This thesis aims to examine how hindbrain spinal projection neurons (SPNs), namely RoV3, MiV1 and MiV2 control tail undulations during turning behaviors. I find that phototaxic turns differ from forward swims by an increased tail bend and a prolonged cycle period during the first undulation, while the later undulations are largely identical. Interestingly, laser ablation of RoV3, MiV1 and MiV2 neurons specifically affects the first undulation cycle by reducing the tail bend and the cycle period. Thus fish without the SPNs mainly perform forward swims in response to the turn-inducing phototaxic cues. These results suggest that the descending motor command that generates turns in larval zebrafish are composed of two pathways: one generates symmetric tail undulations, and the other, mediated by RoV3, MiV1 and MiV2 neurons, provides a brief and biased effect that modulates the first cycle of tail movement. Furthermore, fish whose unilateral SPNs are ablated are unable to perform turns toward the ablated side during the phototaxis, the optomotor response, the dark-flash response, and spontaneous swims, indicating the universal role of the SPNs in controlling visually-induced and spontaneous turns. Simultaneous two-photon calcium imaging and motor nerve recording in paralyzed fish show that RoV3, MiV2 and most MiV1 neurons on the turning side are active during turns, and that these activities are linearly correlated to the vigor of the intended turns. However, some MiV1 neurons are broadly tuned for all swimming directions. Computer simulations suggest that unilateral descending innervations to a specific type of spinal interneurons, namely commissural inhibitory interneurons, can generate a two-fold increase in the spinal network’s cycle period. This suggests that the SPNs could potentially innervate two types of spinal interneurons, namely \(CoBL_{gly}\) and CoLo, in order to control the rhythm during turns. An additional chapter of this thesis examines the ontogeny of operant and classical learning behaviors in zebrafish. Using strategically positioned visual cues paired with electroshocks, I find that both learning behaviors are expressed reliably around week 3, and reach adult performance levels at week 6. These memories are behaviorally expressed in adults for 6 hours and retrievable for 12 hours.

The initiation of the hematopoietic program is orchestrated by key transcription factors that recruit chromatin regulators in order to activate or inhibit blood target gene expression. To generate a complete compendium of chromatin factors that establish the genetic code during developmental hematopoiesis, we conducted a large-scale reverse genetic screen targeting 425 chromatin factors in zebrafish and identified over 30 novel chromatin regulators that function at distinct steps of embryonic hematopoiesis. In vertebrates, developmental hematopoiesis occurs in two waves. During the first and primitive wave, mainly erythrocytes are produced, and we identified at least 15 chromatin factors that decrease or increase formation of \(scl^+\), \(gata1^+\), and \(\beta-globin e3^+\) erythroid progenitors. In the definitive wave, HSCs capable of self-renewal and differentiation into multiple lineages are induced, and we identified at least 18 chromatin factors that decrease or increase the formation of \(c-myb^+\) and \(runx1^+\) stem and progenitor cells in the aorta gonad mesonephros (AGM) region, without disruption of vascular development. The majority of the chromatin factors identified from the screen are involved in histone acetylation, histone methylation, and nucleosome remodeling, the same modifications that are hypothesized to have the most functional impact on the transcriptional status of a gene. Moreover, these factors can be mapped to subunits of chromatin complexes that modify these marks, such as HBO/HAT, HDAC/NuRD, SET1A/MLL, ISWI, and SWI/SNF. One of the strongest phenotypes identified from the screen came from knockdown of chromodomain helicase DNA binding domain 7 (chd7). Morpholino knockdown of chd7 resulted in increased primitive and definitive blood production from the induction of stem and progenitor cells to the differentiation of myeloid and erythroid lineages. This expansion of the blood lineage is cell autonomous as determined by blastula transplantation experiments. Though chromatin factors are believed to function broadly and are often expressed ubiquitously, the combined results of the screen and chd7 analysis show that individual factors have very tissue specific functions. These studies implicate chromatin factors as playing a major role in establishing the programs of gene expression for self-renewal and differentiation of hematopoietic cells.

