Monica Steinmann-Zwicky

Cell-autonomous and inductive signals can determine the sex of the germ line of Drosophila by regulating the gene Sxl

To investigate the mechanism of sex determination in the germ line, we analyzed the fate of XY germ cells in ovaries, and the fate of XX germ cells in testes. In ovaries, germ cells developed according to their X:A ratio, i.e., XX cells underwent oogenesis, XY cells formed spermatocytes. In testes, however, XY and XX germ cells entered the spermatogenic pathway. Thus, to determine their sex, the germ cells of Drosophila have cell-autonomous genetic information, and XX cells respond to inductive signals of the soma. Results obtained with amorphic and constitutive mutations of Sxl show that both the genetic and the somatic signals act through Sxl to achieve sex determination in germ cells.

Sex determination in Drosophila

Abstract Sex determination and differentiation are processes that create morphological, physiological and behavioral differences between the sexes by initiating and completing one of two alternative developmental programs. In Drosophila , as in other organisms whose genetics of sex determination has been studied, this is achieved by a hierarchical control system in which a chromosomal signal affects a small number of regulatory genes whose state of activity then controls the many differentiation genes needed to materialize all the differences between the sexes. The genetic pathway that specifies sexual differentiation can serve as a paradigm of how developmental processes in general are controlled.

Sex determination of the Drosophila germ line: tra and dsx control somatic inductive signals

In Drosophila, the sex of germ cells is determined by cell-autonomous and inductive signals. XY germ cells autonomously enter spermatogenesis when developing in a female host. In contrast, XX germ cells non-autonomously become spermatogenic when developing in a male host. In first instar larvae with two X chromosomes, XX germ cells enter the female or the male pathway depending on the presence or absence of transformer (tra) activity in the surrounding soma. In somatic cells, the product of tra regulates the expression of the gene double sex (dsx) which can form a male-specific or a female-specific product. In dsx mutant larvae, XX and XY germ cells develop abnormally, with a seemingly intersexual phenotype. This indicates that female-specific somatic dsx products feminize XX germ cells, and male-specific somatic dsx products masculinize XX and XY germ cells. The results show that tra and dsx control early inductive signals that determine the sex of XX germ cells and that somatic signals also affect the development of XY germ cells. XX germ cells that develop in pseudomales lacking the sex-determining function of Sxl are spermatogenic. If, however, female-specific tra functions are expressed in these animals, XX germ cells become oogenic. Furthermore, transplanted XX germ cells can become oogenic and form eggs in XY animals that express the female-specific function of tra. Therefore, TRA product present in somatic cells of XY animals or in animals lacking the sex-determining function of Sxl, is sufficient to support developing XX germ cells through oogenesis.

FEMALE GERM CELLS OF DROSOPHILA REQUIRE ZYGOTIC OVO AND OTU PRODUCT FOR SURVIVAL IN LARVAE AND PUPAE RESPECTIVELY

Mutations in the genes ovo or otu can cause abnormal proliferation of XX germ cells, which leads to so-called ovarian tumors, or they can lead to the elimination of XX germ cells, such that adult females possess empty ovaries. Males carrying ovo or otu mutations are unaffected. To find out when this sexual dimorphism affects germ cells, we analyzed the requirement of embryos and larvae for zygotic ovo and otu products. We found that ovo is required for the survival of XX germ cells during larval stages, while XX germ cells lacking otu survive until metamorphosis. Furthermore, we found no sex-transformed mutant larval germ cells and no evidence for an early sex-specific vital process acting in germ cells of the embryo, contrary to what had been suggested earlier.

Cell-autonomous and somatic signals control sex-specific gene expression in XY germ cells of Drosophila.

When XX germ cells develop in a testis they become spermatogenic. Thus, somatic signals determine the sex of genetically female germ cells. In contrast, XY germ cells experimentally transferred to an ovary do not differentiate oogenic cells. Because such cells show some male characteristics when analyzed in adults, it was assumed that XY germ cells autonomously become spermatogenic. Recently, however, evidence showing that a female soma feminizes XY germ cells was reported. The conclusion was drawn that the sex determination of XY germ cells is dictated by the sex of the soma. We monitored the fate of XY germ cells placed in a female environment throughout development. Here we report that such germ cells respond to both cell-autonomous and somatic sex-determining signals, depending on the developmental stage. Analyzing the expression of sex-specific molecular markers, we first detected autonomous male-specific gene expression in XY germ cells embedded in female embryos and larvae. At later stages, however, we found that sex-specific regulation of gene expression within XY germ cells is influenced by somatic gonadal cells. After metamorphosis, XY germ cells developing in a female soma start expressing female-specific and male-specific markers. Transcription of female-specific genes is maintained, while that of male-specific genes is later repressed. We show that in such XY germ cells, the female-specific gene Sex-lethal (Sxl) is activated. Within the germline, Sxl expression is required for the activation of a further female-specific gene and the repression of male-specific genes. We thus report for the first time the existence of downstream targets of the gene Sxl in the germline.

