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Identification of the genetic risks of craniosynostosis in zebrafish modelHe, Xuan Anita 24 January 2024 (has links)
Bones of the cranial vault protect the underlying brain but preserve flexibility to allow brain growth. The edges of skull bones are joined by fibrous sutures, which provide structural support and are the sites of bone growth. Craniosynostosis (CS), premature fusion of bones at the sutures, causes skull deformity and secondary problems in brain development. Bone morphogenetic protein (BMP) signaling is critical in regulating osteoblast differentiation and ossification. Several components of the BMP signaling pathway are associated with increased risk of CS, supporting a central role for BMP signaling in suture formation and development of CS. However, the specific factors involved and disease mechanisms remain largely unknown. To address the gaps in our knowledge, we identified regulatory elements active in skeletal tissues associated with the risk of CS. Briefly, we selected conserved noncoding elements within the genomic regions near the CS risk genes, BMP2 and BMPER (BMP Binding Endothelial Regulator), and performed zebrafish transgenesis to screen for enhancer activities in cranial skeletal tissues. We found multiple enhancers that directed transgene expression consistent with the expression of endogenous bmp2 and bmper genes. Using confocal microscopy, we demonstrated activity of the enhancer, -707BMPER, in cartilage closely associated with developing frontal bones, suggesting its involvement in cranial bone growth, suture formation and the risk of CS. We also performed an enhanced yeast one-hybrid (eY1H) assay to determine the transcription factors that interacted with the identified enhancers, implicating the underlying signaling pathways in regulation of their activity. Compelling human genetic evidence has revealed a role for SMAD6 (mothers against decapentaplegic homolog 6), a negative regulator of BMP signaling, in craniosynostosis. SMAD6 mutations are also associated with cardiovascular abnormalities and nonsyndromic radioulnar synostosis. However, there are significant unanswered questions about the mechanisms linking specific SMAD6 mutations to any of these defects. To create a tractable animal model to address these questions, we used CRISPR targeting to mutate the zebrafish ortholog, smad6a and smad6b. In addition, we developed a zebrafish assay to evaluate the functional consequences of SMAD6 sequence variants, based on the ability of Smad6 to disrupt dorsal-ventral patterning of zebrafish embryos in a dose-dependent manner. We anticipate that the zebrafish assay can provide a convenient approach to verify the disease risk of SMAD6 variants, and that zebrafish lacking smad6 function will be a tractable genetic model to study the role of Smad6 in development.
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Effects of early exposure to fluoxetine on behavioural development in zebrafishForssten, Moa January 2024 (has links)
Many pharmaceuticals are stable molecules and after human excretion they enter the wastewater facilities where roughly 60 % is removed. The remaining residues will affect the concentration in close by streams and at the effluent, where fish like to spawn. Therefore, the development and behaviour of aquatic life is potentially at risk. I studied the effects of fluoxetine on zebrafish (Danio rerio) younger than 1 month which were exposed from fertilized egg until 6 days post fertilization (dpf). The concentrations tested were related to previous findings in the effluent waters, 1 µg/L and 100 µg/L. With Daniovision it was found that there were most differences at 10 dpf, all three variables tested (distance moved, velocity and time in zone) showed differences between the high and low exposure. These results shed light on the rising problem with anti-depressants in the aquatic environment, affecting fish behaviour, withpotential effect on fish population and in the species.
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ALTERED GENE EXPRESSION: A MECHANISM OF REPRODUCTIVE TOXICITY IN ZEBRAFISH (DANIO RERIO) EXPOSED TO BENZO[a]PYRENEHoffmann, Jennifer 19 August 2004 (has links)
No description available.
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Targeted mutagenesis of zebrafish hearing-related genes using ZFN and TALENLiu, Li 21 February 2014 (has links)
No description available.
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Lymphangiogenesis in the Developing ZebrafishCoffindaffer-Wilson, Mikah January 2011 (has links)
No description available.
