Search

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.One-Sentence SummarySex reversal due to Bmp15 deficiency requires macrophage activation by Csf1a, which is expressed by specialized pre-follicle cells in zebrafish.

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.

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.

Maternally provided gene products regulate the earliest events of embryonic life, including formation of the oocyte that will develop into an egg, and eventually an embryo. Forward genetic screens have provided invaluable insights into the molecular regulation of embryonic development, including essential contributions of some genes whose products must be provided to the transcriptionally silent early embryo for normal embryogenesis, 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 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 reproductive and maternal-effect processes, as proof of principal and to assess the efficiency and robustness of mutagenesis we used these transgenic systems to disrupt two genes with known maternal-effect functions,kif5Baandbucky ball.Summary StatementWe report reverse genetic systems to identify essential regulators of reproductive and maternal-effect processes, as proof of principal we used these transgenic systems to disrupt genes with known maternal-effect functions.

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.

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 begins with establishment of a bipotential gonad 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 evidence that the critical female factor, rbpms2 is epistatic to the male factor dmrt1 in terms of adult sex. Moreover, Rbpms2’s role 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 cyp19a1a in the absence of dmrt1. Taken together this work indicates that Cyp19a1a-mediated suppression of Dmrt1 is key to establish a bipotential gonad and initiate female fate acquisition, possibly by promoting rbpms2. Then, after female fate specification, Cyp19a1a regulates subsequent oocyte maturation and sustains female fates independent of Dmrt1 repression.Author SummaryWe show that cyp19a1a-mediated suppression of dmrt1 establishes a bipotential gonad and female fate acquisition, possibly through rbpms2 which is required for female fates, even in the absence of Dmrt1.

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.

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.

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.

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 evolutionary conserved process involves synchronous and incomplete mitotic divisions of a germ cell daughter (cystoblast) to generate sister cells connected by stable intercellular bridges that facilitate exchange of materials to support a large synchronous population of gamete progenitors. Here we investigate cystogenesis in zebrafish and identified Deleted in azoospermia (Dazl), a conserved vertebrate RNA binding protein as a regulator of this process. Analysis ofdazlmutants revealed an essential role for Dazl in regulating incomplete cytokinesis and germline cyst formation before the meiotic transition. Accordingly,dazlmutant 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,dazlfunction is required upstream of germline stem cell establishment and fertility.Summary StatementWe show that zebrafishdazlis required for incomplete cytokinesis to generate germline cysts during cystogenesis, acts upstream of germline stem cell establishment, and is required for meiosis, and fertility.

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.

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.

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., Author summary The earliest stages of animal development are regulated by factors in the egg that are made during oogenesis and are required for the embryo to develop prior to genome activation of the embryo itself. Because eggs are large, the cells of the early embryo are large and so unique mechanisms act during these stages of development. To study these molecular-genetic processes in a vertebrate, we have chemically induced mutations in the zebrafish germline and screened for mutant mothers (maternal-effect mutants) with defects in these processes. We identified three distinct classes of mutations affecting cell division in the early embryo. One of these mutations also exhibits a male-sterile phenotype. We identify the mutated genes in three of these mutants. We expect the remaining mutant lines will serve as important tools for elucidating molecular mechanisms involved in cell organization and/or positioning during the cleavage stage, as well as mechanisms critical for proper patterning of the early embryo.

Vasa is a universal marker of the germ line in animals, yet mutations disrupting vasa cause sexually dimorphic infertility, with impaired development of the ovary in some animals and the testis in others. The basis for this sexually dimorphic requirement for Vasa is not clear; in most animals examined, both the male and female gonad express vasa throughout the life of the germ line. Here we characterized a loss-of-function mutation disrupting zebrafish vasa. We show that maternally provided Vasa is stable through the first ten days of development in zebrafish, and thus likely fulfill any early roles for Vasa during germ-line specification, migration, survival, and maintenance. Although zygotic Vasa is not essential for the development of juvenile gonads, vasa mutants develop exclusively as sterile males. Furthermore, phenotypes of vasa;p53 compound mutants are indistinguishable from those of vasa mutants, therefore the failure of vasa mutants to differentiate as females and to support germ-cell development in the testis is not due to p53-mediated apoptosis. Instead, we found that failure to progress beyond the pachytene stage of meiosis causes the loss of germ-line stem cells, leaving empty somatic tubules. Our studies provide insight into the function of zebrafish vasa during female meiosis, differentiation, and maintenance of germ-line stem cells.

