Epithelial cells called enterocytes line the lumen of the small intestine and are responsible for nutrient processing and barrier maintenance. Enterocytes have highly ordered actin arrays, or brush borders, on their apical surfaces. The brush border is composed of microvilli, membrane based protrusions of parallel actin bundles. Within microvilli, myosin-1a laterally links the actin cytoskeleton to the overlying membrane, and contributes to membrane tension regulation and vesicle shedding. These physiological functions require proper localization of this motor, a process that depends on the membrane binding tail homology 1 (TH1) domain. The goal of this thesis is to provide mechanistic details as to how myosin-1a targets to microvillar membrane and how its cellular dynamics are controlled. We find that in vitro and in cells myosin-1a interacts electrostatically with phosphatidylserine through basic residues in two independent bona fide membrane binding motifs. Because membrane binding controls myosin-1a targeting and previously published solution kinetic studies show the motor/actin interaction is short lived, we hypothesized that TH1 is the master regulator of dynamics for this molecule. We used live cell single molecule total internal reflection fluorescence microscopy in combination with single particle tracking and mean squared displacement analysis to measure membrane bound lateral mobility for myosin-1a and TH1. Many myosin-1a molecules display long-lived low mobility dynamics. Similar events are absent from TH1 analysis, indicating the motor domain makes an unexpected contribution to limiting mobility of myosin-1a at the membrane/cytoskeletal interface. Structure/function analysis confirmed this result and revealed the neck region is also important to controlling myosin-1a dynamics. In the context of full length myosin-1a, the neck region also plays a role in regulating localization, perhaps through a conformational change that involves calmodulin/calcium interactions. This is the first study to examine live cell dynamics for any class I myosin at single molecule resolution. The results presented within this thesis provide novel insight as to how myosin-1a cellular targeting and dynamics are controlled, and how biochemical and biophysical properties of myosin-1a manifest in cells to help this molecule carry out physiological roles in the brush border.

The Notch and TGF13 signaling pathways have been shown to play important roles in regulating endothelial-to-mesenchymal transition (EndMT) during cardiac morphogenesis. EndMT is the process by which endocardial cells of the atrioventricular canal and the outflow tract repress endothelial cell phenotype and upregulate mesenchymal cell phenotype. EndMT is initiated by inductive signals emanating from the overlying myocardium and inter-endothelial signals and generate the cells that form the heart valves and atrioventricular septum. The Notch and TGFf3 pathway are thought to act in parallel to modulate endothelial phenotype and promote EndMT. Vascular endothelial (VE) cadherin is a key regulator of cardiac endothelial cell phenotype and must be downregulated during EndMT. Accordingly, VE-cadherin expression remains stabilized in the atrioventricular canal and outflow tract of Notchl-deficient mouse embryos, while activation of the Notch or TGFP pathways results in decreased VE-cadherin expression in endothelial cells. However, the downstream target gene(s) that are involved in regulating endothelial cell phenotype and VE-cadherin expression remain largely unknown. In this thesis the transcriptional repressor Slug is demonstrated to be expressed by the mesenchymal cells and a subset of endocardial cells of the atrioventricular canal and outflowtract during cardiac morphogenesis. Slug is demonstrated to be required for cardiac development through its role in regulating EndMT in the cardiac cushion. Data presented in Chapter 6 further suggests that Slug-deficiency in the mouse is compensated for by a increase in Snail expression after embryonic day (E) 9.5, which restores EndMT in the cardiac cushions. Additionally, the Notch pathway, via CSL, directly binds and regulates expression of the Slug promoter, while a close Slug family member, Snail is regulated by the TGFB pathway in endothelial cells. While Notch does not directly regulate Snail expression, Notch and TGFB act synergistically to regulate Snail expression in endothelial cells. It is further demonstrated that Slug is required for Notch mediated EndMT, binds to and represses the VE-cadherin promoter, and induces a motile phenotype. Collectively the data demonstrate that Notch signaling directly regulates Slug, but not Snail, expression and that the combined expression of Slug and Snail are required for cardiac cushion morphogenesis.

