The Nodal and Nodal-related morphogens are utilized for the specification of distinct cellular identity throughout development by activating discrete target genes in a concentration-dependant manner. Lefty is the principal extracellular antagonist involved in the spatiotemporal regulation of the Nodal morphogen gradient during mesendoderm induction. The Xenopus Lefty proprotein contains a single N-linked glycosylation motif in the mature domain and two potential cleavage sites that would be expected to produce long (XleftyL) and short (XleftyS) ligand isoforms. Here I demonstrate that both isoforms were secreted from Xenopus oocytes, but that XleftyL is the only isoform detected when embryonic tissues were analyzed. In mesoderm induction assays, XleftyL is the functional blocker of Xnr signaling. When secreted from oocytes, vertebrate Lefty molecules were N-linked glycosylated. However, glycan addition was not required to inhibit Xnr signaling and did not influence its movement through the extracellular space. These findings demonstrate that Lefty molecules undergo post-translational modifications and that some of these modifications are required for the Nodal inhibitory function.

Dissertation under the direction of Professor David M. Miller III Impairment of neurons expressing the neurotransmitter ?-amminobutyric acid (GABA) can result in psychiatric diseases as diverse as schizophrenia, epilepsy, Tourettes syndrome, and autism. Degeneration of specific GABA neuron populations in the adult brain results in the symptoms of Huntingtons disease and Spinocerebellar ataxias. In order to better understand these neurons in development and aging, we performed RNAi studies in the nematode C. elegans to identify genes that are important for GABA neurons throughout the life cycle. We identified genes that affect movement and GABA neuron morphology. These RNAi targets included genes with no previously known neuronal function. Future studies of these genes should provide clues to the genetic specification of GABA neuron differentiation and function. During the course of these studies, we found that knockdown of the coq-1 enzyme resulted in the age-dependent degeneration of GABA neurons. coq-1 is the initial enzyme in the Coenzyme Q (CoQ) biosynthetic pathway. CoQ is a required component of the mitochondrial electron transport chain and essential for normal energy metabolism. CoQ deficiency in humans causes cerebellar ataxia, and myopathy, indicating that selected tissues are especially sensitive to reduced levels of CoQ. We found that RNAi or genetic ablation of coq-1 expression in C. elegans resulted in a progressive uncoordinated, or Unc, phenotype and degeneration of GABA neurons. Both the degenerative and Unc phenotypes emerge during late larval development and progress in adults. Neuron classes in motor and sensory circuits that utilize other neurotransmitters (dopamine, acetylcholine, glutamate, serotonin) and body muscle cells were unaffected morphologically by RNAi depletion of coq-1. The mechanism of GABA neuron cell death depends on release of intracellular calcium stores, and requires the apoptotic genes ced-3 (caspase) and ced-4 (Apaf-1). Additionally, degeneration requires drp-1, implicating mitochondrial fission machinery in the cell death pathway. We conclude that the neuron specificity and developmental progression of the coq-1 knockdown phenotype in C. elegans resembles that of CoQ deficiency in humans, and therefore may provide a useful model system for studies of this and related neurodegenerative diseases.

A conserved biological feature of sexual reproduction in animals is that oocytes arrest in meiotic prophase and resume meiosis in response to extra-ovarian signals. While meiotic maturation signals activate highly conserved pathways (e.g. MAP kinase and CDK/cyclin B), the receptor signaling mechanisms involved are less well defined. In C. elegans, sperm trigger meiotic resumption using the Major Sperm Protein (MSP) signal. MSP signaling involves two parallel genetic pathways, defined by vab-1, which encodes an MSP/Eph receptor protein-tyrosine kinase, and ceh-18, which encodes a POU-homeoprotein expressed in gonadal sheath cells. vab-1 and ceh-18 negatively regulate MAP kinase (MAPK) activation, and MSP relieves this inhibition to promote meiotic maturation. MSP directly binds VAB-1 on oocytes, but as vab-1 null mutants respond normally to MSP, signaling must also involve the function of unidentified receptors. ceh-18 functions in the gonadal sheath cells, indicating that sheath¬-oocyte communication may be important in maintaining meiotic arrest. Thus, additional components of the vab-1 and ceh-18 pathways function to negatively regulate MAPK activation and meiotic maturation in the absence of MSP. Using a genome-wide RNAi screen in a female-sterile genetic background, I identified seventeen conserved genes that maintain meiotic arrest in the absence of the MSP signal. Four conserved proteins, including a disabled protein (DAB-1), a vav family GEF (VAV-1), a protein kinase C (PKC-1), and a STAM homolog (PQN-19), function with the VAB-1 Eph/MSP receptor in oocytes. I show that antagonistic Gás and Gáo/i signaling pathways function in the soma to regulate meiotic maturation in parallel to the VAB-1 pathway. Gás activity is necessary and sufficient to promote meiotic maturation, which it does in part by antagonizing inhibitory sheath/oocyte gap-junctional communication. Further evidence shows that MSP signaling reorganizes oocyte microtubules through a signaling network involving antagonistic Gás and Gáo/i pathways and gap-junctional communication with somatic cells of the gonad. I propose that MSP-dependent microtubule reorganization promotes meiotic spindle assembly by facilitating the search and capture of microtubules by meiotic chromatin following NEBD. My findings show that oocyte Eph receptor and somatic cell G protein signaling pathways control meiotic diapause in C. elegans, highlighting contrasts and parallels between MSP signaling in C. elegans and luteinizing hormone signaling in mammals.

