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 biased. 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.

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.

Macrophage activation drives ovarian failure and masculinization in zebrafish.

BMP15 is a conserved regulator of ovarian development and maintenance in vertebrates. In humans, premature ovarian insufficiency is caused by autoimmunity and genetic factors, including mutation of BMP15. The cellular mechanisms underlying ovarian failure caused by BMP15 mutation and immune contributions are not understood. Using zebrafish, we established a causal link between macrophage activation and ovarian failure, which, in zebrafish, causes sex reversal. We define a germline-soma signaling axis that activates macrophages and drives ovarian failure and female-to-male sex reversal. Germline loss of zebrafish Bmp15 impairs oogenesis and initiates this cascade. Single-cell RNA sequencing and genetic analyses implicate ovarian somatic cells that express conserved macrophage-activating ligands as mediators of ovarian failure and sex reversal. Genetic ablation of macrophages or elimination of Csf1Rb ligands, Il34 or Csf1a, delays or blocks premature oocyte loss and sex reversal. The axis identified here provides insight into the cells and pathways governing oocyte and ovary maintenance and potential therapeutic targets to preserve female fertility.

Macrophage activation drives ovarian failure and masculinization.

In humans, premature ovarian insufficiency (POI) is caused by autoimmunity and genetic factors, such as mutation of BMP15, a key ovarian determining gene. The cellular mechanisms associated with ovarian failure caused by BMP15 mutation and immune contributions to the disorder are not understood. BMP15's role in ovarian follicle development is conserved in vertebrates, including zebrafish. Using zebrafish, we established a causal link between macrophage activation and ovarian failure. We identified a germline-somatic gonadal cell-macrophage axis underlying ovarian atresia. Germline loss of Bmp15 triggers this axis that single-cell RNA sequencing and genetic analyses indicate involves activation of ovarian somatic cells that express conserved macrophage-activating ligands. Genetic ablation of macrophages blocks premature oocyte loss. Thus, the axis identified here represents potential therapeutic targets to preserve female fertility.

Sexual determination in zebrafish.

Zebrafish have emerged as a major model organism to study vertebrate reproduction due to their high fecundity and external development of eggs and embryos. The mechanisms through which zebrafish determine their sex have come under extensive investigation, as they lack a definite sex-determining chromosome and appear to have a highly complex method of sex determination. Single-gene mutagenesis has been employed to isolate the function of genes that determine zebrafish sex and regulate sex-specific differentiation, and to explore the interactions of genes that promote female or male sexual fate. In this review, we focus on recent advances in understanding of the mechanisms, including genetic and environmental factors, governing zebrafish sex development with comparisons to gene functions in other species to highlight conserved and potentially species-specific mechanisms for specifying and maintaining sexual fate.

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.

Zebrafish dazl regulates cystogenesis and germline stem cell specification during the primordial germ cell to germline stem cell transition.

Fertility and gamete reserves are maintained by asymmetric divisions of the germline stem cells to produce new stem cells or daughters that differentiate as gametes. Before entering meiosis, differentiating germ cells (GCs) of sexual animals typically undergo cystogenesis. This evolutionarily conserved process involves synchronous and incomplete mitotic divisions of a GC daughter (cystoblast) to generate sister cells connected by intercellular bridges that facilitate the exchange of materials to support rapid expansion of the gamete progenitor population. Here, we investigated cystogenesis in zebrafish and found that early GCs are connected by ring canals, and show that Deleted in azoospermia-like (Dazl), a conserved vertebrate RNA-binding protein (Rbp), is a regulator of this process. Analysis of dazl mutants revealed the essential role of Dazl in regulating incomplete cytokinesis, germline cyst formation and germline stem cell specification before the meiotic transition. Accordingly, dazl mutant GCs form defective ring canals, and ultimately remain as individual cells that fail to differentiate as meiocytes. In addition to promoting cystoblast divisions and meiotic entry, dazl is required for germline stem cell establishment and fertility.

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.

In Situ Fucosylation of the Wnt Co-receptor LRP6 Increases Its Endocytosis and Reduces Wnt/ß-Catenin Signaling.

Wnt/ß-catenin signaling regulates critical, context-dependent transcription in numerous physiological events. Among the well-documented mechanisms affecting Wnt/ß-catenin activity, modification of N-glycans by L-fucose is the newest and the least understood. Using a combination of Chinese hamster ovary cell mutants with different fucosylation levels and cell-surface fucose editing (in situ fucosylation [ISF]), we report that α(1-3)-fucosylation of N-acetylglucosamine (GlcNAc) in the Galß(1-4)-GlcNAc sequences of complex N-glycans modulates Wnt/ß-catenin activity by regulating the endocytosis of low-density lipoprotein receptor-related protein 6 (LRP6). Pulse-chase experiments reveal that ISF elevates endocytosis of lipid-raft-localized LRP6, leading to the suppression of Wnt/ß-catenin signaling. Remarkably, Wnt activity decreased by ISF is fully reversed by the exogenously added fucose. The combined data show that in situ cell-surface fucosylation can be exploited to regulate a specific signaling pathway via endocytosis promoted by a fucose-binding protein, thereby linking glycosylation of a receptor with its intracellular signaling.

Molecular genetics of maternally-controlled cell divisions.

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.

Setting up for gastrulation in zebrafish.