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Cloning, Expression and Functional Analysis of the Zebrafish Neuronal Nicotinic Acetylcholine ReceptorZirger, Jeffrey M. 05 August 2003 (has links)
No description available.
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Zebrafish deadly seven: neurogenesis, somitogenesis, and neural circuit formationGray, Michelle 04 February 2004 (has links)
No description available.
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Functional analysis of tcf21 and tbx20 in zebrafishBurg, Leonard January 2020 (has links)
In response to cardiac cell death from an injury, zebrafish, as opposed to mammals, are able to regenerate new heart cells without significant scar tissue. Heart attacks, a leading cause of death in the United States, leave behind substantial scar tissue that weakens the heart and leads to a greater chance of a repeated cardiac event. Many genes and major molecular pathways are highly conserved from fish all the way to humans; thus, understanding how the regenerative process works in zebrafish may provide insight into potential therapies for heart attacks in humans. However, we must first understand how heart regeneration occurs in zebrafish at the molecular level. From the time of injury to a zebrafish heart through the completion of regeneration, we want to build a regulatory network showing which genes are up- or down-regulated and how they are interconnected.
Transcription factors, such as tcf21 and tbx20, bind to regulatory elements of DNA and can either upregulate or downregulate nearby genes. To build this gene regulatory network, scientists use a technique called ChIP-seq that can determine where in the genome these transcription factors bind. Nearby genes are potential targets of their regulation, and we can validate these enhancers by testing differences in expression using a fluorescent protein reporter construct.
ChIP-seq requires high quality antibodies capable of specifically recognizing the transcription factor of interest. These are rarely available. Because each different antibody that is used requires validation and optimization for ChIP-seq, it is not easy to scale up the collection of data for different transcription factors. One way to get around these problems is to express a tagged version of the transcription factor. The tag is recognizable by the same antibody; however, expressing the tagged transcription factor in this manner almost inevitably results in higher than normal levels of expression, leading to false positives in the ChIP-seq data.
Using CRISPR/Cas9 technology to target and modify specific sequences in the genome, we developed a novel method to add an epitope tag to these transcription factors at their endogenous loci. This allows us to run ChIP-seq experiments with the transcription factor at physiological levels of expression. We can also use the same antibody to eliminate repeated validation and optimization steps.
We have successfully tagged two genes that may be involved in heart regeneration, tcf21 and tbx20. tcf21 is expressed in the developing epicardium and is required for the proper development of the branchial arches. tbx20 is expressed in the cardiomyocytes and is required for the proper development of the heart, and it has also been shown to be upregulated in response to injury in the zebrafish. With tbx20, we have performed a successful ChIP-seq experiment and have tested several promising target genes.
It is difficult to test if either tcf21 or tbx20 is required for regeneration, as both of these genes are essential for development and mutants do not survive. The solution to this problem is to engineer the gene so that it can be turned off at a specific time and in a specific cell type. A common method of this in the mouse model utilizes the Cre/loxP system: two loxP sites flank a required segment of a gene, and the introduction of the enzyme Cre deletes the DNA between them. Until CRISPRs this was not feasible in zebrafish, which lacked an efficient method of targeted modification in the genome. We adapted our method for integrating epitope tags to add the two loxP sites in the genome. We have made and tested a fully conditional mutant for tbx20, and we have put in the first of the two loxP sites for tcf21. / Biology
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Study of the Functional Role of ATP1A3A in the Vertebrate Nervous SystemMcLaughlin, Brandon Kyle January 2014 (has links)
