<p>Throughout vertebrate evolution, the cardiac outflow vasculature has changed from a branchial arch system to a systemic and pulmonary circulatory system. However, all vertebrate hearts and outflow tracts still develop from a single heart tube. In the chick and mouse, cardiac neural crest cells divide the single outflow tract into the aorta and pulmonary arteries. Additionally, cardiac neural crest cells provide the smooth muscle of the aortic arch arteries, help to remodel the aortic arch arteries into asymmetrical structures, and contribute cardiac ganglia. I review the major contributions of cardiac neural crest cells to the outflow vasculature of the chick and mouse and apply this information to study cardiac neural crest cell contributions to vertebrates that lack a divided circulatory system. I re-evaluate the role of cardiac neural crest cells in zebrafish vasculature and find that these cells do contribute to the gill arch arteries, the ventral aorta and cardiac ganglia, but they do not contribute to myocardium. I also study the outflow tract development of the turtle Trachemys scripta to understand the process of outflow septation in a vertebrate that has a divided outflow tract but an incomplete division of the ventricle. I compare the chick outflow tract to the turtle. The formation of the proximal versus distal cushions and the appearance of smooth muscle cells within the distal cushions of the turtle are very similar to the cushion position and cell types within the cushions of the chick. In the chick, the smooth muscle positive cells in the distal cushions are derived from cardiac neural crest cells. I hypothesize that cardiac neural crest cells are also responsible for the outflow tract septation of reptiles. These results demonstrate that the pattern of cardiac neural crest cell contribution to vertebrate vasculature remains predictable and consistent, enabling future studies to focus on changes in vascular patterning caused by cardiac neural crest cells among different vertebrate lineages.</p> / Dissertation

Myogenesis is the process of skeletal muscle tissue formation where committed muscle progenitor cells differentiate into skeletal muscle fibres. Depending on the instructive cues the muscle progenitor cells receive they will differentiate into specific fibre types with different properties. The skeletal muscle fibres can be broadly classified as fast-twitch fibres or slow-twitch fibres, based on their contractile speed. However, subgroups of fast- and slow-twitch fibres with different metabolic properties, endurance and different isoforms of sarcomeric components have also been identified, adding complexity to the process of muscle tissue patterning. The skeletal muscle tissue has the capacity to regenerate throughout life. Upon muscle tissue damage muscle satellite cells are recruited to the area of injury where they proliferate and either form new fibres similar to those damaged, or fuse with existing fibres. This thesis aims to investigate the process of muscle progenitor cell proliferation and differentiation, as well as the fast-twitch fibre formation and muscle tissue patterning in the zebrafish embryo. I present results identifying the previously uncharacterised gene myl1, encoding an alkali-like myosin light chain, which is specifically expressed in fast-twitch muscle progenitors before fibre formation. Furthermore, I introduce data showing that the transcription factor six1 is expressed in Pax7+ muscle progenitor cells, which has been reported to contribute to part of the fast-twitch muscle tissue as well as to a pool of quiescent muscle satellite cells. With support from the presented data, I hypothesise that six1 keeps the Pax7+ muscle progenitor cells in a proliferative state and consequently prevents them from differentiating into muscle fibres. In addition, I demonstrate that the zebrafish fast-twitch muscle fibres can be divided into different subgroups that express unique forms of fast myosin heavy chain genes along the anterior-posterior (head-tail) axis, and that this subspecification depends on a balance between RA and Wnt signalling. Collectively I propose a previously unknown role for Six1 in zebrafish Pax7+ muscle progenitor cell proliferation and differentiation. Furthermore, I present novel data suggesting that distinct regions of the zebrafish body musculature are composed of different fast-twitch fibre types, and that this regionalisation is conserved in adult zebrafish.

