Sex lethal

DEVELOPMENTAL BIOLOGY

Embryonic

The absence of Sxl protein in unfertilized eggs despite the presence of large quantities of maternal Sxl mRNAs indicates that these mRNAs are not translated prior to fertilization. The transcripts persist for 1-2 hours after fertilization and appear to be evenly distributed in early cleavage embryos. Even though these maternal RNAs are exclusively processed in the female mode, they do not appear to be translated as no protein is detected in early cleavage stage embryos of both sexes. Sxl protein is first detected when Sxl is zygotically activated at the blastoderm stage (Bopp, 1993).

Larval

In both the early and late third instar gonads, high levels of SXL protein is found in the oogonial cells, while much lower levels are detected in the somatic mesodermal cells. Surprisingly, most of the SXL proteins appears to accumulate in the cytoplasm, not in the nucleus. This distribution is in marked contrast to the predominantly nuclear localization seen in somatic cells. The unusual cytoplasmic localization of Sxl protein is also seen in the large cells in the anteriormost region of the adult germarium. This part of the germarium contains two or three stem cells, one or two cystoblasts, and one or two cell cysts. In the posterior half of region 1, which contains the proliferating cystocytes, there is an aburpt transition in the staining pattern. The level of cytoplasmic SXL protein appears to drop precipitously. A little later, when the follicle cells start to invade centripetally and surround the newly formed 16-cell cluster in region 2 of the germarium, a second change in localization occurs. The protein now becomes concentrated in foci within the nuclei of the cystocytes. Later Sxl protein increases and most of it is localized to the cytoplasm of the nurse cells during the previtellogenic stages of oogenesis. In follicle cells, Sxl is present in nuclei. The germ line expression of SXL protein is severely reduced in sans-fille mutants, but messenger RNA is normal. In undifferentiated germ cells of otu mutants Sxl protein is predominantly cytoplasmic (Bopp, 1993).

Adult

A substantial amount of Sxl mRNA is present in all cells of the germarium including the stem cells. During previtellogenesis high levels of Sxl RNA accumulate in the cytoplasm of nurse cells while lower levels are detected in the somatically derived follicle cells tht surround each egg chamber. At the onset of yolk deposition (vitellogenesis), stage 8 of oogenesis, the nurse cells begin transporting a wide range of factors into the maturing egg. Sxl mRNA is included in this process (Bopp, 1993).

Effects of Mutation or Deletion

A pair of muscles span the fifth abdominal segment of male but not female Drosophila melanogaster adults. To establish whether genes involved in the development of other sexually dimorphic tissues controlled the differentiation of sex-specific muscles, flies mutant for five known sex-determining genes were examined for the occurrence of male-specific abdominal muscles. Female flies mutant for alleles of Sex-lethal, defective in sex determination, or null alleles of transformer or transformer-2 are converted into phenotypic males that formed male-specific abdominal muscles. Both male and female flies, when mutant for null alleles of doublesex, develop as nearly identical intersexes in other somatic characteristics. Male doublesex flies produced the male-specific muscles, whereas female doublesex flies lacked them. Female flies, even when they inappropriately expressed the male-specific form of doublesex mRNA, failed to produce the male-specific muscles. Therefore, the wild-type products of the genes Sex-lethal, transformer and transformer-2 act to prevent the differentiation of male-specific muscles in female flies. However, there is no role for the genes doublesex or intersex in either the generation of the male-specific muscles in males or their suppression in females (Taylor, 1992).

Genes known or suspected to be involved in germ-line sex determination in Drosophila melanogaster have been examined to determine if they are required upstream or downstream of Sex lethal, a known germ line sex determination gene. At least seven genes are required for female-specific splicing of germ line Sex lethal pre-mRNA. The genetic hierarchy includes the somatic sex determination genes transformer, transformer-2 and doublesex (and by inference Sex lethal), which control a somatic signal required for female germ line sex determination, and the germ line ovarian tumor genes fused, ovarian tumor, ovo, sans fille, and Sex-lethal, which are involved in either the reception or interpretation of this somatic sex determination signal. The fused, ovarian tumor, ovo and sans fille genes function upstream of Sex lethal in the germ line (Oliver, 1993).

