A molecular and genetic dissection of the stalled grooming mutant was performed to characterize a gene which may be necessary for the assembly of the pupal and adult nervous system of Drosophila melanogaster. To determine whether the grooming deficit observed in stalled resulted from disrupting the development of the sensory nervous system, afferent projections were examined in stalled using two P (Gal4) lines as markers of subsets of femoral chordotonal neurons. These lines were isolated in a screen of P (Gal4) enhancer trap lines to identify those which label distinct subsets of sensory neurons and their afferent projections in the pupal and adult nervous system (Chapter 1). In addition, afferent projections from the posterior tactile neurons were examined in stalled mutants using the en-lacZ marker line. Because the stalled P element insertion site was mapped to the 67C region of the third chromosome (as was the laminin $\gamma$ gene), it was of interest to determine whether stalled had identified $laminin\ \gamma$ since it has been demonstrated that the laminin plays roles during neuronal development. It was possible that stalled would interact with $laminin\ \alpha$ as a result of identifying the $laminin\ \gamma$ gene. Therefore, a genetic analysis was performed by combining stalled with the $laminin\ \alpha$ mutant and examining projections from different classes of sensory receptors in the single and double mutants. It was found that the interaction with stalled and $laminin\ \alpha$ was required for the proper development of at least two classes of sensory receptors (Chapter 3). Therefore, molecular characterization of stalled was performed to determine if stalled had identified the $laminin\ \gamma$ gene. Northern analysis demonstrated that the expression of $laminin\ \gamma$ was decreased in stalled and the $stalled\sp{e71}$ hypomorph. However, mapping experiments demonstrated that the stalled P element insertion site was 20-60 kilobases downstream of $laminin\ \gamma$. Taken together, these results suggest that stalled has identified an enhancer of the $laminin\ \gamma$ gene such that the amount of the laminin heterotrimeric complex is reduced in the $laminin\alpha;stld$ double mutants. This results in significant effects on the development of at least two distinct classes of sensory afferent projections.

The purpose of this thesis was to investigate the firing pattern of single neurons in cerebellar nucleus interpositus (NI) related to a conditioned response (CR) instilled by conditioning with mixed interstimulus intervals (ISIs). The conditioning with mixed ISIs is a protocol of classical conditioning that involves two different interstimulus intervals (ISIs) alternating randomly. The conditioned stimulus (CS) is a tone and the unconditioned stimulus (US) is an electrical shock to periorbital region that causes eyeblink. This protocol resulted in bimodal responses with peaks corresponding to the timing of the US. A related purpose of this investigation was to explore mechanisms of CR timing and temporal integration using a computational method. Sutton and Barto's (1990) temporal difference (TD) model with a complete serial compound (CSC) representation of CS was used to implement the idea of a neuron-like processing element that receives time-tagged inputs and adjusts their associative strength to generate an appropriate output adaptive to the given conditioning environment. The TD model with CSC representation of time can be aligned with the cerebellum. Within this context, there are several scenarios as to how time is segmented in the cerebellum and how this information is integrated to produce the CR. The current investigation presents evidence that single neurons of NI express a firing pattern closely related to the bimodal response. All forty-two CR-related units recorded in NI showed neuronal activity closely related to the time-course of eyeblink CRs, i.e. a neuronal activity pattern with two distinctive increases in firing rate. Most of the units preceded the behavioral response but the degree by which the neuronal activity preceded the behavioral response varied. Among the 42 CR-related units, 9 units responded to the tone CS with short latency(<100 ms), CS-locked activity. Among twenty-four units tested on a US-only trial, 22 units increased firing rate or remained at the same level, and 2 units decreased firing rate after US presentation. The CR topography on short-ISI reinforced trials was unimodal implying that the US has become a conditioned inhibitor. The corresponding neuronal activity of single neurons were also unimodal. The TD (CSC) model and its cerebellar implementation could account for the suppression of the second peak by employing a US-initiated timing cascade. The suppression of the neuronal activity on short-ISI trials suggests that the excitation and the inhibition could be expressed at the level of single neuron.