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

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the authors.The first author, a graduate student, has admitted orally and in writing to fabrication of data that she contributed to this article in which we reported a maternal requirement for the scaffold protein Dazap2 (Mdazap2) in germ granule maintenance by a mechanism that is epistatic to Tdrd7 and that counteracts Dynein activity. Furthermore, we reported that Dazap2 binds to Bucky ball, an essential regulator of oocyte polarity and germ plasm assembly, and colocalizes with the germ plasm in oocytes and in primordial germ cells. Although the protein interactions, expression, and localization results are valid, the first author has admitted orally and in writing to fabrication of all “mutant data” in Figures 1H and 1J, Figure 2, Figure 3, Figure 4, and Figures S2 and S3. Because the data presented in those figures do not reflect the standards of quality that are expected in science, and in order to protect the integrity of science, our laboratories, and institutes, we are retracting the paper. All authors agree with retraction of the paper.

During skeletal morphogenesis diverse mechanisms are used to support bone formation. This can be seen in the bones that require a cartilage template for their development. In mammals the cartilage template is removed, but in zebrafish the cartilage template persists and the bone mineralizes around the cartilage scaffold. Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements; however, the mechanisms that maintain the chondrocyte template that supports perichondral ossification remain unclear. We report double mutants disrupting two zebrafish kinesin-I genes (hereafter kif5Blof) that we generated using CRISPR/Cas9 mutagenesis. We show that zygotic Kif5Bs have a conserved function in maintaining muscle integrity, and are required for cartilage remodeling and maintenance during craniofacial morphogenesis by a PCP-distinct mechanism. Further, kif5Blof does not activate ER stress response genes, but instead disrupts lysosomal function, matrix secretion, and causes deregulated autophagic markers and eventual chondrocyte apoptosis. Ultrastructural and transplantation analysis reveal neighboring cells engulfing extruded kif5Blof chondrocytes. Initial cartilage specification is intact; however, during remodeling, kif5Blof chondrocytes die and the cartilage matrix devoid of hypertrophic chondrocytes remains and impedes normal ossification. Chimeric and mosaic analyses indicate that Kif5B functions cell-autonomously in secretion, nuclear position, cell elongation and maintenance of hypertrophic chondrocytes. Interestingly, large groups of wild-type cells can support elongation of neighboring mutant cells. Finally, mosaic expression of kif5Ba, but not kif5Aa in cartilage rescues the chondrocyte phenotype, further supporting a specific requirement for Kif5B. Cumulatively, we show essential Kif5B functions in promoting cartilage remodeling and chondrocyte maintenance during zebrafish craniofacial morphogenesis., Author summary During skeletal morphogenesis diverse mechanisms are used to support bone formation, for example some bones require a cartilage template. In mammals the cartilage template is removed, but in zebrafish the cartilage template persists and the bone mineralizes around the cartilage scaffold. Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements. We identified a conserved role for the Kinesin-1 heavy chain, kif5B, in maintaining muscle integrity and a novel role in cartilage development during craniofacial morphogenesis. In chondrogenesis Kif5B acts by a distinct mechanism from PCP signaling and does not activate the ER stress response genes, but instead involves regulation of lysosomal function, matrix secretion, and autophagy. When kif5B is lost, autophagic markers are deregulated leading to eventual chondrocyte apoptosis. Chimeric and mosaic analyses indicate that Kif5B functions cell-autonomously in secretion, nuclear position, cell elongation and maintenance. Interestingly, large groups of wild-type cells, likely via their matrix, support elongation of neighboring mutant cells. Cumulatively, our study reveals Kif5B’s essential role in promoting cartilage remodeling and chondrocyte maintenance during craniofacial morphogenesis.

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.

