayscale. Scale bar, 50 μm. (H) Quantification of average membrane-to-cytoplasmic GFP ratio. Mean + s.e.m. Number of follicles quantified are shown inside bars. No statistically significant differences; ordinary one-way ANOVA. See supplementary material Fig. S3 for an explanation of the quantification method.

Fig. 5

GSK-3β, but not FOXO, function is required in adipocytes for proper maintenance of GSCs, whereas early cyst survival and vitellogenesis do not require either insulin signaling effector. (A and B) Adipocytes from females at 10 days of adult adipocyte-specific control (A; lacking UAS transgene) or foxo overexpression (B). FOXO immunoreactivity shows that adipocytes with high levels of FOXO expression have markedly reduced cell sizes (arrows). FOXO (green); E-cadherin (red), cell membranes; DAPI (blue), nuclei. Scale bar, 25 μm. (C and D) Average number of GSCs per germarium at 0 or 10 days of adult adipocyte-specific overexpression of foxo (C) or constitutively active sgg (sgg^S9A) (D) compared to Gal80^ts; Lsp2-Gal4-only control. Mean + s.e.m. *P < 0.05; two-way ANOVA with interaction. See supplementary material Fig. S4 for sample sizes and distribution. (E and F) Percentage of germaria with cleaved Dcp1-positive germline cysts (E) or ovarioles with degenerating vitellogenic follicles (F) in females overexpressing foxo or sgg^S9A in adult adipocytes as described in (A and B). Mean + s.e.m. Total number of germaria (E) or ovarioles (F) quantified are shown inside bars. *P < 0.05; ordinary one-way ANOVA.

Fig. 6

Model for how insulin signaling within adipocytes regulates the GSC lineage. Our previous published work showed that insulin signaling within the ovary (grey arrows) controls GSC maintenance (through the niche) and proliferation, follicle growth, and vitellogenesis (reviewed in Laws and Drummond-Barbosa, 2017). In this study, we show that insulin signaling within adult adipocytes controls multiple steps of oogenesis through distinct effectors: 1) GSC maintenance is modulated by the InR/Akt1/GSK-3β axis (red); 2) early germline cyst survival downstream of Akt1 (blue) and Akt1-independent progression through vitellogenesis (purple) are controlled by as-yet-unidentified downstream effectors of the insulin signaling pathway. As reported previously (DiAngelo and Birnbaum, 2009), the InR/Akt1/FOXO axis controls adipocyte growth (orange).