Excitation and inhibition in the nervous system must be carefully balanced. I utilized the zebrafish (Danio rerio) in order to study this balance in the vertebrate motor system. Zebrafish (Danio rerio) can be used effectively for both forward and reverse genetics. My primary interest lies in genes involved in neural network development and function. Forward genetic mutagenesis screens are particularly powerful as they are unbiased. Using this approach we characterized a mutant based on its abnormal motor behavior. However, mutants identified with abnormal motor behavior may have mutations in muscle proteins. We identified a semi-dominant mutation in the atp2a1 gene, which encodes a protein vital for normal muscle function, from a previously completed mutagenesis screen. In order to investigate genes directly involved in neuronal signaling I used a reverse genetics approach to study GABAA receptors. γ-Aminobutyric acid (GABA) is a major source of inhibition in the motor system. In order to test the effect of blockade of GABAA receptors, I injected gabazine, a GABAA receptor antagonist, into zebrafish embryos. Injected embryos display an abnormal escape response later in development. To determine the GABAA receptor subunits responsible for the observed phenotype, I utilized RNA in situ hybridization to examine the expression of the &agr; class of GABAA receptor subunits. I found that the gabra5 gene, which encodes the GABAA &agr;5 subunit, was expressed in the hindbrain of larvae. To examine the role of the &agr;5 subunit I designed anti-sense morpholinos to target the start codon of the gabra5 gene. Knockdown of the gabra5 gene caused abnormal behavior in larvae similar to that seen in gabazine injected larvae. I have characterized a novel semi-dominant atp2a1 mutant in zebrafish. This mutant completes a zebrafish model system of the human disease Brody's disease. Turning toward a reverse genetic approach I investigated the expression of several GABAA receptor &agr; subunits. I have characterized the behavior of GABAA &agr;5 subunit knockdown embryos and larvae and begun work to generate a stable knockout line. This line will be useful in exploring the function of the &agr;5 subunit and compounds that interact with it.

Thyroid hormone (TH) plays an important role in fetal brain development. Therefore, exogenous factors that interfere with TH signaling may exert potentially important adverse effects on brain development. However the specific roles of TH in brain development are poorly understood. The goal of this research is to delineate the mechanisms of TH and potential mechanisms of endocrine disruption at the TH receptor in the developing brain. The dissertation showed that PCBs can reduce the circulating levels of TH, but simultaneously exerted TH-like effects on TH-responsive genes in fetal brain. To determine whether a specific PCB metabolite, 4-OH-PCB106, could exert a direct agonistic effect on the TRβ1, we employed chromatin immunoprecipitation (ChIP). These studies demonstrate that 4-OH-PCB106 acts as an agonist in GH3 cells, and does not alter the ability of TRβ1 to physically interact with the TRE in the growth hormone (GH) promoter, or with SRC1/NCoR. Interestingly, 4-OH-PCB106 appears to exert actions on gene expression in GH3 cells predominantly through TR, as evidenced by a focused study using differential mRNA display in GH3 cells. A significant impediment in identifying the ability of PCBs to interact with TRs in vivo is that few direct gene targets of TH are known. Therefore, we employed ChIP-on-chip in combination with whole transcriptome expression analysis. We identified 526 direct TH gene targets and these revealed major signaling networks regulated by maternal TH during fetal brain development, including cell-fate specification, cell migration and synaptogenesis. This combination of approaches provides a new look at the role of TH in fetal brain development. In a summary, despite the great deal of research focused on the mechanism of TH action, we do not have comprehensive understanding of the role of TH and its modulators in the brain. Therefore it is important to identify genes that may be direct targets of TH action. This is the first large in vivo database for native TREs in the fetal brain before the onset of fetal thyroid function. Therefore the result will provide profound impact in study of mechanisms of TH as well as endocrine disruptors at the TR in developing brain.

Neural networks in the hindbrain and spinal cord require a proper balance between excitation and inhibition. Identification of zebrafish mutants that have defects in motor output mediated by these networks can allow entrée into underlying network mechanisms. techno trouser ( tnt) mutants demonstrate abnormal motor behavior. Two days after fertilization, wild-type larvae perform an escape response consisting of a large-amplitude body bend away from touch stimuli followed by smaller amplitude body bends to swim away. tnt mutants perform an initial large amplitude body bend away from touch stimuli, but the following smaller amplitude body bends are interrupted by several, abnormal, large amplitude body bends. Four days after fertilization, wild-type larvae exhibit faster escape behavior, whereas tnt mutants are nearly paralyzed and shorter along the rostral-caudal axis. We used meiotic mapping and candidate gene analysis to reveal that the tnt mutation disrupts slc1a2b , which encodes EAAT2, a glutamate transporter expressed in glial cells. Lesion analysis, in situ hybridization, and in vivo electrophysiological recordings all support a model in which reduced slc1a2b function results in exuberant excitation of neurons, initially in the hindbrain and later in development in the spinal cord, to produce the large-amplitude body bends and subsequent paralysis of tnt mutants. Since disruption of human EAAT2 is thought to promote several different neurological diseases, including epilepsy and amyotrophic lateral sclerosis, tnt mutants provide a new tool to understand these disorders.

