Zebrafish, in contrast to mammals, are capable of functional regeneration after complete transection of the spinal cord. In this system I asked: (1) Which spinal cell types regenerate in the lesioned spinal cord? (2) To what extent do the dopaminergic and 5-HT systems regenerate and (3) do dopaminergic axons from the brain influence cellular regeneration in the spinal cord? (1) Lost motor neurons are replaced by newly born motor neurons that mature and are integrated into the spinal circuitry after a spinal lesion in adult zebrafish. Using immunohistochemical and transgenic markers in combination with BrdU labeling, we showed that also 5-HT, parvalbuminergic, Pax2+ and Vsx1+ cells are newly born after lesion. Thus, my work shows that diverse cell types are newly generated in the lesioned spinal cord of adult zebrafish. (2) After spinal cord lesion, zebrafish completely recover locomotion within six weeks. Previous work suggested that axonal regeneration is crucial for functional recovery. Here I analyzed changes in the density of 5-HT and dopaminergic axon terminals in the lesioned spinal cord during recovery. Rostral to the lesion site, I observed die-back and sprouting of dopaminergic axons within two weeks post-lesion. Caudal of the lesion, axons are lost indicating Wallerian degeneration. At six weeks post-lesion I tested functional recovery with a behavioral swim test. In recovered fish, a third of the axonal density was restored just caudal of the lesion site, but not at far caudal levels. In contrast, in fish that had non-recovered, only few axons had bridged the lesion site. Thus dopaminergic axon regrowth correlates with functional recovery. Re-transection of the spinal cord in recovered animals abolished re-gained swimming capability, suggesting that behavioral recovery critically depends on axons that crossed the spinal lesion site and not on an intraspinal circuit. 5-HT axon terminals are of both intra- and supraspinal origin. The overall time course of changes in axon terminal density during recovery is similar to that of dopaminergic axon terminals and also correlates with functional recovery. Overall, the organization of the spinal dopaminergic and 5-HT systems, consisting of neuronal somata in the spinal cord and descending axons, differs significantly from their unlesioned organization. I observe sprouting rostral to the lesion site and limited innervation of the caudal spinal cord, as axons do not regrow into the far distal spinal cord. (3) We further hypothesized that signals released by descending axons are involved in cellular regeneration around the lesion site. Dopaminergic axons of supraspinal origin sprout rostral, but are almost completely absent caudal to the lesion site at two weeks post-lesion. Moreover, we observe that expression of the dopamine receptor drd4a is only increased rostral to the lesion site in the ventricular zone of progenitor cells, including olig2 expressing motor neuron progenitor cells. Correlated with these rostro-caudal differences, numbers of regenerating motor neurons are almost two-fold higher rostral than caudal of the lesion site. To functionally test whether dopamine is involved in motor neuron regeneration, we ablated tyrosine hydroxylase positive, mostly dopaminergic axons by injecting the toxin 6-hydroxydopamine. This treatment significantly reduced motor neuron numbers only rostral to the lesion site. As a gain-of-function experiment, we injected the dopamine agonist NPA after spinal lesion, which increased motor neuron numbers only rostral to the lesion site at two weeks post-lesion. These results suggest that dopamine released by descending axons, augments the generation of motor neurons in the lesioned spinal cord of adult zebrafish. In summary, during spinal cord regeneration I observe generation of various cell types and plastic changes of descending axonal projections. Dopamine released by descending axons is able to increase motor neuron regeneration, showing for the first time that signals from descending axons influence cellular regeneration in the spinal cord.

A sizeable portion of the societal drain from cocaine abuse results from the complications of in utero drug exposure. Because of challenges in using humans and mammalian model organisms as test subjects, much debate remains about the impact of in utero cocaine exposure. Zebrafish offer a number of advantages as a model in longitudinal toxicology studies and are quite sensitive physiologically and behaviorally to cocaine. In this study, we have used zebrafish to model the effects of embryonic pre-exposure to cocaine on development and on subsequent cardiovascular physiology and cocaine-induced conditioned place preference (CPP) in longitudinal adults. Larval fish showed a progressive decrease in telencephalic size with increased doses of cocaine. These treated larvae also showed a dose dependent response in heart rate that persisted 24 h after drug cessation. Embryonic cocaine exposure had little effect on overall health of longitudinal adults, but subtle changes in cardiovascular physiology were seen including decreased sensitivity to isoproterenol and increased sensitivity to cocaine. These longitudinal adult fish also showed an embryonic dose-dependent change in CPP behavior, suggesting an increased sensitivity. These studies clearly show that pre-exposure during embryonic development affects subsequent cocaine sensitivity in longitudinal adults.

