Formation of segments during vertebrate embryogenesis is regulated by a biological clock. Models and experimental data indicate that the core of this clock consists of a cell- autonomous single cell oscillator. This oscillator likely involves a genetic feedback loop of transcriptional repressors belonging to the hairy gene family. In zebrafish, three her genes, her1, hes6 and her7, have been identified as core oscillator components. The main purpose of this project was to study the molecular mechanism of the hairy gene negative feedback oscillator in single cells. To determine whether a single cell oscillator is part of the zebrafish segmentation clock, a cell dissociation protocol was established to track the expression of Her1 ex vivo. Upon dissociation, Her1 expression continued to oscillate for up to three cycles. The period of oscillations was significantly slower than that of the segmentation clock, but appears to speed up in the presence of serum. To test whether the hairy gene interactions are sufficient to generate oscillations in single cells, a protocol was established that uses synthetic biology principles to design, construct and characterize hairy gene networks in yeast. First a library of network parts, containing hairy genes, promoters and Her binding sites was generated and subsequently assembled into simple devices to test their functionality in yeast. The three core oscillator components, Her1, Hes6 and Her7, were characterized and optimized for expression in yeast. In the SWITCH-OFF assay, the Her1 protein, modified with a MigED yeast repressor domain, was found to function as a transcriptional repressor in yeast, while Hes6 with the same modification can not. The dissociation of segmentation clock cells provides the first direct evidence that single cell oscillators exist in zebrafish. In this system, oscillator dynamics can be studied without the interactions of higher level clock components. In parallel, establishing a yeast chassis for hairy gene networks provides a novel technique to directly test predicted oscillator mechanisms by constructing them ’bottom up’.

The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays e.g., in rats or rabbits. Following the 3R principles, the development of alternative methods is encouraged to reduce the number of animal tests. From this perspective, we have developed an in vitro assay (mDarT) using the zebrafish Danio rerio embryo teratogenicity assay (DarT) combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide, ethanol, benzo[a]pyrene and thalidomide were used as test materials to assess the efficiency of this assay. Briefly, the zebrafish embryos were co-cultured at 2 hpf (hours post fertilization) with the test material at varying concentrations, mammalian liver microsomes from different species and NADPH for 60 min at 32°C under moderate agitation in Tris buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterwards fish embryos were transferred individually into 24-well plates filled with fish medium for 48 hours at 26°C with a 12 hour-light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. The test was considered to be valid if a minimum of 90% of fish eggs developed normally for the two controls (test material alone and MAS alone). For each test material, the experiment was repeated three times with the controls satisfying the validation criteria (≤ 10% impaired embryos). Indeed, no significant teratogenic effects were observed compared to controls in fish embryos exposed to the proteratogens alone (i.e., without metabolic activation) or the MAS alone. In contrast, the four test materials induced significant abnormalities in fish embryos when co-incubated with animal liver microsomes. For cyclophosphamide, ethanol and thalidomide a concentration-response relationship was shown and the qualitative nature of the malformations was similar between fish embryos and humans. Benzo[a]pyrene was demonstrated to be significantly teratogenic in fish embryos in spite of no concentration-response and unspecific teratogenic fingerprints. We conclude that the application of animal liver microsomes will improve and refine the DarT as a predictive and valuable alternative method to screen teratogenic substances.

Hox genes encode a conserved family of homeodomain containing transcription factors essential for metazoan development. The establishment of overlapping Hox expression domains specifies tissue identities along the anterior-posterior axis during early embryogenesis and is regulated by chromatin architecture and retinoic acid (RA). Here we present the role nucleosome positioning plays in hox activation during embryogenesis. Using four stages of early embryo development, we map nucleosome positions at 37 zebrafish hox promoters. We find nucleosome arrangement to be progressive, taking place over several stages independent of RA. This progressive change in nucleosome arrangement on invariant sequence suggests that trans-factors play an important role in organizing nucleosomes. To further test the role of trans-factors, we created hoxb1b and hoxb1a mutants to determine if the loss of either protein effected nucleosome positions at the promoter of a known target, hoxb1a. Characterization of these mutations identified hindbrain segmentation defects similar to targeted deletions of mouse orthologs Hoxa1 and Hoxb1 and zebrafish hoxb1b and hoxb1a morpholino (MO) loss-of-function experiments. However, we also identified differences in hindbrain segmentation as well as phenotypes in facial motor neuron migration and reticulospinal neuron formation not previously observed in the MO experiments. Finally, we find that nucleosomes at the hoxb1a promoter are positioned differently in hoxb1b-/- embryos compared to wild-type. Together, our data provides new insight into the roles of hoxb1b and hoxb1a in zebrafish hindbrain segmentation and reticulospinal neuron formation and indicates that nucleosome positioning at hox promoters is dynamic, depending on sequence specific factors such as Hox proteins.

