Selective serotonin (5-HT) reuptake inhibitors (SSRIs) particularly fluoxetine (FLX, Prozac®), are often the first-line of pharmacological treatment for affective disorders in pregnant women. Given that SSRIs readily cross the placenta, a fetus from a SSRI-treated pregnant woman is potentially at risk from the disruptive effects of the SSRIs-induced 5-HT actions during this highly plastic stage of development. One of the prominent roles of 5-HT that we will explore here is its involvement in the development and programming of the stress axis. Pharmaceuticals including FLX and other SSRIs reach aquatic ecosystems through sewage release, so fish may also be inadvertently exposed. We investigated the premise that early-life exposure to FLX induces a transgenerational disruption of the stress axis using the zebrafish (ZF) Danio rerio, as a research model to encompass both the environmental and human health concerns. The FLX concentrations studied were environmentally relevant (0.54 µg·L-1) or comparable to concentrations detected in the cord blood of FLX-treated pregnant women (54 µg·L-1). Exposure to FLX during the first 6 days of life induced a reduction of whole-body cortisol levels in adult ZF (filial generation 0; F0), an effect that persisted across 4 consecutive generations without diminution, even though the descendants (F1 to F4) were not directly exposed to FLX. This effect was more pronounced and persistent in males than females. The in vivo cortisol response of the interrenal cells (the fish ‘adrenal’) to an intraperitoneal injection of adrenocorticotropic hormone was also reduced in the F0 and F3 FLX-exposed males. RNA sequencing of the F0 and F3 male whole kidney containing the interrenal cells detected an array of differentially expressed genes (>500) altered by FLX treatment. Enrichment analysis of these genes revealed that early FLX exposure significantly modified numerous canonical pathways (>40) including key pathways related to steroidogenesis. These findings provide further insights on the underlying mechanism of the transgenerational disruption induced by FLX. We also showed that altered cortisol levels were linked to reduced exploratory behavior in adult males from the F0 to F2 FLX lineage. In contrast, females were susceptible to the effects of FLX-induced hypocortisolism during a different window of development. Exposure to FLX in the sex differentiation period (15 to 42 days post-fertilization) increased exploratory behavior in the adult females. Transcriptional profile of selected steroidogenic genes in the whole-larvae from the F0 varied in magnitude and direction in both treatments, despite the same low cortisol phenotype induced by both concentrations. We also found an up-regulation in the transcript levels of steroidogenic-related genes and a down-regulation of a gene involved in the inactivation of cortisol in the F3 larvae ancestrally exposed to the human-relevant concentration. These findings on the transcript levels of the selected genes in the larvae from F0 and F3 suggest that the larvae adopted specific coping mechanism(s) to the disruptive effects of FLX depending on the exposure concentration and the filial generation. The pigmentation patterns in some of the descendants of the exposed fish (F1 to F3) were reduced by the 6-day early-FLX treatment. In response to a 6-day embryonic exposure to a second antidepressant, venlafaxine, the F4 adult females that were ancestrally exposed (in the F0) to the human-relevant FLX concentration displayed an intensified reduction of cortisol levels. Therefore, FLX exposure of the great-great-grandparents (F0) permanently and most likely epigenetically shaped the response of future generations to other antidepressants. Collectively, our data are cause for concern, given the high-prescription rates of FLX to pregnant women and the potential long-term negative impacts on humans and aquatic organisms exposed to ever rising levels of SSRIs.

