nanos is required for formation of the spectrosome, a germ cell-specific organelle

Authors


Abstract

Germ cell identity and development are controlled by autonomous cues in the germ plasm as well as by interactions between germ cells and somatic cells. Here, we investigate the formation of a germ cell-specific organelle, the spectrosome. We find that spectrosome formation is independent of germ cell–soma interactions and is autonomous to the germ cells. Furthermore, the germ plasm component nanos (nos) is essential for spectrosome formation. The role of nos in spectrosome formation is independent of its role in germ cell survival; nos mutant germ cells that are prevented from undergoing programmed cell death still fail to form spectrosomes. Thus, nos is required to regulate the formation of this germ cell-specific organelle, further supporting a role for nos in promoting germ cell identity. Developmental Dynamics 234:22–27, 2005. © 2005 Wiley-Liss, Inc.

INTRODUCTION

Germ cell identity is initially specified by the germ plasm, a specialized cytoplasm inherited by the germ cells at the time of germ cell formation (reviewed in Williamson and Lehmann, 1996). However, germ cells also require interactions with somatic cells to differentiate properly and produce sperm or eggs. Thus, an important question is which aspects of germ cell identity are controlled autonomously by the germ plasm, and which are regulated by interactions with the soma?

Germ cells initially form at the posterior pole of the developing Drosophila embryo and are carried into the embryo along with the posterior midgut during gastrulation. Germ cells then migrate out of the posterior midgut into the mesoderm, where they make contact with specialized mesodermal derivatives that give rise to the somatic gonad (somatic gonadal precursors, or SGPs, reviewed in Starz-Gaiano and Lehmann, 2001). Finally, germ cells and SGPs coalesce to form the embryonic gonad, a process that involves extensive germ cell–SGP interaction (Jenkins et al., 2003). Around the time of gonad formation, differences between males and females can already be observed in the development of both the somatic gonad (DeFalco et al., 2003) and germ cells (Staab et al., 1996).

A unique aspect of the germ cell developmental program is the formation of a germ cell-specific organelle known as the spectrosome (Lin et al., 1994; Lin and Spradling, 1995). The spectrosome is a vesiculated structure that includes proteins such as Alpha- and Beta-spectrin (Lin et al., 1994; de Cuevas et al., 1996), Hu-li tai shao (Lin et al., 1994), Bag of marbles (McKearin and Ohlstein, 1995), and markers for the endoplasmic reticulum (Snapp et al., 2004). In the Drosophila ovary, spectrosomes are found in germline stem cells and are thought to play an important role in asymmetric division of these cells (Deng and Lin, 1997). Germline stem cells divide to produce cystoblasts, which give rise to the 16 interconnected cells of a germline cyst, one of which will become the oocyte (reviewed in Huynh and St. Johnston, 2004). The spectrosome gives rise to the fusome, which extends through each cell of the cyst and is likely to be critical for oocyte specification (Lin and Spradling, 1995; de Cuevas and Spradling, 1998) and for organizing the microtubule network that transports RNAs and proteins into the developing oocyte (Grieder et al., 2000; Roper and Brown, 2004). Spectrosomes and fusomes are also found in the germline in testes (Lin et al., 1994; Tran et al., 2000). Thus, the spectrosome/fusome is unique to the germ cell lineage in Drosophila and is likely to be important for proper germ cell function in both males and females.

RESULTS

Spectrosome Formation

Previous work has shown that germ cells do not contain spectrosomes at the time of germ cell formation at the posterior pole, but spectrosomes can be observed during germ cell migration and gonad formation (Lin and Spradling, 1997). We first examined spectrosome formation in more detail. Foci of anti-spectrin immunoreactivity (spectrosomes) were not observed in germ cells when they form at the posterior pole of the embryo (Lin and Spradling, 1997; Fig. 1A), or immediately before their migration out of the posterior midgut pocket (Fig. 1B). However, spectrosomes were observed shortly after germ cells begin migrating and enter the mesoderm (Fig. 1C). Spectrosomes were observed uniformly in most or all germ cells at this stage; we did not see variability between germ cells in their ability to form a spectrosome.

