Mitf and Otx transcription factors have been identified as essential to the development of the retinal pigmented epithelium (RPE), but the relationship between these factors and their specific role in the RPE developmental pathway have not been clearly defined. The role of the two Mitf transcription factors (Mitfa and Mitfb) and two Otx transcription factors (Otx1a and Otx2) in zebrafish RPE development was explored in these experiments. The loss of Mitf activity in mitfa, mitfb, or double mitf null mutant fish lines had no effect on RPE pigmentation or development. The loss of Otx2 activity through morpholino knockdown produced a RPE deficient phenotype in a small percentage of embryos, while the additional knockdown of Otx1a caused widespread and severe RPE developmental abnormalities. Analysis of ocular sections revealed that the retinal layers remain unaffected in mitf mutants, as well as in most RPE-deficient otx morphants. Mitf and Otx combined loss of function experiments suggest that Mitfa and Mitfb may still play a role in zebrafish RPE development. Expression analysis through in situ hybridization has demonstrated that Otx transcription factors are necessary for the proper expression of mitfa and mitfb while Mitf transcriptions factors are not required for the expression of otx genes. The transcriptional regulation of Mitf by Otx transcription factors may explain why only Otx transcription factors are necessary for zebrafish RPE development despite the somewhat overlapping functions of Mitf and Otx transcription factors.

MicroRNAs (miRNAs) are a family of small non-coding RNAs that regulate gene expression at both the mRNA and protein levels. MiRNAs have been shown to affect neuronal differentiation, synaptosomal complex localization and synapse plasticity, all functions thought to be disrupted in schizophrenia. We investigated the expression of 667 miRNAs (miRBase v.13) in the prefrontal cortex of individuals with schizophrenia (SZ, N = 35) and bipolar disorder (BP, N =35) using a real-time PCR-based Taqman Low Density Array (TLDA). After extensive QC steps, 441 miRNAs were included in the final analyses. At a FDR of 10%, 22 miRNAs were identified as differentially expressed between cases and controls, 7 dysregulated in SZ and 15 in BP. Using in silico target gene prediction programs, the 22miRNAs were found to target brain-specific genes contained within networks overrepresented for neurodevelopment, behavior, and SZ and BP disease development. Given that miRNAs can bind to their targets with imperfect complementarity, computational prediction of true miRNA:mRNA interactions has been difficult and therefore, functional validation of miRNA:mRNA interactions has been relatively sparse. Thus, it was the goal of this study to demonstrate biological functionality of miRNAs on their targets by evaluating transcriptional and translational levels of gene expression(real-time PCR, western blot) as well as determining miRNA target-site specificity (luciferase reporter gene assays). We investigated two miRNAs, miR-132 and miR-137, both of which have been shown to regulate neuronal function and development, and are believed to be associated with schizophrenia from two distinct avenues of research, miR-132 from expression studies and miR-137 from genetic studies. We demonstrated miR-132 down-regulates NTF3, DISC1, and GRIK5 at the transcript level and down-regulates GRIK5 at the protein level as well. Furthermore, we demonstrated miR-137 down-regulates TCF4, CACNA1C, CDK6, ANK3, and ZNF804A at the transcript level, and down-regulates TCF4, CACNA1C, and CDK6 at the protein level. Going further, we also demonstrated miR-137 binds specifically to target sites in the 3'-UTR of CACNA1C, TCF4, and CDK6, suggesting repression of these genes is directly mediated by miR-137. In total, this study provides strong evidence that miRNA dysregulation may contribute to schizophrenia pathogenesis.

Family, twin and adoption studies consistently suggest that genetic factors strongly influence the risk for alcohol dependence (AD). Although the literature supports the role of genetics in AD, identification of specific genes contributing to the etiology of AD has proven difficult. These difficulties are due in part to the complex set of risk factors contributing to the development of AD. These risk factors include comorbidities with other clinical diagnoses and behavioral phenotypes (e.g., major depression), physiological differences that contribute to the differences between people in their level of response to ethanol (e.g., initial sensitivity) and finally the large number of biological pathways targeted by and involved in the processing of ethanol. These complexities have probably contributed to the limited success of linkage and candidate gene association studies in finding genes underlying AD. The powerful and unbiased genome-wide association study (GWAS) offers promise in the study of complex diseases. However, due to the complexities of known risk factors, GWAS data has yet to provide consistent, replicable results. In light of these difficulties, this dissertation has five specific aims which attempt to investigate genetic risk loci for AD and related phenotypes through improved methods for candidate gene selection, analysis of a pooled genome-wide association study, genome-wide analyses of initial sensitivity and maximum alcohol consumption in a twenty-four hour period and finally, creation of a multivariate AD/internalizing phenotype.

