<p> Evolution&mdash;the great tinkerer&mdash;has produced the astounding diversity of form within and between existing species. It is a fundamental goal of evolutionary biology to understand the origin of such diversity. What types of genes underlie evolved changes in morphology? Are certain types of mutations (notably changes within regulatory regions) more likely to be used to produce adaptive changes in form? When distinct populations evolve similar morphological changes, are the underlying genetic bases changes to the same genes, the same genetic pathways, or largely independent? Are changes in form modular, or are their concerted changes to multiple developmentally similar organs? The ever cheapening cost of sequencing, coupled the availability of high-quality reference genomes, allows high-throughput approaches to identifying the loci of evolution. The emergence of a robust genome engineering system, CRISPR/Cas9, allows for efficient and direct testing of a gene's phenotype. Combining both of these techniques with a model system with naturally evolved phenotypic variation, the threespine stickleback, allows for systems-level answers to the many evolutionary questions. </p><p> Chapter one outlines the field of evolutionary developmental biology. It proposes two alternative viewpoints for thinking about the evolution of form. The first is the view of the `Modern Synthesis', linking Mendelian inheritance with Darwinian natural selection, which explains evolution as the change in allele frequencies over time. The second views evolution through the lens of deep homology, focusing on changes to developmental programs over time, even across related organs within the same animal. It then introduces key concepts within evolutionary and developmental biology, including <i> cis</i>-regulation of gene expression, and gene regulatory networks. It then provides examples of evolution reusing similar gene regulatory networks, including <i>Hox</i> genes, <i>Pax6</i> dependent eye initiation, and ectodermal placode development. Teeth use highly conserved signaling pathways, during both their initiation and replacement. Threespine sticklebacks <i>Gasterosteus aculeatus</i> have repeatedly adapted following a shift from marine to freshwater environments, with many independently derived populations sharing common morphological traits, including a gain in tooth number. The following chapters investigate this gain in tooth number in multiple distinct populations of sticklebacks. </p><p> Chapter two describes the discovery and mapping of a spontaneous stickleback albino mutation, named <i>casper</i>. <i>casper</i> is a sex-linked recessive mutation that results in oculocutaneous albinism, defective swim bladders, and blood clotting defects. Bulked segregant mapping of <i> casper</i> mutants revealed a strong genetic signal on chromosome 19, the stickleback X chromosome, proximal to the gene <i>Hps5</i>. <i> casper</i> mutants had a unique insertion of a G in the 6^th exon on <i>Hps5</i>. As mutants in the human orthologue of <i> Hps5</i> resulted in similar albino and blood clotting phenotypes, <i> Hps5</i> is a strong candidate underlying the <i>casper</i> phenotype. Further supporting this model, genome editing of <i>Hps5</i> phenocopied <i>casper</i>. Lastly, we show that <i>casper</i> is an excellent tool for visualizing the activity of fluorescent transgenes at late developmental stages due to the near-translucent nature of the mutant animals. </p><p> Chapter three details the fine mapping of a quantitative trail locus (QTL) on chromosome 21 controlling increases in tooth number in a Canadian freshwater stickleback population. Recombinant mapping reduced the QTL-containing region to an 884kb window. Repeated QTL mapping experiments showed the presence of this QTL on multiple, but not all, wild derived chromosomes from the Canadian population. Comparative genome sequencing revealed the perfect correlation with genetic data of ten variants, spanning 4.4kb, all within the 4<i> th</i> intron of the gene <i>Bmp6</i>. Transgenic analysis of this intronic region uncovered its role as a robust tooth enhancer. TALEN induced mutations in <i>Bmp6</i> revealed required roles for the gene in stickleback tooth development. Finally, comparative RNA-seq between <i> Bmp6</i> wild-type and mutant dental tissue showed a loss of mouse hair stem cell genes in <i>Bmp6</i> mutant fish teeth, suggesting deep homology of the regeneration of these two organs. </p><p> Chapter four investigates the evolved changes in gene expression that accompany evolved increases in tooth number in two distinct freshwater populations. Independently derived stickleback populations from California and Canada have both evolved increases in tooth number, and previous work suggested that these populations used distinct genetic changes during their shared morphological changes. RNA-seq analysis of dental tissue from both freshwater populations compared to marine revealed a gain in critical regulators of tooth development in both freshwater populations. These evolved changes in gene expression can be partitioned in <i>cis</i> changes (mutations within regulatory elements of a gene) and trans changes (changes to the overall regulatory environment) using phased RNA-seq data from marine-freshwater F1 hybrids. Many genes show evidence for stabilizing selection of expression levels, with <i>cis </i> and <i>trans</i> changes in opposing directions (Abstract shortened by ProQuest.). </p><p>

