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Danio rerio as a model of serious human diseases
Hason, Martina ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
(ENGLISH) Over the last five decades, zebrafish (Danio rerio) has become a useful vertebrate model organism for the field of developmental biology and disease control. Using zebrafish in xenotransplantation studies is becoming more popular and progressed towards drug screening of anti-cancer drugs. Zebrafish are particularly suitable for high-throughput pre-clinical drug screening, due to the small size of embryos and the striking evolutionary conservation of cancer- related pathways between human and zebrafish. The fast, large-scale evaluation of the cancer- drug response in vivo could facilitate progress in personalized cancer therapy. Nevertheless, there is still a lack of methods which would allow for rapid and sensitive evaluation of tumor cell growth to facilitate high-throughput screening of drugs in vivo. In our bioluminescent zebrafish transplantation model, we proposed and validated a new screening platform for pre-clinical drug discovery in zebrafish embryos. In our experiments we used the NanoLuc luciferase, which enabled us to rapidly screen inhibitors of cancer growth in a sensitive and quantitative way with very low background compared to the conventional fluorescence signal. In our screen we evaluated the in vivo drug response of 180 kinase inhibitors in zebrafish embryos...
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The role of Kit ligands in hematopoiesis of Danio rerio
Oltová, Jana
Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...
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Role of transcription factors Meis during embryogenesis Danio rerio
Brežinová, Veronika ; Machoň, Ondřej (advisor) ; Krylov, Vladimír (referee)
Meis transcription factors belong to the group of TALE (three amino acids loop extension) homeodomain proteins. Meis2 proteins have a potential role in regulation of neural crest cells development and in differentiation of their derivates. Zebrafish genome has two paralogues of meis2 gene, meis2a and meis2b. CRISPR/Cas9 technology was used to prepare mutant lines of both paralogues, meis2a and meis2b, for the purpose of study of function of Meis2 transcription factors. Specific morpholinos that reduce the expression of meis2a and meis2b were used as controls. Craniofacial and cardiac development in mutant fish was analyzed in the meis2a line by RNA in situ hybridization, histological cartilage staining, and computed tomography. While we observed impaired craniofacial and cardiac development after injection of specific Morpholinos, we did not detect similar changes in the meis2a KO line. Our genetic approach has not clearly shown that the meis2a paralogue itself plays an important role in craniofacial development and cardiac development. For more detailed analysis, further experiments on fish lines with combined meis2a and meis2b knock-outs are needed. Key words Mutagenesis CRISPR, Danio rerio, neural crest cells, Meis2, transcription factor
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The role of Kit ligands in hematopoiesis of Danio rerio
Oltová, Jana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
Hematopoiesis is a precisely regulated process, dependent on the activity of hematopoietic cytokines and their receptors. Due to an extra round of whole genome duplication in teleost fish, two paralogs of many important genes, including some hematopoietic cytokines and their receptors, are present in the zebrafish (Danio rerio) genome. In this project, we have been investigating the role of zebrafish Kit ligands in hematopoiesis. Kit ligand is a pleiotropic cytokine, which is essential for vertebrate erythropoiesis; however, in zebrafish, no such role has been reported so far. To determine the function of zebrafish paralogs of Kit ligand (Kitlga and Kitlgb) in hematopoiesis, we performed in vivo and ex vivo gain- and loss-of-function experiments. Strikingly, we were the first to report the synergistic cooperation of zebrafish Kitlga with erythropoietin and dexamethasone, enabling the growth of kidney marrow-derived suspension cells and providing optimal conditions for the expansion of adult erythroid progenitors. We assume that by using different cytokine combinations, optimal conditions for the growth of other hematopoietic cell types can be established, and therefore, this new approach now available for the...
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Role of Rnf207b in zebrafish hematopoiesis
Vondráková, Zuzana ; Bartůněk, Petr (advisor) ; Živný, Jan (referee)
Hematopoiesis is the process of proliferation, differentiation and self-renewal of hematopoietic stem cells. Regulation of hematopoiesis is a complex process, which takes place on many different levels and is directed by many signals. RNF207 is one of the perspective genes chosen based on a screen in chicken model, where obtained data show its role in hematopoiesis. The aim of this work was to confirm the role of rnf207b as a new regulator of hematopoiesis in Danio rerio and to find out on which level of hematopoiesis is active. Danio rerio is an excellent model to study the function of genes in vivo, thanks to the easy manipulation of genetic expression and wide range of phenotypes during the development. To study the effect of rnf207b in hematopoiesis of Danio rerio we performed the knock-down of this gene by microinjection of morpholino oligonucleotides into one cell stage embryos. In these injected fish, we saw the effect in both thrombocyte and erythroid lineage, suggesting that rnf207b could be a regulator at the hierarchical level of progenitors or even more upstream. The results of developmental and tissue specific expression analysis then show that expression of rnf207b begins as early as 18 hpf, at the time of primitive hematopoiesis. Although rnf207b is expressed in the kidney (an...