Several genes implicated in Parkinson's disease (PD) encode components of the ubiquitin-proteasome pathway. In a specific form of PD (human Autosomal Recessive Juvenile Parkinsonism, AR-JP), loss of functional Parkin (ubiquitin E3 ligase) results in a selective loss of midbrain dopaminergic neurons and a absence of Lewy bodies (LB) from the surviving dopaminergic neurons. Since cells in patients with AR-JP do not express functional Parkin, it is unclear why most neuronal and non-neuronal populations remain unaffected. One possible explanation is that most cells express a redundant ubiquitin E3 ligase(s) that is absent from dopaminergic neurons. Such candidate(s) redundant E3-ligase would be expected to fulfill several criteria: (1) bind similar E2 Ubiquitin conjugating enzymes; (2) interact with the same cellular substrates; (3) facilitate the formation of aggresome/lewy bodies with similar properties of those induced by Parkin; (4) be expressed in the nervous system but presumably absent (or largely absent) from dopaminergic neurons. In this thesis I have demonstrated that the Human Homolog of Drosophila Ariadne-1 (HHARI) is a candidate for such a redundant E3 ligase. In addition I have shown that even though HHARI induces the formation of LB like aggresomes in cell culture with properties similar to those produced by Parkin, these aggresomes differ in their detergent solubility properties. Using mouse C2C12 primary skeletal muscle cells with altered expressions of Ariadne-1 or Parkin, I determined if HHARI and Parkin may serve redundant protective roles. Using cell viability assays I have shown that HHARI does not confer protection to cells treated with toxic insults like those implicated in PD. On the contrary, using RNA silencing, I have shown that reduced Ariadne-1 expression appears to confer some benefit. Finally, based on phenotypes reported for Ariadne-1-/- escaper and Parkin-/- flies as well as our protein interaction data, I investigated the roles of Parkin and HHARI during myogenesis. Using engineered C2C12 cells I have shown that Ariadne-1 levels are tightly regulated in proliferating and differentiating C2C 12 cells and that increased cellular abundance of Ariadne-1 affects muscle terminal differentiation downstream of myogenin, strongly highlighting the importance of Ariadne-1 and perhaps the Ubiquitin Proteasome Pathway in myogenesis.

Unlike most rodent models, prairie voles, show very few sex differences in social behavior. Despite the similarities in their behavior male and female voles, appear to use different physiological machinery to achieve the same behavioral goals. Therefore, male and female voles are likely to respond differently to pharmacological behavioral modifiers. To test whether the behavioral effects of serotonin potentiation vary by gender and/or reproductive context, I compared the effects of the selective serotonin reuptake inhibitor fluoxetine on parental and aggressive behavior in pairbonded, parentally-experienced male and female voles, and in pairbonded, parentally-inexperienced male and female voles. Because sociosexual experience influences the function of the serotonin system, the target of fluoxetine, I also compared serotonergic function in male and female voles that had mated, remained with a same-sex sibling, or been paired with a novel conspecific of the same sex. Fluoxetine increased the latency to parental behavior in parentally-experienced male and female voles and pairbonded, parentally-inexperienced male voles. Fluoxetine also decreased aggressive behavior in parentally-experienced male voles, but had no effect on the aggressive behavior of parentally-experienced female voles, or pairbonded, parentally-inexperienced voles of either sex. In addition, fluoxetine reduced serotonin turnover in the frontal cortex and hypothalamus/preoptic area of male and female voles. Serotonin turnover was also affected by sociosexual experience. Mating and cohabitation with a mate increased serotonin turnover in the hypothalamus of male and female voles. Furthermore, the levels of serotonin and 5-hydroxy-indoleacetic acid in the frontal cortex and amygdala were sexually dimorphic. Although this thesis focused primarily on the influence of gender and reproductive context on the behavioral effects of fluoxetine, it also revealed important aspects of parental and aggressive behavior that were independent of the effects of fluoxetine. Parentally-inexperienced female voles were infanticidal one week into pregnancy whereas their mates were parental. Furthermore, the aggressive behavior of parentally-inexperienced, pairbonded voles but not parentally-experienced voles was affected by the gender composition of the resident-intruder pair. Parentally-inexperienced, pairbonded voles were more aggressive with opponents of the same sex than opponents of the opposite-sex. The findings described in this thesis suggest that fluoxetine has sexually dimorphic effects on behavior and that sociosexual factors influence the effects of fluoxetine on behavior as well as the function of the serotonin innervation of the brain.

