In many organisms, primordial germ cells (PGCs) are specified at a different location than where the gonad forms, meaning that PGCs must migrate toward the gonad within the early developing embryo. ...Following species-specific paths, PGCs can be passively carried by surrounding tissues and also perform active migration. When PGCs actively migrate through and along a variety of embryonic structures in different organisms, they adopt an ancestral robust migration mode termed “amoeboid motility”, which allows cells to migrate within diverse environments. In this review, we discuss the possible significance of the PGC migration process in facilitating the evolution of animal body shape. In addition, we summarize the latest findings relevant for the molecular and cellular mechanisms controlling the movement and the directed migration of PGCs in different species.
Two waves of DNA methylation reprogramming occur during mammalian embryogenesis; during preimplantation development and during primordial germ cell (PGC) formation. However, it is currently unclear ...how evolutionarily conserved these processes are. Here we characterise the DNA methylomes of zebrafish PGCs at four developmental stages and identify retention of paternal epigenetic memory, in stark contrast to the findings in mammals. Gene expression profiling of zebrafish PGCs at the same developmental stages revealed that the embryonic germline is defined by a small number of markers that display strong developmental stage-specificity and that are independent of DNA methylation-mediated regulation. We identified promoters that are specifically targeted by DNA methylation in somatic and germline tissues during vertebrate embryogenesis and that are frequently misregulated in human cancers. Together, these detailed methylome and transcriptome maps of the zebrafish germline provide insight into vertebrate DNA methylation reprogramming and enhance our understanding of the relationships between germline fate acquisition and oncogenesis.
In the context of development, tissue homeostasis, immune surveillance, and pathological conditions such as cancer metastasis and inflammation, migrating amoeboid cells commonly form protrusions ...called blebs. For these spherical protrusions to inflate, the force for pushing the membrane forward depends on actomyosin contraction rather than active actin assembly. Accordingly, blebs exhibit distinct dynamics and regulation. In this review, we first examine the mechanisms that control the inflation of blebs and bias their formation in the direction of the cell’s leading edge and present current views concerning the role blebs play in promoting cell locomotion. While certain motile amoeboid cells exclusively form blebs, others form blebs as well as other protrusion types. We describe factors in the environment and cell-intrinsic activities that determine the proportion of the different forms of protrusions cells produce.
Chemokines and their receptors were discovered about twenty years ago as mediators of leukocyte traffic. Over the past decade, functional studies of these molecules have revealed their importance for ...cell migration processes during embryogenesis, which, in addition to providing mechanistic insights into embryonic development, could complement information about chemokine function in the immune system. Here, we review the roles of the chemokine stromal cell-derived factor 1 (SDF-1/CXCL12) and its receptor CXCR4 during zebrafish and mouse embryonic development, and discuss their function in regulating the interactions of cells with their extracellular environment, in directing their migration, and in maintaining their location.
Fluorescence resonance energy transfer (FRET)-based molecular biosensors serve as important tools for studying protein activity in live cells and have been widely used for this purpose over the past ...decade. However, FRET biosensors are rarely used in the context of the live organism because of the inherent high cellular complexity and imaging challenges associated with the three-dimensional environment. Here we provide a protocol for using single-chain intramolecular FRET-based biosensors in early development. We provide a general protocol for FRET ratio imaging in embryos, including the data-acquisition conditions and the algorithm for ratio image generation. We then use the pRaichu RacFRET biosensor to exemplify the adaptation and optimization of a particular biosensor for use in live zebrafish embryos. Once an optimized biosensor is available, the complete procedure, including introduction of the probes into embryos, imaging and data analysis, requires 2-3 d.