During skeletal morphogenesis diverse mechanisms are used to support bone formation. This can be seen in the bones that require a cartilage template for their development. In mammals the cartilage template is removed, but in zebrafish the cartilage template persists and the bone mineralizes around the cartilage scaffold. Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements; however, the mechanisms that maintain the chondrocyte template that supports perichondral ossification remain unclear. We report double mutants disrupting two zebrafish kinesin-I genes (hereafter kif5Blof) that we generated using CRISPR/Cas9 mutagenesis. We show that zygotic Kif5Bs have a conserved function in maintaining muscle integrity, and are required for cartilage remodeling and maintenance during craniofacial morphogenesis by a PCP-distinct mechanism. Further, kif5Blof does not activate ER stress response genes, but instead disrupts lysosomal function, matrix secretion, and causes deregulated autophagic markers and eventual chondrocyte apoptosis. Ultrastructural and transplantation analysis reveal neighboring cells engulfing extruded kif5Blof chondrocytes. Initial cartilage specification is intact; however, during remodeling, kif5Blof chondrocytes die and the cartilage matrix devoid of hypertrophic chondrocytes remains and impedes normal ossification. Chimeric and mosaic analyses indicate that Kif5B functions cell-autonomously in secretion, nuclear position, cell elongation and maintenance of hypertrophic chondrocytes. Interestingly, large groups of wild-type cells can support elongation of neighboring mutant cells. Finally, mosaic expression of kif5Ba, but not kif5Aa in cartilage rescues the chondrocyte phenotype, further supporting a specific requirement for Kif5B. Cumulatively, we show essential Kif5B functions in promoting cartilage remodeling and chondrocyte maintenance during zebrafish craniofacial morphogenesis.

Mitochondrial replacement therapy, a procedure to generate embryos with the nuclear genome of a donor mother and the healthy mitochondria of a recipient egg, has recently emerged as a promising strategy to prevent transmission of devastating mitochondrial DNA diseases and infertility. The procedure may produce an embryo that is free of diseased mitochondria. A recent study addresses important fundamental questions about the mechanisms underlying maternal inheritance and translational questions regarding the transgenerational effectiveness of this promising therapeutic strategy. This review considers recent advances in our understanding of maternal inheritance of mitochondria, implications for fertility and mitochondrial disease, and potential roles for the Balbiani body, an ancient oocyte structure, in mitochondrial selection in oocytes, with emphasis on therapies to remedy mitochondrial disorders.

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.

L-fucose, a monosaccharide widely distributed in eukaryotes and certain bacteria, is a determinant of many functional glycans that play central roles in numerous biological processes. The molecular mechanism, however, by which fucosylation mediates these processes remains largely elusive. To study how changes in fucosylation impact embryonic development, we up-regulated N-linked fucosylation via over-expression of a key GDP-Fucose transporter, Slc35c1, in zebrafish. We show that Slc35c1 overexpression causes elevated N-linked fucosylation and disrupts embryonic patterning in a transporter activity dependent manner. We demonstrate that patterning defects associated with enhanced N-linked fucosylation are due to diminished canonical Wnt signaling. Chimeric analyses demonstrate that elevated Slc35c1 expression in receiving cells decreases the signaling range of Wnt8a during zebrafish embryogenesis. Moreover, we provide biochemical evidence that this decrease is associated with reduced Wnt8 ligand and elevated Lrp6 coreceptor, which we show are both substrates for N-linked fucosylation in zebrafish embryos. Strikingly, slc35c1 expression is regulated by canonical Wnt signaling. These results suggest that Wnt limits its own signaling activity in part via up-regulation of a transporter, slc35c1 that promotes terminal fucosylation and thereby limits Wnt activity.

Mutations in Kinesin proteins (Kifs) are linked to various neurological diseases, but the specific and redundant functions of the vertebrate Kifs are incompletely understood. For example, Kif5A, but not other Kinesin-1 heavy-chain family members, is implicated in Charcot-Marie-Tooth disease (CMT) and Hereditary Spastic Paraplegia (HSP), but the mechanism of its involvement in the progressive axonal degeneration characteristic of these diseases is not well understood. We report that zebrafishkif5Aamutants exhibit hyperexcitability, peripheral polyneuropathy, and axonal degeneration reminiscent of CMT and HSP. Strikingly, althoughkif5genes are thought to act largely redundantly in other contexts, and zebrafish peripheral neurons express fivekif5genes,kif5Aamutant peripheral sensory axons lack mitochondria and degenerate. We show that this Kif5Aa-specific function is cell autonomous and is mediated by its C-terminal tail, as only Kif5Aa and chimeric motors containing the Kif5Aa C-tail can rescue deficits. Finally, concurrent loss of thekinesin-3,kif1b, or its adaptorkbp, exacerbates axonal degeneration via a nonmitochondrial cargo common to Kif5Aa. Our results shed light on Kinesin complexity and reveal determinants of specific Kif5A functions in mitochondrial transport, adaptor binding, and axonal maintenance.

