<p> Oriental Weatherfish (<i>Misgurnus anguillicaudatus </i>), native to Southeast Asia, are freshwater fish belonging to the family Cobitidae (loaches). They are benthic fish with the ability to breathe atmospheric air, and exhibit an unusual behavior of swimming vertically in the water column in response to changes in barometric pressure. Oriental Weatherfish appear to be successful invaders to North American waterways. Within the last century they have been sighted in freshwater systems throughout the United States, yet the distribution and source of this invasion are largely unknown. This study investigates the distribution patterns, surveying methods, and population genetics of the Oriental Weatherfish within Illinois and Indiana waterways. These data were collected in 2013 and 2014 and supplemented with publically available data to provide the most up-to-date map of the current distribution in Illinois (IL) and Indiana (IN). Catch rates were calculated and compared across gear types and sampling sites to assess different survey methods. Sequence data from cytochrome c oxidase I (COI) and the control region (D-Loop) were compared among specimens collected from multiple sites throughout IL and IN with those available on GenBank. Results from this study suggest a single introduction to IL and IN before 1987, with a subsequent range expansion. This study is the first to genetically examine this weatherfish population. Data suggest that the population is genetically identical to the weatherfish introduced into Australia before 1984, and that these populations were derived from the same native population in Asia.</p><p>

The diversity of animal shapes and sizes, colors and textures, or behaviors and habitats all depend on specialized cells. A newly fertilized embryo must build all these specialized cell types, a process called differentiation. Much of differentiation depends on appropriately turning genes on and off in each cell type. Cell type specific control of gene expression is encoded in a type of regulatory DNA called enhancers. I am interested in how enhancers control the cell type specific gene expression that enables specialized cell functions. Enhancers read in information from regulatory proteins and output a level of gene expression. This conversion from input regulator concentrations to output expression level is a computation. I use quantitative measurement and computational modeling to study how enhancers compute. In embryos, many regulatory proteins bind to enhancers, and some will turn an enhancer on, while others will turn it off. This complex process is greatly simplified by employing computational models. These models can test whether all regulators have been identified (and if not, find the missing ones) and quantify the relationships between regulators. The relationships between regulators reflect the underlying molecular mechanisms used in the cell; when several models can fit the data, perturbation experiments can be used to distinguish the models and underlying mechanisms. However, most computational models of gene expression in animals have not been rigorously validated by perturbation experiments. A major contribution of my thesis work was developing methods for testing models. To test computational models for how enhancers compute gene expression patterns, I experimentally manipulated the concentrations of regulatory proteins and precisely measured output gene expression patterns. Using the Drosophila melanogaster blastoderm embryo, I first developed efficient and scalable techniques for making perturbations to regulatory protein concentrations. This technique revealed a postulated property of development: that embryos mitigate the impact of perturbations by preventing the creation of new cell types. I then used two perturbations to test computational models of an enhancer, finding they were incomplete and discovering new regulatory connections. My work illustrates how computational modeling and quantitative measurement are powerful tools for untangling how regulatory DNA operates in embryos.

Genomic mutations arise in many forms, varying from single base pair substitutions to complicated sets of overlapping copy number variants (CNVs). While each type of variation contributes to phenotype, complex structural variation, which contains multiple mutations, is difficult to type across many individuals and is largely omitted from genomic studies. This thesis presents methods to type complex structural variation, understand how it evolves, and integrate these complex variants into association studies to phenotypes. We focused on four structurally complex regions in the human genome. The 17q21.31 region contains an inversion, previously uncharacterized overlapping copy number variants, and SNPs that associate to the female meiotic recombination rate and female fertility1. The haptoglobin (HP) gene at chromosome 16q22.2 contains a 1.7 kb tandem duplication2, previously uncharacterized paralogous gene conversion, and nearby SNPs that associate to cholesterol levels3. The haptoglobin related gene (HPR) at chromosome 16q22.2, segregates as a multi-allelic copy number variant (mCNV) specifically in African populations. Lastly, complement component 4 (C4) at chromosome 6p21.3, contains a length polymorphism, paralogous sequence variation, and copy number variation segregating in humans and non-human primates4. We developed methods to characterize the complex structural variation in each of these four regions, type the variation at the population level and integrate it into association studies. Briefly, we determined the breakpoints of each individual structural variant, typed each variant in a population cohort, and learned which variants segregate together through trio inheritance patterns. Once these structural haplotypes were defined, we phased them with surrounding SNP haplotypes and used this data as a reference panel for imputation into disease cohorts, and to better understand their evolutionary history. We found that two overlapping duplications in the 17q21.31 region rose rapidly and independently to high frequency within European populations, and may account for the regional association to female fertility and the female meiotic recombination rate. We also found that a recurrent deletion in the HP gene associates to total cholesterol and LDL cholesterol levels. The methods developed in this thesis enable the integration of structurally complex variation into future association studies so that we can begin to understand their effects on phenotypes.