The Heart of glass-Cerebral Cavernous Malformation (Heg-CCM) pathway is essential for heart development in zebrafish and mouse. In zebrafish, mutants for the Heg-CCM genes ccm1, ccm2, and heg exhibit an extreme dilation of the heart chambers and inflow tract and completely lack blood circulation. The mechanisms by which this pathway regulates heart development are incompletely understood. Two major impediments to our knowledge are the paucity of genes known to participate in the Heg-CCM pathway and a lack of information about how the Heg-CCM pathway interacts with other signaling pathways in live embryos.

I present applications of imaging and spectroscopy to understand mechanical, chemical, and electrical dynamics in biological materials. The first part describes the development and characterization of a protein-based fluorescent calcium and voltage indicator (CaViar). The far-red fluorescence of CaViar faithfully tracks the cardiac action potential in cardiomyocytes. CaViar's green fluorescence reports the resulting calcium transients. I demonstrated the applicability of CaViar in vivo with transgenic zebrafish designed to express CaViar in their hearts. Spinning disk confocal imaging allowed detailed three-dimensional mapping of simultaneous voltage and calcium dynamics throughout the heart of zebrafish embryos, in vivo, as a function of developmental stage. I tested the effect of channel blockers on voltage and calcium dynamics and discovered a chamber-specific transition from a calcium-dependent to a sodium-dependent action potential. I also describe a new measurement technique using a fluorescent voltage indicator to report absolute voltage via the indicator's temporal response. / Physics

Σκοπός της έρευνας ήταν να μελετήσουμε την επίδραση της θερμοκρασίας στο ολικό μεταγράφωμα νυμφών zebrafish (Danio rerio, Hamilton 1822), θέλοντας να εστιάσουμε το ενδιαφέρον μας στη διερεύνηση μηχανισμών και μορίων που εμπλέκονται στο φυλοκαθορισμό του συγκεκριμένου ψαριού. Kατορθώσαμε να επιδράσουμε με τρεις διαφορετικές θερμοκρασίες σε μία θερμοκρασιακά ευαίσθητη οντογενετική περίοδο, η οποία οριοθετείται από την 10 έως την 20dpf. Η επιλογή των θερμοκρασιών έγινε με σημείο αναφοράς τους 28 ºC, η οποία είναι η πρότυπη θερμοκρασία ανάπτυξης των zebrafish. Η μελέτη της επίδρασης της θερμοκρασίας επιτεύχθη με διατήρηση των πειραματικών μας πληθυσμών σε θερμοκρασίες που αποκλίνουν κατά 4-6 ºC από την πρότυπη, δηλαδή στους 32 ºC και 22 ºC αντίστοιχα. Ως εκ τούτου, ολόκληρος ο πειραματικός πληθυσμός αναπτύχθηκε στη πρότυπη θερμοκρασία των 28 ºC μέχρι και την 10 dpf, όπου μετά την πάροδο της χωρίστηκε σε τρεις υπoπληθυσμούς καθένας από τους οποίους εκτέθηκε στους 22 ºC, 28 ºC ή 32 ºC αντίστοιχα για 280 θερμοημέρες (χρονική περίοδος :10-20dpf). Ακολούθησε δειγματοληψία (100 περίπου ατόμων) από κάθε υποπληθυσμό και εν συνεχεία πραγματοποιήθηκε απομόνωση ολικού RNA. Τα δείγματα μας υβριδοποιήθηκαν σε microarrays, τα οποία έφεραν αντιπροσωπευτικές αλληλουχίες για 15.500 γονίδια του zebrafish. Τα δεδομένα επεξεργάστηκαν με τη χρήση δυο εξειδικευμένων βιοπληροφορικών προγραμμάτων, τα Biosystanse και Dchip, με τη βοήθεια των οποίων συγκρίθηκαν οι εξής καταστάσεις: 10dpf _28 ºC με 20dpf _28 ºC, η οποία αποτελεί την αναπτυξιακή σύγκριση ώστε να ελέγξουμε γονίδια των οποίων η έκφραση αυξάνεται ή ελαττώνεται στα δυο αυτά στάδια αλλά και 20dpf_22 ºC με 20dpf_32 ºC, 20dpf_22 ºC με 20dpf_28 ºC και 20dpf_28 ºC με 20dpf_32 ºC, ώστε να παρατηρηθεί η επίδραση του περιβαλλοντικού παράγοντα ″θερμοκρασία″ στο μεταγράφωμα των ιχθυδίων. / The purpsose of this research was to analyse how temperature effects the total trascriptome at juvenile zebrash (Danio rerio, Hamilton 1822). So, we focus our investigation in the inspection of mechanisms and molecules which probably associated with the sex determination at this specific type of fish. We militated in the thermosensitive ontogenetic period ( from 10 to 20 days post fertilization) with three different temperatures. These three temperatures were 22, 28 and 32οC. Exemplary temperature for zebrafish is 28οC (environmental temperature). Initially, experimental population grew up to 28οC until the 10th day post fertilization. After this temporal period, population separated to three smaller experimental populations which of them grew up to 22, 28 and 32οC respectively for 280 thermodays ( chronic period: 10-20dpf). The next step was sampling (about 100 fish from each experimental population) and then isolating total RNA from each one of the samples. Total RNA samples hybridized on microarrays. Microarrays contained complementary sequences for about 15.500 genes of zebrafish genome. The data analyzed with two specific bioinformatics programs, Biosystance and Dchip. The use of these bioinformatics programs helped us comparing the following situations: 10dpf _28 ºC to 20dpf _28 ºC (developmental comparison) over and above 20dpf_22 ºC to 20dpf_32 ºC, 20dpf_22 ºC to 20dpf_28 ºC and 20dpf_28 ºC to 20dpf_32 ºC (temperature comparison).