Effective reproduction is essential for species survival. Sexual reproduction depends upon functional gonads and reproductive ducts. Zebrafish (Danio rerio) is a popular model organism, but the genetic basis of zebrafish sex determination, gonad development, and reproductive tract development are not fully understood, and understanding this basis could inform about the evolutionary conservation of these genes and the use of zebrafish to investigate and treat reproductive diseases. In chapter I, I give a overview of sex determination systems, gonad development, and reproductive duct development in mammals and fish, and ask how sex is determined and how reproductive ducts develop in zebrafish. In chapter II, I used genome wide association studies (GWAS) to investigate if the genetic basis of sex determination in a variety of zebrafish strains -- two ‘wild-type’ strains cultured for about 30 years in the lab, and four ‘natural’ strains, wild-type strains isolated directly or recently from nature in India -- and identified a sex-associated region on zebrafish chromosome 4 in natural zebrafish strains that was lacking in the lab strains. In chapter III, I investigated whether or not wnt4a is important for zebrafish ovarian development, and found that wnt4a is expressed in the early bipotential gonad and that loss of wnt4a results in male-biased sex ratios, indicating that wnt4a is important for zebrafish ovarian development. In chapter IV, I investigated whether or not wnt4a is important for male reproductive duct development, and found that loss of wnt4a slows the formation of male reproductive ducts and prevents the male fused ducti deferens from connecting to the genital orifice in zebrafish males. Results further showed that wnt4a is expressed in tissue around the site where this connection should occur before and after the connection is formed, revealing a novel wnt4a phenotype in zebrafish that hasn’t been seen thus far, and indicating that wnt4a is necessary for proper male reproductive duct development in zebrafish. This dissertation contains both published and unpublished co-authored material.

A segmented vertebral column is one of the major innovations vertebrates. In mice and chicks – amniotes – a subpopulation of the somites, the sclerotome, is sole source of vertebral tissue. It is unclear, however, how applicable this amniote-based ‘canonical’ mechanism is across the vertebrates. In fact, the vast majority and diversity of vertebrates are not amniotes, but are members of ‘fish’ groups where there has been relatively little investigation into vertebral development. Indeed, there is great diversity in vertebra form throughout ‘fish’ groups and fossil evidence suggests that the components of the vertebra, the neural arches and the vertebral bodies, arose separately and that vertebrates have evolved multiple ways of building vertebral bodies. In teleosts fish, the vertebral bodies initially form as mineralised rings within the notochord sheath (chordacentra) and then secondarily, bone is deposited around this (perichordal centra and arches). Notochord cells (chordoblasts) have been implicated in chordacentrum mineralisation and patterning in zebrafish and Atlantic salmon, though the question of how the overtly unsegmented notochord could direct segmental mineralisation still remains. My project first aims to address this dual mechanism in the zebrafish model, by testing whether the chordoblasts can mineralise and pattern the chordacentra. The second aim is to elucidate the role of the sclerotome in teleost vertebral development. To do this, I explored CRISPR knock-in tools to label the sclerotome and used a Gal4 gene trap line to investigate sclerotome ablation. I characterised the chordacentra and chordoblasts in our model system and verified the specificity of a promoter as a chordoblast marker. With this promoter, I established a method to target the chordoblasts for KillerRed-induced phototoxicity. I demonstrated that intact chordoblasts are necessary for chordacentrum formation, but that vertebral arches are unaffected. Fused perichordal centra are still able to form, but the underlying sheath has a very different structure. This supports the ‘duality’ hypothesis that in teleosts the role of the sclerotome in vertebra formation is limited to the arches and perichordal centra, whereas the chordoblasts are responsible for the chordacentra.

MITF is the master melanocyte transcription factor and has a complex role in melanoma. Both gain- and loss-of function mutations in MITF have been identified in melanoma, although its’ role in melanoma development and the effects of targeting MITF are unknown. Using a temperature-sensitive mitf zebrafish mutant I show that low levels of MITF are oncogenic with BRAFV600E in melanoma progression. By pathology and MITF target gene expression, BRAFV600Emitfavc7 tumours are distinct from BRAFV600Ep53M214K tumours, and represent two melanoma subtypes. Melanomagenesis can also be driven independently of BRAFV600E, in a transgenic zebrafish with mutations in mitf and p53, representing a new melanoma model. Abrogating MITF activity in BRAFV600Emitfavc7 melanoma leads to regression of the tumour, characterised by macrophage infiltration and increased apoptosis. This result confirms the dependence on MITF activity in BRAFV600Emitfavc7 melanomas and highlights the role of MITF as a therapeutic target for melanoma. Exome and transcriptome sequencing has been carried out to gain insight into the expression and genomic mutational landscape that is driven by these melanoma transgenic models and results in these genotype-phenotype specific subtypes observed.

