BIOLOGICAL OVERVIEW

The early processes of Drosophila gametogenesis in both ovaries and testes are remarkably similarto one another: germ-line stem cells are sequestered at the anterior end of either organ. When a germ-line cell divides, one of the daughters remains attached and continues to function as a stem cell, whereas the other daughter becomes the 'founder' of a 16-cell syncytial cyst. Four successive divisions of this cystoblast or spermatoblast (within the ovary or testis, respectively) produce a clone of 16 sister germ line cells that remain interconnected due to an unusual process of incomplete cytokinesis, which generates interconnecting ring canals. In the ovary, a complement of somatic cells encloses each 16-cell cyst sometime after it is formed, as it passes through a specific region of the germarium. By comparison, in the testis during spermatogenesis the founding spermatoblast is enclosed by just two somatic cells prior to the four successive spermatoblast divisions. These differences aside, both processes generate cysts of 16 germ-line-derived cells surrounded by a layer of somatic cells. Shortly after this developmental juncture has been reached, the pathways diverge: female cysts go on to produce a single egg, whereas male cysts produce 64 spermatids. (See betaTub85D for a more detailed account of spermatogenesis; see also the oocyte and alpha Spectrin sites for information about oogenesis).

Of particular interest is the fate transition of descendents of the self-renewing stem cell population to become more specialized daughter cells (termed the 'cystoblast' in oogenesis, and the 'primary spermatogonial cell' in spermatogenesis). Several genes have been characterized in Drosophila that carry a common defect in oogenesis. These include ovarian tumor (otu), ovo, benign gonial cell neoplasm, sans fille, Sex lethal and the topic of this site, bag of marbles (bam). Mutations at these loci result in the absence of mature germ cells, and in the overproliferation of small cells with morphological characteristics of undifferentiated germ cells. The tumorous cyst phenotype points to the importance of gene action in the transition from stem cell to daughter cells of distinct fate. Only bag of marbles and benign gonial cell neoplasm (bgcn) act both in oogeneis and spermatogenesis, suggesting that these two genes regulate a pathway shared in the two processes (Gšnczy, 1997).

In bam mutant ovaries, the overproliferating germ cells appear to behave as stem cells, according to two criteria. (1) Germ cells overproliferating in bam mutant ovaries are either not connected cytoplasmically to a neighbouring cell, or are connected to a single neighbouring cell, with a fusome passing between the two cells (see alpha Spectrin for information about the fusome). This pattern of connections is identical to that of wild-type germline stem cells, but different from that of wild-type amplifying germ cells (the differentiating progeny of stem cells), which are connected to several neighbouring germ cells as a result of incomplete cytokineses. Similarly, branched fusomes are rarely observed in bgcn mutant ovaries. (2) Germ cells overproliferating in bam mutant ovaries undergo S phase asynchronously. Again, this behavior is like that observed among germline stem cells, but not among their dividing differentiating progeny, which progress synchronously through the cell cycle. These observations led to the conclusion that bam mutant germ cells behave as stem cells and to the postulate that bam function is normally required in females to assign the fate of the cystoblast (McKearin, 1995).

Does bag of marbles play a similar role in spermatogenesis, that is, does bam mutation result in the overproliferation of germ-line stem cells in the male? Examination of bam testes suggests that gametogenesis is also disrupted at an early stage in males. These testes contain abnormal cysts populated with an excessive number of small cells the size of primary spermatocytes. Normally, the 16 primary spermatocytes, derived from the primary spermatogonial cell by four cell divisions, increase 25-fold in size prior to the onset of meiosis. However, bam spermatocytes remain about the size of early spermatocytes. They never undergo subsequent morphological changes characteristic of meiosis and spermatogenesis. Consistent with an early developmental arrest, expression of betaTub85D (also known as beta2-tubulin) is not detected in mutant testes. Mutant cysts progress into a highly refractile state (McKearin, 1990).

