The midbody component Prc1-like is required for microtubule reorganization during cytokinesis and dorsal determinant segregation in the early zebrafish embryo.

We show that the zebrafish maternal-effect mutation too much information (tmi) corresponds to zebrafish prc1-like (prc1l), which encodes a member of the MAP65/Ase1/PRC1 family of microtubule-associated proteins. Embryos from tmi homozygous mutant mothers display cytokinesis defects in meiotic and mitotic divisions in the early embryo, indicating that Prc1l has a role in midbody formation during cell division at the egg-to-embryo transition. Unexpectedly, maternal Prc1l function is also essential for the reorganization of vegetal pole microtubules required for the segregation of dorsal determinants. Whereas Prc1 is widely regarded to crosslink microtubules in an antiparallel conformation, our studies provide evidence for an additional function of Prc1l in the bundling of parallel microtubules in the vegetal cortex of the early embryo during cortical rotation and prior to mitotic cycling. These findings highlight common yet distinct aspects of microtubule reorganization that occur during the egg-to-embryo transition, driven by maternal product for the midbody component Prc1l and required for embryonic cell division and pattern formation.

Experimental Methodologies to Visualize Early Cell Biology in Zebrafish Embryos.

For organisms to function normally, biological molecules must work at the correct time in the right cells and within the right intracellular compartments of cells. Biological research relies heavily on discovering the cellular locations at which such molecular interactions occur. A mainstay technique in this process of discovery is the visualization of locations of proteins in cells and tissues, known as immunocytochemistry and immunohistochemistry, respectively. If performed correctly, these techniques can provide detailed information regarding the endogenous locations of proteins and their ectopic locations or absence in mutants and in disease states.

Dynamic optima in cell sizes during early development enable normal gastrulation in zebrafish embryos.

Cell migration is the main driver of the evolutionarily conserved process of gastrulation, which shapes metazoan embryo morphology. The molecular and cellular mechanisms of cell migration during gastrulation though well researched lacks an understanding of the contribution of cell sizes to collective cell migration. This is especially important during the early phase of metazoan development, which is dominated by constantly changing cell sizes in the background of which cells migrate en mass to shape the embryo. Here we investigate this phenomenon in zebrafish embryos, a model system in which early cell divisions causes cell sizes to decrease naturally over time as cells migrate collectively to sculpt the embryonic body plan. Because mutations that can perturb cell sizes so early in development do not exist, we generate haploid and tetraploid zebrafish embryos and show that cell sizes in such embryos are smaller and larger than the diploid norm, respectively. Cells in embryos made of smaller or larger than normal cells migrate sub-optimally, leading to gastrulation defects. Gene expression analysis suggests that the observed defects originate from altered cell size, and not from pleiotropic effects of altered ploidy. This interpretation is strengthened when gastrulation defects are rescued by increasing cell sizes in embryos wherein cell sizes are smaller than normal. We show that the migration defects are cell-autonomous by live imaging migrating haploid and tetraploid cells during gastrulation in chimeric diploid embryos. Analysis of membrane protrusion dynamics in single cells shows that cells normally extend protrusions non-uniformly during migration, a phenomenon which is perturbed when cell sizes deviate from the norm. Thus, an optimal range of developmental stage-specific cell sizes appears necessary for collective cell migration to correctly position cells in space and time to shape an amorphous ball of blastoderm into an embryo.

Maternal control of gamete choice during fertilization.

Sexually reproducing organisms generate male and female haploid gametes, which meet and fuse at fertilization to produce a diploid zygote. The evolutionary process of speciation is achieved and maintained by ensuring that gametes undergo productive fusion only within a species. In animals, hybrids from cross-species fertilization events may develop normally, but are usually sterile (Fitzpatrick, 2004). Metazoan sperm and eggs have several features to ensure that the gametes, which have evolved independently and also in conflict with each other, are competent to undergo fertilization (Firman, 2018). Fertilization is a specific process that is ideally supposed to result in randomized fusion of compatible egg and sperm. Here, I will discuss key processes driven by maternal factors in the egg that dictate earliest stages of gamete recognition, gamete choice and fusion in metazoans.

Experimental Manipulation of Ploidy in Zebrafish Embryos and Its Application in Genetic Screens.

