zebrafish
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Notch signalling is critical for the development of the nervous system. In the zebrafish mindbomb mutants, disruption of E3 ubiquitin ligase activity inhibits Notch signalling. In these mutant embryos, precocious development of primary neurons leading to depletion of neural progenitor cells results in a neurogenic phenotype characterized by defects in neural patterning and brain development. Cyclin-dependent kinase 5 (Cdk5), a predominant neuronal kinase, is involved in a variety of essential functions of the nervous system. Most recently, mammalian studies on Notch and Cdk5 regulating each other's function have been emerging. The status of Cdk5 in the mindbomb mutant embryos with excessive primary neurons is not known. In situ hybridization of the zebrafish mindbomb mutant embryos uncovered a robust upregulation in Cdk5 expression but with a reduced Cdk5 activity. The implications of these findings in both the mammalian system and zebrafish are discussed in this mini-review to provide a glimpse into the relationship between Notch and Cdk5 that may explain certain neurodevelopmental defects associated with either mutations in ubiquitin ligase or altered expression of Cdk5.
- MeSH
- biologické modely MeSH
- cyklin-dependentní kinasa 5 metabolismus MeSH
- dánio pruhované metabolismus MeSH
- mutace genetika MeSH
- proteiny dánia pruhovaného genetika MeSH
- receptory Notch metabolismus MeSH
- upregulace genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Tuberculosis is still a global health burden. It is caused by Mycobacterium tuberculosis which afflicts around one third of the world's population and costs around 1.3 million people their lives every year. Bacillus Calmette-Guerin vaccine is inefficient to prevent overt infection. Additionally, the lengthy inconvenient course of treatment, along with the raising issue of antimicrobial resistance, result in incomplete eradication of this infectious disease. The lack of proper animal models that replicate the latent and active courses of human tuberculosis infection remains one of the main reasons behind the poor advancement in tuberculosis research. Danio rerio, commonly known as zebrafish, is catching more attention as an animal model in tuberculosis research field. This shift is based on the histological and pathological similarities between Mycobacterium marinum infection in zebrafish and Mycobacterium tuberculosis infection in humans. Being small, cheap, transparent, and easy to handle have added further advantages to the use of zebrafish model. Besides better understanding of the pathogenesis of tuberculosis, Mycobacterium marinum infected zebrafish model is useful for evaluating novel vaccines against human tuberculosis, high throughput small molecule screening, repurposing established drugs with possible antitubercular activity, and assessing novel antituberculars for hepatotoxicity.
- MeSH
- antituberkulotika farmakologie MeSH
- dánio pruhované * mikrobiologie MeSH
- lidé MeSH
- modely nemocí na zvířatech * MeSH
- Mycobacterium marinum účinky léků fyziologie MeSH
- Mycobacterium tuberculosis účinky léků fyziologie MeSH
- tuberkulóza farmakoterapie mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
We report for the first time, a comparison of two approaches for artificially induced triploidy in zebrafish (Danio rerio) using cold shock and heat shock treatments. Of the two methods, heat shock treatment proved more effective with a triploid production rate of 100% in particular females. Subsequently, triploid zebrafish larvae were used as recipients for intraperitoneal transplantation of ovarian and testicular cells originating from vas:EGFP strain in order to verify their suitability for surrogate reproduction. Production of donor-derived sperm was achieved in 23% of testicular cell recipients and 16% of ovarian cell recipients, indicating the suitability of triploids as surrogate hosts for germ cell transplantation. Success of the transplantation was confirmed by positive GFP signal detected in gonads of dissected fish and stripped sperm. Germline transmission was confirmed by fertilization tests followed by PCR analysis of embryos with GFP specific primers. Reproductive success of germline chimera triploids evaluated as fertilization rate and progeny development was comparable to control groups.
