The zpg locus encodes a 1.6 kb transcript detected in poly A+ mRNA from whole adult males and females but not from agametic animals. Consistent with the transcript being germline dependent, in situ hybridization to embryos revealed that zpg mRNA was concentrated in germ plasm and in the pole cells of wild-type embryos, from the syncitial blastoderm stage through gonad formation. Zpg protein was also detected in pole cells and primordial germ cells throughout embryogenesis. In wild-type testes, zpg mRNA was detected in the spermatogonial region near the apical tip. The level of zpg mRNA decreased sharply to background at the transition from spermatogonia to spermatocytes (Tazuke, 2002).
In testes, Zpg protein was expressed in spermatogonia and early spermatocytes, where it appears to be concentrated at the interface between germ cells and somatic cyst cells. The anti-Zpg antibody detected discrete patches of protein on the surface of early spermatogonia during the mitotic amplification stage. A similar pattern was seen in both larval and adult testes. The appearance of Zpg protein on the surface of early spermatogonia correlates with the stage at which early germ cells are lost in zpg mutant males. In later spermatogonial and early spermatocyte cysts, anti-Zpg staining is distributed more evenly over the germ cell surface but is especially concentrated at the outer surface of the germ cell cluster, where the germ cells interface with the enveloping somatic cyst cells. Staining with the anti-Zpg antibody becomes weaker and more diffuse during the subsequent primary spermatocyte stage. The spatiotemporal correlation between the appearance of Zpg protein on the surface of spermatogonia in wild-type testis and the defective differentiation and loss of spermatogonial cells in zpg mutant testes suggests that gap junctional communication between spermatogonia and somatic cyst cells may be required for normal differentiation and survival of spermatogonia (Tazuke, 2002).
In ovaries, Zpg protein is present on the surface of developing germ cells, at least up to stage 10 of oogenesis. In developing egg chambers, anti-Zpg antibody staining was particularly striking at the germ cell/somatic follicle cell interface, where under conditions of lighter staining, Zpg protein appeared to be concentrated on the germ cell surface in a discrete patch under each follicle cell. The distribution of Zpg protein appeared more continuous at the nurse cell/nurse cell interface. In the germarium, Zpg protein is detected on the surface of all germ cells, including stem cells. Zpg protein appears to be concentrated in discrete patches on the surface of dividing cysts, where germ cells are in contact with cytoplasmic extensions from the somatically derived inner germarium sheath cells (Tazuke, 2002).
In female germline stem cells, Zpg protein also appears to localize to a small plaque adjacent to the spectrosome at the interface between female germline stem cells and somatic apical cap cells, under conditions where less overall anti-Zpg staining was detected. In an experiment where wild-type ovaries were stained with anti-anti-Spectrin and anti-Zpg antibodies, this dot was detected in 258 of the 289 stem cells scored from 10 different ovaries (Tazuke, 2002).
The position of the spot of Zpg detected just apical to the spectrosome in female germline stem cells by immunofluorescence suggests the possibility that there are gap junctions between the female germline stem cells and the overlying somatic cap cells. The presence of gap junctions in early female germ cells was confirmed by ultrastructural studies. In two separate sets of serial sections through the spectrosome region of female germline stem cells, gap junctions with the characteristic 2x10–9 m (20 Å) intermembrane spacing were clearly evident between female germline stem cells and adjacent apical cap cells. It is not known whether these gap junctional structures between female germline stem cells and apical cap cells correspond to the spots of Zpg detected adjacent to the spectrosome by immunofluorescence, although their relative positions are the same. In addition, it was observed that the intercellular space between germline stem cells and apical cap cells directly abutting the spectrosome is large (>200 Å; >2x10–8 m) and filled with lanthanum when stained with this substance. The components of this distinctive space are not known, although the space was characteristic of the five germaria studied by electron microscopy. Adherens junctions were also seen between germline stem cells and apical cap cells. Gap junctions are also observed at the ultrastructural level between adjacent germline stem cells, between cystoblasts, between cysts, between cystoblasts and inner sheath cells, and between adjacent nurse cells. A cluster of multiple gap junction structures was visible by electron microscopy between follicle cells and underlying nurse cells in developing egg chambers, consistent with the patch of Zpg staining at the base of each follicle cell observed by immunofluorescence light microscopy. It is not know whether these gap junctional structures contain Zpg protein (Tazuke, 2002).
