During development, neural crest (NC) cells arise from the neural plate border and are further differentiated into multiple cell types, including melanocytes. Each step of this process is controlled by a gene regulatory network (GRN), and disruption of the GRN governing melanocyte differentiation contributes to the pathogenesis of pigmentation disorders and melanoma. While many of the factors within this network have been well studied, the role of Transcription Factor Activating Enhancer-Binding Protein 2 (TFAP2) paralogs has been unclear. TFAP2A and TFAP2C are required for NC induction. Later, TFAP2A is also expressed in melanocytes, and TFAP2A mutations cause pigmentation phenotypes in humans, mice, and zebrafish. Other paralogs with high homology to TFAP2A, particularly TFAP2B in mouse and Tfap2e in zebrafish, also function redundantly with TFAP2A in the melanocyte lineage. Here, we have used ChIP-seq and expression profiling to identify direct transcriptional targets of TFAP2A in melanocytes, which include genes involved in melanin synthesis and melanosome biology. Furthermore, we show that TFAP2A directly regulates many of the same genes as Microphthalmia-associated Transcription Factor (MITF), the “master regulator” of the melanocyte lineage. MITF activity has been described as a rheostat in melanoma, with high levels promoting differentiation and lower levels promoting invasiveness. The overlap between TFAP2A and MITF transcriptional targets in melanocytes suggests that TFAP2A may influence the MITF rheostat, driving it toward the differentiated state. To study the role of other TFAP2 paralogs in NC and melanocytes, we generated zebrafish lines that are double and triple mutant for tfap2a, tfap2c, and tfap2e and confirm genetic compensation among these paralogs. We also demonstrate that melanocyte-specific inhibition of Tfap2 activity by Kctd15 affects differentiation and that Kctd15 may participate in a negative feedback loop regulating Tfap2 expression. In support of a pro-differentiation role for TFAP2A in melanocytes, we show that overexpression of tfap2a in a zebrafish melanoma model significantly delays tumor formation. Together these results indicate that, in addition to its earlier roles in the NC, TFAP2A acts within the melanocyte GRN to directly regulate differentiation genes in parallel with MITF. This, combined with the tumor-suppressor function of TFAP2A in melanoma, implicates TFAP2A and the factors that regulate it as potential targets for melanoma therapies.

The Transient Receptor Potential, Melastatin-like 7 (TRPM7) protein is composed of a long amino terminus, 6 trans-membrane domains, and a carboxy terminal α-kinase domain; TRPM7 tetramers form non-selective cation channels with unusual permeability to Mg2+. TRPM7 is thought to be expressed in all cell types, and studies conducted primarily on cultured cells have implicated TRPM7 in cellular functions that include cell adhesion, synaptic vesicle release, kidney cation balance, differentiation, survival, and cellular magnesium homeostasis. The full extent of its physiological functions in vivo remains elusive because mouse TRPM7 homozygous null mutants die at embryonic stages. By contrast, zebrafish homozygous for hypomorphic alleles of trpm7 survive for two weeks post fertilization, making it possible to study the physiological consequences of Trpm7 deficiency in a living organism. My work primarily utilizes homozygous animals carrying the trpm7b508 allele, which we suspect encodes a non-functional protein for three reasons. This protein variant is predicted to lack a kinase domain, patch clamp studies fail to detect current, and morpholino knockdown of Trpm7 yields a similar phenotype. Herein, we explore the mechanisms behind each of three phenotypes in trpm7b508 homozygous embryos, i.e., trpm7 mutants. First, we find that cell death of embryonic melanophores in trpm7 mutants is not by apoptosis, and it is dependent upon melanin synthesis and the ion channel Trpm2. Second, we show that paralysis of trpm7 mutants is rescued by surgical opening of the circulatory system to surrounding media, implying that paralysis results from an organismal ion imbalance. Third, we report a variety of findings supporting the model that abnormally low levels of spontaneous swimming in larval trpm7 mutants results from reduced dopamine signaling. We find that specific populations of catecholaminergic neurons are reduced in mutants relative to their unaffected siblings, and that mutants are sensitized to the neurotoxic effects of 1-Methyl-4-phenylpyridinium iodide (MPP+). Together, these results suggest that Trpm7 has a role in ameliorating the toxic effects of reactive oxygen species in certain populations of melanophores and neurons. These findings advance understanding of the function of TRPM7 during embryonic development, and may have relevance to the gene-environment interaction behind certain neurodegenerative conditions.

