Zebrafish最新文献

Early research experiences positively affect students' interest in STEM careers, and develop practical science and critical thinking skills. However, outreach opportunities are not equally accessible for all students. In states like West Virginia, where many students live in rural Appalachian communities, opportunities for engaging in STEM experiences are limited. In addition, rural teachers may not be equipped to provide authentic research experiences for students due to lack of resources or support. For many students in West Virginia, the Health Sciences and Technology Academy (HSTA) is a major opportunity for STEM engagement. Since its inception in 1998, HSTA has spread to 26 of 55 counties in West Virginia. The program recruits first-generation, low-socioeconomic status, rurally living, and African American high school students who are under-represented in STEM fields. Our research laboratory partnered with HSTA to implement an innovative, hands-on research camp using zebrafish for students participating in their annual junior-level biomedical sciences summer camp. Our camp was held in-person and adapted to an online format during the Covid-19 pandemic. We used pre-post surveys in both camps to assess impacts on science confidence and to collect information about general perceptions of zebrafish, research, and STEM fields. We found that students participating in the in-person and online camps experienced similar overall gains in science confidence. We also identified strong interest in zebrafish, research, and STEM degrees among online students. Online students did not prefer virtual learning experiences; however, they still enjoyed our camp. We also surveyed high school teachers volunteering for HSTA to identify factors that would encourage use of zebrafish in classrooms. The most prominent needs include classroom supplies, experience, and funding. Our successful science-education partnership demonstrates that zebrafish research experiences foster positive outcomes for under-represented students, and can inform future outreach efforts and collaborations with teachers.

Zebrafish (Danio rerio) is a widely used vertebrate animal for modeling genetic diseases by targeted editing strategies followed by gross phenotypic and biomarker characterization. While larval transparency permits microscopic detection of anatomical defects, histological adult screening for organ-level defects remains invasive, tedious, inefficient, and subject to technical artifact. Here, we describe a noninvasive magnetic resonance imaging (MRI) approach to systematically screen adult zebrafish for anatomical growth defects. An anatomical atlas of wild-type (WT) zebrafish at 5-31 months post-fertilization was created by ex vivo MRI with a 9.4 T magnet. Volumetric growth over time was measured of animals and major organs, including the brain, spinal cord, heart, eyes, optic nerve, ear, liver, kidneys, and swim bladder. Subsequently, surf1^-/-, fbxl4^-/-, and opa1^+/- mitochondrial disease mutant adult zebrafish were quantitatively studied to compare organ volumes with age-matched WT zebrafish. Results demonstrated that MRI enabled noninvasive, high-resolution, rapid screening of mutant adult zebrafish for overall and organ-specific growth abnormalities. Detailed volumetric analyses of three mitochondrial disease mutants delineated specific organ differences, including significantly increased brain growth in surf1^-/- and opa1^+/-, and marginally significant decreased heart and spinal cord volumes in surf1^-/- mutants. This is interesting as we know neurological involvement can be seen in SURF1^-/- patients with ataxia, dystonia, and lesions in basal ganglia, as well as in OPA1^+/- patients with spasticity, ataxia, and hyperreflexia indicative of neuropathology. Similarly, cardiomyopathy is a known sequelae of cardiac pathology in patients with SURF1^-/--related disease. Future studies will define MRI signaling patterns of organ dysfunction to further delineate specific pathology.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is commonly used to measure the mRNA expression of target genes in zebrafish. Gene expression values from RT-qPCR are typically reported as relative fold-changes, and relative quantification of RT-qPCR data incorporates primer amplification efficiency values for each target gene. We describe the influence of the primer amplification efficiency analysis method on RT-qPCR gene expression fold-change calculations. This report describes (1) a sample analysis demonstrating incorporation of primer amplification efficiency into RT-qPCR analysis for comparing gene expression of a gene of interest between two groups when normalized to multiple reference genes, (2) the influence of differences in primer amplification efficiencies between measured genes on gene expression differences calculated from theoretical delta-C_q (dC_q) values, and (3) an empirical comparison of the influence of three methods of defining primer amplification efficiency in gene expression analyses (delta-delta-C_q [ddC_q], standard curve, LinRegPCR) using mRNA measurements of a set of genes in zebrafish embryonic development. Given the need to account for the influence of primer amplification efficiency along with the simplicity of using software programs (LinRegPCR) to measure primer amplification efficiency from RT-qPCR data, we encourage using empirical measurements of primer amplification efficiency for RT-qPCR analysis of differential gene expression in zebrafish.

Regenerative medicine is an emerging field of research aiming to understand the wound healing mechanisms and to develop new therapeutic strategies. Nanocarriers are used to improve drug bioavailability, solubility, and therapeutic abilities. In this study, we used for the first time curcumin loaded oligo kappa-carrageenan-graft-polycaprolactone (oligoKC-g-PCL) nanomicelles to investigate their regenerative potential using a model of tail amputation in zebrafish eleutheroembryo. First, we showed that curcumin encapsulated oligoKC-g-PCL spherical micelles had a mean size of 92 ± 32 nm and that micelles were successfully loaded with curcumin. These micelles showed a slow and controlled drug release over 72 h. The toxicity of curcumin nanomicelles was then tested on zebrafish eleutheroembryo based on the survival rate after 24 h. At nontoxic concentration, curcumin nanomicelles improved tail regeneration within 3 days postamputation, compared with empty micelles or curcumin alone. Furthermore, we demonstrated that curcumin nanomicelles increased the recruitment of neutrophils and macrophages 6 h postlesion. Finally, our study highlights the efficiency of oligoKC-g-PCL nanomicelles for encapsulation of hydrophobic molecules such as curcumin. Indeed, our study demonstrates that curcumin nanomicelles can modulate inflammatory reactions in vivo and promote regenerative processes. However, further investigations will be required to better understand the mechanisms sustaining regeneration and to develop new therapeutics.

