Disruption of intestinal epithelial homeostasis, including enhanced apoptosis, is a hallmark of inflammatory bowel disease (IBD). We have shown that tumor necrosis factor (TNF) increases the kinase activity of ErbB4, a member of the epidermal growth factor receptor family that is elevated in mucosa of IBD patients and which promotes colon epithelial cell survival. In this study, we tested the hypothesis that TNF transactivates ErbB4 through TNF-alpha converting enzyme (TACE)-mediated ligand release, and that this transactivation is necessary to protect colonic epithelial cells from cytokine-induced apoptosis. Using neutralizing antibodies, we show that HB-EGF is required for ErbB4 phosphorylation in response to TNF. Pharmacological or genetic inhibition of the metalloprotease TACE, which mediates HB-EGF release from cells, blocked TNF-induced ErbB4 activation. MEK, but not Src or p38, was also required for transactivation. TACE activity and ligand binding were required for ErbB4-mediated anti-apoptotic signaling; while mouse colon epithelial cells expressing ErbB4 were resistant to TNF-induced apoptosis, TACE inhibition or blockade of ErbB4 ligand binding reversed the survival advantage. We conclude that TNF transactivates ErbB4 through TACE-dependent HB-EGF release, thus protecting colon epithelial cells from cytokine-induced apoptosis. These findings have important implications for understanding how ErbB4 protects the colon from apoptosis-induced tissue injury in inflammatory conditions such as IBD.

In a screen for cell-cycle regulators, we identified a Drosophila maternal effect-lethal mutant that we named no poles (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. We showed that mutation of a DNA checkpoint kinase, Chk2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint contributes significantly to the nopo phenotype. Chk2-mediated mitotic arrest has been previously shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to completion of DNA replication. NOPO is the Drosophila homolog of mammalian TRAF-interacting protein (TRIP). NOPO and TRIP contain highly similar RING domains that closely resemble that of known E3 ubiquitin ligases. We showed that Bendless (BEN), an E2 ubiquitin conjugating enzyme, interacts with NOPO; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitylation complex consisting of BEN/UEV1A and NOPO is required for preservation of genomic integrity during early embryogenesis. We sought to elucidate the mechanism by which NOPO/TRIP promotes genomic stability by performing a yeast two-hybrid screen to identify NOPO/TRIP interactors. We identified a family of non-canonical DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. Furthermore, we have shown that NOPO interacts with Drosophila Y polymerases and we observe an enhanced ubiquitylation of the Y-family polymerases by TRIP and NOPO E3 ligases. To determine if Y polymerases have a role in Drosophila early embryogenesis, we generated a null mutation in DNApol-eta and observe decreased hatch rates and nopo-like spindle defects in DNApol-eta-derived embryos. Mutation of the human homolog, POLH, results in a variant form of Xeroderma Pigmentosum, a disease characterized by UV sensitivity and skin cancer. We hypothesize that DNApol-eta has a unique role during Drosophila early embryogenesis to promote cell-cycle progression and that NOPO regulates its activity.

Evaluation of late events in cardiovascular development is precluded mid-gestational embryonic lethality associated with most traditional endothelial specific gene knockouts. Thus, it has not been possible to study late gestational events in cardiovascular development using traditional methods. This study utilizes a conditional Tie1 floxed allele in conjunction with the NFAT-c1 P2 Cre specific for the pro-valvular endocardium thus allowing us to bypass the requirement for Tie1 in the developing vasculature. We provide evidence that Tie1 plays a context dependant role in the developing valve. Unlike its role in the vasculature, Tie1 is not required for endothelial cell survival/quiescence in the valve leaflet, but rather as a environmental sensor which relays information from the environment to the valvular interstitial cells. Deletion of Tie1 in the developing valve endocardium leads to an expansion of aortic valve size, as well as perturbations in ECM production and stratification, which is likely due to a miscommunication between the endothelial cells of the valve and the underlying valvular interstitial cells. In addition to being larger in size, aortic valve leaflets lacking Tie1 expression are much more pliable than wild type valves leading to aortic insufficiency and demise in these animals. We are the first to show that Tie1 plays a definitive role in valve remodeling and that Tie1 expression is essential for proper ECM stratification within the leaflet.