Ovo belongs to the ovarian tumor class of genes. Mutants of genes in this class present smaller than normal ovaries and egg chambers filled with an excess of undifferentiated germ cells. The initial observation that female germline cells defective in Sex lethal also form tumorous cysts led to the idea that this phenotype identifies loci involved in germline sex determination. This is supported by the fact that, among tumorous mutants (ovo, ovarian tumor, sans fille, Sex lethal and bag-of-marbles,), only bam functions in the female germline. ovo is a key gene lying upstream of Sxl required for germline cells to respond to the somatic feminization signal (Mével-Nino, 1996 and references).

Female-specific functions of the sex determination gene Sex-lethal (Sxl) regulate sexual behavior and synthesis of the three major sex pheromones that have been identified in normal, sexually mature Drosophilia males and virgin females. Diplo-X flies, heterozygous in trans for two partial loss-of-function Sxl mutations, elicit less courtship than normal females and produce large quantities of the inhibitory pheromones that normal males synthesize. In addition, the mutant flies fail to synthesize the female-predominant aphrodisiac pheromone or make very small quantities of this compound (Tompkins, 1995).

In Drosophila, Sex-lethal (Sxl) controls autoregulation and sexual differentiation by alternative splicing but regulates dosage compensation by translational repression. To elucidate how Sxl functions in splicing and translational regulation, a full-length Sxl protein (Sx.FL) and a protein lacking the N-terminal 40 amino acids (Sx-N) were ectopically expressed. The Sx.FL protein recapitulates the activity of Sxl gain-of-function mutations, since it is both sex transforming and lethal in males. In contrast, the Sx-N protein unlinks the sex-transforming and male-lethal effects of Sxl. The Sx-N proteins are compromised in splicing functions required for sexual differentiation, displaying only partial autoregulatory activity and almost no sex-transforming activity. However, the Sx-N protein does retain substantial dosage compensation function and kills males almost as effectively as the Sx.FL protein. In the course of the analysis of the Sx.FL and Sx-N transgenes, a novel, negative autoregulatory activity, in which Sxl proteins bind to the 3' untranslated region of SXL mRNAs and thereby decrease Sxl protein expression, was also uncovered. This negative autoregulatory activity may be a homeostasis mechanism (Yanowitz, 1999).

While the Sx-N protein retains at least some autoregulatory and dosage compensation activities, the most striking finding is that the protein seems to lack the ability to regulate the tra sexual differentiation pathway. The feminization activities of the Sx.FL and Sx-N transgenes were compared in males that do not contain the endogenous Sxl locus. This background allows alterations in somatic sexual differentiation to the Sxl proteins expressed by the transgenes to be unambiguously ascribed. The extent of sex transformation that is seen in escaper Sx.FL males again appears to resemble that for the strong cF1 lines, in which the Sxl cDNA is expressed under the control of the inducible hsp70 promoter. The males are intersexual; they have lighter abdominal pigmentation, rotated genitalia, and fewer sex combs, and they are sterile. In sharp contrast, Sxl minus; Sx-N males are morphologically indistinguishable from wild-type males and are fertile. The absence of sex transformations in Sx-N males most likely reflects an inability to regulate the tra pathway. To determine if this is the case, a RT-PCR assay was used to examine the splicing pattern of tra mRNAs. Three amplification products can be detected in this assay, and these correspond to unspliced RNA, default (male) spliced RNA, and female spliced RNA. Of these, only unspliced and default spliced tra RNAs are observed in wild-type males, while all three species are found in females. Thus, while the Sx.FL transgene has tra regulatory activity, it is not as effective as the endogenous Sxl gene. These findings confirm the suggestion that the Sx-N protein is defective in its tra regulatory function (Yanowitz, 1999).

Sxl proteins might negatively regulate their own expression by associating with the 3' UTR of the Sxl mRNA. This hypothesis is supported by several findings: (1) Sxl proteins bind to the 3' UTR of Sxl mRNAs in vivo; (2) the regulatory activities of the Sx.FL and Sx-N transgenes are substantially enhanced by deleting most of the 3' UTR from the transgenes; (3) expression of the endogenous Sxl protein is reduced when the Sx.FL and (to a lesser extent) Sx-N proteins are highly expressed; (4) even though the activity of the Sx.FLDelta transgene is by many criteria much stronger than its Sx.FL counterpart, it is impaired in its ability to rescue females from the lethal effects of several hypomorphic Sxl mutations. This latter, paradoxical result could be explained by the fact the Sx.FLDelta transgene may be more efficient than Sx.FL in repressing Sxl protein expression from the endogenous gene (upsetting the normal balance of Sxl protein isoforms) (Yanowitz, 1999).

