In contrast to mammals, adult zebrafish display cellular regeneration of lost motor neurons and achieve functional recovery following a complete spinal cord transection. Using adult zebrafish as a model to study how key developmental pathways can be re-activated to regulate neuroregeneration in cellular recovery, I addressed the following questions: 1) What is the role of Notch signalling during regenerative mechanisms in the lesioned spinal cord of the adult zebrafish? 2) What is the role of Notch overexpression in neurogenesis in the adult zebrafish retina? 3) Which additional signalling pathways are involved in the generation of motor neurons during spinal cord regeneration in adult zebrafish? 1) In the main part of my thesis I have investigated the role of Notch signalling during spinal cord regeneration. The Notch pathway has been shown to regulate neural progenitor maintenance and inhibit neuronal differentiation in the vertebrate nervous system. In the injured mammalian spinal cord, increased Notch signalling is held partly responsible for the low regenerative potential of endogenous progenitors to generate new neurons. However, this is difficult to test in an essentially non-regenerating system. We show that in adult zebrafish, which exhibit lesion-induced neurogenesis, e.g. of motor neurons from endogenous spinal progenitor cells, the Notch pathway is also reactivated. I over-activated the Notch pathway by forced expression of a heat-shock inducible active domain of notch in spinal progenitor cells. I observed that although apparently compatible with functional regeneration in zebrafish, forced activity of the pathway significantly decreased progenitor proliferation and motor neuron generation. Conversely, pharmacological inhibition of the pathway increased proliferation and motor neuron numbers. Thus in summary our work demonstrates that Notch is a negative signal for regenerative neurogenesis in the spinal cord. Importantly, we show for the first time that spinal motor neuron regeneration can be augmented in an adult vertebrate by inhibiting Notch signalling. 2) While in the lesioned spinal cord, over-activation of Notch attenuated neurogenesis, I observed that in the unlesioned retina the same manipulation led to strong proliferation of cells in the inner nuclear layer, presumable Müller glia cells which are the retinal progenitor cells. This coincided with an increase in eye size in adult zebrafish. These preliminary findings provide the first hint that the role of Notch may differ for different adult progenitor cell pools and will lead to future investigations of Notch induced neurogenesis in the retina. 3) We have evidence from previous studies that the dopamine and retinoic acid (RA) signalling pathways may be involved in the generation of motor neurons in the adult lesioned spinal cord. Using in situ hybridisation, I assessed the gene expression patterns a) for all D2-like receptors and b) candidate genes that relate to the RA pathway in the adult lesioned spinal cord to identify the signalling components. a) I found that only the D4a receptor was upregulated in spinal progenitor cells in the ventricular zone rostral to the lesion site, but not caudal to it. This correlates with other results showing that dopamine agonists increase motor neuron regeneration rostral, but not caudal to a spinal lesion site. b) I observed a strong increase in the expression of Cyp26a, a RA catabolising enzyme, in the ventricular progenitor zone caudal to the lesion site, in contrast to the weak expression rostrally. Crabp2a, a cellular retinoic acid binding protein, was also upregulated rostral and in close proximity to the lesion site in a subpopulation of neurons located ventrolaterally in the spinal cord. In summary, we show that the Notch pathway negatively regulates neurogenesis in the spinal cord in contrast to the retina and provide evidence that dopamine from the brain signals via the D4a receptor to promote the generation of motor neurons in addition to RA, which may also play a role in this process. These insights into adult neural progenitor cell activation in zebrafish may ultimately inform therapeutic strategies for spinal cord injury and neurodegenerative diseases such as motor neuron disease.