Epithelial-to-Mesenchymal Transformation (EMT) is an important process in development, and occurs during key steps in both valvular and coronary vessel development. This dissertation uses transcriptional profiling strategies to identify novel regulators of EMT during cardiogenesis. An early step in valvulogenesis occurs when endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo EMT to yield valvular interstitial cells. We developed an unbiased strategy to identify genes important in endocardial EMT using a spatial transcriptional profile. RNA-seq analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick (HH18) and mouse (E11.0) embryos at comparable stages of development was performed. EMT occurs in the AVC and OFT cushions but not the VEN at this time. We identified genes >2-fold enriched in chick and mouse cushions compared to VEN. Gene ontology and Gene Regulatory Network (GRN) analysis of cushion-enriched genes were consistent with cells undergoing EMT. Further analysis accurately identified and validated previously unrecognized novel candidate genes (Meis2, Id1, Hapln1, Foxp2) and the NF-κB pathway as regulators of endocardial cell EMT in vitro. Epicardial EMT is a critical step in coronary vessel formation which is dysregulated in mice lacking TGFβR3. To elucidate the role of TGFβR3 in EMT we developed a strategy to identify genes downstream of TGFβR3 in cultured epicardial cells. Tgfbr3+/+ and Tgfbr3-/- immortalized epicardial cells were incubated with vehicle or ligands known to promote TGFβR3-dependent invasion (TGFβ1, TGFβ2, BMP2) and harvested for RNA-seq analysis. GRN analysis of genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells in each ligand incubation group revealed dysregulated NF-ĸB signaling. TGFβ2 or BMP2 incubation stimulated NF-ĸB activity in Tgfbr3+/+ but not Tgfbr3-/- epicardial cells. Inhibiting NF-ĸB signaling reduced TGFβ2- or BMP2-promoted invasion of Tgfbr3+/+ cell, further supporting a role for NF-ĸB signaling in promoting invasion downstream of TGFβR3. The genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFβR3 signaling. These transcriptional profiling strategies identified and validated novel regulators of endocardial and epicardial EMT.

The molecular mechanisms that regulate survival of embryonic neural precursors are still relatively ill-defined. Here, we have asked whether the p53 family member p63 plays any role during this developmental window, focusing upon the embryonic cerebral cortex. We show that genetic knockdown of p63 either in culture or in the embryonic telencephalon causes apoptosis of cortical precursors and newly-born cortical neurons, and that this can be rescued by expression of ΔNp63, but not TAp63 isoforms. This cortical precursor apoptosis is the consequence of deregulated p53 activity, since both basal precursor apoptosis and that induced by loss of p63 are rescued by coincident genetic silencing of p53. Finally, we demonstrate that the third p53 family member, ΔNp73, does not regulate survival of cortical precursor cells, but that it collaborates with ΔNp63 to ensure the survival of newly-born cortical neurons. Thus, the balance of ΔNp63 versus p53 determines the life versus death of embryonic cortical precursors. To assess if p63 plays a similar role in the adult brain, we examined mice haploinsufficient in p63 (p63+/-); we observed that aging, but not young, mice show deficits in short term memory and vertical exploratory behaviour. These results establish a foundation upon which the role of p63 in aging can be further characterized. Given the importance of p73 in aging and in preventing neurodegeneration, we aimed to create a conditional p73 knock out mouse (floxed p73). This mouse will allow for lineage-specific characterization of p73 function and will circumvent the reduced life span displayed by 4 mice lacking p73 (p73-/-). The work presented in this thesis has advanced our knowledge on the role of the p53 family members (p53, p63, and p73) in regulating survival and death in the nervous system. This knowledge will be important to understand neuropathology and to design appropriate therapeutic interventions.

