Selection and Maintenance of Sexual Identity in the Drosophila Germline

Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transformer 2 gene but, surprisingly, does not appear to require the sex-specific genes, transformer and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-lethal gene.     

Phf7 controls male sex determination in the Drosophila germline

Establishment of germline sexual identity is critical for production of male and female germline stem cells, as well as sperm versus eggs. Here we identify PHD Finger Protein 7 (PHF7) as an important factor for male germline sexual identity in Drosophila. PHF7 exhibits male-specific expression in early germ cells, germline stem cells, and spermatogonia. It is important for germline stem cell maintenance and gametogenesis in males, whereas ectopic expression in female germ cells ablates the germline. Strikingly, expression of PHF7 promotes spermatogenesis in XX germ cells when they are present in a male soma. PHF7 homologs are also specifically expressed in the mammalian testis, and human PHF7 rescues Drosophila Phf7 mutants. PHF7 associates with chromatin, and both the human and fly proteins bind histone H3 N-terminal tails with a preference for dimethyl lysine 4 (H3K4me2). We propose that PHF7 acts as a conserved epigenetic "reader" that activates the male germline sexual program.     

A Genome-Wide Survey of Sexually Dimorphic Expression of Drosophila miRNAs Identifies the Steroid Hormone-Induced miRNA let-7 as a Regulator of Sexual Identity

MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.     

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.     

Somatic control of germ cell development in Caenorhabditis elegans

Germ cell development in Caenorhabditis elegans involves three processes: a shift from the mitotic to the meiotic cell cycle; the adoption of a male or female sexual identity; and differentiation into a functional gamete. All three aspects of germline development appear to be regulated, at least in part, by the soma. We discuss cell ablation, genetic and molecular studies that have shed light on the nature of the signal transduction systems mediating intercellular communication between germline and somatic tissues of the nematode.     

The phenotype of mes-2, mes-3, mes-4 and mes-6, maternal-effect genes required for survival of the germline in Caenorhabditis elegans,is sensitive to chromosome dosage.

Mutations in mes-2, mes-3, mes-4, and mes-6 result in maternal-effect sterility: hermaphrodite offspring of mes/mes mothers are sterile because of underproliferation and death of the germ cells, as well as an absence of gametes. Mutant germ cells do not undergo programmed cell death, but instead undergo a necrotic-type death, and their general poor health apparently prevents surviving germ cells from forming gametes. Male offspring of mes mothers display a significantly less severe germline phenotype than their hermaphrodite siblings, and males are often fertile. This differential response of hermaphrodite and male offspring to the absence of mes+ product is a result of their different X chromosome compositions; regardless of their sexual phenotype, XX worms display a more severe germline phenotype than XO worms, and XXX worms display the most severe phenotype. The sensitivity of the mutant phenotype to chromosome dosage, along with the similarity of two MES proteins to chromatin-associated regulators of gene expression in Drosophila, suggest that the essential role of the mes genes is in control of gene expression in the germline. An additional, nonessential role of the mes genes in the soma is suggested by the surprising finding that mutations in the mes genes, like mutations in dosage compensation genes, feminize animals whose male sexual identity is somewhat ambiguous. We hypothesize that the mes genes encode maternally supplied regulators of chromatin structure and gene expression in the germline and perhaps in somatic cells of the early embryo, and that at least some of their targets are on the X chromosomes.     

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.     

nanos expression at the embryonic posterior pole and the medusa phase in the hydrozoan Podocoryne carnea

Summary The distinction between soma and germline is an important process in the development of animals with sexual reproduction. It is regulated by a number of germline-specific genes, most of which appear conserved in evolution and therefore can be used to study the formation of the germline in diverged animal groups. Here we report the isolation of two orthologs of one such gene, nanos (nos), in the cnidarian Podocoryne carnea, a species with representative zoological features among the hydrozoans. By studying nos gene expression throughout the Podocoryne biphasic life cycle, we find that the germline differentiates exclusively during medusa development, whereas the polyp does not contribute to the process. An early widespread nos expression in developing medusae progressively refines into a mainly germline-specific pattern at terminal stages of medusa formation. Thus, the distinction between germline and soma is a late event in hydrozoan development. Also, we show that the formation of the medusa is a de novo process that relies on active local cell proliferation and differentiation of novel cell and tissue types not present in the polyp, including nos-expressing cells. Finally, we find nos expression at the posterior pole of Podocoryne developing embryos, not related to germline formation. This second aspect of nos expression is also found in Drosophila, where nos functions as a posterior determinant essential for the formation of the fly abdomen. This raises the possibility that nos embryonic expression could play a role in establishing axial polarity in cnidarians.     

Gonadal Dysgenesis Reveals Sexual Dimorphism in the Embryonic Germline of Drosophila

In hybrid dysgenesis, sterility can occur in both males and females. At 27.5 degrees, however, we found that P element-induced germline death was restricted to females. This sex-specific gonadal dysgenesis (GD) is complete by the first larval instar stage. As such, GD at 27.5 degrees reveals the sexually dimorphic character of the embryonic germline. The only other known dimorphic trait of the embryonic germline is the requirement for ovo. ovo is required for germline development in females only and has been implicated in germline sex determination. Dominant mutations of ovo partially suppressed female GD. Although embryonic germ cells are undifferentiated and morphologically indistinguishable between males and females, the functional dimorphism seen in ovo requirement and GD at 27.5 degrees indicates that sexual identity in Drosophila germ cells is established in embryogenesis.     

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.     

Sexual Fate Reprogramming in the Steroid-Induced Bi-Directional Sex Change in the Protogynous Orange-Spotted Grouper,Epinephelus coioides

Androgen administration has been widely used for masculinization in fish. The mechanism of the sex change in sexual fate regulation is not clear. Oral administration or pellet implantation was applied. We orally applied an aromatase inhibitor (AI, to decrease estrogen levels) and 17α-methyltestosterone (MT, to increase androgen levels) to induce masculinization to clarify the mechanism of the sex change in the protogynous orange-spotted grouper. After 3 mo of AI/MT administration, male characteristics were observed in the female-to-male sex change fish. These male characteristics included increased plasma 11-ketotestosterone (11-KT), decreased estradiol (E2) levels, increased male-related gene (dmrt1, sox9, and cyp11b2) expression, and decreased female-related gene (figla, foxl2, and cyp19a1a) expression. However, the reduced male characteristics and male-to-female sex change occurred after AI/MT-termination in the AI- and MT-induced maleness. Furthermore, the MT-induced oocyte-depleted follicle cells (from MT-implantation) had increased proliferating activity, and the sexual fate in a portion of female gonadal soma cells was altered to male function during the female-to-male sex change. In contrast, the gonadal soma cells were not proliferative during the early process of the male-to-female sex change. Additionally, the male gonadal soma cells did not alter to female function during the male-to-female sex change in the AI/MT-terminated fish. After MT termination in the male-to-female sex-changed fish, the differentiated male germ cells showed increased proliferating activities together with dormancy and did not show characteristics of both sexes in the early germ cells. In conclusion, these findings indicate for the first time in a single species that the mechanism involved in the replacement of soma cells is different between the female-to-male and male-to-female sex change processes in grouper. These results also demonstrate that sexual fate determination (secondary sex determination) is regulated by endogenous sex steroid levels.     

Germ cells are not the primary factor for sexual fate determination in goldfish

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.     

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.