In humans, von Willebrand factor (vWF) is a key component in hemostasis and acts as a 'cellular adhesive' by letting the circulating platelets bind to exposed subendothelium. It also acts as a carrier and stabilizer of factor VIII (FVIII). A dysfunction or reduction of vWF leads to von Willebrand disease (vWD), resulting in bleeding phenotype which affects 1% of the population. Currently there are a variety of animal models used for the study of vWF and vWD; however, they do not possess the advantages found in zebrafish. Therefore, we set out to establish zebrafish as a model for the investigation of vWF and vWD through the use of bioinformatics and various molecular techniques. Using bioinformatics we found that the vWF gene is located on chromosome 18, that the GPIb? protein sequence is conserved. Confirmation of vWF production was shown by means of immunostaining and by RT-PCR, in thrombocytes as well as in veins and arteries. Evidence of vWF involvement in hemostasis and thrombosis was shown using MO and VMO technology to produce a vWD like phenotype, resulting in an increase in TTO and TTA, as well as a reduction in FVIII when blood was tested using the kPTT assay, coinciding with a decrease in vWF. Stimate treatment provided opposite results of MO and VMO, showing a decrease in TTO and TTA. Investigation of the role of microparticles in hemostasis and their interaction with vWF resulted in a conclusion that the GPIb? receptor should exist on MPs and that it may interact not only with zebrafish vWF but also with human UL-vWF. Agglutination of MPs in the presence of UL-vWF but in the absence of ristocetin and plasma, treatment with ADAMTS-13 abolishing the interaction between MPs and UL-vWF provided evidence that vWF interacts with MPs probably with the GPIb?. We also found that TMPs agglutinate within the vessel wall in vivo when treated with Stimate. In conclusion, this research provided evidence for the presence of vWF in zebrafish and its conserved role in hemostasis. In addition to this we also showed that MPs also participation in hemostasis.

In order for a human being to process complex thought, cells within the brain must communicate with each other in a very precise manner. The mechanisms which underlie the development of these connections, however, are poorly understood and thus require a thorough investigation. In this dissertation, we attempt to identify components involved in stabilizing synaptic contacts and the mechanisms by which synaptic proteins are trafficked to newly forming contact sites. Interestingly, we also identify a gene involved in the formation of the myotome. To identify proteins involved in stabilizing synaptic contacts, we characterized the function of 4.1B in developing zebrafish embryos. 4.1B is a scaffolding molecule involved in stabilizing protein complexes at sites of cell adhesion. We identified two 4.1B genes in the zebrafish genome, 4.1B-a and 4.1B-b, which are differentially expressed and have evolved divergent functions. 4.1B-a is expressed within the central nervous system, specifically within primary motor neurons. Knockdown studies show a reduction in the number of synapses and altered kinetics of touch evoked-responses, suggesting a role in synaptic stabilization. In contrast, 4.1B-b is primarily expressed in muscle cells. Knockdown of 4.1B-b results in severe muscle fiber disorganization as well as altered locomotor behaviors. Together, these data suggest the basic functions of 4.1B are evolutionarily conserved, with new roles described in the development of synapses and muscle fibers. To determine the mechanisms that underlie protein recruitment to newly forming synapses, we examined the recruitment of three distinct transport packets in the zebrafish spinal cord. During presynaptic assembly, we found synaptic vesicle protein transport vesicles preceded piccolo-containing active zone precursor transport vesicles, which in turn preceded synapsin transport vesicles. We identified the last transport packet as a unique and independent mechanism for the recruitment of synapsin, a protein involved in regulating the reserve pool of synaptic vesicles. Importantly, we found cyclin-dependent kinase 5 regulated the late recruitment of synapsin transport packets to synapses, thus identifying kinases as a key signaling molecule in the formation of synaptic contacts. Together, this work provides new insight into the mechanisms that underlie synaptogenesis. This dissertation includes both previously published and unpublished co-authored material.