Cardiac parasympathetic tone mediates hypoxic bradycardia in fish, however the specific cholinergic mechanisms underlying this response have not been established. In Chapter 2, bradycardia in zebrafish (Danio rerio) larvae experiencing translational knockdown of the M2 muscarinic receptor was either prevented or limited at two different levels of hypoxia (PO2 = 30 or 40 Torr). Also, M2 receptor deficient fish exposed to exogenous procaterol (a presumed β2-adrenergic receptor agonist) had lower heart rates than similarly treated control fish, implying that the β2-adrenergic receptor may have a cardioinhibitory role in this species. Zebrafish have a single β1-adrenergic receptor (β1AR), but express two distinct β2-adrenergic receptor genes (β2aAR and β2bAR). Zebrafish β1AR deficient larvae described in Chapter 3 had lower resting heart rates than control larvae, which conforms to the stereotypical stimulatory nature of this receptor in the vertebrate heart. However, in larvae where loss of β2a/β2bAR and β1/β2bAR function was combined, heart rate was significantly increased. This confirmed my previous observation that the β2-adrenergic receptor has an inhibitory effect on heart rate in vivo. Fish release the catecholamines epinephrine and norepinephrine (the endogenous ligands of adrenergic receptors) into the circulation when exposed to hypoxia, if sufficiently severe. Zebrafish have two genes for tyrosine hydroxylase (TH1 and TH2), the rate limiting enzyme for catecholamine synthesis, which requires molecular oxygen as a cofactor. In Chapter 4, zebrafish larvae exposed to hypoxia for 4 days exhibited increased whole body epinephrine and norepinephrine content. TH2, but not TH1, mRNA expression decreased after 2 days of hypoxic exposure. The results of this thesis provide some of the first data on receptor-specific control of heart rate in fish under normal and hypoxic conditions. It also provides the first observations that catecholamine turnover and the mRNA expression of enzymes required for catecholamine synthesis in larvae are sensitive to hypoxia. Taken together, these data provide an interesting perspective on the balance of adrenergic and cholinergic control of heart rate in zebrafish larvae.

Abstract The Dlx genes play an important role in the differentiation and migration of gamma-aminobutyric acid (GABA) interneurons of mice. GABAergic interneurons are born in the proliferative zones of the ventral telencephalon and migrate to the cortex early during mouse development. Single Dlx mutant mice show only subtle phenotypes. However, the migration of immature interneurons is blocked in the ventral telencephalon of Dlx1/Dlx2 double mutant mice leading to reduction of GABAergic interneurons in the cortex. Also, Dlx5/Dlx6 expression is almost entirely absent in the forebrain, most probably due to cross-regulatory mechanisms. In zebrafish, the role of dlx genes in GABAergic interneuron development is unknown. By injecting Morpholino, we double knocked down dlx1 and dlx2 genes in wildtype zebrafish to investigate the function of the two genes in zebrafish GABAergic interneuron development. By comparing different subsets of GABAergic interneuron development in wildtype and dlx1/2 morphant zebrafish forebrain, we found out that at 3dpf, 4dpf and 7dpf, double knockdown of dlx1 and dlx2 genes in zebrafish remarkably reduced the number of Calbindin-, Somatostatin- and Parvalbumin-positive GABAergic neurons, whereas the development of Calretinin-positive neurons is slightly affected. These results suggest that in zebrafish, dlx1a and dlx2a genes are important for the development of certain subtypes of GABAergic interneurons (Calbindin-, Somatostatin- and Parvalbumin-positive neurons) and may have minor influence on Calretinin-positive neuron development. This also suggests that different regulatory mechanisms are involved in the development of the different subtypes of GABAergic interneurons.

Intracerebral hemorrhage (ICH) is a severe form of stroke, with a high mortality rate and often resulting in irreversible neurological deterioration. Although animal studies have provided insight into the etiology of the disease, many of the causative genes and mechanisms implicated in cerebral-vascular malformations are unknown. Treatment options remain ineffective. With the present models, the pathophysiological consequences of ICH can only be assessed in situ and after histological analysis. Furthermore, common deficiencies of the current models include the heterogeneity, low expression and low reproducibility of the desired phenotype. Hence, there is a requirement for novel approaches to model ICH pathogenesis. Zebrafish (Danio rerio) has gained recognition as a vertebrate model for stroke research. Through a combination of pharmacological blockers, metabolite rescue, genetic approaches, and confocal imaging analysis, I demonstrate a requirement for the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway in regulating developmental cerebral-vascular stabilization. A transient loss in HMGCR function induces ICH, characterised by progressive dilation of blood vessels, vascular permeability and vessel rupture. These effects are likely due to reduced prenylation of Rho GTPases, evidenced by morpholino-mediated blocking of the prenylation pathway and in vivo assessment of endothelial-specific localization of cdc42, a Rho GTPase family protein. These results are in conformity with recent clinical and experimental evidence. I have further shown that this model consistently replicates common pathoghysiological processes associated with ICH. The hemorrhages are associated with the disruption of the blood-brain barrier, vessel disintegration, hematoma expansion and edema into the adjacent brain regions. Also, enhanced apoptosis, activation of inflammatory mediators in the periphery of the hematoma, enriched heme oxygenase 1 (HO-1) expression and localised thrombosis were observed in these embryos. I show that the patterning and distribution of catecholaminergic neurons, response to sensory stimulus and swimming speed were impaired as a consequence of ICH. These results suggest that HMGCR contributes to cerebral-vascular stabilisation through Rho GTPase mediated-signalling and that zebrafish can serve as a powerful paradigm for the systemic analysis of the etiological and pathophysiological underpinnings of ICH and can help establish the basis for future studies into screening for putative therapeutics and elucidating mechanisms aiding functional recovery.

