alpha Spectrin

DEVELOPMENTAL BIOLOGY (part 2/2)

Adult (continued)

When there is no Anillin at the membrane (and presumably no connection) between the stem cell and cystoblast, the stem cell fusome returns to the anterior tip of the cell and regains its spherical shape. In stem cells that appear to have separated only recently from a cystoblast, the fusome looks smaller than in other stem cells; hence it likely accumulates more material before the stem cell divides again. The behavior of the fusome suggests that there is a specific cortical attachment site for the fusome, from which it does not always detach completely. Because most germaria have at least one stem cell, and sometimes several, with a ring canal and an elongated fusome, it was reasoned that these stages of the fusome cycle must occupy a fairly large portion of the cell cycle following mitosis. The ring canal between stem cell and cystoblast stays open until after S-phase of the following cell cycle is complete in both cells. Thus, it is concluded that the exclamation point stage of the fusome cycle occurs in G2 (de Cuevas, 1998).

The results of these experiments also suggest that the stem cell and cystoblast enter and exit S-phase together. By examining the BrdU-labeled stem cell and cystoblast nuclei more closely, it was determined that the BrdU was distributed over the same portion of the nucleus (over euchromatin, over heterochromatin, or over both) in every labeled pair of cells. Thus, it is concluded that the cell cycles of the stem cell and cystoblast are synchronized through S-phase. It is also concluded that the stem cell and cystoblast remain connected and are synchronized at least through S-phase. It is thought that they become asynchronous soon after the cells are separated, however, since a stem cell and a cystoblast are never seen in mitosis at the same time. In stem cells, the ring canal is transient and closes down after the fusome is partitioned through it (de Cuevas, 1998).

The growth and behavior of the fusome in dividing cystoblasts has been studied in ovaries that were triple-labeled with anti-Anillin antibodies, anti-Hts antibodies and DAPI. A new cystoblast, which has separated from the stem cell but not yet entered mitosis, contains a spherical fusome slightly smaller than a stem cell fusome; it is usually located in the anterior half of the cell, near the plasma membrane. In mitotic cystoblasts, the fusome keeps its spherical shape and size but is often located in the posterior half of the cell. Thus, at cytokinesis, the fusome is often retained by the more posterior of the two daughter cells. It was not determined whether the fusome changes its location by migrating through the cystoblast before mitosis begins, or whether the entire cell rotates; it is suggested, however, that the fusome migrates towards the centrosome, which is likely to be located posteriorly. Following mitosis, as in the stem cell, a small plug of fusomal material accumulates in the arrested cleavage furrow. As interphase progresses, the original fusome and plug move closer together, additional fusomal material accumulates between them, and the two pieces of fusome eventually fuse, forming a snowman-shaped structure with its ‘neck’ in the ring canal. In the cystoblast, however, the original fusome does not elongate toward the plug as fusome and plug fuse. Thus, it could not be determined if the original fusome moves toward the plug, or if the plug and ring canal move towards the original fusome. Because it is formed from two smaller fusomes, the fused fusome has also been called a ‘polyfusome’. After fusion, the cell that lacks a fusome at cytokinesis contains less fusomal material than its sister cell and, apparently, it does not accumulate more during the rest of interphase. By the next mitosis, which forms four cells from two, the fusome is still asymmetrically distributed within the cyst (de Cuevas, 1998).

In 4-, 8- and 16-cell cysts, the fusome grows in a similar manner as in 2-cell cysts: after each round of mitosis, plugs form in each nascent ring canal and gradually fuse with the original fusome. Fusion takes place as the plugs move towards the central fusome and as fusomal material accumulates in the gaps between them. All plugs fuse before the next round of mitosis begins. In 16-cell cysts, fusion is always complete before the cyst becomes lens-shaped and the fusome begins to disaggregate. Thus, 16-cell cysts whose fusomes are in the process of fusing are located in region 2a of the germarium. It is likely that some fusomal material is newly synthesized or assembled as the cyst grows, since fusomes in 16-cell cysts are much bigger than cystoblast fusomes. As in 2-cell cysts, after every round of fusome fusion, one cell appears to contain more fusomal material than its sister cystocytes; this cell is always one of the two with the greatest number of ring canals. Based on this observation, the cell with the most fusomal material in a 16-cell cyst is probably the same cell that retained the fusome at cytokinesis in the cystoblast. Thus, the initial asymmetry of the fusome is maintained throughout cyst formation. In 4-, 8- and newly formed 16-cell cysts (found in region 2a of the germarium), the cell with the most fusomal material is positioned at random in the cyst with respect to the axes of the germarium (de Cuevas, 1998).

Fusome morphogenesis was examined in two mutants in which cyst polarity appears to be disrupted. Mutations in Bicaudal-D (Bic-D PA66/R26 ) and egalitarian (egl WU50/RC12 ) cause the formation of cysts that fail to accumulate oocyte-specific factors in a single cell and thus differentiate into 16 nurse cells. Fusomes appear to form normally and are still asymmetrically distributed in these cysts, however, indicating that the polarizing mechanisms that underlie fusome formation are not disrupted by these mutations. Thus, the fact that the oocyte fails to differentiate in these mutants is apparently not caused by a lack of polarity in dividing cysts. Consistent with this result, one of the two cells with four ring canals still localizes to the posterior end of these mutant cysts in region 2b, indicating that some polarity is retained in older cysts as well (de Cuevas, 1998).