The molecular mechanisms that regulate survival of embryonic neural precursors are still relatively ill-defined. Here, we have asked whether the p53 family member p63 plays any role during this developmental window, focusing upon the embryonic cerebral cortex. We show that genetic knockdown of p63 either in culture or in the embryonic telencephalon causes apoptosis of cortical precursors and newly-born cortical neurons, and that this can be rescued by expression of ΔNp63, but not TAp63 isoforms. This cortical precursor apoptosis is the consequence of deregulated p53 activity, since both basal precursor apoptosis and that induced by loss of p63 are rescued by coincident genetic silencing of p53. Finally, we demonstrate that the third p53 family member, ΔNp73, does not regulate survival of cortical precursor cells, but that it collaborates with ΔNp63 to ensure the survival of newly-born cortical neurons. Thus, the balance of ΔNp63 versus p53 determines the life versus death of embryonic cortical precursors. To assess if p63 plays a similar role in the adult brain, we examined mice haploinsufficient in p63 (p63+/-); we observed that aging, but not young, mice show deficits in short term memory and vertical exploratory behaviour. These results establish a foundation upon which the role of p63 in aging can be further characterized. Given the importance of p73 in aging and in preventing neurodegeneration, we aimed to create a conditional p73 knock out mouse (floxed p73). This mouse will allow for lineage-specific characterization of p73 function and will circumvent the reduced life span displayed by 4 mice lacking p73 (p73-/-). The work presented in this thesis has advanced our knowledge on the role of the p53 family members (p53, p63, and p73) in regulating survival and death in the nervous system. This knowledge will be important to understand neuropathology and to design appropriate therapeutic interventions.

A dense array of parallel actin-based protrusions called microvilli extend from the apical surface of intestinal epithelial cells (IECs), collectively called the brush border. In addition to serving as the sole site of nutrient absorption, this domain also contains host defense proteins. As the brush border is located at the interface between intestinal contents and tissue it must prevent translocation of pathogens and toxins. However, little is known about its role in host defense. Using a combination of cell biological and microscopy techniques, we asked how the brush border responds to microbial infection. Microvilli release vesicles laden with the host defense enzyme intestinal alkaline phosphatase (IAP) into the intestinal lumen. Here, we find that IAP on these lumenal vesicles (LVs) is biochemically active, able to detoxify a bacterial toxin and limit inflammation. Independent of IAP activity, LVs inhibit attachment of enteropathogenic E. coli (EPEC) to IECs. LVs also limit growth of bacteria, while the presence of microbes stimulates increased LV shedding. Thus, LVs represent a multi-faceted form of host intestinal defense. When EPEC do reach the host cell surface, the brush border is dramatically remodeled, resulting in microvillar effacement and formation of actin-rich pedestals beneath the bacteria. Though many molecular aspects of attachment have been characterized, contributions of the brush border to the attachment process have not been investigated. We find that while brush border integrity is critical for limiting bacterial attachment, EPEC can utilize this domain to recruit actin bundles to sites of attachment. Using live cell microscopy, we show that EPEC stimulates flow of the brush border across the cell surface to sites of attachment, as well as directed elongation of microvilli towards bacterial cells. Microvillar actin also appears in nascent pedestals, and pedestal formation is inhibited in cells overexpressing an actin bundling protein. This work suggests a novel mechanism wherein EPEC-stimulated pedestal formation does not occur exclusively by de novo formation of a branched actin network, but also progresses through repurposing of existing microvillar parallel actin bundles.

In many organisms, a cytokinetic ring directs daughter cell separation following mitosis. While conserved molecular participants in this process have been defined, the signaling events controlling cytokinetic ring function remain obscure. Using a genetically-tractable fission yeast, Schizosaccharomyces pombe, I have investigated mechanisms involved in such signaling, with a particular interest in kinase and phosphatase networks. Through identification of a new subunit of the S. pombe chromosomal passenger complex, I have found that Aurora B kinase influences cytokinesis by mediating Cdc14-family phosphatase accumulation at the cytokinetic ring. In addition, I have discovered that Sid2, a kinase of the S. pombe septation initiation network, phosphorylates cytokinetic formin Cdc12 to reverse formin multimerization and allow cytokinetic ring maintenance. My studies also indicate that cytokinesis impacts cell cycle-dependent polarized growth, and that phosphosignaling at the cytokinetic ring ensures robust growth following cell division. These studies advance our understanding of molecular cues regulating cytokinesis, and broaden knowledge concerning the consequences of this control.