Chronic liver diseases such as non-alcoholic fatty liver disease and alcoholic liver disease are significant health concerns worldwide. Despite knowledge of disease features and environmental causes, we lack understanding of the genetic factors and molecular mechanisms that can be targeted for liver disease therapeutics. Many of these factors are also essential during embryonic development and organogenesis. Here, we use liver development in zebrafish as a model and paradigm for the discovery of regulators impacting liver disease pathogenesis. A chemical screen in zebrafish identified the endocannabinoid (EC) signaling pathway as a regulator of liver development. This pathway was previously implicated in animal models of chronic liver disease, but little was known about its role in development. We generated cannabinoid receptor mutant zebrafish using genome editing and show that EC signaling is required for hepatic maturation and outgrowth, but not earlier milestones such as hepatic specification. Mutant zebrafish also exhibited defects in lipid processing, and we found that methionine metabolism, involving sterol regulatory element binding proteins (SREBPs), is an integral mediator in this process. In a separate study, we used zebrafish to functionally annotate a panel of candidate genes identified by a genome-wide association study (GWAS) for elevated liver plasma enzymes, which are used as a clinical liver disease marker. We prioritized GWAS candidates for morpholino knockdown in zebrafish and discovered effects on hepatic progenitor and hepatocyte development as well as differences in susceptibility to metabolic and toxic injury. Our approach can be applied to other GWAS data sets to rapidly assess additional characteristics during zebrafish development. The work presented here gives valuable insight into how regulators of hepatogenesis also have roles in metabolism and disease. / Medical Sciences

Sexual selection is rampant in Nature, and has produced some of the most beautiful and bizarre traits on Earth. Because females are often promiscuous, sexual selection can continue even after mating, as the sperm of multiple males race to fertilize a limited number of eggs. Though post-copulatory sexual selection is ubiquitous and drives both rapid adaptation and divergence between lineages, we know little about the genetic basis of phenotypes subject to this force. To illuminate one of the important mechanisms by which evolution produces a remarkable diversity of traits, we must identify the genetic loci targeted by post-copulatory sexual selection. Here we determine the genes or genomic regions underlying particular male reproductive traits—sperm development and morphology, age to male sexual maturity, and segregation distortion—that were likely shaped by post-copulatory sexual selection in the ancestors of mice from the genera Peromyscus and Mus. We measure phenotype at the organismal and cellular levels, and then employ quantitative trait locus mapping, RNA sequencing, and bulk DNA sequencing to pinpoint loci influencing the aforementioned traits. We first identify a single locus of large effect controlling sperm midpiece length, a trait relevant to sperm competition success that differs between promiscuous and monogamous sister species of Peromyscus mice. We then show that regions of the genome underlying polymorphism in sperm morphology within species are entirely distinct from those determining divergence in the same traits between species. Next, we determine differences in age to male sexual maturity in closely related species with disparate mating systems, and characterize the role of cis-regulatory evolution in the timing of male reproductive development. Additionally, we develop a novel method to identify segregation distortion systems that may have been shaped by historical selection at multiple levels, but find none in hybrids of Mus musculus. Finally, we characterize the role of a non-coding RNA locus in male fertility, discovering that it mediates separation of spermatids from collective cytoplasm. In sum, we reveal links between particular genes or genomic regions and male reproductive phenotypes that may influence success in post-copulatory competition, thereby clarifying the mechanistic basis of evolution by sexual selection. / Biology, Organismic and Evolutionary