- MeSH
- chov metody MeSH
- dánio pruhované genetika MeSH
- genetické inženýrství metody veterinární MeSH
- průtoková cytometrie MeSH
- teplota MeSH
- triploidie * MeSH
- zárodečné buňky transplantace MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Calcium plays a variety of vital regulatory functions in many physiological and biochemical events in the cell. The aim of this study was to describe the ultrastructural distribution of calcium during different developmental stages of spermatogenesis in a model organism, the zebrafish (Danio rerio), using a combined oxalate-pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly detectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron-dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The notable changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the process of male gamete development.
- MeSH
- buněčné jádro ultrastruktura MeSH
- dánio pruhované metabolismus MeSH
- spermatidy cytologie ultrastruktura MeSH
- spermatogeneze * MeSH
- subcelulární frakce metabolismus ultrastruktura MeSH
- testis cytologie ultrastruktura MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.
- MeSH
- buněčné kultury MeSH
- cytokiny genetika MeSH
- dánio pruhované krev MeSH
- hematopoetické kmenové buňky cytologie MeSH
- hematopoéza * MeSH
- kapři krev MeSH
- kultivační média chemie MeSH
- molekulární biologie metody MeSH
- proteiny dánia pruhovaného genetika MeSH
- rekombinantní proteiny genetika MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
In nonmammalian vertebrates, the functional units of hemostasis are thrombocytes. Thrombocytes are thought to arise from bipotent thrombocytic/erythroid progenitors (TEPs). TEPs have been experimentally demonstrated in avian models of hematopoiesis, and mammals possess functional equivalents known as megakaryocyte/erythroid progenitors (MEPs). However, the presence of TEPs in teleosts has only been speculated. To identify and prospectively isolate TEPs, we identified, cloned, and generated recombinant zebrafish thrombopoietin (Tpo). Tpo mRNA expanded itga2b:GFP(+) (cd41:GFP(+)) thrombocytes as well as hematopoietic stem and progenitor cells (HSPCs) in the zebrafish embryo. Utilizing Tpo in clonal methylcellulose assays, we describe for the first time the prospective isolation and characterization of TEPs from transgenic zebrafish. Combinatorial use of zebrafish Tpo, erythropoietin, and granulocyte colony stimulating factor (Gcsf) allowed the investigation of HSPCs responsible for erythro-, myelo-, and thrombo-poietic differentiation. Utilizing these assays allowed the visualization and differentiation of hematopoietic progenitors ex vivo in real-time with time-lapse and high-throughput microscopy, allowing analyses of their clonogenic and proliferative capacity. These studies indicate that the functional role of Tpo in the differentiation of thrombocytes from HSPCs is well conserved among vertebrate organisms, positing the zebrafish as an excellent model to investigate diseases caused by dysregulated erythro- and thrombo-poietic differentiation.
- MeSH
- buněčná diferenciace MeSH
- dánio pruhované embryologie fyziologie MeSH
- embryo nesavčí MeSH
- geneticky modifikovaná zvířata MeSH
- hematopoetické kmenové buňky fyziologie MeSH
- hematopoéza genetika MeSH
- kultivované buňky MeSH
- proliferace buněk MeSH
- thrombopoetin genetika MeSH
- trombocyty fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Herbicides are the most widely used group of pesticides but after reaching water bodies they are able to cause adverse effects on non-target organisms. Different formulations using the same active ingredient are frequently available, which raises the issue of potential influence of different formulation types on herbicide toxicity. The present study evaluated the toxicity and teratogenic effects of the active ingredient clomazone and its two formulations (Rampa® EC and GAT Cenit 36 CS, both containing 360g a.i./l of clomazone) on zebrafish embryos. The crucial difference between the two formulation types is the way of active substance release. This investigation is the first report on zebrafish embryotoxicity of both clomazone and its formulations. The technical active ingredient and formulations caused mortality and diverse teratogenic effects, showing different levels of toxicity. The LC50 values for the technical ingredient, Rampa® EC and GAT Cenit 36 CS were 61.4, 9.6 and 92.5mg a.i./l, respectively. Spontaneous movements in 22 hpf embryos decreased under exposure to both the technical ingredient and formulations. A significant number of underdeveloped embryos was detected after exposure to clomazone and Rampa® EC, while no underdevelopment was noted in embryos exposed to GAT Cenit 36 CS. Exposure to the technical ingredient and formulations led also to a series of morphological changes and interfered with the growth of zebrafish embryos. The EC50 based on detection of edemas, spine and tail tip deformations and gas bladder absence (120hpf) was 12.1, 10.1 and 24.1mg/l for technical clomazone, Rampa® EC and GAT Cenit 36 CS, while teratogenicity index (TI) based on LC50/EC50 ratio was 5.1, 1 and 3.8, respectively. The data in this study showed that the emulsifiable concentrate formulation (Rampa® EC) caused statistically significantly higher toxicity, and the aqueous capsule suspension (GAT Cenit 36 CS) lower toxicity than technical clomazone. It indicates that different formulations with the same active ingredient may have different environmental impacts, which is why risk assessment based only on active ingredient toxicity might not be sufficient in terms of preventing formulation effects on the environment.
