Stem cell dynamics in response to nutrient availability

Abstract

When nutrient availability becomes limited, animals must actively adjust their metabolism to allocate limited resources and maintain tissue homeostasis. However, it is poorly understood how tissues maintained by adult stem cells respond to chronic changes in metabolism. To begin to address this question, we fed flies a diet lacking protein (protein starvation) and assayed both germline and intestinal stem cells. Our results revealed a decrease in stem cell proliferation and a reduction in stem cell number; however, a small pool of active stem cells remained. Upon refeeding, stem cell number increased dramatically, indicating that the remaining stem cells are competent to respond quickly to changes in nutritional status. Stem cell maintenance is critically dependent upon intrinsic and extrinsic factors that act to regulate stem cell behavior. Activation of the insulin/IGF signaling pathway in stem cells and adjacent support cells in the germline was sufficient to suppress stem cell loss during starvation. Therefore, our data indicate that stem cells can directly sense changes in the systemic environment to coordinate their behavior with the nutritional status of the animal, providing a paradigm for maintaining tissue homeostasis under metabolic stress.

Figure 1. Starvation causes loss of male GSCs, which is reversed upon re-feeding

(A) Schematic representation of the apical tip of the Drosophila testis. One cyst of germ cells is depicted progressing through mitotic amplification, meiosis and spermatid elongation. (B–C) Immunofluorescence images of testes stained with antibodies to Vasa (green) to mark germ cells and Fasiclin III (FasIII; red) to mark the hub. 4', 6-diamidino-2-phenylindole (DAPI) was used to highlight DNA (blue). Testes are from flies fed for 20 days (B) or starved for 20 days on 10% sucrose (C). (D–E) Immunofluorescence image of wild-type GFP⁺ clones in testes from flies fed (D) or starved (E) for 12 days. Testes are stained with antibodies to FasIII (red); GFP (clones), and DAPI (blue). In each testis, the number of GFP-positive GSCs was counted (arrow indicating GSC), and the number of marked cysts arising from the marked GSC was counted (arrows indicate marked clones numbered 1, 2 and 3) to assay proliferation. (F) Starvation paradigm used. Flies were analyzed following 15 or 20 days of starvation on 10% sucrose and compared to flies fed standard cornmeal molasses medium. Re-fed flies were starved for 15 days then fed for 5 days. (G) Quantification of GSCs, counted at 1 day, 15 and 20 days in fed and starved flies, and in re-fed flies (starved 15 days fed five days). Error bars: 95% confidence interval. Double asterisk: statistically significant difference using Student’s t-test (p<0.001). (H, I) Immunofluorescence images of testes stained with antibodies against Vasa to mark germ cells (green), FasIII (hub, red), and DAPI (blue). Testes are from flies starved for 15 days (H) or starved for 15 days then re-fed for 5 days (I). Scale bars, 20 µm.

Figure 2. Starvation induces loss of ISCs/EBs in the midgut, which is reversed upon re-feeding

(A) Schematic representation of cell types in the Drosophila posterior midgut. Division of an ISC produces one daughter cell that retains stem cell fate and another daughter cell that becomes an EB, both expressing Escargot (Esg). EBs do not divide again and differentiate into either large, polyploid enterocytes that constitute the majority of the gut epithelium or small, diploid enteroendocrine cells that express Prospero (Pros). (B) Quantification of ISCs/EBs using Esg-GFP. GFP⁺ ISCs/EBs were counted in midguts collected from newly eclosed males that were fed 1–2 days, then continuously fed or starved for 15 or 19 days, and from re-fed flies (starved 15 days then fed 4 days) as described in Experimental Procedures. n= total number of guts examined. Error bars represent 95% confidence interval of the mean. Asterisks indicate statistically significant difference using Student’s t-test (p<0.001). (C–E) Immunofluorescence images of posterior midguts from esg-GFP flies stained with antibodies against GFP to mark ISCs/EBs (green), antibodies against Arm to outline ISC/EB boundaries (red), and DAPI to mark DNA (blue). Guts are from flies fed for 15 days (C), starved for 15 days (D) or starved for 15 days then re-fed for 4 days (E). Scale bars, 20 µm. (F) Quantification of the average number of pHH3⁺ cells per posterior midgut under each feeding paradigm. Results are presented as 95% confidence interval of the mean. “n” in parentheses indicated total number of guts counted.

Figure 3. dInR is required for maintenance of male GSCs

(A–C) Immunofluorescence image of testes in which spermatogonia are expressing GFP (green; A,B), stained with antibodies that recognize dInR (red; A,C). dInR is expressed in germ cells including GSCs (GFP⁺, arrows), in somatic cells (GFP⁻, arrowheads) and in the hub (asterisk). Genotype: w; UAS-GFP^mCD8; nanos-GAL4. Scale bars, 20 µm. (D–G) Staining of control (FRT82B) (D,F) or dInR³³⁹/dInR³³⁹ (E,G) marked (GFP⁺) clones at 3 days (D,E) or 10 days (F,G) post-heat shock (PHS). FasIII (red) marks the hub, GFP (green) marks clones, and DAPI (blue) marks DNA. GFP channel alone is shown in insets. GSC clones (GFP⁺, arrows) are not maintained at 10 days PHS if homozygous for dInR³³⁹ (G). Scale bars, 10 µm. (H) Quantification of the number of testes that contain one or more GFP⁺ GSCs at 3 or 10 days PHS, compared to the total number of testes scored (shown in parentheses), as percentages for each genotype.

Figure 4. Simultaneous expression of activated dInR or Dp110 in the hub and germ cells suppresses GSC loss upon starvation

Average number of GSCs per testis from flies fed (dark green) or starved (light green) for 20 days were plotted. Studies are listed across the bottom. Genotypes, average GSC numbers, and the total number of testes analyzed for each study are summarized in Supplemental Table S1. We used nanos-GAL4 to drive expression of transgenes (UAS-dInR^CA and UAS-Dp110^CAAX) in germ cells; upd-GAL4, in hub cells; and a combination of the two drivers (upd, nanos-GAL4) to confer simultaneous expression in both germ cells and hub cells. The difference between fed and starved flies with simultaneous expression of dInR^CA or Dp110^CAAX in the hub and germ cells is significantly smaller than for either of their control studies [analyzed by two way analysis of variance (ANOVA), p<0.005]. Asterisks indicate statistically significant differences at p<0.005. Error bars represent 95% confidence interval of the mean.