Wild-type function of the gene zero population growth is required for early steps in gamete differentiation in both sexes. Although animals carrying a null mutation in zpg were fully viable, they are sterile and have tiny gonads (Tazuke, 2002).
Testes from animals mutant for zpg contained only small numbers of early germ cells up to pre-spermatocyte stage. In wild-type adults, six to nine male germline stem cells lie in a rosette surrounding the cluster of somatic hub cells at the apical tip of the testis. Upon stem cell division, the daughter next to the hub maintains stem cell identity, while the other daughter becomes a gonialblast and initiates four rounds of synchronous mitotic division with incomplete cytokinesis to produce a cyst of 16 interconnected spermatogonial cells, which then differentiate into spermatocytes. Wild-type male germline stem cells and gonialblasts both have a spherical spectrin-rich subcellular structure, the spectrosome. By contrast, interconnected spermatogonia and spermatocytes have a linear and branching spectrin rich fusome. The tiny testes from newly eclosed (0-2 day old) zpg mutant males have only a small number of germ cells, based on immunostaining with germ cell-specific markers. The germ cells usually appear as single or small clusters of cells near the apical tip. Immunostaining revealed that these germ cells contained spherical spectrin rich structures, suggesting stem cell or gonialblast identity. Germ cells in clusters reminiscent of spermatogonia have round or slightly tapered spectrin-rich structures, rather than fusomes, which appear larger than the spectrosomes in stem cells. This suggested that the zpg null mutant spermatogonia attempt, but are unable to complete, differentiation (Tazuke, 2002).
Somatic support cells normally associated with early male germ cells are present in zpg mutant testes, although their morphological arrangement appears abnormal. In wild type, two types of somatic cells, the hub and cyst cells, are in intimate contact with the germ cells. The area and number of the hub cells at the apical tip often appears expanded in zpg-null males compared with wild type. Such abnormalities in the hub may be secondary to a defect in germ cells in zpg mutant testes, since similar abnormalities in hub morphology and cell number are present in testes lacking germ cells altogether. In wild type, a pair of somatic cyst progenitor cells enclose each germline stem cell. Their progeny, the somatic cyst cells, enclose the developing germ cells. Cyst progenitor and cyst cells are present in zpg-null testes, based on the appearance of GFP expressed in these cells under the control of a ptc-GAL4 driver. However, as only a few germ cells were present in the zpg mutant testes, many of the cyst cells do not appear to enclose germ cells, and so do not have the ‘lacy’ appearance characteristic of cyst cells in wild-type testes (Tazuke, 2002).
Wild-type function of zpg was also required for differentiation of early germ cells in females. The tiny ovaries from newly eclosed zpg mutant females lacked the strings of developing egg chambers characteristic of wild type. Instead, germaria from freshly eclosed females commonly contain only a few germ cells, which appear as single cells at the apical tip of the germarium, located where female germline stem cells and cystoblasts reside. As in the male, female germ line stem cells and cystoblasts can be identified by spherical spectrin rich structures, spectrosomes, while the mitotically amplifying interconnected cysts contain branching fusomes. The germ cells remaining in zpg null mutant germaria have spherical spectrosomes rather than branched fusomes, suggesting stem cell or cystoblast identity. Occasionally, in freshly eclosed females, structures resembling egg chambers were observed (one per 2.9 ovaries, n=58 ovaries) further down the ovary, that contain abnormal number of germ cells that appear to be degenerating (Tazuke, 2002).
