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Journal of Visualized Experiments : JoVE Aug 2022Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early...
Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.
Topics: 5-Methylcytosine; Animals; Cytosine; DNA Methylation; Embryonic Development; Oxidation-Reduction; Zebrafish
PubMed: 36063021
DOI: 10.3791/64190 -
Circulation Research Mar 2012Heart development is a complex process that involves cell specification and differentiation, as well as elaborate tissue morphogenesis and remodeling, to generate a... (Review)
Review
Heart development is a complex process that involves cell specification and differentiation, as well as elaborate tissue morphogenesis and remodeling, to generate a functional organ. The zebrafish has emerged as a powerful model system to unravel the basic genetic, molecular, and cellular mechanisms of cardiac development and function. We summarize and discuss recent discoveries on early cardiac specification and the identification of the second heart field in zebrafish. In addition to the inductive signals regulating cardiac specification, these studies have shown that heart development also requires a repressive mechanism imposed by retinoic acid signaling to select cardiac progenitors from a multipotent population. Another recent advance in the study of early zebrafish cardiac development is the identification of the second heart field. These studies suggest that the molecular mechanisms that regulate the second heart field development are conserved between zebrafish and other vertebrates including mammals and provide insight into the evolution of the second heart field and its derivatives.
Topics: Animals; Gene Expression Regulation, Developmental; Heart; Models, Animal; Signal Transduction; Zebrafish
PubMed: 22427324
DOI: 10.1161/CIRCRESAHA.111.246504 -
Human Gene Therapy Apr 2016Since its introduction in early 1980s, the zebrafish (Danio rerio) has become an invaluable vertebrate animal model system to study many human disorders in almost all... (Review)
Review
Since its introduction in early 1980s, the zebrafish (Danio rerio) has become an invaluable vertebrate animal model system to study many human disorders in almost all systems, from hepatic and brain pathology, to autoimmune and psychiatric disorders. Hematopoiesis between zebrafish and mammals is highly conserved, making the zebrafish an attractive model to study hematopoietic development and blood disorders. Unique attributes of the zebrafish include the ability to perform large-scale genetic and chemical screens in vivo, study development at the cellular level, and use transgenic fish to dissect mechanisms of disease or drug effects. This review summarizes major discoveries that helped define molecular control of hematopoiesis in vertebrates and specific contributions from studies in zebrafish.
Topics: Animals; Blood; Genetic Testing; Hematologic Diseases; Hematopoiesis; Humans; Reverse Genetics; Zebrafish
PubMed: 27018965
DOI: 10.1089/hum.2016.024 -
Developmental Biology Dec 1999The study of blood has often defined paradigms that are relevant to the biology of other vertebrate organ systems. As examples, stem cell physiology and the structure of... (Review)
Review
The study of blood has often defined paradigms that are relevant to the biology of other vertebrate organ systems. As examples, stem cell physiology and the structure of the membrane cytoskeleton were first described in hematopoietic cells. Much of the reason for these successes resides in the ease with which blood cells can be isolated and manipulated in vitro. The cell biology of hematopoiesis can also be illuminated by the study of human disease states such as anemia, immunodeficiency, and leukemia. The sequential development of the blood system in vertebrates is characterized by ventral mesoderm induction, hematopoietic stem cell specification, and subsequent cell lineage differentiation. Some of the key regulatory steps in this process have been uncovered by studies in mouse, chicken, and Xenopus. More recently, the genetics of the zebrafish (Danio rerio) have been employed to define novel points of regulation of the hematopoietic program. In this review, we describe the advantages of the zebrafish system for the study of blood cell development and the initial success of the system in this pursuit. The striking similarity of zebrafish mutant phenotypes and human diseases emphasizes the utility of this model system for elucidating pathophysiologic mechanisms. New screens for lineage-specific mutations are beginning, and the availability of transgenics promises a better understanding of lineage-specific gene expression. The infrastructure of the zebrafish system is growing with an NIH-directed genome initiative, providing a detailed map of the zebrafish genome and an increasing number of candidate genes for the mutations. The zebrafish is poised to contribute greatly to our understanding of normal and disease-related hematopoiesis.
Topics: Animals; Disease Models, Animal; Gene Expression Regulation, Developmental; Hematopoiesis; Hematopoietic Stem Cells; Humans; In Situ Hybridization; Zebrafish
PubMed: 10588859
DOI: 10.1006/dbio.1999.9462 -
Experimental Hematology Aug 2014Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation... (Review)
Review
Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor, and effector cell emergence, expansion, and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell types can be identified and characterized. Further, myriad transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of blood stem and progenitor cell biology during development, in response to infection or injury, or in the setting of hematologic malignancy continues to deepen, zebrafish will remain essential for exploring the spatiotemporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease.
Topics: Animals; Cell Lineage; Hematopoiesis; Hematopoietic Stem Cells; Models, Animal; Zebrafish
PubMed: 24816275
DOI: 10.1016/j.exphem.2014.05.002 -
Nature Communications Jan 2023Cardiac valves ensure unidirectional blood flow through the heart, and altering their function can result in heart failure. Flow sensing via wall shear stress and wall...
