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.

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.

It is becoming increasingly clear that chemicals present in the environment can interfere with thyroid hormone (TH) action and signaling. This is of great concern because TH is critical for normal brain development and recent evidence suggests that the developing brain is exquisitely sensitive to perturbations in TH action. Developmental exposure to the ubiquitous environmental toxicants, polychlorinated biphenyls (PCBs), is associated with neurological deficits that may be related to ability of these chemicals to alter TH-mediated brain development by interfering with TH action. Thyroid hormone plays an important role of is on the development and maturation of white matter tracts and its glial constituents. Because PCBs are suspected of interfering with thyroid hormone (TH) signaling in the developing brain, and because TH is important in white matter development, this dissertation set-out to critically test the hypothesis that developmental exposure to PCBs alters white matter development by interfering with TH action. In testing this hypothesis, we addressed four main questions: (1) Do PCBs perturb white matter development by causing a relative state of hypothyroidism? (2) Is the severity of TH insufficiency associated with different effects on white matter? (3) Do PCBs have a TH-like effect on white matter composition? (4) Do PCBs and TH insufficiency alter white matter by affecting the same signaling networks? We focused our experiments on postnatal day 15, a time when both myelination and TH levels are at their peak during development, and effects of hypothyroidism on white matter development have been documented. We evaluated the effect of developmental exposure to the commercial PCB mixture Aroclor 1254, TH insufficiency, and TH excess on markers of oligodendrocyte and astrocytes in two white matter tracts, the corpus callosum (CC) and anterior commissure (AC). The results described in this dissertation are consistent with the idea that developmental exposure to PCBs alters white matter composition independent of ability to interfere with TH action. Furthermore, the nature of the experimental designs used in testing our hypothesis provided fundamental information on the role of TH in the differentiation of oligodendrocytes and astrocyte from common precursors.

Synapses are the functional units of neuronal circuits and the sites of integration for multiple signaling pathways. Understanding the molecular basis of synaptic function is critical to understanding the bigger picture of how we think, learn, remember and how neurological diseases and disorders disrupt these faculties. Here we characterize the role of bendless (ben) in central synapse formation by utilizing the giant fiber system (GFS), a well established neuronal network in Drosophila melanogaster. Ben is an E2 conjugase and a key member of the enzyme cascade involved in ubiquitin dependent regulation. Ben was originally identified more than two decades ago and was believed to be involved in axon guidance as mutants lacked a synaptic connection between the giant fiber (GF) and its target, the jump motor neuron (TTMn). We have been able to redefine Ben function by demonstrating that an incipient GF-TTMn synaptic connection is present in ben mutants. We have also analyzed the synapse with the help of synaptic markers as well as studied its features at an ultrastructural level. By conducting cell autonomous rescue experiments we have spatially determined that ben has a presynaptic function in the GFS. We then used the TARGET system to temporally characterize the gene and have isolated a critical period for Ben function during development. We further assayed protein localization by generating GFP-tagged Ben constructs and have found the protein to be nuclear as well as cytoplasmic. Subsequent studies have identified two multifunctional proteins—Semaphorin1a and Distracted—to be putative targets of Ben action. We have also carried out a preliminary characterization of the synaptic roles other components of the ubiquitin system have in the GFS, such as the ubiquitin ligase highwire and the deubiquitinating proteases, fat facets and UBP2. In summary, we have found the ubiquitin conjugase Ben to have a novel and distinct role as a developmental switch in the establishment of a central synapse. The identification of likely downstream targets of Ben and comparison with related ubiquitin associated proteins suggest that a delicate regulatory balance has to be maintained in order for a synapse that is functionally and morphologically normal to be sculpted.

The zebrafish belladonna (bel) mutation was identified in a large-scale mutagenesis screen to identify genes involved in retino-tectal pathfinding in Tubingen, Germany. In bel mutants, after exiting the eye, retinal axons grow ipsilaterally instead of crossing the midline to form optic chiasm. bel mutants are semi-viable and live bel embryos at 5 days show a "dilated pupil" phenotype after which the mutation was named. Later work showed that bel mutants have functional eyes although the optokinetic response is reversed in the mutants. Previous work in our lab showed that most retinal axons in the mutants initially grow towards the midline but later turn ipsilaterally. Also, two major forebrain commissures, the anterior commissure (AC) and the post-optic commissure (POC) also failed to form in bel mutants. These studies showed that bel defects are restricted to forebrain. Detailed analysis of eye sections showed defects in bel eye morphology during embryonic and adult stages. Initial work also mapped the bel locus on chromosome 8 and finer mapping linked one z-marker on either side of bel locus (z24272 and z44909). My dissertation project was to clone the bel gene and understand its role in forebrain patterning and axon guidance. I identified that bel locus encodes a zebrafish lim-homeodomain transcription factor, Lhx2. To further understand how bel(lhx2) might affect axon guidance, I first showed that bel mutants have subtle defects in forebrain patterning in the regions where axons cross the midline. I also showed that these forebrain patterning defects lead to defects in expression of proper cellular and molecular axon guidance cues at the midline in bel mutants. Finally, I showed that bel(lhx2) is required for cell proliferation in the diencephalon. Thus my detailed analysis of bel mutants has revealed new roles for lhx2 in diencephalon patterning and axon guidance.

Mice with a deletion of the hypothalamic basic helix-loop-helix transcription factor Nhlh2 (N2KO) display adult onset obesity, implicating Nhlh2 in the neuronal circuits regulating energy availability. Nhlh2 co-localizes with the hypothalamic thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus (PVN) and proopiomelanocortin (POMC) neurons in the arcuate nucleus. N2KO mice become obese due to reduced physical activity in the absence of hyperphagia making them a unique mouse model for the study weight gain, obesity and energy expenditure. Signals that regulate Nhlh2 and the effects of Nhlh2 on peripheral tissues remain largely unknown. The research presented here utilized numerous techniques to investigate the effects of changes in energy availability on Nhlh2 expression. We show that Nhlh2 expression decreases significantly with food deprivation and cold exposure. Nhlh2 expression is stimulated with food return or leptin injection following food deprivation or return to room temperature following cold exposure. These data suggest that Nhlh2 gene expression responds positively to increased energy availability and negatively to reduced energy availability. These findings combined with the phenotype of N2KO mice led us to propose that Nhlh2 integrates energy availability inputs in various hypothalamic nuclei to drive expression of genes required for body weight maintenance. Investigation into peripheral tissues in N2KO mice revealed that responses of genes in the hypothalamus-pituitary-thyroid axis, muscle, and brown and white adipose tissue to changes in energy availability require Nhlh2 expression. The responses of serum total T4 levels and UCP1 mRNA and UCP3 mRNA to energy availability signals are altered in N2KO mice. In addition, N2KO mice maintain body temperature with cold exposure but are unable to maintain body weight. In summary, my work provides new insight into the role of Nhlh2 in coordinating energy availability signals to downstream genes required for body weight maintenance and thermoregulation.