c-Myc is frequently deregulated in human cancers. While deregulated c-Myc leads to tumor growth, it also triggers apoptosis in partnership with tumor suppressors such as ARF and p53. Apoptosis induced by c-Myc is a critical fail-safe mechanism for the cell to protect against unrestrained proliferation. Despite the plethora of information on c-Myc, the molecular mechanism of how c-Myc induces both transformation and apoptosis is unclear. Oncogenic c-Myc can indirectly induce the expression of the tumor suppressor ARF, which leads to apoptosis through the stabilization of p53, but both c-Myc and ARF have apoptotic activities that are independent of p53. In cells without p53, ARF directly binds to c-Myc protein and inhibits c-Myc-induced hyperproliferation and transformation with a concomitant inhibition of canonical c-Myc target gene induction. However, ARF is an essential cofactor for p53-independent c-Myc-induced apoptosis. Here we show that ARF is necessary for c-Myc to drive transcription of a novel noncanonical target gene, Egr1. In contrast, c-Myc induces another family member, Egr2, through a canonical mechanism that is inhibited by ARF. We further demonstrate that Egr1 is essential for p53-independent c-Myc-induced apoptosis, but not ARF-independent c-Myc-induced apoptosis. Therefore, ARF binding switches the inherent activity of c-Myc from a proliferative to apoptotic protein without p53 through a novel noncanonical transcriptional mechanism. These findings also provide evidence that cofactors can differentially regulate specific transcriptional programs of c-Myc leading to different biological outcomes.

Epithelial cells establish apical and basolateral (BL) membranes with distinct protein and lipid compositions. To achieve this spatial asymmetry, the cell utilizes a variety of mechanisms for differential sorting, delivery and retention of cell surface proteins. The EGF receptor (EGFR) and its ligand amphiregulin (AREG) are transmembrane proteins that are delivered to the BL membrane in polarized epithelial cells. Herein, I demonstrate that the cytoplasmic domain of AREG contains dominant BL sorting information capable of redirecting an apical protein to the BL surface. By sequential truncations and site-directed mutagenesis of the AREG cytoplasmic domain, I identify a BL sorting motif consisting of a mono-leucine preceded by an acidic cluster (EExxxL). This sorting motif differs from the other reported mono-leucine BL sorting motif (EEDxxxxxL) in CD147 and stem cell factor. In LLC-PK1 and MDCK cells lacking AP-1B, AREG is detected on the apical surface, demonstrating that steady state BL distribution of AREG is AP-1B-dependent. In LLC-PK1 cells, AREG is transcytosed from the BL surface to the apical surface, suggesting AP-1B plays a role in recycling of AREG from an endosomal compartment to the BL membrane. AREG is present in exosomes in a signaling competent topology and is enriched in exosomes from cells expressing mutant KRAS. Exosomal AREG appears to be post-translationally modified. I provide data supporting the hypothesis that this modification is ubiquitin and may be necessary for efficient delivery of AREG to exosomes. Signaling competent exosomal AREG may act as a novel EGFR signaling platform. Using two methods, a split ubiquitin yeast two-hybrid screen and a crosslinked AREG IP mass spectral analysis, I identified potential AREG interacting proteins. Several of the proteins identified are interesting candidates for future work. Combined, the data presented in this dissertation demonstrates the mode of AREG BL delivery, provides insight into the regulation and effects of exosomal AREG localization, and reveals potential AREG interacting partners.

The <i>C. elegans</i> genome has been completely sequenced, and the developmental anatomy of this model organism is described at single-cell resolution. Here we utilize strategies that exploit this precisely defined architecture to link gene expression to cell type. We obtained RNAs from specific cells and from each developmental stage using tissue-specific promoters to mark cells for isolation by FACS or for mRNA extraction by the mRNA-tagging method. We then generated gene expression profiles of more than 30 different cells and developmental stages using tiling arrays. Machine-learningbased analysis detected transcripts corresponding to established gene models and revealed novel transcriptionally active regions (TARs) in noncoding domains that comprise at least 10% of the total <i>C. elegans</i> genome. Our results show that about 75% of transcripts with detectable expression are differentially expressed among developmental stages and across cell types. Additionally, we used self-organizing maps to define groups of co-regulated transcripts and applied regulatory element analysis to identify known transcription factor and miRNA-binding sites, as well as novel motifs that likely function to control subsets of these genes. By using cell-specific, whole-genome profiling strategies, we have detected a large number of novel transcripts and produced high-resolution gene expression maps that provide a basis for establishing the roles of individual genes in cellular differentiation. In a second project, I have identified an immunoglobulin-domain containing cell adhesion molecule that promotes synaptic-connectivity between the AVA command interneuron and A-class motor neurons in the <i>C. elegans</i> motor circuit. Animals carrying a mutation in <i>rig-3</i> show moderate backward locomotion defects. Additionally, <i>rig-3</i> mutants show minor AVA axon guidance defects and most synapses between AVA and A-class motor neurons are lost. These data suggest <i>rig-3</i> plays a critical role in synapse formation. It will be interesting to determine the involvement of <i>rig-3</i> function in connectivity between other neurons in the motor circuit and the entire nervous system.

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.