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doi: 10.1242/10.1242/dev.00365

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A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis

Howard Hughes Medical Institute Research Laboratories, Department of Embryology, Carnegie Institution of Washington, 115 W. University Parkway, Baltimore, MD 21210, USA

View larger version (100K): [in a new window]   Fig. 1. High resolution detection of mitochondria. (A) The germarium in cross-section; at the anterior tip (region 1, left) reside two germline stem cells (blue) as well as cystoblasts and growing cysts (gray). Region 2a contains 16-cell germline cysts that enter meiosis and begin centrosome migration, while in region 2b, more mature cysts associate with follicle cells (green), and stretch to span the entire width of the germarium. Subsequently, in region 3, a new follicle with a well-defined oocyte (white) prepares to bud off. The fusome (red) grows, branches and eventually breaks down, leaving a remnant in the oocyte; it provides a distinctive marker for each stage. (B) A transmission electron micrograph showing the nucleus (nu) and mitochondria (m) of a region 1 cystocyte. The inset shows a cross-section of a ring canal containing a fusome plug surrounded by mitochondria. (C) Another region 1 cystocyte sectioned facing a ring canal (arrowhead): mitochondria (anti-ATP synthase) are green, the fusome (1B1 antibody) is blue and ring canal actin (rhodamine phalloidin) is red. (D,E) Mitochondria within living 16-cell cystocytes (broken circles) are labeled with the mito-GFP fusion gene (D) or using Mitotracker (E). Scale bars: in E, 1 µm for B-E; 100 nm in inset in B.

View larger version (80K): [in a new window]   Fig. 2. Equal distribution of mitochondria during cyst formation. (A) Asymmetric fusome behavior during stem cell and cystocyte divisions. The fusome (red) remains in the stem cell (GSC) following mitosis (green, mitotic spindle; blue, chromosomes), while a smaller fusome grows in the cystoblast (CB) before cell separation is complete. The fusome branches during subsequent divisions that produce two-, four-, eight- and 16-cell cysts. The letters under each diagram correspond to figure panels illustrating the indicated stage. (B) A germline stem cell (broken circle) in G2 phase. Mitochondria (green) vary in length but most associate with the fusome (blue). Green, ATPsynthase; red, phalloidin; blue, 1B1. (C) Another GSC (broken circle), revealing the clustering of microtubules (green) around the fusome (red). Green, tubulin; red, 1B1; blue, DNA. (D) A mitotic GSC (broken circle). Round mitochondria associate with the outer region of the mitotic spindle (red) and segregate equally (see Table 1). Green, ATP synthase; red, tubulin; blue, DNA. (E) A stem cell (broken circle, left) and daughter cystoblast (broken circle, right) in S phase after mitosis remain connected via a ring canal (red) containing the fusome (blue). Mitochondria aggregate around the stem cell fusome (upper left), but show little or no association with the cystoblast fusome (lower right). Green, ATPsynthase; red, phalloidin; blue, 1B1. (F) Spherical mitochondria (green) within an eight-cell cyst (broken circle) do not associate with the fusome (blue). Green, ATPsynthase; red, phalloidin; blue, 1B1. (G) The anterior region of a living germarium labeled with Mitotracker to reveal the position and movement of mitochondria. Mitochondria in region 1 cysts do not associate with the fusome (arrowhead) while mitochondria in region 2 do (arrow). B-F are projections of multiple confocal z-sections. Scale bars: in F, 5 µm for B-F; 10 µm for G.

View larger version (92K): [in a new window]   Fig. 3. Fusome-dependent formation of a Balbiani body. (A-D) Green, mitochondria (ATPsynthase); red, fusome (1B1). (A) Mitochondria in a 16-cell cyst (broken outline) midway in region 2a begin to associate with the fusome (red); in an older adjacent cyst (not outlined), they move toward the center of the fusome (yellow). (B) Mitochondrial movement along the fusome has progressed further in these two region 2b cysts (broken outline). (C) In cysts just entering region 3, clouds of mitochondria (arrowhead) accumulate near the ring canals that connect to the oocyte (small broken circle). (D) In region 3 follicles and young budded egg chambers, a Balbiani body containing many aggregated mitochondria (arrow) is visible in the anterior of the oocyte (small broken circle). (E) Electron micrograph of a region 3 follicle (large outline) reveals mitochondria entering the oocyte (small outline) via a ring canal (arrowheads) to form the Balbiani body (arrow). (F,G) Balbiani bodies fail to form in cysts from hts mutant females that lack fusomes. Arrowheads show mitochondrial clusters around presumptive centrosomes in the stem cells. Green, mitochondria (ATPsynthase); red, germ cells (Vasa); blue, DNA. (G) A stage 1 hts cyst (broken outline) showing the absence of a Balbiani body or mitochondrial clusters near presumptive ring canals. (H) Cysts mutant for egalitarian (egl) contain a normal fusome (red) but mitochondrial aggregates (green) of reduced size arise in all 16 cells at stage 1. Green, mitochondria (ATPsynthase); red, fusome (1B1). Scale bars: in H, 10 µm for A-D,F-H; in E, 2 µm for E.