The major goals of my research were to characterize the hypothalamic neural progenitors and to understand how Hh/Gli signaling plays a role in regulating cell proliferation in the hypothalamic neurogenic zone. In contrast to mammals, the zebrafish brain has a life-long potential to grow continuously. Thus, for comparative neurogenesis studies, zebrafish become an indispensible model organism to understand adult neurogenesis and regulatory signaling pathways. Identification of the regulatory mechanisms underlying the controlled cell proliferation in adult zebrafish brain will pave the way to manipulate the healing potential of the mammalian brain. Using immunohistochemistry and in situ hybridization techniques to label known markers for neural stem/ and progenitor cells I have identified three different populations of cells with radial glia (RG) like morphology in the adult zebrafish hypothalamic ventricular zone. In adult zebrafish, cells with RG-like morphology in the ventricular regions are thought to be the neurogenic population. The first population of cells I identified was positive for the neural stem cell marker NESTIN and showed additional characteristics of neural stem cells. Using a label retention assay we showed that Nestin(+) cells are slow cycling. The second population of RG-like cells was Hh responsive, and expressed markers of neural progenitor/transit amplifier cells. Double labeling experiments reveal that the Hh responsive cells were distinct from the Nestin(+) cells These cells were proliferative and cycled faster compared to nestin(+) neural stem cells. The third population of cells with RG morphology in the hypothalamic ventricular zone expressed shh ligand, indicating a regulatory role for Hh signaling in the hypothalamic ventricular zone. Down-regulation of Hh signaling at larval and adult stages reduced proliferation in the hypothalamic ventricle, indicating that Hh acts as a positive regulator of proliferation, as in the dorsal brain. According to our working model, nestin(+) cells are slow cycling, and/or quiescent neural stem cell population in the hypothalamic ventricular zone, whereas Hh responsive cells are the fast cycling transit amplifier cells which proliferate and give rise to new neurons and glia in the adult. My comprehensive analysis of the neural stem/progenitors in the adult zebrafish hypothalamic ventricular zone provides a starting point for the continued study of the mammalian hypothalamic ventricular zone. This study also demonstrates Hh signaling functions as a positive regulator of cell proliferation in the post-embryonic zebrafish hypothalamus consistent with its role in the dorsal brain. (Abstract shortened by UMI.)

Parkinson’s disease (PD) is a movement disorder characterized by a massive loss of dopaminergic neurons of nigrostriatal origins. Several genes associated with familial cases of PD encode proteins that are direct components of the 26S Ubiquitin Proteasome System (UPS) or interact with enzymes involved in the UPS. Of these genes, parkin, and its product Parkin, an ubiquitin E3 ligase, is the most well characterized. Loss-of-function mutations in parkin result in the “early onset” PD known as Autosomal Recessive Juvenile Parkinsonism (AR-JP). Most research has focused on studying in what ways do nigrostriatal dopaminergic neurons differ from other neurons in the brain and how and why do these cells die in PD. In the following report I describe studies addressing the equally important alternative question: How do other neurons of the brain differ from nigrostriatal dopaminergic neurons that allow them to survive in AR-JP? I hypothesize that another E3 ligase provides redundant functions to Parkin in surviving neurons but that this redundant UPS enzyme is absent from dopaminergic neurons of the SNC. One protein that could possibly provide such a redundant function is the Parkin-like E3 ligase, Ariadne-1. Ariadne-1 and Parkin share significant sequence identity and similarity; they share the RING-IBR-RING signature domain; they share some UPS E2 enzymes; and they bind some of the substrates. In this dissertation I show Ariadne-1 to be a component of LB in post-mortem human tissue of various neurodegenerative disease. Then, in rats, I determine that Ariadne-1 is present as both mRNA and protein in cells of the SNC. Furthermore, Ariadne-1 is globally expressed throughout the mammalian brain and this expression is restricted to neurons and absent from glial cells and white matter tracts. I also find that only a subset of nigrostriatal dopaminergic neurons express Ariadne-1. Then, using the PD model of unilateral striatal lesioning of mice, I determine that Ariadne-1 expression actually correlates more closely with an increased susceptibility to oxidative stress-induced cell death. Lastly, using two different parkin-/- mice, I determine that, in the absence of Parkin, Ariadne-1 expression correlates with a measurable advantage to dopaminergic neurons of the SNC.