Doctor of Philosophy / Department of Division of Biology / Rollie J. Clem / Mosquitoes are carriers of a variety of harmful human pathogens, including viruses. In order to be successfully transmitted, a virus must evade mosquito immune responses. In this work, the innate immune role of apoptosis in mosquito-virus interactions was examined utilizing the disease vector Aedes aegypti and Sindbis virus. Ae. aegypti is the main vector for yellow fever and dengue virus, which result in over 100 million infections per year. Sindbis virus (Togaviridae) can be transmitted to vertebrates by Ae. aegypti in the laboratory. Sindbis is also well characterized molecularly, making it a good model system for understanding virus-vector interactions. Sindbis MRE-16 recombinant virus clones were utilized to express either an antiapoptotic or pro-apoptotic gene during virus replication. Mosquitoes were infected with recombinant virus clones during a blood meal or by intrathoracic injection. Midgut tissue and whole body samples were analyzed for virus infection and dissemination. Virus was also quantified in saliva and mosquito survival was assayed. Decreased infection in the midgut and delayed virus replication were observed in mosquitoes that were infected with virus expressing a pro-apoptotic gene. Infection with this virus clone also resulted in less virus in the saliva and reduced survival of infected mosquitoes. In addition, negative selection against pro-apoptotic gene expression during virus replication was observed. Collectively, these data suggest that apoptosis can serve as an antiviral defense in Ae. aegypti and may potentially be exploited to control virus transmission. An additional study included in this dissertation focused on zebrafish development and migration of somitic precursors from the tailbud. The tailbud consists of a population of stem cells at the posterior tip of the embryonic tail. The exit of these stem cells from the tailbud is required for the formation of tail somites. A novel double mutant was identified that lacked the t-box transcription factor spadetail and the BMP inhibitor chordin. Double mutants completely lacked somites and had an enlarged tailbud due to accumulation of stem cells that were unable to exit the tailbud. This study indicates the importance of BMP inhibition and spadetail expression in the proper exit of muscle precursors from the tailbud.