Summary Vasa is a universal marker of the germ line in animals, yet mutations disrupting vasa cause sexually dimorphic infertility, with impaired development of the ovary in some animals and the testis in others. The basis for this sexually dimorphic requirement for Vasa is not clear; in most animals examined, both the male and female gonad express vasa throughout the life of the germ line. Here we characterized a loss-of-function mutation disrupting zebrafish vasa. We show that maternally provided Vasa is stable through the first ten days of development in zebrafish, and thus likely fulfills any early roles for Vasa during germ-line specification, migration, survival, and maintenance. Although zygotic Vasa is not essential for the development of juvenile gonads, vasa mutants develop exclusively as sterile males. Furthermore, phenotypes of vasa;p53 compound mutants are indistinguishable from those of vasa mutants, therefore the failure of vasa mutants to differentiate as females and to support germ-cell development in the testis is not due to p53-mediated apoptosis. Instead, we found that failure to progress beyond the pachytene stage of meiosis causes the loss of germ-line stem cells, leaving empty somatic tubules. Our studies provide insight into the function of zebrafish vasa during female meiosis, differentiation, and maintenance of germ-line stem cells. Mol. Reprod. Dev. 81: 946–961, 2014. © 2014 Wiley Periodicals, Inc.

To monitor the kinetics of biological processes that take place within the minute time scale, simple and fast analytical methods are required. In this article, we present our discovery of an azide with an internal Cu(I)-chelating motif that enabled the development of the fastest protocol for Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to date, and its application toward following the dynamic process of glycan biosynthesis. We discovered that an electron-donating picolyl azide boosted the efficiency of the ligand-accelerated CuAAC 20-38-fold in living systems with no apparent toxicity. With a combination of this azide and BTTPS, a tris(triazolylmethyl)amine-based ligand for Cu(I), we were able to detect newly synthesized cell-surface glycans by flow cytometry using as low as 1 nM of a metabolic precursor. This supersensitive chemistry enabled us to monitor the dynamic glycan biosynthesis in mammalian cells and in early zebrafish embryogenesis. In live mammalian cells, we discovered that it takes approximately 30-45 min for a monosaccharide building block to be metabolized and incorporated into cell-surface glycoconjugates. In zebrafish embryos, the labeled glycans could be detected as early as the two-cell stage. To our knowledge, this was the first time that newly synthesized glycans were detected at the cleavage period (0.75-2 hpf) in an animal model using bioorthogonal chemistry.

In vertebrates, the first asymmetries are established along the animal-vegetal axis during oogenesis, but the underlying molecular mechanisms are poorly understood. Bucky ball (Buc) was identified in zebrafish as a novel vertebrate-specific regulator of oocyte polarity, acting through unknown molecular interactions. Here we show that endogenous Buc protein localizes to the Balbiani body, a conserved, asymmetric structure in oocytes that requires Buc for its formation. Asymmetric distribution of Buc in oocytes precedes Balbiani body formation, defining Buc as the earliest marker of oocyte polarity in zebrafish. Through a transgenic strategy, we determined that excess Buc disrupts polarity and results in supernumerary Balbiani bodies in a 3′UTR-dependent manner, and we identified roles for the buc introns in regulating Buc activity. Analyses of mosaic ovaries indicate that oocyte pattern determines the number of animal pole-specific micropylar cells that are associated with an egg via a close-range signal or direct cell contact. We demonstrate interactions between Buc protein and buc mRNA with two conserved RNA-binding proteins (RNAbps) that are localized to the Balbiani body: RNA binding protein with multiple splice isoforms 2 (Rbpms2) and Deleted in azoospermia-like (Dazl). Buc protein and buc mRNA interact with Rbpms2; buc and dazl mRNAs interact with Dazl protein. Cumulatively, these studies indicate that oocyte polarization depends on tight regulation of buc: Buc establishes oocyte polarity through interactions with RNAbps, initiating a feedback amplification mechanism in which Buc protein recruits RNAbps that in turn recruit buc and other RNAs to the Balbiani body.

