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In sex determination, globally acting genes control a spectrum of tissue-specific regulators to coordinate the overall development of an animal into one sex or the other. In mammals, primary sex determination initially occurs in the gonad, with the sex of other tissues specified as a secondary event. In insects and nematodes, globally acting regulatory pathways have been elucidated, but the more tissue- and organ-specific downstream effectors of these pathways remain largely unknown. We focus on the control of sexual dimorphism in the C. elegans gonad. We find that the forkhead transcription factor FKH-6 promotes male gonadal cell fates in XO animals. Loss-of-function fkh-6 mutant males have feminized gonads and often develop a vulva. In these mutant males, sex-specific cell divisions and migrations in the early gonad occur in the hermaphrodite mode, and hermaphrodite-specific gonadal markers are expressed. However, sexual transformation is not complete and the male gonad is malformed. By contrast, fkh-6 mutant hermaphrodites exhibit no sign of sex reversal. Most fkh-6 hermaphrodites form a two-armed symmetrical gonad resembling that of the wild type, but differentiation of the spermatheca and uterus is variably abnormal. The function of fkh-6 appears to be restricted to the gonad: fkh-6 mutants have no detectable defects in extra-gonadal tissues, and expression of a rescuing fkh-6 reporter is gonad-specific. Genetic and molecular analyses place fkh-6 downstream of tra-1, the terminal regulator of the global sex determination pathway, with respect to the first gonadal cell division. We conclude that fkh-6 regulates gonadogenesis in both sexes, but is male specific in establishing sexual dimorphism in the early gonad.

The posterior pole plasm of the Drosophila egg contains the determinants of abdominal and germ-cell fates of the embryo. Pole plasm assembly is induced by oskar RNA localized to the posterior pole of the oocyte. Genetics has revealed three additional genes, staufen, vasa, and tudor, that are also essential for pole plasm formation. Staufen protein is required for both oskar RNA localization and translation. Vasa and Tudor are localized dependent on Oskar protein and are required to accumulate Oskar protein stably at the posterior pole. We have explored interactions between these gene products at the molecular level and find that Oskar interacts directly with Vasa and Staufen, in a yeast two-hybrid assay. These interactions also occur in vitro and are affected by mutations in Oskar that abolish pole plasm formation in vivo. Finally, we show that in the pole plasm, Oskar protein, like Vasa and Tudor, is a component of polar granules, the germ-line-specific RNP structures. These results suggest that the Oskar-Vasa interaction constitutes an initial step in polar granule assembly. In addition, we discuss the possible biological role of the Oskar-Staufen interaction.

The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. In this study we demonstrate that, when oskar mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5′ region of the mRNA. We demonstrate that this novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the oskar 3′ UTR is required for activation of oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction betweentrans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. We also show specific interaction of two proteins with the oskar mRNA 5′ region; one of these also recognizes the 3′ repressor element. We discuss the possible involvement of these factors as well as known genes in the process of localization-dependent translation.

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.