Blebs are cellular protrusions that have been shown to be instrumental for cell migration in development and disease. Bleb expansion is driven by hydrostatic pressure generated in the cytoplasm by ...the contractile actomyosin cortex. The mechanisms of bleb formation thus fundamentally differ from the actin polymerization-based mechanisms responsible for lamellipodia expansion. In this review, we summarize recent findings relevant for the mechanics of bleb formation and the underlying molecular pathways. We then review the processes involved in determining the type of protrusion formed by migrating cells, in particular in vivo , in the context of embryonic development. Finally, we discuss how cells utilize blebs for their forward movement in the presence or absence of strong substrate attachment.
We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) ...development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies.
Primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the ...inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types. PGCs specification in zebrafish appears to depend on inheritance of germ plasm in which several RNA molecules such as vasa and nanos reside. Whether the specification mode of PGCs found in zebrafish is general for other fish species was brought into question upon analysis of olvas expression--the vasa homologue in another teleost, medaka (Oryzias latipes). Here, in contrast to the findings in zebrafish, the PGCs are found in a predictable position relative to a somatic structure, the embryonic shield. This finding, coupled with the fact that vasa mRNA, which is localized to the germ plasm of zebrafish but does not label a similar structure in medaka opened the possibility of fundamentally different mechanisms governing PGC specification in these two fish species.
In this study we addressed the question concerning the mode of PGC specification in medaka using embryological experiments, analysis of RNA stability in the PGCs and electron microscopy observations. Dramatic alterations in the somatic environment, i.e. induction of a secondary axis or mesoderm formation alteration, did not affect the PGC number. Furthermore, the PGCs of medaka are capable of protecting specific RNA molecules from degradation and could therefore exhibit a specific mRNA expression pattern controlled by posttrancriptional mechanisms. Subsequent analysis of 4-cell stage medaka embryos using electron microscopy revealed germ plasm-like structures located at a region corresponding to that of zebrafish germ plasm.
Taken together, these results are consistent with the idea that in medaka the inheritance of maternally provided asymmetrically-localized cytoplasmic determinants directs cells to assume the germ line fate similar to zebrafish PGCs.
Primordial germ cells follow a characteristic developmental path that is manifested in the specialized regulation of basic cell functions and behaviour. Recent studies in zebrafish have greatly ...enhanced our understanding of the mode of specification of primordial germ cells, cell-fate maintenance and the migration of these cells towards their target, the gonad, where they differentiate into gametes.
In sexually reproducing organisms, primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. These cells follow a ...characteristic developmental path that is manifested in specialized regulation of basic cell functions and behavior making them an attractive system for studying cell fate specification, differentiation and migration. This review summarizes studies aimed at understanding the development of this cell population in zebrafish and compares these results with those obtained in other model organisms.
An important mechanism for the specification and development of the animal germ line is the localization of specific molecules to the germ plasm 1. Restriction of these molecules to the germ line is ...considered to be critical for proper development of the germ line as well as the soma. Cytoplasmic localization alone, however, may not be sufficient to achieve germ line-specific expression. While zebrafish vasa mRNA is localized to the germ plasm 2, 3, the Vasa protein is initially distributed uniformly in the embryo, and its expression becomes restricted to the PGCs only later in development 2, 4. Here, we demonstrate that, in addition to vasa RNA localization, multiple cell type-specific posttranscriptional mechanisms act on vasa mRNA and Vasa protein. We show that the portion of the maternal vasa mRNA, which is partitioned to somatic cells, is rapidly degraded, whereas vasa RNA is stabilized in the PGCs in a process that is mediated by cis-acting elements within the molecule. Similarly, the Vasa protein is highly unstable in somatic cells, but not in the PGCs. Finally, we demonstrate that subcellular localization of Vasa protein involves cis-acting domains within the protein. In conclusion, we show that posttranscriptional degradation-protection mechanisms acting on RNA and protein function in a vertebrate to enrich for specific molecules in the PGCs.
Whole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for ...detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules.
The protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies.
By fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers.
Primordial Germ Cell (PGC) migration in zebrafish is guided by SDF-1a. Binding of this chemokine to its receptor CXCR4b activates downstream signalling cascades leading to cell polarization and ...directed migration towards the attractant source. Despite the detailed information available concerning the role of SDF-1 in guiding the PGCs to their targets, little was known regarding the molecular mechanisms controlling the distribution of SDF-1a within the tissue. We have recently shown that the activity of a second SDF-1/CXCL12 receptor, CXCR7 is crucial for proper migration of PGCs. Although CXCR4 and CXCR7 are structurally related and serve as receptors for the same ligand, they appear to serve very different functions during PGC migration. Here we discuss a model according to which CXCR4b translates the polarized distribution of SDF-1 into directed PGC migration, while CXCR7 acts as a high-affinity decoy receptor and facilitates the migration of PGCs by shaping the distribution of the chemokine in the environment.
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