Mutations in BBS6 cause two clinically distinct syndromes, Bardet-Biedl syndrome (BBS), a syndrome caused by defects in cilia transport and function, as well as McKusick-Kaufman syndrome, a genetic disorder characterized by congenital heart defects. Congenital heart defects are rare in BBS, and McKusick-Kaufman syndrome patients do not develop retinitis pigmentosa. Therefore, the McKusick-Kaufman syndrome allele may highlight cellular functions of BBS6 distinct from the presently understood functions in the cilia. In support, we find that the McKusick-Kaufman syndrome disease-associated allele, BBS6H84Y; A242S, maintains cilia function. We demonstrate that BBS6 is actively transported between the cytoplasm and nucleus, and that BBS6H84Y; A242S, is defective in this transport. We developed a transgenic zebrafish with inducible bbs6 to identify novel binding partners of BBS6, and we find interaction with the SWI/SNF chromatin remodeling protein Smarcc1a (SMARCC1 in humans). We demonstrate that through this interaction, BBS6 modulates the sub-cellular localization of SMARCC1 and find, by transcriptional profiling, similar transcriptional changes following smarcc1a and bbs6 manipulation. Our work identifies a new function for BBS6 in nuclear-cytoplasmic transport, and provides insight into the disease mechanism underlying the congenital heart defects in McKusick-Kaufman syndrome patients.

The process of vertebrate development requires communication among many cells of the embryo in order to define the body axis (front/back, head/tail or left/right). The Wnt signaling network plays a key role in a vast array of cellular processes including body axis patterning and cell polarity. One arm of the Wnt signaling network, the non-canonical Wnt pathway, mediates intracellular Ca2+ release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GAPs and are uniquely situated to control the amplitude of a Wnt signal. I hypothesize that individual RGS proteins are critical in modulating the frequency and amplitude of Wnt/Ca2+ signaling in different tissues and at different developmental stages and this modulation is essential for developmental patterning events. To this end, this thesis is focused on the effects G protein regulation has on intracellular Ca2+ release dynamics and developmental patterning events. I combine cellular, molecular and genetic analyses with high resolution Ca2+ imaging to provide new understanding of the role of RGS proteins on Wnt mediated Ca2+ release dynamics and developmental patterning events. In chapter 2, I describe endogenous Ca2+ dynamics from the very first cell divisions through early somitogenesis in zebrafish embryos. I find that each phase of zebrafish development has a distinct pattern of Ca2+ release, highlighting the complexity of Ca2+ ion and cellular physiology. In Chapter 3, I identify rgs3 as potential modulator of Ca2+ dynamics and Chapter 4 expands upon these observations by providing data supporting that Rgs3 function is necessary for appropriate frequency and amplitude of Ca2+ release during somitogenesis and that Rgs3 functions downstream of Wnt5 activity. My results provide new evidence that a member of the RGS protein family is essential for modulating the non-canonical Wnt network to assure normal tissue patterning during development. In Chapter 5, I report the identification and characterization of Rgs3b, a paralogue to Rgs3, in zebrafish. I describe results indicating that Rgs3b is poised to interact with Wnt11 indicating that individual RGS genes may have unique roles in modulating Wnt/Ca2+ signaling in different tissues or different stages. In conclusion, this thesis provides data supporting that individual RGS proteins are critical in modulating the frequency and amplitude of Wnt/Ca2+ signaling in different tissues and at different developmental stages and this is a substantial breakthrough in understanding how RGS proteins function to fine-tune known signaling pathways

Eukaryotic transcriptional activators stimulate transcription of genes otherwise expressed at low levels. The typical activator operates by binding to specific sites on DNA with its activating region contacting the multiprotein machinery that directs transcription. SBF/Staf is a transcriptional activator that binds to the SPH element found in the promoters of genes for snRNAs and genes that code for mRNAs. SBF/Staf binds to SPH through a reiterated zinc finger DNA binding domain and also contains two distinct activation domains, one for snRNA genes and one for mRNA genes. To test the role of SBF/Staf in vivo, morpholino antisense oligos were used to knock down SBF/Staf expression in zebrafish. A high percentage of developing zebrafish embryos exhibited abnormalities. Co-injection of a synthetic mRNA construct rescued the morpholino-induced knockdown. Furthermore, both the mRNA and snRNA activation domains have significant roles in the function of SBF/Staf because when each domain was removed separately, partial rescue of the knockdown phenotype was obtained. When both domains were removed, no rescue of the phenotype was observed. Unexpectedly, knockdown of SBF/Staf expression in zebrafish embryos caused an increase in steady-state levels of all endogenous mRNAs tested, as well as transcripts produced from co-injected U6 maxigenes. However, quantitative RT-PCR analysis showed a relatively smaller increase in the steady-state levels of several mRNAs from genes that contain a SPH element in their promoters. In zebrafish U6 genes, the SPH element is in the unique location of being next to the TATA box, instead of ~220 bp upstream of the start site as in mammals. To determine the significance of the proximally-located SPH element for transcription of the zebrafish U6 snRNA gene, the SPH element was mutated. Transcription of a zebrafish U6 maxigene was reduced to 20.6% in transfected ZF4 cells and 26.8% in injected embryos, compared to that of the U6 maxigene with a normal promoter. This work indicates a more global role of SBF/Staf in mRNA gene transcription, instead of only activating the transcription of snRNA and a few mRNA genes, leading to an increased importance of the role of SBF/Staf in transcriptional control.