SummaryMutations in the human NOTCH3 gene cause CADASIL syndrome (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). CADASIL is an inherited small vessel disease characterized by diverse clinical manifestations including vasculopathy, neurodegeneration and dementia. Here we report two mutations in the zebrafish notch3 gene, one identified in a previous screen for mutations with reduced expression of myelin basic protein (mbp) and another caused by a retroviral insertion. Reduced mbp expression in notch3 mutant embryos is associated with fewer oligodendrocyte precursor cells (OPCs). Despite an early neurogenic phenotype, mbp expression recovered at later developmental stages and some notch3 homozygous mutants survived to adulthood. These mutants, as well as adult zebrafish carrying both mutant alleles together, displayed a striking stress-associated accumulation of blood in the head and fins. Histological analysis of mutant vessels revealed vasculopathy, including: an enlargement (dilation) of vessels in the telencephalon and fin, disorganization of the normal stereotyped arrangement of vessels in the fin, and an apparent loss of arterial morphological structure. Expression of hey1, a well-known transcriptional target of Notch signaling, was greatly reduced in notch3 mutant fins, suggesting that Notch3 acts via a canonical Notch signaling pathway to promote normal vessel structure. Ultrastructural analysis confirmed the presence of dilated vessels in notch3 mutant fins and revealed that the vessel walls of presumed arteries showed signs of deterioration. Gaps evident in the arterial wall and the presence of blood cells outside of vessels in mutants indicated that compromised vessel structure led to hemorrhage. In notch3 heterozygotes, we found elevated expression of both notch3 itself and target genes, indicating that specific alterations in gene expression caused by a partial loss of Notch3 function might contribute to the abnormalities observed in heterozygous larvae and adults. Our analysis of zebrafish notch3 mutants indicates that Notch3 regulates OPC development and mbp gene expression in larvae, and maintains vascular integrity in adults.

During vertebrate gastrulation, Wnt/planar cell polarity (PCP) signaling orchestrates polarized cell behaviors underlying convergence and extension (C&E) movements to narrow embryonic tissues mediolaterally and lengthen them anteroposteriorly. Here, we have identified Gpr125, an adhesion G protein-coupled receptor, as a novel modulator of the Wnt/PCP signaling system. Excess Gpr125 impaired C&E movements and the underlying cell and molecular polarities. Reduced Gpr125 function exacerbated the C&E and facial branchiomotor neuron (FBMN) migration defects of embryos with reduced Wnt/PCP signaling. At the molecular level, Gpr125 recruited Dishevelled to the cell membrane, a prerequisite for Wnt/PCP activation. Moreover, Gpr125 and Dvl mutually clustered one another to form discrete membrane subdomains, and the Gpr125 intracellular domain directly interacted with Dvl in pull-down assays. Intriguingly, Dvl and Gpr125 were able to recruit a subset of PCP components into membrane subdomains, suggesting that Gpr125 may modulate the composition of Wnt/PCP membrane complexes. Our study reveals a role for Gpr125 in PCP-mediated processes and provides mechanistic insight into Wnt/PCP signaling.

Fmlns belong to the Formin family, catalysts of linear actin polymerization with mostly unknown roles in vivo. In cell culture Fmnls are involved in cell migration and adhesion and the formation of different types of protrusions including filopodia and blebs, suggesting important roles during development. Moreover, Fmnls can act downstream of Rac and Cdc42, mediators of cytoskeletal changes as targets of important pathways required for shaping tissues. The zebrafish genome encodes five Fmnls. Here we report their tissue specific expression patterns during early development and pharyngula stages. The fmnls show overlapping and distinct expression patterns, which suggest that they could regulate similar processes during development, but may also have independent functions. In particular, we find a strong maternal contribution of all fmnls, but distinct expression patterns in the developing brain eye, ear, heart and vascular system.

(Cell Reports 12, 49–57; July 7, 2015)The first author, a graduate student, has admitted orally and in writing to fabrication of data that she contributed to this article in which we reported a maternal requirement for the scaffold protein Dazap2 (Mdazap2) in germ granule maintenance by a mechanism that is epistatic to Tdrd7 and that counteracts Dynein activity. Furthermore, we reported that Dazap2 binds to Bucky ball, an essential regulator of oocyte polarity and germ plasm assembly, and colocalizes with the germ plasm in oocytes and in primordial germ cells. Although the protein interactions, expression, and localization results are valid, the first author has admitted orally and in writing to fabrication of all “mutant data” in Figures 1H and 1J, Figure 2, Figure 3, Figure 4, and Figures S2 and S3. Because the data presented in those figures do not reflect the standards of quality that are expected in science, and in order to protect the integrity of science, our laboratories, and institutes, we are retracting the paper. All authors agree with retraction of the paper.

