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

Description of the vaginal microbiota in nulliparous ewes during natural mating and pregnancy: preliminary signs of the male preputial microbiota modulation

The vaginal microbiota plays a key role in animals' health. Understanding its diversity and composition and associated changes occurring through the reproductive cycle represents valuable knowledge to disclose the mechanisms leading to dysbiosis and eventually to infection. Even if the human vaginal microbiota has been thoroughly studied, scarce research has been conducted on the vaginal microbiota of livestock. In this study, 16S rRNA gene-based sequencing was performed on vaginal samples of ten nulliparous ewes at three different sampling points: before the estrus synchronization protocol (T0), at the time of estrus before mating (Testrus), and the day of the pregnancy diagnosis (Tpreg). Preputial samples from the three males collected pre and post-mating were also analyzed. Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria were the most abundant phyla in vaginal samples. The most abundant genera were Porphyromonas, Anaerococcus, and Peptinophilius. Vaginal microbiota biodiversity decreased during pregnancy. Tenericutes (Ureaplasma spp.) increased significantly at Tpreg in both pregnant and non-pregnant ewes. Differences were observed between pregnant and non-pregnant ewes at Tpreg where pregnant ewes had a significantly higher abundance of Actinobacillus spp. and Ureaplasma spp. Ewes that were diagnosed with pregnancy at Tpreg showed a decreased abundance of gram-negative bacteria such as Bacteroidales, Campylobacterales, and Enterobacteriales. In addition, a significant decrease in the relative abundances of genera within Firmicutes, such as Alloicoccus (Lactobacillales), Atopostipes (Lactobacillales), and an uncultured bacteria W5053 from Family XI (Firmicutes, Clostridiales) was observed in non-pregnant ewes at Tpreg. The four most abundant phyla in the rams' prepuce were the same as in the ewes' vagina. The most abundant genus was Corynebacterium. No major differences were observed in the ram's preputial microbiota between pre and post-mating samples. Nevertheless, the differences in the taxonomic composition of ewes' vaginal microbiota between Testrus and Tpreg could be explained by the exposure to the preputial microbiota. This study offers new insights into the effects of several key steps of the ewe's reproductive cycle such as estrus-synchronization protocol, mating, and pregnancy on ovine vaginal microbiota. The knowledge of the microbiota dynamics during the reproductive cycle can help improve the reproductive outcomes of dams by identifying biomarkers and putative probiotics.Este trabajo fue financiado y aprobado por la subvención para la realización de proyectos de investigación, desarrollo e innovación de grupos de investigación emergentes 2020 (GVA/2020/026), Generalitat Valenciana (España). Además, por el proyecto del Ministerio de Ciencia e Innovación de España (Referencia del proyecto PID2020-119462RA-I00/AEI/10.13039/501100011033).Microbiota reproductivareproductive microbiotaovinesheepmicrobiomepregnantintravaginal spongePublishe

Age-associated B cells predict impaired humoral immunity after COVID-19 vaccination in patients receiving immune checkpoint blockade

Age-associated B cells (ABC) accumulate with age and in individuals with different immunological disorders, including cancer patients treated with immune checkpoint blockade and those with inborn errors of immunity. Here, we investigate whether ABCs from different conditions are similar and how they impact the longitudinal level of the COVID-19 vaccine response. Single-cell RNA sequencing indicates that ABCs with distinct aetiologies have common transcriptional profiles and can be categorised according to their expression of immune genes, such as the autoimmune regulator (AIRE). Furthermore, higher baseline ABC frequency correlates with decreased levels of antigen-specific memory B cells and reduced neutralising capacity against SARS-CoV-2. ABCs express high levels of the inhibitory FcγRIIB receptor and are distinctive in their ability to bind immune complexes, which could contribute to diminish vaccine responses either directly, or indirectly via enhanced clearance of immune complexed-antigen. Expansion of ABCs may, therefore, serve as a biomarker identifying individuals at risk of suboptimal responses to vaccination

Nouvelles technologies d'édition du génome chez le poisson zèbre : de la modification génétique précise et efficace à la modélisation de maladies et au génie génétique

