Gametogenesis in males and females differs in many ways. An important difference in Drosophila is that recombination between homologous chromosomes occurs only in female meiosis. Here, we report that this process relies on the correct functioning of Sex-lethal (Sxl) which is primarily known as the master gene in somatic sex determination. Certain alleles of this gene (Sxlfs) disrupt the germline, but not the somatic function of Sxl and cause an arrest of germ cell development during cystocyte proliferation. Using dominant suppressor mutations that relieve this early block in Sxlfs mutant females, we discovered additional requirements of Sxl for normal meiotic differentiation of the oocyte. Females mutant for Sxlfs and carrying a suppressor become fertile, but pairing of homologous chromosomes and formation of chiasmata is severely perturbed, resulting in an almost complete lack of recombinants and a high incidence of non-disjunction events. Similar results were obtained when germline expression of wild-type Sxl was compromised by mutations in virilizer (vir), a positive regulator of Sxl. Ectopic expression of a Sxl transgene in premeiotic stages of male germline development, on the other hand, is not sufficient to allow recombination to take place, which suggests that Sxl does not have a discriminatory role in this female-specific process. We propose that Sxl performs at least two tasks in oogenesis: an ‘early’ function in formation of the egg chamber, and a ‘late’ function in progression of the meiotic cell cycle, suggesting that both events are coordinated by a common mechanism.

Germ cells undergo a specialized meiotic cell cycle which coordinates differentiation with nuclear events necessary for production of haploid gametes. Although certain aspects of meiotic cell cycle regulation in higher organisms are understood (Sagata, 1996), little is known about how germ cell progression through meiosis is regulated and coordinated with gametogenesis. The fruitfly Drosophila melanogaster offers a suitable system to experimentally approach this question because many facets of early germ cell regulation and differentiation have already been analyzed in detail. In particular, studies in male gametogenesis led to the discovery of a number of genes that are involved in the coordinate control of the meiotic cycle and spermatid differentiation (reviewed in Maines and Wasserman, 1998). Less is known about the complementary process in females. Like the gonial cells in testes, their female counterparts, the cystoblasts, first undergo four mitotic divisions with incomplete cytokinesis before entering the meiotic prophase. In the resulting 16-cell cysts, cystocytes start assembling structures of the synaptonemal complex (Schmekel et al., 1993) with two cells, the pro-oocytes, attaining full pachytene, but only one cell, the oocyte, is destined to complete the meiotic program. The other 15 cells of the cluster will amplify their genomes endomitotically to support further growth and differentiation of the oocyte (see Fig. 1A,B; reviewed in Spradling, 1993).

Fig. 1.

Differentiation of the adult ovary and biphasic expression of Sxl. (A) Germarial events leading to the formation of the egg chamber. Stem cells (green) give rise to a cystoblast daughter cell (red) which in turn undergoes four rounds of cystocyte division (different shades of violet) to form a 16-cell cluster located in the middle of the germarium. This cluster becomes enveloped by infiltrating follicle cells and moves to the posterior end to form a stage 1 cyst. One pro-oocyte is determined (indicated by the darker coloured cell in the pink cluster) and occupies the most posterior position. (B) The nuclei of the nurse cells (pink) undergo endomitosis and become polyploid, whereas the nucleus of the oocyte (red) arrests in the prophase of meiosis, indicated by the densely condensed karyosome. (C,D) Expression of SXL protein in wild-type germarium (C) and at early previtellogenic stages (D). Cytoplasmic expression is observed in the anterior-most germarial cells and drastically declines during cystocyte divisions. SXL protein (red) reappears as nuclear foci in cells of the 16-cell cyst (arrows in C). In later stages, SXL protein accumulates in nurse cells. (E,F) Germarium and previtellogenic stages of a vir2f clone. Expression of SXL is seen in the anterior-most cells of the germarium, but not in germ cells of the early formed cysts or late stages. (G) Stage 10 of a vir6 clone. SXL is only detected in the somatically derived follicle cells (fc) surrounding the egg chamber, but not in the nurse cells (nc) or oocyte (oc).

Fig. 1.

Differentiation of the adult ovary and biphasic expression of Sxl. (A) Germarial events leading to the formation of the egg chamber. Stem cells (green) give rise to a cystoblast daughter cell (red) which in turn undergoes four rounds of cystocyte division (different shades of violet) to form a 16-cell cluster located in the middle of the germarium. This cluster becomes enveloped by infiltrating follicle cells and moves to the posterior end to form a stage 1 cyst. One pro-oocyte is determined (indicated by the darker coloured cell in the pink cluster) and occupies the most posterior position. (B) The nuclei of the nurse cells (pink) undergo endomitosis and become polyploid, whereas the nucleus of the oocyte (red) arrests in the prophase of meiosis, indicated by the densely condensed karyosome. (C,D) Expression of SXL protein in wild-type germarium (C) and at early previtellogenic stages (D). Cytoplasmic expression is observed in the anterior-most germarial cells and drastically declines during cystocyte divisions. SXL protein (red) reappears as nuclear foci in cells of the 16-cell cyst (arrows in C). In later stages, SXL protein accumulates in nurse cells. (E,F) Germarium and previtellogenic stages of a vir2f clone. Expression of SXL is seen in the anterior-most cells of the germarium, but not in germ cells of the early formed cysts or late stages. (G) Stage 10 of a vir6 clone. SXL is only detected in the somatically derived follicle cells (fc) surrounding the egg chamber, but not in the nurse cells (nc) or oocyte (oc).

