Sex determination in the Drosophila germline is dictated by the sexual identity of the surrounding soma

It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. In the studies reported here, we have examined the role of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway. We asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. We find that Tra(F) is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by Tra(F) can be blocked by the male form of the Dsx protein (Dsx(M)). Expression of the female form of dsx, Dsx(F), in XY animals also induced germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, our findings indicate that the somatic signaling pathway is redundant at the level tra and dsx. Finally, our studies call into question the idea that a cell-autonomous X chromosome counting system plays a central role in germline sex determination.     

Germ cell sex determination: a collaboration between soma and germline

Sex determination is regulated very differently in the soma vs. the germline, yet both processes are critical for the creation of the male and female gametes. In general, the soma plays an essential role in regulating sexual identity of the germline. However, in some species, such as Drosophila and mouse, the sex chromosome constitution of the germ cells makes an autonomous contribution to germline sexual development. Here we review how the soma and germline cooperate to determine germline sexual identity for some important model systems, the fly, the worm and the mouse, and discuss some of the implications of 'dual control' (soma plus germline) as compared to species where germline sex is dictated only by the surrounding soma.     

Sexual back talk with evolutionary implications: stimulation of the Drosophila sex-determination gene sex-lethal by its target transformer

We describe a surprising new regulatory relationship between two key genes of the Drosophila sex-determination gene hierarchy, Sex-lethal (Sxl) and transformer (tra). A positive autoregulatory feedback loop for Sxl was known to maintain somatic cell female identity by producing SXL-F protein to continually instruct the target gene transformer (tra) to make its feminizing product, TRA-F. We discovered the reciprocal regulatory effect by studying genetically sensitized females: TRA-F from either maternal or zygotic tra expression stimulates Sxl-positive autoregulation. We found female-specific tra mRNA in eggs as predicted by this tra maternal effect, but not predicted by the prevailing view that tra has no germline function. TRA-F stimulation of Sxl seems to be direct at some point, since Sxl harbors highly conserved predicted TRA-F binding sites. Nevertheless, TRA-F stimulation of Sxl autoregulation in the gonadal soma also appears to have a cell-nonautonomous aspect, unprecedented for somatic Sxl regulation. This tra-Sxl retrograde regulatory circuit has evolutionary implications. In some Diptera, tra occupies Sxl's position as the gene that epigenetically maintains female identity through direct positive feedback on pre-mRNA splicing. The tra-mediated Sxl feedback in Drosophila may be a vestige of regulatory redundancy that facilitated the evolutionary transition from tra to Sxl as the master sex switch.     

Sex determination in the germ line of Drosophila depends on genetic signals and inductive somatic factors

We have analyzed the mechanism of sex determination in the germ line of Drosophila by manipulating three parameters: (1) the ratio of X-chromosomes to sets of autosomes (X:A); (2) the state of activity of the gene Sex-lethal (Sxl), and (3) the sex of the gonadal soma. To this end, animals with a ratio of 2X:2A and 2X:3A were sexually transformed into pseudomales by mutations at the sex-determining genes Sxl (Sex-lethal), tra (transformer), tra-2 (transformer-2), or dsx (double-sex). Animals with the karyotype 2X;3A were also transformed into pseudofemales by the constitutive mutation SxlM1. The sexual phenotype of the gonads and of the germ cells was assessed by phase-contrast microscopy. Confirming the conclusions of Steinmann-Zwicky et al. (Cell 57, 157, 1989), we found that all three parameters affect sex determination in germ cells. In contrast to the soma in which sex determination is completely cell-autonomous, sex determination in the germ line has a non-autonomous component inasmuch as the sex of the soma can influence the sexual pathway of the germ cells. Somatic induction has a clear effect on 2X;2A germ cells that carry a Sxl+ allele. These cells, which form eggs in an ovary, can enter spermatogenesis in testes. Mutations that cause partial loss of function or gain of function of Sxl thwart somatic induction and, independently of the sex of the soma, dictate spermatogenesis or oogenesis, respectively. Somatic induction has a much weaker effect on 2X;3A germ cells. This ratio is essentially a male signal for germ cells which consistently enter spermatogenesis in testes, even when they carry SxlM1. In a female soma, however, SxlM1 enables the 2X;3A germ cells to form almost normal eggs. Our results show that sex determination in the germ line is more complex than in the soma. They provide further evidence that the state of Sxl, the key gene for sex determination and dosage compensation in the soma, also determines the sex of the germ cells, and that, in the germ line, the state of activity of Sxl is regulated not only by the X:A ratio, but also by somatic inductive stimuli.     

