Zebrafish最新文献

Stromal interaction molecules (STIMs) are endoplasmic reticulum-resident proteins that regulate Ca²⁺ homeostasis and signaling by store-operated calcium entry (SOCE). The different properties and functions of STIM1 and STIM2 have been described mostly based on work in vitro. STIM2 knockout mice do not survive until adulthood. Therefore, we generated and characterized stim2a and stim2b double-knockout zebrafish. The (stim2a;stim2b)^-/- zebrafish did not have any apparent morphological phenotype. However, RNA sequencing revealed 1424 differentially expressed genes. One of the most upregulated genes was annexin A3a, which is a marker of activated microglia. This corresponded well to an increase in Neutral Red staining in the in vivo imaging of the (stim2a;stim2b)^-/- zebrafish brain. The lack of Stim2 decreased zebrafish survival under low oxygen conditions. Behavioral tests, such as the visual-motor response test and dark-light preference test, indicated that (stim2a;stim2b)^-/- larvae might have problems with vision. This was consistent with the downregulation of many genes that are related to light perception. The periodic acid-Schiff staining of retina sections from adult zebrafish revealed alterations of the stratum pigmentosum, suggesting the involvement of a Stim2-dependent process in visual perception. Altogether, these data reveal new functions for Stim2 in the nervous system.

One of the greatest expenses in running a zebrafish laboratory is the aquatic systems used for housing. These critical pieces of equipment are essential and incorporate components undergoing constant activity in pumping water, monitoring, dosing, and filtration. The systems available on the market are robust, yet ongoing activity eventually leads to the need for repair or replacement. Moreover, some systems are no longer commercially available, impairing the ability to service this critical infrastructure. In this study, we demonstrate a do it yourself (DIY) method to re-engineer an aquatic system's pumps and plumbing, which hybridizes a system no longer commercially available with components used by active vendors. This transition from a two external pump Aquatic Habitat/Pentair design to an individual submerged pump Aquaneering-like plan saves funds by expanding infrastructure longevity. Our hybridized configuration has been in uninterrupted use for >3 years, supporting zebrafish health and high fecundity.

Zebrafish are widely used as experimental animal models. They are small and move fast in the water. Real-time imaging of fast-moving zebrafish is a challenge, and it requires that the imaging technique has higher spatiotemporal resolution and penetration ability. The purpose of this study was to evaluate the feasibility of dynamic phase retrieval (PR)-based phase-contrast imaging (PCI) for real-time displaying of the breathing and swimming process in unanesthetized free-moving zebrafish, and to evaluate the feasibility of PR-based phase-contrast CT (PCCT) for visualizing the soft tissues in anesthetized living zebrafish. PR was performed using the phase-attenuation duality (PAD) method with the δ/β values (PAD property) of 100 and 1000 for dynamic PR-based PCI and PR-based PCCT, respectively. The contrast-to-noise ratio (CNR) was used for quantitatively assessing the visibility of the adipose tissue and muscle tissue. The skeleton and swim bladder chambers in fast-moving zebrafish were clearly shown. The dynamic processes of breathing and swimming were visibly recorded. The respiratory intensity and frequency and the movement flexibility of the zebrafish could be dynamically evaluated. By producing more obvious image contrast, PR-based PCCT clearly showed the adipose tissue and muscle tissue. The CNRs from PR-based PCCT were significantly higher than those from PR-free PCCT for both adipose tissue (9.256 ± 2.037 vs. 0.429 ± 0.426, p < 0.0001) and muscle tissue (7.095 ± 1.443 vs. 0.324 ± 0.267, p < 0.0001). Dynamic PR-based PCI holds the potential for investigating both morphological abnormalities and motor disorders. PR-based PCCT offers clear visualization and the potential for quantification of soft tissues in living zebrafish.

The liver plays a very important role in physiological processes of the human body. Liver regeneration has developed into an important area of study in liver disease. The Mtz (metronidazole)/NTR (nitroreductase)-mediated cell ablation system has been widely used to study the processes and mechanisms of liver injury and regeneration. However, high concentrations and toxic side effects of Mtz severely limit the application of the Mtz/NTR system. Therefore, screening new analogs to replace Mtz has become an important means to optimize the NTR ablation system. In this study, we screened five Mtz analogs including furazolidone, ronidazole, ornidazole, nitromide, and tinidazole. We compared their toxicity on the transgenic fish line Tg(fabp10a: mCherry-NTR) and their specific ablation ability on liver cells. The results showed that Ronidazole at a lower concentration (2 mM) had the same ability to ablate liver cells comparable with that of Mtz (10 mM), almost without toxic side effects on juvenile fish. Further study found that zebrafish hepatocyte injury caused by the Ronidazole/NTR system achieved the same liver regenerative effect as the Mtz/NTR system. The above results show that Ronidazole can replace Mtz with NTR to achieve superior damage and ablation effects in zebrafish liver.