Figure 1.

Analysis of spectrosome development during embryogenesis. Wild-type embryos coimmunostained with anti-Vasa (red) and anti-Spectrin (green). A,B: During early germline development, spectrosomes are not detected at embryonic stage 5 in germ cells at the posterior pole (A) or at stage 10 in germ cells before or during migration out of the midgut pocket (B). CF: Foci of Spectrin immunoreactivity are first detected at embryonic stage 11 in germ cells associated with the somatic mesoderm (C) and increase throughout embryonic development (compare C–F; stages 11–17). G,H: Under limited staining conditions, spectrosomes are only detected at a later stage (compare G and H; stages 11 and 15, respectively). I,J: Spectrosome immunoreactivity is equivalent in male and female germ cells (compare I and J; stage 15). Scale bar = 12.5 μm in A (applies to A–J).

After formation, foci of anti-spectrin immunoreactivity increase in size and intensity throughout later stages of gonad formation and development (Lin and Spradling, 1997). Just before gonad formation (Fig. 1D) spectrosomes were observed but were noticeably smaller than spectrosomes in the fully coalesced embryonic gonad (Lin and Spradling, 1997; Fig. 1E). Spectrosomes continued to grow in later stage embryonic gonads (Fig. 1F) and eventually reach their full size during larval stages (Lin and Spradling, 1997). To further address changes in spectrosome development, we performed anti-spectrin immunostaining using a shorter, and presumably limiting, primary antibody incubation (see Experimental Procedures section). Under these conditions, spectrosomes were no longer visible during stages of germ cell migration (Fig. 1G) and only became visible in the coalesced gonad (Fig. 1H). This finding provides further evidence that spectrosomes continue to develop during these stages and contain either more spectrin protein or spectrin is more accessible to immunostaining at later time points. Finally, we examined spectrosome formation in embryos whose sex could be identified and observed no differences in spectrosomes between the two sexes in embryonic gonads (Fig. 1I,J).

Spectrosome Development Is Not Dependent on Germ Cell–Soma Interactions

We next investigated whether the SGPs or other somatic cells influence spectrosome formation. In mutants for HMG Coenzyme A reductase (hmgcr), SGPs still form but many germ cells fail to contact SGPs because they no longer navigate correctly to the somatic gonad (Van Doren et al., 1998a). Spectrosomes were still seen in germ cells mislocalized in different regions of hmgcr mutant embryos (Fig. 2A,B). Furthermore, when the somatic gonad was genetically ablated using tinman, zinc finger homeodomain protein-1 (tin zfh-1) double mutants (Broihier et al., 1998), spectrosomes were still observed in the germ cells (Fig. 2C,D). Thus, neither contact with nor the presence of the somatic gonad is required for spectrosome formation.

Figure 2.

Spectrosome formation is independent of the soma. Stage 15 embryos coimmunostained with anti-Vasa (red) and anti-Spectrin (green). B and F are enlargements of A and E, respectively. A,B: Spectrosome formation is detected in mislocalized germ cells in hmgcr mutant embryos. C,D: Spectrosome formation is observed in tin zfh-1 mutant embryos lacking somatic gonadal precursor cells. E,F: Spectrosomes are detected in germ cells trapped in the gut in srp mutant embryos. Scale bars = 25 μm in A (applies to A,C,E), 12.5 μm in B (applies to B,D,F).

Finally, we investigated whether any of the germ cells' other normal somatic cell contacts are required for spectrosome formation. In serpent (srp) mutants, the midgut is transformed into hindgut, which prevents many germ cells from migrating out of the gut (Reuter, 1994). These germ cells cannot make contact with any mesodermal derivatives, and the posterior midgut endoderm that they normally contact is also no longer properly specified. Spectrosomes were still seen in germ cells that are trapped in the gut in srp mutants (Fig. 2E,F). We conclude that spectrosome formation does not require germ cell–soma contact and, therefore, is likely to be dependent on germ cell autonomous cues.