The temporal and spatial patterning that underlies morphogenetic events is controlled by gene regulatory networks (GRNs). These operate through a combinatorial code of DNA – binding transcription factor proteins, and non – coding DNA sequences (cis-regulatory elements, or CREs), that specifically bind transcription factors and regulate nearby genes. By comparatively studying the development of different species, we can illuminate lineage – specific changes in gene regulation that account for morphological evolution. The central nervous system of vertebrates is composed of diverse neural cells that undergo highly coordinated programs of specialization, migration and differentiation during development. Approximately 20% of neurons in the cerebral cortex are GABAergic inhibitory interneurons, which release the neurotransmitter gamma-aminobutyric acid (GABA). Diseases such as autism, schizophrenia and epilepsy are associated with defects in GABAergic interneuron function. Several members of the distal-less homeobox (Dlx) transcription factor family are implicated in a GRN underlying early GABAergic interneuron development in the forebrain. I examined the role played by orthologous dlx genes in the development of GABAergic interneurons in the zebrafish forebrain. I found that when ascl1a transcription factor is down-regulated through the micro-injection of translation – blocking morpholino oligonucleotides, Dlx gene transcription is decreased in the diencephalon, but not the telencephalon. Similarly, gad1a transcription is also decreased in this region for these morphants. As gad1a encodes an enzyme necessary for the production of GABA, these genes are implicated in a cascade underlying GABAergic interneuron development in the diencephalon.

These studies present a series of novel roles for glucocorticoid signaling in the developing zebrafish embryo. The best-characterized roles of cortisol, the primary circulating corticosteroid in teleost fish, are known to occur by the activation of the glucocorticoid receptor (GR) in the post-hatch animal to mobilize energy reserves for response and recovery from stressful situations. For the first time, evidence is presented that GR and cortisol are key developmental regulators in the pre-hatch zebrafish embryo and that glucocorticoid signaling modulates multiple critical developmental pathways and affects embryogenesis in diverse ways. Prior to these experiments, very little was known regarding the developmental role of glucocorticoids in lower vertebrates. In mammalian models, there has been extensive study of the action of these steroids in late-stage organ maturation, and they have a variety of clinical and biomedical applications. However, in fish, there was a relative dearth of information regarding the basic dynamics and potential functional roles of cortisol and GR in embryogenesis. Zebrafish are a popular model for developmental study, with optically transparent embryos that allow for reliable observation. Additionally, the zebrafish genome is fully sequenced and extensively annotated, and a variety of molecular biology techniques are well-established in the existing literature. The zebrafish is also now recognized as an advantageous model for endocrine and stress axis studies, as it expresses a single GR gene, unique among teleosts but comparable to mammals. Preliminary studies published in the literature described cortisol and GR as deposited in the zebrafish embryo prior to fertilization, and showed their expression declining prior to hatch, then rising significantly as larvae approach the stage of first feeding. This dynamic expression of both ligand and receptor during embryogenesis, combined with knowledge from mammalian models, led to the hypothesis that glucocorticoid signaling may be functionally relevant in zebrafish development. A variety of techniques were used to examine the roles of cortisol and GR in the zebrafish embryo. Morpholino oligonucleotides were injected into one-cell embryos to block GR protein translation, allowing for the identification of GR-responsive developmental events and putative GR target genes. High-density microarray analysis of GR morphants presented numerous novel genes and pathways that are modulated by glucocorticoid signaling in the embryo. The ability to microinject molecules into a newly-fertilized zygote also allowed for other manipulations, including the addition of exogenous cortisol or the use of a cortisol-specific antibody to sequester maternally deposited cortisol. These studies provided the first evidence regarding the functional importance of the maternal cortisol deposition in the zebrafish oocyte prior to fertilization. The detailed temporal and spatial expression of GR mRNA and protein in the developing embryo has been characterized for the first time. GR expression is widespread, especially in developing mesoderm, and de novo GR transcription in the zebrafish embryo begins within 12 hours post fertilization. Lack of GR protein expression in the developing zebrafish embryo causes reduced growth, delayed somitogenesis, altered myogenesis, and severely reduces post-hatch survival. Additionally, GR modulates the expression of bone morphogenetic proteins, a family of morphogens that are involved in major developmental events including dorsoventral patterning, somitogenesis, myogenesis, and organogenesis. Reduction in GR protein content in the developing embryo is also linked to other major developmental processes including neurogenesis, eye formation, cellular adhesion, and development and function of the hypothalamic-pituitary-interrenal (HPI) axis. Cortisol in the early embryo, which is contributed entirely by maternal deposition prior to fertilization, is an important regulator of cardiogenesis and development of the HPI axis. Modulation of cortisol content in the early embryo causes an impairment of the post-hatch response to a physical stressor, as larvae exposed to increased cortisol during embryogenesis displayed an inability to increase heart rate in response to an acute physical stress, and did not display the classical increase in cortisol that follows a stressor challenge. Embryos that experience lowered glucocorticoid signaling in development tend to have a heightened post-hatch response to stress, further supporting the conclusion that HPI axis development is regulated by glucocorticoid signaling. These studies have identified key cardiogenic and HPI axis genes that are GR-responsive, providing mechanistic explanations for these phenotypic changes. Together these findings indicate that maternal deposition of cortisol in the embryo can pattern the post-hatch larva and has definitive impacts on performance as the offspring begin locomotion and approach feeding stages. In total, these studies demonstrate that glucocorticoid signaling is critically important to zebrafish embryogenesis, defining novel roles that are completely independent of the classical vertebrate stress response. These functions have significant effects on diverse developmental pathways and processes, and with the potential applicability of the zebrafish model to studies in higher vertebrates, may have important biomedical applications.