Soon after fertilization the zebrafish embryo generates the pool of cells that will give rise to the germline and the three somatic germ layers of the embryo (ectoderm, mesoderm and endoderm). As the basic body plan of the vertebrate embryo emerges, evolutionarily conserved developmental signaling pathways, including Bmp, Nodal, Wnt, and Fgf, direct the nearly totipotent cells of the early embryo to adopt gene expression profiles and patterns of cell behavior specific to their eventual fates. Several decades of molecular genetics research in zebrafish has yielded significant insight into the maternal and zygotic contributions and mechanisms that pattern this vertebrate embryo. This new understanding is the product of advances in genetic manipulations and imaging technologies that have allowed the field to probe the cellular, molecular and biophysical aspects underlying early patterning. The current state of the field indicates that patterning is governed by the integration of key signaling pathways and physical interactions between cells, rather than a patterning system in which distinct pathways are deployed to specify a particular cell fate. This chapter focuses on recent advances in our understanding of the genetic and molecular control of the events that impart cell identity and initiate the patterning of tissues that are prerequisites for or concurrent with movements of gastrulation.

Visualizing the Balbiani Body in Zebrafish Oocytes.

Approaches to visualize the Balbiani body of zebrafish primary oocytes using protein, RNA, and mitochondrial markers are described. The method involves isolation, histology, staining, and microscopic examination of early zebrafish oocytes. These techniques can be applied to visualize gene products that are localized to the Balbiani body, and when applied to mutants can be used to decipher molecular and genetic pathways acting in Balbiani body development in early oocytes.

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.

Recent advances in understanding oogenesis: interactions with the cytoskeleton, microtubule organization, and meiotic spindle assembly in oocytes.

Maternal control of development begins with production of the oocyte during oogenesis. All of the factors necessary to complete oocyte maturation, meiosis, fertilization, and early development are produced in the transcriptionally active early oocyte. Active transcription of the maternal genome is a mechanism to ensure that the oocyte and development of the early embryo begin with all of the factors needed for successful embryonic development. To achieve the maximum maternal store, only one functional cell is produced from the meiotic divisions that produce the oocyte. The oocyte receives the bulk of the maternal cytoplasm and thus is significantly larger than its sister cells, the tiny polar bodies, which receive a copy of the maternal genome but essentially none of the maternal cytoplasm. This asymmetric division is accomplished by an enormous cell that is depleted of centrosomes in early oogenesis; thus, meiotic divisions in oocytes are distinct from those of mitotic cells. Therefore, these cells must partition the chromosomes faithfully to ensure euploidy by using mechanisms that do not rely on a conventional centrosome-based mitotic spindle. Several mechanisms that contribute to assembly and maintenance of the meiotic spindle in oocytes have been identified; however, none is fully understood. In recent years, there have been many exciting and significant advances in oogenesis, contributed by studies using a myriad of systems. Regrettably, I cannot adequately cover all of the important advances here and so I apologize to those whose beautiful work has not been included. This review focuses on a few of the most recent studies, conducted by several groups, using invertebrate and vertebrate systems, that have provided mechanistic insight into how microtubule assembly and meiotic spindle morphogenesis are controlled in the absence of centrosomes.

G protein-coupled receptor gpr34l mutation affects thrombocyte function in zebrafish.

Haemostasis is a defence mechanism that has evolved to protect organisms from losing their circulating fluid. We have previously introduced zebrafish as a model to study the genetics of haemostasis to identify novel genes that play a role in haemostasis. Here, we identify a zebrafish mutant that showed prolonged time to occlusion (TTO) in the laser injury venous thrombosis assay. By linkage analysis and fine mapping, we found a mutation in the orphan G protein-coupled receptor 34 like gene (gpr34l) causing a change of Val to Glu in the third external loop of Gpr34l. We have shown that injection of zebrafish gpr34l RNA rescues the prolonged TTO defect. The thrombocytes from the mutant showed elevated levels of cAMP that supports the defective thrombocyte function. We also have demonstrated that knockdown of this gene by intravenous Vivo-Morpholino injections yielded a phenotype similar to the gpr34l mutation. These results suggest that the lack of functional Gpr34l leads to increased cAMP levels that result in defective thrombocyte aggregation.

Correction: Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis.

[This corrects the article DOI: 10.1371/journal.pgen.1006918.].

Acquisition of Oocyte Polarity.

Acquisition of oocyte polarity involves complex translocation and aggregation of intracellular organelles, RNAs, and proteins, along with strict posttranscriptional regulation. While much is still unknown regarding the formation of the animal-vegetal axis, an early marker of polarity, animal models have contributed to our understanding of these early processes controlling normal oogenesis and embryo development. In recent years, it has become clear that proteins with self-assembling properties are involved in assembling discrete subcellular compartments or domains underlying subcellular asymmetries in the early mitotic and meiotic cells of the female germline. These include asymmetries in duplication of the centrioles and formation of centrosomes and assembly of the organelle and RNA-rich Balbiani body, which plays a critical role in oocyte polarity. Notably, at specific stages of germline development, these transient structures in oocytes are temporally coincident and align with asymmetries in the position and arrangement of nuclear components, such as the nuclear pore and the chromosomal bouquet and the centrioles and cytoskeleton in the cytoplasm. Formation of these critical, transient structures and arrangements involves microtubule pathways, intrinsically disordered proteins (proteins with domains that tend to be fluid or lack a rigid ordered three-dimensional structure ranging from random coils, globular domains, to completely unstructured proteins), and translational repressors and activators. This review aims to examine recent literature and key players in oocyte polarity.