The caudal fin of zebrafish (danio rerio) is often used to study regeneration thanks to its extraordinary regenerative ability, easy access, and relative simplicity in structure. Branching morphogenesis is observed in many organs, including lungs and salivary glands in mammals, as well as the fin rays in zebrafish and is thought to follow unifying principles. An important developmental gene, sonic hedgehog a (shha), has been shown in other studies to play an essential role in the branch formation. Previous studies in our lab have shown that the transient depletion of the shha-expressing cells following laser ablation of the shha-expressing cells in the regenerating caudal fin results in a delay of fin rays branch formation. In order to study the long-term effect of ablating the shha-expressing cells, I generated a new zebrafish transgenic line (Tg)(2.4shha:CFP-NTR-ABC) to perform a conditional cell ablation using the Metronidazole/Nitroreductase (Mtz/NTR) system. Preliminary data suggest that cell ablation using the Mtz/NTR system is successful in the Tg(2.4shha:CFP-NTR-ABC) embryos. In addition, short-term ablation of the shha-expressing cells through Mtz/NTR system delays branch formation during caudal fin regeneration of the Tg(2.4shha:CFP-NTR-ABC) adult fish. Further work will involve the analysis of the effects of the long-term ablation of the shha-expressing cells and the involvement of other signaling pathways in the ray branching formation during zebrafish caudal fin regeneration. This study can provide insights into understanding of the molecular mechanisms underlying branching morphogenesis in various organs. During the course of the above project, I have observed an organ-wide response to local injury in the zebrafish caudal fin. In this study, I have shown, for the first time, an immediate organ-wide response to partial fin amputation characterized by the damage of blood vessels, nerve fibers and the activation of inflammatory response in the non-amputated tissues. I established that the adult zebrafish caudal fin serves as an excellent model for the study of the organ-wide response to local injury, and such study may provide new insights into the field of regenerative medicine in which stimulating regeneration locally may trigger responses in unintended locations.   Résumé La nageoire caudale du poisson zèbre (danio rerio) est souvent utilisée pour étudier les mécanismes de régénération à cause de son extraordinaire capacité de régénération, son accès facile, et sa relative simplicité structurale. La morphogenèse de branches est observée dans plusieurs organes incluant les poumons et les glandes salivaires chez les mammifères ainsi que les rayons des nageoires du poisson zèbre et est supposée suivre des principes communs. Un important gène de développement, sonic hedgehog a (shha), joue un rôle essentiel dans la formation des branches. Des études précédentes effectuées dans notre laboratoire ont montré que l’absence transitoire des cellules exprimant shha dans des expériences d’ablation au rayon laser induit un délai de la formation des branches dans les rayons au cours de la régénération de la nageoire caudale. Afin d’étudier les effets de l’ablation à long terme des cellules exprimant shha, j’ai fait un nouvelle lignée transgénique de poisson zèbre Tg(2.4shha:CFP-NTR-ABC) pour effectuer une ablation cellulaire conditionnelle à l’aide du système Métronidazole / Nitroréductase (Mtz/NTR). Mes données préliminaires suggèrent que l’ablation cellulaire à l’aide du système Mtz/NTR fonctionne sur les embryons Tg(2.4shha:CFP-NTR-ABC). De plus, l’ablation à court terme des cellules exprimant shha à l’aide du système Mtz/NTR induit un délai de la formation des branches au cours de la régénération des rayons la nageoire caudale des poissons adultes Tg(2.4shha:CFP-NTR-ABC). Des études supplémentaires incluront l’analyse des effets de l’ablation à long terme des cellules exprimant shha et le rôle d’autres cascades de signalisation dans la formation des branches des rayons au cours de la régénération de la nageoire caudale du poisson zèbre. Cette étude pourrait fournir des informations concernant la compréhension des mécanismes moléculaires sous-jacents à la formation de branches dans des organes variés. Au cours de l’étude décrite ci-dessus, j’ai fait l’observation d’une réponse globale de toute la nageoire caudale à une blessure locale. Dans cette étude, j’ai montré pour la première fois, une réponse immédiate et globale après amputation partielle de la nageoire. Cette réponse est caractérisée par des lésions des vaisseaux sanguins, des fibres nerveuses et par l’activation d’une réponse inflammatoire dans les tissus non-amputés. J’ai établi que la nageoire caudale du poisson zèbre adulte est un excellent modèle pour l’étude de la réponse globale d’un organe à une lésion locale. Une telle étude pourrait fournir de nouvelles informations pertinentes à la médecine régénérative qui, en visant à stimuler la régénération de façon locale, peut entraîner des réponses dans des domaines non voulus.