The phenotypic analysis of freshly eclosed zpg-null mutant ovaries suggests that Zpg function is not required for the initial placement of germ cells in the stem cell niche during ovary morphogenesis, although the possibility cannot be ruled out that a minute undetectable amount of maternally derived Zpg product perdures during the larval stages. Analysis of older zpg-null mutant females revealed that the number of germaria with early germ cells in the stem cell niche at the apical tip decreases with age. In three separate experiments, 70%-93% of germaria from newly eclosed zpg/Df females had at least one and usually two or more early germ cells in the stem cell position at the apical tip. By contrast, in 3-week-old zpg-null mutant females of the same genotype, only 17%-24% of germaria had even one Vasa-positive cell at the apical tip, when compared with 100% of wild-type control germaria. In some ovaries, ovarioles that lacked germ cells at the tip of the germarium had one or a few differentiating egg chambers further down the ovariole, as if germline stem cells were not maintained but instead initiated differentiation in the absence of zpg function in older females. The differentiating egg chambers in aged zpg females commonly appeared abnormal (Tazuke, 2002).
Most ovarioles from newly eclosed female flies that carry a strong mutation in the gene zpg have only a few germ cells, located at the anterior tip of the ovariole; the others contain no germ cells. zpg ovarioles that were occupied by germ line harbored 3.8 germ cells on average, as determined by anti-Vasa antibody labeling to mark the germ line. In comparison, wild-type germaria are filled with dividing cysts, the products of cystoblast divisions. The stem cell positions (close to cap cells) were occupied by zpg cells, but some single germ cells or clusters of germ cells were also located away from the tip. The single cells in the zpg mutant contained a round spectrosome, and could therefore be a mixed population of GSCs, cystoblasts or an intermediate between the two. To determine the developmental state of zpg germ cells, wild-type and zpg ovaries were double-labelled with anti-Vasa antibody and an antibody against cytoplasmic Bam protein (BamC), which stains cystoblasts and early cysts, but not GSCs. Of the 169 zpg ovarioles that had germ cells, only 2 contained a single cell that was BamC-positive. By contrast, 13 out of 50 wild-type ovarioles had a single cell stained with anti-BamC (26%). It is concluded that most zpg ovarioles lack cystoblasts (Gilboa, 2003).
In some zpg ovarioles clusters of 2-3 cells were observed that stained positive with anti-BamC antibodies. These may be the developing cysts that give rise to the rare egg chambers observed in these mutants (Tazuke, 2002). As in wild type, the rare developing zpg cysts are interconnected by ring canals. However, the fusome, which normally spreads through the dividing cyst, is either aberrant or absent. The rare egg chambers were almost always composed of less than 16 germ line cells, and DAPI staining indicated degeneration of the chamber. Since the fusome controls the divisions of the cyst, the abnormal number of germ cells in the egg chamber is likely to be the result of a severed fusome in the mutants (Gilboa, 2003).
Using an antibody raised against the cytoplasmic tail of Zpg (Tazuke, 2002), staining was observed in germ cells of every stage of development from GSCs to budding cysts of wild-type germaria. The staining appeared to be at the membrane of the cells and was somewhat stronger in region 2. The presence of Zpg staining in GSCs and dividing cysts correlates with the early defect in germ cell differentiation in zpg mutants and suggests that Zpg acts in the germ line (Gilboa, 2003).
The low number of cystoblasts in zpg ovarioles could be the result of defects in GSC division or in survival of the GSC daughter cell destined to differentiate. To distinguish between these two possibilities, zpg or wild-type germ cells were stained for the mitotic marker phospho-Histone H3. The percentage of marked zpg cells at the stem cell position per ovariole was not statistically different from that of wild-type GSCs. Similarly, the number of wild-type GSCs per ovariole in S phase (detected by BrdU labeling) was comparable to that of zpg cells. Taken together, these data show that zpg and wild-type cells spend a similar proportion of time in M and S phase. Although the possibility that zpg cells have an overall slower cell cycle, as compared to wild-type GSCs, cannot be ruled out, the data show that zpg cells divide. The lack of differentiating cells in zpg ovarioles may therefore indicate that zpg germ cells die when they commence differentiation (Gilboa, 2003).