Zebrafish is a popular animal model in regeneration studies due to their ability to regenerate the heart. Primary cardiomyocytes could be an alternative tool for studying the intrinsic mechanisms of cardiovascular disease in vitro. Thus, our objective is to develop an efficient protocol to isolate primary cardiomyocytes from zebrafish hearts. Low concentration of digestive enzyme (0.5 mg/mL collagenase type II) was utilized in our protocol to obtain single-cell suspension. The ventricles were fragmented, mechanically pipetted, and constantly shaken to ensure adequate contact between the tissues and the enzyme. Preplating the cell suspension onto culture plates for 2 h helped remove cardiac fibroblasts. The purity of isolated cells was validated by flow cytometry analysis of transgenic zebrafish with cardiomyocyte-specific expression of enhanced green fluorescent protein (EGFP) or endothelial cell-specific expression of mCherry. Quantitative real-time PCR analysis revealed a high level of the purity, with cardiac fibroblasts, endothelial cells, and epicardial cell markers scarcely detected in the purified cells. Altogether, this study established a reproducible protocol for isolating primary cardiomyocytes with high purity and activity from adult zebrafish hearts that can be cultured in vitro for up to 4 weeks. This protocol provides a valuable tool for studying the intrinsic mechanisms of cardiovascular disease in vitro using primary cardiomyocytes.

Despite advances in research employing zebrafish (Danio rerio) as an animal model, it is still difficult to obtain animals with attested sanitary qualities from commercial breeders. This study reports the first-time occurrence of Eustrongylides spp. parasitism in a zebrafish colony recently acquired from a pet store supplier to begin a research colony at a scientific facility. This parasite has not been reported in any current zebrafish health-monitoring guidelines to date. Thus, this report is a warning to breeders and researchers that this nematode can parasitize zebrafish, causing high lethality rates and compromising research outcomes.

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa^-/- null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa^-/- zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa^-/- mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa^-/- mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa^-/- mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.

Population aging is a global problem worldwide, and the discovery of antiaging drugs and knowledge of their potential molecular mechanisms are research hotspots in biomedical field. Tetrahydroxystilbene glucoside (TSG) is a natural component isolated from Heshouwu (Polygonum multiflorum Thunb.). It has been widely used to treat various chronic diseases for its remarkable biological activities. In this study, we successfully established aging larval zebrafish by exposing larvae to 2 mM hydrogen peroxide (H₂O₂). Using this aging model, we assessed the antiaging effect of TSG with different concentrations (25-100 μg/mL). After being treated with H₂O₂, zebrafish showed the obvious aging-associated phenotypes characterized by higher senescence-associated β-galactosidase activity, significantly downregulated expression of sirtuin 1 (sirt1) and telomerase reverse transcriptase (tert), and upregulated serpine1 mRNA level compared to the control group. TSG pretreatment delayed the aging process of oxidative stress-induced zebrafish, indicative of the reduced positive rate of senescence-associated β-galactosidase, improved swimming velocity, and stimulus-response capacity. Further studies proved that TSG could suppress reactive oxygen species production and enhance the activity of antioxidant enzymes superoxide dismutase and catalase. TSG also inhibited the H₂O₂-induced expressions of inflammation-related genes il-1β, il-6, cxcl-c1c, and il-8 in aging zebrafish, but it did not affect apoptosis-related genes (bcl-2, bax, and caspase-3) of aging zebrafish. In conclusion, TSG can protect against aging by regulating the antioxidative genes and enzyme activity, as well as inflammation in larval zebrafish, providing insight into the application of TSG for clinical treatment of aging or aging-related diseases.

Gymnotiformes a monophyletic group of fish endemic to the Neotropics, represent an important component of the freshwater ichthyofauna that presents relevant taxonomic problems. Thus, in view of the morphological complexity involving Eigenmannia (Gymnotiformes) fish species, this study aimed to characterize Eigenmannia aff. desantanai of the upper Paraguay River basin through cytogenetic and molecular analyses, to help in the correct identification and delimitation of species. This study reports a multiple sex system of the type ZW₁W₂/ZZ, with 2n = 31 for females and 2n = 30 for males. A single pair of chromosomes carrying the nucleolar organizing regions (NORs) was detected. The heterochromatin was colocated in NOR sites and mainly located in the centromeric regions of chromosomes. Besides that, individual sequences COI from the specimens of E. aff. desantanai were obtained, totalizing three haplotypes. The distance p between the haplotypes in E. aff. desantanai, ranged from 0.2% to 7.1%. Species delimitation tests indicated the existence of two possible operational taxonomic units of E. aff. desantanai. Thus, this study reports a new multiple sex system in Gymnotiformes and these specimens previously identified as E. aff. desantanai may belong to two distinct species.