Olfaction is vitally important to animals in all environments and is used to identify food, habitat, conspecifics and predators. Some odors, like pheromones or the pungent smell of spoiled foods, can trigger pre-existing behavioral responses that appear to require no learning. Most odors, however, are only attended to as a result of prior experience. It is believed that different types of odors are processed in different olfactory pathways in the forebrain. This thesis examines the relationship between innate and learned olfactory behaviors and the anatomy of the neural pathways that underlie them, using the zebrafish olfactory system as a model. I first characterized an appetitive olfactory behavior, which is displayed promptly by zebrafish when they encounter amino acid odors. A similar appetitive behavior can also be learned by the fish for another, initially neutral odorant, if it is repeatedly paired with food rewards. Zebrafish can therefore respond to, and learn to respond to certain odors. I then conducted an in-depth anatomical analysis of the structure and distribution of glomeruli in the zebrafish olfactory system. Glomeruli are spheroidal synaptic aggregates that organize and shape olfactory information that arrives in the brain. Throughout the development of zebrafish, I identified two distinct populations of glomeruli. One population consisted of 25 individually identifiable, anatomically stereotypic glomeruli that closely resembled specialized glomeruli in mammals and insects. These glomeruli were already formed during embryonic development and persisted in remarkably stable configurations throughout later developmental stages. I hypothesize that the 25 individually identifiable glomeruli constitute stable olfactory pathways (i.e., for innate olfactory behaviors). Most glomeruli, however, were anatomically variable and displayed different distributions within coarsely circumscribed regions in the zebrafish olfactory bulbs. The development of these glomeruli could be modified by sensory experience, suggesting that they may comprise plastic olfactory pathways that subserve the establishment of learned olfactory behaviors. Collectively my results show that innate and learned olfactory behaviors may indeed be represented in different olfactory pathways, and that these types of pathways may be located in both main and accessory olfactory systems.

The purpose of this study was to characterize the endocrine stress response of zebrafish (Danio rerio) and to test if this response is modified by sex or the major sex steroids 17β-estradiol (E2) and 11-ketotestosterone (11KT). A standardized stress of having zebrafish swim in a beaker of stirring water resulted in rapid and transient changes in whole-body cortisol. While the cortisol response of males and females were comparable, exposure to E2 (100 ng/L) for 48 h inhibited the cortisol response of males and exposure to 11KT (100ng/L) for 48 h stimulated the cortisol response of females. The inhibitory actions of E2 were mediated in part by decreases in corticotropin releasing factor (crf) expression and decreases in the synthesis of cortisol by interrenal tissue. This research adds to the increasing knowledge of zebrafish physiology and will be useful for future studies concerning the endocrine stress response in fish.