Hedgehog (Hh) signaling is important for patterning and cell differentiation within many embryonic tissues. Hh is expressed in the notochord and the floor plate and acts as a morphogen in patterning of the ventral central nervous system (CNS), pituitary gland, somites, pancreas, and many other tissues. Gli transcription factors act as main mediators of Hh signaling in vertebrates. Their unique and overlapping functions lead to differential outcome of Hh signaling in different cells and tissues. The goal of my dissertation is to determine how zebrafish Gli proteins work together to transduce Hh signals and to activate or repress the transcription of Hh target genes. I have shown that Gli1 acts only as an activator of Hh signaling similar to what is known from other species, while Gli2 and Gli3 act as both activators and repressors. Gli2 functions as a co-activator of Gli1 in subset of ventral cells in the posterior diencephalon and in the adaxial cells in the embryonic trunk. In contrast, activator role of Gli3 overlaps with Gli1 throughout the ventral CNS during early development of the embryo. Later, GO represses Hh targets in the dorsal spinal cord, but not in the forebrain. In contrast, Gli2 represses genes in both, the dorsal telencephalon and the spinal cord. I also demonstrate that Gli3 repressor function is temporally regulated by active Hh signaling, unlike Hh independent Gli2 repressor function. Thus, my detailed analysis of zebrafish Gli functions reveals complex interactions between Gli proteins in embryonic patterning. I also studied a novel zebrafish mutation umleitung (uml) that was identified because of defects in axon guidance and neural patterning in the ventral forebrain. My detailed phenotypic analysis showed that the uml mutation disrupts Hh signaling and forebrain patterning. I have genetically mapped uml on zebrafish chromosome 24 near the Zmarker z10372. My linkage analysis indicates there are no known components of the Hh signaling cascade in the uml genetic region. This leads to the exciting possibility that uml may encode a previously undefined regulator of Hh signaling. I have initiated a genomic walk toward finding a gene that encodes uml.

Genetic screens which assay behavior have been successfully used to identify genes required for neural function. This thesis is the analysis of midline uncoordinated (muc), a mutation identified for its effect on grooming behavior. This mutation was caused by a single P (lac W) insertion at position 28A. A number of additional muc alleles have been generated by excision of the P element. Using markers for two types of femoral chordotonal neurons we have been able to show that muc disrupts the axon trajectories of these cells. In addition to grooming behavior and neuroanatomy, many muc alleles also affect midline parting of the thoracic microchaetae, flightlessness, lethality and male sterility. Genetic analysis of the various muc mutations suggest that they form a unique complementation group. Three transcripts were found near the area of the muc mutation. The most likely gene affected by muc is the Drosophila homolog of dihydrolipoamide acetyltransferase, component E$\sb2$ of the mitochondrial pyruvate dehydrogenase complex. The P (lac W) element sits in an intron of this gene. We have found that the most severe grooming alleles retain all or almost of all of the P element used to cause the original mutation. In addition to severe grooming behavior, these alleles also have severe axon projection defects. Revertant alleles which have cleanly excised the P element have wild type grooming behavior and normal axon projection patterns.

