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.

Primordial Germ Cell Specification in Vertebrate Embryos: Phylogenetic Distribution and Conserved Molecular Features of Preformation and Induction.

The differentiation of primordial germ cells (PGCs) occurs during early embryonic development and is critical for the survival and fitness of sexually reproducing species. Here, we review the two main mechanisms of PGC specification, induction, and preformation, in the context of four model vertebrate species: mouse, axolotl, Xenopus frogs, and zebrafish. We additionally discuss some notable molecular characteristics shared across PGC specification pathways, including the shared expression of products from three conserved germline gene families, DAZ (Deleted in Azoospermia) genes, nanos-related genes, and DEAD-box RNA helicases. Then, we summarize the current state of knowledge of the distribution of germ cell determination systems across kingdom Animalia, with particular attention to vertebrate species, but include several categories of invertebrates - ranging from the "proto-vertebrate" cephalochordates to arthropods, cnidarians, and ctenophores. We also briefly highlight ongoing investigations and potential lines of inquiry that aim to understand the evolutionary relationships between these modes of specification.

Conserved germ plasm characteristics across the Danio and Devario lineages.

In many animal species, germ cell specification requires the inheritance of germ plasm, a biomolecular condensate containing maternally derived RNAs and proteins. Most studies of germ plasm composition and function have been performed in widely evolutionarily divergent model organisms, such as Caenorhabditis elegans, Drosophila, Xenopus laevis, and Danio rerio (zebrafish). In zebrafish, 12 RNAs localize to germ plasm at the furrows of the early embryo. Here, we tested for the presence of these RNAs in three additional species within the Danionin clade: Danio kyathit, Danio albolineatus, and Devario aequipinnatus. By visualizing nanos RNA, we find that germ plasm segregation patterns during early embryogenesis are conserved across these species. Ten additional germ plasm RNAs exhibit localization at the furrows of early embryos in all three non-zebrafish Danionin species, consistent with germ plasm localization. One component of zebrafish germ plasm, ca15b, lacked specific localization in embryos of the more distantly related D. aequipinnatus. Our findings show that within a subset of closely related Danionin species, the vast majority of germ plasm RNA components are conserved. At the same time, the lack of ca15b localization in D. aequipinnatus germ plasm highlights the potential for the divergence of germ plasm composition across a restricted phylogenetic space.

Methods for Visualization of RNA and Cytoskeletal Elements in the Early Zebrafish Embryo.

Zebrafish embryos, with their large size (>0.5 mm) and accessibility, are valuable tools for investigating core cellular processes. Many of those processes, such as cell division, asymmetric inheritance of cellular components, and structural dynamics involved in cell motility and morphology rely on cytoskeletal rearrangements and associated macromolecules. In addition to the protein-rich cytoskeleton, the early embryo is packed with maternally deposited RNA, which serves essential roles in establishing cell polarity, cell fate, and cell organization. Here, we present methods for visualizing endogenous RNA along with cytoskeletal structures, including microtubules and filamentous actin (F-actin) in the context of an intact vertebrate embryo. Each of the four protocols described herein (embryo fixation, RNA probe design/synthesis, double fluorescent in situ hybridization with tubulin immunofluorescence, and fluorescent in situ hybridization with phalloidin labeling of F-actin) are intended for optimal preservation and visualization of both the cytoskeleton and RNAs of interest. These methods can also be modified and applied to a broad range of other uses.

Aggregation, segregation, and dispersal of homotypic germ plasm RNPs in the early zebrafish embryo.

BACKGROUND: In zebrafish and many other organisms, specification of primordial germ cells (PGCs) requires the transmission of maternally-derived germ plasm. Zebrafish germ plasm ribonucleoparticles (RNPs) aggregate along the cleavage furrows during the first several cell cycles, segregate asymmetrically during the cleavage stages, and undergo cytoplasmic dispersal in the late blastula. RESULTS: For all tested germ plasm RNAs [carbonic anhydrase 15b (ca15b), deleted in azoospermia-like (dazl), dead end (dnd), nanos 3 (nos3), regulator of G-protein signaling14a (rgs14a), and vasa/DEAD box polypeptide 4 (vasa/ddx4)], RNPs are homotypic (containing a single RNA type), with RNPs packing tightly yet remaining distinct within germ plasm aggregates. Homotypic clustering of RNAs within RNPs is observed before aggregation in the cortex and is maintained through germ plasm recruitment, asymmetric segregation and RNP dispersal. We also identify a step of germ plasm fragmentation during the cleavage stages that precedes RNP dispersal. CONCLUSIONS: Our findings suggest that germ plasm aggregates act as subcellular compartments that temporarily collect and carry single RNA-type RNPs from fertilization until their cytoplasmic dispersal in PGCs at the end of the blastula period, and describe a previously unknown fragmentation step that allows for an increase in the pool of germ plasm-carrying cells, presumably PGCs. Developmental Dynamics 248:306-318, 2019. © 2019 Wiley Periodicals, Inc.

Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae.

In mitosis, the centromeres of sister chromosomes are pulled toward opposite poles of the spindle. In meiosis I, the opposite is true: the sister centromeres move together to the same pole, and the homologous chromosomes are pulled apart. This change in segregation patterns demands that between the final mitosis preceding meiosis and the first meiotic division, the kinetochores must be restructured. In budding yeast, unlike mammals, kinetochores are largely stable throughout the mitotic cycle. In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis. We use quantitative mass spectrometry methods and imaging approaches to explore the kinetochore restructuring process that occurs in meiosis I in budding yeast. The Ndc80 outer kinetochore complex, but not other subcomplexes, is shed upon meiotic entry. This shedding is regulated by the conserved protein kinase Ipl1/Aurora-B and promotes the subsequent assembly of a kinetochore that will confer meiosis-specific segregation patterns on the chromosome.