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This volume details a wide range of methods, ranging from beginner through advanced, used to further study zebrafish and fish germline. Chapter guide readers through cultivating and manipulating germ cells, imaging of germline processes and the molecular analysis of their, protein, and RNA. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Germline Development in the Zebrafish: Methods and Protocols aims to ensure successful results in the further study of this vital field.
In zebrafish (Danio rerio) and many other organisms, the differentiation of primordial germ cells (PGCs) requires the reliable transmission of maternally-derived germline determinants, collectively termed germ plasm. PGCs give rise to the germline; thus, the fidelity of PGC specification and development is intimately tied to the survival and fitness of sexually-reproducing species. In this dissertation, I systematically investigate the formation, structure, and composition of germ plasm aggregates in zebrafish and related fish species in the Danio and Devario genera. I provide a comprehensive overview of preformation (germ plasm-dependent) and induction (non-germ plasm) as mechanisms of PGC specification, including major findings in several model vertebrate species. PGC specification is also discussed in a phylogenetic context, with particular attention to conserved gene families and the ongoing efforts to characterize germline determination in non-model vertebrate species. In Chapters 3 and 4, I use advanced methods of fluorescence in situ hybridization (detailed in Appendix I), microscopy, and image analysis to characterize the formation and structure of zebrafish germ plasm aggregates at a macromolecular-level, from the identification of homotypic germline-determinant RNA particles (Chapter 3) to the description of and proposed mechanism for organized supramolecular assembly of germ plasm in cleavage-stage embryos (Chapter 4). Appendix II presents the creation and initial characterization of mutations in bucky ball 2-like, the only zebrafish paralog of the germ plasm organizing protein Bucky ball. In Chapter 5, I identify germ plasm in three non-zebrafish species within the Danionin clade: Danio kyathit, Danio albolineatus, and Devario aequipinnatus, and find that eleven RNAs, including several not previously reported as germ plasm components in any species beyond zebrafish, exhibit conserved localization patterns within the embryos of all three examined non-zebrafish species. One component of zebrafish germ plasm, ca15b, was observed in the germ plasm of the two non-zebrafish Danio species but lacked specific localization in Devario aequipinnatus embryos. Together, these works present an improved understanding of the molecular components and behavior of maternally-derived germ plasm in zebrafish and related species.
The zebrafish is the most important fish model in developmental andgenetic analyses. This book contains 19 review articles covering abroad spectrum of topics, from development to genetic tools. Thecontents range from early development, the role of maternal factorsand gastrulation, to tissue differentiation and organogenesis, such asdevelopment of the organizer, notochord, floor plate, nervous system, somites, muscle, skeleton and endoderm
The Zebrafish: Cellular and Developmental Biology, Part B Developmental Biology, the second volume on the topic in the Methods in Cell Biology series, looks at methods for analyzing cellular and developmental biology of zebrafish. Chapters cover such topics as cell biology and developmental and neural biology. Covers sections on model systems and functional studies, imaging-based approaches, and emerging studies Chapters written by experts in the field Contains cutting-edge material on the topic of zebrafish and developments relating to their cellular and developmental biology New, two part Fourth Edition in this important volume
This second edition provides updated and new chapters on selected genetic, molecular, biochemical, and cell biological techniques. Chapter’s guide readers through methods and principles on primordial germ cells and germline stem cells, however many of these principles can be applied to different types of adult stem cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Germline Stem Cells: Second Edition aims to present the new findings and techniques that have developed since the first edition.
Due to its prolific reproduction and the external development of the transparent embryo, the zebrafish is the prime model for genetic and developmental studies, as well as research in genomics. While genetically distant from humans, nonetheless the vertebrate zebrafish has comparable organs and tissues that make it the model organism for study of vertebrate development.This book, one of two new volumes in the Reliable Lab Solutions series dealing with zebrafish, brings together a robust and up-to-date collection of time-tested methods presented by the world’s leading scientists. Culled from previously published chapters in Methods in Cell Biology and updated by the original authors where relevant, it provides a comprehensive collection of protocols describing the most widely used techniques relevant to the study of the cellular and developmental biology of zebrafish. The methods in this volume were hand-selected by the editors, whose goal was to a provide a handy and cost-effective collection of fail-safe methods, tips, and “tricks of the trade to both experienced researchers and more junior members in the lab. Provides busy researchers a quick reference for time-tested methods and protocols that really work, updated where possible by the original authors Gives pragmatic wisdom to the non-specialist from experts in the field with years of experience with trial and error