In normal embryonic development, cells generated from a fertilised oocyte lose their pluripotent status and become restricted to a particular differentiation pathway. This production of functionally distinct cell lineages is thought to be mediated by epigenetic processes that help control gene expression both temporally and spatially without any changes to the DNA sequence. These epigenetic changes consist of posttranslational modifications of the N-terminal tails of histones and differential DNA methylation. Together these act by altering local chromatin structure, which in turn directs gene transcription by regulating the accessibility of the underlying DNA. To examine the potential developmental roles of these modifications, we determined the global cellular patterns of DNA methylation, as well as histone H3 lysine 9 (H3K9) and histone H4 lysine 20 (H4K20) methylation in the developing zebrafish embryo. These modifications are seen as hallmarks of heterochromatin, which consists of DNA that is tightly packaged, gene-poor and transcriptionally silent. Thus using immunostaining techniques, we confirmed the occurrence of genome-wide DNA methylation changes during zebrafish embryogenesis, as well as observing the unique localisation of this mark around the nuclear periphery in conjunction with pericentric heterochromatin. For mono-, di- and tri-methylated H3K9, it was observed by both immunostaining and immunoblotting that these marks became apparent after the onset of zygotic transcription. Ultimately their levels increased as development progressed, in a fashion similar to that of DNA methylation, consistent with a link between these epigenetic marks. Using the same methodology, the three methylation states of H4K20 were seen to vary differentially during zebrafish development, where in particular the levels of H4K20me1 decreased in concert with a potentially sumoylated form. In contrast, the levels of H4K20me2 increased progressively during embryogenesis, while those of H4K20me3 decreased rapidly after the mid-blastula transition. Together, these findings demonstrate that both DNA and histone lysine methylation take place in a highly dynamic manner, further supporting their roles in augmenting chromatin structure and directing cellular differentiation, while also providing a valuable comparison to the developmental epigenetics of other model organisms characterised to date. Preparatory work for somatic cell nuclear transfer in zebrafish was also undertaken. In future studies, the dynamics of these marks could be compared with those of cloned embryos, so that the specific epigenetic profiles necessary for development can be elucidated. Epigenetically, a homologous process occurs within pluripotent embryonic stem cells (ESCs), which can differentiate into any cell type or undergo indefinite self-renewal. Advantageously, we were able to derive zebrafish ESC-like clusters which were morphologically similar to those derived from mice. These clusters were alkaline phosphatase-positive and expressed key ESC markers as detected by RT-PCR and immunofluorescence. In pilot studies, GFP-expressing ESC-like clusters have so far also contributed to ectodermal tissues when transplanted into wild type zebrafish embryos. Subsequently, these ESC-like clusters were epigenetically profiled using immunofluorescence, which showed that they had a similar complement of modifications to ESCs derived from mice. The derivation and initial characterisation of these ESC-like clusters from zebrafish, in addition to the development of somatic cell nuclear transfer in this species, will help pave the way for future studies involving tissue repair and regeneration, as well as opening up the potential of targeted genetic manipulation in this valuable model organism.

Quaking (QKI) is an RNA binding protein involved in the post-transcriptional regulation of gene expression. Originally identified as the cause of hypomyelination in a mouse mutant, it has since been consistently implicated in a wide range of neurological diseases. As a gene exclusively expressed in glial cells of the central nervous system, such associations emphasise the importance of an indirect, or non-neuronal link to aberrant neural function. A role in early neural development has also been suggested from the viable and embryonic lethal mouse mutants, yet detailed and in vivo study has been precluded thus far by the murine uterine gestation, and mutant lethality prior to oligodendrogenesis. This thesis examines the role of QKI in human neurological disease, and explores the use of the zebrafish as a model organism to allow the unimpeded study of neural development. We first examined the expression of QKI in human post-mortem brain samples, in separate studies of Alzheimer’s disease (AD) and schizophrenia. In AD we found that QKI and the splice variants QKI5, QKI6, and QKI7 were all significantly upregulated, and were additionally implicated in the regulation of genes related to AD pathogenesis. Within schizophrenic samples, we explored the expression of QKI6B, a newly identified splice variant of QKI, alongside GFAP. We found that both were significantly upregulated, and a previously implicated regulation of GFAP by QKI was supported. In order to advance investigations of the potential of QKI to disturb neural development, we established the suitability of zebrafish for studying qki. This was achieved through phylogenetic and syntenic analysis, coupled with examination of the qki genes expression patterns. We found that qkib and qki2 are orthologues of human QKI, and both have distinct, yet overlapping expression patterns in neural progenitors, and are not found in differentiated neurons. Following from this, we explored the effects of knockdown to qkib and qki2, finding that qkib exclusively led to aberrant motor neuron development, cerebellar abnormalities, and alterations to the progenitor domain. This clearly demonstrated the crucial role of qki in early neural development, and confirms a previously speculated, yet occluded, function prior to oligodendrogenesis.