Role of nanos in Spectrosome Formation

Because spectrosome formation appears to be a germ cell autonomous process, it is likely to be regulated by the germ plasm. nanos (nos) is a key germ plasm component in Drosophila (Wang and Lehmann, 1991) and has been shown to regulate germ cell development in species as diverse as flies and mice (Kobayashi et al., 1996; Tsuda et al., 2003). Maternal (germ plasm) nos has been reported to regulate several aspects of germ cell development in Drosophila, including germ cell gene expression, proliferation, and migration (Kobayashi et al., 1996; Asaoka et al., 1998; Forbes and Lehmann, 1998; Asaoka-Taguchi et al., 1999; Deshpande et al., 1999; Sano et al., 2001). However, it was demonstrated recently that maternal nos is also required for germ cell survival (Hayashi et al., 2004). Therefore, it is unclear which aspects of germ cell development are directly affected by nos and which are only secondarily affected due to programmed cell death of the germ cells.

We examined whether nos is required for spectrosome formation and found that the vast majority of germ cells in nos mutant embryos (embryos from nos homozygous mutant mothers) completely lacked spectrosomes (Fig. 3A). Only a small percentage of germ cells in nos mutants (9.3%, n = 778 for nosBN/nosRC) still retained foci of anti-spectrin immunoreactivity (Fig. 3A, inset), which in general labeled less intensely than wild-type spectrosomes. A similar effect was observed for two different nos allele combinations (nosBN/nosBN and nosBN/nosRC), and it is possible that the low level of residual spectrosome formation reflects a small amount of nos activity remaining in the nosBN allele (null allele combinations of nos were not analyzed because they exhibit oogenesis defects). Because nos is also required for posterior patterning of the embryo (Lehmann and Nüsslein-Volhard, 1991), we wanted to verify that these defects in the soma were not responsible for the defects in spectrosome formation. To determine this, we used nos, hunchback double mutants in which the effects of nos on posterior patterning are eliminated (Hülskamp et al., 1989; Irish et al., 1989; Struhl, 1989), while germ cells still lack nos function. Again, spectrosomes were not observed in the vast majority of germ cells in these embryos either at the time spectrosomes normally form (Fig. 3B) or at later stages when spectrosomes are normally more robust (Fig. 3C, percent of germ cells with foci of anti-Spectrin immunoreactivity was 13% in nos, hb, n = 914, vs. 100% in wild-type, n = 180). To confirm that nos mutants exhibit a general defect in spectrosome formation, we examined a second fusome component, Hu-li tai shao (HTS; Yue and Spradling, 1992; Lin et al., 1994). In contrast to wild-type (Fig. 3D), most germ cells in nos, hb mutants lack foci of anti-HTS immunoreactivity (Fig. 3E). Thus, nos appears to be required in germ cells for proper spectrosome formation and development.

Figure 3.

Maternal nanos is required for spectrosome formation. Embryos coimmunostained with anti-Vasa (red) and either anti-Spectrin (A–C,F, green) or anti-HTS (D,E, green). Spectrosomes are detected in wild-type germ cells using both anti-Spectrin (see Fig. 1) and anti-HTS antibodies (D). AF: Spectrosomes are absent from the vast majority of germ cells in embryos mutant for maternal nanos (nosBN) aged 12–16 hr at 25°C (∼ stage 15–16; A), embryos mutant for maternal nanos and hunchback at stage 11 (B) and stage 15 (C,E), and embryos mutant for maternal nanos and zygotic Df(H99) aged 12–16 hr at 25°C (∼ stage 15–16; F). Scale bar = 10 μm in B (applies to A–F).

