Browsing by Subject "Zebrafish"

Now showing 1 - 8 of 8
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    Development of the larval zebrafish as a genetic model for the nicotine response.
    (2010-08) Petzold, Andrew Michael
    Tobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21st century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish (Danio rerio) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav/ cct8 and hbog/gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. Additionally, we developed a conditioned place preference assay for use with larval zebrafish. This assay allows for the perturbation of the differences in genetic function between the physiological and learned response representing one of the first associative learning based assays in the larval zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning.
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    High-throughput automated detection and analyses of locomotor and hunting sequences in larval zebrafish unveil the role of a conserved dopaminergic diencephalospinal tract in locomotor development and goal-directed behavior
    (2015-03) Lambert, Aaron Mattthew
    The dopaminergic diencephalospinal tract (DDT), and its source orthopedia- specified dopaminergic (DAergic) population, is the most conserved part of the vertebrate DAergic system. The source somata of the DDT have widespread ascending and descending projections that span and have potential to integrate the entire rostro-caudal axis of the central nervous system, from telencephalon to spinal cord. Mammalian studies confirm that the extensive DDT network is multifunctional, even via its direct influence within the spinal cord. While specific mechanosensory and nociceptive functions of the DDT acting in the spinal cord in vivo have been elucidated in adult mammals, whether or not the DDT also exerts locomotor influences in the spinal cord in vivo, as well as whether the DDT plays an early role during development, has remained unknown despite suggestive in vitro studies. My thesis explored the role of the vertebrate DDT in locomotor development and goal-directed behavior in zebrafish larvae, a premiere model to elucidate the neural bases of such behaviors at organismal, systems, circuit, cellular, and subcellular levels in vivo. To this aim, I developed new methodologies for high-throughput, unbiased, automated detection and analyses of locomotion and goal-directed hunting. This approach was combined with peripheral nerve recordings of the neural locomotor output of the spinal cord and employed during: DAergic pharmacological perturbations, demarcated transections at varying locations of the nervous system, selective chemogenetic ablation of orthopedia neurons, and laser ablations of the DDT. Collectively, this thesis reveals that the DDT acts specifically through endogenous dopamine receptor 4 (D4R) signaling to mediate locomotor development and provide a multifunctional modulation of multiple locomotor parameters in a separable manner, by putatively influencing disparate neuronal targets concurrently. Moreover, this thesis elucidates that endogenous D4R signaling is crucial to goal-directed prey capture via a specific motor-centric role in shifting prey- directed motor strategies and providing precision of speed control during execution of advancing hunting maneuvers. From the collective elucidations of my thesis, I posit the existence of a modular organization subserving a versatile locomotor network, wherein separate neural modules are recruited spontaneously and during hunting and that the hunting module is further subdivided into separable orienting and advancing regimes. Integrated into this granular network, endogenous D4R signaling, perhaps through a widespread integrative impetus at disparate regions via the extensive DDT network, differentially influences multiple modules concurrently. These findings, integrated with the mammalian literature, suggest that the conserved vertebrate DDT is crucial for locomotor development, as well as motor planning and execution of goal-directed behavior.
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    Identifying Morphological Differences of Anterior Neural Tube Formation in Zebrafish
    (2019-04) Schroer, Emily; Campbell, Hannah; Hergert, Polla; Liang, Jennifer;
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    Impacts of SDF-1 and radiation dose-rate in an adult zebrafish model of hematopoietic cell transplant
    (2013-05) Glass, Tiffany
    Despite a history of refinements, Hematopoietic Cell Transplant (HCT) remains a potentially difficult treatment that can have high risks for complications and mortality. We used adult zebrafish models of HCT to study two broad biological processes that occur during HCT; homing and early donor-derived hematopoietic reconstitution. In the first case, we validated the adult zebrafish model for the study of the chemokine SDF-1 in HCT, developed a transgenic sdf-1 reporter zebrafish line, and used it to determine sites of high sdf-1 expression in recipient organisms. These sites were discovered both in the hematopoietic tissue as well as in previously un-described structures throughout the skin, and were found to consistently attract donor-derived cells after transplant. Ultimately, this allowed the identification of new putative HSC-niche cells which can be isolated with relative ease. Secondly, we assessed the effects of high conditioning radiation dose-rates on the process of hematopoietic engraftment after transplant. In groups of adult zebrafish given the same total dose of preconditioning radiation, we found that recipients irradiated at a high rate show significantly faster engraftment compared to those irradiated at a lower rate. Insights offered by this work will contribute to future efforts identifying endogenous factors promoting rapid engraftment, as well as to future reassessments of therapeutic opportunities offered by biologically informed refinements of preconditioning radiation strategies.