Evidence points to different consequences for bam mutation, depending on whether it occurs in spermatogenesis or oogenesis. In either case, mutations in bam and its kindred gene bgcn result in the overproliferation of undifferentiated germ cells. In males, bam and bgcn appear to be required to restrict the proliferation of amplifying germ cells (the progeny of primary spermatogonial cells) or to promote their entry into the meiotic cell cycle, in the case of spermatogenesis. Germ cells accumulating in bam and bgcn mutant testes have several characteristics of amplifying germ cells: (1) cytoplasmic Bam protein is expressed in bgcn mutant germ cells; (2) bgcn and bam mutant germ cells undergo incomplete cytokinesis, and (3) they proliferate in synchrony within a cyst. All three aspects are characteristic of amplifying germ cells, but not of germline stem cells. It is concluded that bam and bgcn most likely do not regulate the decision to adopt a primary spermatogonial cell fate; rather, they act to restrict the proliferation of amplifying germ cells or promote their entry into the meiotic cell cycle (Gšnczy, 1997).

In females, overexpression of bam has a deleterious effect on oogenic germline stem cells, one that is not evident in spermatogenic germline stem cells. Female germ cells are progressively depleted with bam overexpression, suggesting that ectopic bam expression blocks stem cell function. It is though that ectopic bam causes stem cells to divide as committed cystoblasts: as contractile ring formation and closure takes place, fusomes grow and cell-cycle coordination is evident. It is suggested that a presumptive cystoblast daughter (pre-cystoblast) of the stem cell division undergoes a maturation process during which bam+ activation initiates cystoblast/cystocyte mitoses by modifying the germ-cell division cycle. Surprisingly, ectopic bam+ has no deleterious consequences for male germline cells, suggesting again that Bam may regulate somewhat different steps of germ-cell development in oogenesis as compared to spermatogenesis (Ohlstein, 1997).

What is the basis of the difference between functions of Bam in males and females? The function of bam and bgcn may have been adapted to play an essential role in the cystoblast due to a unique requirement of the female germline stem cell lineage. In females, only one of the sixteen amplifying germ cells adopts the oocyte fate, while its fifteen sisters become nurse cells. Oocyte determination relies on polarized microtubule-dependent intercellular transport from the nurse cells into the oocyte through ring canals. The basis for this polarity appears to be established as early as in the cystoblast. Since the fusome is associated with microtubules, it has been suggested that the fusome helps establish and maintain polarity during cystoblast divisions and the amplifying of germ cell divisions. Since a form of Bam protein is associated with the fusome (McKearin, 1995), it is possible that Bam itself plays a role in establishing polarity in the cystoblast. In bam mutant ovaries, polarity cannot be established, and the cystoblast cell fate cannot be assigned, resulting in an overproliferation of cells with stem cell characteristics. By contrast, in males, all sixteen amplifying germ cells are equivalent; there is no evidence of polarity analogous to that observed in females. This might explain why bam and bgcn are not strictly necessary for assigning primary spermatogonial cell fate (Gšnczy, 1997).

What is the biochemical function of Bag of marbles? Little can be gleaned from the protein sequence since Bam is a novel protein. Some information is provided by the phenotype especially with regard to fusome appearance. In the female, each round of cystocyte mitosis is accompanied by the growth of a germ cell-specific organelle, the fusome. The fusome contains four membrane cytoskeletal proteins: alpha-Spectrin, beta-Spectrin, the adducin-like Hu-li tai shao and Ankyrin. Stem cells and cystocytes contain a large sphere of fusomal material, termed the spectrosome. During the four cystocyte mitoses, one pole of each spindle associates with the fusome, and following each mitosis, as the spindles disaggregate, additional fusomal material accumulates. Thus, by the fourth division, the fusome forms one large branched structure that extends though the ring canals into all the cells in a cyst. alpha-Spectrin deficient cells were generated in fly ovaries and the effects on cyst formation and oocyte differentiation were observed. This work shows that the fusome acts as a pole for each mitotic spindle by capturing a centriole and, in this capacity, serves to orient the planes of cell division at each cystocyte mitosis (reviewed in McKearin, 1997).

Bam is a component of the fusome. It has been noted the bam mutant fusomes are deficient in the membranous tubular reticulum that normally fills the fusome core. This observation prompts the suggestion that Bam protein might be required to recruit vesicular material into the reticulum. A role for the fusome reticulum in directing a switch from stem cell to cystoblast-like divisions could explain both the bam loss-of-function and ectopic expression phenotypes. It is proposed that fusome biogenesis is an obligate step for cystoblast cell fate and that Bam is the limiting factor for fusome maturation in female germ cells (Ohlstein, 1997).

