Learning and memory are complex phenomena; numerous biochemical and neurobiological mechanisms subserving these functions have been identified. A key molecular component involved in learning and memory, the NMDA-R (N-Methyl-D-Aspartate Receptor) is impaired by MK-801(dizocilpine), an antagonist compound. Here I analyze the effects of MK-801 on the performance characteristics of zebrafish (Danio rerio) as these in turn can significantly influence the outcome of learning tasks. Subsequently, I study the effects of MK-801 on the acquisition, consolidation, and recall of memory in a plus maze, a new task I adapted from zebrafish literature. Although MK-801 seemed to have no effect on acquisition of memory in zebrafish, it disrupted their ability to consolidate and recall in the plus maze, echoing results found in rodent literature. Combined, these results suggest that zebrafish can be used as a tool to further advance the discovery of learning and memory.

Inhibitor of differentiation (Id) family helix-loop-helix proteins regulate the proliferation, survival and differentiation of numerous cell types during development, however their function during retinal development has not been analyzed. Using loss-of-function and overexpression assays in zebrafish, I demonstrate that Id2a levels modulate retinoblast cell cycle kinetics and thereby influence neuron and glia formation in the retina. Id2a-deficient retinas possess increased numbers of cells occupying S phase, at the expense of mitotic cells, and kinetic analyses demonstrate that Id2a is required for S-phase progression and/or the transition from S to M phase. Id2a-dependent defects in retinoblast proliferation lead to microphthalmia and to an absence of nearly all differentiated inner and outer nuclear layer cell types. Overexpression of id2a has the opposite effect on retinoblast cell cycle kinetics: id2a-overexpressing retinoblasts progress from S to M phase more rapidly and they undergo mitosis more frequently, which results in macrophthalmia. Mosaic analyses reveal that Id2a function in facilitating both cell cycle progression and neuronal differentiation in the retina is non-cell-autonomous, suggesting that Id2a functions upstream of the extrinsic pathways that regulate retinogenesis. In an effort to identify which extrinsic pathways function downstream of Id2a, I discovered that Id2a function is necessary and sufficient to limit Notch pathway activity during retinogenesis. Id2a-deficient retinae possess elevated levels of Notch pathway component gene expression, while retinae overexpressing id2a possess reduced expression of Notch pathway component genes. Attenuation of Notch signaling activity by DAPT or by morpholino knockdown of Notch1a is sufficient to rescue both the proliferative and differentiation defects in Id2a-deficient retinae. In addition to regulating Notch pathway activity, through an RNA-Seq and differential gene expression analysis of Id2a-deficient retinae, I identified a number of additional intrinsic and extrinsic regulatory pathway components whose expression is regulated by Id2a. These data highlight the integral role played by Id2a in the gene regulatory network governing the transition from retinoblast proliferation to terminal differentiation during vertebrate retinogenesis. / text

During embryogenesis, hematopoietic cells arise from the lateral plate mesoderm (LPM) following gastrulation. The transcriptional program required for this LPM to blood switch is not fully understood. Previous work on a zebrafish mutant with a deletion in the cdx4 gene demonstrated the importance of this caudal transcription factor in the LPM to blood transition. To explain how cdx4 regulates embryonic hematopoiesis, two main approaches were taken in this thesis. The first part of the thesis describes a chemical genetics screen that identified cdx4 interacting pathways. To find small molecules that could rescue the loss of red blood cells caused by the cdx4 deletion, cdx4 mutant embryos were incubated with 2640 compounds from the beginning of the gastrula stage to the 10-somite stage. Two related psoralen compounds, Bergapten (Ber) and 8-methoxypsoralen (8-MOP), rescued the erythroid progenitors in the cdx4 mutants. This rescue is closely linked to the compounds' effects on anteriorposterior patterning, reminiscent of retinoic acid pathway compounds. The second part of my thesis identifies a Cdx4-Sall4 transcriptional module in the LPM. Chromatin-immunoprecipitation coupled to sequencing (ChIP-seq) and microarray analysis revealed that Cdx4 directly regulates cdx4 and a zinc finger transcription factor spalt-like 4 (sall4) transcription. Sall4 ChIP-seq showed that Sall4 also binds to its own locus and to the cdx4 locus, suggesting an auto- and cross-regulation between two transcription factors. In addition, Cdx4 and Sall4 bind to common genomic regions proximal to mesodermal progenitor (tbx16 and mespa) and hematopoietic genes (scl, gata2a, and ldb1a), indicating Cdx4 and Sall4 co-regulate key genes that are required for LPM and blood specification. sall4 knockdown in the cdx4 mutants demonstrated that Sall4 synergizes with Cdx4 in regulating embryonic hematopoiesis. These findings suggest that auto- and cross-regulation of Cdx4 and Sall4 establish a stable circuit in the LPM that facilitates the activation of blood-specific program as development proceeds. How undifferentiated germ layers transition into various tissues is a key question in developmental biology. My thesis establishes a model based on LPM to blood transition, which is also applicable to other studies on germ layer specification.

