CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of ...knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish.
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•Urod inactivation using CRISPR yields a fluorescent phenotype in zebrafish embryos•A CRISPR-based vector system enables tissue-specific gene inactivation in zebrafish•The targeting vector can be easily modified for any gene and tissue-specific promoter
Ablain et al. describe a vector system for spatial control of gene disruption in zebrafish. Based on the CRISPR technology, this modular vector, which only requires a tissue-specific promoter and a gene-specific target sequence, greatly expands the range of loss-of-function studies in vivo.
Hematopoiesis Jagannathan-Bogdan, Madhumita; Zon, Leonard I
Development (Cambridge),
06/2013, Letnik:
140, Številka:
12
Journal Article
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Hematopoiesis - the process by which blood cells are formed - has been studied intensely for over a century using a variety of model systems. There is conservation of the overall hematopoietic ...process between vertebrates, although some differences do exist. Over the last decade, the zebrafish has come to the forefront as a new model in hematopoiesis research, as it allows the use of large-scale genetics, chemical screens and transgenics. This comparative approach to understanding hematopoiesis has led to fundamental knowledge about the process and to the development of new therapies for disease. Here, we provide a broad overview of vertebrate hematopoiesis. We also highlight the benefits of using zebrafish as a model.
Zebrafish have been widely used as a model system for studying developmental processes, but in the last decade, they have also emerged as a valuable system for modeling human disease. The development ...and function of zebrafish organs are strikingly similar to those of humans, and the ease of creating mutant or transgenic fish has facilitated the generation of disease models. Here, we highlight the use of zebrafish for defining disease pathways and for discovering new therapies.
When stem cells divide, they can generate progeny with the same developmental potential as the original cell, a process referred to as self-renewal. Self-renewal is driven intrinsically by gene ...expression in a cell-type-specific manner and is modulated through interactions with extrinsic cues from the environment, such as growth factors. However, despite the prevalence of the term self-renewal in the scientific literature, this process has not been defined at the molecular level. Haematopoietic stem cells are an excellent model for the study of self-renewal because they can be isolated prospectively, manipulated relatively easily and assessed by using well-defined assays. Establishing the principles of self-renewal in haematopoietic stem cells will lead to insights into the mechanisms of self-renewal in other tissues.
Long restricted to the field of developmental biology, the use of the zebrafish ( Danio rerio ) has extended to the study of human pathogenesis. Fostered by the rapid adaptation of new technologies, ...the design and analysis of fish models of human diseases have contributed important findings that are now making their way from aquariums to clinics. Here we outline the clinical relevance of the zebrafish as a model organism.
Use of Zebrafish in Drug Discovery Toxicology Cassar, Steven; Adatto, Isaac; Freeman, Jennifer L ...
Chemical research in toxicology,
01/2020, Letnik:
33, Številka:
1
Journal Article
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Unpredicted human safety events in clinical trials for new drugs are costly in terms of human health and money. The drug discovery industry attempts to minimize those events with diligent preclinical ...safety testing. Current standard practices are good at preventing toxic compounds from being tested in the clinic; however, false negative preclinical toxicity results are still a reality. Continual improvement must be pursued in the preclinical realm. Higher-quality therapies can be brought forward with more information about potential toxicities and associated mechanisms. The zebrafish model is a bridge between in vitro assays and mammalian in vivo studies. This model is powerful in its breadth of application and tractability for research. In the past two decades, our understanding of disease biology and drug toxicity has grown significantly owing to thousands of studies on this tiny vertebrate. This Review summarizes challenges and strengths of the model, discusses the 3Rs value that it can deliver, highlights translatable and untranslatable biology, and brings together reports from recent studies with zebrafish focusing on new drug discovery toxicology.