Germline stem cell number in the ovary is regulated by mechanisms that control Dpp signaling: Bam blocks Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes

The available experimental data support the hypothesis that the cap cells (CpCs) at the anterior tip of the germarium form an environmental niche for germline stem cells (GSCs) of the Drosophila ovary. Each GSC undergoes an asymmetric self-renewal division that gives rise to both a GSC, which remains associated with the CpCs, and a more posterior located cystoblast (CB). The CB upregulates expression of the novel gene, bag of marbles (bam), which is necessary for germline differentiation. Decapentaplegic (Dpp), a BMP2/4 homolog, has been postulated to act as a highly localized niche signal that maintains a GSC fate solely by repressing bam transcription. The role of Dpp in GSC maintenance has been examined in more detail. In contrast to the above model, it is found that an enhancer trap inserted near the Dpp target gene, Daughters against Dpp (Dad), is expressed in additional somatic cells within the germarium, suggesting that Dpp protein may be distributed throughout the anterior germarium. However, Dad-lacZ expression within the germline is present only in GSCs and to a lower level in CBs, suggesting there are mechanisms that actively restrict Dpp signaling in germ cells. One function of Bam is to block Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes. Moreover, in females doubly mutant for bam and the ubiquitin protein ligase Smurf, the number of germ cells responsive to Dpp is greatly increased relative to the number observed in either single mutant. These data indicate that there are multiple, genetically redundant mechanisms that act within the germline to downregulate Dpp signaling in the Cb and its descendants, and raise the possibility that a Cb and its descendants must become refractory to Dpp signaling in order for germline differentiation to occur (Casanueva, 2004).

The prevalent model for Dpp action within the ovary is that it is a local niche signal whose activity is permissive for GSC maintenance. In this model, only GSCs within the niche are exposed to Dpp protein and removal of the CB from the niche lessens or eliminates exposure to the ligand. Moreover, the only postulated function of Dpp is to repress the transcription of bam within the GSCs. The data presented in this paper reveal additional aspects of Dpp function in GSC maintenance. The results strongly suggest that Dpp ligand is not restricted to the niche but rather is present throughout the anterior germarium. Data is presented that the observed specificity of Dpp signaling to the GSCs and CBs is due to functionally redundant mechanisms that operate in the germline to actively downregulate Dpp signaling during GSC differentiation. One of these mechanisms is Bam itself, thus establishing a negative feedback loop between the actions of the two genes. These findings indicate GSC differentiation is correlated with downregulation of Dpp signaling, raising the possibility that Dpp signaling plays an active role in GSC maintenance, and that GSC differentiation requires both the presence of Bam and the absence of Dpp signaling (Casanueva, 2004).

If GSCs and CBs are exposed to equivalent amounts of Dpp protein, as is suggested by both the transcription pattern of the Dpp gene and the expression of Dad-lacZ in the CpCs of the niche and the ISCs posterior to the niche, then it is likely that the observed reduction in Dad-lacZ expression between the GSC and the CB results from intracellular modulation of the strength of the Dpp signal. One hallmark of the GSC is its invariant plane of division. It is proposed that the differential Dpp signaling between the GSC and CB sign results from an intracellular modulation of Dpp signal strength between the two daughter cells, either by the asymmetric segregation of one or more cellular components that modulate Dpp signaling, or by loss of a contact-based niche signal that elevates Dpp signaling preferentially within the GSCs. Removal of the CB cell from the niche thus results in partial downregulation of Dpp signaling. A lower level of Dpp signaling in the CB cell results in the transcription of Bam, which plays multiple roles in CB differentiation, one of which is to cause the daughters of the CB cell to become refractory to further Dpp signaling. Thus, sequential regulatory mechanisms cooperate to ensure an irreversible change in the fate of the GSC cell within two generations (Casanueva, 2004).

Loss-of-function mutations in Smurf and gain-of-function mutations in sax increase the number of GSCs, suggesting these genes may perturb the proposed intracellular modulation of Dpp signaling that occurs between the GSC and CB. However, these data are not sufficient to determine whether this proposed modulatory pathway acts through direct regulation of the functions of one or both of these gene products, or whether the proposed pathway acts in parallel to these genes. In the embryo, loss of Smurf activity results in a ligand-dependent elevation of Dpp signaling that has greater, but not indefinite, perdurance (Podos, 2001), suggesting that Dpp signaling in Smurf mutants, and by inference sax mutants, is still responsive both to the amount of ligand and to the presence of other negative regulatory mechanisms. In the ovary, the Dad-lacZ-expressing germ cells in the Smurf and sax mutants fill the region of the anterior germarium that roughly corresponds to the spatial extent of Dad-lacZ expression in the somatic cells of region 1 and 2A of a wild-type germarium, suggesting that potentially all germ cells in region 1 and 2A of the Smurf and sax germaria are equally and fully responsive to the Dpp ligand. It is proposed that GSCs in the Smurf and sax germaria ultimately undergo normal differentiation because in the more posterior regions of the germaria the amount of Dpp ligand may be reduced to a level that allows bam transcription, which further reduces Dpp signaling and causes cyst differentiation (Casanueva, 2004).