Fibronectin (fn) and integrin α5 (itgα5) are both key players in cell adhesion and intracellar signaling, however the specific in vivo role of these proteins has never been analyzed in the vertebrate lens. The results presented here indicate that Fn1 and Itgα5 proteins are essential for the proper development of the lens. The loss of Fn1 protein in the zebrafish embryo results in distinct adhesion defects, defects in lens fiber morphogenesis, and cataracts. These results were phenocopied in zebrafish itga5 mutants, thereby indicating an essential role for Fn1 and Itgα5 during lens development. Furthermore, embryos with reduced levels of ptk2.1 (focal adhesion kinase – FAK) also phenocopied the defective fn1 and itgα5 lens, suggesting that FAK is a major player in the intracellular signaling mediated by Fn1/Itgα5 interactions in the lens. / text

The Hedgehog receptor Patched (Ptch) is a well-studied tumor suppressor. Mutations in Ptch have been linked to mis-regulation of stem cell proliferation and tumorigenesis in numerous contexts. To study the role of Ptch function during retinal development and homeostasis, I analyzed retinal growth and patterning in the embryonic and post-embryonic (juvenile) zebrafish ptc2 mutant line. ptc2 deficiency in zebrafish results in an expansion of the stem/progenitor population of the ciliary marginal zone (CMZ), as well as ectopic proliferation within the neural retina at juvenile stages. ptc2-/- mutants also possess vitreo-retinal abnormalities that appear to be embryonic in origin. These phenotypes are similar to the ocular abnormalities previously reported in human patients suffering from Basal Cell Naevus Syndrome (BCNS), a disorder that has been linked to mutations in the human PTCH gene (the orthologue of the zebrafish ptc2), and point to the utility of the ptc2 mutant line as a model for the study of BCNS-related ocular pathologies. In addition, peripheral retinal dysplasias that include ectopic neuronal clusters and disrupted lamination were observed at later, juvenile stages. It has been previously proposed that retinal over-proliferation might contribute to retinal dysplasias observed in the post-natal Ptch1 /- mice (an established model for BCNS); however, this potential relationship has yet to be established experimentally. I demonstrated that a population of ectopically proliferating cells give rise to the ectopic neuronal clusters in the INL of ptc2-/- mutants and established ectopic proliferation as the likely cellular underpinning of retinal dysplasia in juvenile ptc2-/- mutants. / text

Neuroepithelial cells (NECs) of the zebrafish gill filament have been previously identified as bimodal O2 and CO2/H+ sensors that depolarize in response to chemostimuli via inhibition of background K+ channels. To further elucidate the signaling pathway underlying CO2/H+ chemoreception in the NECs we employed microspectrofluorometric techniques to examine the effects of hypercapnia on [Ca2+]i and pHi. NECs increased their [Ca2+]i in response to acidic hypercapnia (5% CO2, pH 6.6) and isocapnic acidosis (normocapnia, pH 6.6), but not to isohydric hypercapnia (5% CO2, pH 7.8). The acid- induced increase in [Ca2+]i persisted in the absence of extracellular Ca2+, and Ca2+ channel blockers (Cd2+, Ni2+ and nifedipine). NECs exhibited a rapid and reversible drop in pHi in response to acidic hypercapnia and isohydric hypercapnia. Isocapnic acidosis also induced intracellular acidification within NECs, but it was less severe than the drop in pHi elicited by acidic hypercapnia and isohydric hypercapnia. The rate and magnitude of intracellular acidification was reduced by the CA-inhibitor, acetazolamide, without effect on the acid-induced increase in [Ca2+]i. Acetate was used to investigate the relationship between pHi and [Ca2+]i. Acetate induced intracellular acidification without augmentation of [Ca2+]i. The results of this thesis demonstrate that (1) extracellular acidification, but not CO2, is critical to the hypercapnia-induced increase in [Ca2+]i (2) the increase in [Ca2+]i is independent of the drop in pHi (3) the increase in [Ca2+]i is not mediated by the influx of Ca2+ across the plasma membrane.