<p> Arteriogenesis, the expansion of collateral arteries, is vital process for compensatory blood flow to tissues in response to vascular occlusions. In patients with peripheral arterial disease (PAD), arteriogenesis is crucial for overcoming limb ischemia, but for many, further treatment is required. In the United States alone, approximately 80,000 individuals lose limbs to this disease each year. Thus, it is critical to understand the mechanisms regulating arteriogenesis. Indirect evidence suggests that mitogen-activated protein kinase kinases 1 and 2 (MEK1 and 2) are involved in arteriogenesis, but this has not been directly tested. To do this, we used a mouse model of hindlimb ischemia, femoral artery ligation (FAL), treated with a specific allosteric MEK1 and 2 inhibitor (PD0325901). Whereas control mice showed increased myogenesis, angiogenesis, and arteriogenesis, mice dosed daily with PD0325901 failed to recover. In order to examine for any temporal regulation, mice were treated days 8 to 28 post surgery. Interestingly, mean arterial luminal area increased. In a complimentary experiment, mice were treated out to 7 days post surgery, treatment was stopped, and tissues were collected at day 28 post surgery. Although muscle tissue had recovered by this time, mean arterial luminal area remained low relative to controls, suggesting a critical window of MEK1 and 2 signaling being necessary for recovery. Surprisingly, results from MEK2^{&ndash;/&ndash;} mice also fail to undergo arteriogenesis after surgery, indicating this effect may be specific to MEK2 alone. BrDU-injected mice co-stained with either CD31 or &alpha;SMA show that loss of MEK2 predominantly affects endothelial cells within the arteries. Lastly, equivalent results are shown in mice lacking tumor endothelial marker 8 (TEM8). These results indicate MEK2 activity is required for arteriogenesis, and show the first known physiological role for TEM8. In addition, the results have implications in the current use of MEK1 and 2 inhibitors for anti-cancer therapy, as these drugs may affect remodeling arteries. Our results also have potential implications for future therapies for PAD, as MEK2 activation after blockage could stimulate arterial growth, preventing the need for amputation.</p><p>

Gene therapy is a promising approach for the treatment of Duchenne Muscular Dystrophy (DMD). Adenoviral vectors (Ad) are the most commonly used vectors in gene therapy studies however, the overall large size of the Ad particles (162nm), due in part to the fiber proteins that extrude from the surface of the virion, prevent their efficient distribution in muscle. The objective of this project was to evaluate the transduction performance of Ad5 based vectors genetically modified to encode shorter fiber proteins derived from Ad serotypes 35 and 9. Optimal transduction was dependent on fiber length in some cell lines and in mdx muscle. However, fiber-modified viruses have an improved viral dispersion and improved transduction up to 10-fold in normal muscle. In addition, an optimized non-immunogenic reporter gene ideal for monitoring vector function in murine disease models was presented. The results of these experiments will contribute to the understanding of Ad transduction in muscle and aid in the design of efficient vectors for DMD therapy.