Cadmium (Cd) is a toxic metal known to accumulate in and have adverse effects on the olfactory systems of fish. The objective of this project was to investigate the effect of cadmium on zebrafish larvae, specifically the effects on the olfactory system at cellular and functional levels. Zebrafish larvae (72 hours post fertilization) were exposed to sublethal concentrations of cadmium (0.5, 1, 5, and 10 µM) for 96 h. Whole-body cadmium accumulation during this exposure period as quantified using GFAAS (graphite furnace atomic absorption spectroscopy) was found to increase with both exposure length and concentration. Using a transgenic strain of hsp70/eGFP (heat shock protein 70/enhanced green fluorescent protein reporter gene) zebrafish, dose-dependent induction of the heat shock response was observed in the olfactory epithelium. Expression of hsp70/eGFP in the olfactory epithelium was a highly sensitive biomarker for the effects of cadmium in the olfactory system with a lowest observed effects concentration (LOEC) of 0.5 µM Cd. Strong induction of the transgenic reporter gene correlated closely with cell death (LOEC of 5 µM Cd) and histological alterations (LOEC of 1 µM Cd) in the olfactory epithelium of zebrafish larvae following cadmium exposure. Additionally, loss of sensory cilia from the surface of the olfactory epithelium was observed in larvae exposed to 5 and 10 µM Cd. Furthermore, behaviour tests to assess olfactory function revealed sensory deficits, likely due in part to the cadmium-induced degeneration of the olfactory epithelium (p<0.05 for 1 µM; p<0.001 for 5 and 10 µM Cd). <p>To determine if cadmium was entering the cells of the olfactory epithelium by acting as a calcium (Ca) antagonist, zebrafish larvae were co-exposed to 1, 5, or 10 µM Cd with 1 or 5 mM Ca for 96 h. Whole-body cadmium accumulation as quantified using ICP-MS (inductively coupled plasma mass spectrometry) was decreased in larvae co-exposed to cadmium and calcium. Additionally, induction of the heat shock response was reduced in the presence of increasing calcium co-treatment. These ameliorating effects of calcium were further revealed in cell death and histological analyses of the olfactory epithelium. Also, larvae co-exposed to cadmium and calcium exhibited greater olfactory sensory function compared to larvae exposed to cadmium only. Significant increases in aversion response were observed in larvae exposed to 5 µM Cd with 1 and 5 mM Ca (p<0.05). These results indicate that cadmium gains entry to the olfactory epithelium via calcium uptake systems, wherein it causes damage to the olfactory system and can lead to sensory impairment.