Tuberculosis (TB) is still a global epidemic disease despite its discovery over 100 years ago. It is caused by Mycobacterium tuberculosis, which invades and replicates within macrophages, key cells of the innate immune system. The hallmark of tuberculosis is the granuloma which is an accumulation of Mycobacterium-infected cells surrounded by immune cells, and the containment of the bacteria is assured as long as the host immune response remains intact. Despite a well-developed immune response in the infected host, reactivation of latent tuberculosis infection (LTBI) may occur through the introduction of other bacterial pathogens, re-infection with M. tuberculosis or due to other immunosuppression, e.g. AIDS or cancer. The zebrafish–M. marinum model provides an ideal system for examining the pathogenesis of tuberculosis and the associated immune response of the host due to its vertebrate-like immune system, and the close phylogenetic relationship of M. marinum to M. tuberculosis. Granuloma formation and immune response to M. marinum have been investigated mainly in zebrafish embryos or larvae, which lack an adaptive immune response, and little work has been performed in adult fish. This complicates the transfer of findings in these models to chronic, latent or re-activated disease stages in humans, where adaptive immunity plays an important part. The aim of the research presented here was to investigate the immune response of the adult zebrafish to M. marinum infection, with the focus on the kidney as one of the major immune organs in fish. The results obtained support further use of the adult zebrafish-M. marinum model for human tuberculosis infections in the future. In the present study, adult zebrafish were infected with low doses of M. marinum (NCIMB 1297 or NCIMB 1298) and the kidney was investigated for histopathological changes in the form of granulomas over a period of two months(Chapter 3). No granulomas were detected in the fish infected with M. marinum NCIMB 1298 while in zebrafish infected with NCIMB 1297, macrophage aggregation and granuloma formation were detected as early as day 11 post-infection. Occurrence and severity of granulomas and the presence of replicating bacteria increased over time, resulting in a high density of non-caseating and caseating granulomas in the head and posterior kidney after two months of infection. Interleukin 1 beta (IL-1β), Interleukin-12 (IL-12), Tumor necrosis factor alpha (TNFα) and Interferon gamma (IFNγ) have been shown to be important cytokines functioning in defence against tuberculosis, especially IFNγ which is considered to play an important part in acute, chronic and latent tuberculosis. Changes in gene expression of these immune genes in adult zebrafish were investigated over the first two weeks of infection with M. marinum NCIMB 1298 and NCIMB 1297. The results obtained in the first week after infection were inconclusive for both strains investigated. In agreement with the results presented in Chapter 3, no specific immune response was detectable in fish infected with M. marinum NCIMB 1298. However, after 14 days, a high-fold change in IL-12 and TNFα expression were detected in fish infected with M. marinum NCIMB 1297, while IL-1β showed no changes compared to the control fish. Furthermore, no IFNγ expression was detectable over the first two weeks of infection. The delay in the expression of IL-12 and the lack of IFNγ expression can be explained by the ability of M. marinum to manipulate the host immune response, as described for M. tuberculosis and other intracellular bacteria. Besides in vivo investigations of the host-pathogen interactions, in vitro primary macrophage cultures from individual zebrafish kidneys were developed to investigate macrophage-specific gene expression to M. marinum infection (Chapter 4). Although the results looked promising, further optimization is required before the results of the in vitro assays can be fully compared to the in vivo results. Our understanding of reactivation in latent tuberculosis infection (LTBI) both in healthy and immune compromised individuals is insufficient and is delaying the development of treatments for the disease. Therefore, the transcriptome profile of long-term infections (26 weeks) with M. marinum NCIMB 1297 in adult zebrafish was investigated to determine whether the gene expression in this model is comparable to LTBI in humans or other vertebrate model organisms (Chapter 5). In addition, transcriptome profiling was investigated in a group of long-term infected zebrafish exposed to stress to induce re-activation of the disease. Expression profiles in the long-term infected fish and the infected plus stressed fish differed from each other and displayed similar gene profiles to those found in the latent or re-activated disease states, respectively, in human and other vertebrate models. Infected fish displayed a profile highlighted by IFNγ, TNFα, NOS2b and IL-8 expression alongside activating and regulatory T cell responses, including involvement of cytotoxic T cells (CTLs). The transcriptome profile of the group of fish that had been infected and then stressed was distinguished by the lack of IFNγ expression and reduction in TNFα and NOS2b expression, as well as a lack of T cell response compared to the infected fish. In conclusion, the results obtained from Chapters 3 and 4 showed that M. marinum NCIMB 1298 is non-pathogenic to zebrafish. Infection with M. marinum NCIMB 1297, on the other hand, resulted in a similar immune response to that described for human and other mammalian vertebrate models (Chapters 3-5). These results support the use of the adult zebrafish-M. marinum model to investigate LTBI and disease reactivation, and will aid our understanding host-pathogen interactions for tuberculosis in the future.

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programs for endangered species. Many studies have been devoted to the mechanisms of cryodamage. However, in spite of growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells often remains poor. It is known that cryopreservation causes extensive damage to membrane, results in decreased metabolism of cells, and disturbs the bioenergetical processes of cells by damage to mitochondria. Nonetheless, it has not yet been identified clearly if cryopreservation causes some disruption in the genetic integrity of reproductive cells and the safety of this approach still needs to be confirmed. The present study was undertaken on the spermatozoa of weather loach (Misgurnus tassilis) and blastomeres cells of zebrafish (Danio rerio). It was shown that survival was decreased for embryos derived from sperm after cryoprotectant treatment or cryopreservation. Some evidence has emerged that this decrease is more likely to reflect some genetic instability caused by cryopreservation of sperm. The present study showed for the first time that the DNA repair system of oocytes was activated after fertilisation with cryopreserved sperm. The effect of DNA repair system was also studied. It was found that incubation of fertilised eggs in caffeine could reverse the detrimental effects of cryopreservation of loach sperm on subsequent embryo development. On the other hand incubation of fertilised eggs with 3-aminobenzamide - inhibitor of the poly (ADP-ribose) polymerase (PARP)- brought further decrease in the survival of embryos derived from cryopreserved sperm. The effect of individual donors of sperm and eggs on overall embryo survival was also studied and these investigations revealed significant differences between different donors. Effect of cryopreservation on zebrafish blastomeres was studied at the DNA molecular level. Mitochondrial DNA was sequenced after cryopreservation and increased level of frequency of the mutation was observed. This finding showed that cryopreservation might potentially increase the instability of mtDNA genome. The significance of these changes on the subsequent function of the cells is to be elucidated. Meanwhile this study suggests that it is important to be cautious in making judgements on the safety of cryopreservation techniques in reproduction.