To test this hypothesis, zpg germ cells were forced to differentiate by inducing expression of Bam protein in these cells, using a heat shock construct. In wild type, overexpression of Bam in GSCs induces the cystoblast differentiation program, resulting in a chain of egg chambers connected to a germarium depleted of germ line. When Bam was induced in zpg flies, no germ cells could be observed with anti-Vasa staining. Heat shock itself was not the cause of germ cell death, since ovaries of heat-shocked zpg animals, which did not carry the hs-bam transgene, still possessed germ cells. Thus, zpg germ cells die when forced to differentiate. This indicates that Zpg is required for the survival of differentiating germ cells. Taken together, these data indicate that zpg cells divide, and that the daughter cells that are destined to differentiate die (Gilboa, 2003).
To further explore the role of Zpg in early germ cell differentiation and survival, zpg alleles were recombined with mutant alleles of the gene pumilio (pum). pum mutant ovaries exhibit a compound phenotype. Many ovarioles lack germ line completely, a defect that may be attributed to preoogenic defects. Ovarioles with germ line have a germ line-depleted germarium connected to a few defective egg chambers. This phenotype suggests that Pum has roles in GSC maintenance. In ovaries from zpg, pum double-mutant females, many ovarioles were empty. This is consistent with the embryonic and larval requirement for pum. Ovarioles occupied by germ line exhibited a phenotype more similar to zpg than to pum mutants: few germ cells at the tip of the ovariole. Thus Zpg function is required for the differentiation of pum mutant germ cells. The apparent difference between the zpg, pum and hs-Bam; zpg phenotypes may reflect the different roles of Pum and Bam in GSC differentiation. Pum, as a translational repressor may permit GSC maintenance by repressing differentiation, which requires Zpg. By contrast, Bam may have a more instructive role in GSC differentiation, such that its overexpression can overrule GSC-maintenance cues emanating from the niche, independent of zpg (Gilboa, 2003).
In addition to defects in GSC maintenance, pum mutants also show defects in cyst development. This function is also evident in the zpg, pum phenotype. Although zpg, pum ovarioles occupied by germ line mostly resemble the zpg phenotype, they contain more germ cells, and have more dividing cysts and differentiating egg chambers, than those of the single zpg mutant. The double mutant had an average of 0.23 egg chambers per ovariole (n=290), compared with 0.02 (n=500) in flies homozygous for zpg and heterozygous for pum. The higher number of single cells and egg chambers in zpg, pum double mutants may indicate that Zpg function is less essential when cyst development is abrogated, as is the case in pum mutants (Gilboa, 2003).
This analysis suggests that Zpg wild-type function is required for the differentiation of GSCs. At least two other genes, bam and bgcn, are required for early germ cell differentiation. However, the phenotype of bam and bgcn mutant ovaries is strikingly different from that of zpg mutants. Ovaries mutant for bam or bgcn are filled with many undifferentiated single germ cells harboring a spherical spectrosome, which have been described as GSC tumors. By contrast, ovaries from zpg flies have only a few germ cells at the tip of the ovariole. To determine the functional relationship between these genes, flies were made doubly mutant for zpg with either bam or bgcn. Ovaries from newly eclosed females were stained to visualize the germ line and the spectrosomes. In the double mutant lacking both zpg and bam, only a few germ cells were detected at the ovariole tip. However, most double-mutant ovarioles had somewhat more germ cells than zpg ovarioles. Similar results were obtained with double mutants of zpg and bgcn (Gilboa, 2003).
Since GSCs can survive in a zpg background, the predicted phenotype of a zpg, bam or bgcn; zpg double mutant would be similar to a bam (or bgcn) phenotype (i.e. a germarium filled with undifferentiated GSCs). By contrast, the double-mutant phenotype more closely resembles the zpg phenotype. To test whether slow division of zpg cells accounts for the lack of tumors in young females, older (1- to 2-week-old) females were analyzed; a similar phenotype to that of young females was found. Thus, wild-type Zpg function is required for the accumulation of bam or bgcn mutant germ cell tumors removed from the niche (Gilboa, 2003).