Naked family members (Drosophila Naked Cuticle and mammalian Naked1 and Naked2) have been identified as inducible antagonists of canonical Wnt signaling. We previously reported that Naked2, but not Naked1, interacts with the cytoplasmic tail of transforming growth factor Ñ (TGFÑw, thereby coating TGFÑ-containing exocytic vesicles and directing these vesicles to the basolateral corner of polarized epithelial cells. We have designated Naked2 a cargo recognition and targeting (CaRT) protein required for TGFÑ basolateral cell surface delivery. Despite the importance of its role in both Wnt signaling and TGFÑ trafficking, the regulation of Naked2 has not been characterized. Moreover, in vivo roles for Naked2 in these biological processes are largely unknown. To address these deficiencies, I have examined the regulation of Naked2 expression in the context of canonical Wnt signaling and epidermal growth factor receptor (EGFR) signaling, and I have generated a targeted conditional disruption of Naked2 in the mouse.

The maintenance of the intestinal epithelium is dependent upon controlled regulation of cell proliferation and apoptosis, or programmed cell death. Dysregulation of either of these processes can compromise the integrity of the intestinal epithelium leading to the development of inflammatory bowel disease (IBD). Although the cause of IBD remains unknown, increased production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF), exacerbates the inflammatory response contributing to epithelial damage. One known target of TNF signaling is Raf-1 kinase, a serine/threonine kinase that is a key regulator of cell proliferation, differentiation and survival. An intestinal epithelium-specific conditional Raf knockout mouse (Raf KOIE) was generated to address the role of Raf in the intestinal epithelium following inflammation-induced injury. Using this mouse model, we demonstrated that Raf expression protects against dextran sulfate sodium (DSS)-induced colitis by promoting colon epithelial cell survival in response to injury and inflammation. Following DSS treatment of colon epithelial cells, Raf stimulates anti-apoptotic signaling pathways through nuclear factor (NF)-kappa B in a novel MEK-independent manner. Raf has previously been identified as a target of TNF receptor (R) signaling; therefore, studies were conducted to determine the requirement for Raf in colon epithelial cell survival following TNF exposure. Further analysis of TNF signaling pathways showed that Raf promotes NF-kappa B p65 phosphorylation downstream of TNFR1 activation. Similar to our findings in the DSS model, MEK inhibition did not effect anti-apoptotic signaling in response to TNF indicating that Raf-mediated cell survival occurs through a MEK-independent mechanism. In summary, we have demonstrated a protective role for Raf kinase in the colon epithelium following acute colitis or TNF exposure. While we show that Raf promotes anti-apoptotic signaling through NF-kappa B, inhibition of MEK kinase activity has no effect colon epithelial cell survival. These findings indicate a novel mechanism in which Raf contributes to maintenance of the colon epithelium under both non-inflammatory conditions and during acute colitis, which may lead to the development of novel therapeutics for the treatment of IBD.

