Zebrafish as a Model for the Study of Live in vivo Processive Transport in Neurons

Motor proteins are responsible for transport of vesicles and organelles within the cell cytoplasm. They interact with the actin cytoskeleton and with microtubules to ensure communication and supply throughout the cell. Much work has been done in vitro and in silico to unravel the key players, including the dynein motor complex, the kinesin and myosin superfamilies, and their interacting regulatory complexes, but there is a clear need for in vivo data as recent evidence suggests previous models might not recapitulate physiological conditions. The zebrafish embryo provides an excellent system to study these processes in intact animals due to the ease of genetic manipulation and the optical transparency allowing live imaging. We present here the advantages of the zebrafish embryo as a system to study live in vivo processive transport in neurons and provide technical recommendations for successful analysis

EMT 2.0: shaping epithelia through collective migration

Epithelial-mesenchymal transitions (EMTs) drive epithelial remodelling by converting cohesive, stable epithelial layers into individual, motile mesenchymal cells. It is now becoming clear that, from being an all-or-nothing switch, EMT can be applied in a fine-tuned manner to allow the efficient migration of cohesive epithelia that maintain their internal organisation. Recent work suggests that such collective motility involves a complex balance between epithelial and mesenchyme-like cell states that is driven by internal and external cues. Although this cohesive mode requires more complex control than single cell migration, it creates opportunities in term of tissue guidance and shaping that are starting to be unravelled

Stabilité et dynamique de la microvillosité instestinale (fonction des protéines de mise en faisceau de l'actine)

Les invalidations individuelles et simultanées des protéines de mise en faisceau de l actine, villine, I-fimbrine et espine, n abolissent pas la formation de microvillosités intestinales. Leur formation en absence de protéines de mise en faisceau est discutée. Quelle est alors la fonction de ces protéines ? L étude du rôle de la villine démontre que son activité de fragmentation augmente la dynamique d un mouvement dépendant de l actine. La I-fimbrine apparaît comme un acteur central de la polarité et de la stabilité du réseau apical des entérocytes. Enfin, un modèle est envisagé selon lequel la villine agirait comme un facteur déstabilisant du cytosquelette de la microvillosité compensé en partie ou en totalité par le rôle stabilisateur de la I-fimbrine. Ces deux protéines seraient des protagonistes permettant d une part le maintien de la microvillosité en équilibre dynamique en conditions normales, et d autre part son remodelage rapide en réponse à des situations de stress.The individual and the simultaneous invalidations of the genes encoding for the bundling proteins, villin, I-fimbrin ans espin do not abolish the formation of intestinal microvilli in mice. The possibilities of formation of this structure in the absence of bundling proteins is discussed. What is then the role of these proteins in the enterocytes ? We demonstrate that villin actin severing property enhances an actin-based movement. We also report polarity and stability defects of the apical network of the enterocytes lacking I-fimbrin. A model in which villin acts as a destabilising factor of the cytoskeleton of the microvilli that would be compensated by the stabilising effect of I-fimbrin is proposed. These two proteins would be two protagonists, on the one hand maintaining the microvilli in a dynamic equilibrium in normal conditions, and on the other hand allowing a fast remodelling of the structure in response to stresses.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish

International audienceWith its variety of applications, the CRISPR/Cas9 genome editing technology has been rapidly evolving in the last few years. In the zebrafish community, knock-out reports are constantly increasing but insertion studies have been so far more challenging. With this review, we aim at giving an overview of the homologous directed repair (HDR)-based knock-in generation in zebrafish. We address the critical points and limitations of the procedure such as cutting efficiency of the chosen single guide RNA, use of cas9 mRNA or Cas9 protein, homology arm size etc. but also ways to circumvent encountered issues with HDR insertions by the development of non-homologous dependent strategies. While imprecise, these homology-independent mechanisms based on non-homologous-end-joining (NHEJ) repair have been employed in zebrafish to generate reporter lines or to accurately edit an open reading frame by the use of intron-targeting modifications. Therefore, with higher efficiency and insertion rate, NHEJ-based knock-in seems to be a promising approach to target endogenous loci and to circumvent the limitations of HDR whenever it is possible and appropriate. In this perspective, we propose new strategies to generate cDNA edited or tagged insertions, which once established will constitute a new and versatile toolbox for CRISPR/Cas9-based knock-ins in zebrafish

Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation

The directed migration of cell collectives drives the formation of complex organ systems. A characteristic feature of many migrating collectives is a 'tissue-scale' polarity, whereby 'leader' cells at the edge of the tissue guide trailing 'followers' that become assembled into polarised epithelial tissues en route. Here, we combine quantitative imaging and perturbation approaches to investigate epithelial cell state transitions during collective migration and organogenesis, using the zebrafish lateral line primordium as an in vivo model. A readout of three-dimensional cell polarity, based on centrosomal-nucleus axes, allows the transition from migrating leaders to assembled followers to be quantitatively resolved for the first time in vivo. Using live reporters and a novel fluorescent protein timer approach, we investigate changes in cell-cell adhesion underlying this transition by monitoring cadherin receptor localisation and stability. This reveals that while cadherin 2 is expressed across the entire tissue, functional apical junctions are first assembled in the transition zone and become progressively more stable across the leader-follower axis of the tissue. Perturbation experiments demonstrate that the formation of these apical adherens junctions requires dynamic microtubules. However, once stabilised, adherens junction maintenance is microtubule independent. Combined, these data identify a mechanism for regulating leader-to-follower transitions within migrating collectives, based on the relocation and stabilisation of cadherins, and reveal a key role for dynamic microtubules in this process