To investigate further a possible role for Zpg-mediated intercellular communication in the development of germ cell tumors, the genetic interaction between dpp and zpg was analyzed. It has been proposed that an increased Dpp signal induces over-proliferation of GSC-like cells. It was therefore reasoned that an increased Dpp signal could induce zpg cells to over-proliferate. To test this, flies carrying several insertions of a heat-shock dpp transgene (hs-dpp) were crossed into a zpg background. Flies were heat shocked, and then dissected and stained to reveal the germ line and spectrosomes. Control animals heterozygous for zpg, carrying a subset of the hs-dpp transgenes, had more single germ cells with spherical spectrosomes than did wild type. Homozygous zpg animals, which carried at least the same number of hs-dpp transgenes as the control, did not show an increase in germ cell number. To test whether zpg cells could respond to a Dpp signal, ovaries of zpg animals were stained with antibodies against phosphorylated Mad (p-Mad). Mad is a component of the Dpp signal transduction pathway and is phosphorylated upon activation of the pathway. In wild type, p-Mad staining can be detected in GSCs, cystoblasts and dividing cysts in region 1 of the germarium. The highest level of staining is observed in early germ cells; staining then gradually declines towards the posterior. p-Mad is also detected in the single cells that accumulate following Dpp overexpression. Similarly, p-Mad staining is detected in zpg germ cells, suggesting that the mutant cells are able to receive the Dpp signal. There may be several explanations for the failure of zpg cells to proliferate or survive in response to a Dpp signal. (1) The Dpp pathway could be blocked downstream of Mad in zpg cells. (2) zpg cells may not be able to survive when unattached to the tip of the ovary. (3) Proliferating cells in hs-dpp flies, that move away from the niche, are in a more differentiated state than the cells in the niche, and therefore die in a zpg background (Gilboa, 2003).
bam tumor cells and germ cells proliferating after Dpp overexpression (hs-dpp tumor cells) are considered to be GSCs because of their round spectrosomes and lack of BamC staining. Yet, these cells do not accumulate in a zpg background. One possible explanation for this observation is that bam and hs-dpp cells, as they move away from the niche, are at an intermediate state (pre-cystoblast) between a stem cell and a cystoblast, and that cystoblast development and survival requires Zpg. To determine whether an intermediate state between GSCs and cystoblasts exists in wild type, ovarioles were triple-labeled with anti-Vasa, 1B1 monoclonal antibody and anti-BamC, to mark the germ line, spectrosomes and cystoblasts, respectively. BamC antibody stains cysts of 4 or 8 cells strongly. Two-cell cysts had notably weaker staining. Only rarely were cystoblasts, i.e., single cells, stained with anti-BamC. In many ovarioles, single cells with a spherical spectrosome were observed that were removed from the stem cell position yet did not stain for the cystoblast marker BamC. The number of cells were counted that carried a spherical spectrosome and did not stain with anti-BamC. These cells would comprise the GSC population plus the presumptive intermediate population. Of 100 ovarioles scored, most had between 3 and 5 single cells that did not stain with anti-BamC. The average number of these cells was 3.9. This is a greater number than the average number of GSCs that populate an ovariole (between 2 and 3), as determined by cell-lineage analysis and electron microscopy. These data support the hypothesis that an intermediate state between a stem cell and a cystoblast exists in wild type (Gilboa, 2003).
