Hormone receptor 4 is required in muscles and distinct ovarian cell types to regulate specific steps of Drosophila oogenesis

Abstract

The conserved nuclear receptor superfamily has crucial roles in many processes, including reproduction. Nuclear receptors with known roles in oogenesis have been studied mostly in the context of their ovary-intrinsic requirement. Recent studies in Drosophila, however, have begun to reveal new roles of nuclear receptor signaling in peripheral tissues in controlling reproduction. Here, we identified Hormone receptor 4 (Hr4) as an oogenesis regulator required in the ovary and muscles. Global Hr4 knockdown leads to increased germline stem cell (GSC) loss, reduced GSC proliferation, early germline cyst death, slowed follicle growth and vitellogenic follicle degeneration. Tissue-specific knockdown experiments uncovered ovary-intrinsic and peripheral tissue requirements for Hr4 In the ovary, Hr4 is required in the niche for GSC proliferation and in the germline for GSC maintenance. Hr4 functions in muscles to promote GSC maintenance and follicle growth. The specific tissues that require Hr4 for survival of early germline cysts and vitellogenic follicles remain unidentified. These results add to the few examples of muscles controlling gametogenesis and expand our understanding of the complexity of nuclear receptor regulation of various aspects of oogenesis.

Keywords: Drosophila; Germline; Hr4; Muscle; Oogenesis; Stem cell.

Fig. 1.

The nuclear receptor Hr4 is required in adult somatic cells for normal rates of egg production. (A) Diagram of Drosophila ovariole showing the anterior germarium followed by progressively older follicles, each of which contains a 16-cell germline cyst (15 nurse cells and one oocyte; light blue) surrounded by follicle cells (gray). (B) Diagram of germarium, which contains 2-3 germline stem cells (GSCs, dark blue) residing in a niche composed of cap cells (pink), terminal filament cells (green) and a subset of escort cells (purple). Each GSC divides asymmetrically to self-renew and generate a cystoblast that divides to form a 16-cell cyst. Early germline cysts remain closely associated with escort cells (purple) until they become enveloped by follicle cells (gray) to bud a new follicle. GSCs and their early progeny are identified based on the morphology and position of their fusome (orange), a germline-specific organelle. (C) Females carrying tub-Gal4^ts and UAS-hairpin transgenes against Luc control or Hr4 were raised at 18°C and switched to 29°C for adult-specific ubiquitous somatic RNAi for 6, 10 or 15 days. Somatic Hr4 knockdown caused a significant decrease in the average number of eggs laid per female per day. (D) Control females carrying UAS-hairpin transgenes against Luc or Hr4 in the absence of tub-Gal4^ts were raised at 18°C and switched to 29°C for 4, 10 or 15 days. The UAS transgenes alone do not alter egg-laying rates. Data are shown as mean±s.e.m. **P<0.01, ***P<0.001 (paired two-tailed Student's t-test).

Fig. 2.

Ubiquitous somatic Hr4 knockdown in adult females increases GSC loss. (A,B) Germaria from females at 14 days of adult-specific ubiquitous somatic Luc control (A) or Hr4 (B) RNAi knockdown. α-Spectrin (magenta), fusome; LamC (magenta), cap cell nuclear lamina; Vasa (green), germ cells; DAPI (blue), nuclei. GSCs are outlined. (C,D) Average number of GSCs (C) or cap cells (D) per germarium in females with Luc control or Hr4 RNAi driven by tub-Gal4^ts over time. Data are shown as mean±s.e.m. ***P<0.001, ****P<0.0001 (two-way ANOVA with interaction). Scale bar: 10 µm.

Fig. 3.

Ubiquitous somatic knockdown of Hr4 decreases GSC proliferation. (A,B) Examples of anterior portion of germaria (from Luc control RNAi females) showing GSCs (outlined) labeled with EdU (green; A) and phospho-histone H3 (pHH3, green; B). α-Spectrin (magenta), fusome; LamC (magenta), cap cell nuclear lamina; DAPI (blue), nuclei. (C,D) Average frequencies of EdU-positive (C) or pHH3-positive (D) GSCs in adult females at 0 and 7 days of ubiquitous somatic RNAi against Luc control or Hr4. Data are shown as mean±s.e.m. **P<0.01, ***P<0.001 (paired two-tailed Student's t-test). Numbers of GSCs analyzed are shown inside bars. Scale bar: 2.5 µm.

Fig. 4.