Several mutations are known to affect the three major features of meiosis, first synapsis, then exchange, and finally disjunction of meiotic chromosomes (Lindsley and Sandler, 1977). For instance, the kinesin-like products of claret-nondisjunctional (cand) (Endow et al., 1990) and no distributive disjunction (nod) (Zhang and Hawley 1990) are required for chromosome segregation in meiosis I, while mei-S332 and ord appear to play a specific role in keeping sister chromatids together during the first division (Kerrebrock et al., 1992; Miyazaki and Orr-Weaver, 1992). Recently, it has been reported that members of the RAD52 DNA repair pathway, okra and spindle-B, participate in the meiotic DNA metabolism of female germ cells (Ghabrial et al., 1998). Mutations in these genes affect both recombination and disjunction. It is a common feature that meiotic mutations that disrupt recombination also affect disjunction because this process normally relies on cross-overs to align the homologs on the spindle. Some mutations seem to act early in the meiotic prophase. For instance, in mutant c(3)G germ cells, the process of synapsis and the formation of the synaptonemal complex are absent (Hall, 1972). As a result, recombination is abolished and non-disjunction highly elevated.

Our report adds an unexpected member to the list of mutations that affect early pachytene functions of meiosis. The corresponding gene, Sxl, is primarily known for its key role in somatic sex determination. In this tissue, it imposes female development when activated whereas male development follows when the gene is inactive. The gene encodes an RNA-binding protein which performs its regulatory function at the post-transcriptional level, either by forcing female-specific splicing pattern or by translational repression of male-specific target genes (Cline and Meyer, 1996; Bashaw and Baker, 1997; Kelley et al., 1997). Little is understood about its function in the germline. First evidence for an involvement in female germline development came from analysis of loss-of-function alleles in genetic mosaics (Schüpbach, 1985; Steinmann-Zwicky et al., 1989). When mutant germ cells are incorporated in a wild-type ovary, they fail to differentiate and instead display an overgrowth phenotype. Also, a class of sterile alleles exists that are specifically defective for germline, but not for somatic functions (Salz et al., 1987). Germ cells mutant for the recessive alleles Sxlf4, Sxlf5 and Sxlf18 (collectively called Sxlfs) remain small and undifferentiated and continue to proliferate excessively, forming large cysts, a phenotype which is commonly referred to as ovarian tumors or multicellular cysts. The overproliferation phenotype implicated Sxl in playing an essential role in the control of the cystocyte mitotic cycle and subsequent cyst formation.

The cellular mechanism by which Sxl executes this function is not understood. None of the known somatic target genes of Sxl is required in oogenesis (Marsh and Wieschaus, 1978; Schüpbach, 1982). Also, regulation and expression of this gene in the female germline differ from those in the soma. For instance, unlike the soma, germline activity of Sxl depends on non-autonomous cues emanating from the surrounding soma (Steinmann-Zwicky, 1992; Oliver et al., 1993; Horabin et al., 1995). Furthermore, its expression pattern in the germline appears biphasic with a striking change in intracellular distribution (Fig. 1C). Sxl is first expressed in female embryonic germ cells and maintains a high level of cytoplasmic expression until the first cystocyte divisions in the adult ovary (Bopp et al., 1993; Horabin et al., 1995). The level of SXL protein then precipitously declines and reappears in a second phase as nuclear foci in the newly formed 16-cell cyst. From stage 1 onward, it steadily increases in level and localizes to the cytoplasm and nuclei of nurse cells (Fig. 1D). The biphasic nature of this expression pattern is also evident from the differential effects of some positive regulators of Sxl. In germ cells mutant for snf1621 or for otu alleles of the ONC class, early Sxl expression is prevented (Bopp et al., 1993; Oliver et al., 1993). These cells cannot differentiate and they display the same tumorous phenotype as seen in Sxl mutant germ cells. However, in germ cells mutant for vir, the first phase of cytoplasmic expression is still maintained, but the second expression phase, which commences in early germarial cysts, is abolished (Fig. 1E-F). Absence of this ‘late’ SXL does not affect formation and differentiation of the egg chamber. The affected cells complete oogenesis and give rise to progeny. It was therefore concluded that early cytoplasmic activity is sufficient to allow formation of a functional egg chamber (Schütt et al., 1998).

In this report, we describe an additional requirement for Sxl activity in the normal progression of the meiotic cell cycle once the egg chamber has been formed. We propose that Sxl plays a role in the coordination of the mitotic and subsequent meiotic cell cycle.

Culture conditions and mutations

Flies were reared on standard food (cornmeal, sugar, yeast, agar, Nipagin). All crosses were done at 25°C under controlled population density. For genetic symbols, see Lindsley and Zimm (1992). vir2f causes female-specific lethality, and vir6 is lethal for females and semilethal for males (Hilfiker et al., 1995). Sxlf4, Sxlf5 and Sxlf18 (formerly Sxlfs3) cause female sterility and are described by Salz et al. (1987). The identification and characterization of the two suppressor mutations Su(Sxlfs)X2 and Su(Sxlfs)46 which were used in this study will be described elsewhere (Benjamin I. Arthur and D. B. unpublished results). In Sxl wild-type flies, these mutations did not display a detectable recessive or dominant phenotype.

Generating clones in the germline

Cell clones homozygous for vir were generated by the FLP-DFS technique (Chou and Perrimon, 1996). The complete genotype of experimental females is y w FL122.16/Xmarker; P[ry+; hs-neo; FRT]42D, P[ry+; y+]44B, vir bw/P[ry+; hs-neo; FRT]42D, P[ry+; y+]44B, P[mini w+; ovoD1]. These females are sterile due to ovoD1 unless they lose this dominant female-sterile mutation by mitotic recombination that simultaneously renders such stem cells homozygous for vir. To induce mitotic recombination, the females were given a single heat shock of 30 minutes at 37°C as early first instar larvae. Adult flies were crossed to tester males, and their offspring was analyzed for non-disjunction and crossing over. Crossing over frequencies were measured between the three markers w (position 1-1.5), ct (1-20.0), and f (1-56.7) on the X chromosome.