While the positive (splicing) autoregulatory feedback loop provides a mechanism for maintaining the Sxl gene in the 'on' state in females, it is possible that this feedback loop, operating unchecked, would produce toxic levels of Sxl protein (especially if the protein directly down-regulates expression of X-linked genes). A negative autoregulatory feedback loop would maintain homeostasis, keeping the levels of Sxl protein just high enough to maintain the positive autoregulatory feedback loop but below the level where the proteins could begin to have detrimental effects. Favoring the idea that this feedback loop is likely to have a role in fine-tuning the amount of Sxl protein, the Sx.FLDelta and Sx-NDelta transgenes do not have dominant effects in wild-type females. Such a model is not without precedent; it is thought that the snf homolog in mammals (U1A) and the poly(A) binding protein in yeast control their own rates of accumulation by binding to the 3' UTRs of their respective mRNAs and down-regulating translation. It is also possible that Sxl negative autoregulation is a vital process. In this case, the two- to three-fold induction over background of the transgenes would not be sufficient to reveal this essential role. Perhaps drastically higher levels of Sxl protein might be obtained by removing the additional Sxl binding sites from the 3' UTR of Sx.FLDelta; this hypothesis should be tested (Yanowitz, 1999).

This model would also help explain why the 3' UTR profile of the Sxl mRNAs changes during development. The Sxl mRNA profile is dynamic throughout development. During early embryogenesis, when Sxl protein must be rapidly synthesized to ensure that the positive (splicing) autoregulatory feedback loop is activated in all female cells, there is a preponderance of Sxl mRNAs with short 3' UTRs, and few Sxl protein binding sites. Later in development, when the Sxl gene is stably activated and a high rate of Sxl protein accumulation would no longer be required, the major Sxl mRNA species have long 3' UTRs. Negative autoregulation mediated by Sxl protein binding to multiple sites in the long 3' UTRs of these RNAs would ensure that the concentration of Sxl protein is maintained at a constant level. This concentration should be high enough to sustain the positive autoregulatory feedback loop but low enough to avoid toxic effects (Yanowitz, 1999).

While these results are consistent with the idea that Sxl proteins negatively regulate their own synthesis through binding sites in the 3' UTRs of the Sxl mRNAs, the molecular mechanism(s) of repression remain unclear. Northern blots indicate that the level of RNA from the transgenes with short 3' UTRs (Sx.FLDelta and Sx-NDelta) is higher than from the transgenes with long 3' UTRs. This could mean that the RNAs with the longer UTRs turn over more rapidly (in the model this would be a consequence of Sxl protein binding). Alternatively, the reduction might be an indirect consequence of reduced translation. This puzzle is not unique to the Sxl transgene RNAs; for example, the amount of msl-2 RNA is less in females than in males. An additional complication with the transgene data is that the RNAs encoded by these constructs are not spliced. Since 3'-end processing is often coupled to splicing, it is possible that the postulated regulation of the transgene RNAs by Sxl proteins follows a pathway that is different in some respects from that of RNAs (like msl-2 or the endogenous Sxl mRNAs) which are subject to splicing. Further studies will clearly be required to elucidate how Sxl is able to reduce protein expression and to show conclusively that Sxl negatively autoregulates its own expression (Yanowitz, 1999).

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. 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 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. Several mutations are known to affect the three major features of meiosis, first synapsis, then exchange, and finally disjunction of meiotic chromosomes. For instance, the kinesin-like products of claret-nondisjunctional (ca^nd) and no distributive disjunction (nod) 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. 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. 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. As a result, recombination is abolished and non-disjunction highly elevated (Bopp, 1999 and references therein).

