Recently, the zebrafish has been considered an important animal model that can be used to investigate human diseases and drug development. Parkinson¡¦s disease (PD), an important neurodegenerative disorder, is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra and movement defects, including bradykinesia, tremor, and postural imbalance. However, current treatments for PD are limited and mainly improve only the clinical symptoms of the disease. Thus, a neurodegenerative rat model has been widely used for a long while to search for a new treatment for PD. However, the use of rats as an animal model has certain limitations such as breeding, efficiency, and high dosage. Recently, researchers indicated that neurotoxins such as rotenone, 6-hydroxydopamine (6-OHDA), and paraquat can induce Parkinson¡¦s-like symptoms in zebrafish, and this may be a useful PD model because of the complete development of the zebrafish nervous system, low costs, and low dosage. In this study, we treated zebrafish with 6-OHDA and analyzed their locomotor activity to establish an in vivo animal model of PD. Then, we analyzed the mRNA expression of parkin and PINK1 by reverse transcription¡Vpolymerase chain reaction (RT-PCR).Moreover, we observed tyrosine hydroxylase (TH) expression by immunohistochemical (IHC) staining to confirm if this can be used as a PD model. Finally, we found that treatment with 6-OHDA significantly reduced TH expression. We observed a similar declining trend in the case of mammals. Likewise, parkin and PINK1 mRNA expressions were also decreased after treatment with 6-OHDA. In summary, our study provides a feasible in vivo Parkinson¡¦s model, and a small volume of drugs or compounds can be screened using this model.

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.

Bisphenol A (BPA) is an alkylphenol compound used in plastic manufacturing, which enters the aquatic environment through wastewater treatment plants and landfill leachates. Exposure of fish to BPA results in developmental defects, decreased hatching, and increased occurrence of the egg yolk precursor protein vitellogenin in male fish. BPA is an estrogenic like compound (xenoestrogen) that can bind and activate the nuclear estrogen receptors, ERα and ERβ, causing changes in gene transcription (genomic mechanism). However, the ability of BPA to activate rapid estrogen signaling and to disrupt nongenomic physiological responses to estrogens is not known. One important nongenomic estrogen action in fish is to maintain meiotic arrest of oocytes and to prevent precocious oocyte maturation through activation of the transmembrane G-protein coupled estrogen receptor (Gper). Binding of estrogens to Gper on zebrafish oocytes results in rapid activation of epidermal growth factor receptor (Egfr) signaling and subsequent Mapk3/1 activation. We show here that BPA and three structurally-related chemicals, tetrachlorobisphenol A, tetrabromobisphenol A and nonylphenol, can mimic estrogen by activating this mechanism of meiotic arrest through Gper in zebrafish (Danio rerio). BPA bound to zebrafish Gper and inhibited spontaneous oocyte maturation (OM) of denuded oocytes. Treatment of oocytes with Actinomycin D did not block the effects of BPA, suggesting that this inhibition of OM is through a nongenomic mechanism. Incubation of oocytes with a selective GPER antibody and the specific GPER antagonist G-15 blocked the effects of BPA on OM, further suggesting that BPA inhibition of OM is through its interaction with the receptor. Various inhibitors of the EGFR pathway were utilized to determine if the inhibition of OM by BPA is mediated through this mechanism. BPA activation of the Egfr pathway resulted in Mapk3/1 (also known as Erk 1/2) phosphorylation. The results show that BPA disrupts oocyte maturation through a novel mechanism involving activation of a Gper/Egfr/Mapk3/1 pathway with potential adverse impacts on reproductive success. / text

Epigenetics, or the reversible and heritable marks of gene regulation not including DNA sequence, encompasses modifications on both the DNA and histones and is as important as the DNA sequence itself. Gene transcription, DNA repair, DNA replication, and the cell cycle are each impacted by the chromatin structure. A variety of enzymes modulate these modifications, and a suite of factors interacts with them to aid in promoting or inhibiting cellular functions. Many of these chromatin-modifying factors are deregulated in cancer, making them novel therapeutic targets. This dissertation describes the identification of an H3K9 histone methyltransferase, SUV39H1, as a suppressor of rhabdomyosarcoma formation in zebrafish. This suppressor is dependent on the methyltransferase domain of the enzyme, ruling out any scaffold effects since this enzyme is a part of a multiprotein complex. SUV39H1-overexpressing and control tumors share many of the same characteristics, including proliferation rate, muscle differentiation state, and tumor growth rate. The tumor suppressive phenotype cannot be rescued by alterations in the downstream muscle program alone. However, SUV39H1-overexpressing fish initiate fewer tumors, which results in the observed suppressive phenotype. This initiation defect occurs between 5 and 7 days of life in the zebrafish, likely by impacting cyclin B1 expression. This dissertation also describes the development of a novel F1 transgenic screening strategy in the zebrafish. This approach was utilized to screen a variety of chromatin-modifying factors for their effects on hematopoietic development. The developed strategy will have future applications as a zebrafish screening tool. Our data suggest that chromatin-modifying factors play an important role in rhabdomyosarcoma and illustrate the use of the zebrafish in discovering genes involved in tumorigenesis and hematopoiesis.

