Dopamine exerts an important role in the regulation of motor activity in humans. During the progression of Parkinson’s disease, patients are faced with the progressive neurodegeneration of nigro-striatal dopamine neurons resulting in an array of pathological symptoms characteristic of the disease. Current treatment relies on targeting symptomatic aspects of the disease but currently Parkinson’s disease is incurable. Targeting the regeneration of DA neurons in PD patients could offer an alternative therapeutic approach that could stall and perhaps even revert the progression of the disease and improve the quality of life for patients. Here, I describe the generation of a transgenic zebrafish line for the non-invasive, conditional and specific ablation of dopaminergic neurons in both larval and adult zebrafish. Understanding the endogenous regenerative ability of the zebrafish may in the future contribute to the development of novel therapeutic approaches targeting DA neuron regeneration in humans. The Tg(dat:CFP-NTR) line efficiently labels and ablates most clusters of DA neurons in both the larval and the adult zebrafish brain. Neuronal ablation is followed by a locomotor and tail bend phenotype as well as by an increase in exploratory behavior. Using double transgenic larvae, we showed through live imaging that loss of DA neurons induces an increase in nestin expression; in addition we show an increase in the number of proliferating cells and an up regulation of genes involved in neurogenesis and tissue repair. Adult zebrafish were able to fully recover their DA neuronal population in the olfactory bulb within 45 days post ablation. Overall the Tg(dat:CFP-NTR) zebrafish offers a novel tool for the study of the molecular and cellular mechanisms driving the regeneration of DA neurons in the zebrafish brain and will be a useful tool for the field of regenerative medicine.

The zebrafish (Danio rerio) has emerged as a model to study vertebrate development due to rapid ontogenetic processes with external embryonic development. It is also an excellent model to study the mechanisms of regeneration and in this respect, the caudal fin is particularly convenient because it is easily accessible for experimental manipulation. Collection of quantitative data and postulation of theoretical models have become an attractive practice to explain complex biological problems. These models are used to test hypothetical mechanisms and predict results, but they also require calibration and validation with analysis of experimental data. This thesis aims at studying the developmental control of fin joint formation, which determines segment patterns of the rays in the caudal fin of zebrafish, before and after an amputation event, through a computational approach and imaging morpho-dynamics. We used a computational approach based on a quantitative framework developed for the analysis of fish fin development and regeneration and more specifically focused our analysis on the pattern of bone segments forming the ray. This allowed us to generate visual maps of the developing and regenerating caudal fins based on average fin data. The results from our experimental set show that bone segments at the amputation plane are longer after regeneration than segments at the same position in non-amputated fins. We also optimized a previously proposed morphogen driven model for fin growth and regeneration to accurately recreate segment numbers based on experimental data. Finally, we collected segment regression data that could be integrated into a new visual map method to analyse fin bony segment patterns.

During fin regeneration, a blastema, a group of de-differentiated cells, forms underneath the wound epidermis. As regeneration proceeds, cells leave the proximal blastema and enter the differentiation zone. Adjacent to the differentiation zone, a subset of cells in the basal epidermal layer (BEL) express sonic hedgehog a (shha). Cells that come in contact with BEL differentiate into osteoblasts and joint cells, enabling the formation of bone segments at the end of each fin ray. Generally, fin regeneration occurs similarly in males and females. However, breeding tubercles (BT), keratinized epidermal structures on the male pectoral fin, result in regenerative differences when compared to females. In this thesis, three aspects of zebrafish fin regeneration were studied: 1) Cell lineage tracing of shha-expressing cells in the caudal fin regenerate; 2) The differentiation of joint cells and osteoblasts in the caudal fin regenerate; 3) Regeneration of pectoral fin BTs. Studies on caudal fin regenerates suggest osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Joint cells follow a genetic pathway in which evx1 occurs downstream or parallel to hoxa13a and upstream of pthrp1. In the absence of Evx1, presumptive joint cells are committed to an osteoblast cell fate. Furthermore, joint cells do not regenerate following laser cell ablation, suggesting joint cell differentiation occurs only at specific intervals during osteoblast regeneration. Collectively, these results suggest a mechanism for joint cell differentiation during caudal fin regeneration. Studies on pectoral fins indicate androgens induce and estrogens inhibit BT formation. BT regeneration in males and androgen-treated females follows the initiation of revascularization, but occurs concomitantly with a novel second wave of angiogenesis. The inhibition of angiogenesis in androgen-treated females prevents BT formation. Altogether, these results suggest the growth and regeneration of BTs requires a v hormonal stimulus and the presence of an additional blood vessel network naturally found in males. In conclusion, these studies have increased the overall knowledge of key aspects of zebrafish fin regeneration. A gain in understanding zebrafish regeneration provides a basis in which treatments can be developed to induce regeneration in species with limited regenerative capabilities.