Development and regeneration of the vertebrate eye are the result of complex interactions of regulatory networks and spatiotemporally controlled gene expression events. During embryonic retinal development, the coordination of cell signaling and transcriptional regulation allows for a relatively homogenous sheet of neuroepithelial cells to proliferate and differentiate in-to a multilayered, light sensitive retinal tissue. Following injury, the retinas of many cold-blooded vertebrates, such as the zebrafish, undergo a proliferative response that results not only in new retinal cells of the correct type in the correct location, but also functional integration of these cells and restoration of vision. In order for embryonic retinal neurogenesis to proceed correctly, systems must be in place that restrict subsets of progenitor cells from differentiation. Pools of actively proliferating retinal progenitor cells are maintained to fill the needs of developmental processes and normal growth of the retina. In addition, subsets of radial glia in the retina retain the ability to de-differentiate into proliferating progenitor cells to meet the demands of the regenerating retina. All of these processes rely on the tight coordination of extrinsic and intrinsic cues, as well as regulation of gene expression by transcription factors. Although a considerable amount of work has been conducted to identify key regulators of retinal development and regeneration, many gene regulatory networks which include both master signaling pathways as well as individual transcription factors remain poorly characterized. Some of these factors implicated in retinal development and regeneration are members of the Hairy/Enhancer of Split (Hes) superfamily of genes, including the Hairy-related (Her) factors Her4 and Her9. Her transcription factors are basic-helix-loop-helix-orange (bHLH-O) transcription factors that bind to palindromic E- and N-box canonical sequences in the promoters of target genes. Her factors have been previously shown to play roles in a diverse array of developmental and neurogenic processes, including neural tube closure, floor plate development, somitogenesis, and development of various components of the central nervous system as well as the cranial sensory placodes. The roles of her4 and her9 in retinogenesis, however, remain undefined. To determine the possible roles of her4 and her9 factors in the retina, I characterized the expression patterns of these factors during developmental retinal neurogenesis and/or regeneration, examined loss of function phenotypes, and identified signaling pathways that modulate expression of these factors. Chapter 1 of this dissertation provides an overview of vertebrate retina and retinal development, the known functions of her4 in other tissues, and the Notch-Delta signaling pathway. Chapter 2 provides evidence that her4 is a primary effector of the Notch pathway during retinal development, and examines the role of her4 expressing cells during regeneration of the mature zebrafish retina within the context of both chronic and acute photoreceptor damage paradigms. In addition, generation and validation of the transgenic her4:Kaede zebrafish which was used to identify the lineage of her4-expressing cells is described. Characterization of her9 during retinal development, identification of the retinoic acid signaling pathway as a regulator of her9 expression in the retina, and the role her9 plays during retinal vasculogenesis are discussed in Chapter 3. Chapter 4 discusses the generation of her9 knock-out zebrafish lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterization of mutant phenotypes in mosaic her9 mutant F0 fish. In addition, in Chapter 4 I also discuss the screening processes used to identify and characterize genetic lesions in the her9 allele and establish various lines that stably transmit deleterious her9 alleles in the germline, and provide preliminary data of the her9 mutant phenotype. Finally, in Chapter 5 I discuss conclusions from the data generated from this dissertation, additional studies that would expand upon this work, and the implications of these results on the broader understanding of retinal development and regeneration. My dissertation incorporates reverse genetic analysis in zebrafish, biochemical analysis, transgenesis, and various molecular approaches to help better understand the roles of her4 and her9 during retinal neurogenesis. Moreover, these studies may also contribute to a better understanding of retinal development, and disease pathogenesis. It is my hope that this work could also ultimately contribute, even if in some small way, to the goal of enabling human patients who have suffered from vision loss a means by which a damaged retina could be regenerated and functional vision restored.