Chemotherapy frequently fails to cure cancer patients due to toxicity or resistance to treatment. Variability in toxicity and resistance is influenced by polymorphisms and mutations in the individual's genome (Pharmacogenetics). Zebrafish has extensive evolutionary conservation with human, its genetics is a powerful gene discovery tool, and it has been described as a suitable model in cancer research. To study chemotherapy resistance, we used ENU-mutagenised zebrafish in a forward genetic screen to identify genes that modify responses to cancer chemotherapy. Zebrafish larvae were challenged with two chemotherapeutic drugs and stained with acridine orange (AO) to detect apoptosis and reveal hypo- or hyper-responders to chemotherapy. A mutation, conferring an increased uptake of AO, was identified by genetic mapping as a premature stop codon truncating the ribonuclease T2 (rnaset2) gene. Human RNASET2 encodes a putative lysosomal RNase. Lysosomal storage disorders, due to deficiencies in lysosomal hydrolases and resultant accumulation of macromolecules within lysosomes, are collectively among the commonest genetic diseases. RNASET2 deficiency in man results in a static encephalopathy arising in infancy and characterized by multifocal bilateral white matter lesions, subcortical cysts and focal enlargement of the anterior inferior horn. This doctoral thesis demonstrates that rnaset2 deficient zebrafish embryos suffer from a lysosomal storage disorder accumulating undigested ribosomal RNA (rRNA) in enlarged lysosomes within neurons of the brain. Moreover, high-field intensity μMRI revealed white matter lesions in the brain of adult rnase2 mutant animals. Thus, this zebrafish mutant can be used as a model to study the abnormalities observed in RNASET2 deficient individuals. This model suggests that the leukoencephalopathy results from a lysosomal storage disorder and provides a preclinical model for further elaborating disease mechanisms and evaluating candidate therapeutics.RNASET2 has also been advanced as a candidate tumour suppressor in several solid tumours. Recombinant rnaset2 protein has been tested in the clinic as an anti-cancer cytotoxic agent, with anti-angiogenic properties. By combining the rnaset2 mutant presented here with a transgenic melanoma model developed in the laboratory, the tumour suppressor and angiogenic role for rnaset2 was refuted.

The ontogeny of carbon dioxide (CO2) sensing in zebrafish (Danio rerio) has not been studied. In this thesis, CO2-mediated increases in heart rate were used to gauge the capacity of zebrafish larvae to sense CO2. CO2 is thought to be sensed through neuroepithelial cells (NECs), which are homologous to mammalian carotid body glomus cells. Owing to its role in facilitating intracellular acidification during exposure to hypercapnia, it was hypothesized that carbonic anhydrase (CA) is involved in CO2 sensing, and that inhibition of CA would blunt the downstream responses. The cardiac response to hypercapnia (0.75% CO2) was reduced in fish exposed to acetazolamide, a CA inhibitor, and in fish experiencing CA knockdown. Based on pharmacological evidence using β-adrenergic receptor (ß-AR) antagonists, and confirmed by β1AR gene knockdown, the efferent limb of the reflex tachycardia accompanying hypercapnia is probably mediated by sympathetic adrenergic neurons interacting with cardiac β1 receptors.

The increasing demand for fish in the food industry has resulted in the extensive overfishing of wild fisheries. In efforts to alleviate the demand from the food industry, genetically modified (GM) fish were developed possessing traits such as larger mass, faster growth, and increased resistance to disease. However, the greater fitness advantage of GM fish presents potential risks for wild type populations in the event of release or escape from a confined fish facility. In addition to physical barriers, it is critical to develop a genetic mechanism in order to ensure that the spread of GM transgene to the natural populations does not occur. RNA interference (RNAi) is an endogenous mechanism used to regulate gene expression by destroying targeted mRNA molecules. Manipulation of this biological process has been successfully utilized to knockdown specific genes through the introduction of synthetic transgenes in organisms such as C. elegans, M. musculus, and D. melanogaster. Although the use of RNAi as a biological tool is still relatively new in zebrafish, recent work has explored elements of this mechanism allowing for greater knockdown efficiencies. The deadend (dnd) gene is required for primordial germ cell (PGC) development and survival. Previous studies have shown that zebrafish dnd knockouts develop into sterile adults without disrupting somatic development. In efforts to induce sterility in zebrafish, short hairpin RNA (shRNA) constructs targeting dnd were designed to exploit the endogenous RNAi pathway. Upon qualitative analysis in transient and transgenic zebrafish subjected to the synthetic RNAi construct, a reduction in the germ cell population at early stages of development was observed. However, quantification of dnd mRNA in fish from the same time points did not show significant changes in expression levels compared to their wildtype counterparts. Adult fish subjected to the transgene construct produced viable gametes. The use of RNAi as a tool for bioconfinement relies on sterility among all individuals subjected to the shRNA bearing transgene. Based on the results obtained, the verdict is still unclear as to whether shRNA is a viable mechanism for large scale bioconfinement.