Little is understood about Sex-lethal's function in the germline. Sxl expression during oogenesis appears biphasic, with a striking change in intracellular distribution. 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. 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. The first evidence for an involvement in female germline development came from analysis of loss-of-function alleles in genetic mosaics. 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. Germ cells mutant for certain recessive alleles (collectively called Sxl^fs) remain small and undifferentiated and continue to proliferate excessively, forming large cysts, a phenotype that is commonly referred to as 'ovarian tumors' or 'multicellular cysts'. The overproliferation phenotype implicates Sxl in playing an essential role in the control of the cystocyte mitotic cycle and subsequent cyst formation. The meiotic process relies on the correct functioning of Sex-lethal. Sxl^fs alleles disrupt the germline, but not the somatic function of Sxl and causes an arrest of germ cell development during cystocyte proliferation. Using dominant suppressor mutations that relieve this early block in Sxl^fs mutant females, additional requirements of Sxl for normal meiotic differentiation of the oocyte have been discovered. Females mutant for Sxl^fs that carry a suppressor, become fertile, but pairing of homologous chromosomes and formation of chiasmata are 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 is compromised by mutations in virilizer (vir), a positive regulator of Sxl. In contrast, ectopic expression of a Sxl transgene in premeiotic stages of male germline development is not sufficient to allow recombination to take place; this suggests that Sxl does not have a discriminatory role in this female-specific process. It is proposed that Sxl performs at least two tasks in oogenesis: an 'early' function in the formation of the egg chamber, and a 'late' function in the progression of the meiotic cell cycle, suggesting that both events are coordinated by a common mechanism (Bopp, 1999).

A rather unexpected result was observed when the block in cystocyte divisions was relieved by dominant suppressor mutations. Although these suppressors allow Sxl^fs 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, similar results are produced when the expression of normal Sxl protein is compromised. 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. The temporal parallels are intriguing: it is speculated that the early cytoplasmic expression that 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, 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 (Bopp, 1999).

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. These genes in turn are believed to be controlled by other components of the Twine class, pelota, and boule. Mutations in either 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 (Bopp, 1999 and references therein).

The cell cycle regulators, twine and pelota, are also required in oogenesis. 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. 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 Sxl^fsSu(Sxl^fs) 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 is genetically separable from cyst formation. The apparent lack of pachytene configurations in early cysts of Sxl^fsSu(Sxl^fs) 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 that are necessary to coordinate the transition from the mitotic to the meiotic cell cycle. A checkpoint mechanism can thus be envisioned that is responsible for this transition after four mitotic divisions. It is proposed that this mechanism includes Sxl as an important regulatory component. A complete loss of Sxl activity would prevent entrance into the postmitotic stage, because no cues are provided to trigger the transition, a situation that is observed in Sxl^fs mutant ovaries. However, in Sxl^fsSu(Sxl^fs) and in vir ovaries, 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 that 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 Sxl^fs defect in cyst formation, but cannot supply the cues for correct meiotic differentiation of the oocyte (Bopp, 1999 and references therein).

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. (1) 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. (2) 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; these genes are not needed in oogenesis. However, in females, it has been shown that three members of the RAD52 DNA repair pathway coordinate meiotic DNA metabolism and patterning of the oocyte. 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, no recombination occurs in male germ cells expressing Sxl at the premeiotic stage. Therefore, the lack of recombination in male germ cells is due to the absence of other female-specific activities that 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 nor for female-specific traits such as recombination (Bopp, 1999 and references therein).

It remains to be investigated what the precise regulatory role of Sxl is in the germline. 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, and ca^nd. Since 1968, systematic searches have discovered a still growing number of new mei mutations. 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. Since 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 homolog of Musca domestica is expressed in germarial germ cells. Of particular interest is the finding that the Sxl homolog in Megaselia scalaris is expressed only in the germline, but not in the soma of adult flies. 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 for Sxl in germline development. Comparing the expression and, whenever possible, the 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 (Bopp, 1999).

In this report, an examination was carried out of how mutations in the principal sex determination gene, Sex lethal (Sxl), impact the H4 acetylation and gene expression on both the X and autosomes. When Sxl expression is missing in females, the sequestration occurs concordantly with reductions in autosomal H4Lys16 acetylation and gene expression on the whole. When Sxl is ectopically expressed in Sxl^M mutant males, the sequestration is disrupted, leading to an increase in autosomal H4Lys16 acetylation and overall gene expression. In both cases relatively little effect is found on X chromosomal gene expression (Bhadra, 2000).