<p>The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. While these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a, and H+-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role for notochord vacuoles in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.</p> / Dissertation

Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as global endocrine and physiological effectors. In recent years the zebrafish has been utilized as a favorable animal model as a step towards dissecting and better understanding these postembryonic developmental processes. One of the more powerful methods utilized in zebrafish has been the identification of new gene functions through the use of mutant screens. The nutria mutant was recovered from one such screen to identify postembryonic defects in pigment pattern, growth and metamorphosis. These mutants exhibited a pigment cell defect, touch unresponsiveness and severe growth retardation. Here I will discuss my work towards dissecting the underlying developmental processes governing the phenotypic changes in nutria mutants. I characterize gross alterations in skeletal development in nutria mutants that lead to accelerated endochondral ossification but delayed intramembranous ossification. I show that the nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and a kinase. I find trpm7 expression in the fish-specific, ion homeostasis-regulating gland known as the corpuscles of Stannius (CS), and in the mesonephric kidney. I show that mutants also develop kidney stones. Together these results suggest a role for trpm7 activity in regulation of physiological ion homeostasis. Next I confirm that role by identifying late-embryonic and early larval defects in the CS and the kidney, two organs that regulate physiological ion homeostasis. I demonstrate the early larval detection of kidney stones in trpm7 mutants and show that their appearance is presaged by decreased levels of total calcium and magnesium. Furthermore I establish a link between trpm7 function in the CS and stanniocalcin1 (stc1), a potent molecular regulator of calcium homeostasis. Finally, using transgenic overexpression and morpholino-oligonucleotide knockdown, I demonstrate that stc1 modulates calcium and magnesium levels in trpm7 mutant and wild-type backgrounds. Together these analyses establish postembryonic roles for trpm7 function in growth, skeletogenesis, kidney function, and physiological ion homeostasis. / text

The neural cell adhesion molecule L1 is a member of the immunoglobulin superfamily and it mediates many adhesive interactions during brain development. Mutations in the L1 gene are associated with a spectrum of X-linked neurological disorders known as CRASH or L1 syndrome. The objective of this thesis was to use the zebrafish model to investigate the molecular mechanisms of L1 functions and the pathological effects of its mutations. Zebrafish has two L1 homologs, L1.1 and L1.2. Inhibition of L1.1 expression by antisense morpholino oligonucleotides resulted in phenotypes that showed resemblances to L1 patients. However, knockdown of L1.2 expression did not result in notable neural defects. Furthermore, analysis of the expression pattern of L1.1 has led to the discovery of a novel soluble L1.1 isoform, L1.1s. L1.1s is an alternatively spliced form of L1.1, consisting of the first four Ig-like domains and thus a soluble secreted protein. L1.1 morphants exhibited disorganized brain structures with many having an enlarged fourth/hindbrain ventricle. Further characterization revealed aberrations in ventricular polarity, cell patterning and proliferation and helped differentiate the functions of L1.1 and L1.1s. While L1.1 plays a pivotal role in axonal outgrowth and guidance, L1.1s is crucial to brain ventricle formation. Significantly, L1.1s mRNA rescued many anomalies in the morphant brain, but not the trunk phenotypes. Receptor analysis confirmed that L1.1 undergoes heterophilic interactions with neuropilin-1a (Nrp1a). Peptide inhibition studies demonstrated further the involvement of L1.1s in neuroepithelial cell migration during ventricle formation. In the spinal cord, spinal primary motoneurons expressed exclusively the full-length L1.1, and abnormalities in axonal projections of morphants could be rescued only by L1.1 mRNA. Further studies showed that a novel interaction between the Ig3 domain of L1.1 and Unplugged, the zebrafish muscle specific kinase (MuSK), is crucial to motor axonal growth. Together, these results demonstrate that the different parts of L1.1 contribute to the diverse functions of L1.1 in neural development.