The question of how a single cell can grow, divide, and ultimately acquire a distinct function within an adult animal is central to the field of developmental biology. An elegant way to address this question is by studying the specification of a specific cell type, for example, vertebrate motoneurons. For an animal to be able to move and behave appropriately, individual motoneurons (MNs) must correctly innervate specific muscles. For this to happen, MNs must first be specified and then must differentiate into distinct subtypes, each of which is classified in part by the muscle it innervates. MN subtype specification is dependent on both the acquisition of MN-specific characteristics as well as the failure to acquire characteristics specific to interneurons, cells that only innervate other neurons. The entire process of specification is initiated in progenitor cells and relies on the correct spatial and temporal expression of specific genes. Previous work in various vertebrate models has identified some of the key genes involved in MN specification, most notably transcription factors such as olig2, nkx6s, lhxs, mnxs, and islet1. In this dissertation, I use the zebrafish model to demonstrate novel roles in MN specification for two of these families of transcription factors - the lhxs and the mnxs. I provide evidence that both lhx3 and lhx4 are necessary for normal MN and ventral interneuron (IN) development and work by preventing MNs from expressing IN-specific characteristics. I also show that mnx1, mnx2a, and mnx2b are necessary in MNs both to promote the acquisition of some MN subtype-specific characteristics and to prevent the acquisition of some IN-specific characteristics and appear to be working in part through interactions with islet1. Finally, I identify an intermediate filament gene, inab, as being expressed in a subset of zebrafish MNs and a ventral IN and as having a potential role in the axon outgrowth of a specific MN subtype. Together, this work provides evidence for a mechanism of MN specification dependent on the expression of genes that both promote aspects of MN fate and inhibit aspects of IN fate. This dissertation includes previously unpublished co-authored material.

Nearly all individual members of the animal kingdom have gastrointestinal tracts which feature unique cellular compositions, geometries, and temporal dynamics. These guts are distinct enough from one another, even across siblings or even across the same individual at different points in space and time, that defining meaningful scientific representations of those features is difficult. Studying these guts is also innately challenging as it requires accessing to the insides of the enclosed 3D volumes. The work presented here describes tools and methodologies designed to address these difficulties. To investigate gut motility, we constructed a combined light sheet fluorescence and differential interference contrast microscope to obtain videos of larval zebrafish (Danio rerio) gut motility and to obtain 3D information about nearby fluorescently tagged cells. Using advanced computer vision algorithms, we quantified aspects of zebrafish gut motility which have never before been characterized, then used that information to identify the effects of different genetic, chemical, and physiological states of zebrafish gut motility. Finally, we designed and constructed an instrument for automating 3D microscopy for future studies. This dissertation includes previously published and unpublished co-authored material.

