Visualizing Fusome Morphology via Tubulin Immunofluorescence in Drosophila Ovarian Germ Cells

Abstract

In many species, oocytes are initially formed by the mitotic divisions of germline stem cells and their differentiating daughters. These progenitor cells are frequently interconnected in structures called cysts, which may function to safeguard oocyte quality. In Drosophila, an essential germline-specific organelle called the fusome helps maintain and coordinate the mitotic divisions of both germline stem cells and cyst cells. The fusome also serves as a useful experimental marker to identify germ cells during their mitotic divisions. Fusomes are cytoplasmic organelles composed of microtubules, endoplasmic reticulum-derived vesicles, and a meshwork of membrane skeleton proteins. The fusome branches as mitotic divisions progress, traversing the intercellular bridges of germline stem cell/cystoblast pairs and cysts. Here, we provide a protocol to visualize fusome morphology in fixed tissue by stabilizing microtubules and immunostaining for α-Tubulin and other protein constituents of the fusome. We identify a variety of fluorophore-tagged proteins that are useful for visualizing the fusome and describe how these might be combined experimentally. Taken together, these tools provide a valuable resource to interrogate the genetic control of germline stem cell function, oocyte selection, and asymmetric division.

Keywords: Cyst; Germarium; Germline; Germline stem cell; Microtubules; Oocyte.

Fig. 1

Germ cell mitotic division in adult Drosophila melanogaster. Drosophila ovaries are composed of 14 to 16 ovarioles, or strings of progressively older egg chambers. (a) Schematic of a germarium. Germline stem cells (GSCs, pink) are located at the anterior tip of the germarium, which resides at the anterior-most tip of each ovariole. GSCs lie adjacent to somatic cap cells (yellow) and escort cells (green) which support GSC self-renewal. GSC division gives rise to another GSC and a differentiating daughter cell, called a cystoblast (CB), which forms posterior to the GSC and continues to divide into 2-cell, 4-cell, 8-cell, and 16-cell cysts (blue). Within each cyst, one germ cell is specified as the oocyte (dark blue), while others become nurse cells. At the posterior of the germarium, cysts are surrounded by somatic follicle cells (tan), which descend from follicle stem cells (FSC). (b) Germ cells divide in a stereotypical fashion. GSCs undergo asymmetric mitotic divisions with complete cytokinesis, while cystoblasts divide four times (M1-M4) with incomplete cytokinesis, giving rise to the interconnected cells of the cyst. Individual cells remain connected by small ring canals (yellow), through which the fusome (red) branches. (c) Schematic diagram of the GSC cell cycle. GSCs divide approximately every 15 h; however, abscission (the final stage of cytokinesis) is delayed well into the G2 phase of the next cell cycle [9, 14, 23, 24, 26]. Fusome morphology (red) can be used for identifying GSCs generally, and more specifically, as an indirect indicator of the cell cycle stage of the GSC [9, 14, 22]. G1 gap phase 1; S synthesis phase; G2 gap phase 2; M mitosis

Fig. 2

Visualizing fusome and spindle morphology. (a, b) GSCs and cystoblasts from Jupiter-GFP (a) and nos-Gal4::VP16 > tub-GFP (b) germaria immunostained for GFP (green; mitotic spindle), Hts and LaminC (LamC; red; fusomes and nuclear membrane of cap cells), and DAPI (blue; DNA). (c) GSC in metaphase of mitosis from Scrib-GFP germarium immunostained for GFP (green; cell membrane), α-Tubulin (red; mitotic spindle), Hts and LamC (red), and DAPI (blue; DNA). (d-d”) Dividing cystoblast from Scrib-GFP germarium co-immunostained for GFP (green; cell membrane), α-Tubulin (magenta; mitotic spindle remnants), Hts and LamC (red; fusome), and DAPI (blue; DNA). Images in d’-d” display Tubulin (d’) or Hts/LamC (d”) channels only. Asterisks indicate cap cells; dashed white lines indicate GSCs; arrows indicate fusomes; arrowheads indicate mitotic spindles or spindle remnants. (Images a–c were acquired with a Zeiss LSM 700 laser scanning confocal; images d-d” acquired with a Zeiss LSM 800 with Airyscan. Scale bars = 5 μm)