Metazoan animals are typically diploid, possessing two sets of a chromosome in the somatic cells of an organism. In naturally diploid species, alteration from the endogenous diploid state is usually embryonic lethal. However, the ability to experimentally manipulate ploidy of animal embryos has fundamental as well as applied biology advantages. In this chapter we describe experimental procedures to convert normally diploid zebrafish embryos into haploid or tetraploid states. We also describe methodologies to verify the ploidy of embryos and the utility of ploidy manipulation in expediting the isolation of mutations using both forward and reverse genetic strategies in zebrafish.

Transient window of resilience during early development minimizes teratogenic effects of heat in zebrafish embryos.

BACKGROUND: Transient heat shock during early development is an established experimental paradigm for doubling the genome of the zebrafish zygote, which has practical applications in expedited identification of recessive mutations in genetic screens. Despite the simplicity of the strategy and the genetic tractability of zebrafish, heat shock has not been used for genome doubling since the proof-of-principle experiments done in the 1980s. This is because of poor survival of embryos that ensue from transient heat shocks and gross developmental abnormalities in the few survivors, which is incompatible with phenotype driven screens. RESULTS: We show that heat shocks during early zebrafish development uncouple the second cycle of DNA and centrosome duplication. Interestingly, the developmental time of the heat shock that triggers the dissociation between DNA and centrosome duplication cycles significantly affect the potential of embryos to survive and attain normal morphology. The potential to develop normally after a heat shock alters in a developmental time span of 2 min in zebrafish embryos, a phenomenon that has not been reported in any species. CONCLUSIONS: The existence of heat resilient developmental windows and reduced heat teratogenicity during these windows could be an effective step forward in practical application of transient heat for experimental manipulation of ploidy in zebrafish. More broadly, heat resilience before zygotic genome activation suggests that metazoan embryos may possess innate protective features against heat beyond the canonical heat shock response. Developmental Dynamics 247:992-1004, 2018. © 2018 Wiley Periodicals, Inc.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Effective Generation of Gynogenic Haploid Zebrafish Embryos Using Low Dosage of UV Rays.

The phenomenon of phenotype manifestation when the single allele in a haploid is affected is desirable for uncovering recessive mutations expeditiously in a diploid organism. However, experimentally generated haploids manifest extensive lethality and a cluster of non-specific developmental defects known as the haploid syndrome. This precludes the use of experimentally generated haploids for genetic screens due to an insufficient number of embryos for screening and the possibility of phenotypes due to the affected gene being masked by the haploid syndrome. We show here that gynogenic haploid zebrafish can be generated by irradiation of spermatozoa with a lower UV dosage than is currently used. This strategy results in reduced haploid lethality, incidence and severity of haploid syndrome. When viewed in the context of zebrafish as a genetically tractable model organism for forward and reverse genetic strategies, these results place zebrafish in a unique niche as a vertebrate in which haploid genetic screens for developmental phenotypes could be successfully attempted.

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.

In vitro oocyte culture-based manipulation of zebrafish maternal genes.

In animals, females deposit gene products into developing oocytes, which drive early cellular events in embryos immediately after fertilization. As maternal gene products are present before fertilization, the functional manipulation of maternal genes is often challenging to implement, requiring gene expression or targeting during oogenesis. Maternal expression can be achieved through transgenesis, but transgenic approaches are time consuming and subject to undesired epigenetic effects. Here, we have implemented in vitro culturing of experimentally manipulated immature oocytes to study maternal gene contribution to early embryonic development in the zebrafish. We demonstrate phenotypic rescue of a maternal-effect mutation by expressing wild-type product in cultured oocytes. We also generate loss-of-function phenotypes in embryos through either the expression of a dominant-negative transcript or injection of translation-blocking morpholino oligonucleotides. Finally, we demonstrate subcellular localization during the early cell divisions immediately after fertilization of an exogenously provided maternal product fused to a fluorescent protein. These manipulations extend the potential to carry out genetic and imaging studies of zebrafish maternal genes during the egg-to-embryo transition.

Practical approaches for implementing forward genetic strategies in zebrafish.