<p>As vertebrate embryos grow and develop into adults, their organs must acquire mass and mature tissue architecture to maintain proper homeostasis. While juvenile growth encompasses a significant portion of life, relatively little is known about how individual cells proliferate, with respect to one another, to orchestrate this final maturation. For its simplicity and ease of genetic manipulations, the teleost zebrafish (Danio rerio) was used to understand how the proliferative outputs of individual cells generate an organ from embryogenesis into adulthood. </p><p>To define the proliferative outputs of individual cells, a multicolor clonal labeling approach was taken that visualized a large number of cardiomyocyte clones within the zebrafish heart. This Brainbow technique utilizes Cre-loxP mediated recombination to assign cells upwards of ~90 unique genetic tags. These tags are comprised of the differential expression of 3 fluorescent proteins, which combine to give rise to spectrally distinct colors that represent these genetic tags. Tagging of individual cardiomyocytes was induced early in development, when the wall of the cardiac ventricle is a single myocyte thick. Single cell cardiomyocyte clones within this layer expanded laterally in a developmentally plastic manner into patches of variable shapes and sizes as animals grew into juveniles. As maturation continued into adulthood, a new lineage of cortical muscle appeared at the base of the ventricle and enveloped the ventricle in a wave of proliferation that fortified the wall to make it several myocytes thick. This outer cortical layer was formed from a small number (~8) of dominant cortical myocyte clones that originated from trabecular myocytes. These trabecular myocytes were found to gain access to the ventricular surface through rare breaches within the single cell thick ventricular wall, before proliferating over the surface of the ventricle.</p><p> </p><p>These results demonstrated an unappreciated dynamic juvenile remodeling event that generated the adult ventricular wall. During adult zebrafish heart regeneration, the primary source of regenerating cardiomyocytes stems from this outer wall of muscle. Regenerating cardiomyocytes within this outer layer of muscle are specifically marked by the cardiac transcription factor gene gata4, which they continue to express as they proliferate into the wound area.</p><p>Using heart regeneration to guide investigation of juvenile cortical layer formation, we found that both processes shared similar molecular and tissue specific responses including expression and requirement of gata4. Additional markers suggested that juvenile hearts were under stress and that this stress could play a role to initiate cortical morphogenesis. Indeed, experimental injury or a physiologic increase in stress to juvenile hearts caused the ectopic appearance of cortical muscle, demonstrating that injury could trigger premature morphogenesis.</p><p>These studies detail the cardiomyocyte proliferative events that shape the heart and identify molecular parallels that exist between regeneration and cortical layer formation. They show that adult zebrafish heart regeneration utilizes an injury/stress responsive program that was first used to remodel the heart during juvenile growth.</p> / Dissertation

In recent years, numerous studies have linked cryopreservation with increased occurrence of mutations, DNA fragmentation and the event of apoptosis in biological objects. However, the evidence emerged from such studies is somewhat inconclusive. The current study, therefore, aimed to analyse the DNA damage response (DDR) from the cryopreserved cells in order to characterise the nature of the putative DNA damage. The study set out to investigate the effects of different cryopreservation parameters on the genome in terms of double strand breaks (DSBs), single strand breaks (SSBs), and various forms of sequence alteration using 5¼ hour post fertilisation (hpf) zebrafish (Danio rerio) embryos. The experimental conditions under which the investigation was carried out were short term chilling at 0˚C, treatment with two cryoprotective agents (CPA), namely, MeOH and Me2SO, and cooling to -35˚C. Assays for detecting DSB-activated DDR proteins and SSB-activated DDR proteins in 5¼ hpf zebrafish (Danio rerio) were developed and then utilised to investigate the occurrence of DSBs and SSBs in the genome of the embryos treated with the experimental conditions. The study then analysed the expression profiles of a set of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) pathways as indicators of the occurrence of various forms of sequence alterations in the genome of the embryos treated with the experimental conditions. It was found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER, respectively. CPA treatment also down-regulated the NER and the MMR mechanisms. Cooling, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling also up-regulated the NER and the BER mechanisms in the embryos. The overall finding of the study indicated that the experimental conditions increased the occurrence of various single stranded DNA lesions in the genome of the embryos. The present study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the current knowledge of the effects of cryopreservation on the genome of biological objects.

