The cerebellum, which forms from anterior hindbrain, coordinates motor movements and balance. Sensory input from the periphery is relayed and modulated by cerebellar interneurons, which are organized into layers. The mechanisms that specify the different neurons of the cerebellum and direct its layered organization remain poorly understood. Drawing from investigations of spinal cord, we hypothesized that the embryonic cerebellum is patterned on the dorsoventral axis by opposing morphogens. We tested this using zebrafish. Here we show that expression of olig2, which encodes a bHLH transcription factor, marks a subset of PNs. In combination with other markers, olig2 reveals a dorsoventral organization of cerebellar neurons in embryos. Disruption of Hedgehog signaling, which patterns the ventral neural tube, produced a two-fold increase in the number of olig2+ PNs. By contrast, olig2+ PNs did not develop in embryos deficient for Wnt signaling, which patterns dorsal neural tube, nor did they develop in embryos deficient for both Hedgehog and Wnt signaling. Our data indicate that Hedgehog and Wnt work in opposition across the dorsoventral axis of the cerebellum to regulate formation of olig2+ PNs. Specifically, we propose that Hedgehog limits the range of Wnt signaling, which is necessary for olig2+ PN development.

In most eukaryotes, telomere length is maintained by telomerase, a ribonucleoprotein that adds TG-rich telomeric repeats de novo to chromosome ends. Telomeric binding proteins (for example, Rap1p) affect telomere length by negatively regulating the access of telomerase to the telomeres. However it has been unclear whether telomerase directly participates in this regulation. In S. cerevisiae, telomerase has at least four components, among which Est2p (Telomerase Reverse Transcriptase subunit) and TLC1 RNA compose the catalytic core. This thesis describes analysis of the mechanism through which several mutations identified in the N-terminus of Est2p (est2-LT) cause telomere over-elongation and uncovers a novel role of telomerase in influencing the association of Rap1p with telomeres. est2-LT mutants behave normally in standard primer extension assays, and have normal telomerase composition and telomere end structure. These data suggest that the LT mutations might affect a regulatory function of telomerase without impairing its intrinsic enzymatic activity. Here I show that the association of Rap1p (per nucleotide) is reduced in est2-LT mutants. Telomere over-lengthening in est2-LT is dependent on downstream effectors of the Rap1p pathway (RIF1, RIF2 and TEL1), consistent with the hypothesis that the reduced binding of Rap1p in vivo allows increased access of telomerase to telomeres. This behavior contrasts with a mutation in EST2 (est2-up34) that increases the association of Rap1p as expected for a strain with long telomeres. Although I observe changes in the telomere sequences of est2-LT strains, est2-up34 strain shows the same alterations, suggesting that the sequence changes are a consequence, rather than a cause of telomere over-elongation. Consistent with this idea, there are no significant differences in the association of Rap1p with WT or est2-LT telomeres in vitro. Taken together, I propose that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, and that telomerase has an additional role upstream of Rap1p in telomere length homeostasis. A mutation within the same region of S. pombe TERT subunit causes telomere lengthening and alters telomeric sequences, suggesting that this function of TERT might be evolutionarily conserved.

Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. In this thesis, I describe my work using a Drosophila model to study cellular, molecular and genetic mechanisms that underlie the cognitive dysfunction in FXS. First, I used the MARCM technique to generate clones of Drosophila fragile X mutant retardation 1 (dfmr1) mutant neurons within an otherwise wild-type brain. I focused on the Mushroom Body, a well-characterized brain region of learning and memory. The dfmr1 null mutant neurons display overgrowth and overbranching in cell bodies, dendrites and axons. Consistently, dfmr1 over-expression neurons results in simpler cellular architecture. These results indicate that dfmr1 is a negative regulator of neuronal architectural complexity. Second, using immunocytochemistry and confocal imaging fluorescence intensity quantification, I investigated the regulatory function of the dfmr1 protein (dFMRP) on ionotropic glutamate receptors (iGluR) at the Drosophila NMJ synapses, and the relationship between dFMRP function and Drosophila metabotropic glutamate receptor (DmGluRA) synaptic signaling. I found that dFMRP regulates two iGluR classes in opposite directions. In contrast, DmGluRA negatively regulates both iGluR classes in common. Double null mutants of dfmr1 and dmGluRA always display an additive effect of the two single mutant phenotypes, which suggests independent, convergent pathways between dFMRP and DmGluRA regulation. Thirdly, I examined mechanistic relationships between dFMRP and DmGluRA by assaying protein expression, behavior and neuron structure in both the brain and NMJ synapse; in single mutants, double mutants and with an mGluR antagonist. These results show that DmGluRA and dFMRP convergently regulate presynaptic properties. Taken together, my work has clarified the cellular function of dFMRP on neuronal and synaptic architecture, uncovered new molecular mechanisms showing that dFMRP regulates class-specific iGluR levels in synaptic terminals, and elucidated the mechanistic relationship between dFMRP function and DmGluRA signaling.

Splicing is the regulated removal of introns and the concurrent ligation of exons to produce mature mRNA transcripts. Variability in this tightly regulated process is responsible for an extraordinarily diverse proteome from a relatively small mammalian genome. Alternative splicing can lead to differential exon inclusion or exclusion, as can aberrant splicing, and such transcripts therefore differ from constitutively spliced transcripts. Where mistakes in splicing cause disease, the resulting mutant transcripts appear to be ideal targets for RNA interference (RNAi). In the case of inappropriate exon inclusion, small interfering RNAs (siRNAs) can be targeted to specific exons. When exon skipping prevails, siRNAs can be designed complementary to the specific exon-exon junctions that are not present in normal transcripts. The human growth hormone gene, GH-1, nicely illustrates these points. Constitutive splicing of all 5 exons produces the normal hormone but aberrant skipping of exon 3 can lead to the production of a dominant negative isoform and associated Isolated Growth Hormone Deficiency type II (IGHD II). This thesis describes research into understanding the causes of exon 3 skipping and shows that siRNAs targeting the unique exon 2-exon 4 sequence in mutant transcripts can prevent onset of IGHD II in a mouse model.

Dopamine plays key roles in many basic functions in the central nervous system. In order to study developmental and functional roles of dopaminergic cells in the zebrafish, we have examined a transgenic line of zebrafish expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (TH) promoter. TH-driven GFP was expressed in cells located in the inner nuclear layer. Immunocytochemistry with antibodies for GFP and TH showed that 29 ± 2% of GFP-labeled cells also expressed TH. Loose-patch voltage-clamp recording from GFPlabeled cells revealed that these dopaminergic neurons are spontaneously active in darkness. This transgenic line provides a useful tool to target retinal dopaminergic cells in vivo and in situ. The vertebrate retina is profoundly influenced by circadian rhythmicity, yet little is known about the mechanisms of the zebrafish retinal circadian clock. To further the study of the zebrafish retinal clock, we have constructed a recombinant BAC in which short-half life GFP is under the control of the zebrafish circadian gene Per3 promoter. Expression of the modified BAC clone was observed in injected zebrafish embryos. Further intercrossing of injected fish and screening of their progeny may identify transgenic Per3::d2GFP fish. Meanwhile, by using transgenic Per3::LUC fish (generated by G. Cahill, University of Houston), we are able to examine the effect of light stimuli on bioluminescence rhythm at different times of a circadian cycle. Our results demonstrate that cultured zebrafish retina shows large phase shifts with phaseresponse curve close to type 0.