Chan Chung Yiu Patrick. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-151). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xv / Chapter CHAPTER 1 --- Literature review --- p.1 / Chapter 1.1 --- Aquatic heavy metal contaminations --- p.1 / Chapter 1.1.1 --- Biology of heavy metals --- p.1 / Chapter 1.1.2 --- Mechanism of heavy metal toxicity --- p.2 / Chapter 1.2 --- Environmental monitoring of aquatic heavy metal contaminations --- p.4 / Chapter 1.2.1 --- Bioconcentration effects of heavy metals --- p.4 / Chapter 1.2.2. --- The concept of bioindicator in pollution assessment --- p.5 / Chapter 1.3 --- Metallothioneins --- p.7 / Chapter 1.3.1 --- Biological functions of MT and its metal inducibility --- p.7 / Chapter 1.4 --- Zebrafish (Daino reio) as an animal model --- p.11 / Chapter 1.4.1 --- Biology of zebrafish --- p.11 / Chapter 1.4.2 --- Current applications of transgenic zebrafish --- p.12 / Chapter 1.5 --- The use of cell culture systems in toxicology research --- p.13 / Chapter 1.6 --- Project aims --- p.16 / Chapter CHAPTER 2 --- Quantification of zMT mRNA levels using real-time PCR --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.1.1 --- The use of zebrafish embryos in toxicity assessment --- p.17 / Chapter 2.1.2 --- MT mRNA as a bioindicator of metal exposure --- p.18 / Chapter 2.1.3 --- Quantification of gene transcripts by RT-PCR --- p.19 / Chapter 2.1.4 --- Specific aims of this chapter --- p.27 / Chapter 2.2 --- Materials and methods --- p.28 / Chapter 2.2.1 --- Animal --- p.28 / Chapter 2.2.2 --- Cell culture --- p.31 / Chapter 2.2.3 --- General molecular biology techniques --- p.33 / Chapter 2.2.4 --- mRNA quantification by Real-time PCR --- p.35 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Heavy metal toxicity --- p.42 / Chapter 2.3.1.1 --- In vivo metal toxicity in zebrafish adult --- p.43 / Chapter 2.3.1.2 --- In vivo metal toxicity in zebrafish embryos at late epiboly --- p.44 / Chapter 2.3.1.3 --- In vivo metal toxicity in zebrafish eleutheroembryo --- p.46 / Chapter 2.3.1.4 --- In vitro metal toxicity on ZFL cell line --- p.48 / Chapter 2.3.2 --- Optimization of real-time PCR conditions --- p.50 / Chapter 2.3.2.1 --- Construction of relative standard curve --- p.50 / Chapter 2.3.2.2 --- Optimization of primer concentration --- p.52 / Chapter 2.3.2.3 --- Melting curve analysis for PCR specificity --- p.53 / Chapter 2.2.3 --- Quantification of zMT mRNA by Real-time PCR --- p.54 / Chapter 2.2.3.1 --- Relative zMT mRNA induction in zebrafish embryos at late epiboly --- p.54 / Chapter 2.2.3.2 --- Relative zMT mRNA induction in zebrafish eleutherombryos --- p.57 / Chapter 2.2.3.3 --- Relative zMT mRNA induction in SJD. 1 cell line --- p.58 / Chapter 2.2.2.4 --- In vitro zMT mRNA induction in ZFL cell line --- p.62 / Chapter 2.4 --- Discussions --- p.64 / Chapter 2.4.1 --- Change in metal sensitivity during zebrafish embryo development --- p.64 / Chapter 2.4.2 --- Developmental stage-specfic inducibility of zMT gene expression and metal toxicity --- p.65 / Chapter 2.4.3 --- In vitro zMT regulation by heavy metal ions --- p.67 / Chapter 2.4.4 --- The potential use of zMT expression as exposure biomarker --- p.69 / Chapter CHAPTER 3 --- Functional analysis of a cloned zMT-II gene promoter in zebrafish cell-lines: SJD.1 and ZFL --- p.71 / Chapter 3.1 --- Zebrafish MT gene promoter --- p.71 / Chapter 3.1.1 --- The structure of zMT promoter --- p.71 / Chapter 3.1.2 --- Functional analysis of cloned zMT-II promoter region in HepG2 cells --- p.72 / Chapter 3.1.3 --- Specific aims of this chapter --- p.77 / Chapter 3.2 --- Materials and methods --- p.78 / Chapter 3.2.1 --- General molecular biology techniques --- p.78 / Chapter 3.2.2 --- Cell culture --- p.79 / Chapter 3.2.3 --- Transient transfection assay --- p.81 / Chapter 3.3 --- Results --- p.84 / Chapter 3.3.1 --- Metal responsiveness of zMT-II promoter by transient transfection --- p.84 / Chapter 3.3.2 --- Deletion analysis --- p.88 / Chapter 3.3.2.1 --- Deletion analysis of zMT-II gene promoter in SJD. 1 cell line --- p.88 / Chapter 3.3.2.2 --- Deletion analysis of zMT-II gene promoter in ZFL cell line --- p.90 / Chapter 3.4 --- Discussions --- p.91 / Chapter 3.4.1 --- Structure of zMT-II gene promoter --- p.91 / Chapter 3.4.2 --- Metal responsiveness of zMT-II promoter --- p.94 / Chapter 3.4.3 --- Deletion analysis of zMT-IIgene promoter --- p.95 / Chapter CHAPTER 4 --- Transgenic zebrafish model for in vivo zMT gene regulation study --- p.97 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.1.1 --- Development of transgenic fish --- p.97 / Chapter 4.1.2 --- The principle of gene delivery --- p.98 / Chapter 4.1.3 --- The application of transgenic zebrafish model --- p.99 / Chapter 4.1.4 --- Specific aim of this chapter --- p.101 / Chapter 4.2 --- Materials and methods --- p.102 / Chapter 4.2.1 --- General molecular biology techniques --- p.102 / Chapter 4.2.2 --- Sequence of primers used --- p.103 / Chapter 4.2.3 --- Engineering of constructs for transgenic zebrafish study --- p.103 / Chapter 4.2.4 --- In vitro efficacy test of the GFP constructs --- p.105 / Chapter 4.2.5 --- Gene transfer into zebrafish embryos by electroporation --- p.107 / Chapter 4.2.6 --- Screening of transgenic candidates --- p.108 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- Engineering of DNA constructs for transgenic zebrafish production --- p.110 / Chapter 4.3.2 --- In vitro efficacy test of the DNA constructs --- p.111 / Chapter 4.3.3 --- Optimization of electroporation voltage --- p.117 / Chapter 4.4.4 --- Screening of transgenic candidates --- p.118 / Chapter 4.4 --- Discussion --- p.120 / Chapter 4.4.1 --- Potential application of the zMT promoter transgenic mode --- p.120 / Chapter 4.4.2 --- The use of GFP transgenic zebrafish model in developmental gene regulation study --- p.121 / Chapter 4.4.3 --- In vitro efficacy of the GFP constructs --- p.122 / Chapter 4.4.4 --- Transgene expression in zebrafish --- p.122 / Chapter CHAPTER 5 --- General discussion --- p.126 / REFERENCES --- p.134

