Francisco Pelegri

Maternal factors in zebrafish development

All processes that occur before the activation of the zygotic genome at the midblastula transition are driven by maternal products, which are produced during oogenesis and stored in the mature oocyte. Upon egg activation and fertilization, these maternal factors initiate developmental cascades that carry out the embryonic developmental program. Even after the initiation of zygotic gene expression, perduring maternal products continue performing essential functions, either together with other maternal factors or through interactions with newly expressed zygotic products. Advances in zebrafish research have placed this organism in a unique position to contribute to a detailed understanding of the role of maternal factors in early vertebrate development. This review summarizes our knowledge on the processes involved in the production and redistribution of maternal factors during zebrafish oogenesis and early development, as well as our understanding of the function of these factors in axis formation, germ layer and germ cell specification, and other early embryonic processes. Developmental Dynamics, 2003.

A mutation in the zebrafish maternal-effect gene nebel affects furrow formation and vasa RNA localization

BACKGROUND In many animals, embryonic patterning depends on a careful interplay between cell division and the segregation of localized cellular components. Both of these processes in turn rely on cytoskeletal elements and motor proteins. A type of localized cellular component found in most animals is the germ plasm, a specialized region of cytoplasm that specifies the germ-cell fate. The gene vasa has been shown in Drosophila to encode an essential component of the germ plasm and is thought to have a similar function in other organisms. In the zebrafish embryo, the vasa RNA is localized to the furrows of the early cellular divisions. RESULTS We identified the gene nebel in a pilot screen for zebrafish maternal-effect mutations. Embryos from females homozygous for a mutation in nebel exhibit defects in cell adhesion. Our analysis provides genetic evidence for a function of the microtubule array that normally develops at the furrow in the deposition of adhesive membrane at the cleavage plane. In addition, nebel mutant embryos show defects in the early localization of vasa RNA. The vasa RNA localization phenotype could be mimicked with microtubule-inhibiting drugs, and confocal microscopy suggests an interaction between microtubules and vasa-RNA-containing aggregates. CONCLUSIONS Our data support two functions for the microtubule reorganization at the furrow, one for the exocytosis of adhesive membrane, and another for the translocation of vasa RNA along the forming furrow.

The maternal-effect gene futile cycle is essential for pronuclear congression and mitotic spindle assembly in the zebrafish zygote

Embryos have been successfully used for the general study of the cell cycle. Although there are significant differences between the early embryonic and the somatic cell cycle in vertebrates, the existence of specialised factors that play a role during the early cell cycles has remained elusive. We analysed a lethal recessive maternal-effect mutant, futile cycle (fue), isolated in a maternal-effect screen for nuclear division defects in the zebrafish (Danio rerio). The pronuclei fail to congress in zygotes derived from homozygous fue mothers. In addition, a defect in the formation of chromosomal microtubules prevents mitotic spindle assembly and thus chromosome segregation in fue zygotes. However, centrosomal functions do not appear to be affected in fue embryos, suggesting this mutant blocks a subset of microtubule functions. Cleavage occurs normally for several divisions resulting in many anucleate cells, thus showing that nuclear- and cell division can be uncoupled genetically. Therefore, we propose that in mitotic spindle assembly chromosome-dependent microtubule nucleation is essential for the coupling of nuclear and cell division.

Vertebrate maternal-effect genes: Insights into fertilization, early cleavage divisions, and germ cell determinant localization from studies in the zebrafish

In the earliest stages of animal development prior to the commencement of zygotic transcription, all critical cellular processes are carried out by maternally‐provided molecular products accumulated in the egg during oogenesis. Disruption of these maternal products can lead to defective embryogenesis. In this review, we focus on maternal genes with roles in the fundamental processes of fertilization, cell division, centrosome regulation, and germ cell development with emphasis on findings from the zebrafish, as this is a unique and valuable model system for vertebrate reproduction. Mol. Reprod. Dev. 77: 299–313, 2010.

