Rbpms2 promotes female fate upstream of the nutrient sensing Gator2 complex component, Mios.

Reproductive success relies on proper establishment and maintenance of biological sex. In many animals, including mammals, the primary gonad is initially ovary in character. We previously showed the RNA binding protein (RNAbp), Rbpms2, is required for ovary fate in zebrafish. Here, we identified Rbpms2 targets in oocytes (Rbpms2-bound oocyte RNAs; rboRNAs). We identify Rbpms2 as a translational regulator of rboRNAs, which include testis factors and ribosome biogenesis factors. Further, genetic analyses indicate that Rbpms2 promotes nucleolar amplification via the mTorc1 signaling pathway, specifically through the mTorc1-activating Gap activity towards Rags 2 (Gator2) component, Missing oocyte (Mios). Cumulatively, our findings indicate that early gonocytes are in a dual poised, bipotential state in which Rbpms2 acts as a binary fate-switch. Specifically, Rbpms2 represses testis factors and promotes oocyte factors to promote oocyte progression through an essential Gator2-mediated checkpoint, thereby integrating regulation of sexual differentiation factors and nutritional availability pathways in zebrafish oogenesis.

A transgenic system for targeted ablation of reproductive and maternal-effect genes.

Maternally provided gene products regulate the earliest events of embryonic life, including formation of the oocyte that will develop into an egg, and eventually into an embryo. Forward genetic screens have provided invaluable insights into the molecular regulation of embryonic development, including the essential contributions of some genes whose products must be provided to the transcriptionally silent early embryo for normal embryogenesis, called maternal-effect genes. However, other maternal-effect genes are not accessible due to their essential zygotic functions during embryonic development. Identifying these regulators is essential to fill the large gaps in our understanding of the mechanisms and molecular pathways contributing to fertility and to maternally regulated developmental processes. To identify these maternal factors, it is necessary to bypass the earlier requirement for these genes so that their potential later functions can be investigated. Here, we report reverse genetic systems to identify genes with essential roles in zebrafish reproductive and maternal-effect processes. As proof of principle and to assess the efficiency and robustness of mutagenesis, we used these transgenic systems to disrupt two genes with known maternal-effect functions: kif5ba and bucky ball.

Loss of dmrt1 restores zebrafish female fates in the absence of cyp19a1a but not rbpms2a/b.

Sex determination and differentiation is a complex process regulated by multiple factors, including factors from the germline or surrounding somatic tissue. In zebrafish, sex-determination involves establishment of a bipotential ovary that undergoes sex-specific differentiation and maintenance to form the functional adult gonad. However, the relationships among these factors are not fully understood. Here, we identify potential Rbpms2 targets and apply genetic epistasis experiments to decipher the genetic hierarchy of regulators of sex-specific differentiation. We provide genetic evidence that the crucial female factor rbpms2 is epistatic to the male factor dmrt1 in terms of adult sex. Moreover, the role of Rbpms2 in promoting female fates extends beyond repression of Dmrt1, as Rbpms2 is essential for female differentiation even in the absence of Dmrt1. In contrast, female fates can be restored in mutants lacking both cyp19a1a and dmrt1, and prolonged in bmp15 mutants in the absence of dmrt1. Taken together, this work indicates that cyp19a1a-mediated suppression of dmrt1 establishes a bipotential ovary and initiates female fate acquisition. Then, after female fate specification, Cyp19a1a regulates subsequent oocyte maturation and sustains female fates independently of Dmrt1 repression.

Correction: rbpms2 functions in Balbiani body architecture and ovary fate.

[This corrects the article DOI: 10.1371/journal.pgen.1007489.].

rbpms2 functions in Balbiani body architecture and ovary fate.

The most prominent developmental regulators in oocytes are RNA-binding proteins (RNAbps) that assemble their targets into ribonucleoprotein granules where they are stored, transported and translationally regulated. RNA-binding protein of multiple splice forms 2, or Rbpms2, interacts with molecules that are essential to reproduction and egg patterning, including bucky ball, a key factor for Bb formation. Rbpms2 is localized to germ granules in primordial germ cells (PGCs) and to the Balbiani body (Bb) of oocytes, although the mechanisms regulating Rbpms2 localization to these structures are unknown. Using mutant Rbpms2 proteins, we show that Rbpms2 requires distinct protein domains to localize within germ cells and somatic cells. Accumulation and localization to subcellular compartments in the germline requires an intact RNA binding domain. Whereas in zebrafish somatic blastula cells, the conserved C-terminal domain promotes localization to the bipolar centrosomes/spindle. To investigate Rbpms2 functions, we mutated the duplicated and functionally redundant zebrafish rbpms2 genes. The gonads of rbpms2a;2b (rbpms2) mutants initially contain early oocytes, however definitive oogenesis ultimately fails during sexual differentiation and, rbpms2 mutants develop as fertile males. Unlike other genes that promote oogenesis, failure to maintain oocytes in rbpms2 mutants was not suppressed by mutation of Tp53. These findings reveal a novel and essential role for rbpms2 in oogenesis. Ultrastructural and immunohistochemical analyses revealed that rbpms2 is not required for the asymmetric accumulation of mitochondria and Buc protein in oocytes, however its absence resulted in formation of abnormal Buc aggregates and atypical electron-dense cytoplasmic inclusions. Our findings reveal novel and essential roles for rbpms2 in Buc organization and oocyte differentiation.