Zebrafish, Danio rerio, are able to survive exposure to extreme acidity (pH 4). Because previous studies demonstrated that disruption of ionic balance during exposure to acidic water is the major cause of mortality in acid-sensitive freshwater species, the focus of this thesis was to characterize the molecular mechanisms enabling zebrafish to maintain their Na+ homeostasis following exposure to acidic water. Initial findings (Chapter 2) demonstrated that branchial mRNA expression of selected isoforms of claudins, major components of tight junctions, are altered in an isoform-dependent manner, suggesting the potential regulation of epithelial permeability to minimize ion loss. Concurrently, a marked stimulation of Na+ uptake was observed in adults and larvae following acid-exposure. Because of the uniqueness of this response (increasing Na+ uptake in acidic water) among freshwater teleosts, the mechanisms related to Na+ uptake and its stimulation were investigated further (Chapters 3 - 7). Pharmacological treatments and gene knockdown approaches revealed that a functional metabolon consisting of an apically expressed Na+-H+-exchanger (NHE3b) in association with an apically expressed ammonia-conducting channel (Rhcg1), enables Na+ uptake in acidic water. During chronic (>1 day) exposure to acidic water, cortisol (via glucocorticoid receptors) and catecholamines (via β-adrenergic receptors) are involved in stimulating Na+ uptake. Although catecholamines may act on both NHE3b and Na+-Cl- co-transporter (NCC), the effects of cortisol on Na+ uptake are mediated primarily by activation of NHE3b. On the other hand, during acute (<3 h) exposure to acidic water, cortisol does not appear to affect Na+ uptake; rather, the stimulation of Na+ uptake appears to be mediated by angiotensin II and catecholamines. Cyclic AMP (cAMP), a signalling molecule synthesized following the activation of β-adrenergic receptors, is critically involved in stimulating Na+ uptake, likely via activation of NHE3b and NCC. In agreement with this idea, ionocytes that express NHE3b also express high levels of β-adrenergic receptor (propranolol binding sites) as well as trans-membrane adenylyl cyclase (forskolin binding sites). Taken together, the results of this thesis provide fresh insight into the mechanisms of osmoregulation in freshwater (FW) fish. In particular, the data reveal the presence of complex pathways regulating Na+ uptake in zebrafish exposed to acidic water. The relative importance of the various pathways depends in part on the duration of exposure; acute versus chronic.

Previous in vitro studies have demonstrated that AQP1, traditionally viewed as a water channel, also facilitates the passage of CO2 and ammonia across cell membranes. This thesis summarizes the first in vivo studies confirming a physiologically-relevant role for AQP1 in acid-base balance and nitrogenous waste excretion. Zebrafish embryos were microinjected with a translation-blocking morpholino oligonucleotide targeted to the zebrafish AQP1 paralog, AQP1a. Closed-system respirometry, total CO2 analysis, tritiated water fluxes and measurement of ammonia excretion were performed on larvae at 4 days post-fertilization (dpf). Knockdown of AQP1a significantly reduced rates of water, CO2 and ammonia excretion. Use of phenylhydrazine, a haemolytic agent, provided evidence that the yolk sac epithelium AQP1a (and not erythrocyte AQP1a) is the major site of CO2 and ammonia movements. Further, the hypothesis that AQP1a and the Rh glycoprotein Rhcg1, another multi-functional gas channel, act in concert to regulate CO2 and ammonia excretion was explored. Exposure to conditions impairing ammonia excretion (such as high external ammonia (HEA) or alkaline water) modulated AQP1a protein expression in 4 dpf zebrafish larvae experiencing knockdown of Rhcg1. Chronic HEA exposure triggered a significant compensatory increase in AQP1a protein abundance in Rhcg1 morphants. Exposure of Rhcg1 morphants to pH 10 water, however, caused a significant decrease in AQP1a protein expression. Interestingly, when AQP1a mRNA and protein levels were examined in Rhcg1 morphants and vice versa, no changes were observed. Overall, zebrafish AQP1a was found to be a multi-functional transporter of water, CO2 and ammonia, though the exact relationship it holds with other such gas channels bears further exploration.