In Sxl^M males, in which Sxl is ectopically expressed, the slow accumulation of the Sxl protein during development eventually prevents significant Msl-2 expression and hence reduces the Msl complex association with the X chromosome. This results in X and autosomal gene expression quite similar to that found in the mle mutant, i.e., little response of the X-linked genes, but an overall increase in autosomal expression. Similarly, the association of the Msl proteins in ~50% of the cells of heteroallelic Sxl females causes sequestration of MOF to the two X chromosomes. This sequestration reduces the H4Ac16 on the autosomal loci, resulting in a lowered expression. There is a concomitant increase of acetylation on the X chromosome, but little overall response of the X-linked genes (Bhadra, 2000).

In the Sxl^M and Sxl^f;mle genotypes a low level of Msl-1/Msl-2 shows chromosomal binding to some degree, although binding takes present in distinct patterns in the two cases. This low level of binding, however, is insufficient to sequester all the available Males absent on the first (Mof) present in the cell. Previous and present data suggest that in the absence of a functional Msl complex, Mof still associates with the chromosomes and is active in modifying H4. The reduced amount of Msl-1/Msl-2 appears saturated with Mof, allowing the remainder to be uniformly distributed across the genome, which modulates gene expression (Bhadra, 2000).

The general trends of X and autosomal gene expression in Sxl^M and Sxl^f mutants match the published autoradiographic data when the latter is considered as absolute levels rather than relative X to autosomal ratios. Autoradiographic grain counts over the X chromosome were changed very little in Sxl^M larvae compared to normal, but the counts over the autosomes were increased. Conversely, in Sxl^f females, the autosomal counts were lower than in normal females with little change over the X. Because the data reported here are anchored to rRNA levels, which in turn do not vary per unit of DNA, the 'per cell' expression trends can be determined on an absolute rather than a relative comparison basis; they indicate greater changes of the autosomes compared to the X chromosome (Bhadra, 2000 and references therein).

The loss of individual components of the Msl complex in the msl mutant males releases the Mof acetylase from the X and a uniform H4 acetylation distribution results. Accordingly, autosomal gene expression is generally increased, reflecting an inverse effect of the X on the autosomes, because the normally sequestered acetylase is now dispersed: this results in higher acetylation levels on the autosomes. An increase or decrease of acetylation level on the X is not reflected in major changes in gene expression, suggesting that some member of the Msl complex insulates genes on the X from responding to the much increased acetylation level (Bhadra, 2000 and references therein).

The collective data indicate that models that posit an association of the Msl complex with a gene for dosage compensation to occur are not supported. First of all, genetic destruction of the complex does not eliminate dosage compensation of most X-linked genes. However, one could perhaps argue that this action eliminates compensation of the regulatory genes on the X, which, because they are now dosage dependent, will compensate most of the 'housekeeping' genes that were assayed. This alternative is not favored for three reasons. (1) Ectopic expression of MSL2 in females as a transgene or in Sxl^f mutants (present study) have the Msl complex present on their Xs but gene expression in general is not increased as predicted if the MSL complex alone conditions hyperactivation. (2) Dosage compensation also occurs in metafemales (3X chromosomes with diploid autosomes), where there is no complete complex and this compensation is related to that occurring in males. (3) Autosomal insertions of many X-derived genes still exhibit some degree of compensation despite the fact that these genes have no association with the Msl complex. Thus, there are several circumstances known in which compensation occurs without the Msl complex. The function of the Msl complex on the X chromosome appears to be to inhibit the response of most X-linked genes to high levels of histone acetylation (Bhadra, 2000 and references therein).

When these data are taken together along with previous studies on mle, a consistent model is supported, indicating that the effect of the Sex lethal gene is mediated through its control of the presence or absence of Msl-2. When Msl-2 protein is expressed, the sequestration of the MSL complex occurs with a resultant increase of H4Lys16 acetylation on the X at the expense of acetylation on the autosomes. In the absence of MSL-2, there is a uniform genomewide distribution of Mof and H4Lys16 acetylation. In general, gene expression on the autosomes responds positively to the level of acetylation, but the X is refractory to it in the presence of the Msl complex. In this way, the twofold inverse-dosage effect of the X is used to achieve a proper level of dosage compensation, but the effect on the autosomes is diminished. Thus, as the heteromorphic sex chromosomes have evolved, both the X and the autosomes have maintained nearly equal expression between the sexes (Bhadra, 2000).