Immunocytochemical and cytological analysis of egg chambers

Ovaries of adult females were dissected 4-7 days after eclosion and prepared as described by Bopp et al. (1993). For SXL staining, ovaries were incubated with 1:10 of hybridoma supernatant of mSXL18; for HTS staining, a 1:200 dilution of ascites fluid of an anti-HTS monoclonal line was used (Yue and Spradling, 1992). The antigen-antibody complex was detected with lissamine-rhodamine-conjugated anti-mouse antibody (Jackson Immunoresearch). For nuclei detection, samples were stained with 0.2 μM of YOPRO-1 (Molecular Probes, Inc.).

For cytological analysis of meiotic chromosomes, we essentially followed the protocol described by Puro and Nokkala (1977). Schiff’s reagent used for Feulgen treatment contained 1 g of basic Fuchsin (Gurr), 8 ml of 1 N HCl, and 0.8 g of K2 S2O5 per 200 ml of staining solution.

Construction of mst-Sxl transgene

First, a mst59dDa-lacZ transgene was constructed by fusing a NdeI-BglII fragment (−480 bp to +35 bp) of the mst59Da promoter region (H. H., unpublished data) upstream of the Adh-lacZ fusion gene in the pCaSpeR-lacZ vector described in Thummel et al. (1988). A KpnI-XbaI fragment (2.6 kb) was excised from SxlcF1 (Bell et al., 1988) carrying pBMS-3B1 plasmid and replaced the Adh-lacZ portion of the mst59Da-lacZ vector. The resulting mst-59Da-Sxl construct was introduced into w1118 embryos by P-element-mediated transformation and selected for stable insertion by the expression of the mini-white gene.

Immunoblot analysis

Tissue material was collected in microfuge tubes and frozen in liquid N2. While thawing, material was homogenised in 2× SDS loading buffer. Samples were then boiled for 5 minutes to denature protein, and insoluble material was removed by centrifugation. Equal volume of the supernatant was loaded per lane on a 12% SDS-polyacrylamide gel. After blotting, anti-SXL antibody was applied as a 1:200 dilution of affinity-purified anti-SXL polyclonal serum (Bopp et al., 1993). For detection of antibody-antigen complexes, we used a secondary antibody conjugated to alkaline-phosphatase (Promega).

Dominant suppressors restore fertility in Sxlfsmutant females

The recessive allele Sxlf4 produces mutant proteins with a single missense mutation downstream of the two RNA-binding RRM domains (Bopp et al., 1993). This change in the protein sequence does not affect somatic functions in females, but renders these sterile. In Sxlf4 mutant ovaries, germ cells are largely arranged in small clusters of 2 or 4 cells connected by intercellular branches, as seen by antibody staining (Fig. 2). The detected adducin-like protein of Hu-li tai shao (Hts) is a component of the fusome, a structure that has been proposed to play a central role in early cyst formation (Yue et al., 1992). It first appears as a spherical organelle in stem cells and cystoblasts and then elongates and branches to connect the dividing cystocytes (Fig. 2D; reviewed in McKearin, 1997). The staining pattern observed in Sxl mutant ovaries is typical for cystocytes that have undergone one or two rounds of cystocyte divisions (Fig. 2E). The prevalence of these stages suggests that mutant cells are unable to complete the four mitotic cycles and, therefore, cannot form a functional cyst nor advance into the next stages of oogenic differentiation.

Fig. 2.

Su(Sxlfs)46 restores differentiation of Sxlf4 mutant ovaries. (A-C) Optical sections of nuclear staining of ovaries from wild-type OrR females (A), Sxlf4/Sxlf4 females (B), and Sxlf4/Sxlf4; Su(Sxlfs)46/+ females (C). g: germarium, nc: nurse-cell nuclei, fc: follicle cells, oc: oocyte. (D-F) Anti-HTS-staining of germaria of the same genotypes: OrR (D), Sxlf4/Sxlf4 (E) and Sxlf4/Sxlf4; Su(Sxlfs)46/+ females (F). Arrowheads point to spherical fusome organelles in stem cells and cystoblasts (D-F), and small arrows to the branched extensions of fusomes in proliferating cystocytes. 16-cell cysts are surrounded by follicle cells stained with anti-HTS. Note absence of staining in germ cells of the stage 1 cysts (S1).

Fig. 2.

Su(Sxlfs)46 restores differentiation of Sxlf4 mutant ovaries. (A-C) Optical sections of nuclear staining of ovaries from wild-type OrR females (A), Sxlf4/Sxlf4 females (B), and Sxlf4/Sxlf4; Su(Sxlfs)46/+ females (C). g: germarium, nc: nurse-cell nuclei, fc: follicle cells, oc: oocyte. (D-F) Anti-HTS-staining of germaria of the same genotypes: OrR (D), Sxlf4/Sxlf4 (E) and Sxlf4/Sxlf4; Su(Sxlfs)46/+ females (F). Arrowheads point to spherical fusome organelles in stem cells and cystoblasts (D-F), and small arrows to the branched extensions of fusomes in proliferating cystocytes. 16-cell cysts are surrounded by follicle cells stained with anti-HTS. Note absence of staining in germ cells of the stage 1 cysts (S1).