Zebrafish as a Model for the Study of Live in vivo Processive Transport in Neurons

Motor proteins are responsible for transport of vesicles and organelles within the cell cytoplasm. They interact with the actin cytoskeleton and with microtubules to ensure communication and supply throughout the cell. Much work has been done in vitro and in silico to unravel the key players, including the dynein motor complex, the kinesin and myosin superfamilies, and their interacting regulatory complexes, but there is a clear need for in vivo data as recent evidence suggests previous models might not recapitulate physiological conditions. The zebrafish embryo provides an excellent system to study these processes in intact animals due to the ease of genetic manipulation and the optical transparency allowing live imaging. We present here the advantages of the zebrafish embryo as a system to study live in vivo processive transport in neurons and provide technical recommendations for successful analysis.status: publishe

Brush border structure defects in I-plastin deficient mice

Abstract of Annual Meeting of the German Societey for Cell Biology (2007).Plastins (or fimbrins) are actin-bundling proteins that consist of two actin-binding domains in tandem preceded by two calcium-binding EF-hands. In mammals three different isoforms are expressed in a cell-type specific manner. I-plastin is specifically expressed in the small intestine, colon and kidney, whereas the isoforms L- and T-plastin are expressed in leukocytes and in all other tissues, respectively. I-plastin localises in microvilli, specialised surface structures with highly ordered microfilament bundles, together with villin and espin, two other actin-bundling proteins. We have generated knockout mice lacking I-plastin. Deficient mice showed no overt phenotype at the whole animal level: growth rate, reproductive rate and litter size were normal. Lacking of I-plastin is not compensated by another plastin isoform. In HE stained sections an increased number of intraepithelial lymphocytes was observed. Transmission and scanning electron microscopy studies revealed morphological alterations in the microvilli of the small intestine. In immunofluorescence studies of intestinal mucosa a discontinous actin, ezrin and villin staining was observed. We are also analysing the distribution of proliferation, apoptosis and other markers. I-plastin seems to be an important regulator of the morphological structure and stability of microvilli of the intestinal brush border barrier.Peer Reviewe

I-plastin, a central element for brush border integrity and proper apical transport

1-plastin, one of three mammalian plastin isoforms, is an actinbundling protein of the brush border (BB) of the small intestine, colon and kidney. To investigate the contribution of 1-plastin to the microvillar architecture we have generated a KO mouse strain. Although at the whole animal level KO mice do not show any overt phenotype, ultrastructural studies revealed important morphological alterations. In particular the rootlet that extends the microvillar core actin bundle into the. terminal web (TW) is missing, microvilli are shorter and the organelle free zone occupied by the TW is thinner. Inununohistochemistry studies revealed a depletion of important TW components like myosin 11, spectrin and tropomyosin and an altered localization of cytokeratins. These alterations result in increased fragility of the BB, altered targeting of apical components and decreased transepithelial resistance. 1-plastin emerges as a central regulator of the morphological structure and stability of the intestinal BB.Peer Reviewe

A minimally invasive fin scratching protocol for fast genotyping and early selection of zebrafish embryos

Abstract Current genetic modification and phenotyping methods in teleost fish allow detailed investigation of vertebrate mechanisms of development, modeling of specific aspects of human diseases and efficient testing of drugs at an organ/organismal level in an unparalleled fast and large-scale mode. Fish-based experimental approaches have boosted the in vivo verification and implementation of scientific advances, offering the quality guaranteed by animal models that ultimately benefit human health, and are not yet fully replaceable by even the most sophisticated in vitro alternatives. Thanks to highly efficient and constantly advancing genetic engineering as well as non-invasive phenotyping methods, the small zebrafish is quickly becoming a popular alternative to large animals’ experimentation. This approach is commonly associated to invasive procedures and increased burden. Here, we present a rapid and minimally invasive method to obtain sufficient genomic material from single zebrafish embryos by simple and precise tail fin scratching that can be robustly used for at least two rounds of genotyping already from embryos within 48 h of development. The described protocol betters currently available methods (such as fin clipping), by minimizing the relative animal distress associated with biopsy at later or adult stages. It allows early selection of embryos with desired genotypes for strategizing culturing or genotype–phenotype correlation experiments, resulting in a net reduction of “surplus” animals used for mutant line generation

Brush border structure defects in I-plastin deficient mice

Abstract of Annual Meeting of the German Society for Cell Biology (29.3.-1.4. 2006)Plastin (or fimbrin) is an actin-bundling protein consisting of two actin-binding domains in tandem preceded by two calcium-binding EF-hands. In mammals three different isoforms are expressed in a cell-type specific manner. Iplastin is specifi cally expressed in the small intestine,colon and kidney, where it localizes in microvilli, specialized surface structures with highly ordered microfi lament bundles. We have generated knockout mice lacking I-plastin. I-plastin deficient mice showed no overt phenotype at the whole animal level: growth rate, reproductive rate and litter size were normal. Antibodies specific for T- and L-plastin were used to demonstrate that lacking of I-plastin is not compensated by another plastin isoform. Transmission electron microscopy studies revealed morphological differences between wildtype and I-plastin deficient mice in the microvilli. Brush border preparations of knockout mice are sensitive and seem to degrade easily. In immuno fluorescence studies of intestinal mucosa cryosections decreased actin staining of the apical membrane was observed. Another abundant actin cross-linker of the brush border, villin, was found to be absent from the apical membrane. I-plastin seems to be an important regulator of the morphological structure and stability of microvilli.Peer Reviewe