<p> Many adult stem cells are characterized by prolonged quiescence, promoted by cues from their niche. Upon tissue damage, a coordinated transition to the activated state is necessary for successful repair. Non-physiological breaks in quiescence often lead to stem cell depletion and impaired tissue restoration. Here, I identify cadherin-mediated adhesion and signaling between muscle stem cells (satellite cells; SCs) and their myofiber niche as a mechanism that orchestrates the quiescence-to-activation transition. Conditional removal of N-cadherin and M-cadherin in mice leads to a break in SC quiescence with long-term expansion of a regeneration-proficient SC pool. These SCs have an incomplete disruption of the myofiber-SC adhesive junction, and maintain niche residence and cell polarity, yet show properties of SCs in a state of transition from quiescence towards full activation. Among these properties is nuclear localization of b- catenin, which is necessary for this phenotype. These findings are consistent with the conclusion that injury-induced perturbation of niche adhesive junctions is a first step in the quiescence-to-activation transition. </p><p>

<p> Anal squamous cell carcinoma (SCC) which is strongly associated with human papilloma virus (HPV) infection is a rare cancer but its incidence is increasing throughout the world. Even though it represents just 0.4% of all new cancer cases in the US, the mortality rate is estimated at 14%, which is comparable to both breast and prostate cancer mortality rates. To decrease the high rate of mortality and morbidity of anal cancer there is an enormous need for early detection and prevention strategies. Besides understanding the role of HPV infection, we also need to comprehend the basics of genetics and epigenetics involved in anal cancer progression. With both the highest incidence rate and a lower survival rate among African-American men, we are interested in understanding the relationship of HPV, miRNAs and somatic mutations associated with the African-American population in anal cancers. This was accomplished by (1) identifying and determining HPV genotypes associated with anal condylomas, pre-malignant/dysplastic lesions and malignant anal SCC through type specific genotyping, (2) profiling miRNAs in anal SCC based on gender and type of HPV infection to identify novel biomarkers using Nanostring technology, and (3) by identifying oncogenic mutations associated with anal lesions, transformation and progression using novel next generation sequencing methods. Common HPV genotypes associated with our samples included HPV-11, 16, 6, 32, 35, 51, 58, 59, and 68, of which HPV-32, 51, 59 and 68 are not protected by the current FDA approved nonavalent vaccine. Furthermore, 10 of 800 known human miRNAs were significantly dysregulated in SCC samples; these miRNAs (miR-451a, miR-1185-13p, miR-637, miR-4525a-5p, miR-1275, miR-1303, miR-600, miR-892b, miR-297 and miR-944) target tumor suppressor and oncogenes and potentially play an oncomir role in cancer progression. <i>TP53, PIK3CA, PDGFRA, HRAS</i>, and <i> RET</i> were some of the most frequently found somatic mutations in the sample set and it was observed that the accumulation of mutations begin at the condyloma stage. In conclusion, it was determined that three key factors determine the possible progression of anal cancer and can therefore aid in future development of novel targeted therapy approaches: type of HPV infection, epigenetic factors involving miRNAs, and genetic factors such as &lsquo;driver&rsquo; somatic mutations that an individual accumulates over their lifetime.</p><p>