Proper embryonic development requires precise cell movements, which are coordinated by multiple signaling pathways. Formation of specific organs is initiated during gastrulation when organ precursors acquire their initial cell fates. Additionally, precursors move to specific locations where they engage in additional inductive interactions to continue their differentiation, and form the specialized tissues required for organ functions. Impaired cell movements can cause severe embryonic malformation, developmental arrest and also many related diseases. In this work, I used the zebrafish, Danio rerio, a well-established vertebrate model system to study the involvement of G-protein coupled receptor (GPCR) signaling in cell movements. Apelin and its GPCR receptor Agtrl1 regulate adult physiology, in particular cardiovascular functions, and blood vessel development. Here we show that the zebrafish Apelin and Agtrl1b homologs control heart field formation during gastrulation. Cardiac precursors, specified in the lateral plate mesoderm territories, converge toward the embryonic midline during gastrulation and extend rostrally to form bilateral heart fields. We found that agtrl1b is expressed in the forming mesendoderm before gastrulation, and in the lateral plate mesoderm later, while apelin expression is confined to the midline. Suppressing the function of Agtrl1b or its ligand Apelin using morpholino antisense oligonucleotides resulted in a deficiency of cardiac precursors and a subsequent absence or reduction of heart. Embryos injected with apelin RNA formed no heart. Our cell tracing experiments demonstrated that in embryos with excess Apelin, cardiac precursors failed to move to the correct location and to express heart markers. Time-lapse analyses of Apelin overexpressing gastrulae revealed reduced migration and defective morphology of mesodermal cells including cardiac precursors. Moreover, in Apelin deficient gastrulae, the cardiac precursors moved less efficiently to the correct location, and showed broadened and ectopic distribution. Our results demonstrate an essential developmental role for the Apelin-Agtrl1b GPCR signaling system in mesodermal cell movements and migration of cardiac precursors to form the heart field during vertebrate gastrulation. During our investigation, we found that Apelin also regulates the migration of zebrafish primordial germ cells (PGCs), a process previously shown to be regulated by Sdf1a/Cxcr4b GPCR signaling. During gastrulation and somitogenesis, apelin mRNA is expressed in the dorsal midline, while its receptor agtrl1b gene is broadly expressed in the mesendoderm, where PGCs are localized. Manipulating Apelin function by misexpression throughout the embryo or by overexpression specifically in primordial germ cells impaired movements of PGCs towards their target tissues. Suppressing Apelin function by injections of antisense morpholino oligonucleotides also resulted in a phenotype of mis-localized PGCs. The abnormal PGC movements in these loss and gain of function scenarios are not a consequence of altered sdf1a expression. Using transplantation experiments, we showed that the cells expressing Apelin in ectopic locations attracted PGCs. However, in these experiments the PGCs stopped short of Apelin overexpressing cells. Interaction between both Apelin and Sdf1a signaling was also investigated. In odysseus (ody) (-/-) mutants, which harbor a null mutation in cxcr4b gene, the majority of ectopic PGCs aggregate in the dorsal midline where apelin is expressed. Interference with both signaling pathways, by injecting apelin MO into ody (-/-) mutant embryos, significantly reduced the dorsal aggregation of PGCs. Based on the preliminary data, I hypothesize that Apelin provides an attractive cue for PGCs migration during gastrulation and segmentation stages, in addition to the previously discovered Sdf1a/Cxcr4b signaling pathway.