We next wanted to determine whether nos is required for spectrosome formation, or spectrosomes fail to form in nos mutants because germ cells are undergoing programmed cell death. To address this question, we examined nos mutant embryos in which cell death has been prevented by eliminating a group of genes required for programmed cell death (using the H99 deficiency, which removes head involution defective, reaper, and grim [White et al., 1994; Grether et al., 1995; Chen et al., 1996]). In these embryos (nos maternal mutant, Df(H99) zygotic mutant), germ cells now survive and no longer exhibit characteristics of programmed cell death such as fragmented DNA (Hayashi et al., 2004) or the presence of activated caspases (data not shown). However, the vast majority of germ cells still lacked spectrosomes in these embryos (Fig. 3F). Therefore, we conclude that nos is required for spectrosome formation independently of effects of nos on germ cell survival, supporting the view that nos can directly influence germ cell identity and development.

DISCUSSION

Our data indicate that spectrosome formation is autonomous to the germ cells and dependent on the germ plasm. Germ cells do not require any of their normal somatic cell contacts for proper formation of spectrosomes but do require the germ plasm component nos. Thus, part of the information laid down by the mother to specify germ cell identity includes a program for forming this germ cell-specific organelle. One possibility is that the components necessary for spectrosome formation are already present in the germ plasm, either as mRNAs or proteins. Alternatively, spectrosome formation might be dependent on the germ cell-specific pattern of gene expression. Indeed, germ cell-specific gene expression begins before spectrosome formation and is independent of soma–germline interaction (Van Doren et al., 1998b) and, therefore, may contribute to spectrosome formation.

An interesting question is whether all germ cells are created equal, or there are differences within the pool of germ cells after their formation at the posterior pole of the embryo. Heterogeneity has been observed within the germ cell population for other characteristics, such as the ability to process the P element transposase mRNA in a germline-specific manner (Kobayashi et al., 1993). This finding may reflect differences between germ cells, possibly due to the amount of germ plasm they inherit. We find little or no variability in the ability of wild-type germ cells to form spectrosomes or in the timing of spectrosome formation; germ cells appear equivalent by this assay. However, the total number of germ cells decreases during early germ cell development and gonad formation (Sonnenblick, 1950; Hay et al., 1988). Thus, it is still possible that there are differences between germ cells in their ability to form spectrosomes, but this ability is somehow related to their likelihood of survival; either all germ cells that survive have the ability to form spectrosomes or spectrosome formation is required for germ cell survival.

We have found that nos is a critical regulator of spectrosome formation; How might nos regulate this process? If NOS acts as an mRNA-specific translational repressor, as it does in posterior somatic patterning, NOS might repress the translation of a protein that specifically interferes with spectrosome formation. Alternatively, NOS might more generally promote germ cell identity, which in turn leads to spectrosome formation. Recently, it has been shown that nos mutant germ cells, which have been prevented from undergoing apoptosis, still do not develop properly (Hayashi et al., 2004). When these cells are incorporated within a gonad, they cannot differentiate and give rise to gametes. These nos-mutant germ cells can also integrate into somatic tissues, such as the gut, and express somatic genes. This work strongly indicates that nos is a critical regulator of germ cell identity, apart from regulating germ cell survival (Hayashi et al., 2004). Our work demonstrates that the role for nos in promoting germ cell identity extends to the formation of a germ cell-specific organelle, the spectrosome. Although it is likely that spectrosome formation is downstream of some of the effects of nos on germ cell identity, there may also be important aspects of germ cell development that require the spectrosome. Thus, some of the effects of nos on germ cell identity may be caused by defects in spectrosome formation.