<p> Crohn&rsquo;s Disease (CD) is a type of Inflammatory Bowel Disease (IBD) with increasing incidence worldwide. In the US alone, prevalence of CD is shown to be 214 per 100,000. CD is not fatal, but deeply impacts the quality of life of an individual. While existing therapies alleviate the symptoms of the disease, there is no cure yet. Even with proper treatment, as many as two-thirds to three-quarters of people with CD will require surgery, which involves high risk of compromised bowel function and malnutrition. Therefore, there is a great need for the development of novel therapies for treatment and prevention of the disease. A better understanding of how CD-associated genetic mutations impact biological pathways involved in disease pathogenesis could offer insights into the development of new therapeutic strategies. </p><p> An autophagy gene associated with CD is Leucine-rich repeat kinase 2 (<i>LRRK2</i>). LRRK2 is a multidomain protein with numerous suggested biological functions including vesicular traffic, immune response regulation and autophagy. While <i>LRRK2</i>&rsquo;s involvement in Parkinson&rsquo;s disease (PD) development has been investigated for over a decade, only recently have studies detected associations between <i>LRRK2</i> mutations and susceptibility to CD. Recently, our group identified novel association signals at <i>LRRK2</i> conferring CD risk, N2081D; or protection, N551K, tagging the R1398H-associated haplotype. Interestingly, the <i> LRRK2</i> N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. </p><p> The main goal of my thesis was to elucidate the functional consequences of these novel CD-associated <i>LRRK2</i> mutations, N2081D and N551K+R1398H, on the autophagy process. To study autophagy and its role in regulating inflammation, the major component of CD, we utilized macrophages, white blood cells, important in the process of phagocytosis that highly express <i> LRRK2</i>. Through our study of human-derived macrophages, we found that carriers of the <i>LRRK2</i> N2081D mutation exhibited impaired autophagy when compared to N551K+R1398H carriers and non-carriers. We also found that impaired autophagy in N2081D carriers was associated with increased total LRRK2 protein levels and defective lysosomal pH and tubulin acetylation regulation. Additionally, we studied autophagy in macrophages from wildtype (WT) and <i>Lrrk2</i> knockout (KO) mice and found that the regulation of lysosomal pH in KO macrophages was significantly affected, further suggesting that <i>LRRK2</i> may play a role in autophagy through regulation of lysosomal pH. Another experimental goal was to determine if knocking out <i>Lrrk2</i> would result in changes to the microbiota in a mouse model with induced colitis and without. Specifically, we explored <i> Lrrk2</i>&rsquo;s role in association with the microbiota and found that <i> Lrrk2</i> participates in regulating microbial taxa under inflammatory circumstances. </p><p> In summary, our findings suggest that CD&ndash;associated mutations in the <i>LRRK2</i> gene conferring risk (N2081D) or protection (N551K+R1398H) affect gene expression, lysosomal acidity and tubulin acetylation in monocyte-derived macrophages (MDM) implicating them in the process of autophagy. We also propose <i> Lrrk2</i> as a modulator of microbiota changes under inflammation. These results should warrant future studies to explore <i>LRRK2</i> as a potential target for new therapies to treat CD.</p><p>

<p> Skeletal muscle generates contractile forces that allow the body to execute movements for walking, speaking and breathing. Although we understand a great deal about the steps of muscle formation, the mechanisms that control muscle size are poorly understood. Even less is known about how muscles interact with skeletal elements, including connective tissue, tendon and bone. This dissertation describes a novel role for Abelson tyrosine-protein kinase 2, a non-receptor tyrosine kinase, during muscle development. First, I characterize the defects in skeletal muscle of <i>abl2</i> mutant mice and show that muscle fibers in the diaphragm and other muscles are extraordinarily long in <i>abl2</i> mutant mice. As a consequence of expansion of the diaphragm muscle, the central tendon of the diaphragm is proportionally reduced in size. Second, I demonstrate that <i>abl2</i> controls muscle size by regulating myoblast proliferation. Third, I show that Abl2 acts in myoblasts to attenuate their proliferation, thereby limiting myoblast fusion and muscle fiber size. Fourth, I show that the exercise endurance of <i> abl2</i> mutant mice is diminished, likely due to the compensatory reduction in size of the diaphragm central tendon. Finally, I provide evidence for signaling between muscle cells and tendon cells that induces tendon cell differentiation. </p><p>