Variation in the shape, size, and number of segments along the vertebral column underlies a vast amount of vertebrate diversity. Although the molecular pathways controlling vertebrate segmentation during normal development are well understood, the genetic and developmental underpinnings responsible for the tremendous variation in size and number of vertebrae are relatively unexplored. The main goal of this dissertation is to explore the genetic and developmental mechanisms influencing naturally occurring variation in the vertebral column. To this end, I focus on intraspecific skeletal variation, with an emphasis on tail length, in the deer mouse, Peromyscus maniculatus. In Chapter 1, I employ a phylogeographic framework to show that longer tails have evolved independently in different populations of forest-dwelling mice. Closer investigation of the underlying morphology shows that long-tailed mice have both (1) a greater number of tail vertebrae and (2) individually longer vertebrae, compared to ancestral short-tailed mice. Chapter 2 explores the genetic basis of tail length variation. I use quantitative trait locus mapping to uncover six loci that influence differences in total tail length (3 associated with vertebral length and 3 with vertebrae number). Finally, in Chapter 3 I combine comparative data from quantitative measurements of tissue dynamics during somitogenesis in fixed embryos and ex vivo explant culture to show that embryos of forest mice make more segments because they produce more presomitic mesoderm, and not because of any significant difference in the timing of somitogenesis. Together, this work integrates phylogeographic, genetic, and developmental studies to pinpoint the ways that natural selection modifies development to produce the repeated evolution of an evolutionarily important trait, and suggests that there are a limited number of ways that long tails can evolve. / Biology, Organismic and Evolutionary

Hundreds of human genes reside in structurally complex loci that elude molecular analysis and assessment in genome-wide association studies (GWAS). One such locus contains the three different amylase genes (AMY2B, AMY2A, and AMY1) responsible for digesting starch into sugar. The copy number of AMY1 is reported to be the genome’s largest influence on obesity, yet has gone undetected in GWAS. Using droplet digital PCR (ddPCR), sequence analysis, and optical mapping, we characterized eight common structural forms of the amylase locus, their mutational histories, and their relationships to SNPs. We found that AMY1 copy number has a unique distribution undetectable to earlier methods that can be understood from an underlying set of structural forms and their allele frequencies. Despite a history of recurrent structural mutations, AMY1 copy number has maintained partial correlations to nearby SNPs; these SNPs do not associate with body mass index (BMI). To directly test for association, we measured amylase gene copy number using ddPCR in 1,000 Estonians selected for being either obese or lean and in two cohorts totaling ~3,500 individuals using sequence analysis. We had 99% power to detect even the lower bound of the reported effects on BMI and obesity, yet found no association. This study model of using multiple methods to analyze the copy number, structural haplotypes, and surrounding SNP haplotypes of multi-allelic variants will likely facilitate more robust disease association results in future studies. / Medical Sciences

The identification of genetic events that cause tumorigenesis is a key goal of cancer research. While recent sequencing efforts have provided unprecedented illumination of cancer genomes, the inherent genomic instability of tumors results in vast numbers of somatic mutations and copy number alterations (CNAs), hampering identification of causative driver events. Functional genetic screens provide a complementary approach to genomics in the search for cancer drivers. While many studies have focused on loss-of-function studies in cancer cells using RNAi technology, we present a platform for gain-of-function screening using sequence-verified human open reading frames (ORFs). We have paired genome-scale ORF collections with unique DNA barcodes of uniform length, facilitating quantitative readout by next generation sequencing. ORFs are expressed from an inducible promoter, allowing precise control of screening conditions. We have used our libraries to identify regulators of proliferation in non-transformed human mammary epithelial cells (HMECs), seeking to uncover early driving events in tumorigenesis. We rediscovered many known oncogenes and observe significant enrichment of our pro-proliferative gene set within regions of recurrent focal amplification in human cancers, indicating the efficacy of our method for cancer gene driver discovery. We have identified a novel family of pro-proliferative genes, the keratin associated proteins, which strongly promote proliferation in HMECs and may contribute to human cancers. Additionally, we report several common sources of error that contribute to noise in pooled genetic screens in mammalian cells. We have observed that library plasmid DNA present in viral supernatants can contaminate screen samples resulting in inaccurate reference measurements of the abundance of library elements. A further artifact of this contamination is a perceived bias towards enrichment of library elements with low GC content. Additionally, libraries containing multiple unique elements carried on a single retroviral genome are subject to recombination during reverse transcription. Our inducible ORF libraries circumvent these problems, allowing for highly accurate, reproducible screen results. As CNAs are considered to be key initiating events in tumorigenesis, and many oncogenes are activated by amplification, we believe that modeling increased gene expression in untransformed human cells will provide critical functional data facilitating the discovery of bona fide cancer drivers. / Medical Sciences