Na+, K+ ATPases are a group of transmembrane-bound pumps found in all animal cell types. The primary functions of Na+, K+ ATPases are to maintain electrochemical gradients across cell membranes by actively transporting Na+ and K+ ions between intracellular and extracellular spaces using ATP hydrolysis. In vertebrates, Na+, K+ ATPases come in a variety of different isoforms that are expressed in a variety of tissues. Specifically, the α3 isoform, encoded by gene ATP1A3A in humans, has been shown to be expressed in neurons. Mutations in ATP1A3A have been linked to rapid-onset dystonia-parkinsonism and alternating hemiplegia of childhood in humans and has also been shown to cause motor deficits, neuronal excitability, and perinatal death in various animal models. Our lab has generated a mutant in zebrafish (Danio rerio) containing a gene trap that prematurely stops transcription of the ATP1A3A homolog, atp1a3a. We found that larvae homozygous for the gene trap mutation do not survive past 10 days post fertilization. Further analysis revealed that homozygous mutants show vision deficits. We attempted to rescue these phenotypes by expressing atp1a3a in neurons exclusively. However, no rescue of the larval death or vision abnormality was observed, suggesting that atp1a3a presence in cells other than neurons may be critical for survival and proper visual function of the animal. / Biology
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Toxicity of Cd, Cu, Pb, Ni, and Zn to Chironomids, and Trophic Transfer of Cd from Chironomids to Zebrafish / Toxicity of Metals to Chironomids, Cd Trophic Transfer from Chironomids to ZebrafishBechard, Karen M. 08 1900 (has links)
The toxicity of the metals: Cd, Cu, Pb, Ni, and Zn to the freshwater aquatic larvae of the midge fly Chironomus riparius was investigated using 24 h waterborne exposures. Even at the most sensitive life stage, first instar, the chironomids were extremely metal tolerant, with LC50 values for all metals being orders of magnitudes above both the CCME Canadian water quality guidelines for the protection of aquatic life, and the USEP A Water Quality Criteria. This high tolerance of C. riparius to metal toxicity, combined with an exceptional ability to accumulate and tolerate high internal metal burdens makes the chironomid an ideal organism to use in studies on factors affecting the trophic transfer of Cd.
Zebrafish were fed with Cd-contaminated chironomids for 7 days, followed by 3 days of gut clearance with clean chironomids. Chironomids loaded with Cd by exposure to Cd-contaminated sediments exhibited a significantly higher trophic transfer efficiency (TTE) than did zebrafish 1 fed chironomids contaminated with Cd by waterborne exposure, although in both cases the TTE's were low (<2%). The majority ofCd transferred to zebrafish was stored in the gut and carcass, irrespective of ingestion of a natural diet (chironomids loaded with Cd), or a manufactured pellet diet of identical Cd concentrations. On a tissue concentration basis, the highest tissue accumulations in zebrafish were (in decreasing order): kidney> gut> liver> gill> carcass; this accumulation pattern w;ls also independent of concentration of Cd in the diet or of prey exposure route.
Subcellular fractionation of chironomids found most of the Cd in the metal rich
granule fraction followed by the organelle fraction. It also revealed that sediment exposed chironomids had significantly more Cd in the metallothionein-like protein fraction, and significantly less Cd in the cellular debris fraction than water-borne exposed chironomids, although these fractions accounted for only a small percent (~7%) of the total accumulated Cd. Despite this difference in prey subcellular fractionation, the subcellular storage of Cd in zebrafish fed on sediment-exposed chironomids and zebrafish fed on water-borne exposed chironomids was the same, with the highest accumulations in the organelles, enzymes, and metal rich granules fractions. Main areas of subcellular storage in zebrafish fed on a manufactured pellet food were identical to those ofzebrafish fed on chironomids. However, zebrafish fed on chironomids had significantly more Cd in the metallothionein-like protein fraction (5-10%) than did zebrafish fed on pellets :o%).
Overall, TTE's were independent of concentration, but were dependent on route of prey exposure. Tissue-specific accumulations and tissue-specific distributions in zebrafish were independent of both concentration and route of prey exposure, and the gut consistently accounted for the highest proportion of overall body burden, and had the highest accumulation of all tissues; verifying its importance in preventing the internalization of Cd. Subcellular distributions were also independent of concentration, although zebrafish fed a biological food (chironomids) had more Cd in the metallothionein-like protein fraction than zebrafish fed a manufactured food. This has biological implications for Cd detoxification. / Thesis / Candidate in Philosophy
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