EXPERIMENTAL PROCEDURES

Fly Stocks

w1118 and ru st faf-lacZ e ca (Moore et al., 1998) flies were used as wild-type. nos Df(H99) flies were generated by recombination of nosBN (Wang et al., 1994) and Df(H99). Other stocks include nosRC, Dfd-lacZ-HZ2.7 (Bergson and McGinnis, 1990), yw; FRT hbFBnosBN (Forbes and Lehmann, 1998), yw hs-FLP; FRT ovoD (Chou et al., 1993), foi20.71 (Van Doren et al., 2003), wunenCE (Zhang et al., 1996), hmgcrclb1 (Van Doren et al., 1998a), tinGC14zfh-175.26 (Broihier et al., 1998) and srp3. Any unspecified stocks were obtained from the Bloomington Stock Center (http://flybase.bio.indiana.edu/). Homozygous maternal hbFBnosBN mutant embryos were generated by inducing germ line clones in yw hs-FLP; FRT hbFBnosBN/FRT ovoD females as described (Forbes and Lehmann, 1998) and mating to wild-type males.

Immunocytochemistry and Antibodies

For immunohistochemistry, embryos were fixed, devitellinized, and immunostained as described (DeFalco et al., 2003), unless otherwise noted. To examine gonads in stage 17 embryos, where cuticle formation blocks antibody penetration, embryos were cut in half with a razor blade just before immunostaining. For spectrin immunocytochemistry, staining was performed sequentially by first incubating with mouse anti–Alpha-spectrin 3A9 (Developmental Studies Hybridoma Bank [DSHB]; D. Branton) at a 1:2 dilution overnight at 4°C, followed by incubation with goat anti-mouse Alexa488 secondary antibody (see below) for 4 hr at room temperature, then with other primary and secondary antibodies. Under “limiting” conditions, mouse anti–Alpha-spectrin 3A9 was diluted 1:2 together with other primary antibodies and stained for 2–3 hr at room temperature followed by secondary antibodies as per usual. Additional primary antibodies include mouse anti-HTS (DSHB; H. Lipshitz) at 1:4, mouse anti–β-gal (Promega) at 1:1,000, chick anti-Vasa (K. Howard) at 1:10,000, rabbit anti-Vasa (R. Lehmann) at 1:10,000, rabbit anti-cleaved Caspase-3 (Cell Signaling Technologies) at 1:50, rabbit anti–green fluorescent protein (GFP; Torrey Pines) at 1:3,000, and rabbit anti–β-gal (Cappel) at 1:10,000. Secondary antibodies at 1:500 include Alexa488 and Alexa546 goat anti-rabbit and Alexa488 and Alexa568 goat anti-mouse, Alexa546 goat anti-chick (all from Molecular Probes), and Cy5- goat anti-chick (Rockland).

Analysis of Whole-Mount Embryos

After staining, embryos were placed in 2.5% DABCO (Sigma) in 70% glycerol, then mounted onto slides. Samples were viewed with a Zeiss LSM510 Meta confocal microscope. When possible, staging of embryos was conducted according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1985). nos, nos Df(H99), tin zfh-1, and srp mutant embryos were maintained at 25°C until they were 12–16 hr old (a time when control embryos are stage 15–16). Genotype of embryos was determined by the presence of balancer chromosomes carrying either lacZ or GFP transgenes, unless otherwise noted: P{ftz lacZ-CyO}, P{Ubx-lacZ-TM3}, or P{Kr-GFP-TM3} were used to distinguish homozygous mutants from balancer-carrying siblings. To genotype Df(H99) embryos, anti-cleaved Caspase-3 was used to assess absence of programmed cell death in the soma of homozygous Df(H99) mutants. To determine sex of embryos, we used an X-chromosome carrying P{Dfd-lacZ-HZ2.7} (Bergson and McGinnis, 1990) as described above.

Acknowledgements

The authors thank Ken Howard, Ruth Lehmann, Bill McGinnis, and the Bloomington Drosophila Stock Center for providing fly stocks and antibodies. We acknowledge the Developmental Studies Hybridoma Bank (under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242) and all our colleagues who generously have contributed antibodies to this bank. We thank Michael McCaffery and the JHU Integrated Imaging Center for help with confocal microscopy. We thank Allan Spradling for helpful discussions, and Maggie de Cuevas and members of the Van Doren Lab for critical reading of the manuscript.