To study the disrupted cellular process, we isolated suppressor mutations that can relieve this block during early oogenesis. From an initial screen using EMS-mutagenized males, we recovered several dominant suppressors that restore fertility of Sxlf4 and Sxlf5 mutant females to a significant level (Benjamin I. Arthur and D. B., unpublished results). One of these suppressors, Su(Sxlfs)46, was mapped to 3-[85]. This extragenic suppressor mutation exhibits the strongest activity in our collection and allows practically normal oogenesis in Sxlf4 and Sxlf5 mutant females. Because a significant number of fertile progeny is produced by these females, the mutant stock can be readily propagated. A cytological analysis of the affected ovaries shows mostly normal looking cysts that proceed through all stages of oogenesis without any apparent morphological defects (Fig. 2C). Only rare cases of abnormal morphology were observed, such as for instance mislocalization of the oocyte to the medial region of the egg chamber rather than to the posterior end. The germarial amplification steps leading to the formation of a 16-cell cyst appear normal as judged from the fusome architecture revealed by anti-HTS staining (Fig. 2F).

Though no morphological defect was evident in mutant ovaries carrying Su(Sxlfs)46, we noticed that recombination and proper segregation of homologous chromosomes were severely impaired. Females with a heteroallelic combination of Sxlf4 and Sxlf5 carrying Su(Sxlfs)46 produced only non-recombinant progeny when tested for the w-f interval on the X chromosomes (Table 1A). Similarly, the recombination values in Sxlf4/Sxlf4; Su(Sxlfs)46 females obtained for markers on the second chromosome were substantially reduced to about 10% of the expected values (Table 1B). This reduction is caused by lack of Sxl wild-type activity and is not due to the presence of the suppressor mutation: replacing one mutant allele by a wild-type copy of Sxl restores recombination frequencies to almost normal values (Table 1B). Moreover, the same results were obtained with a different suppressor Su(Sxlfs)X2 which maps to the X chromosome. Testing a heteroallelic combination of Sxlf4 and Sxlf18 carrying Su(Sxlfs)X2 on the Sxlf18 chromosome, the absence of recombination was even more striking, as none of the experimental females produced any recombinant progeny.

Table 1.

Recombination frequencies in Sxlfs; Su(Sxlfs)46 females

Recombination frequencies in Sxlfs; Su(Sxlfs)46 females
Recombination frequencies in Sxlfs; Su(Sxlfs)46 females

We performed the recombination tests independently with different intervals on the X, second or third chromosome (Table 1C-E). Again, presence of one wild-type copy of Sxl was sufficient to restore almost normal recombination frequencies in Su(Sxlfs)X2 females (Table 1C).

In addition to a severely impaired recombination, we also observed a high incidence of non-disjunction (Table 2). For instance, among 178 descendants of females of the genotype Sxlf4/Sxlf18Su(Sxlfs)X2, 39% were products of X-chromosomal non-disjunction events. Besides the occurrence of XXY and XO individuals, we observed a substantial number of nonviable embryos. This lethality is most likely caused by aneuploidy resulting from non-disjunction of autosomes. Adding a single wild-type copy of Sxl in the mother significantly reduced the frequency of non-disjunctional offspring (Table 2B) which indicates an involvement of Sxl in proper segregation. The still elevated frequency of non-disjunction which is observed even in presence of a wild-type copy of Sxl could be due to the occasional presence of an extra Y chromosome (Fig. 4B,E) resulting from a non-disjunctional event in a previous generation. An extra Y chromosome causes secondary non-disjunction of non-exchange X chromosomes. This phenomenon could also explain the rather high frequency of X-chromosomal non-disjunction in Su(Sxlfs)46 females carrying one wild-type copy of Sxl (Table 2A). In the presence of two wild-type copies, however, no single case of X chromosomal non-disjunction was recovered among 820 descendants (Table 2A). This result excludes the possibility that Su(Sxlfs)46 itself is responsible for the segregation defects. The results from this analysis indicate that Sxl wild-type activity is needed in the female germline for exchange and normal progression of the meiotic cell cycle.

Table 2.

X-chromosome non-disjunction in Sxlfs; Su(Sxlfs)46 females

X-chromosome non-disjunction in Sxlfs; Su(Sxlfs)46 females
X-chromosome non-disjunction in Sxlfs; Su(Sxlfs)46 females
Fig. 4.

Stages of meiosis I in wild-type and mutant oocytes. (A) Chiasmata are visible in large autosomal bivalents of prometaphase in stage 13 oocytes of control females. (B) Corresponding prometaphase in a Sxlf4/Sxlf4; Su(Sxlfs)46/+ stage 13 oocyte. All chromosomes appear as univalents. (C,D) Stage 14 wild-type oocytes demonstrating the arrangement of bivalents (X and autosomes 2 and 3) in the spindle with terminal chiasmata on the equatorial plane. The univalent chromosomes 4 appear as dots and locate near the spindle poles. (E,F) Stage 14 oocytes of Sxlf4/Sxlf4; Su(Sxlfs)46/+ and Sxlf4/Sxlf18Su(Sxlfs)X2 ovaries, respectively. Univalent chromosomes arrange themselves in anaphase-like configurations and not on the equatorial plane as seen in wild-type. Segregation in F results in an aneuploid 2 to 4 distribution of major chromosomes while segregation in E results in 3 to 4 distribution, suggesting the presence of a Y chromosome in the latter. The 4th chromosomes segregate normally in both cases. (G) Aberrant meiotic division I in a stage 14 oocyte of Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovary. The chromosomes are distributed between two separate spindles, two major autosomes (probably homologues) plus chromosomes 4 on the left and the other two major autosomes plus a pair of the X chromosomes on the right. It is assumed that such a ‘cleft spindle’ derived from a ‘split karyosome’-type oocyte by the two spindles having been assembled independently around two separate karyosomes. Scale bar, 10 μm.

Fig. 4.