Previous studies exposing fish to xenoestrogens have demonstrated vitellogenin (VTG) induction, delayed gametogenesis, altered sex distribution, and decreased reproductive performance, with a majority of those studies focusing on exposure to single chemicals. The objective of this study was to determine the effects of binary mixtures of a weak estrogen receptor agonist, nonylphenol (NP) and a potent estrogen receptor agonist, 17α-ethinylestradiol (EE) on sex distribution, gametogenesis, VTG induction, heat shock protein 70 (HSP70) expression and reproductive capacity in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to nominal concentrations of 10 or 100 µg/l NP (NP10 or NP100, respectively), 1 or 10 ng/l EE (EE1 or EE10, respectively), 1 ng/l EE + 10 or 100 µg/l NP (EE1+NP10 or EE1+NP100, respectively), 10 ng/l EE + 10 or 100 µg/l NP (EE10+NP10 or EE10+NP100, respectively) or solvent control (0.01% acetone v/v) in a static-renewal system with replacement every 48h. At 60 dph, fish from each treatment were euthanized for histological examination of gonads, and whole body VTG and HSP70 levels. Remaining fish were reared in clean water until adulthood (240 dph) for breeding studies. In all EE10 exposure groups (EE10, EE10+NP10 and EE10+NP100), increasing NP concentration acted less than additively to the action of EE in terms of VTG induction at 60 dph. Similarly, a less than additivity of effect was observed with egg production, where EE1+NP100 exposure resulted in significantly more eggs produced per breeding trial than EE1 alone. Histological staging of oogenesis revealed suppressed gametogenesis in females at 60 dph. There were no differences among treatment groups in whole body HSP70 expression in 60 dph fish or in gonadal HSP70 expression in adult fish. Although there was no statistical evidence of non-additivity, breeding trials in adults revealed significant reductions in egg viability, egg hatchability and/or F1 swim-up success, suggesting that developmental exposures to xenoestrogens may cause irreversible effects on egg quality and progeny even after depuration. In conclusion, these results suggest that environmentally relevant mixtures of NP and EE can produce additive or non-additive effects depending on the particular response being determined and the respective exposure concentrations of each chemical. Thus, it is recommended that caution be exercised in ecological risk assessments when assuming additivity in piscine responses to xenoestrogen mixtures.

Previous studies exposing fish to xenoestrogens have demonstrated vitellogenin (VTG) induction, delayed gametogenesis, altered sex distribution, and decreased reproductive performance, with a majority of those studies focusing on exposure to single chemicals. The objective of this study was to determine the effects of binary mixtures of a weak estrogen receptor agonist, nonylphenol (NP) and a potent estrogen receptor agonist, 17α-ethinylestradiol (EE) on sex distribution, gametogenesis, VTG induction, heat shock protein 70 (HSP70) expression and reproductive capacity in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to nominal concentrations of 10 or 100 µg/l NP (NP10 or NP100, respectively), 1 or 10 ng/l EE (EE1 or EE10, respectively), 1 ng/l EE + 10 or 100 µg/l NP (EE1+NP10 or EE1+NP100, respectively), 10 ng/l EE + 10 or 100 µg/l NP (EE10+NP10 or EE10+NP100, respectively) or solvent control (0.01% acetone v/v) in a static-renewal system with replacement every 48h. At 60 dph, fish from each treatment were euthanized for histological examination of gonads, and whole body VTG and HSP70 levels. Remaining fish were reared in clean water until adulthood (240 dph) for breeding studies. In all EE10 exposure groups (EE10, EE10+NP10 and EE10+NP100), increasing NP concentration acted less than additively to the action of EE in terms of VTG induction at 60 dph. Similarly, a less than additivity of effect was observed with egg production, where EE1+NP100 exposure resulted in significantly more eggs produced per breeding trial than EE1 alone. Histological staging of oogenesis revealed suppressed gametogenesis in females at 60 dph. There were no differences among treatment groups in whole body HSP70 expression in 60 dph fish or in gonadal HSP70 expression in adult fish. Although there was no statistical evidence of non-additivity, breeding trials in adults revealed significant reductions in egg viability, egg hatchability and/or F1 swim-up success, suggesting that developmental exposures to xenoestrogens may cause irreversible effects on egg quality and progeny even after depuration. In conclusion, these results suggest that environmentally relevant mixtures of NP and EE can produce additive or non-additive effects depending on the particular response being determined and the respective exposure concentrations of each chemical. Thus, it is recommended that caution be exercised in ecological risk assessments when assuming additivity in piscine responses to xenoestrogen mixtures.