An intermediate cell population between slowly dividing stem cells and differentiating cells, described as transit amplifying cells, is a common feature in stem cell systems, including those giving rise to blood cells, skin and the gut epithelium. These are the products of stem cell division and have limited potential to divide prior to differentiation. In principle, the existence of a transit amplifying cell population in the Drosophila germ line could be suggested if the GSC division rate is too low to account for the number of cysts/egg chambers produced (including dying ones) in a set period of time. Acquiring this information, especially in region 1 of the germarium has proved to be difficult. Therefore the products of possible division of cells were directly examined at the transition state. If a pre-cystoblast cell amplifies, it should give rise to more than one egg chamber. By contrast, a cystoblast divides incompletely, giving rise to a two-cell cyst and, eventually, to one egg chamber. To distinguish between 'intermediate-state' divisions and cystoblast divisions, mosaic analysis was conducted in wild-type germaria using the FLP-FRT marker system, in which cells that are produced by mitotic recombination are marked as twin-spots by the copy number of the gene (lacZ), encoding the marker ß-Galactosidase (0 or 2 copies). Recombination in GSCs would result in a marked GSC, a string of marked cysts arising from its subsequent division, and one cyst that is the twin-spot of the original recombination event. A recombination event in the cystoblast would not be observed under the experimental conditions because marked cells within a cyst share the diffusable ß-Gal. It was reasoned that, if a cell at the transition state divides, twin-spot cysts would be observed even in germaria where GSCs are not marked. Such a situation could also arise if a marked GSC left the niche and differentiated. However, GSC loss is unlikely to affect this analysis, since the half-life of wild-type GSCs is 4.6 weeks, whereas the females were dissected 2-3 days after induction of Flipase. Of 177 ovarioles scored, 104 contained no marked GSCs and no marked cysts (~60%). 73 ovarioles contained marked cysts and a marked GSC (~40%). Ovarioles were observed that contained only marked cysts and marked GSCs. These results led to the conclusion that cells at the transition state in wild type do not divide at an appreciable rate but proceed to differentiate to a cystoblast. Thus, they do not constitute the equivalent of a transient amplifying population. By contrast, tumor cells in bam mutants, or when Dpp is overexpressed, continue to divide, since their differentiation is blocked (Gilboa, 2003).
Thus, zpg ovarioles contain single germ cells at the anterior tip. Most of these cells do not reach the cystoblast stage. Since zpg cells are not arrested at a particular stage of their cell cycle, and can divide, it is concluded that GSCs that lack zpg divide to give another stem cell and a daughter cell that dies at early stages of differentiation. Accordingly, overexpression of bam, which is necessary and sufficient to promote germ cell differentiation, kills zpg cells (Gilboa, 2003).
zpg encodes innexin 4, one of eight innexins in Drosophila (Phelan, 2001; Stebbings, 2002; Tazuke, 2002). Innexins are the functional homologs of the vertebrate connexins, or gap junction proteins (Phelan, 2001). In mammalian oogenesis, gap junctions have been implicated in cell-cell signaling. Early luteinization of granulosa cells is observed either when the oocyte is physically removed from immature wild-type follicles, or in mice lacking connexin 37 (Simon, 1997), suggesting a gap junction-mediated signaling mechanism between the oocyte and granulosa cells (Gilboa, 2003).
Zpg is required for the survival of the germ line stem cell daughter as it moves away from the niche and begins to differentiate. From its expression pattern, and the specificity of its function, it is clear that Zpg acts in a germ line autonomous way. However, it is not yet known whether Zpg facilitates communication between germ cells, or between germ line and soma. The germarium is a compact structure where early germ cells contact each other, the somatic cap cells contact GSCs, and inner-sheath cells contact GSCs and their daughters. Further study is needed to clarify which cells communicate with germ cells through Zpg-gap junctions. Likewise, the nature of the requirement for zpg in GSC differentiation is still uncertain. Gap junctions may be used to supply the GSC daughter cell with nutrients, or with a survival factor required for its subsequent growth. Alternatively, gap junctions may be used to deposit a factor that is required for the differentiation process itself, rather than for survival, while an accessory mechanism eliminates cells that begin differentiating without that factor. The major argument in favor of a role for Zpg in differentiation at the stem cell stage comes from the phenotypic analysis of zpg, pum double mutants in which, unlike in pum single mutants, GSCs do not differentiate. Although Pum-mediated repression is removed in the double mutants, GSCs cannot differentiate as they may lack a differentiation signal provided by Zpg. It is harder to imagine how nutritional deficiency per se could block differentiation of the double-mutant cells because single-mutant zpg cells do begin to differentiate (and then die) (Gilboa, 2003).