Injury, infection and tissue malfunction are triggers of inflammation which if not regulated may acquire new characteristics that result in pathological outcomes. Since innate immunity plays a key role in the resolution of acute inflammation knowledge of the regulation of this component of the host response is relevant to understanding processes in disease progression and therefore has potential clinical benefits. In this thesis I have applied zebrafish as a model organism to investigate the response of innate immune cells to qualitatively distinct inflammatory signals in the absence of adaptive immunity. Using a zebrafish embryo wound injury model I have investigated leukocyte migration profiles by in vivo imaging. In response to wound alone leukocytes migrated to the site of injury with predominantly random walk behaviour. However, the addition of lipopolysaccharide (LPS) enhanced recruitment and influenced the directionality of leukocyte migration to the wound. I demonstrate that leukocyte dynamic behaviour is also dependent on the location of the cells. The LPS enhanced directionality and reduced the random walk behaviour of the leukocytes, and these effects were ablated in the presence of the p38 mitogenactivated protein kinase (MAPK) specific inhibitor SB203580. Cytokine gene profiling in adult zebrafish leukocytes reveals that LPS can stimulate a pro-inflammatory response via the activation of p38 MAPK characteristic of mammalian innate immune responses. It is documented in mammalian innate immune cells that LPS can modulate Notch mediated signalling and thereby cell function. Using zebrafish with null mutations in Notch, which provide an unbiased in vivo model, I have investigated the influence of Notch signalling on leukocyte recruitment and demonstrate that migration to a wound injury is reduced. However, this effect is due to decreased cell numbers and not altered function as the Notch signalling inhibitor DAPT had no effect of recruitment to wound injury. The defect in myelomonocyte numbers was also present in adult zebrafish and this was partially compensated for by an increase in lymphocytes. The experimental results that I report here highlight zebrafish as a model 2 organism for studying the function and regulation of innate immunity. The unique optical translucency, which permits in vivo imaging of host responses in real-time, facilitates the analysis of the innate immune response to different inflammatory signals and immune modulators.

The aim of environmental enrichment is to alter the environment of a captive animal in a way that results in improved mental and physical welfare. The technique has been utilised effectively for many years for captive mammals in a variety of settings. However, until now it has never been considered as a way of improving the welfare of aquatic animals such as fish. Fish that are used in regulatory toxicology studies are at present maintained solely in barren tank environments. Little is known about how these types of environments affect the well-being of the animals residing there and whether they impact either physiological heath or behavioural repertoire. This thesis aims to address this gap in the knowledge regarding the potential for environmental enrichment to improve the welfare of fish used in regulatory toxicology. More specifically it looks at two types of enrichment and the effects of these on the commonly used model species, the zebrafish (Danio rerio). The first type of enrichment studied was glass rod structures of varying heights provided to increase tank complexity and provide refuge. The glass structures did not produce any quantifiable benefits in unstressed fish and appeared to delay the formation of stable social hierarchies. When fish were stressed by a period of chasing, the presence of the glass rods appeared to reduce the magnitude of the cortisol response. Whilst this could be viewed as a potential benefit, it was felt that it would not outweigh the costs of this type of enrichment. The second type of enrichment studied was provision of airstones. Again, no clear evidence was found that fish in tanks with airstones experienced an improvement in welfare. The main observation was the vast increase in mortality in tanks containing these airstones, in particular, those of a smaller size. Regardless of the physiological cause underlying this result, this can only be viewed as a negative consequence and one that appears to rule out airstones as an effective form of enrichment for this species and strain of fish. It was also observed that both stress and the presence of enrichment influenced the absolute deviation from the mean in several endpoints. Since changes in endpoint variation will have effects both on the number of animals required to statistically measure environmentally relevant effects this is a factor that should be considered when researching methods of environmental enrichment. Finally, results from these studies suggest the possibility that laboratory zebrafish do not require the addition of environmental enrichment to tanks in order to promote maximum welfare. Furthermore, as considerable costs would be involved in implementing many types of enrichment (relating to manufacture, cleaning, incompatibility of results with previous studies etc.) it is likely that observed benefits would have to be both substantial and well established in order for changes in regulatory guidelines to take place. For a species such as zebrafish that are extremely easy to breed and maintain in the laboratory with minimal amounts of disease, social problems or mortalities, it may be that current conditions are satisfactory.