The reduction in Dpp signaling between the GSC and the CB releases Bam from Dpp-dependent transcriptional repression, and one, but not the only, function of Bam is to downregulate Dpp signaling downstream of receptor activation prior to overt GSC differentiation. This is the first molecular action ascribed to Bam, and these data could provide an entry point to elucidate the biochemical basis of the function of Bam in CB differentiation. Further work will be necessary to determine whether the action of Bam on the Dpp pathway is direct or indirect, whether Bam action results in the reduction or complete elimination of Dpp signaling in the developing cysts, and which step in the intracellular Dpp signal transduction pathway or expression of Dpp target genes is affected by Bam action. However, it is possible that initial insights into Bam function can be made by comparing the thresholds for Dpp signaling readouts in the developing wing disc of the larva to the data obtained in the germarium. In the wing disc, Dpp diffuses from a limited source to form a gradient throughout the disc that displays different thresholds for multiple signaling readouts. Specifically, Dad-lacZ is transcribed in response to high and intermediate levels of Dpp, but does not respond to the lowest levels of ligand. An antibody exists that recognizes the active phosphorylated form of Mad, pMad. In the wing disc, high level staining with the pMad antibody is present in only a subset of cells that express high levels of Dad-lacZ, suggesting that in this tissue the pMad antibody is less sensitive to Dpp signaling than is Dad-lacZ expression. Intriguingly, in the ovariole pMad staining is visible in the GSCs, CBs and the developing cysts. Because Dad-lacZ expression was never observed in the developing cysts, these results could suggest that the relative sensitivities of these two reagents are reversed within the germline. Alternatively, if the reagents have the same relative sensitivities in the two tissues, the data suggest that Bam could act, probably at a post-transcriptional level, to downregulate Dpp signaling downstream of Mad activation (Casanueva, 2004).

The pattern of Dad-lacZ expression observed in the Smurf; bam and sax; bam double mutant ovarioles is qualitatively different from that observed in any of the single mutant ovarioles. Although Dad-lacZ expression is observed only at the anterior tip of the germarium of each single mutant, many, but not all, of the double mutant ovarioles contain germ cells throughout the ovariole that express high levels of Dad-lacZ. From these data, it is concluded that two redundant pathways downregulate Dpp signaling in the germline, and that in the single mutants, the action of the remaining active pathway is sufficient to constrain Dpp responsiveness to the anterior tip of the germarium. However, not all doubly mutant ovarioles display a spatial expansion of Dpp signaling, and this variability can even be observed in ovarioles from a single female. It is proposed that the observed variability results because the Smurf and sax mutations have modulatory effects on Dpp signaling that are both dependent on the presence of ligand and are sensitive to additional mechanisms that downregulate Dpp signaling. In both the Smurf; bam and sax; bam ovarioles, the germ cells that express Dad-lacZ are observed throughout the ovariole, but are more likely to be near somatic cells. It is possible that the variability in Dad-lacZ expression occurs because of a non-uniform distribution of the Dpp ligand. Nevertheless, there is not a consistent correlation between the domains of Dad-lacZ expression in the somatic and germ cells, suggesting that there may be additional germline intrinsic factors that affect Dpp signaling (Casanueva, 2004).

GENE STRUCTURE

Bases in 5' UTR - 112

Exons - 3

Bases in 3' UTR - 479

PROTEIN STRUCTURE

Amino Acids - 442

Structural Domains

Bam has some sequence homology to the ovarian tumor gene of Drosophila. Weak similarity is detected within three regions spanning about 150 amino acids in the central region of the Bam protein. About 20% of the residues are identical and 15% correspond to conserved replacements within these regions (McKearin, 1990).

date revised: 20 NOV 97

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