Fluorescent-based methods for gene knockdown and functional cardiac imaging in zebrafish.
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ABSTRACT:
SUBMITTER: Umemoto N
PROVIDER: S-EPMC3825550 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants often present in aquatic systems as complex mixtures. Embryonic fish are sensitive to the developmental toxicity of some PAHs, but the exact mechanisms involved in this toxicity are still unknown. This study explored the role of the aryl hydrocarbon receptor (AHR) in the oxidative stress response of zebrafish to the embryotoxicity of select PAHs. Embryos were exposed to two PAHs, benzo[k]fluoranthene (BkF; a strong AHR agonist) and fluoranthene (FL; a cytochrome P4501A (CYP1A) inhibitor), alone and in combination. CYP1A, CYP1B1, CYP1C1, and redox-responsive genes glutathione s-transferase pi 2 (GSTp2), glutathione peroxidase 1 (GPx1), the glutamate-cysteine ligase catalytic subunit (GCLc), MnSOD and CuZnSOD mRNA expression was examined. CYP1 activity was measured via an in vivo ethoxyresorufin-O-deethlyase (EROD) activity assay, and the area of the pericardium was measured as an index of cardiotoxicity. BkF or FL alone caused no deformities whereas BkF+FL resulted in extreme pericardial effusion. BkF induced CYP activity above controls and co-exposure with FL inhibited this activity. BkF induced expression of all three CYPs, GSTp2, and GCLc. BkF+FL caused greater than additive induction of the three CYPs, GSTp2, GPx1, and GCLc but had no effect on MnSOD or CuZnSOD. AHR2 knockdown protected against the cardiac deformities caused by BkF+FL and significantly inhibited the induction of the CYPs, GSTp2, GPx1, and GCLc after BkF+FL compared to non-injected controls. These results further show the protective role of AHR2 knockdown against cardiotoxic PAHs and the role of AHR2 as a mediator of redox-responsive gene induction.
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Project description:BACKGROUND: Reduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. However, methodology that allows spatio-temporally knockdown of gene expression in various model organisms such as zebrafish has not been well established, which largely limits the potential of zebrafish as a vertebrate model of human malignant disorders. PRINCIPAL FINDING: Here, we report that multiple copies of small hairpin RNA (shRNA) are expressed from a single transcript that mimics the natural microRNA-30e precursor (mir-shRNA). The mir-shRNA, when microinjected into zebrafish embryos, induced an efficient knockdown of two developmentally essential genes chordin and alpha-catenin in a dose-controllable fashion. Furthermore, we designed a novel cassette vector to simultaneously express an intronic mir-shRNA and a chimeric red fluorescent protein driven by lineage-specific promoter, which efficiently reduced the expression of a chromosomally integrated reporter gene and an endogenously expressed gata-1 gene in the developing erythroid progenitors and hemangioblasts, respectively. SIGNIFICANCE: This methodology provides an invaluable tool to knockdown developmental important genes in a tissue-specific manner or to establish animal models, in which the gene dosage is critically important in the pathogenesis of human disorders. The strategy should be also applicable to other model organisms.
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Project description:Although zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. Here we investigate the knockdown efficiency of three synthetic miRNA-expressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency. Using this approach, we reproduce spinal muscular atrophy (SMA) in zebrafish by targeting the smn1 gene. We also generate different transgenic lines, with severity and age of onset correlated to the level of smn1 inhibition, recapitulating for the first time the different forms of SMA in zebrafish. These lines are proof-of-concept that miRNA-based approaches can be used to generate potent heritable gene knockdown in zebrafish.
