Maintenance of Proper Germline Stem Cell Number Requires Adipocyte Collagen in Adult Drosophila Females

Abstract

Stem cells reside in specialized niches and are regulated by a variety of physiological inputs. Adipocytes influence whole-body physiology and stem cell lineages; however, the molecular mechanisms linking adipocytes to stem cells are poorly understood. Here, we report that collagen IV produced in adipocytes is transported to the ovary to maintain proper germline stem cell (GSC) number in adult Drosophila females. Adipocyte-derived collagen IV acts through β-integrin signaling to maintain normal levels of E-cadherin at the niche, thereby ensuring proper adhesion to GSCs. These findings demonstrate that extracellular matrix components produced in adipocytes can be transported to and incorporated into an established adult tissue to influence stem cell number.

Keywords: Drosophila; adipocytes; collagen IV; germline stem cells; oogenesis.

Figure 1

Collagen IV produced in adult adipocytes is required for GSC maintenance. (A) The Drosophila ovary is composed of 16–20 ovarioles that contain progressively older follicles. Each follicle consists of a germline cyst (one oocyte and 15 nurse cells) surrounded by somatic follicle cells (blue), and they are produced in the germarium at the anterior tip of each ovariole. (B) Each germarium contains two–three GSCs that reside in a somatic niche composed primarily of cap cells. GSCs divide asymmetrically to self-renew and generate a cystoblast that forms a 16-cell cyst. GSCs and germline cysts are identified based on the morphology and position of the fusome, a germline-specific organelle (de Cuevas and Spradling 1998). Follicle cells envelop the germline cyst to form a follicle. (C) The adult fat body (composed of adipocytes and hepatocyte-like oenocytes) surrounds the ovary. (D) Germaria at 14 days of adult adipocyte-specific Luc RNAi control or vkg RNAi. Vasa (red), germ cells; α-Spectrin (green), fusome; LamC (green), cap cell nuclear lamina; DAPI (blue), nuclei. GSCs are outlined. Bar, 10 µm. (E–G) Average number of GSCs per germarium over time of control, Cg25C RNAi (E), vkg RNAi (F), or SPARC RNAi (G) (mean ± SEM, * P < 0.05 and **** P < 0.0001, two-way ANOVA with interaction). See also Figures S1 and S2. GSC, germline stem cell; RNAi, RNA interference; st., stage.

Figure 2

Collagen IV is transported from adipocytes to the GSC region in adult females. (A) Experimental design for adipocyte-specific GFP RNAi and analysis of Vkg::GFP distribution. Vkg::GFP was knocked down specifically during development using the 3.1Lsp2-Gal4 driver and a previously described UAS-GFP shRNA. After eclosion, females were immediately dissected, maintained at 29° (to maintain GFP RNAi), or switched to 18° (GFP RNAi off). After 21 days, females at 29 or 18° were dissected and analyzed for GFP fluorescence intensity. (B) Adipocytes from newly eclosed females (0d) and 21-day old (21d) females maintained at 29 or 18° showing higher levels of Vkg::GFP (arrows) at 18° (when adipocyte-specific GFP RNAi is off). GFP (green), Vkg::GFP; DAPI (blue), nuclei. Vkg::GFP shown in grayscale in bottom panels. Arrowheads indicate trachea. Bar, 10 µm. (C) Box-and-whisker plot of mean Vkg::GFP intensity in adipocytes for experiment shown in (B). (D) Germaria from females as in (B) showing that higher levels of Vkg::GFP in the GSC niche region result specifically from Vkg::GFP expression in adult adipocytes. GFP (green), Vkg::GFP; α-Spectrin (red), fusome; LamC (red), cap cell nuclear lamina; DAPI (blue), nuclei. Bar, 5 µm. (E) Box-and-whisker plot of mean Vkg::GFP intensity in the GSC region for experiment shown in (D). (F) Representative images of stage 6–8 egg chambers from females showing that levels of Vkg::GFP in developing egg chambers do not depend on Vkg::GFP expression in adult adipocytes. Bar, 25 µm. (G) Box-and-whisker plot of mean Vkg::GFP intensity for experiment in (F). **** P < 0.0001, Mann–Whitney U-test. Sample sizes are included above. See also Figure S3. GSC, germline stem cell; n.s., not significant; RNAi, RNA interference; shRNA, short hairpin RNA; UAS, upstream activating sequence.