Parkinson’s disease is a highly prevalent multifactorial neurodegenerative disorder caused by a complex cascade of interactions between various genetic and environmental factors. Due to this, the majority of cases are termed idiopathic. However, about 10% of PD cases are due to defined genetic factors. Interestingly, both idiopathic and familial cases of PD share mitochondrial dysfunction as a central component in the pathology of the disease. The mitochondrial protease, presenilin-associated rhomboid-like (PARL), is one such Parkinson's disease-linked gene, and is associated with diverse processes including mitochondrial dynamics, active inhibition of unnecessary apoptosis and mitophagy in Drosophila and yeast. Here, I investigated the role of the two zebrafish parl paralogs, parla and parlb, through stable CRISPR-Cas9 mediated mutagenesis. I injected wild type embryos with sgRNAs targeting parla and parlb loci, successfully producing indel mutations in parlb and multi-exon deletions in parla at mutation efficiencies of 74% and 40%, respectively. Through whole mount in situ hybridization experiments against th1, I saw no change in dopaminergic (DA) neuron development displayed by parlb mutants compared to wild types. Injection of parla splice blocking morpholinos into parlb mutants indicates that proper DA neuron development may depend principally on Parla function and loss of both Parla and Parlb function increases larval mortality. These results suggest a negative epistatic relationship between the parl paralogs as seen by the more severe phenotype observed in the loss of both Parla and Parlb function compared to the individual effects.

For organisms relying on an aerobic metabolism, a constant oxygen (O₂) supply must be available to energy demanding tissues. In this thesis. the effects of hypoxia exposure and altered ionoregulatory demands on O₂ uptake of the larval zebrafish (Danio rerio) were evaluated. In Chapter 2, it was hypothesized that a pre-exposure to hypoxia would alter the O₂ uptake capacity of 4- and 7-days post-fertilisation (dpf) larvae through a modified vasculature system. Additionally, using a genetic knockout line, the role of Hif-1α in regulating cutaneous O₂ flux (JO₂) was tested. It was predicted that hypoxia-exposed larvae would display a higher JO₂ across the body due to a hypoxic, acclimatory response, explained by an increased vascularity and supported by an increased whole-body O₂ consumption (ṀO₂) and decreased critical O₂ tension (Pcrit). Consequently, this response was expected to be negated in the Hif1aa⁻/⁻ab⁻/⁻ larvae. Ultimately, JO₂ measured using the scanning micro-optrode technique (SMOT) remained unchanged between WT and Hif1aa⁻/⁻ab⁻/⁻ and normoxia- and hypoxia-exposed larvae, a finding which was supported by an unchanged vascularity across all treatments. The results from this chapter suggest that changes in hypoxia performance mediated by Hif-1α are unrelated to cutaneous JO₂ and vascularity. In Chapter 3, the aerobic costs of ion transport in 4 dpf larval zebrafish was assessed. We hypothesized that changes in rates of Na⁺ uptake evoked by acidic or low Na⁺ rearing would result in changes in ṀO₂ and/or JO₂, measured at the ionocyte-expressing yolk sac epithelium using SMOT. Ultimately, it was found that the measured JO₂ and ṀO₂ did not correlate with the corresponding Na⁺ uptake rate triggered by the acidic and low Na⁺ rearing environment. Thus, we conclude that the aerobic costs of ion uptake by ionocytes in larval zebrafish, at least in the case of Na⁺, are below detection using whole-body respirometry or cutaneous SMOT scans, providing evidence for a low aerobic cost for ion regulation in zebrafish larvae.