A host–parasite interaction rescues Drosophila oogenesis defects

The cytoplasmically inherited bacterium Wolbachia pipientis is a widespread parasite of arthropods that manipulates the reproductive biology of its hosts, often to their detriment, in order to foster its own transmission through egg cytoplasm. Infection by Wolbachia restores fertility to Drosophila melanogaster mutant females prevented from making eggs by protein-coding lesions in Sex-lethal (Sxl), the master regulator of sex determination. Suppression of sterility by Wolbachia discriminates markedly among similar germline-specific Sxl alleles, and is not observed for mutations in other genes that produce similar 'tumorous ovary' phenotypes, including one that blocks Sxl germline expression. This allele and gene specificity indicates that suppression probably results from a specific interaction with Sxl protein, rather than from a bypass of the normal germline requirement for this developmental regulator or from an effect on Sxl expression. The Sxl–Wolbachia interaction provides a rare opportunity to explore host–parasite relationships at the molecular level in a model insect. Furthermore, demonstration that a parasite infection can counteract the deleterious effects of mutations in host genes illustrates how hosts might become dependent on parasites (Starr, 2002).

Because Wolbachia depends on obligate maternal transmission, it manipulates the reproduction of its host to increase the number of infected females. Reproductive phenotypes caused by Wolbachia infection include parthenogenesis, feminization, male killing, and cytoplasmic incompatibility (inviability of offspring from a mating of infected males with uninfected females). These effects can skew sex ratios and may even promote speciation. Although Wolbachia infection generally has no obvious benefit to the host, the parasitic wasp Asobara tabida can be considered an exception. In a remarkable natural parallel to the laboratory phenomenon described here, Asobara requires Wolbachia infection for oogenesis. Little is known about the mechanisms Wolbachia uses to affect reproduction, in part because the host species that display strong phenotypes have not been readily amenable to genetic analysis. Although infection in D. melanogaster has been reported to cause low levels of cytoplasmic incompatibility and a shortening of adult life span, suppression of mutant Sex-lethal alleles represents a robust phenotype for Wolbachia infection in Drosophila (Starr, 2002).

Sex-lethal (Sxl) is an X-linked, female-specific master switch gene that controls somatic sex determination and the vital process of X-chromosome dosage compensation in Drosophila melanogaster. It is also required in the female germ line for oogenesis and meiotic recombination. Complete loss-of-function Sxl alleles are recessive, female-specific lethal; however, a small subset of partial loss-of-function point mutant alleles are disrupted specifically with respect to female germline functions of Sxl and hence are fully viable but sterile. Females mutant for such alleles fail to produce eggs owing to a germline-autonomous block in oogenesis that leads to overproliferation of undifferentiated germ cells. The fact that the positive feedback loop for Sxl pre-messenger RNA splicing is engaged and mutant protein is produced in these ovarian tumors suggests that the overall level of Sxl protein in these mutant cells is normal (Starr, 2002).

A stock constructed for a suppressor screen of the female-sterile allele Sxl^f4 was unusual in that homozygous mutant females were weakly fertile even before the screen began. These Sxl^f4 females produced large numbers of eggs but few offspring. Three observations showed that the serendipitous suppressor carried in this stock was maternally inherited: (1) suppression could be passed through all mothers over several generations of outcrossing; (2) no chromosome of the original suppressed stock was required zygotically or maternally for suppression; and (3) males could not transmit suppression (Starr, 2002).

Knowledge of maternal inheritance of suppression led to a consideration of Wolbachia as a causative agent. The fertile Sxl^f4 stock tested positive for Wolbachia by a polymerase chain reaction assay, whereas the mutant stock from which the Sxl^f4 chromosome had originally been obtained tested negative. Curing the fertile mutant stock of its bacterial infection by tetracycline treatment eliminates suppression. Without tetracycline treatment, ovaries of homozygous mutant Sxl^f4 females are full of eggs like the ovaries of their heterozygous Sxl⁺ sisters. With tetracycline treatment, most Sxl^f4 ovaries make no mature eggs, whereas the Sxl⁺ ovaries of their sisters continue to produce normal numbers of eggs (Starr, 2002).