Ovarian tumor expression is dependent on the functions of the somatic sex regulatory genes transformer-2 and doublesex

The doublesex-dependent sex regulatory pathway in Drosophila controls major aspects of somatic sexual differentiation, but its expression is not required in the X/X germline. Nevertheless, mutations in doublesex and in the genes that directly regulate its expression, transformer and transformer-2, disrupt early stages of oogenic differentiation to produce gonads containing immature germ cells. This indicates a critical, but uncharacterized, set of soma-germline interactions essential for oogenesis. In this paper, we examined the effects of mutations in transformer-2 on the expression and function of the germline-specific ovarian tumor gene. We demonstrated that in transformer-2 mutants, there was a marked reduction in the activity of the ovarian tumor promoter in the mutant germline. In addition, the phenotypic effects on the arrested germline could be partially suppressed by the simultaneous over-expression of both ovarian tumor and a second germline gene, Sex-lethal. This differs from transformer mutations, in which the over-expression of ovarian tumor alone is sufficient for a similar improvement in germline differentiation. In contrast to transformer-2, doublesex activity was not required for ovarian tumor promoter activity and we found indirect evidence that the doublesex male-specific function might have a negative regulatory effect. These data indicate that the components of the genetic pathway regulating somatic sexual differentiation have specific and differential effects on germline gene activity.     

Evolutionary conservation of Ceratitis capitata transformer gene function

Transformer functions as a binary switch gene in the sex determination and sexual differentiation of Drosophila melanogaster and Ceratitis capitata, two insect species that separated nearly 100 million years ago. The TRA protein is required for female differentiation of XX individuals, while XY individuals express smaller, presumably nonfunctional TRA peptides and consequently develop into adult males. In both species, tra confers female sexual identity through a well-conserved double-sex gene. However, unlike Drosophila tra, which is regulated by the upstream Sex-lethal gene, Ceratitis tra itself is likely to control a feedback loop that ensures the maintenance of the female sexual state. The putative CcTRA protein shares a very low degree of sequence identity with the TRA proteins from Drosophila species. However, in this study we show that a female-specific Ceratitis Cctra cDNA encoding the putative full-length CcTRA protein is able to support the female somatic and germline sexual differentiation of D. melanogaster XX; tra mutant adults. Although highly divergent, CcTRA can functionally substitute for DmTRA and induce the female-specific expression of both Dmdsx and Dmfru genes. These data demonstrate the unusual plasticity of the TRA protein that retains a conserved function despite the high evolutionary rate. We suggest that transformer plays an important role in providing a molecular basis for the variety of sex-determining systems seen among insects.     

Genetic Evidence That the Ovo Locus Is Involved in Drosophila Germ Line Sex Determination

Zygotically contributed ovo gene product is required for the survival of female germ cells in Drosophila melanogaster. Trans-allelic combinations of weak and dominant ovo mutations (ovoD) result in viable germ cells that appear to be partially transformed from female to male sexual identity. The ovoD2 mutation is partially suppressed by many Sex-lethal alleles that affect the soma, while those that affect only the germ line fail to interact with ovoD2. One of two loss-of-function ovo alleles is suppressed by a loss-of-function Sex-lethal allele. Because ovo mutations are germ line dependent, it is likely that ovo is suppressed by way of communication between the somatic and germ lines. A loss-of-function allele of ovo is epistatic to germ line dependent mutations in Sex-lethal. The germ line dependent sex determination mutation, sans fille, and ovoD mutations show a dominant synergistic interaction resulting in partial transformation of germ line sexual identity. The ovo locus appears to be involved in germ line sex determination and is linked in some manner to sex determination in the soma.     

The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and act as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. We propose that this repression is achieved by the Y-linked male-determining factor (M).     