No description available.

In this study, a novel method based on biotinylated antibodies and streptavidin coated magnetic beads was used to separate the thrombocyte subpopulations from zebrafish whole blood. DiI-C18, a lipophilic dye, labels only young thrombocytes when used at low concentrations. Commercially available biotinylated anti-Cy3 antibody was used to label the chromophore of DiI-C18 on the young thrombocytes and streptavidin coated magnetic beads were added subsequently, to separate young thrombocytes. The remaining blood cells were probed with custom-made biotinylated anti-GPIIb antibody and streptavidin magnetic beads to separate them from other cells. Further, thrombocytes are equivalents of mammalian platelets. Platelets play a crucial role in thrombus formation. The G-protein coupled receptors (GPCRs) present on the platelet surface are involved during platelet activation and aggregation processes. So, thrombocytes were studied for the presence of GPCRs. The GPCR mRNA transcripts expressed in the young and mature thrombocytes were subjected to densitometry analysis and pixel intensities of the bands were compared using one way ANOVA. This analysis did not show significant differences between the young and mature GPCR mRNA transcripts but identified a novel GPCR, GPR18 that was not reported in platelets earlier. To study the function of this GPCR, it was knocked down using GPR18 specific antisense morpholino and vivo morpholino. The immunofluorescence experiment indicated the presence of GPR18 on thrombocytes. The results of the assays, such as, time to occlusion (TTO) and time to aggregation (TTA) in response to N-arachidonyl glycine (NAG) as an agonist, showed prolongation of time in GPR18 larval and adult morphants respectively, suggesting that GPR18 plays a role in thrombus formation in zebrafish. In conclusion, our results indicate that GPR18 may be present in zebrafish thrombocytes, it may be involved in thrombus formation and that NAG may be an agonist at GPR18 on thrombocytes.

Secretoneurin (SN) is a 31-42 amino acid neuropeptide, derived from the proteolytic processing of the precursor protein secretogranin-II (Scg2). In zebrafish, SNa and SNb are respectively 34 and 31 amino acids long, deriving from selective processing of the distinct Scg2a and Scg2b precursors. Our lab recently reported that frameshift mutations in Scg2 leads to reduces sexual behavior and disrupted spawning. This defect was partially rescued by injection of SNa. In my work, we determined the distribution of SNa in relation to other known reproductive hormones in zebrafish brain and pituitary by double immunofluorescent staining. SNa-immunoreactivity (ir) was observed in neuronal cell bodies in the ventral telencephalon, preoptic area (POA) and hypothalamus. Neuronal fibers staining for SNa projecting from the magnocellular POA passed through the pituitary stalk and terminated largely in the neurointermediate lobe (NIL). The SNa-ir fibers were less abundant but clearly present in the pars distalis. Moreover, SNa colocalized with isotocin in cell bodies in the POA and fibers in the NIL. Using the lhb-RFP x fshb-eGFP transgenic zebrafish line, we observed SNa-ir near gonadotroph cell bodies but not in them. Peptidomic analysis uncovered shorter processed fragments of both in SNa and SNb in whole brain and pituitary. We performed mass spectrometry to determine natural periovulatory variations and studied their potential bioactivities. Both SNa1-34 and SNa1-14 in the brain varied during the ovulatory cycle, while SNb-related peptides were relatively stable. The levels of SNa1-34 in brain peaked coincident with increased Gnrh3 at the time of the luteinizing hormone (Lh) surge. The levels of SNa1-14 in brain and ovaries peaked at the time of ovulation. To investigate the potential bioactivity of SNa1-34 and SNa1-14, we performed intraperitoneal injections and analyzed the expression numerous reproductive genes. The results suggested that SNa1-34 could induce ovulation by stimulating time-dependent expression of gnrh3 in brain, cga and lhb in pituitary and npr in ovaries. In contrast, SNa1-14 exhibited far fewer effects, but stimulated the expression of gnrh2 but suppressed gnrhr2, so its natural biological function remains unknown. After a single injection of SNa1-34 in females isolated from males, 61% (11/18) zebrafish ovulated. This compares favorably with the effects of the Lh analog human chorionic gonadotropin, inducing ovulation in 72% (13/18) of females. Natural variations in levels of SN in relation to other well-known neuropeptides and biological activity data in the zebrafish model support the hypothesis that SNa is a new stimulatory reproductive hormone. The SN peptides are conserved in evolution so what we uncover in fish may help us speculate on its importance in other vertebrates.