Fluorescence in situ hybridization (FISH) is a powerful assay that can visualize the position of DNA and RNA molecules in individual cells. Here, I describe the development of a method that utilizes complex oligonucleotide (oligo) libraries as a renewable source of FISH probes, which we term ‘Oligopaints’. Our novel FISH platform includes a reliable and robust protocol for the bulk production of fluorescently labeled, strand-specific, single-stranded DNA (ssDNA) probe sets and a bioinformatic pipeline able to identify optimal target sequences for in situ hybridization on a genome-wide scale. A key advantage of Oligopaints is that it permits the researcher to precisely define the genomic sequence contained within each probe molecule, specify the placement of fluorophores, and engineer ssDNA overhangs to which activities can be targeted. We harness this control to make two significant technological advances in FISH- based imaging. In one, Oligopaint probes are programmed to carry 5’ ssDNA overhangs that enable stochastic super-resolution microscopy via two methodologies, STORM and DNA- PAINT. We have used these probes to produce <25 nm resolution images of developmentally regulated chromatin in Drosophila and mouse, which are to our knowledge the first images at this resolution of single-copy chromosomal regions produced by FISH. In the second, we utilize single nucleotide polymorphism (SNP) data to generate FISH probes that can for the first time visually distinguish single-copy regions of the maternal and paternal homologous chromosomes, thus allowing the examination of parent-of-origin dependent effects on chromosome positioning and gene expression in individual cells.

Mycobacterium tuberculosis (Mtb) mutants lacking the operon Rv1411c-1410c encoding a lipoprotein, Rv1411c (LprG) and a putative transporter, Rv1410c (Rv1410) are dramatically attenuated for growth in mice. Previous work in our lab, using the model organism Mycobacterium smegmatis, suggested that this operon regulated the lipid content of the cell wall. Work in other laboratories characterizing LprG as a lipid-binding lipoprotein lead us to hypothesize that these bacteria grew poorly due to loss of a key lipid important in the host-pathogen interaction. Based on structural and biochemical studies we hypothesized that this attenuation was due to a lipid transport defect. Using whole cell lipidomic analysis, we found changes in LprG-1410 mutants including accumulation of triacylglyceride (TAG) species in the absence of the transport system. We have identified TAG in outer membrane fractions and supernatants of Mtb, have demonstrated the ability of LprG to transport TAG in an in vitro vesicle transfer assay, and have co-crystallized LprG with TAG. Moreover, accumulation of intracellular TAG substantially decreases growth under carbon stress in vitro and in vivo in the mouse model. Our results suggest a far different model – that TAG is ordinarily transported out of the cell and, in the absence of a transporter, limits cell proliferation independent of the host immune response. This suggests that TAG is a key metabolic regulator of cellular growth within the host.

How the size of an organ is established and altered during evolution is poorly understood. The ovary of fruit flies of the genus Drosophila serves as an interesting model for understanding organ size evolution, as the number of egg-producing structures called ovarioles determines the ovary’s functional ‘size’. Species with more ovarioles can lay more eggs, and ovariole number can evolve rapidly between closely related species. However, the developmental and genetic mechanisms that determine and alter ovariole number were poorly characterized at the beginning of this thesis. I first analyzed the developmental basis of plasticity and species-specific ovariole number changes in D. melanogaster and closely related species. This analysis revealed distinct developmental mechanisms that alter ovariole number via changes in one cell type (terminal filament cells) in the developing ovary. To characterize the genetic mechanisms underlying proliferation patterns and potential cell-type interactions within the ovary, I then studied the role of the Hippo pathway in the somatic and germ cells of D. melanogaster. I uncovered a complex interaction between somatic cells and germ line cells, where proportional growth of these cell types is maintained by the Hippo pathway via interactions with the EGFR and JAK/STAT pathways. Finally, I expanded this work to investigate the physical, ecological, and developmental parameters that influence ovariole number evolution in Hawaiian Drosophila, where previous studies suggested that ovariole number correlated with larval food substrate. I describe my ongoing efforts to test correlations of ecology and ovariole number in a phylogenetic context in Hawaiian Drosophila. Primary differences in ovariole number between species occur through changes in cell number. / Biology, Organismic and Evolutionary