We utilized the powerful molecular and genetic tools available for the analysis of neural development in Drosophila to characterize a mutation for its effects on imaginal sensory axons. Previous analysis demonstrated that loss of function alleles caused defects in axon anatomy. Molecular analysis revealed that these axon defects were due to a disruption in the 10.4 kDa cytoplasmic dynein light chain gene (Cdlc1). This molecular analysis involved the recovery of both genomic and cDNA clones, characterization of the transcription unit in both wild type and mutants, and sequence analysis. As verification that mutations in the cytoplasmic dynein light chain gene caused the axon defects, we generated transgenic flies in which expression of the gene was targeted to specific sensory neurons. When this targeting system was introduced into a Cdlc1$\rm\sp{null}$ genetic background the axon phenotype of the targeted neurons was rescued. Further, the rescuing effects of the targeted expression was restricted to the Cdlc1-positive neurons. Other sensory neurons that were not expressing the transgene in this system retained mutant axon phenotypes. The specificity of the rescue demonstrated that Cdlc1 function is cell autonomous, and that sensory neurons require Cdlc1 function for proper development of their axon projections. The dynein light chain has been shown to associate with the cytoplasmic dynein complex, myosin V and nitric oxide synthase. All of these molecules have neural function so it is important to determine whether any of these partners were involved in producing the axon defects we observed in Cdlc1 mutants. In Drosophila, mutant alleles are only available for genes encoding components of the cytoplasmic dynein complex. We used these alleles in a double mutant analysis to determine whether the axon phenotype was influenced by genetic interactions between mutations affecting the light chain and the dynein heavy chain and p150$\rm\sp{Glued}.$ Our data showed that loss of function mutations in the heavy chain gene acted as dominant suppressors of the Cdlc1 axon phenotype, while loss of function mutations in Glued acted as dominant enhancers of the phenotype. These results support a model in which the light chain functions with the cytoplasmic dynein complex during axon development.

Recent clinical evidence indicates that thyroid hormone plays an essential role in fetal brain development. However, the mechanism by which thyroid hormone affects development has been largely unexplored. Because thyroid hormone receptors (TRs) are ligand-activated transcription factors, the TR-mediated effects of thyroid hormone in the fetal brain will necessarily be manifested first by changes in gene expression. Therefore, I used differential display to identify genes expressed in the fetal brain that are affected by acute thyroxine administration to the dam before the onset of fetal thyroid function. I identified 11 putative thyroid hormone-regulated genes using differential display. Eight of these genes are selectively expressed in areas of the gestational day (G) 16 brain that contain TRs, indicating that these genes may be directly regulated by maternal thyroid hormone. Next, the distributions of three of these genes, neuroendocrine-specific protein (NSP), Oct-1, and a known thyroid hormone-regulated gene, RC3/neurogranin, were characterized. All mRNAs are expressed from at least G14 until adulthood in brain areas that contain TRs and their regulation by maternal thyroid hormone was confirmed using in situ hybridization in the G16 cortex. Additionally, I examined the effects of thyroid hormone on NSP and Oct-1 in the adult brain. I demonstrated that NSP and Oct-1 are expressed in the adult brain and are regulated by thyroid hormone. These studies provide the first evidence that maternal thyroid hormone directly affects fetal brain development by regulating the expression of specific genes in vivo. These data support the concept that maternal thyroid hormone exerts a direct action on the expression of genes that are important for normal neurological development of the fetus. Collectively, these data have clinical importance because thyroid hormone affects NSP, Oct-1, and RC3/neurogranin expression in brain regions affected in cretinism and congenital hypothyroidism. These three genes are regulated by thyroid hormone from at least G14 to adulthood and are expressed in brain areas known to be affected by hypothyroidism. These areas include the cortex, hippocampus, and cerebellum. The data presented in this dissertation provide experimental evidence that NSP, Oct-1, and RC3/neurogranin may be partially responsible for the detrimental effects of hypothyroidism in developing brain and support several recent clinical studies indicating that untreated fetomaternal hypothyoidism adversely affects fetal brain development.