Adaptation to environmental fluctuations, through sensing and appropriate physiological responses, is crucial to homeostasis. Neuroepithelial cells (NECs) are putative chemoreceptors resembling mammalian Type I (glomus) cells. They have been shown to respond in vitro to changes in O2, CO2, NH3 and pH. Cytosolic carbonic anhydrase (Ca17a) is thought to be involved in CO2 sensing owing to its presence in NECs. A mutant line of zebrafish (Danio rerio) lacking functional Ca17a was generated using CRISPR/Cas9 technology and used to assess the role of Ca17a in initiating the cardiorespiratory responses to elevated CO2 (hypercapnia). Unfortunately, the homozygous knockout mutants (ca17a-/-) did not survive longer than ~12-14 days post fertilization (dpf), restricting experiments to early developmental stages (4-8 dpf). Changes in ventilation (fV) and cardiac (fH) frequency in response to hypercapnia (1% CO2) in wild type (ca17a+/+), heterozygous (ca17a+/-) and ca17a-/- fish were used to investigate Ca17a-dependent CO2 sensing and downstream signalling. Wild type fish exhibited hyperventilation during hypercapnia as indicated by an increase in fV. In the ca17a-/- fish, the hyperventilatory response was attenuated markedly, but only at 8 dpf. Hypercapnic tachycardia was observed for all genotypes and did not appear to be influenced by the absence of Ca17a. Interestingly, ca17a-/- fish exhibited a significantly reduced resting fH¬. This effect of knockout became more pronounced as the fish aged. Anesthesia did not contribute to the decreased fH in the ca17a-/- fish, nor did changes in cardiac adrenergic or cholinergic tone, which were probed using propranolol (-adrenergic receptor blocker) or atropine (muscarinic receptor blocker). The decrease in resting fH was prevented (“rescued”) when ca17a-/- embryos were injected with ca17a mRNA. Collectively, the results of this thesis support a role for Ca17a in promoting hyperventilation during hypercapnia in larval zebrafish and suggest a previously unrecognized role for Ca17a in determining resting heart rate.

Zebrafish are part of the teleost infraclass (bony fish) of the ray-finned fish. Like other teleosts, zebrafish possess the ability to regenerate most tissues, including their fins. Zebrafish fins contain segmented bony fin rays that longitudinally span the fin. The segments of fin ray are separated by fibrous joints at regularly spaced intervals providing segmentation and flexibility for the fin. Based on gene expression and changes in cell morphology, joint cell differentiation during development and regeneration proceeds through three stages: presumptive joint, joint-forming, and mature joint cells. Our lab has shown that new joint formation correlates with the upregulation of 5’ hoxa gene, hoxa13a. The hox genes encode transcription factors important for patterning in development. In mice, phenotypes resulting from loss- and gain-of-function mutations in Hox genes have revealed that the spatiotemporal expression of these genes is critical for the correct morphogenesis of the limb, a homologous structure to the fin. The first experiments in this thesis use the NTR/MTZ mechanism to partially ablate hoxa13a-expressing cells in the joints and blastema of the regenerating caudal fin. Partial ablation of the hoxa13a-expressing cells results in shorter bone segments following regeneration of the fin. This experiment draws the conclusion that hoxa13a-expressing cells are involved in the regulation of segment length. To examine the function of the 5’ hoxa/d genes in zebrafish, our lab created CRISPR/Cas9 mutations that inactivate hoxa13a, hoxa13b, and hoxd13a. The triple mutants created through serial breeding, show fin-specific defects in the formation and patterning of joints, as well as general defects in the morphology of the ray and in the actinotrichia, collagenous fibres found at the distal edge of the fin. Overall, our data suggest that hox13 genes are necessary for joint formation and proper fin ray growth. With further phenotypic and genotypic analyses our lab proposes that the dosage of hox13 alleles is responsible for anomalies in fin ray formation found in hox13 mutants.