To establish that Wolbachia is indeed the bacterial agent causing suppression, a female-sterile Sxl^f4 line was infected by crossing it to a wild-type (Canton S) line carrying a characterized strain of Wolbachia. Because only the mother can transmit Wolbachia, reciprocal crosses could be used to create chromosomally identical Sxl^f4/Sxl^f4 females differing only in their Wolbachia infection status. More than 60% of the mutant ovaries from twenty females infected this way produced more than two eggs, whereas only 3% of the mutant ovaries from twenty chromosomally identical but uninfected Sxl^f4 females produced even one egg (Starr, 2002).

Although most infected Sxl^f4 females laid eggs, only a minority actually generated viable adult progeny over a ten-day period. Among 610 eggs collected from a group mating of such females, 42% of the embryos reached at least the germband elongation stage, but only 7% hatched into larvae. This high level of embryonic lethality is probably caused at least in part by aneuploidy arising from meiotic chromosome segregation defects in Sxl^f4 females that are not suppressed by Wolbachia infection. The X-chromosome non-disjunction rate [(2XXY + 2X0)/(XX + XY + 2XXY + 2X0), where 0 indicates a missing Y chromosome] was 30% for Sxl^f4/Sxl^f4 females compared with less than 0.3% for their Sxl^f4/Sxl^f4; Dp (Sxl⁺) sisters. Such meiotic defects have been seen for a variety of heteroallelic mutant Sxl genotypes, and a similar phenotype has been reported for mutant Sxl females whose sterility was suppressed by uncharacterized chromosomal mutations. The fact that Wolbachia suppresses germline proliferation defects but not meiotic defects is evidence that the interaction with Sxl is functionally specific even within a single cell type (Starr, 2002).

The ability of Wolbachia to suppress female-sterile Sxl alleles depends on the specific molecular nature of those mutant alleles, rather than the severity of their germline defect. Such allele specificity is the strongest evidence for a direct involvement of the Sxl protein in suppression and argues against the possibility that Wolbachia suppresses by simply increasing the level of Sxl germline expression. Similar to Sxl^f4, the homozygous viable, female-sterile allele Sxl^f5 is altered within a poly-proline tract that is present in all of the many Sxl protein isoforms. In Sxl^f5, a leucine has replaced a proline only three residues away from the proline to serine change in Sxl^f4. Wolbachia suppressed the oogenesis defect in Sxl^f5 but less than in Sxl^f4, and it failed to suppress the germline phenotype of Sxl^f5 when the allele also carried the phenotypically subtle, partial-loss-of-function mutation Sxl^fHa in cis. Sxl^fHa is a spontaneous insertion of a hobo transposon in intron 2 at base pair 7,476 that arose unexpectedly in a Sxl^f5 stock. Females homozygous for the double-mutant Sxf^f5,fHa are viable but sterile, like those homozygous for Sxl^f5; however, although Sxl^f5 is fully viable in trans to a null allele, the double mutant is only poorly viable. As predicted, the non-suppressible double mutant allele had an effect on suppression in heteroallelic combination with Sxl^f4 equivalent to that of the null allele in trans to Sxl^f4, excluding the possibility that some genetic factor other than the difference in Sxl alleles is responsible for the difference in suppression. Because the Sxl^fHa transposon insertion is in non-protein-coding DNA, its effects presumably stem from a reduction in Sxl^f5 protein levels. Hence it seems that Wolbachia cannot suppress oogenesis defects if Sxl^f5 germline protein levels fall below a certain threshold (Starr, 2002).

