Dynamic assembly of brambleberry mediates nuclear envelope fusion during early development.

To 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.

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.

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.

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.

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.

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.

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

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

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

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

Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis.

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.

Split top: a maternal cathepsin B that regulates dorsoventral patterning and morphogenesis.

The vertebrate embryonic dorsoventral axis is established and patterned by Wnt and bone morphogenetic protein (BMP) signaling pathways, respectively. Whereas Wnt signaling establishes the dorsal side of the embryo and induces the dorsal organizer, a BMP signaling gradient patterns tissues along the dorsoventral axis. Early Wnt signaling is provided maternally, whereas BMP ligand expression in the zebrafish is zygotic, but regulated by maternal factors. Concomitant with BMP activity patterning dorsoventral axial tissues, the embryo also undergoes dramatic morphogenetic processes, including the cell movements of gastrulation, epiboly and dorsal convergence. Although the zygotic regulation of these cell migration processes is increasingly understood, far less is known of the maternal regulators of these processes. Similarly, the maternal regulation of dorsoventral patterning, and in particular the maternal control of ventral tissue specification, is poorly understood. We identified split top, a recessive maternal-effect zebrafish mutant that disrupts embryonic patterning upstream of endogenous BMP signaling. Embryos from split top mutant females exhibit a dorsalized embryonic axis, which can be rescued by BMP misexpression or by derepressing endogenous BMP signaling. In addition to dorsoventral patterning defects, split top mutants display morphogenesis defects that are both BMP dependent and independent. These morphogenesis defects include incomplete dorsal convergence, delayed epiboly progression and an early lysis phenotype during gastrula stages. The latter two morphogenesis defects are associated with disruption of the actin and microtubule cytoskeleton within the yolk cell and defects in the outer enveloping cell layer, which are both known mediators of epiboly movements. Through chromosomal mapping and RNA sequencing analysis, we identified the lysosomal endopeptidase cathepsin Ba (ctsba) as the gene deficient in split top embryos. Our results identify a novel role for Ctsba in morphogenesis and expand our understanding of the maternal regulation of dorsoventral patterning.

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.

Kinesin-1 interacts with Bucky ball to form germ cells and is required to pattern the zebrafish body axis.

In animals, specification of the primordial germ cells (PGCs), the stem cells of the germ line, is required to transmit genetic information from one generation to the next. Bucky ball (Buc) is essential for germ plasm (GP) assembly in oocytes, and its overexpression results in excess PGCs in zebrafish embryos. However, the mechanistic basis for the excess PGCs in response to Buc overexpression, and whether endogenous Buc functions during embryogenesis, are unknown. Here, we show that endogenous Buc, like GP and overexpressed Buc-GFP, accumulates at embryonic cleavage furrows. Furthermore, we show that the maternally expressed zebrafish Kinesin-1 Kif5Ba is a binding partner of Buc and that maternal kif5Ba (Mkif5Ba) plays an essential role in germline specification in vivo. Specifically, Mkif5Ba is required to recruit GP to cleavage furrows and thereby specifies PGCs. Moreover, Mkif5Ba is required to enrich Buc at cleavage furrows and for the ability of Buc to promote excess PGCs, providing mechanistic insight into how Buc functions to assemble embryonic GP. In addition, we show that Mkif5Ba is also essential for dorsoventral (DV) patterning. Specifically, Mkif5Ba promotes formation of the parallel vegetal microtubule array required to asymmetrically position dorsal determinants (DDs) towards the prospective dorsal side. Interestingly, whereas Syntabulin and wnt8a translocation depend on kif5Ba, grip2a translocation does not, providing evidence for two distinct mechanisms by which DDs might be asymmetrically distributed. These studies identify essential roles for maternal Kif5Ba in PGC specification and DV patterning, and provide mechanistic insight into Buc functions during early embryogenesis.

Dynamic visualization of transcription and RNA subcellular localization in zebrafish.

Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes--optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gateway-compatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the ßactin promoter at ∼ 3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3'UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes.

Dachsous1b cadherin regulates actin and microtubule cytoskeleton during early zebrafish embryogenesis.

Dachsous (Dchs), an atypical cadherin, is an evolutionarily conserved regulator of planar cell polarity, tissue size and cell adhesion. In humans, DCHS1 mutations cause pleiotropic Van Maldergem syndrome. Here, we report that mutations in zebrafish dchs1b and dchs2 disrupt several aspects of embryogenesis, including gastrulation. Unexpectedly, maternal zygotic (MZ) dchs1b mutants show defects in the earliest developmental stage, egg activation, including abnormal cortical granule exocytosis (CGE), cytoplasmic segregation, cleavages and maternal mRNA translocation, in transcriptionally quiescent embryos. Later, MZdchs1b mutants exhibit altered dorsal organizer and mesendodermal gene expression, due to impaired dorsal determinant transport and Nodal signaling. Mechanistically, MZdchs1b phenotypes can be explained in part by defective actin or microtubule networks, which appear bundled in mutants. Accordingly, disruption of actin cytoskeleton in wild-type embryos phenocopied MZdchs1b mutant defects in cytoplasmic segregation and CGE, whereas interfering with microtubules in wild-type embryos impaired dorsal organizer and mesodermal gene expression without perceptible earlier phenotypes. Moreover, the bundled microtubule phenotype was partially rescued by expressing either full-length Dchs1b or its intracellular domain, suggesting that Dchs1b affects microtubules and some developmental processes independent of its known ligand Fat. Our results indicate novel roles for vertebrate Dchs in actin and microtubule cytoskeleton regulation in the unanticipated context of the single-celled embryo.

Oocyte polarity requires a Bucky ball-dependent feedback amplification loop.

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.

Hecate/Grip2a acts to reorganize the cytoskeleton in the symmetry-breaking event of embryonic axis induction.

Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis.

Gpr125 modulates Dishevelled distribution and planar cell polarity signaling.

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.

The integrator complex subunit 6 (Ints6) confines the dorsal organizer in vertebrate embryogenesis.

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of ß-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.