My study of three signaling pathways in the context of three gastrointestinal diseases is presented here. Firstly, the epidermal growth factor receptor (EGFR) pathway and its role in the pathogenesis of the hypertrophic gastropathy Ménétrier's disease will be introduced. I will discuss the results of a clinical trial demonstrating that cetuximab, an EGFR pathway inhibitor, is the first effective medical therapy against Ménétrier's disease. I will then introduce the transforming growth factor-β (TGFB) pathway, and its role in the disorder juvenile polyposis syndrome (JPS). I will compare the clinico-pathological presentation of JPS to that of Ménétrier's disease, and suggest that there is a role for aberrant EGFR signaling in the pathogenesis of JPS. Finally, I will discuss my studies of the canonical WNT signaling pathway in the context of colorectal cancer (CRC). This part of the dissertation will center on my findings that the E3 ubiquitin ligase, NEDD4L, is a canonical WNT pathway inhibitor that is downregulated early in CRC, and may serve a tumor-suppressive role in CRC.

Neurodegenerative diseases are characterized by inappropriate death of distinct neuronal populations. Although symptoms vary, these diseases share pathogenic features such as age-dependent onset and progressive death of specific neuronal subtypes. Additionally, dysfunction of mitochondria (essential energy-providing organelles) is widely associated with neurodegenerative disorders. Mitochondria are also key regulators of cell death pathways, further underscoring the need to understand how this organelle contributes to disease pathogenesis. This dissertation characterizes a novel neurodegeneration pathway induced by Coenzyme Q (CoQ) deficiency in C. elegans. CoQ depletion results in an age-related loss in motor coordination and selective degeneration of GABA neurons. My work establishes C. elegans as a model to study CoQ deficiency in the adult nervous system. Age-related onset, selective neuronal vulnerability and mitochondrial dysfunction are features of neurodegenerative diseases that are also prominent in our model of CoQ deficiency. This study also describes aspects of human CoQ deficiency that are conserved in C. elegans. My work supports a model of CoQ transport between tissues. This experimental paradigm has translational implications that could provide a genetic system to study regulators of CoQ uptake. Additionally, I show that localization and function of human CoQ biosynthetic enzymes is conserved in C. elegans. Together, this work emphasizes the strength of using C. elegans as a genetic model to study mechanisms that control CoQ uptake and function. I further investigate the mechanism of GABA neurodegeneration and discover novel roles for canonical apoptotic regulators. In response to CoQ depletion, GABA neurons activate a death pathway that requires CED-4/Apaf-1 but is antagonized by CED-3/caspase. This finding indicates that these components of the core cell death machinery adopt alternative roles in a degeneration pathway arising from CoQ depletion. I also address the role of necrotic proteases in this neurodegeneration pathway. This work revealed that the CoQ depletion-induced death pathway is controlled by proteases with death-promoting or death-preventing functions. Lastly, ultrastructural analysis identified features of necrosis in CoQ-deficient neurons. Together my work provides a foundation for studying neuronal responses to mitochondrial dysfunction.

Ninjurins are a conserved family of transmembrane proteins that increase expression in response to injury and stress. There are few in vivo studies of Ninjurin, and little is known about Ninjurin function. In this thesis I investigated the immune function of a recently generated null mutant of NijA, which did not display a detectable phenotype. Expression studies of the NijA protein show redistribution to the cell surface in larval immune tissues after septic injury. The NijA protein is also upregulated by constitutive activation of the Toll pathway. Ectopic expression of NijA induced cell death. These dying cells appeared to die by nonapoptotic cell death because they did not display hallmarks of apoptotic cells including TUNEL staining and inhibition by p35. Preliminary studies investigating UV-irradiation induced cell death in the NijA null mutant suggests that NijA maybe required to induce stress-activated cell death. These results suggest a role for NijA in stress-activated nonapoptoic cell death.