Wolbachia also suppresses the sterility of a molecularly different, homozygous viable, female-sterile allele, Sxl^f18. However, Sxl^f18 was suppressed more poorly than Sxl^f4, despite being less defective. The point lesion in Sxl^f18 leaves the poly-proline tract unchanged, but substitutes aspartic acid for glycine in exon-8 carboxy-terminal isoforms and blocks alternative splicing that generates exon-10 C-terminal isoforms. Although none of the Sxl C-terminal isoforms seem to be germline-specific in their expression, the genetic behavior of Sxl^f18 indicates that exon-10 C-terminal isoforms are specifically required for oogenesis. The fact that egg production in uninfected Sxl^f18 females is clearly better than that in uninfected Sxl^f4 females shows that Sxl^f18 is the less deleterious germline mutation; nevertheless, Wolbachia-infected Sxl^f18 females made significantly fewer eggs than infected Sxl^f4 females. That the more functional mutant allele is the allele more weakly suppressed is also apparent from the behavior of hemizygous mutant females -- individuals who carry only a single copy of the mutant allele in trans to a completely non-functional (null) allele. Egg production in infected Sxl^f18/Sxl^null females was far below that for infected Sxl^f4/Sxl^null females. Clearly, for both Sxl^f18 and Sxl^f4, the dose of the mutant allele matters, as egg production for infected homozygous mutant females is much greater than for infected hemizygous mutant females (Starr, 2002).

The fact that the mutant allele with the stronger oogenesis defect when uninfected would be the allele with phenotype closer to the wild type when infected argues against the possibility that suppression by Wolbachia arises from a general elevation of Sxl germline activity. The argument is even more compelling when one looks more carefully at the mutant allele dose effect. Egg production for infected Sxl^f18/Sxl^null females was far below that for even uninfected Sxl^f18/Sxl^f18 females. If infection had been able to even double Sxl^f18 expression, egg production for these two genotypes would have been the same. In contrast, although egg production for infected Sxl^f4/Sxl^null females is below that for infected Sxl^f4/Sxl^f4 females, it is far above that for uninfected Sxl^f4/Sxl^f4 females. Hence to account for the level of oogenesis in infected Sxl^f4/Sxl^null females, one would have to argue that Wolbachia increases Sxl^f4 expression by much more than 100%, a proposal at odds with the results with Sxl^f18 (Starr, 2002).

The conclusion that Wolbachia does not bypass or reduce the requirement for Sxl in the germline in a general way, nor increase overall germline Sxl expression, is also supported by its failure to suppress female-sterile mutations of other genes, particularly sans-fille. Ovarian tumors resembling at least superficially those in Sxl mutants can be observed in females mutant for sans-fille, ovarian tumors or mei-P26. The fact that none of these germline phenotypes are suppressed by Wolbachia infection is additional evidence for the specificity of the interaction with Sxl. Lack of suppression of snf¹⁶²¹ tumors is particularly significant, as these tumors are caused by an absence of germline Sxl activity, as shown by the fact that they can be suppressed by constitutively active mutant Sxl alleles, or even simply by an extra copy of Sxl⁺. If suppression of Sxl female-sterile mutations by Wolbachia were due to an increase in the expression of Sxl, rather than an effect on mutant Sxl protein function, Wolbachia should have suppressed snf¹⁶²¹, as do extra copies of Sxl⁺ (Starr, 2002).

The effect of Wolbachia on Sxl functioning in D. melanogaster seems to be restricted to the germ line, since there is no evidence for the sex-specific lethality that would accompany inappropriate somatic expression of Sxl. Moreover, using a very sensitive test, it was determined that infection does not alter the effectiveness of the primary sex-determination signal, perturbations of which can cause sex-specific lethality owing to inappropriate somatic expression of Sxl. Nevertheless, the possibility should be explored that Wolbachia-induced male killing reported for other Drosophila species may be caused by inappropriate activation of Sxl (Starr, 2002).

Although it may seem surprising that infection with a parasite would reverse the deleterious effect of a mutation in the host genome, particularly when the isolation of that mutation had nothing to do with infection, such surprise should be tempered by the fact that the interaction described here between host and parasite mimics a naturally occurring situation that has been reported for the parasitic wasp Asobara tabida. Moreover, in light of the fact that Wolbachia is a parasite that is known to manipulate host reproductive and sex-determination systems, it does not seem unreasonable that the host gene with which it interacts in Drosophila is the master regulator of sex-determination and a gene essential for oogenesis. The fact that the interacting gene in this case has been studied so extensively and belongs to a model experimental organism can be exploited to yield further insights into the mechanism by which this parasite takes advantage of its various arthropod hosts (Starr, 2002).

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