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Project description:The ribosomal proteins (RPs) form the majority of cellular proteins and are mandatory for cellular growth. RP genes have been linked, either directly or indirectly, to various diseases in humans. Mutations in RP genes are also associated with tissue-specific phenotypes, suggesting a possible role in organ development during early embryogenesis. However, it is not yet known how mutations in a particular RP gene result in specific cellular changes, or how RP genes might contribute to human diseases. The development of animal models with defects in RP genes will be essential for studying these questions. In this study, we knocked down 21 RP genes in zebrafish by using morpholino antisense oligos to inhibit their translation. Of these 21, knockdown of 19 RPs resulted in the development of morphants with obvious deformities. Although mutations in RP genes, like other housekeeping genes, would be expected to result in nonspecific developmental defects with widespread phenotypes, we found that knockdown of some RP genes resulted in phenotypes specific to each gene, with varying degrees of abnormality in the brain, body trunk, eyes, and ears at about 25 hours post fertilization. We focused further on the organogenesis of the brain. Each knocked-down gene that affected the morphogenesis of the brain produced a different pattern of abnormality. Among the 7 RP genes whose knockdown produced severe brain phenotypes, 3 human orthologs are located within chromosomal regions that have been linked to brain-associated diseases, suggesting a possible involvement of RP genes in brain or neurological diseases. The RP gene knockdown system developed in this study could be a powerful tool for studying the roles of ribosomes in human diseases.
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Project description:The zebrafish (Danio rerio) has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology) or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis). The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.
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Project description:BackgroundThe zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae.ResultsWe screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1?g/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells.ConclusionsThe coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.
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Project description:ABSTRACT The zebrafish is a widely used model organism for biomedical research due to its ease of maintenance, external fertilization of embryos, rapid embryonic development, and availability of established genetic tools. One notable advantage of using zebrafish is the transparency of the embryos, which enables high-resolution imaging of specific cells, tissues, and structures through the use of transgenic and knock-in lines. However, as the embryo develops, multiple layers of tissue wrap around the lipid-enriched yolk, which can create a challenge to image tissues located deep within the embryo. While various methods are available, such as two-photon imaging, cryosectioning, vibratome sectioning, and micro-surgery, each of these has limitations. In this study, we present a novel deyolking method that allows for high-quality imaging of tissues that are obscured by other tissues and the yolk. Embryos are lightly fixed in 1% PFA to remove the yolk without damaging embryonic tissues and are then refixed in 4% PFA and mounted on custom-made bridged slides. This method offers a simple way to prepare imaging samples that can be subjected to further preparation, such as immunostaining. Furthermore, the bridged slides described in this study can be used for imaging tissue and organ preparations from various model organisms. Summary: A new deyolking method for zebrafish embryos enables high-resolution confocal imaging of deep tissues, offering a simple and effective technique for biomedical research.
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Project description:The heart is the most important muscular organ of the cardiovascular system, which pumps blood and circulates, supplying oxygen and nutrients to peripheral tissues. Zebrafish have been widely explored in cardiotoxicity research. For example, the zebrafish embryo has been used as a human heart model due to its body transparency, surviving several days without circulation, and facilitating mutant identification to recapitulate human diseases. On the other hand, adult zebrafish can exhibit the amazing regenerative heart muscle capacity, while adult mammalian hearts lack this potential. This review paper offers a brief description of the major methodologies used to detect zebrafish cardiac rhythm at both embryonic and adult stages. The dynamic pixel change method was mostly performed for the embryonic stage. Other techniques, such as kymography, laser confocal microscopy, artificial intelligence, and electrocardiography (ECG) have also been applied to study heartbeat in zebrafish embryos. Nevertheless, ECG is widely used for heartbeat detection in adult zebrafish since ECG waveforms' similarity between zebrafish and humans is prominent. High-frequency ultrasound imaging (echocardiography) and modern electronic sensor tag also have been proposed. Despite the fact that each method has its benefits and limitations, it is proved that zebrafish have become a promising animal model for human cardiovascular disease, drug pharmaceutical, and toxicological research. Using those tools, we conclude that zebrafish behaviors as an excellent small animal model to perform real-time monitoring for the developmental heart process with transparent body appearance, to conduct the in vivo cardiovascular performance and gene function assays, as well as to perform high-throughput/high content drug screening.
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