Stages of meiosis I in wild-type and mutant oocytes. (A) Chiasmata are visible in large autosomal bivalents of prometaphase in stage 13 oocytes of control females. (B) Corresponding prometaphase in a Sxlf4/Sxlf4; Su(Sxlfs)46/+ stage 13 oocyte. All chromosomes appear as univalents. (C,D) Stage 14 wild-type oocytes demonstrating the arrangement of bivalents (X and autosomes 2 and 3) in the spindle with terminal chiasmata on the equatorial plane. The univalent chromosomes 4 appear as dots and locate near the spindle poles. (E,F) Stage 14 oocytes of Sxlf4/Sxlf4; Su(Sxlfs)46/+ and Sxlf4/Sxlf18Su(Sxlfs)X2 ovaries, respectively. Univalent chromosomes arrange themselves in anaphase-like configurations and not on the equatorial plane as seen in wild-type. Segregation in F results in an aneuploid 2 to 4 distribution of major chromosomes while segregation in E results in 3 to 4 distribution, suggesting the presence of a Y chromosome in the latter. The 4th chromosomes segregate normally in both cases. (G) Aberrant meiotic division I in a stage 14 oocyte of Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovary. The chromosomes are distributed between two separate spindles, two major autosomes (probably homologues) plus chromosomes 4 on the left and the other two major autosomes plus a pair of the X chromosomes on the right. It is assumed that such a ‘cleft spindle’ derived from a ‘split karyosome’-type oocyte by the two spindles having been assembled independently around two separate karyosomes. Scale bar, 10 μm.

Oocyte nuclei in Sxlfs; Su ovaries display multiple abnormalities in meiotic stages

To investigate whether the failure to proceed through normal meiosis is accompanied by a cytologically visible defect, we microscopically examined squashed preparations from different oogenic stages. The first conspicuous difference between germarial cysts of wild type and those of the Sxlf4/Sxlf4; Su(Sxlfs)46 genotype is an apparent lack in the latter of nuclei that could be identified as pro-oocytes. Unlike in wild-type cysts (Fig. 3A), no distinction can be made between the presumptive oocyte and the nurse cell nuclei. Late pachytene nuclei which are typical for stage 3 wild-type egg chambers were not found (Fig. 3B). The first signs of meiotic differentiation appear around stage 7. However, the appearance of oocyte nuclei is accompanied by a variety of defects in karyosomal organisation. Chromosome threads seem to be decondensed and dispersed (Fig. 3D) or defectively packed in the karyosome (Fig. 3E). Occasionally, two or three loose bodies of chromatin are present instead of a single compact karyosome which is typical for wild-type oocytes (Fig. 3F-J). Such a loose organisation could be due to a defect in pairing and/or formation of the chromocentre during meiotic prophase. The same types of aberrations were also detected in vitellogenic cysts of the Sxlf4/Sxlf18Su(Sxlfs)X2 genotype.

Fig. 3.

Different meiotic stages of karyosome formation in wild-type and mutant oocytes. Pachytene chromosomes in wild-type germarial pro-oocytes (A) and stage 3 oocyte (B). These meiotic stages were not found in Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovaries. Arrow in B points to the loop probably consisting of paired chromosomes 4. Fibres connecting it and the centromeric regions of the major chromosomes constitute the chromocentral system bringing all chromosomes together. (C) Wild-type stage 7 oocyte nucleus showing a compact and densely stained karyosome. (D) Comparable stage in Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovary. Uncondensed chromatin fibres are evenly distributed in the nuclear space of the oocyte. This is the earliest stage of meiotic differentiation that can be observed in these ovaries. The densely stained sphere (arrow in D) is a follicle cell nucleus that lies below the oocyte nucleus. (E-G) Aberrant nuclei in vitellarial oocytes (stages 7-9) in Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovaries. Loops of chromosome fibres protrude from the chromocentre (E). Occasionally, chromatin is distributed between two (F) or three (G, arrows) loosely organized karyosomes indicating a defect in the formation of a single chromocentre. (H-J) Stage 10 oocyte nuclei. (H) A loosely organized karyosome in Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovary next to a follicle cell nucleus that lies above the oocyte nucleus. (I) Diffuse chromatin staining in a Sxlf4/Sxlf18Su(Sxlfs)X2 ovary. (J) Stage 10 wild-type oocyte nucleus for comparison.

Fig. 3.

Different meiotic stages of karyosome formation in wild-type and mutant oocytes. Pachytene chromosomes in wild-type germarial pro-oocytes (A) and stage 3 oocyte (B). These meiotic stages were not found in Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovaries. Arrow in B points to the loop probably consisting of paired chromosomes 4. Fibres connecting it and the centromeric regions of the major chromosomes constitute the chromocentral system bringing all chromosomes together. (C) Wild-type stage 7 oocyte nucleus showing a compact and densely stained karyosome. (D) Comparable stage in Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovary. Uncondensed chromatin fibres are evenly distributed in the nuclear space of the oocyte. This is the earliest stage of meiotic differentiation that can be observed in these ovaries. The densely stained sphere (arrow in D) is a follicle cell nucleus that lies below the oocyte nucleus. (E-G) Aberrant nuclei in vitellarial oocytes (stages 7-9) in Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovaries. Loops of chromosome fibres protrude from the chromocentre (E). Occasionally, chromatin is distributed between two (F) or three (G, arrows) loosely organized karyosomes indicating a defect in the formation of a single chromocentre. (H-J) Stage 10 oocyte nuclei. (H) A loosely organized karyosome in Sxlf4/Sxlf4; Su(Sxlfs)46/+ ovary next to a follicle cell nucleus that lies above the oocyte nucleus. (I) Diffuse chromatin staining in a Sxlf4/Sxlf18Su(Sxlfs)X2 ovary. (J) Stage 10 wild-type oocyte nucleus for comparison.