The influence of the mammalian retinal circadian clock on retinal physiology and function has been widely recognized, yet the cellular locations and neural regulation of retinal circadian pacemaking remains unclear. Therefore, the focus of this study is to localize the retinal circadian clocks within the mouse retina and to determine the key neural mechanisms regulating the mouse retinal clock. By combining several experimental strategies including single-cell real-time PCR, quantitative PCR, and real-time gene expression reporting, I have demonstrated that retinal neurons in the inner nuclear layer (INL) and ganglion cell layer express the complete molecular basis for rhythms generation and comprise an endogenous circadian clock that oscillates independent of the photoreceptors and of the brain. To study the neural regulation of retinal clock, I have developed a protocol for long-term culture (> 10 days) of intact retinas from adult mice, which allows retinal circadian rhythms to be monitored in real-time as luminescence rhythms from a clock gene reporter (PERIOD2::LUCIFERASE). With this assay, I have studied the characteristics and location within the retina of circadian PER2::LUC rhythms and the influence of major retinal neurotransmitters. Imaging of vertical retinal slices demonstrated that the rhythmic luminescence signals were concentrated in the INL. Interruption of cell communication via the major neurotransmitter systems of photoreceptors and ganglion cells (melatonin and glutamate), and the INL (dopamine, acetylcholine, GABA, glycine, glutamate), did not disrupt generation of retinal circadian PER2::LUC rhythms, nor did interruption of intercellular communication through sodium-dependent action potentials or connexin 36-containing gap junctions, indicating that PER2::LUC rhythms generation in the INL is likely cell-autonomous. However, dopamine, acting through D1 receptors, and GABA, acting through membrane hyperpolarization, set the phase and amplitude of retinal PER2::LUC rhythms, respectively. Collectively, my dissertation studies have shown that the inner retina is the primary location of mouse retinal circadian clock, and have indicated that dopamine and GABA act at the molecular level of PER proteins to play key roles in the organization of the retinal circadian clock, reinforcing the autonomous generation of retinal day and night states.

This thesis is concerned with the study of olfactory systems in both larval and adult mosquitoes. In this dissertation I cloned the first odorant receptor from mosquito Culex quinquefasciatus and localized the receptor proteins to the olfactory organs of the mosquito. By utilizing a sensitive behavioral assay, I also identified a range of odorant-specific responses that are dependent on the integrity of the Anopheles gambiae larval antennae. Parallel molecular studies further identified a subset of odorant receptors localized to larval antennae and these receptors could confer responses to all behaviorally active compounds when expressed in xenopus oocytes. My research may ultimately enhance the development of vector control strategies, targeting olfactory pathways in both larval and adult mosquitoes to reduce the catastrophic effects of malaria and other mosquito-borne diseases.

One of the greatest factors contributing to the worldwide decline in biodiversity is habitat destruction leading to the loss and fragmentation of populations of many species. Knowledge of the genetic and demographic factors that are affected by and influence rarity advances our understanding of the consequences of habitat degradation, and this knowledge is crucial for creating management plans for rare or endangered species. In this dissertation, I address these factors in a rare sunflower species, Helianthus verticillatus, through studies of its population genetics and taxonomic status, the assessment of population size, and the evaluation of fitness. A population genetic study, employing a novel genetic marker, demonstrated that this species is not the product of recent hybridization and, surprisingly, harbors high levels of genetic diversity despite its small number of populations and disjunct range. A study of the clonal diversity and structure in this species revealed far fewer numbers of individuals than were previously reported; these results led to the upgrading of the species priority status for the Endangered Species Act. Finally, populations differed with respect to phenotypic fitness related traits; this was not predicted by population genetic data and further highlights the need for comprehensive studies of endangered species in order to fully evaluate the effects of rarity and fragmentation on population viability.

Structural, biological, and phenotypic analyses of proteins that are involved in transcriptional regulation in the pathogenic bacterium Helicobacter pylori are presented. A high-resolution structure of the C-terminal domain of the á subunit of RNA polymerase, determined using nuclear magnetic resonance methods, is presented, along with a model showing species-specific differences compared to the Escherichia coli protein. The structure of the protein encoded by the HP0564 gene is also presented, suggesting that it is a novel transcriptional regulator in H. pylori. In order to determine the specific functions of HP0564 and HP0222, another novel transcriptional regulator discovered in our laboratory, several experimental approaches were used to analyze mutant strains with disrupted HP0222 or HP0564 genes. Based on results of growth and motility assays of our mutant strains, as well as microarray data, we propose that HP0222 is a regulator of motility in H. pylori and that it may play an important role in the adhesion response.