Cell-to-cell communication is one of the fundamental requisites of making multicellular organisms. G protein-coupled receptors (GPCRs) are one of the most abundant receptor-types within vertebrates. They canonically mediate their signal via hetrotrimeric G proteins, and G protein signaling is regulated by regulators of G protein-signaling (RGS). One of these RGS proteins, RGS4, is preferentially expressed in the central nervous system of humans and has been strongly connected to dopaminergic signaling, along with a number of severe neuronal diseases. rgs4 is not well studied in the model organism Danio rerio, the zebrafish, with only two publications. In this project, a newly constructed transgenic line, tg(rgs4:mCherry), with the fluorophore mCherry regulated by the promoter element of rgs4 was characterized in order to investigate fidelity to endogenous rgs4 expression and the utility of the transgenic line. The mCherry expression is apparent by 48 hours post fertilization, and expression is found mainly in neuronal tissue. Cell bodies are visible only in some labeled areas, while other areas show a more diffuse signal indicative of projections. There is only one transgenically labeled area that also unambiguously expresses rgs4; the pronephric tubule. This line is therefore not particularly well suited for rgs4-specifc studies, but this does not discredit the fidelity of the construct. A transgenic line made with a site-directed technique would most likely confer the fidelity of the promoter to the expression of the fluorophore. A way of increasing the labeling resolution includes exchanging the mCherry fluorophore for one with stronger signal and a lower tendency to aggregate, e.g. eGFP. Increasing the resolution of the characterization, e.g. to the level of sub-nuclei or neuronal types, would serve to enhance the utility of the line. As it is, the tg(rgs4:mCherry) zebrafish line has limited uses, and yet it is not without them.

The goal of this study was to identify critical regions of a novel gene, Nance-Horan syndrome-like 1b (nhsl1b). It was previously discovered that C-terminal truncation of the Nhsl1b protein in nhsl1b mutants resulted in a loss of migration in the facial motor neurons of the hindbrain (Walsh et al. 2011). As nhsl1b expresses many isoforms, multiple targets were investigated in order to determine which transcript bears the largest impact on the motor neurons. Using confocal microscopy to observe immunostained embryos, we examined a mutation in an nhsl1b transcript that encodes a WHD, a domain that is known to function within the actin nucleation and polymerization pathways. In situ hybridization and injection of antisense morpholino oligonucleotides indicate that it is not the WHD but another transcript (ex1bnhsl1b) that is necessary for migration. The control experiments for rescuing the mutant phenotype have successfully been performed, but inducing expression of full length nhsl1b in the nhsl1b mutants is proving difficult.

In order for oligodendrocyte progenitor cells (OPCs) to differentiate into fully mature, myelinating oligodendrocytes, they must be specified at the correct times and undergo coordinated changes in both gene expression and morphology. As oligodendrocytes differentiate, they transition from a bipolar morphology into a morphology characterized by a complex network of multiple processes, which will eventually generate membranous structures necessary for myelination of axonal segments. As changes are observed in cellular morphology, oligodendrocytes also undergo changes in their gene expression profile and express genes necessary for both early and later stages of development such as olig1 and myelin basic protein (mbp), respectively. Data from our laboratory demonstrate that autotaxin (ATX), also referred to as phosphodiesterase Iα/autotaxin (PD-Iα/ATX), is involved in all of these processes as a multifunctional protein by regulating lysophospholipid signaling and cell-extracellular matrix interactions. Previously, our laboratory has identified ATX as an oligodendrocyte-secreted factor present in the extracellular environment that via a newly-identified functional domain, named the MORFO domain (modulator of oligodendrocyte remodeling and focal adhesion organization), can regulate adhesion of oligodendrocytes to naturally occurring extracellular matrix (ECM) proteins and ultimately the establishment of the oligodendrocyte’s complex process network. In vitro data presented in this dissertation suggest that lysophosphatidic acid (LPA), via production from ATX’s well characterized lysophospholipase D (lysoPLD) domain, can induce the expression of myelin basic protein (mbp) and the establishment of membranous structures by differentiating oligodendrocytes, both necessary for the initial stages of myelination. Interested in relating these functions to an in vivo model and due to the early embryonic lethality of atx-null mice, we utilized the zebrafish as an in vivo model. The in vivo data presented in this dissertation demonstrate that atx expression in the zebrafish is evolutionarily conserved within vertebrates. Interestingly, in both mouse and the zebrafish, atx was found expressed by cells of the cephalic floor plate in addition to differentiating oligodendrocytes. Functionally the in vivo data presented in this dissertation confirmed ATX’s role in stimulating mbp expression during later stages of oligodendrocyte development. In addition, a novel function for ATX was revealed by the studies undertaken as part of this dissertation that has never been described before. More specifically, based on the timing of atx expression and the phenotype seen upon atx knock-down, the data presented here suggest that ATX, released by the cephalic floor plate, regulates early oligodendrocyte development and/or specification. Taken together, these data identify ATX as a major regulator for early as well as late developmental stages of the oligodendrocyte lineage.