The congenital myopathies are a diverse group of inherited neuromuscular disorders that manifest as skeletal muscle weakness at birth or in infancy, and are classically defined by the predominant morphological features observed on muscle biopsy. The goals of this dissertation were to better understand the pathophysiology behind these devastating diseases and to identify new therapeutic approaches through the use of faithful vertebrate models. Due to their proliferative capacity, transparency, and well-characterized genome, zebrafish represent a robust vertebrate model system to study muscle development. In the first part of this work, we created and characterized a novel zebrafish model of centronuclear myopathy using antisense morpholinos targeting the bridging integrator 1 (bin1) gene. Bin1 morphant skeletal muscles revealed structural defects reported in human biopsies, and live calcium imaging offered new mechanistic insights linking abnormal triads to impairments in intracellular signaling. Later studies focused on two forms of core myopathy, and utilized stable zebrafish models to guide development of targeted and effective therapies. We began by using TALE nucleases to generate germ line mutations in the zebrafish selenoprotein N (sepn1) gene, and in doing so created the first vertebrate to accurately model human SEPN1-related myopathy (SEPN1-RM). Sepn1 zebrafish mutants exhibited morphological abnormalities, reduced contractile strength, and skeletal muscle “cores” under electron microscopy. We then showed that the sepn1 phenotype could be ameliorated by pharmacological inhibition of a thiol oxidase localized at the sarcoplasmic reticulum. These data served as the first in vivo evidence to indicate that reactive oxygen species significantly contribute to SEPN1-RM, and may do so by impairing calcium re-uptake following muscle contraction. Finally, we performed a medium-throughput chemical screen on the closely related relatively relaxed (ryr1b) zebrafish, and identified JAK-STAT cytokine signaling as a druggable molecular pathway relevant to these pathologies. In summary, these studies increase our knowledge of the affected systems in both centronuclear and core myopathies, and provide strong in vivo support that these conditions arise from defects in skeletal muscle excitation-contraction coupling. This work also further establishes zebrafish-based small molecule screens as a powerful tool for lead compound identification and drug development in human genetic disease. / Medical Sciences

Argonautes and their small RNA guides form an ancient partnership with diverse roles in controlling gene expression and preserving genome stability. In the fission yeast Schizosaccharomyces pombe, the Argonaute Ago1 acts within the RITS complex to target the repetitive DNA elements that flank each centromere for heterochromatic silencing, which is necessary for faithful chromosome transmission. A separate Ago1-containing complex, termed ARC, is also required for pericentromeric silencing but its precise function, and the mechanisms that regulate the movement of Ago1 between ARC and RITS, have remained unclear. This dissertation investigates both of these questions. By combining distinct approaches, we have defined the role of ARC as that of enabling Ago1 to be programmed with small RNA guides. In an in vitro assay using immunopurified proteins, we found that loading of synthetic double-stranded small RNAs into Ago1 requires the ARC subunit Arb1 but not the RITS subunit Tas3. In parallel, we isolated cellular Ago1- associated small RNAs and, by high-throughput sequencing, observed that deletion of ARC components produced read features indicative of nonspecifically-interacting small RNAs. Together, these data indicate that the small-RNA-loading capability of Ago1 is conferred by ARC. We also discovered using co-immunoprecipitation that the ARC subunits Arb1 and Arb2 are required for the proper association between RITS subunits Ago1 and Tas3, suggesting that small-RNA loading by ARC might license Ago1 for assembly into RITS. Indeed, we went on to show that Ago1 mutants deficient for small-RNA loading universally fail to interact with Tas3, whereas other non-functional Ago1 variants maintain Tas3 association. We conclude that Tas3 distinguishes between loaded and unloaded Ago1, admitting only the former into RITS. Our studies have delineated the mechanisms that control the programming and assembly of the RITS complex. The results illuminate the role of ARC in heterochromatic silencing and identify this complex as the machinery required for loading small RNAs into Argonaute in S. pombe. Furthermore, we have uncovered small-RNA loading as a checkpoint for the entry of Argonaute into RITS, which may reflect a common discriminatory function of GW-repeat proteins such as Tas3 that precludes the formation of inactive and potentially deleterious complexes. / Medical Sciences