Chemical exposure during the early life stages of development may have long lasting effects on organisms that are rarely studied. The present work intended to evaluate the effect of embryonic exposure to the pesticide carbaryl on adult fish behavior. Zebrafish (Danio rerio) embryos were exposed, for 4 days, to sublethal concentrations of carbaryl (0.01, 0.1 and 1.0 mg/L) plus a control and then kept in standard cultivation conditions until adulthood. A battery of behavioral tests was then performed to assess anxiety-like behavior (locomotor activity, thigmotaxis and novel tank diving test), social behavior, and feeding. Developmental exposure of zebrafish to sublethal concentrations of carbaryl produced important behavioral alterations in the adulthood. Main effects included decreased locomotion/hypoactivity (increase in slow movements and decrease of medium and rapid movements), especially in the light periods. Moreover, spatial pattern also changed: while during dark periods control fish increased activity in the outer zone of the tank, this was not observed in exposed fish. Overall, this demonstrated the importance of life stage exposure, clearly demonstrating long lasting effects of a (chemical) stress event at embryonic stages. This data supports the need of considering this scenario in environmental risk evaluations. Further work should focus on the mechanistic effects of developmental disruption responsible for the effects observed.
- MeSH
- chování zvířat účinky léků MeSH
- dánio pruhované embryologie MeSH
- embryo nesavčí účinky léků MeSH
- insekticidy toxicita MeSH
- karbaryl toxicita MeSH
- lokomoce účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Calcium plays prominent roles in regulating a broad range of physiological events in reproduction. The aim of this study was to describe the subcellular distribution of calcium deposits during stages of oogenesis in zebrafish using a combined oxalate-pyroantimonate technique. The oocyte development of zebrafish was categorized into four stages: primary growth, cortical-alveolus, vitellogenic, and maturation, based on morphological criteria. Calcium deposits in the primary growth stage were detected in the cytoplasm, mitochondria, nucleus, and follicular cells. At the cortical-alveolus stage, calcium particles were transported from follicular cells and deposited in the cortical alveoli. In the vitellogenic stage, some cortical alveoli were compacted and transformed from flocculent electron-lucent to electron-dense objects with the progression of the stage. Calcium deposits were transformed from larger to smaller particles, coinciding with compaction of cortical alveoli. In the maturation stage, calcium deposits in all oocyte compartments decreased, with the exception of those in mitochondria. The proportion of area covered by calcium deposits in the mitochondria and cortical alveoli of oocytes at different stages of development was significantly different (p<0.05). The extent of calcium deposits in the cortical alveoli of mature oocytes was substantially lower than in earlier stages. Basic information about calcium distribution during zebrafish oogenesis may contribute to better understanding of its role in oogenesis.
- MeSH
- dánio pruhované fyziologie MeSH
- mikroskopie metody MeSH
- oocyty chemie MeSH
- oogeneze * MeSH
- počítačové zpracování obrazu metody MeSH
- vápník analýza MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