Dissertation under the direction of Professor Stephen R. Hann<p> c-Myc is a transcription factor whose deregulation has been implicated in numerous cancers. The biological responses resulting from Myc deregulation include hyperproliferation, apoptosis upon serum withdrawal, changes in cellular size, blocking of cellular differentiation, and transformation in conjunction with constitutively activated Ras. c-Myc contains three highly conserved regions within the N-terminal transcriptional transregulatory domain, termed Myc Box I, Myc Box II and Myc Box III. MycS, which is missing the first 100 amino acids, retains several biological functions; however it is unable to transform primary fibroblasts or induce cell cycle entry from quiescence. This suggests that the first 100 amino acids are necessary for some biological functions. It also indicates that specific Myc boxes can each mediate separable and distinct functions through distinct cofactor interactions. I hypothesize that the first 100 amino acids have a role in primary cell transformation and cell cycle progression. Dissection of the transregulatory domain has uncovered a region spanning the first 62 amino acids containing Myc Box I that has the highest transactivation activity. The transactivation activity can be inhibited by interaction with the p19ARF tumor suppressor and be modulated by post-translational modification. When the transactivation domain was fused to the c-Myc C-terminal DNA binding and heterodimerization domain, the resulting protein induced hyperproliferation of immortalized cells, but not Myc-mediated apoptosis. Further assessment of the transactivation activity revealed that a smaller region containing only the first 46 amino acids had substantial transactivation activity. Sequence analysis of this region compared to other Myc proteins uncovered a small sequence of high sequence similarity within the first 46 amino acids, termed MB0. Deletion of this new Myc box demonstrated that this domain is critical for transactivation, cotransformation of primary rat fibroblasts and upregulation of the target genes examined.

Recovery from acute kidney injury (AKI) requires renal tubule cell regeneration. The population of cells that repair the damaged proximal tubule epithelial cells (PTC) has been proposed to be derived from an external population circulating in the blood stream, an adjacent less-injured renal cell population, or a resident self-renewing PTC population. The identification of renal progenitor cells has been difficult because such a population remains phenotypically indistinguishable from their terminally differentiated counterparts and would require creation and characterization of transgenic reporters. For my dissertation research, I investigated the role of NFATc1 in PTCs. A single dose of mercuric chloride (HgCl2) was administered to induce AKI. The data presented here show that NFATc1 plays a role in PTC regeneration following AKI and genetic and/or pharmacologic attenuation of NFATc1 results in increased PTC apoptosis, increased serum creatinine, decreased proliferation, and even death. Using novel NFATc1 transgenic lines that reports activation of an NFATc1 enhancer domain important for autoamplification of NFATc1, we identified a subpopulation of proximal tubule progenitor cells that were resistant to apoptosis following HgCl2 injury. Lineage analysis documented that the NFATc1 labeled PTCs proliferate to repair the damaged proximal tubule segment. The expression profile of this labeled cell population and their labeled progeny was compared to unlabeled PTCs and showed that the labeled population were differentiated proximal tubule cells that had increased transcription of pluripotent stem cell and tubule development markers. To our knowledge, the delayed regeneration after AKI is the first example of a phenotype identified in the Nfatc1+/- mouse and proposes a role for NFATc1 in the regeneration of injured proximal tubule cells by as resident population of progenitor proximal tubules accentuated by NFATc1 expression.

This project aims to better understand the role of Src kinase on the development of colon cancer. Enhanced Src expression and activity are commonly elevated in colon cancer, with progressively higher activity observed as tumors progress and metastasize, however, the specific mechanism by which Src promotes cancer progression are still under investigation. First, we examined the potential for biological cooperativity between APC (adenomatous polyposis coli), a tumor suppressor often mutated in early stages of colon cancer, and elevated Src activity, using conditionally immortalized mouse colon epithelial cell lines, IMCE (APC +/min) and YAMC (APC +/+) as a model. The APC genotype did not affect the ability of Src to disrupt cell-cell junctions and promote cell invasiveness. However, IMCE cells exhibited increased capacity for oncogenic Src-mediated anchorage-independent proliferation as compared to YAMC cells. This property was correlated with enhanced â-catenin expression and activity, but not with enhanced ERK phosphorylation. The selective Src inhibitor, AZD0530, was found to be effective in blocking both cell invasion and proliferation. Second, we investigated the possible role for one of the major Src substrates, CAS (Crk associated substrate) in epithelial cell polarization. Stable depletion of CAS did not affect proliferation on the colon epithelial cell lines Caco2 and HCA7, however it increased thee transepithelial cell resistance of Caco2 cells compared to control cells. Furthermore, fluorescently tagged CAS was shown to localize to cell-cell adhesions in polarized Caco2 cells.