Inbred species are useful resources for a variety of biomedical research applications. To create isogenic zebrafish, it is feasible to stop meiosis II (repeatedly) or mitosis (two times) in a haploid embryo by applying pressure or by delivering a heat shock, respectively. In this study, to improve the repeatability, we suggest a less complicated approach based on sperm ultraviolet-C (UV-C) exposure for a shorter period followed by heat shock at various temperatures, eliminating the use of pressure in meiotic therapy since heat shock is more accessible to laboratories. In this study, the survivability rates of meiotic (Mei) and mitotic (Mit) gynogenesis offspring produced by various combinations of irradiation (28.5, 105, and 210 mJ/cm²) and temperature (Mei: 40.40°C, 40.60°C, or 40.90°C; Mt: 41.40°C, 41.90°C, or 42.45°C) were compared with diploid (C) and haploid (H) controls. Our findings demonstrated that 40.60°C and 41.90°C were the most suitable temperatures to produce meiotic and mitotic gynogenesis, respectively, whereas 28.5 mJ/cm² was more successful in ensuring haploid embryos. As a result, we deduced that meiotic gynogenesis produces more viable offspring than the mitotic approach and requires a lower temperature to maintain the second polar body.

Emergency granulopoiesis (EG) is a response to severe inflammation in which increased neutrophils are generated in the hematopoietic tissue. Photolabeling is utilized to distinguish newly developed neutrophils from existing neutrophils. However, this technique requires a strong laser line and labels subsets of the existing neutrophils. Here we create a transgenic zebrafish line that expresses a time-dependent switch from green fluorescent protein (GFP) to red fluorescent protein (RFP) in neutrophils, which allows quantification of EG using simple GFP/RFP ratiometric imaging.

The effects of manganese (Mn) toxicity in different organs and tissues in humans and other vertebrates have been studied since the beginning of the past century, but most of its cellular effects remain largely unknown. In this study, we studied the effects of Mn in zebrafish, at the cellular level, due to the transparent nature of zebrafish larvae that enables a powerful analysis under the light microscope. The collection of our results shows that environmental concentrations of 0.5 mg/L affect swim bladder inflation; at concentration of 50 and 100 mg/L Mn (1) induces alterations in viability, swim bladder, heart, and size of zebrafish larvae, (2) induces an increase in melanocyte area and the formation of cellular aggregates in the skin, and (3) induces an accumulation of β-Catenin in mesenchymal cells in the caudal fin of zebrafish larvae. Our data suggest that increased levels of Mn induce cell aggregate formation in the skin and the presence of more melanocytes in the zebrafish caudal fin. Interestingly, the adhesion protein β-Catenin was activated in mesenchymal cells near the cell aggregates. These results open important new questions on the role of Mn toxicity on cellular organization and β-Catenin responses in fishes.

Power outages can happen anywhere and anytime for various reasons. This threat affects also scientific work of biologists. Especially problematic area is aquatic animal husbandry, where life support of the animals is dependent on continuous electricity supply and years of scientific work may depend on the well-being of these animal stocks. Therefore, tools to estimate and control these risks are needed. In this study, I have used modeling to estimate aquarium water temperature changes during power outages and constructed simplified models for zebrafish aquaria. A calculation worksheet is also provided to help to model kinetics of water temperature changes in zebrafish facilities.

The use of larval zebrafish developmental testing and assessment, specifically larval zebrafish locomotor activity, has been recognized as a higher throughput testing strategy to identify developmentally toxic and neurotoxic chemicals. There are, however, no standardized protocols for this type of assay, which could result in confounding variables being overlooked. Two chemicals commonly employed during early-life stage zebrafish assays, methylene blue (antifungal agent) and dimethyl sulfoxide (DMSO, a commonly used vehicle) have been reported to affect the morphology and behavior of freshwater fish. In this study, we conducted developmental toxicity (morphology) and neurotoxicity (behavior) assessments of commonly employed concentrations for both chemicals (0.6-10.0 μM methylene blue; 0.3%-1.0% v/v DMSO). A light-dark transition behavioral testing paradigm was applied to morphologically normal, 6 days postfertilization (dpf) zebrafish larvae kept at 26°C. Additionally, an acute DMSO challenge was administered based on early-life stage zebrafish assays typically used in this research area. Results from developmental toxicity screens were similar between both chemicals with no morphological abnormalities detected at any of the concentrations tested. However, neurodevelopmental results were mixed between the two chemicals of interest. Methylene blue resulted in no behavioral changes up to the highest concentration tested, 10.0 μM. By contrast, DMSO altered larval behavior following developmental exposure at concentrations as low as 0.5% (v/v) and exhibited differential concentration-response patterns in the light and dark photoperiods. These results indicate that developmental DMSO exposure can affect larval zebrafish locomotor activity at routinely used concentrations in developmental neurotoxicity assessments, whereas methylene blue does not appear to be developmentally or neurodevelopmentally toxic to larval zebrafish at routinely used concentrations. These results also highlight the importance of understanding the influence of experimental conditions on larval zebrafish locomotor activity that may ultimately confound the interpretation of results.