Krüppel-like factors KLF1 and KLF2 are closely related transcription factors with three zinc finger domains in their carboxy-termini. KLF1 (erythroid Krüppel-like factor, or EKLF) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β- globin genes. KLF1 and KLF2 have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double knockout (KO) mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, and double KO embryos are more anemic and die sooner than either single KO. We have shown that KLF1 and KLF2 positively regulate the human ϵ- (embryonic) and γ-globin (fetal) genes during embryonic erythropoiesis. Chromatin immunoprecipitation assays (ChIP) show that KLF1 and KLF2 bind to the promoters of the human ϵ- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region (LCR) in mouse embryonic erythroid cells. ChIP assays show that KLF1 but not KLF2 ablation results in abnormal histone modifications in the β-globin locus in mouse embryonic erythroid cells. H3K9Ac and H3K4me3, which correlate with open chromatin and active transcription, are both reduced in KLF1-/- primitive erythroid cells. Human CD34+ hematopoietic stem cells obtained from umbilical cord blood were in vitro differentiated along the erythroid lineage. ChIP assays indicate that both KLF1 and KLF2 bind to the promoter of γ-globin gene in this fetal erythroid model. KLF1 knockdown in these cells affects mainly adult β- globin gene expression. However, the decrease in β- globin gene expression in KLF1 knockdown also affects the ratio of γ- to β- globin in these cells. H3K9Ac and H3K4me3 were decreased only at the β- globin gene which coincides with lower recruitment of RNA polymerase II and its active form, RNA polymerase II phospho-serine 2. In conclusion, we showed using mouse primitive erythroid cells and cord blood definitive cells that KLF1 and KLF2 coordinate the regulation of the mouse and human β- globin genes by direct binding to the promoters and LCR in the β- globin locus. In conclusion, cord blood hematopoietic cells could serve as a complimentary system in addition to the transgenic mouse models to study the regulation of γ- globin gene expression.

The genetic pathways influencing alcohol abuse and dependence are poorly characterized. Many critical discoveries about the interactions between ethanol-related behaviors and genetics have been made in the fruit fly Drosophila melanogaster. Coupling the statistical power of model organism studies to human association studies bolsters the analytical efficacy of these genomic approaches. A variety of behavioral assays are available for assessing behavioral responses to ethanol in Drosophila. However, we find our previously described eRING assay is influenced by the commonly used transgenic marker mini-white. We developed a Simple Sedation Assay (SSA) that is insensitive to the effects of white and mini-white. In SSAs, expression of endogenous wild-type white was not necessary for normal responses to ethanol. Neither expression nor RNAi-mediated knockdown of the transgenic mini-white influenced the effects of ethanol in flies. Critically, mini-white expression did not affect the phenotypes of flies with known alterations in ethanol sensitivity. Also, loss of function mutations in Clic show decreased sensitivity to ethanol in both eRING assays (as previously reported) and SSAs. Therefore, we explored the role of the known Clic interactors, TGF-β and ryanodine receptors. These studies were inconclusive but do not exclude the need for future work. Finally, using bioinformatic tools we constructed a mutli-species network of genes predicted to interact with Clic. Our RNAi screen against the Clic network serves as an important proof-of-concept and holds great potential for uncovering important therapeutic targets for alcohol use disorders.

Colobomas are a type of eye defect characterized by the presence of a hole in certain eye structures. In this study, the roles of the zebrafish Otx genes, otx2 and otx1a, as well as the Rx family gene, rx3, in choroid fissure closure, the disruption of which leads to the onset of colobomas, were studied. It was observed that while the otx2 loss-of-function mutant, otx2hu3237 displayed small colobomas and the otx1a mutant, otx1a6del, did not exhibit any morphological eye defects, zebrafish possessing both mutations presented with a range of colobomas, some of which were more severe than otx2 single mutants and the size of the coloboma corresponded with the gene dosage of otx1a. Furthermore, it was also observed that additional knockdown of otx1b using morpholinos worsened the coloboma phenotype. Moreover, it was observed that rx3, while involved in RPE pigmentation, does not contribute to choroid fissure closure. Additionally, it appears that otx2 does not affect the rudimentary lens formation which is seen in loss-of-function rx3 mutants, i.e., eyeless mutants.

Intercellular junctions and cell-matrix adhesions play important roles in the maintenance of epithelial integrity. Assembly and remodeling of the plasma membrane complexes are regulated by membrane trafficking and fusion. This thesis is aimed to elucidate the roles of an important membrane fusion protein, Ykt6, in the regulation of epithelial cell adhesion and migration. For the first time, we show that Ykt6 is essential for assembly of adherens junctions and tight junctions in human prostate epithelial cells. We also observed that Ykt6 negatively regulates both collective epithelial cell migration and cell invasion into Matrigel. The effects of YKT6 on epithelial junctions involves expressional regulation of key junctional proteins, E-cadherin and claudin-4, whereas its effects on cell motility can be explained by antagonizing functions of junctional adhesion molecule-A. Overall, this study identifies YKT6 as a novel regulator of epithelial cell adhesions and motility.