Wnt/beta-catenin signaling controls various cell fates in metazoan development and is misregulated in several cancers and developmental disorders. Binding of a Wnt ligand to its transmembrane coreceptors, Frizzled (Fz) and LRP5/6, inhibits phosphorylation and degradation of the transcriptional coactivator beta-catenin, which then translocates to the nucleus to regulate target gene expression. To understand how Wnt signaling prevents beta-catenin degradation, I focused on the Wnt coreceptor LRP6, which is required for signal transduction and is sufficient to activate Wnt signaling when overexpressed. LRP6 has been proposed to stabilize beta-catenin by stimulating degradation of Axin, a scaffold protein required for beta-catenin degradation. In certain systems, however, Wnt-mediated Axin turnover is not detected until after beta-catenin has been stabilized. Thus, LRP6 may also signal through a mechanism distinct from Axin degradation. To establish a biochemically tractable system to test this hypothesis, I expressed and purified the LRP6 intracellular domain from bacteria and show that it promotes beta-catenin stabilization and Axin degradation in Xenopus egg extract. Using an Axin mutant that does not degrade in response to LRP6, I demonstrate that LRP6 can stabilize beta-catenin in the absence of Axin turnover. Through experiments in egg extract and reconstitution with purified proteins, I identify a mechanism whereby LRP6 stabilizes beta-catenin independently of Axin degradation by directly inhibiting GSK3's phosphorylation of beta-catenin. In addition to studies of LRP6, I explore the role of the other Wnt coreceptor Fz, which has been suggested to be a G protein coupled receptor. Through biochemical studies in Xenopus egg extract, I demonstrate that Galphao, Galphai, Galphaq, and Gbetagamma promote beta-catenin stabilization by inhibiting GSK3s phosphorylation of beta-catenin. Independently of studies on Wnt signaling, I find that two enzymes involved in glycosylation, NAGK and DPAGT1, regulate anteroposterior patterning in Xenopus embryogenesis. I discover that these enzymes involved in N-glycosylation specifically regulate FGF-mediated events in Xenopus development. Because partial loss-of-function mutations in global regulators of N-glycosylation cause a group of human developmental disorders called Congenital Disorders of Glycosylation (CDGs), I suggest the use of Xenopus as a model organism to study the molecular etiology of CDGs.

A conserved biological feature of sexual reproduction in animals is that oocytes arrest in meiotic prophase and resume meiosis in response to extra-ovarian signals. In Caenorhabditis. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation. Sperm release the major sperm protein (MSP) signal to promotes meiotic maturation by antagonizing Eph receptor signaling and counteracting inhibitory inputs from the gonadal sheath cells. I show that MSP promotes oocyte meiotic maturation in part through direct interaction with the VAB-1/Eph receptor. Four conserved proteins, including a disabled protein (DAB-1), a vav family GEF (VAV-1), a protein kinase C (PKC-1), and a STAM homolog (PQN-19), function with VAB-1 in oocytes. We also show that antagonistic Gáo/i and Gás signaling pathways function in the soma to regulate meiotic maturation in parallel to the VAB-1 pathway. Furthermore, I show that in the absence of MSP VAB-1 inhibits meiotic maturation while either in or in transit to the endocytic recycling compartment (ERC). VAB-1::GFP localization to the RAB-11-positive ERC is antagonized by MSP signaling. Two negative regulators of oocyte meiotic maturation, DAB-1/Disabled and RAN-1, interact with the VAB-1 receptor and are required for its accumulation in the ERC in the absence of MSP/sperm. Inactivation of the endosomal recycling regulators rme-1 or rab-11.1 causes a vab-1-dependent reduction in the meiotic maturation rate in the presence of MSP/sperm. In addition, I show that Gás signaling in the gonadal sheath cells, affects VAB-1::GFP trafficking in oocytes. Taken together, our findings show that oocyte Eph receptor and somatic cell G protein signaling pathways control meiotic diapause in C. elegans, highlighting contrasts and parallels between MSP signaling in C. elegans and luteinizing hormone signaling in mammals. Moreover, my finding suggests that regulated endocytic trafficking of the VAB-1/Eph receptor contributes to the control of oocyte meiotic maturation in C. elegans. Eph receptor trafficking in other systems may be influenced by the conserved proteins DAB-1/Disabled and RAN-1 and by cross-talk with G-protein signaling in neighboring cells.