Consistent with the lack of recombination, no chiasmata are present in stage 13-14 oocytes of either Sxlf4/Sxlf18Su(Sxlfs)X2 or the Sxlf4/Sxlf4; Su(Sxlfs)46 females (Fig. 4A-F). Consequently, chromosomes are usually seen in anaphase-like arrangement in the first division spindle. Another unusual feature in these oocytes is the occurrence of ‘cleft spindles’. In many instances, chromosomes are arranged in two or three spindles instead of one (Fig. 4G). This abnormality probably relates to the loose organisation of chromatin in defective karyosomes and suggests that more than one spindle-organising center may be present. An extra chromosome, presumably a Y, is seen in some meiotic plates and must derive from a previous non-disjunction (Fig. 4E,F).

The described results may all be attributed to pairing defects of early meiotic prophase chromosomes which in turn would explain the absence of chiasmata and defects in segregation during the first meiotic division.

Compromised germline expression of Sxl leads to reduced recombination and increased non-disjunction frequencies

The described meiotic abnormalities usually occur when a specific class of sterile Sxl alleles is suppressed by our dominant suppressor mutations. It is thus possible that these mutant SXL proteins interfere with a process in meiosis that is normally not regulated by Sxl. As an additional test for a role of Sxl in meiotic differentiation, a female genotype was constructed in which germline activity of Sxl is affected not by a mutation in the coding sequence, but at the level of its expression.

It was previously reported that germline expression of Sxl is in part controlled by vir. Germ cells mutant for vir differentiate into functional eggs, although expression of SXL protein is no longer maintained after the apparently normal expression in early germarial stages (Schütt et al., 1998). The vir-independent expression of Sxl in the germarium is sufficient for germ cells to advance into and to complete the oogenic pathway. It was of obvious interest to test if absence of vir-dependent expression of Sxl in these germ cells may have any bearings on normal meiotic differentiation of the oocyte. To this end, vir mutant germ cells were generated using the FLP-DFS system, and the derived progeny was examined for possible defects in recombination and segregation. In germ cells homozygous for the female-specific mutation vir2f, recombination frequencies for X chromosomal intervals appeared only slightly reduced when compared to those in heterozygous control females (Table 3A). A clear and significant reduction in recombination frequencies, however, was obtained with the stronger allele vir6. In this case, recombination values for at least one interval collapsed to 25% of that obtained in control females (Table 3B). The reduction in recombination was accompanied by a substantial increase in the frequency of non-disjunction (XO males in Table 3B).

Table 3.

Recombination and non-disjunction in vir mutant germ cells

Recombination and non-disjunction in vir mutant germ cells
Recombination and non-disjunction in vir mutant germ cells

The frequency of recombination was further lowered by removing one copy of Sxl+ in vir2f mutant germ cells (compare lines 1 and 3 in Table 3A). Concurrently, non-disjunction events increased almost tenfold. A dose-dependent effect of Sxl could not be tested in the mutant vir6 background, because, when combined with Sxlf1, the viability of these females was severely reduced, and escapers rarely produced eggs. The dose-dependent interaction with Sxl suggests that recombination and segregation defects in vir mutant germ cells are primarily based on insufficient expression of Sxl activity. Thus, the normal process of meiosis is not only disturbed by presence of mutant SXL protein as shown before, but also by compromising its wild-type expression. Taken together, these results convincingly prove Sxl to be required in normal meiosis.

Ectopic expression of Sxl does not allow recombination in male germ cells

In Drosophila, recombination is a female-specific trait. Cross-over events do not occur in spermatogenesis. As shown in this report, recombination in females depends on the activity of Sxl, a gene which is not active in the male germline. This raises the possibility that Sxl acts as a discriminating factor that permits meiotic recombination in germ cells to occur when the gene is active. If so, we expect that forced Sxl expression in the premeiotic stages of male germline development may provoke exchange between homologous chromosomes. Misexpression studies by Hager and Cline (1997) have shown that, in contrast to the soma, ectopic SXL is tolerated in male germ cells and does not affect the normal course of spermatogenic differentiation.

We constructed a transgene that expresses the female-specific activity of Sxl in spermatocytes. The construct contains the SxlcF1 open reading frame (Bell et al., 1988) under the control of mst59Da promoter sequences, a potent driver of male- and germline-specific transcription (Fig. 5 and H. H., unpublished results). 18 independent lines were obtained carrying a stable insertion of this construct. All lines show substantial expression of the corresponding 38 kDa SXL protein variant in testes extracts, but not in extracts prepared from the remaining carcasses, confirming the germline specificity of the mst59Da promoter (Fig. 5C). As shown by immunolocalization studies in larval testes, transgenic males produce abundant amounts of SXL protein in early primary spermatocytes (Fig. 5B). The expression is maintained into the late stages of meiotic prophase. As in the cases reported by Hager and Cline (1997), ectopic SXL does not have any adverse effect on spermatogenesis; males are fertile and their testes and spermatogenic contents show a cytologically normal appearance. We examined the progeny of these males for the occurrence of recombination between markers on the second or third chromosomes. In none of the cases tested were paternally derived recombinants found (Table 4). The absence of recombination in male germ cells expressing SXL protein during the meiotic prophase demonstrates that ectopic Sxl activity is not sufficient to allow recombination and, thus, argues against a discriminatory role of Sxl in the sex-specific control of this process.

Table 4.

Recombination in male germ cells expressing SXL

Recombination in male germ cells expressing SXL
Recombination in male germ cells expressing SXL
Fig. 5.

