The nuclear receptor seven up functions in adipocytes and oenocytes to control distinct steps of Drosophila oogenesis

Abstract

Reproduction is intimately linked to the physiology of an organism. Nuclear receptors are widely expressed transcription factors that mediate the effects of many circulating molecules on physiology and reproduction. While multiple studies have focused on the roles of nuclear receptors intrinsically in the ovary, it remains largely unknown how the actions of nuclear receptors in peripheral tissues influence oogenesis. We identified the nuclear receptor encoded by svp as a novel regulator of oogenesis in adult Drosophila. Global somatic knockdown of svp reduces egg production by increasing GSC loss, death of early germline cysts, and degeneration of vitellogenic follicles. Tissue-specific knockdown experiments revealed that svp remotely controls these different steps of oogenesis through separate mechanisms involving distinct tissues. Specifically, adipocyte-specific svp knockdown impairs GSC maintenance and early germline cyst survival, whereas oenocyte-specific svp knockdown increases the death of vitellogenic follicles without any effects on GSCs or early cysts. These results illustrate that nuclear receptors can control reproduction through a variety of mechanisms involving peripheral tissues.

Keywords: Adipocytes; Drosophila; Germline; Oenocytes; Oogenesis; Seven up.

Fig. 1.. svp is required in somatic cells of adult females for normal egg production.

(A) The Drosophila ovary has 16-20 ovarioles, each comprising an anterior germarium followed by developing follicles arranged in chronological order. Each follicle consists of a germline cyst (15 nurse cells and one oocyte; light blue) surrounded by somatic follicle cells (brown). (B) The germarium contains 2-3 germline stem cells (GSCs; dark blue) residing in a niche composed primarily of cap cells (orange). Each asymmetric GSC division renews the GSC and gives rise to a cystoblast that further divides incompletely to form a 16-cell cyst. GSCs and germline cysts are identified based on their characteristic fusome morphology (pink), a germline-specific organelle. Follicle cells (brown) surround the developing cyst to bud a new follicle. (C) Females carrying Gal80^ts; tub-Gal4 (tub-Gal4^ts) and UAS-hairpin transgenes against Luc (for control RNAi) or svp raised at 18°C were switched to 29°C (in the presence of y w males) for adult-specific ubiquitous somatic RNAi knockdown for five, 10, or 15 days. Knockdown of svp caused a significant decrease in the average number of eggs laid per female per day. (D) Control females carrying UAS-hairpin transgenes against Luc control or svp in the absence of tub-Gal4^ts were subjected to the same conditions as in (C), showing that the UAS alone does not alter egg-laying rates. Note that UAS alone controls were tested in a separate experiment than (C). Variations among experiments (e.g. batch of yeast paste) may lead to variations in egg counts; therefore, genotypes should be compared within the same experiment. Data shown as mean±SEM. ***P<0.001, two-tailed Student’s t test.

Fig. 2.. Ubiquitous somatic knockdown of svp in adult females increases the rate of GSC loss.

(A,B) Germaria from females at 14 days of adult-specific ubiquitous somatic RNAi knockdown of Luc (control) or svp. Vasa (red), germ cells; α-Spectrin (green), fusome; LamC (green), cap cell nuclear lamina; DAPI (blue), nuclei. GSCs are outlined. Scale bar, 10 μm. (C,D) Average number of GSCs (C) or cap cells (D) per germarium of females with tub-Gal4^ts-driven control or svp RNAi over time. Data shown as mean±SEM. *P<0.05; ****P<0.0001, two-way ANOVA with interaction.

Fig. 3.. svp functions in somatic cells of adult females to promote survival of early germline cysts.

(A,B) Germaria from females at 10 days of adult-specific ubiquitous somatic RNAi against Luc (control) (A) or svp (B). α-Spectrin (red), fusome; LamC (red), cap cell nuclear lamina; ApopTag (green), dying cells; DAPI (blue), nuclei. Scale bar, 10 μm. (C) Average percentage of germaria containing ApopTag-positive dying cysts in Regions 1 or 2 from adult females at zero and 10 days of ubiquitous somatic RNAi against Luc (control) or svp. Data shown as mean+SEM. **P < 0.01. The numbers of germaria analyzed are shown above bars.