The tropical fresh water minnow, Danio rerio, more commonly known as zebrafish, has emerged rapidly over the last decade as a powerful tool for developmental geneticists. External fertilization, high fecundity, a short generation time, and optical transparency of embryos during early development combined with the amenability to a variety of genetic manipulations constitute in the zebrafish the convergence of several unique advantages for a vertebrate model system. Traditional forward genetic screens, which employ the use of a chemical mutagen such as N-ethyl-N-nitrosourea to induce mutations in the male genome, have also proven to be highly successful in the zebrafish. This chapter provides experimental approaches to successfully induce pre-meiotic mutations in the male zebrafish germline and genetic strategies to recover and maintain such mutations in subsequent generations (Section 3.1). Though discussed specifically in the context of zebrafish research in this chapter, many of these genetic approaches may also be broadly applicable in other model systems. We also discuss experimental techniques to manipulate the ploidy of zebrafish embryos, which when used in combination with the standard mutagenesis protocol significantly expedite the identification of the induced mutations (Section 3.2). Additional stand-alone procedures are provided in Section 3.3, which are also required for the execution of the experiments discussed in its preceding sections.

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.

Chemokine signaling controls endodermal migration during zebrafish gastrulation.

Directed cell movements during gastrulation establish the germ layers of the vertebrate embryo and coordinate their contributions to different tissues and organs. Anterior migration of the mesoderm and endoderm has largely been interpreted to result from epiboly and convergent-extension movements that drive body elongation. We show that the chemokine Cxcl12b and its receptor Cxcr4a restrict anterior migration of the endoderm during zebrafish gastrulation, thereby coordinating its movements with those of the mesoderm. Depletion of either gene product causes disruption of integrin-dependent cell adhesion, resulting in separation of the endoderm from the mesoderm; the endoderm then migrates farther anteriorly than it normally would, resulting in bilateral duplication of endodermal organs. This process may have relevance to human gastrointestinal bifurcations and other organ defects.

Requirements for Endothelin type-A receptors and Endothelin-1 signaling in the facial ectoderm for the patterning of skeletogenic neural crest cells in zebrafish.

Genetic studies in mice and zebrafish have revealed conserved requirements for Endothelin 1 (Edn1) signaling in craniofacial development. Edn1 acts through its cognate type-A receptor (Ednra) to promote ventral skeletal fates and lower-jaw formation. Here, we describe the isolation and characterization of two zebrafish ednra genes - ednra1 and ednra2 - both of which are expressed in skeletal progenitors in the embryonic neural crest. We show that they play partially redundant roles in lower-jaw formation and development of the jaw joint. Knockdown of Ednra1 leads to fusions between upper- and lower-jaw cartilages, whereas the combined loss of Ednra1 and Ednra2 eliminates the lower jaw, similar to edn1-/- mutants. edn1 is expressed in pharyngeal arch ectoderm, mesoderm and endoderm. Tissue-mosaic studies indicate that, among these tissues, a crucial source of Edn1 is the surface ectoderm. This ectoderm also expresses ednrA1 in an edn1-dependent manner, suggesting that edn1 autoregulates its own expression. Collectively, our results indicate that Edn1 from the pharyngeal ectoderm signals through Ednra proteins to direct early dorsoventral patterning of the skeletogenic neural crest.

lockjaw encodes a zebrafish tfap2a required for early neural crest development.

The neural crest is a uniquely vertebrate cell type that gives rise to much of the craniofacial skeleton, pigment cells and peripheral nervous system, yet its specification and diversification during embryogenesis are poorly understood. Zebrafish homozygous for the lockjaw (low) mutation show defects in all of these derivatives and we show that low (allelic with montblanc) encodes a zebrafish tfap2a, one of a small family of transcription factors implicated in epidermal and neural crest development. A point mutation in low truncates the DNA binding and dimerization domains of tfap2a, causing a loss of function. Consistent with this, injection of antisense morpholino oligonucleotides directed against splice sites in tfap2a into wild-type embryos produces a phenotype identical to low. Analysis of early ectodermal markers revealed that neural crest specification and migration are disrupted in low mutant embryos. TUNEL labeling of dying cells in mutants revealed a transient period of apoptosis in crest cells prior to and during their migration. In the cranial neural crest, gene expression in the mandibular arch is unaffected in low mutants, in contrast to the hyoid arch, which shows severe reductions in dlx2 and hoxa2 expression. Mosaic analysis, using cell transplantation, demonstrated that neural crest defects in low are cell autonomous and secondarily cause disruptions in surrounding mesoderm. These studies demonstrate that low is required for early steps in neural crest development and suggest that tfap2a is essential for the survival of a subset of neural crest derivatives.

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans.

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.