The maternal-effect gene cellular island encodes aurora B kinase and is essential for furrow formation in the early zebrafish embryo.

Females homozygous for a mutation in cellular island (cei) produce embryos with defects in cytokinesis during early development. Analysis of the cytoskeletal events associated with furrow formation reveal that these defects include a general delay in furrow initiation as well as a complete failure to form furrow-associated structures in distal regions of the blastodisc. A linkage mapping-based candidate gene approach, including transgenic rescue, shows that cei encodes the zebrafish Aurora B kinase homologue. Genetic complementation analysis between the cei mutation and aurB zygotic lethal mutations corroborate gene assignment and reveal a complex nature of the maternal-effect cei allele, which appears to preferentially affect a function important for cytokinesis in the early blastomeres. Surprisingly, in cei mutant embryos a short yet otherwise normal furrow forms in the center of the blastodisc. Furrow formation is absent throughout the width of the blastodisc in cei mutant embryos additionally mutant for futile cycle, which lack a spindle apparatus, showing that the residual furrow signal present in cei mutants is derived from the mitotic spindle. Our analysis suggests that partially redundant signals derived from the spindle and astral apparatus mediate furrow formation in medial and distal regions of the early embryonic blastomeres, respectively, possibly as a spatial specialization to achieve furrow formation in these large cells. In addition, our data also suggest a role for Cei/AurB function in the reorganization of the furrow-associated microtubules in both early cleavage- and somite-stage embryos. In accordance with the requirement for cei/aurB in furrow induction in the early cleavage embryo, germ plasm recruitment to the forming furrow is also affected in embryos lacking normal cei/aurB function.

A role for non-muscle myosin II function in furrow maturation in the early zebrafish embryo

Cytokinesis in early zebrafish embryos involves coordinated changes in the f-actin- and microtubule-based cytoskeleton, and the recruitment of adhesion junction components to the furrow. We show that exposure to inhibitors of non-muscle myosin II function does not affect furrow ingression during the early cleavage cycles but interferes with the recruitment of pericleavage f-actin and cortical β-catenin aggregates to the furrow, as well as the remodeling of the furrow microtubule array. This remodeling is in turn required for the distal aggregation of the zebrafish germ plasm. Embryos with reduced myosin activity also exhibit at late stages of cytokinesis a stabilized contractile ring apparatus that appears as a ladder-like pattern of short f-actin cables, supporting a role for myosin function in the disassembly of the contractile ring after furrow formation. Our studies support a role for myosin function in furrow maturation that is independent of furrow ingression and which is essential for the recruitment of furrow components and the remodeling of the cytoskeleton during cytokinesis.

Identification of recessive maternal-effect mutations in the zebrafish using a gynogenesis-based method.

In animal species, early developmental processes are driven by maternally derived factors. Here, we describe a forward genetics approach to identify recessive mutations in genes encoding such maternal factors in the zebrafish. We used a gynogenesis‐based approach to identify 14 recessive maternal‐effect mutations. Homozygosity for these mutations in adult females leads to the inviability of their offspring. Confocal microscopy of embryos labeled with a DNA dye and a membrane marker allowed us to further analyze mutant embryos for defects in nuclear and cellular divisions. The mutations result in a range of defects in early developmental processes, including egg activation, early nuclear events, mitosis, cytokinesis, axial patterning, and gastrulation. Our effort constitutes a systematic attempt to identify maternal‐effect genes in a vertebrate species. The sample of mutations that we have identified reflects the diversity of maternally driven functions in early development and underscores the importance of maternal factors in this process. Developmental Dynamics 231:324–335, 2004.