Poon, Shui-Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 78-98). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of content --- p.vii / List of figures --- p.x / Symbols and abbreviations --- p.xii / Chapter 1 General Introduction / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Folliculogenesis and its control --- p.1 / Chapter 1.2.1 --- History of inhibin discovery --- p.4 / Chapter 1.2.2 --- Inhibin and its related proteins --- p.5 / Chapter 1.3 --- Inhibin --- p.6 / Chapter 1.3.1 --- Structure --- p.6 / Chapter 1.3.2 --- Function --- p.7 / Chapter 1.3.3 --- Signaling --- p.11 / Chapter 1.3.4 --- Expression --- p.14 / Chapter 1.3.5 --- Regulation --- p.15 / Chapter 1.4 --- Objectives of the present study --- p.19 / Chapter Chapter 2 --- Spatiotemperoal Expression Profiles of Inhibin a in the Zebrafish Ovary / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Animals --- p.27 / Chapter 2.2.2 --- Chemicals --- p.27 / Chapter 2.2.3 --- Separation of oocytes and follicular layers --- p.28 / Chapter 2.2.4 --- Isolation of ovarian follicles --- p.28 / Chapter 2.2.5 --- RNA isolation and reverse transcription --- p.28 / Chapter 2.2.6 --- Real-time and semi-quantitative RT-PCR quantification of expression --- p.29 / Chapter 2.2.4 --- Data analysis --- p.30 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Validation of semi-quantitative RT-PCR quantification --- p.30 / Chapter 2.3.2 --- Tissue distribution of inha expression --- p.30 / Chapter 2.3.3 --- Localization of inha expression within the ovarian follicle --- p.31 / Chapter 2.3.4 --- Further evidence for inha expression in the follicle layer --- p.31 / Chapter 2.3.5 --- Stage-dependent expression of inha in the ovarian follicles --- p.32 / Chapter 2.4 --- Discussion --- p.33 / Chapter Chapter 3 --- Regulation of Inhibin a Expression in vitro and in vivo and the Effect of Inhibin on Final Oocyte Maturation / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Animals --- p.46 / Chapter 3.2.2 --- Chemicals and hormones --- p.46 / Chapter 3.2.3 --- Preparation of goldfish pituitary extract --- p.47 / Chapter 3.2.4 --- Ovarian fragment incubation --- p.47 / Chapter 3.2.5 --- Preparation of spontaneously matured follicles --- p.48 / Chapter 3.2.6 --- Intra-peritoneal injection --- p.48 / Chapter 3.2.7 --- RNA isolation and reverse transcription --- p.48 / Chapter 3.2.8 --- Real-time and semi-quantitative RT-PCR quantification of expression --- p.48 / Chapter 3.2.9 --- Data analysis --- p.49 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Validation of semi-quantitative RT-PCR quantification --- p.49 / Chapter 3.3.2 --- Temporal change of basal inha expression in cultured ovarian fragments --- p.49 / Chapter 3.3.3 --- Effect of pituitary extract on the expression of inha --- p.50 / Chapter 3.3.4 --- Effects of recombinant zebrafish FSH and LH on inha expression --- p.51 / Chapter 3.3.5 --- Effect of forskolin on inha expression --- p.52 / Chapter 3.3.6 --- Involvement of PKA and p38MAPK in the pituitary extract and forskolin-induced up-regulation of inha expression --- p.52 / Chapter 3.3.7 --- Effect of recombinant zfFSH on the expression of inha in vivo --- p.53 / Chapter 3.3.8 --- Effects of inhibin on the basal and DHP-induced final oocyte maturation --- p.53 / Chapter 3.4 --- Discussion --- p.54 / Chapter Chapter 4 --- General Discussion --- p.71 / References --- p.78