Analysis of a null mutation in the Drosophila splicing regulator Tra2 suggests its function is restricted to sexual differentiation

Tra2 is a regulator of pre-mRNA splicing and a key component of the Drosophila somatic sex determination pathway. Functional orthologs of this protein are thought to perform nonsex-specific functions essential for viability in both vertebrates and nematodes. Although Drosophila Tra2 is expressed throughout the soma of both sexes, studies on it have focused only on the sex-specific phenotypes of known viable alleles. Here we show that that widely used tra2 mutant alleles have residual activity and are not suitable for evaluating its effect on viability. To test whether Tra2 has an essential role in development, we generated a transposon-induced deletion in critical coding sequences. We find that tra2 deletion adults can survive as well as their heterozygous siblings. Thus, in contrast to other organisms, Tra2 is not required in Drosophila for general viability under laboratory conditions.     

Germ-line sex determination in Drosophila melanogaster

In Drosophila melanogaster, the mechanism of sex determination is substantially different in the germ line and in the soma. In the germ line, the process is not completely cell-autonomous, but requires some signals from the soma. Only some of the genes involved in somatic sex determination are also needed for germ cell development. Recent genetic studies have identified loci required for germ-line sex determination.     

Rap-GEF signaling controls stem cell anchoring to their niche through regulating DE-cadherin-mediated cell adhesion in the Drosophila testis

Stem cells will undergo self-renewal to produce new stem cells if they are maintained in their niches. The regulatory mechanisms that recruit and maintain stem cells in their niches are not well understood. In Drosophila testes, a group of 12 nondividing somatic cells, called the hub, identifies the stem cell niche by producing the growth factor Unpaired (Upd). Here, we show that Rap-GEF/Rap signaling controls stem cell anchoring to the niche through regulating DE-cadherin-mediated cell adhesion. Loss of function of a Drosophila Rap-GEF (Gef26) results in loss of both germline and somatic stem cells. The Gef26 mutation specifically impairs adherens junctions at the hub-stem cell interface, which results in the stem cells "drifting away" from the niche and losing stem cell identity. Thus, the Rap signaling/E-cadherin pathway may represent one mechanism that regulates polarized niche formation and stem cell anchoring.     

Nonautonomous sex determination controls sexually dimorphic development of the Drosophila gonad

Sex determination in Drosophila is commonly thought to be a cell-autonomous process, where each cell decides its own sexual fate based on its sex chromosome constitution (XX versus XY). This is in contrast to sex determination in mammals, which largely acts nonautonomously through cell-cell signaling. Here we examine how sexual dimorphism is created in the Drosophila gonad by investigating the formation of the pigment cell precursors, a male-specific cell type in the embryonic gonad. Surprisingly, we find that sex determination in the pigment cell precursors, as well as the male-specific somatic gonadal precursors, is non-cell autonomous. Male-specific expression of Wnt2 within the somatic gonad triggers pigment cell precursor formation from surrounding cells. Our results indicate that nonautonomous sex determination is important for creating sexual dimorphism in the Drosophila gonad, similar to the manner in which sex-specific gonad formation is controlled in mammals.     

Sxl in the germline of Drosophila: A target for somatic late induction

In Drosophila, the sex of germ cells is determined by autonomous and inductive signals. Somatic inductive signals can drive XX germ cells into oogenesis or into spermatogenesis. An autonomous signal makes XY germ cells male and unresponsive to sex determination by induction. The elements forming the X:A ratio in the soma and the genes tra, tra2, dsx, and ix that determine the sex of somatic cells have no similar role in the germline. The gene Sxl, however, is required for female differentiation of somatic and germ cells. Inductive signals that are dependent on somatic tra and dsx expression already affect the sex-specific development of germ cells of first instar larvae. At this early stage, however, germline expression of Sxl does not appear to affect the sexual characteristics of germ cells. Since inductive signals dependent on tra and dsx nevertheless influence the choice of sex-specific splicing of Sxl, it can be concluded that Sxl is a target of the inductive signal, but that its product is required late for oogenesis. Other genes must therefore control the early sexual dimorphism of larval germ cells. © 1994 Wiley-Liss, Inc.