Navigation in complex environments requires selection of appropriate actions as a function of local cues. To gain a quantitative and mechanistic understanding of zebrafish thermal navigation, we have developed a novel assay that requires animals to rely exclusively on thermosensory information in the absence of other cues such as vision or mechanosensation. We show that zebrafish use both absolute and relative temperature information to restrict their locomotor trajectories to a preferred temperature range. We identify components of movement that are modulated solely by absolute temperature, as well as components that are modulated by both absolute and relative temperature. Specifically, we find that dwell time between movements and displacement per movement depend solely on absolute temperature, whereas turn magnitude and turn direction bias are modulated by absolute and relative temperature. To evaluate whether these sensorimotor relationships could explain thermal restriction in our navigation assay, we performed Monte Carlo simulations of locomotor trajectories based on all or subsets of these relationships. We find that thermosensory modulation of turn magnitude and turn direction bias constitute the core navigation strategy in larval zebrafish, while modulation of dwell time accelerates the execution of this strategy at noxious temperatures. Modulation of turn direction bias represents a novel strategy not found in invertebrate models, whereby animals correct unfavorable headings by preferentially turning in a preferred turn direction until they obtain a favorable heading. Modulating turn direction bias in response to recent sensory experience is an effective strategy for selecting favorable headings in organisms that do not have a dedicated sampling phase before each reorientation event.

Hematopoietic Stem Cells (HSCs) are characterized by their ability to both self-renew and give rise to all lineages of the blood system. A recent chemical genetic screen identified 17β-estradiol (estrogen) as a novel modifier of the expression of the conserved HSC markers runx1 and cmyb in the Aorta-Gonad-Mesonephros of developing zebrafish. Exposure to exogenous estrogen during the development of the hematopoietic niche impeded specification of hemogenic endothelium and the subsequent emergence of HSCs via antagonism of somitic-derived VEGF signaling. Conversely, inhibition of endogenous estrogen activity increased the number of functional HSCs present in the embryo and resulted in higher expression of VEGF target genes, suggesting that endogenous estrogen acts to define the ventral limit of VEGF activity and hemogenic endothelial specification. In contrast, when embryos were exposed to estrogen after niche specification, markers of HSCs were increased, indicating that estrogen has a biphasic effect on HSC formation; this effect appears to be at least partially mediated by enhanced cell cycling of the HSC population. Estrogen exposure during primitive erythropoiesis likewise increased the number of erythroid progenitors in the embryo, but their maturation into functional erythrocytes was impaired. Inhibition of erythrocyte maturation is also conserved in a mammalian model of in utero excess estrogen, causing propensity for embryonic lethality. Treatment of adult zebrafish with exogenous estrogen after ablation of the hematopoietic system by irradiation revealed that elevated estrogen levels improved hematopoietic regeneration. Consistent with a role for hormonal regulation of HSC homeostasis, accelerated recovery of hematopoietic stem and progenitor numbers was observed in female fish compared to males, suggesting an endogenous difference in regenerative capacity between the sexes. Together, these data identify multiple distinct roles for estrogen in HSC biology and indicate it is a physiologically relevant regulator of HSC development and homeostasis.

Hemoglobin switching is a developmental process involving the dynamic transcriptional regulation of multiple globin genes. This molecular process involves multiple layer of complexity, and elucidating new mechanisms in this process will result in a more complete understanding of general gene regulation and will likely have direct clinical implications for hemoglobinopathies, such as sickle cell anemia. In this dissertation, I develop and characterize a new model for hemoglobin switching, the zebrafish. I defined and fully annotated the two zebrafish globin loci, termed major and minor loci. Both loci contain α– and β–genes oriented in a head–to–head fashion. Characterization of the globin expression pattern precisely defined the timing of normal switching and demonstrated that zebrafish, like humans, have two globin switches. The locus control region for the major locus was identified and in conjunction with a proximal promoter was able to generate robust, erythroid–specific expression in a transgenic line.

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Satellite cells (SCs) are muscle stem cells that stay in a metabolically and mitotically quiescent state in adult skeletal muscle until activated. In mammals, SCs are activated and enter into the cell cycle for growth and regeneration. The mechanism initiating SC activation in vivo and in vitro, mediated by nitric oxide (NO) and hepatocyte growth factor (HGF) is described in the mouse model, but not in other species. Here, we assessed SC activation by counting bromodeoxyuridine (BrdU)-immuno-positive cells, and found that SC activation in zebrafish single muscle-fibre cultures is also NO and HGF dependent, peaking at 1 mM isosorbide dinitrate (ISDN, an NO donor drug) and 10 ng/mL HGF respectively, using dose-response experiments. Moreover, HGF signalling via the c-Met receptor is involved in the SC activation pathway and is considerably affected by temperature (i.e., 21 °C). Overall, understanding NO-HGF-c-Met signalling in SC activation gives new insights on fish muscle growth and conservation of regulatory pathways between species.

To determine whether exposure to simulated microgravity (SMG) affects cranial neural crest (CNC)-derived tissues, zebrafish embryos were exposed to SMG starting at one of three developmental stages corresponding to CNC migration. Juvenile and adult fish were analyzed after exposure to SMG using statistics and geometric morphometrics for changes in melanophore surface area and number, and changes in skull morphology. Analyses reveal an initial increase in the surface area of melanophores present on the dorsal view of the juvenile skull and a decrease in melanophore number over the period of a week. Additionally, buckling is observed in CNC-derived frontal bones in juvenile fish after exposure. The effects on the melanophores are transient and the effects on CNC-derived bones are short-term. Surprisingly, severe long-term effects occurred in mesoderm-derived bones, such as the parasphenoid. In summary, exposure to SMG affects both CNC- and mesoderm-derived tissues in the juvenile and adult zebrafish head.