View larger version (127K): [in a new window]   Fig. 4. Multiple organelles associate with the Balbiani body. (A) An electron micrograph of the Balbiani body in the anterior region of a young oocyte. The residual fusome (white arrow), still rich in ER-like vesicles, lies at the anterior near the ring canals (white arrowheads) it formerly occupied. Numerous mitochondria are located adjacent to the fusome, while scattered Golgi stacks lie further below (asterisks). (B,C) Electron micrographs showing nearby sections of a single ring canal (white arrowhead) connecting a nurse cell (NC) and a young ooctye (O). Five centrioles or centriole pairs (numbered) lie within the material moving in to form the Balbiani body. (D) The anterior portion of an ovariole stained with anti--mannosidase 2 to reveal Golgi vesicles (green) and with 1B1 to show the fusome (red). The Golgi elements associate with the fusome beginning in region 2b (yellow, arrowhead), move into the oocyte with the Balbiani body (arrow), but in a slightly older oocyte (broken line) spread throughout the ooplasm. (E) COXI-reactive vesicles (green) associate with the fusome (red) in region 2b (yellow, arrowhead) and move into the oocyte as part of the Balbiani body (arrow). (F) COXI-positive particles (green) appear to be mitochondria because they are co-labeled with the mitochondrial ATP synthase marker (red). Broken circle, oocyte. (G) COXI-positive particles (green) are not co-labeled with a Golgi marker (red, -mannosidase 2). Broken circle, oocyte. Scale bars: in E, 10 µm for D,E; in A, 1 µm for A; in C, 1 µm for B,C; in G, 1 µm for F,G.

View larger version (119K): [in a new window]   Fig. 5. Localized RNAs and proteins associate with the fusome and Balbiani body. (A) In situ hybridization reveals that osk RNA (green) associates with the central fusome (red) within the future oocyte beginning in region 2 (arrowhead). The RNA moves to the posterior of the oocyte during stage 1 (rightmost follicle). (B) An early region 3 oocyte (broken line) shown at higher magnification, reveals that osk RNA (green) partially overlaps (yellow) the Balbiani body on the posterior side of the mitochondrial mass (red, anti-ATP synthase). The arrowhead indicates the fusome (blue, 1B1). B' is the same image as B without the green channel, in order to better show the Balbiani body (arrow). (C) orb RNA (green), like osk RNA, associates with the center of the fusome (red) beginning early in region 2 (arrowheads), and then moves to the oocyte posterior in stage 1 (rightmost follicle). (D) Like osk RNA, orb RNA (green) also overlaps with the Balbiani body (red). D' is the same as D without the green channel, in order to better show the Balbiani body (arrow). (E,F,H) Cup protein (red) associates with the center of the fusome (blue) and with mitochondria (green) in region 2 cysts. (G,I) Orb protein (red) localizes to a similar position as Cup (green, mitochondria). (H,I) Both Cup (red, H) and Orb (red, I) briefly associate with the edge of the Balbiani body mitochondria (green) in early region 3 oocytes and then move to the oocyte posterior (not shown). Scale bars: in G, 10 µm for A,C,E-G; in D' 1 µm for B,B',D,D'; in I, 1 µm for H,I.

View larger version (120K): [in a new window]   Fig. 6. Balbiani body-derived mitochondria associate with the germ plasm prior to nurse cell dumping. (A) Mitochondria labeled with anti-ATP synthase (green) congregate on the posterior side of the GV (arrowhead) in stage 6-7 egg chambers. Green, ATP synthase; red, germ cells (Vasa); blue, ring canals (anti-Phosphotyrosine). (B) By contrast, mitochondria (green) in the nurse cells adjacent to the oocyte accumulate (arrowheads) at the ring canals (blue), but fail to enter the oocyte until nurse cell dumping (see Table 2). Green, ATPsynthase; red, Vasa; blue, phosphotyrosine. (C) At stage 9, a few mitochondria (green) associate with the germ plasm, which has begun to coalesce at the oocyte posterior as revealed by Vasa staining (red). Green, ATPsynthase; blue, Phosphotyrosine. (D,D'). By stage 10A, mito-GFP-labeled mitochondria (green) are abundant in the germ plasm (arrowheads in D'). D' shows a higher magnification. Green, mito-GFP; red, Vasa; blue, actin-rich membranes (phalloidin). (E) A summary of the structure and assembly of the Drosophila Balbiani body. Green arrows indicate direction of movement along the fusome (red) towards the oocyte. Scale bars: in C, 10 µm for A-D; in D', 5 µm for D'.