Fig. 4.. Ubiquitous somatic knockdown of svp causes death of vitellogenic follicles.

(A,B) Ovarioles at 10 days of adult specific ubiquitous Luc RNAi (A) or svp RNAi (B) stained with DAPI (white, nuclei). Scale bar, 50 μm. Arrow points to a dying vitellogenic follicle. (C) Average percentage of dying ovarioles observed by pyknotic nuclei. Data shown as mean+SEM. ***P<0.001. The numbers of ovarioles analyzed are shown above bars.

Fig. 5.. svp is required in adult female adipocytes, but not oenocytes, to regulate GSC maintenance independently of niche E-cadherin or BMP signaling.

(A,B) Fat bodies from adult females labeled with DAPI (blue, nuclei) and nuclear GFP (nucGFP, green) driven by adipocyte-specific 3.1Lps2-Gal4 (A) or oenocyte-specific PromE(800)-Gal4 (B) in combination with tub-Gal80^ts for adult-specific expression. The dotted line separates the adipocytes (right) from oenocytes (left, identified by their autofluorescence in the green channel). Scale bar, 25 μm. (C,D) Average number of GSCs per germarium over time for females with adult adipocyte-specific (C) or oenocyte-specific (D) RNAi against Luc (control) or svp. Data shown as mean±SEM. **P<0.01; ****P<0.0001, two-way ANOVA with interaction. (E) Germaria from females at 10 days of adult adipocyte-specific Luc or svp RNAi. α-Spectrin (red), fusome; LamC (red), cap cell nuclear lamina; E-Cadherin (green). GSCs are outlined. (F) Dot plot of total cap cell E-Cadherin intensity per germarium for experiment in (E). (G) Germaria from females as described in E. α-Spectrin (red), fusome; LamC (red), cap cell nuclear lamina; Dad::nlsGFP (green), reporter of BMP signaling. GSC nuclei are outlined. Scale bars in (E) and (G), 5 μm. (H) Dot plot of mean Dad::nlsGFP intensity per germarium for experiment in (G). Black lines in (F) and (H) indicate mean±SEM for each experiment. No statistically significant differences, Mann Whitney U-test.

Fig. 6.. Knockdown of svp in adult female adipocytes, but not oenocytes, increases 16-cell cyst death.

(A) Average percentages of germaria containing dying cysts in Region 2 in females with adipocyte- or oenocyte-specific RNAi knockdown of Luc (control) or svp at 10 days of transgene expression. The numbers above each bar represent the number of analyzed germaria. (B-F) Average number of cystoblasts (B), two-cell cysts (C), four-cell cysts (D), eight-cell cysts (E), and 16-cell cysts per GSC per germarium of females at zero or 10 days of adult adipocyte-specific RNAi against Luc (control) or svp. Data shown as mean+SEM. *P<0.05; **P<0.01, Student’s t test. 75 germaria were analyzed for each condition in (B-E).

Fig. 7.. Knockdown of svp in adult female oenocytes, but not adipocytes, increases vitellogenic follicle death.

Average percentages of ovarioles containing dying follicles in females with adult adipocyte- or oenocyte-specific knockdown of Luc (control) or svp at 10 days of transgene expression. Data shown as mean+SEM. *P<0.05; **P<0.01, Student’s t test. The numbers above each bar represent the number of analyzed ovarioles.

Fig. 8.. Model for how Svp signaling in different somatic tissues regulates specific steps of oogenesis.

This study shows that svp is required in adult female adipocytes to regulate GSC number and germline cyst survival at the 16-cell cyst stage. In addition, Svp activity is required specifically in adult oenocytes for survival of vitellogenic follicles. Future studies should identify the specific downstream effectors of Svp in adipocytes (Target A and B) versus oenocytes (Target C) that function in a complementary way to regulate distinct processes in oogenesis.