<p>Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants formed from the incomplete combustion of fossil fuels and are found in the environment as complex mixtures. PAHs are developmentally toxic to fish, causing yolk sac edema, hemorrhaging, craniofacial malformations and cardiac defects including impaired heart looping, elongated heart, decreased blood flow, and pericardial effusion. Previous research has shown that many of the toxic effects of PAHs are mediated through the aryl hydrocarbon receptor (AHR), which upregulates phase I and II metabolic genes, but the underlying mechanisms of PAH-induced toxicity are not yet known. The primary goal of this dissertation was to better understand the molecular mechanisms by which PAH mixtures cause developmental toxicity in fish. To this end, the zebrafish (Danio rerio) was used as a developmental model. Simple mixtures consisting of a PAH that is an AHR agonist (benzo[a]pyrene or benzo[k]fluoranthene) and a PAH that is a cytochrome P450 1 (CYP1) inhibitor (fluoranthene) were used in these experiments along with the dioxin-like compound 3,3',4,4',5-pentachlorobiphenyl (PCB-126). Morpholino gene knockdown was used to examine the role of specific genes in response to PAHs, gene expression changes in response to PAH exposures were examined via QPCR, quantification of pericardial effusion was used as a metric for cardiac toxicity, and CYP1 activity was measured as an indication of AHR pathway induction. First, PAH mixtures consisting of an AHR agonist (BkF) and a CYP1 inhibitor (FL) induced cardiac toxicity that was preceded by upregulation of CYP1 and redox-responsive gene expression, and these effects were dependent upon the AHR2. Second, knockdown of glutathione s-transferase pi class 2 (GSTp2), part of phase II metabolism, exacerbated PAH-induced toxicity but did not affect PCB-126-induced toxicity. Third, knockdown of another isoform of the AHR, AHR1, exacerbated PAH- and PCB-126-induced toxicity and increased CYP1 activity but did not affect CYP expression in response to these agonists. Simultaneous knockdown of AHR1A and AHR2 did not exacerbate nor ameliorate PAH-induced toxicity but did prevent PCB-126-induced toxicity. Fourth, to examine AHR2-dependent and AHR2-independent gene induction in zebrafish hearts in response to PAHs, microarrays were used. Gene expression changes caused by PAHs were largely AHR2-dependent and consisted of genes involved in cell adhesion, oxidation-reduction, and TGF-&beta signaling processes as well as genes involved in heart structure and function. These findings help to elucidate how PAHs elicit deformities during development and highlight differences between PAHs and other AHR agonists. Additionally, these experiments have identified other genes in addition to AHR2 that are involved in mediating or responding to the toxicity of PAHs.</p> / Dissertation

Discovering novel compounds that stimulate or abrogate angiogenesis can lead to development of new therapeutic agents that may effectively treat diseases with pathological angiogenesis. The zebrafish model allows for a whole-organism approach to drug discovery. Advantages over other animal models include small embryo size, fecundity, rapid embryonic development, optical clarity and easy accessibility of the embryos. My goal is to validate the therapeutic efficacy and identify the molecular mechanisms of action of three compounds identified from our previous chemical genetic screens. Fenretinide promoted angiogenesis in zebrafish embryos but inhibited the angiogenesis-dependent process of fin regeneration. The pro-angiogenic effects of fenretinide appear secondary to the stimulation of somitogenesis. I3M potently inhibited angiogenesis and fin regeneration, and may act partially through the notch pathway. Lastly, I validated the anti-angiogenic effect of a novel compound DHM. Comprehensively, my studies support the utility of zebrafish as a versatile tool for anti-angiogenic drug discovery.