The Drosophila dorsoventral determinant pipe contains ten copies of a variable domain homologous to mammalian heparan sulfate 2-sulfotransferase

In Drosophila, the gene pipe is expressed in follicle cells, the somatic cells that surround the forming egg during maturation, specifically on one side of the egg chamber. This asymmetry establishes the dorsoventral axis of the future embryo. Through the action of pipe, the ligand spätzle, that is located in the perivitelline fluid of the embryo, is activated ventrally. This signal activates Toll, a membrane‐bound receptor. According to present knowledge, pipe encodes two different transcripts, one of which restored ventral pattern elements to embryos when introduced into mutant pipe females. Here we show that pipe is far more complex than previously reported. It encodes not two, but at least ten different transcripts, two of which are localized to ventral follicle cells. The transcripts contain one of ten copies of a variable domain, all homologous to heparan sulfate 2‐sulfotransferase, an enzyme known to modify heparan sulfate proteoglycans, which are molecules that can bind ligands. The complex gene structure of pipe thus evolved by duplications of one exon, a strategy used by genes of the immunoglobulin superfamily to generate molecular diversity. We show that pipe transcripts can be eliminated by RNAi, although in this method double‐stranded RNA is injected in embryos, while pipe transcripts appear in the adult ovary. Our data suggest that at least two different pipe transcripts redundantly provide the ventralizing pipe function. 3′ of pipe we identified an enhancer element that drives a lacZ reporter gene specifically in ventral follicle cells. Since pipe transcripts are found in salivary glands, and since expression of salivary gland genes is dependent on signaling molecules, we speculate that pipe became localized to ventral follicle cells by a preexisting control system after acquiring a follicle cell enhancer.

A small region on the X chromosome of Drosophila regulates a key gene that controls sex determination and dosage compensation

In Drosophila, flies with two X chromosomes are females, with one X chromosome, males. We investigated the presence of sex determining factors on the X chromosome by constructing genotypes with one X and various X-chromosomal duplications. We found that female determining factors are not evenly distributed along the X chromosome as had been previously postulated. A distal duplication covering 35% of the X chromosome promotes female differentiation, a much larger proximal duplication of 60% results in male differentiation. The strong feminizing effect of distal duplications originates from a small segment that, when present in two doses, activates Sxl, a key gene for sex determination and dosage compensation. Our results suggest that Sxl can be activated to intermediate levels.

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in Drosophila

BackgroundMetal-responsive transcription factor 1 (MTF-1), which binds to metal response elements (MREs), plays a central role in transition metal detoxification and homeostasis. A Drosophila interactome analysis revealed two candidate dMTF-1 interactors, both of which are related to the small regulatory protein Dumpy-30 (Dpy-30) of the worm C. elegans. Dpy-30 is the founding member of a protein family involved in chromatin modifications, notably histone methylation. Mutants affect mating type in yeast and male mating in C. elegans.ResultsConstitutive expression of the stronger interactor, Dpy-30L1 (CG6444), in transgenic flies inhibits MTF-1 activity and results in elevated sensitivity to Cd(II) and Zn(II), an effect that could be rescued by co-overexpression of dMTF-1. Electrophoretic mobility shift assays (EMSA) suggest that Dpy-30L1 interferes with the binding of MTF-1 to its cognate MRE binding site. Dpy-30L1 is expressed in the larval brain, gonads, imaginal discs, salivary glands and in the brain, testes, ovaries and salivary glands of adult flies. Expression of the second interactor, Dpy-30L2 (CG11591), is restricted to larval male gonads, and to the testes of adult males. Consistent with these findings, dpy-30-like transcripts are also prominently expressed in mouse testes. Targeted gene disruption by homologous recombination revealed that dpy-30L1 knockout flies are viable and show no overt disruption of metal homeostasis. In contrast, the knockout of the male-specific dpy-30L2 gene results in male sterility, as does the double knockout of dpy-30L1 and dpy-30L2. A closer inspection showed that Dpy-30L2 is expressed in elongated spermatids but not in early or mature sperm. Mutant sperm had impaired motility and failed to accumulate in sperm storage organs of females.ConclusionOur studies help to elucidate the physiological roles of the Dumpy-30 proteins, which are conserved from yeast to humans and typically act in concert with other nuclear proteins to modify chromatin structure and gene expression. The results from these studies reveal an inhibitory effect of Dpy-30L1 on MTF-1 and an essential role for Dpy-30L2 in male fertility.

Genetics of sex determination in eukaryotes.

The biologically significant consequence of sex is that a genetic variant is no longer the unique property of an individual and its clonal descendents, but becomes part of a gene pool that is shared by the members of a population. Variants can spread through the population, leading, without additional mutations, to the rapid production of new genetic combinations. The essence of sexual processes thus is the recombination of existing genetic, information.

No premature gene expression in germ cells of embryos deriving from nos females

The product of nos is required at the posterior pole of the embryo for the differentiation of abdominal structures, but not for pole cell formation. A previous analysis that reported the expression of germline-specific enhancer-trap lines suggested that nos also controls the timing of the initiation of transcription of germline-specific genes. Here we repeat the experiments that led to this hypothesis and we report further experiments. Our results show that, contrary to what had been reported, germ cells of embryos deriving from nos females do not show premature gene expression. Germ cells of such embryos, however, often show artefactual lacZ staining even in the absence of a lacZ gene.