Recent studies suggest that the niche promotes stem cell fate through Dpp signaling. This may be achieved through repression of bam. GSCs are also tightly tethered to the niche via adherens junctions. Other, as-yet unidentified mechanisms may be used by the niche to protect GSCs (Gilboa, 2003).
Once germ cells leave the niche they activate the differentiation pathway. It is proposed that differentiation of GCSs to cystoblasts is not direct but proceeds via an intermediate state. Most wild-type cells, which by their position within the germarium were judged to be cystoblasts, are not stained with BamC antibody. This finding concurs with Ohlstein (1997), who observed cytoplasmic Bam just before the cystoblasts divide to form a two-cell cyst and proposed the existence of an intermediate/pre-cystoblast state between GSCs and cystoblasts. In other stem cell systems, the intermediate cell population has a biological function, namely increasing the progeny of a single stem cell division. The results indicate that in Drosophila females, cells at the intermediate state do not constitute a transit-amplifying cell population. However, the 'pre-cystoblast state' may have a different biological significance. A vacant niche can be filled by a neighboring GSC that divides 'sideways' instead of along the anteroposterior axis. An alternative for filling a vacant GSC position might be for a cell at the intermediate state to reoccupy the niche (Gilboa, 2003).
The suggested model, adding a transitory state between the stem cell and the cystoblast, raises an interesting question. Is the differentiation of a GSC to a cystoblast a continuous process or a discrete one? It is notable that none of the markers that are currently used is specific to the stem cell, the cystoblast or the intermediate. Bam is present (in its fusomal form) already in the stem cell, and BamC gradually accumulates in the cystoblast. Pumilio protein and phosphorylated Mad are also detected from GSCs to cystoblasts and early cysts. In other stem cell systems, including mammalian hematopoiesis, many intermediate cell types are known, and can be isolated by specific marker combinations. Due to the relative lack of markers, the isolation of these 'cell types' from Drosophila ovaries is currently impossible. The overlap of expression patterns of the proteins that are known to have a role in stem cell maintenance and differentiation may indicate that differentiation is gradual, and possibly reversible (Gilboa, 2003).
It has been suggested that in bam or bgcn mutant ovaries, or when Dpp is overexpressed, the germaria are filled with GSC tumor cells. The findings of an intermediate cell population in wild type raises the possibility that GSC tumor cells share some properties with precystoblasts. Both these populations are single, do not stain for BamC, but exist outside of the niche. Some support to the analogy between pre-cystoblasts and 'GSC tumors' is evident in the fact that the latter do not survive past the niche in a zpg background. Thus, the zpg double mutants allow two cell populations in 'GSC tumors' to be distinguish - cells that are inside or outside of the niche. It is suggest that when Dpp is overexpressed, or in bam/bgcn mutants, cells outside the niche cannot fully activate the differentiation program and are at an intermediate state between a GSC and a cystoblast. These cells die in a zpg background, whereas the tumor cells in contact with the niche survive. The results thus suggest that beyond Zpg gap junctions and Dpp signaling, there must be additional signaling between GSCs and the niche, which helps maintain GSCs. Additional markers are needed to determine unequivocally whether bam tumors are similar to Dpp tumors, and whether they share properties with wild-type precystoblasts (Gilboa, 2003).
Differentiation of the stem cell daughter requires gap junctions. It is assumed that zpg is acting in parallel to pum, dpp, bam and bgcn because the double mutants showed incomplete epistasis of zpg over each of these genes. Although a role for gap junction proteins has been established in mammalian oogenesis (Ackert, 2001; Carabatsos, 2000; Juneja, 1999; Simon, 1997), this is thought to be the first instance where a gap junction protein is shown to control stem cell differentiation. What passes through these gap junctions, and which cells are connected to GSCs through Zpg channels, is still unknown. One intriguing option is that Zpg forms part of the channels that connect GSCs to the surrounding somatic niche cells. If so, that would suggest that the niche in the Drosophila germarium is necessary, not only for stem cell maintenance, but also for stem cell differentiation (Gilboa, 2003).
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date revised: 27 April 2004
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
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