About the Authors: Didier Y. R. Stainier * E-mail: didier.stainier@mpi-bn.mpg.de (DYRS); cmoens@fredhutch.org (CBM) Affiliation: Department of Developmental Genetics, Max Planck Institute for Heart ...and Lung Research, Bad Nauheim, Germany ORCID http://orcid.org/0000-0002-0382-0026 Erez Raz Affiliation: Institute of Cell Biology, ZBME, University of Münster, Münster, Germany ORCID http://orcid.org/0000-0002-6347-3302 Nathan D. Lawson Affiliation: Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America Stephen C. Ekker Affiliation: Mayo Clinic, Rochester, Minnesota, United States of America Rebecca D. Burdine Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America Judith S. Eisen Affiliation: Institute of Neuroscience, University of Oregon, Eugene, Oregon, United States of America ORCID http://orcid.org/0000-0003-1229-1696 Philip W. Ingham Affiliations Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, The Living Systems Institute, University of Exeter, Exeter, United Kingdom Stefan Schulte-Merker Affiliation: Institute of Cardiovascular Organogenesis and Regeneration, WWU Münster, Faculty of Medicine, Münster, Germany Deborah Yelon Affiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, California, United States of America Brant M. Weinstein Affiliation: Division of Developmental Biology, NICHD, NIH, Bethesda, Maryland, United States of America Mary C. Mullins Affiliation: Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America ORCID http://orcid.org/0000-0002-9979-1564 Stephen W. Wilson Affiliation: Department of Cell and Developmental Biology, University College London, London, United Kingdom ORCID http://orcid.org/0000-0002-8557-5940 Lalita Ramakrishnan Affiliation: Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Sharon L. Amacher Affiliation: Departments of Molecular Genetics and Biological Chemistry and Pharmacology, Ohio State University, Columbus, Ohio, United States of America Stephan C. F. Neuhauss Affiliation: Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland ORCID http://orcid.org/0000-0002-9615-480X Anming Meng Affiliation: School of Life Sciences, Tsinghua University, Beijing, China Naoki Mochizuki Affiliation: National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan ORCID http://orcid.org/0000-0002-3938-9602 Pertti Panula Affiliation: Department of Anatomy and Neuroscience Center, University of Helsinki, Helsinki, Finland Cecilia B. Moens * E-mail: didier.stainier@mpi-bn.mpg.de (DYRS); cmoens@fredhutch.org (CBM) Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of AmericaCitation: Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, et al. Additionally, mutant alleles for many genes are now readily available through zebrafish community resource centers. ...MOs should be used alongside mutant(s) for the corresponding gene. ...a word of caution that previous publication of MOs is not a guarantee of their fidelity, particularly if a new phenotype is being described. ...we hope that these brief and mostly conceptual guidelines will assist scientists working with zebrafish as well as those assessing manuscripts and grant proposals based on experiments using zebrafish.
Abstract
The migration of many cell types relies on the formation of actomyosin-dependent protrusions called blebs, but the mechanisms responsible for focusing this kind of protrusive activity to the ...cell front are largely unknown. Here, we employ zebrafish primordial germ cells (PGCs) as a model to study the role of cell-cell adhesion in bleb-driven single-cell migration in vivo. Utilizing a range of genetic, reverse genetic and mathematical tools, we define a previously unknown role for E-cadherin in confining bleb-type protrusions to the leading edge of the cell. We show that E-cadherin-mediated frictional forces impede the backwards flow of actomyosin-rich structures that define the domain where protrusions are preferentially generated. In this way, E-cadherin confines the bleb-forming region to a restricted area at the cell front and reinforces the front-rear axis of migrating cells. Accordingly, when E-cadherin activity is reduced, the bleb-forming area expands, thus compromising the directional persistence of the cells.
Paracrine Wnt/β-catenin signalling is important during developmental processes, tissue regeneration and stem cell regulation. Wnt proteins are morphogens, which form concentration gradients across ...responsive tissues. Little is known about the transport mechanism for these lipid-modified signalling proteins in vertebrates. Here we show that Wnt8a is transported on actin-based filopodia to contact responding cells and activate signalling during neural plate formation in zebrafish. Cdc42/N-Wasp regulates the formation of these Wnt-positive filopodia. Enhanced formation of filopodia increases the effective signalling range of Wnt by facilitating spreading. Consistently, reduction in filopodia leads to a restricted distribution of the ligand and a limited signalling range. Using a simulation, we provide evidence that such a short-range transport system for Wnt has a long-range signalling function. Indeed, we show that a filopodia-based transport system for Wnt8a controls anteroposterior patterning of the neural plate during vertebrate gastrulation.
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.
Maintaining cell fate relies on robust mechanisms that prevent the differentiation of specified cells into other cell types. This is especially critical during embryogenesis, when extensive cell ...proliferation, patterning, and migration events take place. Here we show that vertebrate primordial germ cells (PGCs) are protected from reprogramming into other cell types by the RNA-binding protein Dead end (Dnd). PGCs knocked down for Dnd lose their characteristic morphology and adopt various somatic cell fates. Concomitantly, they gain a gene expression profile reflecting differentiation into cells of different germ layers, in a process that we could direct by expression of specific cell-fate determinants. Importantly, we visualized these events within live zebrafish embryos, which provide temporal information regarding cell reprogramming. Our results shed light on the mechanisms controlling germ cell fate maintenance and are relevant for the formation of teratoma, a tumor class composed of cells from more than one germ layer.
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•During their migration through the embryo, PGCs maintain their germline fate•Dnd-deficient zebrafish PGCs transdifferentiate into somatic cells•Dnd-deficient PGCs gain somatic gene expression profile and morphology•Germline-to-soma transdifferentiation can be visualized in vivo
Primordial germ cells (PGCs) maintain their fate during their migration within the embryo. Through monitoring PGCs in live zebrafish embryos, Gross-Thebing et al. uncover a key role for the vertebrate-specific RNA-binding protein Dead end (Dnd) in protecting and maintaining germ cell fate. Dnd-deficient germ cells transdifferentiate into somatic cells.
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