Figure 3

Adipocyte-derived collagen IV positively regulates E-cadherin levels at the GSC-niche interface through FAK. (A) Germaria from females at 10 days of adipocyte-specific Luc control or vkg^GD5246 RNAi. GFP (green), Dad::nlsGFP; LamC (red), cap cell nuclear lamina; α-Spectrin (red), fusome. Dad::nls shown in grayscale in panels on the right. GSC nuclei are outlined. Bar, 5 µm. (B) Box-and-whisker plot of mean Dad::nlsGFP intensity for experiment in (A). (C) Germaria from females described in (A). E-cadherin (red); LamC (green), cap cell nuclear lamina; α-Spectrin (green), fusome; DAPI (blue), nuclei. E-Cadherin shown in grayscale in panels on the right and asterisks indicate cap cells. Bar, 5 µm. (D) Box-and-whisker plot of mean E-cadherin intensity for experiment in (C). Sample sizes in (B and D) are included above boxes. **** P < 0.0001, Mann–Whitney U-test. (E) Germaria from females at 14 days of knockdown of Luc or Fak in the adult niche showing Vasa (germ cells, red), α-spectrin (fusome, green), LamC (nuclear lamina, green), and DAPI (DNA, blue). Bar, 10 µm. (F) Average number of GSCs per germarium at 0, 10, and 14 days of bab1^ts-mediated induction of RNAi against Fak or Luc (mean $\pm$ SEM; ** P < 0.01 and **** P < 0.0001, two-way ANOVA with interaction). (G) Germaria at 10 days of adipocyte-specific Luc or Fak RNAi labeled for E-cadherin (red), LamC (nuclear lamina, green), and α-spectrin (fusome, green). E-cadherin shown in grayscale in panels on the right and asterisks indicate cap cells. Bar, 2.5 µm. (H) Box-and-whisker plot of mean E-cadherin intensity. Sample sizes are included above. **** P < 0.0001, Mann–Whitney U-test. The number of germaria analyzed is shown above each box. See also Figure S4. FAK, focal adhesion kinase; GSC, germline stem cell; n.s., not significant; RNAi, RNA interference.

Figure 4

FAK interacts genetically with adipocyte collagen IV to regulate GSC numbers. (A) RT-PCR of Fak in w¹¹¹⁸ or Fak^KG00304 homozygous females showing that the Fak^KG00304 hypomorphic allele has reduced levels of Fak mRNA expression. Rp49 is used as a control. Normalized band intensities are indicated. (B) Frequencies of germaria containing zero-or-one, two, three, or four-or-more GSCs at 1 and 10 days of adipocyte-specific RNAi against Luc (control) or vkg RNAi in females carrying one copy of Fak^KG000304 compared to their balancer sibling controls without Fak^KG000304. The average number of GSCs per germarium is plotted in green. (C and D) Frequencies of germaria containing zero-or-one, two, three, or four-or-more GSCs in newly eclosed (0d) and 10-day-old (10d) double heterozygous dpp^e87 +/+ vkg⁰¹²⁰⁹, vkg⁰¹²⁰⁹ +/+ scb², or vkg⁰¹²⁰⁹ +/+ shg² females compared to their balancer sibling (single heterozygous) controls are shown on the left y-axis. The right y-axis shows the average number of GSCs per germarium. The number of germaria analyzed is shown inside bars. Mean $\pm$ SEM. * P < 0.05, ** P < 0.01, and **** P < 0.0001, two-way ANOVA with interaction. FAK, focal adhesion kinase; GSC, germline stem cell; n.s., difference in slopes not statistically significant; RNAi, RNA interference.