SummaryTo accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, mitotic intermediates wherein individual chromatin masses are surrounded by nuclear envelope; the karyomeres then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion, resulting in formation of multiple micronuclei. As karyomeres form, Brambleberry protein localizes to the nuclear envelope, with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. brambleberry corresponds to an unannotated gene with similarity to Kar5p, a protein that participates in nuclear fusion in yeast. We also demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. Our studies provide insight into the machinery required for karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres.

Retraction of: Development doi: [10.1242/dev.129023][1]. The corresponding author of this article contacted the journal shortly after the authors' accepted version of the manuscript was posted online, regarding potential issues with a number of the figures in this paper. Specifically, the first

Raising the bar: the efficacy of bioorthogonal reactions for bioconjugation has been thoroughly evaluated in four different biological settings. Powered by the development of new biocompatible ligands, the copper-catalyzed azide-alkyne cycloaddition has brought about unsurpassed bioconjugation efficiency, and thus it holds great promise as a highly potent and adaptive tool for a broader spectrum of biological applications.

The glycome, the totality of glycans produced by a cell, is a dynamic indicator of the cell's physiology.[1] Changes in the glycome reflect a cell's developmental stage and the transformation state of a cell. Recently, imaging glycans in vivo has been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharide precursors.[2, 3] The modified monosaccharides, when taken up by cells, are activated in the cytoplasm to form nucleotide sugars, substrates of glycosyltransferases that generate complex glycans in the endoplasmic reticulum and Golgi. Once incorporated into cell surface glycoconjugates, the bioorthogonal chemical tags allow covalent conjugation with fluorescent probes for visualization,[2] or with affinity probes for enrichment and glycomic analysis.[4] This approach has been successfully used for the detection and imaging of mucin O-linked glycans,[2] sialylated[2] and fucosylated glycans,[5] and cytosolic O-GlcNAcylated proteins.[2] However, only monosaccharides are tracked by this strategy, and each monosaccharide is usually found on a plethora of glycans.[6] Higher order glycans, i.e. disaccharides or trisaccharides, of specific composition cannot be uniquely labeled by hijacking their biosynthetic pathways with unnatural monosaccharides (Figure 1a). Here we report a rapid and highly specific chemoenzymatic method for labeling cell surface glycans bearing a ubiquitous disaccharide—N-acetyllactosamine (LacNAc, Galβ1,4GlcNAc)—with biophysical probes for imaging or glycomic analysis.

The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the standard method for bioorthogonal conjugation. However, current Cu(I) catalyst formulations are toxic, hindering their use in living systems. Here we report that BTTES, a tris(triazolylmethyl)amine-based ligand for Cu(I), promotes the cycloaddition reaction rapidly in living systems without apparent toxicity. This catalyst allows, for the first time, noninvasive imaging of fucosylated glycans during zebrafish early embryogenesis. We microinjected embryos with alkyne-bearing GDP-fucose at the one-cell stage and detected the metabolically incorporated unnatural sugars using the biocompatible click chemistry. Labeled glycans could be imaged in the enveloping layer of zebrafish embryos between blastula and early larval stages. This new method paves the way for rapid, noninvasive imaging of biomolecules in living organisms.

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., Author summary Oocyte development relies on posttranscriptional regulation by RNA binding proteins (RNAbps). RNAbps form large multi-molecular structures called RNPs (ribonucleoproteins) that further aggregate into regulatory granules within germ cells. In zebrafish primary oocytes, a large transient RNP aggregate called the Balbiani body (Bb) is essential for localizing patterning molecules and germline determinants within oocytes. RNA-binding protein of multiple splice forms 2, or Rbpms2, localizes to germ granules and the Bb, and interacts with bucky ball, a key factor for Bb formation. We show that Rbpms2 requires RNA binding for localization within germ cells, and that the C-term and RRM contribute to Rbpms2 subcellular localization in distinct somatic cell types. To investigate Rbpms2 functions we mutated the duplicated zebrafish rbpms2 genes. Consistent with redundant functions, rbpms2a and rbpms2b gene expression overlaps, and single mutants have no discernible phenotypes. Although rbpms2a;2b double mutants have cardiac phenotypes, those that reach adulthood are exclusively fertile males. Genetic analysis shows that rbpms2 mutant oocytes are not maintained even when Tp53, a regulator of cell death is absent. Initial oocyte polarity is established in rbpms2 mutants based on asymmetric distribution of Buc protein and mitochondria; however, abnormal Buc structures and atypical cytoplasmic inclusions form. This work reveals independent Rbpms2 functions in promoting Bb integrity, and as a novel regulator of ovary fate.