Localized products of futile cycle/lrmp promote centrosome-nucleus attachment in the zebrafish zygote

BACKGROUND The centrosome has a well-established role as a microtubule organizer during mitosis and cytokinesis. In addition, it facilitates the union of parental haploid genomes following fertilization by nucleating a microtubule aster along which the female pronucleus migrates toward the male pronucleus. Stable associations between the sperm aster and the pronuclei are essential during this directed movement. RESULTS Our studies reveal that the zebrafish gene futile cycle (fue) is required in the zygote for male pronucleus-centrosome attachment and female pronuclear migration. We show that fue encodes a novel, maternally-provided long form of lymphoid-restricted membrane protein (lrmp), a vertebrate-specific gene of unknown function. Both maternal lrmp messenger RNA (mRNA) and protein are highly localized in the zygote, in a largely overlapping pattern at nuclear membranes, centrosomes, and spindles. Truncated Lrmp-EGFP fusion proteins identified subcellular targeting signals in the C terminus of Lrmp; however, endogenous mRNA localization is likely important to ensure strict spatial expression of the protein. Localization of both Lrmp protein and lrmp RNA is defective in fue mutant embryos, indicating that correct targeting of lrmp gene products is dependent on Lrmp function. CONCLUSIONS Lrmp is a conserved vertebrate gene whose maternally inherited products are essential for nucleus-centrosome attachment and pronuclear congression during fertilization. Precise subcellular localization of lrmp products also suggests a requirement for strict spatiotemporal regulation of their function in the early embryo.

The Chromosomal Passenger Protein Birc5b Organizes Microfilaments and Germ Plasm in the Zebrafish Embryo

Microtubule-microfilament interactions are important for cytokinesis and subcellular localization of proteins and mRNAs. In the early zebrafish embryo, astral microtubule-microfilament interactions also facilitate a stereotypic segregation pattern of germ plasm ribonucleoparticles (GP RNPs), which is critical for their eventual selective inheritance by germ cells. The precise mechanisms and molecular mediators for both cytoskeletal interactions and GP RNPs segregation are the focus of intense research. Here, we report the molecular identification of a zebrafish maternal-effect mutation motley as Birc5b, a homolog of the mammalian Chromosomal Passenger Complex (CPC) component Survivin. The meiosis and mitosis defects in motley/birc5b mutant embryos are consistent with failed CPC function, and additional defects in astral microtubule remodeling contribute to failures in the initiation of cytokinesis furrow ingression. Unexpectedly, the motley/birc5b mutation also disrupts cortical microfilaments and GP RNP aggregation during early cell divisions. Birc5b localizes to the tips of astral microtubules along with polymerizing cortical F-actin and the GP RNPs. Mutant Birc5b co-localizes with cortical F-actin and GP RNPs, but fails to associate with astral microtubule tips, leading to disorganized microfilaments and GP RNP aggregation defects. Thus, maternal Birc5b localizes to astral microtubule tips and associates with cortical F-actin and GP RNPs, potentially linking the two cytoskeletons to mediate microtubule-microfilament reorganization and GP RNP aggregation during early embryonic cell cycles in zebrafish. In addition to the known mitotic function of CPC components, our analyses reveal a non-canonical role for an evolutionarily conserved CPC protein in microfilament reorganization and germ plasm aggregation.

Bone Morphogenetic Protein 1 Prodomain Specifically Binds and Regulates Signaling by Bone Morphogenetic Proteins 2 and 4

Highly purified fractions of bone extracts capable of inducing ectopic bone formation have been reported to contain peptides corresponding to the mature active regions of the TGF-β-like bone morphogenetic proteins (BMPs) 2–7, and to the prodomain region of the metalloproteinase BMP1. Co-purification of BMPs 2–7 with BMP1 prodomain sequences through the multiple biochemical steps used in these previous reports has suggested the possibility of interactions between the BMP1 prodomain and BMPs 2–7. Here we demonstrate that the BMP1 prodomain binds BMPs 2 and 4 with high specificity and with a KD of ∼11 nm, in the physiological range. It is further demonstrated that the BMP1 prodomain is capable of modulating signaling by BMPs 2 and 4 in vitro and in vivo, and that endogenous BMP1 prodomain-BMP4 complexes exist in cell culture media and in tissues.