Boston University. University Professors Program Senior theses. / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / 2031-01-02

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 153-179).

Neuroepithelial cells (NECs) of the zebrafish gill filament have been previously identified as bimodal O2 and CO2/H+ sensors that depolarize in response to chemostimuli via inhibition of background K+ channels. To further elucidate the signaling pathway underlying CO2/H+ chemoreception in the NECs we employed microspectrofluorometric techniques to examine the effects of hypercapnia on [Ca2+]i and pHi. NECs increased their [Ca2+]i in response to acidic hypercapnia (5% CO2, pH 6.6) and isocapnic acidosis (normocapnia, pH 6.6), but not to isohydric hypercapnia (5% CO2, pH 7.8). The acid- induced increase in [Ca2+]i persisted in the absence of extracellular Ca2+, and Ca2+ channel blockers (Cd2+, Ni2+ and nifedipine). NECs exhibited a rapid and reversible drop in pHi in response to acidic hypercapnia and isohydric hypercapnia. Isocapnic acidosis also induced intracellular acidification within NECs, but it was less severe than the drop in pHi elicited by acidic hypercapnia and isohydric hypercapnia. The rate and magnitude of intracellular acidification was reduced by the CA-inhibitor, acetazolamide, without effect on the acid-induced increase in [Ca2+]i. Acetate was used to investigate the relationship between pHi and [Ca2+]i. Acetate induced intracellular acidification without augmentation of [Ca2+]i. The results of this thesis demonstrate that (1) extracellular acidification, but not CO2, is critical to the hypercapnia-induced increase in [Ca2+]i (2) the increase in [Ca2+]i is independent of the drop in pHi (3) the increase in [Ca2+]i is not mediated by the influx of Ca2+ across the plasma membrane.

Learning and memory are complex phenomena; numerous biochemical and neurobiological mechanisms subserving these functions have been identified. A key molecular component involved in learning and memory, the NMDA-R (N-Methyl-D-Aspartate Receptor) is impaired by MK-801(dizocilpine), an antagonist compound. Here I analyze the effects of MK-801 on the performance characteristics of zebrafish (Danio rerio) as these in turn can significantly influence the outcome of learning tasks. Subsequently, I study the effects of MK-801 on the acquisition, consolidation, and recall of memory in a plus maze, a new task I adapted from zebrafish literature. Although MK-801 seemed to have no effect on acquisition of memory in zebrafish, it disrupted their ability to consolidate and recall in the plus maze, echoing results found in rodent literature. Combined, these results suggest that zebrafish can be used as a tool to further advance the discovery of learning and memory.