DNA methylation at the 5 position of cytosine (5-mC) is a key epigenetic mark that is critical for various biological and pathological processes. 5-mC can be converted to 5-hydroxymethylcytosine ...(5-hmC) by the ten-eleven translocation (TET) family of DNA hydroxylases. Here, we report that “loss of 5-hmC” is an epigenetic hallmark of melanoma, with diagnostic and prognostic implications. Genome-wide mapping of 5-hmC reveals loss of the 5-hmC landscape in the melanoma epigenome. We show that downregulation of isocitrate dehydrogenase 2 (IDH2) and TET family enzymes is likely one of the mechanisms underlying 5-hmC loss in melanoma. Rebuilding the 5-hmC landscape in melanoma cells by reintroducing active TET2 or IDH2 suppresses melanoma growth and increases tumor-free survival in animal models. Thus, our study reveals a critical function of 5-hmC in melanoma development and directly links the IDH and TET activity-dependent epigenetic pathway to 5-hmC-mediated suppression of melanoma progression, suggesting a new strategy for epigenetic cancer therapy.
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► Loss of 5-hmC is an epigenetic hallmark of melanoma, with diagnostic/prognostic value ► Genome-wide mapping reveals a demolished 5-hmC landscape in human melanoma epigenome ► Downregulating IDH2 and TETs suggests a mechanism underlying 5-hmC loss in melanoma ► TET2 and IDH2 set the 5-hmC landscape, suppress melanoma growth, and increase survival
Genome-wide mapping of 5-hmC reveals that loss of 5-hmC is an epigenetic hallmark of melanoma, with diagnostic and prognostic implications. Downregulation of IDH2 and TET family enzymes likely underlies 5-hmC loss in melanoma, and rebuilding the 5-hmC landscape suppresses melanoma growth in animal models.
Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to ...perform high-resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization.
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•Direct observation of endogenous blood stem cells using highly specific transgenics•Perivascular lodgement of blood stem cells triggers active remodeling of the niche•Correlative light and electron microscopy reveals high-resolution niche architecture•Chemical treatment of the embryo increases the size of the stem cell pool in adults
Live imaging reveals that hematopoietic stem cell lodgement in the perivascular niche triggers remodeling of endothelial cells into a surrounding pocket. The stem cell becomes anchored to a single mesenchymal stromal cell that orients its division plane.
Numerous drug treatments that have recently entered the clinic or clinical trials have their genesis in zebrafish. Zebrafish are well established for their contribution to developmental biology and ...have now emerged as a powerful preclinical model for human disease, as their disease characteristics, aetiology and progression, and molecular mechanisms are clinically relevant and highly conserved. Zebrafish respond to small molecules and drug treatments at physiologically relevant dose ranges and, when combined with cell-specific or tissue-specific reporters and gene editing technologies, drug activity can be studied at single-cell resolution within the complexity of a whole animal, across tissues and over an extended timescale. These features enable high-throughput and high-content phenotypic drug screening, repurposing of available drugs for personalized and compassionate use, and even the development of new drug classes. Often, drugs and drug leads explored in zebrafish have an inter-organ mechanism of action and would otherwise not be identified through targeted screening approaches. Here, we discuss how zebrafish is an important model for drug discovery, the process of how these discoveries emerge and future opportunities for maximizing zebrafish potential in medical discoveries.
Modeling Cancer with Flies and Fish Cagan, Ross L.; Zon, Leonard I.; White, Richard M.
Developmental cell,
05/2019, Letnik:
49, Številka:
3
Journal Article
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Cancer has joined heart disease as the leading source of mortality in the US. In an era of organoids, patient-derived xenografts, and organs on a chip, model organisms continue to thrive with a ...combination of powerful genetic tools, rapid pace of discovery, and affordability. Model organisms enable the analysis of both the tumor and its associated microenvironment, aspects that are particularly relevant to our understanding of metastasis and drug resistance. In this Perspective, we explore some of the strengths of fruit flies and zebrafish for addressing fundamental cancer questions and how these two organisms can contribute to identifying promising therapeutic candidates.
In this Perspective, Cagan et al. discuss how powerful genetic tools, a rapid pace of discovery, and affordability contribute to the strength of flies and zebrafish as models for cancer research. They highlight studies addressing fundamental cancer questions and consider how these organisms can contribute to identifying promising therapeutic candidates.
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