Class I myosins are monomeric actin-binding, ATP hydrolyzing molecular motors that are expressed in a variety of cell types, and function at the membrane-actin interface. Myosin-1d, one of eight vertebrate class I myosins, is expressed in polarized cells of the small intestine and nervous system, but subcellular localization and function for the motor remains largely unexplored. Intriguingly, myosin-1d is coexpressed in epithelial cells of the small intestine with myosin-a, where both motors target to the well-defined apical actin array of the brush border. However, how similar class I motors compartmentalize subcellularly is unknown, and raises the question of functional overlap. Interestingly, we found that myosin-1d and myosin1a exhibit differential localization and this partitioning can be explained by differential dynamics. Moreover, myosin-1d redistributes along the microvillar actin bundle in the absence of myosin-1a in MYO1A knockout animals. This suggests that class I myosins do have unique functions in wildtype, but may compensate for loss of activity. Interestingly, our data demonstrates that myosin-1d has a different subcellular localization in the nervous system. In these polarized cells, myosin-1d exhibits a punctate distribution in neuronal dendrites, cell bodies, and axons. We observed prominent expression in Purkinje cells and a subset of granule cells, with both patterns developmentally regulated. However, myosin-1d was not detectable in oligodendrocytes during early development. In the PNS, we observed that myosin-1d is present in neurons, and myelinating Schwann cells. This suggests a differential role for the motor in myelinating cells between the two nervous systems. Our studies also revealed that myosin-1d interacts with aspartoacylase, a catalytic enzyme involved in fatty acid synthesis that is widely expressed in similar polarized cells as myosin-1d. Together, these studies suggest that myosin-1d has distinct localization patterns in different polarized cell types, but may modulate aspartoacylase activity.

Diabetes mellitus affects approximately 150 million people worldwide. This disease is characterized by hyperglycemia resulting from dysfunctional pancreatic beta cells. Current treatments for diabetics are inadequate because they often do not prevent complications associated with the disease; therefore, considerable efforts are focused on derivation of beta cells from embryonic stem cells. Accomplishing this requires a precise understanding of beta cell development and the molecular control of beta cell expansion in vivo. We addressed these approaches in two ways: first, we analyzed the requirement for neural crest (NC) derivatives in regulating beta cell maturation and second, we determined that the transcription factor Foxd3 is required for beta cell mass expansion during pregnancy. The pancreas develops through a coordinated system of signals from both the endoderm and surrounding mesoderm. Little effort has been devoted to analyzing the role of ectodermally-derived NC that innervates the pancreas during embryogenesis. Our work illustrated that NC enters the pancreatic primordium around 10.25 dpc, shortly after pancreatic evagination from the foregut epithelium. Using a genetic ablation of NC derivatives in the pancreas, we showed, in agreement with published data, increased beta cell proliferation and insulin-positive area. Additionally, our work illustrated a novel requirement for this lineage; NC derivatives are required for beta cell maturation. Beta cell proliferation in adult mice is rare unless the mice are metabolically challenged, such as during pregnancy. Therefore, I chose to analyze the requirement for Foxd3 during pregnancy. Foxd3 is expressed in the pancreatic primordium beginning at 10.5 dpc and is localized predominantly to beta cells after birth. Virgin mice carrying a pancreas-specific deletion of Foxd3 are euglycemic; however, during pregnancy these mice become glucose intolerant. Several genes required for cell proliferation are misregulated in the absence of Foxd3 resulting in decreased beta cell proliferation and beta cell mass during pregnancy. Together, my thesis research illustrated the requirement for NC derivatives in controlling beta cell maturation and demonstrated a novel role for Foxd3 in beta cell mass expansion during pregnancy. The findings from both studies can be applied to cell-based therapies to treat diabetics.