No description available.

The extracellular matrix is a vital component of the cardiovascular system, in that, it not only provides structural support but also plays a critical role in the maintenance of cellular stability. One of the major components of the vascular matrix is elastin, which confers vessels with the specialized property of stretch and recoil. Elastin deficiency has been implicated in many vascular diseases and determined experimentally to be a negative regulator of smooth muscle cell proliferation. In zebrafish, two elastin genes have been identified, which are actively expressed during development. Based on this finding, protein production and spatial localization for the two elastin proteins was studied by immunohistochemistry with specific antibodies. Results revealed a global distribution for elastin 1 in the ventral aorta and swim bladder, whereas elastin 2 was preferentially localized to the bulbus arteriosus indicating a possible specialized function of elastin 2 in this structure. This observation, and the unique physiological property of this structure, suggests a possible reason for the preservation of both elastin genes during evolution. / In the second part of this study, elastin-null mice were studied to uncover the impact of the loss of elastin on the expression of other elastic fiber-associated proteins. The expression of fibrillin-1, the major component of microfibrils, was not altered in the absence of elastin, implying that elastin is not necessary for the formation of microfibrils. On the other hand, both fibulin-2 and -5 were upregulated in the absence of elastin, suggesting that expression of these genes are controlled by elastin. Overall, this study highlights the importance of elastin in evolution, as well as its potential role in the regulation of expression of other matrix molecules.

More than one million people worldwide receive some kind of bone graft each year. Grafts are often needed following bone tumour removal or traumatic fractures to fill voids in the bone and to aid in the healing process. The most common method involves bone transplantation, in which bone tissue is taken from one site to fill the defect in another site. The procedure thus involves two surgeries, which leads to an increased risk of complications. New, synthetic graft materials that can be used to fill defects and minimise the complications associated with bone tissue harvesting are therefore necessary. The synthetic materials available today lack the inherent biological factors of bone that stimulate the bone regeneration process. Much of today’s research concerning synthetic bone graft materials aims to solve this issue and researchers have suggested several different strategies. The purpose of this thesis is to improve the performance of acidic calcium phosphate cements, which are materials used as synthetic bone grafts. By combining these cements with drugs or ion additives, local delivery could be achieved with the potential to stimulate bone formation. Two different combinations were attempted in this thesis: cement in combination with simvastatin, or cement in combination with strontium halide salts. Both simvastatin and strontium are known to positively affect bone formation. The efficacy of the cements with the additives was evaluated using different bone cell cultures. The results regarding simvastatin showed that the cement’s mechanical property was not affected upon drug loading, and that the drug was released by a diffusion-controlled mechanism. Moreover, results showed that simvastatin stimulated the bone-forming cells (osteoblasts) to produce more bone tissue, while it inhibited bone-degrading cells (osteoclasts) from degrading the cement. These findings suggest that simvastatin could aid in the bone regeneration process in the local area surrounding the cement. The main purpose of the study using strontium halide salts was to increase the cement’s X-ray contrast, which is a property used to monitor cement during injection. In addition, strontium is believed to positively affect bone cells. The X-ray contrast did increase after the addition of 10 wt% strontium bromide or strontium iodide, while the cell study results did not indicate any significant effects on the bone-forming cells. In the last section of this thesis, zebrafish were used as a model to evaluate bone formation upon treatment with degradation products from synthetic bone grafts. The zebrafish is a small organism with 70 % gene homology to humans; due to its transparency, fast development and ease of handling, it is an interesting model for high-throughput studies. Silicate, which is an ionic degradation product of many different bone substitute materials, was used as a proof-of-concept to visualise bone formation in these fish. The results showed an increased bone formation upon treatment with 0.625 μM silicate ions. The results suggest that this model could be used as a complement to bone cell culture studies in pre-clinical evaluations of the degradation products of bone substitute materials, thus helping researchers to design materials with degradation products that could stimulate bone formation.