Expression of Sxl in male germ cells. (A,B) Anti-SXL staining of larval testes of a wild-type (A) and a transgenic third instar larva (B). Arrows point to the apical pole of the testis. Only larvae that carry the construct mst59D-SxlcF1 (B) exhibit specific staining. High levels of SXL expression are detected in all germ cells of the larval gonad except for the most apical region which is populated by stem cells, gonial cells and early stages of spermatocyte amplification. (C) Immunoblot analysis of extracts prepared from adult wild-type ovaries (lane 1), adult wild-type testes (lane 2), adult transgenic testes (lane 3) and the corresponding gonadectomized carcasses of the same males (lane 4). Blot was probed with a polyclonal anti-SXL rabbit serum. In lane 1, two major products of the endogenous gene (36 and 38 kDa) are detected; and in lane 3, substantial levels of the 38 kDa variant are found in testes of transgenic males. No SXL expression, except for two minor fast-running variants, are observed in the remaining body parts of these males. These minor products are derivatives of the endogenous gene since they do not appear in Sxl-deficient males.

Fig. 5.

Expression of Sxl in male germ cells. (A,B) Anti-SXL staining of larval testes of a wild-type (A) and a transgenic third instar larva (B). Arrows point to the apical pole of the testis. Only larvae that carry the construct mst59D-SxlcF1 (B) exhibit specific staining. High levels of SXL expression are detected in all germ cells of the larval gonad except for the most apical region which is populated by stem cells, gonial cells and early stages of spermatocyte amplification. (C) Immunoblot analysis of extracts prepared from adult wild-type ovaries (lane 1), adult wild-type testes (lane 2), adult transgenic testes (lane 3) and the corresponding gonadectomized carcasses of the same males (lane 4). Blot was probed with a polyclonal anti-SXL rabbit serum. In lane 1, two major products of the endogenous gene (36 and 38 kDa) are detected; and in lane 3, substantial levels of the 38 kDa variant are found in testes of transgenic males. No SXL expression, except for two minor fast-running variants, are observed in the remaining body parts of these males. These minor products are derivatives of the endogenous gene since they do not appear in Sxl-deficient males.

Sxl controls two distinct processes of oogenesis

A requirement for Sxl for normal female germline development has been demonstrated by Schüpbach (1985) who produced Sxl mutant germline clones in a wild-type ovary by pole cell transplantation. The failure of these cells to form an egg chamber and, in particular, the overgrowth phenotype associated with a lack in differentiation hinted at an early function in the progression of the mitotic cycle of cystocytes. Our data, using anti-HTS staining as a morphological marker for stages in cystocyte proliferation, are consistent with Sxlfs causing a block in cell cycle progression after the first or second round of division (Fig. 2E). As a result, clusters of 2 or 4 cells are produced which become enveloped by follicle cells into one multicellular cyst. This precocious exit from the normal course of cystocyte amplification prevents the cells from reaching the stage of a 16-cell cyst, and, thus, no oogenic differentiation takes place.

We observed a rather unexpected result when the block in cystocyte divisions was relieved by dominant suppressor mutations. Although these suppressors now allow Sxlfs mutant germ cells to form functional eggs, the oocyte nucleus fails to undergo normal synapsis, recombination and segregation of homologous chromosomes.

Using mutations in vir, a positive regulator of Sxl in the germline (Schütt et al., 1998), we show that similar results are produced when the expression of normal SXL protein is compromised (Table 3). Hence, the two types of mutation permit a genetic dissection of the role of Sxl in the germline into two temporally distinct steps of ovarian development: an ‘early’ role for cyst formation, and a ‘late’ role for proper meiotic differentiation. This correlates remarkably well with the two phases of Sxl expression (Bopp et al., 1993). The temporal parallels are intriguing, and we may speculate that the early cytoplasmic expression which persists into the first rounds of cystocyte divisions is necessary and sufficient for the formation of a 16-cell cyst and subsequent differentiation of the oocyte. If this early expression fails, as for example, in germ cells mutant for snf1621 or for the ONC class of otu alleles (Bopp et al., 1993; Oliver et al., 1993), differentiation is prevented and multicellular cysts are formed. The reappearance of protein in nuclear foci of the early 16-cell cyst in wild-type ovaries and subsequent expression in differentiating cysts might then be essential for proper meiotic development. In line with this idea is the observation (Schütt et al., 1998) that early cytoplasmic expression of Sxl in the germarium is not discernibly affected in vir mutant cells, thus permitting normal cyst formation and differentiation, whereas the second phase of expression is strongly reduced or even abolished, affecting meiotic differentiation. In a preliminary report Cook et al. (1992) noticed weak but significant reduction of recombination frequencies in females with only one wild-type copy of Sxl suggesting a dose-dependent requirement for this ‘late’ function.

What is the function of Sxl in the meiotic cell cycle?

Genetic evidence shows that meiotic cell division in the male germline is governed by well known regulators of the mitotic cell cycle. For instance, Dmcdc2 and the cdc25 homolog twine, components of the p34/cdc2 kinase family and the cdc25 phosphatase family, are essential for regulating the onset of the meiotic cell divisions, most likely by triggering the G2/M transition (Jiménez et al., 1990; Lehner and O’Farrell, 1990; Alphey et al., 1992; Courtot et al., 1992; Stern et al., 1993). These genes in turn are believed to be controlled by other components of the Twine class, pelota (Eberhart and Wasserman, 1995), and boule (Eberhart et al., 1996). Mutations in pelota or boule prevent execution of meiosis, but still allow a remarkable degree of (postmeiotic) spermatid differentiation. Some weaker alleles of pelota, which allow the production of sperm, cause defects in segregation and spindle formation.

The cell cycle regulators, twine and pelota, are also required in oogenesis (Alphey et al., 1992; Courtot et al., 1992; Eberhart and Wasserman, 1995). Meiotic spindles, although abnormal in appearance, do form in twine mutant females. However, the meiotic divisions are not arrested at metaphase I as in wild type, but continue repeatedly, leading to high frequencies of non-disjunction (White-Cooper et al., 1993). Thus, unlike the situation in males, where meiosis is completely thwarted, twine in females affects only certain aspects of normal progression of the meiotic cell cycle.