Synaptic transmission between a neuron and its target is crucially dependent upon the precise spatial arrangement of proteins in the pre- and postsynaptic apparatus. PDZ domain-containing proteins such as the Drosophila tumor suppressor Discs-Large (DLG) play critical roles in synapse maturation by regulating the assembly of synaptic protein complexes. DLG is composed of a number of modular domains, including three PDZ-domains, an SH3 domain and an enzymatically inactive Guanylate Kinase-like (GUK) domain. Previous studies have shown that the PDZ domains of DLG mediate clustering of Shaker K+ channels and of the cell adhesion molecule Fasciclin II. However, the function of the GUK domain has been unclear. To understand the role of the GUK domain, we carried out a yeast-two hybrid screen for interacting partners of the DLG GUK domain. This screen lead to the identification of a novel synapse-associated protein, GUKHolder (GUKH). GUKH is a 1044 amino acid protein with a molecular weight of 110 kDa. Its sequence includes a GUK-holding domain, a region homologous to the C-terminal of the long isoform of Kelch, a WH1-like domain, and a PDZ-domain binding motif. These latter two features suggest that GUKH may interact not only with DLG, but also with other proteins, including proteins containing PDZ domains. GUKH is expressed at the larval neuromuscular junction and at epithelial cell borders in partial colocalization with DLG. Further, DLG can be co-immunoprecipitated with GUKH from Drosophila extracts, indicating an in vivo interaction between the two proteins. GUKH has also been shown to interact directly with SCRIBBLE (SCRIB), another synaptically expressed PDZ-domain protein known to have a genetic interaction with dlg in epithelial tissues. Synaptic SCRIB immunoreactivity is mislocalized in both gukh and dlg mutants. gukh, scrib, and dlg mutants all exhibit synaptic bouton defects at the ultrastructural level. These data indicate that all three proteins are required for proper synapse maturation, and support a model that the three proteins exist in a tripartite complex, with GUKH forming a link between the other two proteins, and further, between the protein scaffolds organized by the two proteins.

Fetal alcohol exposure is the leading known cause of mental retardation in the western world. However, the mechanisms underlying alcohol-induced damage in fetal brain are largely unknown. The goal of this dissertation is to identify ethanol-responsive genes during brain development to provide more insights into the mechanisms. I chose a well-established animal model for all the studies in this dissertation. First, I demonstrated that this ethanol paradigm increased the mRNA of cellular retinol binding protein I (CRBP-1) in gestational day 13 (G13) brain and the incidence of apoptosis in G16 brain. Second, I identified 12 putative ethanol-responsive genes using mRNA differential display. After the quantitative analysis by Northern blot, in situ hyridization, western blot and relative quantitative RT-PCR, ribosomal protein S6 (rpS6), neuroendocrine-specific protein-A (NSP-A) and a novel gene were verified as ethanol-responsive genes. Third, I isolated 32 putative ethanol-responsive genes using cDNA microarray analysis. They encode proteins engaged in cell signaling, cell cycle regulation, metabolism, stress response and cell structure. Among all the putative genes, alcohol dehydrogenase 3 (Adh3) and glutathione S transferase pi 2 (GST pi 2) are previously known ethanol-responsive genes. Additionally, bone morphogenetic protein receptor type IA (BMPR-IA) showed the largest change induced by ethanol, 2.1-fold, and the ethanol effect on its expression was confirmed by relative quantitative RT-PCR. Fourth, because NSP-A is also a thyroid hormone-regulated gene, I analyzed the expression of two other thyroid hormone-regulated genes, Oct-1 and Hes-1, in my ethanol-treated animal model. However, ethanol did not affect thyroid hormone regulation of these two genes in G16 cerebral cortex. Fifth, I examined the effects of ethanol on protein expression and phosphoralytion using two-dimensional (2D) electrophoresis and western blotting. Ethanol was shown not to robustly change the abundance of individual proteins, but may change the post translation modifications of some proteins during brain development, such as glycosylation. In conclusion, these studies systematically and thoroughly examined the effects of fetal alcohol exposure on gene expression during brain development. They provide useful insights for analyzing the complex pathways leading to CNS damage in the children born to mothers who drank heavily during pregnancy.