At the hypothalamo-pituitary interface, multiple neurotransmitters and neuropeptides interact to control luteinizing hormone and follicle stimulating hormone release from gonadotrophs. The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. While it is well-established that gonadotropin-releasing hormone and/or kisspeptin are required for pulsatile and surge release of luteinizing hormone in mammalian species, their essentiality is challenged by studies showing knockouts in zebrafish and medaka do not block reproduction. In goldfish, secretoneurin-a, a neuropeptide derived from secretogranin-IIa processing, stimulates luteinizing hormone release in vivo and from dispersed pituitary cells in vitro. Secretoneurin does not bind to the human gonadotropin releasing hormone receptor and can enhance luteinizing hormone release when applied directly to mouse LbetaT2 cells. Our study indicates the presence of secretogranin-IIa and secretogranin-IIb mRNA in specific regions of the zebrafish brain and pituitary that have been implicated in the control of reproductive processes. I also observed that secretogranin-II knockout disrupts normal morphology of the pectoral fins by reducing the number of breeding tubercule rows, breeding tubercule width and breeding tubercule cluster length which could potentially lead to a reduced spawning success. Knocking out the secretogranin-IIa and secretogranin-IIb genes using TALENs in zebrafish indicates that these genes are required for optimal reproduction. Rates of oviposition for double secretogranin-II knockout females are 6% and 11% when crossed with double secretogranin-II knockout and wild-type males, respectively, compared to 62% in virgin wild-type pairings. Comprehensive video analysis demonstrates that secretogranin-II knockout reduces all stereotypical male courtship behaviours. Severe reductions in the expression of gonadotropin releasing hormone 3 in the hypothalamus and luteinizing hormone in the pituitary suggest that secretogranin-II-derived peptides drive the gonadotropin releasing hormone-luteinizing hormone control system. Spawning success is rescued in double secretogranin-II knockouts following one injection of synthetic secretoneurin-a in which it increases from 11% to 30% thereby supporting the hypothesis that secretoneurin-a is a key reproductive hormone. However, embryo survival rate of secretoneurin-a injected double secretogranin-II knockout was lower than saline-injected wild-type pairings. Injection of human chorionic gonadotropin, a hormone that mimics the action of luteinizing hormone on binding and activating the luteinizing hormone receptor in fish, increased double secretogranin-II knockout spawning success to 38%, thus comparing favourably to 47% in saline-injected wild-type controls. My data provides support that secretogranin-II is required for optimal reproductive functions although the precise mechanisms behind this reduced capacity in zebrafish lacking the secretogranin-II genes remain to be investigated. Moreover, the high conservation of secretoneurin from lamprey to human suggests a broader importance of this emerging peptide family.

There have been dramatic advances in melanoma therapy in the last 10 years, yet there is still a demand for effective and affordable therapies. To identify novel therapeutic pathways a transcriptome analysis was performed on zebrafish melanoma models representing the different stages of melanoma progression. Transcriptomic differences between pre-malignant and malignant conditions highlighted lipid metabolism as a potential mediator of progression. A mass spectrometry analysis confirmed multiple changes in lipid composition between wild type fish, pre-malignant and advanced melanoma models. To better investigate metabolism a positron emission tomography (PET) technique was developed in zebrafish. Tumours in the zebrafish were successfully scanned with FDG used to detect human tumours. A novel tracer of unconjugated FA was then developed and, consistent with inferences from the transcriptome and mass spectrometry, was shown to be incorporated into tumours. Demonstrating the feasibility of PET in zebrafish now opens the way to systematic use of this organism in tracer development with potential time-saving and cost benefits. One of the most significantly up-regulated genes exclusive to the malignant state encodes lipoprotein lipase (LPL). LPL is involved in the release and uptake of FA from circulating triglyceride. LPL was found to increase the rate of tumour appearance and tumour growth in a zebrafish tumour assay. LPL was expressed in human tumours and expression correlated with progression. Melanoma cell lines expressed LPL and knocking-down LPL resulted in reduced cell numbers. The effect was most dramatic in WM852 cells. A novel role for LPL in autophagy was identified. WM852 cells treated with LPL siRNA showed a stabilisation of p62/SQSTM and induction of LC3B II. Electron microscopy revealed large autolysosomal vacuoles in the cytoplasm. Additionally many cells showed damaged mitochondria with absent cristae. The dependency of cells on LPL seemed to be modified by the co-expression of fatty acid synthase (FASN) required for de novo FA synthesis, as the magnitude of the effect of LPL-knockdown was dependent on the levels of FASN expressed in melanoma cell lines. Moreover, combining LPL and FASN inhibitors synergised to kill cells previously less sensitive to LPL inhibitor. FASN and LPL co-inhibition could provide a unique combinatorial therapeutic strategy.