Zebrafish is an ideal model organism to study developmental biology and has become a powerful system for disease modeling in vivo. Since the emergence of the CRISPR/Cas9 technology, several disease models have been generated to introduce loss-of-function mutations, putting this vertebrate model at the forefront of genetic studies. However, the generation of mutant lines harboring precise pathological mutations is still challenging as knock-in approaches are still poorly efficient. In this study we investigated the capacities of several new cytosine base editor variants engineered in introducing precise C:G to T:A mutations into the genome. Using these tools, we broadened the gene editing possibilities in zebrafish by obtaining mutagenesis up to 100% of efficiency and we developed new approaches to perform multiplex mutagenesis and co-selection. We further explored biological applications of this technology such as endogenous activation of signaling pathway and disease modeling. In addition, we were able to validate in zebrafish the pathogenicity of mutations found in patients. Finally, we aimed at developing the prime editing technology in this animal model, a recent technology that although highly promising remains to date poorly efficient. To conclude, this study contributed in providing a large panel of genetic tools working in zebrafish without generating DNA double strand break, assessing their limitations and complementarity. Through the accessibility and ease of genome manipulations in this animal model, this work further highlights the use of zebrafish as a good platform to engineer new genome editing approaches in animals and to study genetic disorders.Le poisson zèbre est un organisme modèle idéal pour étudier la biologie du développement et est devenu un système puissant pour modéliser des maladies in vivo, notamment grâce à la technologie d’édition du génome CRISPR/Cas9. Cependant, la génération de lignées portant des mutations pathologiques humaines précises reste un défi majeur. Dans cette étude, nous avons étudié les capacités de plusieurs éditeurs de bases conçus pour introduire de manière précise dans le génome les mutations de C:G en T:A. En utilisant ces outils génétiques, nous avons élargi les possibilités d'édition de gènes chez le poisson zèbre en obtenant une mutagenèse jusqu'à 100% d'efficacité et nous avons développé de nouvelles approches pour effectuer une mutagenèse multiplexe et co-sélectionner les embryons les plus édités. Nous avons également exploré les applications biologiques de cette technologie, telles que l'activation endogène de voies de signalisation et la modélisation de cancer ou de syndromes rares. De plus, nous avons validé la pathogénicité de mutations trouvées chez de jeunes patients présentant un syndrome polymalformatif. Enfin, nous cherchons à développer le « Prime Editing » chez ce modèle animal, une technologie qui bien que très prometteuse reste à ce jour peu efficace. En conclusion, cette étude a contribué à fournir un large panel d'outils génétiques fonctionnant chez le poisson zèbre sans générer de cassures double brin d’ADN et d’en évaluer leurs limites ainsi que leur complémentarité chez cet organisme. Ce travail met en évidence que le poisson zèbre est un système puissant pour concevoir de nouvelles approches d'édition du génome et pour étudier les maladies génétiques

Nouvelles technologies d'édition du génome chez le poisson zèbre : de la modification génétique précise et efficace à la modélisation de maladies et au génie génétique