Zebrafish, danio rerio, are bony fish in the Teleost class. Zebrafish can regenerate most organs, including their fins. The caudal fin of zebrafish adults has bundles of rigid, uncalcified fibrils, termed actinotrichia, at the distal end of each lepidotrichia. During embryonic and early larval stages of fin development, actinotrichia are synthesized between the fin fold epidermis. Actinotrichia remain present in the fins of adult fish and during fin regeneration. They provide structural support and facilitate mesenchymal cell migration during fin development and regeneration. The importance of the role of actinotrichia for fin formation and regeneration can be studied through loss of function analysis of structural components of the actinotrichia. Our lab identified two genes, actinodin1 and actinodin2 (and1, and2), that are coding for structural proteins of the actinotrichia. CRISPR/Cas9 mediated knockout of and1 and and2 lead to loss of actinotrichia in the embryonic fins. Immunohistochemistry with anti-And1 and anti-ColII antibodies, in addition to controlled tissue degradation to observe actinotrichia fibers revealed the absence of actinotrichia in the caudal fins of adult and1/2 double homozygous mutants. We hypothesized that loss of actinotrichia will lead to the disruption of mesenchymal cell migration during development and regeneration, resulting in fin growth and morphology defects. The intact adult caudal fin of and1/2 double mutants have fewer rays (lepidotrichia) and these lepidotrichia are shorter and wavy when compared to WT. Along the lepidotrichia of actinodin mutants, there is an uneven distribution of joints revealing that there are defects in ray patterning. We carried out a regeneration time course analysis to compare caudal fin regeneration in WT and and1/2 double homozygous mutants. The and1/2 double homozygous mutants have a delay in bone re-formation and defects in joint patterning are observed in regenerating rays. From these results, and1 and and2 are shown to be required for actinotrichia formation. In addition, morphological analyses identified that actinotrichia is required for proper caudal fin growth and patterning during development and regeneration.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most abundant environmental pharmaceutical contaminants. In this study, a proteomic analysis was conducted to identify proteins differentially expressed in gill tissue of zebrafish (Danio rerio) after a 14-day exposure to the NSAIDs ibuprofen or naproxen. A total of 104 proteins with altered expression as indicated by 2-dimensional electrophoresis were analyzed by liquid chromatography with ion trap mass spectrometry (MS/MS). A total of 14 proteins fulfilled our requirements for identification which included consistency among replicate gels as well as successful MS/MS ion searches with the MASCOT database. The most prominent feature of the differential protein expression observed after NSAID exposure was an up-regulation of proteins belonging to the globin family which are involved in the transport of oxygen from gills and availability of heme molecules required for synthesis of cyclooxygenase. Differential expression was observed at exposure concentrations as low as 1-10 µg/L indicating that altered gene expression may occur in fish subjected to environmentally realistic levels of NSAID exposure.

It has been shown previously in our laboratory that zebrafish produce trypsin from their gills when they are under stress, and this trypsin is involved in thrombocyte activation via PAR2 during gill bleeding. In this study, I investigated another role of the trypsin that is secreted from zebrafish. This investigation has demonstrated a novel role of trypsin in zebrafish. Not only did this investigation demonstrate the role of trypsin in zebrafish behavior, but also it showed that PAR2 might be the receptor that is involved in trypsin-mediated behavioral response. In addition, we have shown that Gq and ERK inhibitors are able to block the trypsin pathway and prevent the escaping behavior. Finally, the results of this investigation suggest that the cells that respond to trypsin are surface cells, which have an appearance similar to that of neuromast cells.