Cardiac valves ensure unidirectional blood flow through the heart, and altering their function can result in heart failure. Flow sensing via wall shear stress and wall stretching through the action of mechanosensors can modulate cardiac valve formation. However, the identity and precise role of the key mechanosensors and their effectors remain mostly unknown. Here, we genetically dissect the role of Pkd1a and other mechanosensors in atrioventricular (AV) valve formation in zebrafish and identify a role for several pkd and piezo gene family members in this process. We show that Pkd1a, together with Pkd2, Pkd1l1, and Piezo2a, promotes AV valve elongation and cardiac morphogenesis. Mechanistically, Pkd1a, Pkd2, and Pkd1l1 all repress the expression of klf2a and klf2b, transcription factor genes implicated in AV valve development. Furthermore, we find that the calcium-dependent protein kinase Camk2g is required downstream of Pkd function to repress klf2a expression. Altogether, these data identify, and dissect the role of, several mechanosensors required for AV valve formation, thereby broadening our understanding of cardiac valvulogenesis.
Topics: Animals; Zebrafish; Animals, Genetically Modified; Heart Valves; Zebrafish Proteins; Organogenesis
PubMed: 36639367
DOI: 10.1038/s41467-023-35843-3 -
The International Journal of... 2009Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include... (Review)
Review
Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.
Topics: Amino Acid Sequence; Animals; Automation; Body Patterning; Computational Biology; Developmental Biology; Gene Library; Humans; Immune System; Inflammation; Models, Biological; Molecular Sequence Data; Phenotype; Sequence Homology, Amino Acid; Zebrafish
PubMed: 19557689
DOI: 10.1387/ijdb.082615sb -
Journal of Neurogenetics Jun 2016Over the course of each day, animals prioritize different objectives. Immediate goals may reflect fluctuating internal homeostatic demands, prompting individuals to seek... (Review)
Review
Over the course of each day, animals prioritize different objectives. Immediate goals may reflect fluctuating internal homeostatic demands, prompting individuals to seek out energy supplies or warmth. At other times, the environment may present temporary challenges or opportunities. Homeostatic demands and environmental signals often elicit persistent changes in an animal's behavior to meet needs and challenges over extended periods of time. These changes reflect the underlying motivational state of the animal. The larval zebrafish has been established as an effective genetically tractable vertebrate system to study neural circuits for sensory-motor reflexes. Fewer studies have exploited zebrafish to study brain circuits that control motivated behavior. In part this is because appropriate conceptual frameworks, anatomical knowledge, and behavioral paradigms are not yet well established. This review sketches a general conceptual framework for studying motivated state control in animal models, how this applies to larval zebrafish, and the current knowledge on neuroanatomical substrates for state control in this model.
Topics: Animals; Behavior, Animal; Brain; Larva; Motivation; Neural Pathways; Zebrafish
PubMed: 27293113
DOI: 10.1080/01677063.2016.1177048 -
Current Biology : CB Apr 2010A central goal of modern neuroscience is to obtain a mechanistic understanding of higher brain functions under healthy and diseased conditions. Addressing this challenge... (Review)
Review
A central goal of modern neuroscience is to obtain a mechanistic understanding of higher brain functions under healthy and diseased conditions. Addressing this challenge requires rigorous experimental and theoretical analysis of neuronal circuits. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of synaptic wiring diagrams have created new opportunities to achieve this goal. To fully harness these methods, model organisms should allow for a combination of genetic and neurophysiological approaches in vivo. Moreover, the brain should be small in terms of neuron numbers and physical size. A promising vertebrate organism is the zebrafish because it is small, it is transparent at larval stages and it offers a wide range of genetic tools and advantages for neurophysiological approaches. Recent studies have highlighted the potential of zebrafish for exhaustive measurements of neuronal activity patterns, for manipulations of defined cell types in vivo and for studies of causal relationships between circuit function and behavior. In this article, we summarize background information on the zebrafish as a model in modern systems neuroscience and discuss recent results.
Topics: Animals; Behavior, Animal; Brain; Gene Expression; Models, Animal; Nerve Net; Neurosciences; Olfactory Pathways; Visual Pathways; Zebrafish
PubMed: 21749961
DOI: 10.1016/j.cub.2010.02.039 -
Journal of Cardiovascular Translational... Dec 2011Proper atrioventricular canal (AVC) patterning and subsequent valvulogenesis is a complex process, and defects can result in disease or early death. The zebrafish Danio... (Review)
Review
Proper atrioventricular canal (AVC) patterning and subsequent valvulogenesis is a complex process, and defects can result in disease or early death. The zebrafish Danio rerio has become a useful model system for studying AVC development, and much progress has been made in dissecting out the critical steps. Here, we review the recent advances in the field and highlight the cellular and molecular changes observed during zebrafish AVC development.
Topics: Animals; Body Patterning; Endocardial Cushions; Gene Expression Regulation, Developmental; Heart Valves; Morphogenesis; Signal Transduction; Zebrafish
PubMed: 21948390
DOI: 10.1007/s12265-011-9313-z