Le poisson zèbre est un organisme modèle idéal pour étudier la biologie du développement et est devenu un système puissant pour modéliser des maladies in vivo, notamment grâce à la technologie d’édition du génome CRISPR/Cas9. Cependant, la génération de lignées portant des mutations pathologiques humaines précises reste un défi majeur. Dans cette étude, nous avons étudié les capacités de plusieurs éditeurs de bases conçus pour introduire de manière précise dans le génome les mutations de C:G en T:A. En utilisant ces outils génétiques, nous avons élargi les possibilités d'édition de gènes chez le poisson zèbre en obtenant une mutagenèse jusqu'à 100% d'efficacité et nous avons développé de nouvelles approches pour effectuer une mutagenèse multiplexe et co-sélectionner les embryons les plus édités. Nous avons également exploré les applications biologiques de cette technologie, telles que l'activation endogène de voies de signalisation et la modélisation de cancer ou de syndromes rares. De plus, nous avons validé la pathogénicité de mutations trouvées chez de jeunes patients présentant un syndrome polymalformatif. Enfin, nous cherchons à développer le « Prime Editing » chez ce modèle animal, une technologie qui bien que très prometteuse reste à ce jour peu efficace. En conclusion, cette étude a contribué à fournir un large panel d'outils génétiques fonctionnant chez le poisson zèbre sans générer de cassures double brin d’ADN et d’en évaluer leurs limites ainsi que leur complémentarité chez cet organisme. Ce travail met en évidence que le poisson zèbre est un système puissant pour concevoir de nouvelles approches d'édition du génome et pour étudier les maladies génétiques.Zebrafish is an ideal model organism to study developmental biology and has become a powerful system for disease modeling in vivo. Since the emergence of the CRISPR/Cas9 technology, several disease models have been generated to introduce loss-of-function mutations, putting this vertebrate model at the forefront of genetic studies. However, the generation of mutant lines harboring precise pathological mutations is still challenging as knock-in approaches are still poorly efficient. In this study we investigated the capacities of several new cytosine base editor variants engineered in introducing precise C:G to T:A mutations into the genome. Using these tools, we broadened the gene editing possibilities in zebrafish by obtaining mutagenesis up to 100% of efficiency and we developed new approaches to perform multiplex mutagenesis and co-selection. We further explored biological applications of this technology such as endogenous activation of signaling pathway and disease modeling. In addition, we were able to validate in zebrafish the pathogenicity of mutations found in patients. Finally, we aimed at developing the prime editing technology in this animal model, a recent technology that although highly promising remains to date poorly efficient. To conclude, this study contributed in providing a large panel of genetic tools working in zebrafish without generating DNA double strand break, assessing their limitations and complementarity. Through the accessibility and ease of genome manipulations in this animal model, this work further highlights the use of zebrafish as a good platform to engineer new genome editing approaches in animals and to study genetic disorders

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

Regulation of the apical extension morphogenesis tunes the mechanosensory response of microvilliated neurons.

Multiple types of microvilliated sensory cells exhibit an apical extension thought to be instrumental in the detection of sensory cues. The investigation of the mechanisms underlying morphogenesis of sensory apparatus is critical to understand the biology of sensation. Most of what we currently know comes from the study of the hair bundle of the inner ear sensory cells, but morphogenesis and function of other sensory microvilliated apical extensions remain poorly understood. We focused on spinal sensory neurons that contact the cerebrospinal fluid (CSF) through the projection of a microvilliated apical process in the central canal, referred to as cerebrospinal fluid-contacting neurons (CSF-cNs). CSF-cNs respond to pH and osmolarity changes as well as mechanical stimuli associated with changes of flow and tail bending. In vivo time-lapse imaging in zebrafish embryos revealed that CSF-cNs are atypical neurons that do not lose their apical attachment and form a ring of actin at the apical junctional complexes (AJCs) that they retain during differentiation. We show that the actin-based protrusions constituting the microvilliated apical extension arise and elongate from this ring of actin, and we identify candidate molecular factors underlying every step of CSF-cN morphogenesis. We demonstrate that Crumbs 1 (Crb1), Myosin 3b (Myo3b), and Espin orchestrate the morphogenesis of CSF-cN apical extension. Using calcium imaging in crb1 and espin mutants, we further show that the size of the apical extension modulates the amplitude of CSF-cN sensory response to bending of the spinal cord. Based on our results, we propose that the apical actin ring could be a common site of initiation of actin-based protrusions in microvilliated sensory cells. Furthermore, our work provides a set of actors underlying actin-based protrusion elongation shared by different sensory cell types and highlights the critical role of the apical extension shape in sensory detection

Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish

International audienceWhile zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-toT point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA protospacer adjacent motif, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic an mutation of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes including cbl. With this last target, we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic diseaseassociated mutations

Disease modeling by efficient genome editing using a near PAM-less base editor in vivo

Abstract Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the base editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing nearly all PAM sequences, we could introduce point mutations for the first time in an animal model with high efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and we developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we apply our method to create a zebrafish model for melanoma predisposition based on the simultaneous base editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish

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