This behavior resembles that found in females mutant for SxlfsSu(Sxlfs) or vir where some aspects of meiosis are disturbed, but neither meiosis itself is abolished nor formation of the egg. It is thus conceivable that the ‘late’ function of Sxl acts in the same pathway that controls entry into meiosis, a process that we have shown to be genetically separable from cyst formation. The apparent lack of pachytene configurations in early cysts of SxlfsSu(Sxlfs) ovaries suggests that the meiotic function of Sxl may be to provide correct cues for the oocyte nucleus to enter the extended prophase of meiosis. Sxl may elicit these cues by regulating meiosis-promoting factors which are necessary to coordinate the transition from the mitotic to the meiotic cell cycle. A checkpoint mechanism can thus be envisioned which is responsible for this transition after four mitotic divisions. We propose that this mechanism includes Sxl as an important regulatory component. A complete loss of Sxl activity would prevent entrance into the postmitotic stage as no cues are provided to trigger the transition, a situation that is observed in Sxlfs mutant ovaries. In SxlfsSu(Sxlfs) and in vir ovaries, however, the transition does take place, but may be retarded compared to wild type. Thus, the pairing and condensation defects observed during karyosome formation may be a consequence of impeded or diminished function of Sxl which affects the correct timing of this process. Alternatively, the process of cystocyte mitoses and entry into meiosis may not be coupled and may be controlled by different cues. In this case, the suppressor mutations may specifically rescue the Sxlfs defect in cyst formation, but cannot supply the cues for correct meiotic differentiation of the oocyte.

Meiosis in females versus males

Differentiation of gametes and the process of generating a haploid genome follow different pathways in males and females. Differences in the architecture of the end products, eggs and sperm, and their differential contribution of nutrients and information to the next generation are traits that account for the need of distinct pathways. Less obvious is why the process of meiosis is different in males and females. In Drosophila, two major female-specific features stand out. First, only one cell of the 16 cells of a cyst will enter and complete meiosis, while the 15 sister cells undergo endomitosis. Thus, in contrast to the male where all cells execute the meiotic program, different fates have to be assigned to cells of the female cluster. Second, recombination between paired chromosomes only occurs in females, not in males. The differences in meiotic pathways are also in part reflected by the need for different sets of genes. For instance, coordinate control of meiotic cell cycle and spermatid differentiation is achieved by four genes, spermatocyte arrest, cannonball, always early and meiosis I arrest (Lin et al., 1996) which are not needed in oogenesis. In females, on the other hand, it has been shown that three members of the RAD52 DNA repair pathway coordinate meiotic DNA metabolism and patterning of the oocyte (Ghabrial et al., 1998). This report now adds Sxl to the list of genes specifically required in the female germline for meiosis, in particular for recombination and segregation.

In view of its master-switch role in sexual development of the soma, it is conceivable that, in the germline, an active Sxl gene is also sufficient to impose certain female-specific traits, such as recombination. However, our results show that no recombination occurs in male germ cells expressing SXL at the premeiotic stage. It can be argued that our cDNA construct was not active early enough or that it did not express the specific protein variants needed for this process. The latter argument may be less of a concern, as it has been shown that expression of a Sxl transgene in the male germline can transactivate the otherwise silent endogenous gene (Hager and Cline, 1997). We consider it more likely that the lack of recombination in male germ cells is due to absence of other female-specific activities which normally participate in this process. Thus, it is possible that Sxl is neither the main switch for the choice of the sexual pathway of germ cells (Steinmann-Zwicky, 1992; Horabin et al., 1995; Hager and Cline, 1997) nor for female-specific traits such as recombination.

It remains to be investigated what the precise regulatory role of Sxl is in the germline. Not only would we like to understand how it performs this function, but also which target genes are controlled. Candidate genes are cell cycle regulators that allow transition from the mitotic phase into meiosis, and those involved in normal progression through the meiotic prophase. For decades, only two meiotic mutations were known, c(3)G (Gowen, 1928), and cand (Sturtevant, 1929). Since 1968, systematic searches have discovered a still growing number of new mei mutations (Sandler et al., 1968; Baker and Carpenter, 1972; Sekelsky et al., 1999). Testing whether germline activity of Sxl and vir is required for correct expression of these meiotic genes should eventually help to understand the genetic system regulating female meiosis and to define the position of Sxl in this pathway.

As this process is fundamentally different from that regulated by Sxl in the soma, it is conceivable that these tissue-specific functions evolved independently. It will therefore be interesting to investigate whether the germline function of Sxl is conserved in other dipteran insects. In line with a conserved role in this tissue is the observation that the Sxl homologue of Musca domestica is expressed in germarial germ cells (D. B., unpublished results). Of particular interest is the finding that the Sxl homologue in Megaselia scalaris is expressed only in the germline, but not in the soma of adult flies (Sievert et al., 1997). This result suggests an exclusive role in the germline of this fly. Unlike the phylogenetically recent acquisition of a sex-determining function in the soma of Drosophila, the germline function may thus be more widely conserved indicating a possible ancestral role of Sxl in germline development. Comparing expression and, whenever possible, function of this gene in other dipteran species should give insights into the evolution of the pathways regulating gametogenesis and sexual differentiation and into the mechanisms of how genes are recruited for various tasks in different developmental pathways.

Our thanks go to Benjamin I. Arthur for sharing unpublished results and for many stimulating discussions. We thank Dennis McKearin for the gift of anti-HTS antibodies. We are grateful to Markus Niessen and Adrian Streit for critical comments on the manuscript and many other colleagues in our laboratories for their interest in this work. This work was supported by grants from the Swiss National Foundation (31-47180.96) and from the Julius Klaus-Stiftung.

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