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Mutagenesis in Danio rerio using CRISPR technology
Nickl, Petr ; Machoň, Ondřej (advisor) ; Soukup, Vladimír (referee)
CRISPR/Cas9 technology is currently one of the most important tools of genome engineering. This technology allows a precise site-specific gene editing and such ability was applied to study the role of TALE (TALE - three amino acids loop extension) homeodomain transcription factors during neural crest cells development. The main genes of interest, belonging to sub-family of TALE proteins, are Meis1 transcription factors that are present in the zebrafish genome as two paralogous genes, meis1a and meis1b. Their function was assessed by mutating their DNA-binding domain - homeodomain to abrogate the ability of transcription factor to bind DNA and by that disturb regulatory network, in which Meis1 proteins operate in. Phenotype analysis of mutant fish would reveal a potential role of Meis1 proteins in regulation of neural crest cells development and outline the functional significance of the homeodomain in regulatory operations. To determine the regulatory relationship between meis1a and meis1b genes morpholino-based knock-down of the genes was performed. Preliminary results suggest a dominant role of Meis1b in neural crest cells regulation and importance of its homeodomain. Furthermore, knock-down of Meis1a indicates its contribution to regulation of craniofacial development. However, a detailed...
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Optimization of the rearing of the species Danio rerio (Hamilton, 1822)
Gottwald, Milan ; Kalous, Lukáš (advisor) ; Martin, Martin (referee)
The diploma thesis summarizes the experimental method for the rearing of zebrafish Danio rerio (Hamilton, 1822) screed. The work of figuring out which of the methods of rearing fry in the transitional period of the ontogenetic development is best for the survival rate and growth of the standard length of the surveyed individuals.
The test was carried out on two basic lines of zebrafish Wild Type and Casper. At the beginning of the test were created four groups divided by feeding mode. The control group was fed once a day with pellet feed GEMMA Micro 75 and one day from the 10th day after fertilization the fed with nauplii stages of brine shrimps Artemia salina (Linnaeus, 1758). The second group was fed only once a day with pellet feed GEMMA Micro 75. The last two groups were fed five times a day saltwater rotifer species Brachionus plicatilis (Müller, 1786), and only one of them and the other was still three times daily nourished with nauplii stages of brine shrimps. For each group of four representative have been created initially, thirty tanks of fish at the age of five days after fertilization.
The results of this method have shown that the methods they use to feed the rotifers, did not have a statistically significant impact on the survival rate of individuals. In the growth of this method has proven to be statistically significant result in the group where the fed in combination with brine shrimps, where to achieve the average standard size of juvenile fish 16.02 +/- 0.80 mm for the Wild type and 17.39 +/- 0.81 mm for Casper, compared to the control group, that has been the standard length 11.63 +/- 0.64 mm for the Wild Type and 9.54 +/- 0.56 mm for Casper.
This method has great potential and breeding is therefore necessary to further develop this method and to adapt it to individual zebrafish facilities.
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Do humic substances reduce nitrite toxicity to fish?
VANĚČEK, Miloslav
The aim of this work was to verify the hypothesis that humic substances reduce toxicity of nitrite to fish. Under laboratory conditions, tests of acute toxicity were performed on aquarium fish: zebrafish (Danio rerio) and guppy (Poecilia reticulata). Fish were exposed to nitrite in the presence or absence of humic substance HS 1500. Nitrite was dosed as NaNO2. Preparation Dupla Cur (manufacturer Dohse Aquaristik KG) was a source of the humic substance (HS 1500). The preparation was tested in two different concentrations: 0.5 ml.l-1 and 1 ml.l-1. Toxicity tests were performed according to Czech standard ČSN EN ISO 7346-2. Based on the observed mortality in the acute toxicity tests, 96hLC50 values of sodium nitrite for selected species were calculated in the presence or absence of the tested preparation Dupla Cur. Tests on zebrafish showed a positive effect of Dupla Cur preparation at a concentration of 0.5 ml.l-1 on decreasing of nitrite toxicity. In this case 96hLC50 value was about 73 % higher than the value found in the test where the preparation was omitted. At a concentration of 1 ml.l-1 of the preparation a significantly positive effect was not observed. In the tests on guppy, an effect of Dupla Cur preparation on the toxicity of sodium nitrite was not observed. The tested humic substance does not have the same effect on all fish species. The effect depends on its concentration in the water.
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Different ways of reproduction of laboratory fish Danio rerio
KOMENDOVÁ, Jana
Danio rerio is often used in laboratories all over the world in varieties of fields. The demand for this fish is the precondition for building up a perfect system of its reproduction. The aim of the work was to find out the most suitable rate between the sexes of Danio rerio used for reproduction. The experiments were carried out on seven rates of sexes who represented the differences between big and small shoals. Males and females were rear separately. Fish in specific rates were used to spawn. The spawn were counted. Big shoals with one female staned in the spawn aquarium for one day. Little shoals for two days, the big ones for three days. The results were put in graphs. The most successful spawn was considered the one with the rate of one male to one female. Big shoals didn´t appear to be economic. The big number of fish had negative influence on the number of the spawn of one female of the shoal.
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Verification of possibility to breed chosen aquarium fish species in outdoor basins
DENKOVÁ, Adéla
The aim of the work was to find out the possibilities of outside breeding of chosen fish species: Phalloceros caudimaculatus, Gambusia affinis, Danio rerio, Macropodus opercularis and Tanichthys albonubes. Fish were placed into basins and left there for five months (may ? october). Water temperature was measured once a week, water parameters ( pH, temperature, conductivity, oxygen) were measured once a month. Fish were netted after air temperatures decreased nearly on the zero point. They were counted, results were put into graphs or tabs.
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