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    In vivo assessment of hematopoietic cell homing and engraftment in zebrafish using bioluminescence imaging
    (2013-05) Astuti, Yuliana Sakti Dwi
    Hematopoietic cell transplantation (HCT) has been increasingly used as treatment for malignant and non-malignant diseases over the past 40 years. Despite many advancements made in the field, this procedure continues to face some major challenges, including graft versus host disease and the limited donor availability. Current researches are directed at expanding the number of hematopoietic stem cells and/or improving the HSC homing, especially when the donor cell number is low. Zebrafish has emerged as a versatile organism to study HCT due to its cost-effectiveness and high degree of genetic similarities to mammals. As post-mortem analysis of engraftment following HCT does not offer rapid and long-term continuous monitoring of the engraftment process, we sought to use bioluminescence imaging (BLI) in our HCT study. We generated transgenic zebrafish line that ubiquitously expressed the firefly luciferase. We optimized the anesthesia regiments and the BLI settings used in our system. Finally, using in vivo BLI, we were able to longitudinally monitor the dynamic of donor cells in the transplant recipients and observe the effect of ex vivo prostaglandin E2 treatment on the early donor cell engraftment and the survival of the transplant recipients.
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    Temporal and Spatial Requirement of the Nodal induced head mesendoderm in neurulation AND An inexpensive, efficient method for regular egg collection from a zebrafish in a recirculating system
    (2013-07) Gonsar, Ngawang Youdon
    Chapter 2 Zebrafish in our laboratory are usually bred by removing the fish from the recirculating aquatic system and placing them into 1-2 L spawning tanks. These spawning tanks consist of a bottom reservoir, a lid, and an insert that fits in closely into the bottom reservoir. When the fish breed, the eggs fall through holes of the insert and into the reservoir, thus preventing them from being cannibalized. Because fish in these spawning tanks are not fed and do not get fresh water, they are bred only once a week. During a period where we had high demand for embryos, we instead tried breeding the fish for multiple consecutive days on the recirculating system. Fish were placed into the spawning insert as usual, but the insert was placed into the home tank instead of into the bottom reservoir. We found that there was no significant difference in the number of fertilized eggs produced between the spawning tank and home tank breeding methods. Further, the fish in the home tanks regularly produced fertile embryos over a 28-day time course, with the highest number of eggs per pair produced by the tank with only one pair of adult fish. This method is time-saving as fish bred in home tanks only require to be set up once. It is also an effective way to collect embryos over long periods from the same pair or group of fish and to more easily obtain embryos from stocks with low spawning frequency. Chapter 3 The neural tube is the precursor to the brain and spinal cord. Failure of neural tube closure in humans is one of the most common causes of birth defects. Zebrafish with a decrease in Nodal signaling have a phenotype that is analogous to the fatal human birth defect anencephaly, which is caused by an open anterior neural tube. Previous work in our laboratory has found that Nodal signaling acts through the induction of the head mesendoderm and anterior mesoderm, which underlie the anterior neural tube. Using a pharmacological approach, we determined that Nodal signaling is required up to the late blastula stage of 4.3 hpf for a closed neural tube. This falls within the developmental period when Nodal signaling is most active in mesendoderm and mesoderm induction, supporting the model that Nodal acts through the induction of these tissues. We also found there was a strong correlation between the presence of multiple anterior mesendodermal and mesoderm tissues and neural tube closure. However, no individual tissue was required for neural tube closure. Our finding identifies a specific time window of when Nodal is required for the process of neurulation. This time occurs before the neuroectoderm starts to form, suggesting that Nodal and anterior mesendoderm/mesoderm act very early in the process of neurulation. Further, the finding that multiple mesendodermal/mesodermal tissues are involved suggests that wide region of tissue helps promote closure of the adjacent neural tube.
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    Universal robot for automated microinjection with applications in transgenesis and cryopreservation
    (2023-01) Joshi, Amey
    Microinjection is the process of injecting a small amount of solution into biological organisms at a microscopic level using a glass micropipette. It is a widely utilized technique with a wide range of applications in both fundamental research and clinical settings. However, microinjection is an extremely laborious and manual procedure, which makes it a critical bottleneck in the field and thus ripe for automation. In this thesis, we introduce a simple computer vision-guided robot that uses off-the-shelf components to fully automate the microinjection procedure in different model organisms. The robot uses machine learning models that have been trained to detect individual embryos on agar plates and serially performs microinjection at a particular site in each detected embryo with no human interaction. We deployed three such robots operated by expert and novice users to perform automated microinjections in zebrafish (Danio rerio) and Drosophila melanogaster. We conducted survivability studies to better understand the impact of microinjection on zebrafish embryos and the fundamental mechanisms by which microinjection affects zebrafish embryos. We were able to use the robot to examine the speed of the micropipette, the volume of the microinjectant, the micropipette geometry, and the rate of the volume delivered. These results helped us in determining the optimum settings for automated microinjection into zebrafish embryos. We used transgenesis studies to compare microinjection efficiency to manual microinjection utilizing optimum settings for automated microinjection. Further, we demonstrated that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and post-thaw survivability of cryopreserved embryos compared to manual microinjection, opening the door to large-scale cryo-banking of various aquatic species on an industrial scale. We anticipate that this robotic microinjection can be readily adapted to other organisms and applications.