Ubiquitous somatic Hr4 knockdown in adult females leads to increased death of early germline cysts. (A,B) Germaria from females at 10 days of adult-specific ubiquitous somatic RNAi against Luc control (A) or Hr4 (B). ApopTag (green), dying cells; α-Spectrin (magenta), fusome; LamC (magenta), cap cell nuclear lamina; DAPI (blue), nuclei. Arrow points to a dying germline cyst. (C) Average percentage of germaria containing ApopTag-positive germline cysts in Region 2 in adult females at 0 and 7 days of ubiquitous somatic RNAi against Luc control or Hr4. Data are shown as mean±s.e.m. *P<0.05, **P<0.01 (paired two-tailed Student's t-test). Numbers of germaria analyzed are shown inside bars. Scale bar: 10 µm.

Fig. 5.

Ubiquitous somatic Hr4 knockdown slows down follicle growth. (A,B) Stage 4-6 follicles from females at 7 days of ubiquitous somatic RNAi against Luc control (A) or Hr4 (B). EdU (magenta), follicle cells in S-phase; phospho-histone H3 (pHH3, green), follicle cells in mitosis; DAPI (blue), nuclei. (C,D) Average percentages of EdU-positive (C) or pHH3-positive (D) follicle cells from adult females at 0 and 7 days of ubiquitous somatic RNAi against Luc control or Hr4. Data are shown as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 (paired two-tailed Student's t-test). For each condition, 75 ovarioles were analyzed. Scale bar: 25 µm.

Fig. 6.

Hr4 knockdown in adult somatic cells leads to degeneration of vitellogenic follicles. (A,B) Ovarioles at 10 days of adult-specific ubiquitous Luc control (A) or Hr4 (B) RNAi. DAPI (white), nuclei. Arrowheads point to healthy vitellogenic follicles with yolk accumulation. Arrows point to dying vitellogenic follicles, recognized by the presence of pyknotic nuclei. (C) Average percentages of ovarioles containing dying vitellogenic follicles in females at 0 and 10 days of ubiquitous somatic RNAi against Luc control or Hr4. Data are shown as mean±s.e.m. **P<0.01, ***P<0.001 (paired Student's two-tailed t-test). Numbers of ovarioles analyzed are shown above bars. Scale bar: 100 µm.

Fig. 7.

Hr4 is required in adult female muscles for proper GSC maintenance and follicle growth. (A) RT-PCR analysis of whole females or individual organs showing expression of Hr4 transcripts relative to Rp49 control. (B) Average number of GSCs per germarium over time in females with adult muscle-specific RNAi against Luc control or Hr4 driven by MHC-Gal4^ts. Data are shown as mean±s.e.m. *P<0.05, ***P<0.001 (two-way ANOVA with interaction). (C,D) Average percentages of EdU-positive (C) or pHH3-positive (D) follicle cells in adult females at 0 and 7 days of muscle-specific RNAi against Luc (control) or Hr4. (Follicle cell proliferation was used as a proxy for follicle growth; see text.) Data are shown as mean± s.e.m. *P<0.05, **P<0.01, **P<0.001 (paired two-tailed Student's t-test). Numbers of ovarioles analyzed are shown inside bars.

Fig. 8.

Hr4 is required in the niche for normal rates of GSC proliferation and in the germline for GSC maintenance. (A,B) Average frequencies of EdU-positive (C) or pHH3-positive (D) GSCs in adult females at 0 and 7 days of RNAi against Luc control or Hr4 in the GSC niche driven by hh-Gal4. Data are shown as mean±s.e.m. *P<0.05, **P<0.01 (paired two-tailed Student's t-test). Numbers of GSCs analyzed are shown inside bars. (C,D) Genetic mosaic germaria showing GFP-negative control (from ‘mock’ mosaics; C) or Hr4^W728X homozygous (D) cystoblasts and cysts (dashed outlines) at 7 days after heat shock. Although the GFP-negative mother GSC (solid outline; from which GFP-negative cystoblasts/cysts are derived) is still present in C, it is absent in D, which indicates a GSC loss event. GFP (green), wild-type cell nuclei; α-Spectrin (magenta), fusomes; LamC (magenta), cap cell nuclear lamina; DAPI (blue), DNA. (E) Percentages of mosaic germaria showing GSC loss events at 7 days after heat shock. Data are shown as mean±s.e.m. **P<0.01 (paired two-tailed Student's t-test). Numbers of mosaic germaria analyzed are shown inside bars. (F) Average number of cystoblasts and/or germline cysts per GSC in mosaic germaria 7 days after heat shock. Data are shown as mean±s.e.m. For each genotype, 50 mosaic germaria were analyzed. (G) Model for how Hr4 signaling in the muscle and ovarian cell types influences distinct processes of oogenesis (see text for details). Scale bar: 10 µm.