Information flow in nervous systems depends on the asymmetric organization of neurons; neurotransmitters are released from presynaptic domains and stimulate receptors localized to postsynaptic regions. These specialized signaling domains can be reorganized within neurons during development or in response to injury. Although transcription factors are known to regulate synaptic plasticity, downstream genes that contribute to remodeling are largely undefined. To identify these factors, we have studied an example of synaptic remodeling in the nematode Caenorhabditis elegans. In this case, GABAergic Dorsal D (DD) motor neuron synapses are relocated to new sites during larval development. This remodeling program is blocked in Ventral D (VD) GABAergic motor neurons by the COUP nuclear hormone receptor, UNC-55. We exploited this UNC-55 function to identify downstream remodeling genes that encode a diverse array of protein types including ion channels, cytoskeletal components, and transcription factors. <p> We show that one of these targets, the Iroquois-like homeodomain protein, IRX-1, functions as a key regulator of synaptic reorganization. IRX-1 is required for remodeling both wild-type DD and unc-55 mutant VD motor neurons, and ectopic IRX-1 expression in VD motor neurons is sufficient to induce remodeling. Our discovery of IRX-1 as an UNC-55-regulated target defines a transcriptional pathway that orchestrates an intricate synaptic remodeling program. The established roles of these conserved transcription factors in mammalian neural development suggest that a similar cascade may also control synaptic plasticity in more complex nervous systems. <p> Our study of UNC-55-regulated genes also revealed a role for UNC-8, a degenerin-family acid-sensing ion channel, in synaptic remodeling. Expression of UNC-8 is necessary for execution of the UNC-55-regulated synaptic remodeling program in VD motor neurons. Furthermore, UNC-8 promotes the disassembly of immature ventral synapses during DD remodeling. The potential that UNC-8 channel activity locally destabilizes synapses led us to investigate whether the GABAergic synaptic remodeling program is activity-dependent. We show that both calcium influx and synaptic activity drive remodeling of GABAergic synapses. Thus, we have defined a genetic program that employs both transcriptional and local activity-dependent factors to promote re-organization of synaptic patterning during development.

Epicardial development is a complex process that involves tightly regulated coordination of concurrent cellular behaviors ranging from sheet migration to secretion. The regulation of these behaviors is poorly understood, and epicardial cell biological studies will improve the understanding of heart development and subsequent function. Here, a novel small organic molecule screening methodology of epicardial behaviors is used to elucidate regulatory relationships governing this developmental program. As proof-of-principle, a novel signaling relationship was identified in which TGFβ and BMP signal cascades cooperatively regulate epicardial sheet migration. It is further demonstrated here that epicardial cells participate in a newly identified cellular behavior: regulated cell movement through autocrine extracellular matrix (ECM) deposition. The studies identify two novel regulators of autocrine ECM deposition, Bves and NDRG4. Additionally, we demonstrate that Bves regulates cell surface trafficking of the focal adhesion component β1-integrin through an interaction with VAMP3, a v-SNARE recycling endosome component. These studies in combination with previous work indicate that Bves functions in epicardial cells and globally to traffic adhesion components to the cell surface. This positions Bves as a general regulator of cell-cell and cell-matrix adhesion. Taken together, these studies elucidate regulation of epicardial cell behaviors, reveal a novel epicardial behavior, and suggest a global mechanism for Bves diverse effects of development and disease.

Pathological ocular angiogenesis, or ocular neovascularization (NV) is a central feature of retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and age-related macular degeneration (AMD). In the developed world, these diseases are the leading causes of blindness in infants, working-age individuals, and the elderly, respectively. As of today, there are two FDA-approved angiostatic agents being used to treat conditions characterized by ocular NV. Both angiostatic agents inhibit vascular endothelial growth factor (VEGF), the principle growth factor mediating ocular NV. Although VEGF-centric therapies reduce NV, they do not completely eliminate it. In order to more effectively prevent and/or treat these conditions, a more thorough understanding of the key players involved in the angiogenic cascade is needed. One enzyme that holds promise for therapeutic intervention is cyclooxygenase-2 (COX-2). We examined the involvement of COX-2 and COX-2-derived prostanoids in order to 1) understand their role in ocular angiogenic disease, and 2) develop more specific therapeutic targets for diseases comprised of an angiogenic component. We have shown that non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit the activity of COX, significantly reduced the severity of NV in an animal model or oxygen-induced retinopathy (OIR). This anti-angiogenic effect was likely due to inhibition of VEGF-induced endothelial cell proliferation and tube formation. Furthermore, genetic deletion of COX-2 significantly reduced hypoxia-induced VEGF production by Müller cells. This effect was mediated by one of the five COX-derived prostanoids, PGE2. Additional experiments confirmed the role of PGE2 and, more specifically, the EP4 receptor, in angiogenic Müller cell and endothelial cell behaviors. Importantly, EP4 antagonism significantly reduced VEGF production by hypoxic Müller cells, VEGF-induced proliferation and tube formation in endothelial cells, and the severity of NV in rodent models of OIR and laser-induced choroidal NV (LCNV). Our findings are significant because they demonstrate that the EP4 receptor affects the ocular angiogenic cascade at more than one point, with the potential to be a powerful and effective therapeutic target for angiogenic diseases of the eye and other tissues.