After each round of mitosis, as the fusome plugs move toward the central fusome, their associated ring canals move with them. To quantify this movement, the distances between new and old ring canals were measured both before movement (in cysts with newly arrested cleavage furrows, which did not yet contain a distinct plug) and after movement (in cysts whose plugs had all fused with the central fusome). After plug fusion, the newest ring canal in each cell can still be distinguished by its smaller size. In cells with more than one old ring canal, only the distance from the new ring canal to the closest old ring canal was measured. The results of this analysis show that new ring canals move about 2-3 mm closer to old ring canals after each round of mitosis. It could not be determined from this experiment if new ring canals actually flow through the plasma membrane, or if they are brought closer to old ring canals by removal of plasma membrane from one side of the cell and its replacement elsewhere. Regardless of how it happens, the centripetal ring canal ‘movement’ results in a change in the cyst’s geometry. This change occurs after every round of mitosis but is best illustrated in 4-cell cysts. In newly formed 4-cell cysts, just after cytokinesis, the cells are arranged in a linear or V-shaped structure. In older 4-cell cysts, however, the cells radiate out like flower petals from the central clump of ring canals. This arrangement, called a ‘rosette’, can be seen most clearly in cysts that are just entering mitosis, when Anillin outlines the cortex of the cells. Rosettes have been found in many other insect species and their formation appears to be a general characteristic of cyst morphogenesis. The effect of rosette formation is to shorten the distances between cells that are not directly connected; thus, it is tempting to speculate that this change in cyst geometry facilitates synchronization or communication between cystocytes. It is also suggested that ring canal movement and rosette formation might be facilitated by the reduced amounts of membrane skeletal proteins found at the plasma membrane of dividing cystocytes (de Cuevas, 1998).

To characterize fusome formation in more detail, ovaries were labelled with antibodies against two other fusome components, alpha-Spectrin and Bag-of-marbles (Bam) protein. Both of these proteins, like Hts protein, localize to fusome plugs in nascent ring canals and are distributed asymmetrically within the cyst throughout its formation. No difference in composition has been found between the plugs and central fusome, but it is possible that other components might associate with only one part of the fusome. The plugs are formed initially by material that appears to approach the ring canal from both sides. Moreover, especially when labeled with anti-Hts antibodies, this material appears to approach the ring canal in lines, as if it is travelling along fibers. Thus, it is suggested that the fusome plugs are assembled from material that is transported to the site of the spindle midbody, around which the ring canal forms, perhaps along microtubules that are remnants of the mitotic spindle (de Cuevas, 1998).

Anillin is also present in the somatic ring canals that link ovarian follicle cells. These ring canals, which are first visible in region 2 of the germarium, increase in number as the follicle cells divide; thus by late oogenesis many follicle cells have multiple ring canals associated with them. Unlike germline ring canals, somatic ring canals are very small - about 0.5 mm in diameter - and do not enlarge as oogenesis progresses. They also continue to stain with anti-Anillin antibodies throughout oogenesis. Ring canals are not seen between the cells that comprise the interfollicular stalks (except when the stalks are forming, in stages 1-2) but they are present in the follicle cells that remain over the nurse cells after the others migrate posteriorly in stage 10. Somatic ring canals in later egg chambers also contain actin. In dividing follicle cells that were double-labeled with anti-Anillin and anti-Hts antibodies, no fusome-like structures were seen spanning the ring canals (de Cuevas, 1998).

The work presented in this paper strongly suggests that ovarian cysts are polarized at the first division, that this polarity is maintained during the subsequent rounds of division, and that the fusome is a sensitive indicator of polarity in dividing cysts. Thus, these observations support the idea that the oocyte is specified early, during the cyst divisions, rather than later, after the completion of the cyst mitoses. Previous studies had shown that the fusome associates with only one pole of the spindle in dividing cystoblasts. Since the two cells that are produced by this division give rise to the two cells with four ring canals (at the 16 cell stage), one of which becomes the oocyte, this result suggested that the fusome might mark the future oocyte. It was not clear, however, how or even if the original asymmetry between these two cells was maintained as the cyst continued to divide. The results presented in this paper provide the first evidence suggesting that the initial polarity of the cyst is maintained throughout the cell division process that produces the final 16 cell cyst (de Cuevas, 1998).

The fusome grows by a regular, polarized process throughout the stem cell and cyst cell cycles. Each polarization cycle begins in mitosis, when the fusome segregates to a single daughter cell of each pair. Following mitosis, a 'plug' of fusomal material forms in each nascent ring canal and gradually fuses with the pre-existing fusome. In dividing cysts, as the fusome plugs move toward the pre-existing fusome, their associated ring canals also move, changing the geometry of the cyst. At the end of each cycle of cyst growth, the fusome remains asymmetrically distributed within the cyst; one of the two cells with four ring canals retains a bigger piece of fusome than any other cell, including the other cell with four ring canals. Based on these observations, it is argued that the oocyte is specified at the first cyst division (de Cuevas, 1998).

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