Adherens junctions (AJs) and tight junctions (TJs) are characteristic features of differentiated epithelial cells and are critical for regulation of epithelial barriers and cell polarity. Integrity and remodeling of epithelial junctions depend on their interactions with underlying actomyosin cytoskeleton. Anillin is a multifunctional scaffold able to interact with different cytoskeletal proteins including F-actin and Myosin II. This project aimed to investigate roles of anillin in regulating epithelial AJs and TJs. Using A549 human lung epithelial and DU145 human prostate epithelial cells, we demonstrated the anillin depletion-induced loss of AJs and TJs. This was accompanied by disorganization of perijunctional actomyosin belt and disruption of the adducin-based membrane skeleton that links actin filaments to the plasma membrane and epithelial junctions. Depletion of anillin decreased protein levels of γ-adducin and downregulation of γ-adducin mimicked effects anillin knockdown on AJ and TJ integrity. These findings suggest a novel role for anillin in the assembly of epithelial junctions.

Smith-Magenis Syndrome (SMS) [OMIM, #182290] is a congenital anomaly and mental retardation (MCA/MR) syndrome associated with deletion of chromosome17p11.2 [1]. The clinical phenotype has been well described and includes minor craniofacial anomalies, self-injurious behaviors as well as sleep disturbances, speech delays, and obesity [1,2,3]. The incidence of SMS is estimated to be ~ 1:15,000 - 25,000 births [2,6]. Among SMS patients, ~90% are comprised of 17p11.2 deletions, while ~10% have RAI1 mutations [8]. All 17p11.2 deletions associated with SMS include RAI1 deletion [10]. RAI1 is thought to function as a transcriptional factor although its cellular role is still unclear. First, in order to better understand the role of RAI1 as a transcriptional factor and its relation to SMS, we confirmed that RAI1 regulates BDNF within an intronic region. This sequence was further narrowed down by utilizing the luciferase reporter assay. This test confirmed what was previously found using ChIP-chip assay and microarray analysis of Rai1+/- mice hypothalami. Next, in order to evaluate the role of Bdnf, an ampakine drug was administered to the Rai1+/- mouse model. A mouse model is a powerful tool for studying a specific gene. Rai1+/- mice exhibit the SMS phenotypes of obesity, craniofacial abnormalities, reduced pain sensitivities, seizures and others. Many physical, neurological, and behavioral tests were performed on the mice to see if any of the phenotypes can be rescued. Interestingly, twice-daily injections of ampakine CX1837 restored the pain sensitivities in Rai1+/- mice. The hot plate data suggest that BDNF potentially has a role in regulating the SMS phenotype of decreased pain sensitivity. In order to evaluate other genes that are altered as a result of the CX1837 ampakine drug, the whole brain's global gene expression was evaluated via microarray analysis. Two potential pain-related genes were identified to be upregulated due to drug administration, which could account for the pain phenotypes observed. One of the genes upregulated in treated mice was Osm, which is interesting because Osm is responsible for pain sensitivity. Further analysis is needed to confirm that an ampakine drug can potentially be used to treat SMS patients.

Smith-Magenis syndrome (SMS) is a complex mental retardation syndrome caused by deletion of 17p11.2 region or mutation of the RAI1 gene (retinoic acid induced 1). Individuals with SMS typically exhibit speech and motor delays, mental retardation, characteristic craniofacial and skeletal anomalies, and a distinct neurobehavioral phenotype that includes sleep disturbances, stereotypes, and maladaptive and self-injurious behaviors. RAI1 is thought to be a transcription factor modulating the expression of genes involved in a variety of cellular functions. Previous studies have shown the RAI1 gene being induced by retinoic acid (RA), a derivative of vitamin A. RA plays a significant role in many processes such as immune function, neurogenesis and reproduction, and deprivation of RA causes craniofacial defects. We hypothesized that RA could be inducing RAI1 which then acts as a transcription factor in modulating the expression of multiple genes. To understand the consequences of clinical variation of RAI1 gene, we performed mutation screening and identified the first case of SMS without mental retardation. Using a zebrafish model, full-length rai1 gene was cloned and spatial and temporal expression of rai1 by in-situ hybridization was evaluated and the effect of RA on rai1 expression was subsequently analyzed. The data show rai1 expression in forebrain (diencephalon) and midbrain. A rai1 antisense morpholino will eventually be created to perform knockdown studies and rescue experiments. These studies will help in determining the significance of the rai1 gene, and its interacting molecular pathways responsible for growth, development, and behavior.