Wound healing, an essential function to the survival of all multicellular organisms, requires the precise orchestration of multiple cell types in order to repair the damaged tissue. Wound healing involves three overlapping phases: inflammation, re-epithelialization, and scar formation. Matrix metalloproteinases (MMPs) are endopeptidases of the metzincin family, which have been shown to function throughout wound healing, but their precise functions and regulatory mechanisms are unclear <em>in vivo</em> largely due to the complications of redundancy. In mammals there are 24 MMPs (seventeen secreted type MMPs and seven membrane-anchored type MMPs). MMPs are inhibited by Tissue Inhibitors of Metalloproteinases (TIMP), of which there are four in mammals. To bypass the complications of redundancy, we utilized <em>Drosophila melanogaster</em> as a model system to study MMPs <em>in vivo</em> during wound healing. <em>Drosophila</em> serve as a simple system to not only study wound healing, but the <em>Drosophila</em> genome encodes only two MMPs, one secreted (<em>Mmp1</em>) and one membrane-anchored (<em>Mmp2</em>), providing a system to elucidate the functions of each MMP class <em>in vivo</em>. Our results indicate that <em>Mmp1</em> and <em>Mmp2</em>, as well as the inhibitor, <em>Timp</em> are required for re-epithelialization, where they may function together to promote cell migration. During re-epithelialization, <em>Mmp1</em>, under the control of the JNK signaling pathway, functions to promote cell migration by facilitating collagen IV remodeling, promoting actin cytoskeleton reorganization, and inducing RTK signaling. <em>Mmp1</em> from the hemocytes may function to limit the area of both JNK and RTK signaling to the immediate vicinity of the wound. Preliminary results suggest that <em>Mmp1</em> and <em>Mmp2</em> may regulate hemostasis and the melanization cascade, as we observe both clotting and melanization defects in <em>Mmp1</em> and <em>Mmp2</em> mutants. In unwounded tissue, both <em>Mmp1</em> and <em>Timp</em> are required for basement membrane maintenance, a function they do not share with <em>Mmp2</em>. The combination of both shared and independent phenotypes between <em>Mmp1</em>, <em>Mmp2</em>, and <em>Timp</em> mutants suggest that Mmp1, Mmp2, and Timp may form a complex to promote wound healing; however, if such a complex does form in vivo it is context-specific.

Canonical Wnt signaling regulates many fundamental developmental processes and is misregulated in a variety of disease states in humans. Ubiquitylation has been shown to play critical roles in the regulation of Wnt signal transduction, and many components of the Wnt pathway are known to be ubiquitylated. At the time I began my thesis work, however, little was known about how the ubiquitin system regulates Wnt signaling. Thus, I designed a RNA interference (RNAi) screen in Drosophila S2 cells to identify novel E3 ligases and deubiquitylases involved in Wg/Wnt signaling and identified XIAP (an E3 ligase) and USP47 (a deubiquitylase) as novel Wnt pathway components. <P>A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, I describe an unanticipated role for X-linked Inhibitor of Apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its anti-apoptotic function. I identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen and show that XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. I also demonstrate that upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it mono-ubiquitylates Groucho/TLE: this modification decreases the affinity of Groucho/TLE for TCF/Lef. These data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Groucho/TLE that facilitates its removal from TCF/Lef, thus allowing assembly of &#946-catenin-TCF/Lef complexes and initiation of a Wnt-specific transcriptional program.</P> <P>In addition to the discovery of XIAP as a novel Wnt pathway component, I also identified the de-ubiquitylase Ubp64E as a positive regulator of Wingless signaling in the Drosophila S2 cell-based RNAi screen. USP47, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. My data indicate that USP47 likely functions at the level of transcription in the nucleus potentially through its interaction with the E3 ligases &#946-TRCP or XIAP or an as yet unidentified target.</P>