The work contained in this document describes the generation of new immunoreagents for Bves study, and our identification of a Bves-interacting protein. After generating new monoclonal antibodies to the Bves protein, I here describe the characterization of Bves expression throughout mouse embryogenesis, clearly demonstrating that Bves is widely expressed in many tissue types throughout gestation. I also describe the isolation of Geft as a directly interacting protein for Bves, and biochemically confirm and characterize the interaction between these two proteins. I then demonstrate a role for Bves in control of cellular motility via modulation of the Rac1/Cdc42 GTPases, likely through the Geft interaction. These findings represent both the first identification of a Bves-interacting protein and the first placement of Bves into a molecular pathway within the cell. The data presented here will undoubtedly serve as a departure point for many future investigations into the Bves protein, and represent seminal findings in the study of Bves function.

Animal movement is controlled by the motor circuit, which features an axial nerve cord where motor neurons transmit signals from the brain to specific muscles. The development of this circuit depends on differential gene expression in the specific cells that contribute to its function. The identification of these genes should lead to a better understanding of the developmental programs that generate each of these essential cell types. To identify the genes that specify this network, I have developed a genomic strategy, MAPCeL (Microarray Profiling <i>C. elegans</i> Cells) and used it to obtain gene expression profiles of the body wall muscle cells and the excitatory cholinergic motor neurons from <i>C. elegans</i>. Bioinformatic analysis and GFP reporters were used to validate MAPCeL profiles obtained from these experiments. In addition, we show that <i>acr-16</i>, a nicotinic acetylcholine receptor gene identified in the MAPCeL profile of body muscle cells, is required for normal locomotion. <p> In a second project, I used MAPCeL to identify genes that regulate synaptic assembly and function in the <i>C. elegans</i> motor circuit. The UNC-4 homeodomain protein is expressed in DA and VA class motor neurons (i.e., A-class motor neurons) where it functions with its transcriptional co-repressor, UNC-37/Groucho to define the specificity of synaptic input and the strength of synaptic output of these motor neurons. In <i>unc-4</i> mutants, VA motor neurons are miswired with inputs normally reserved for their VB sister cells; both DAs and VAs show decreased numbers of synaptic vesicles. We propose that UNC-4 specifies A-motor neuron traits by repressing B-motor neuron genes. Using the MAPCeL approach, I have generated a list of candidate UNC-4 regulated genes and shown that one of these targets, CEH-12/Hb9, functions downstream of UNC-4. <p> In flies and vertebrates, the Hb9 transcription factor is expressed in the developing spinal cord where it specifies motor neuron fate. We show that the <i>C. elegans</i> Hb9 homolog, <i>ceh-12</i>, is normally restricted to VB motor neurons but is also expressed in DAs and VAs in <i>unc-4</i> and <i>unc-37</i> mutants. Ectopic expression of CEH-12 in VAs is sufficient to induce the Unc-4 movement phenotype. Furthermore, <i>ceh-12(0)</i> mutants partially suppress the Unc-4 movement phenotype and fully suppress the synaptic vesicle defect. These data suggest that <i>ceh-12</i> functions downstream of UNC-4 to regulate synaptic input to A-class motor neurons as well as the strength of signaling output to body muscles.

SNAP-25 and syntaxin 4 are SNARE proteins that are involved in membrane transport. In order for proteins to traffic properly through membranous organelles, a series of budding and fusion events must occur between donor and acceptor membranes. Therefore, determining the precise complex of proteins that are responsible for these events within the cell is critical in understanding this fundamental cellular process. In this document, I show that cytLEK1, a relatively large protein that contains numerous leucine zippers, directly binds both SNAP-25 and syntaxin 4. Through this association identified by a yeast two-hybrid screen, the protein complex regulates plasma membrane trafficking. I identified the binding domain within each of the proteins that is responsible for interaction, and performed co-immunoprecipitation and colocalization studies to confirm their association. Further analyses show that VAMP2, also a member of the SNARE complex, in contained within the cytLEK1-SNAP-25-syntaxin 4 complex. Using cytLEK1 dominant negative and knock-down approaches, I show that cytLEK1 functions in two processes that regulate the recycling endosome network: transferrin and GLUT4-trafficking. Previous work has shown that cytLEK1 interacts with the microtubule cytoskeleton. We postulate that cytLEK1 links recycling endosomes with the microtubule network. This is the first report linking these two subcelluar systems. I have also created a conditional Lek1 knock-out mouse line. By utilizing a mouse line that expresses heart specific Cre, I am able to examine Lek1 loss-of-function during heart organogensis. Through my pilot studies, I am able to show that both myocardial wall structure and function are severely altered in conditional Lek1 knock-out mice. My data show that the phenotypes may be due to the inability of cardiomyocytes to traffic proteins properly, therefore altering cell coupling and overall heart function. Taken together, my studies show that cytLEK1 is an integral member of the plasma membrane recycling pathway, and cytLEK1 function is critical in heart development.

The spindle pole body, the yeast analog of the centrosome, serves not only to nucleate and organize microtubules but also as a signaling center to coordinate events in mitosis and cytokinesis. It does so by localizing proteins responsible for chromosome segregation, spindle formation and cytokinesis. The first part of my study focuses on the signaling pathway, SIN (septation initiation network), located at the spindle pole body and is responsible for initiating actomyosin ring constriction, septation and cell division. In an effort to identify novel components or tethers of the in the SIN to the SPB, we performed a TAP (tandem purification analysis) analysis on Cdc11p, an essential SIN scaffolding protein and identified a previously uncharacterized protein, Ppc89. Ppc89 localizes constitutively to the SPB and interacts directly with Sid4. ppc89? cells are inviable and exhibit defects in SPB integrity, and hence in spindle formation, chromosome segregation, and SIN localization. Ppc89 overproduction is lethal, resulting primarily in a G2 arrest accompanied by massive enlargement of the SPB and increased SPB MT nucleation. These results suggest a fundamental role for Ppc89 in organization of the S. pombe SPB. The second part of my studies focused on characterizing the role of phosphorylation on Mto2, a protein that activates the ?-TuC and localizes it to iMTOCs and eMTOCs during interphase. However, it is not known how Mto2 performs this function. Based on previous studies, we hypothesized that Mto2 could possibly be phospho-regulated in a cell-cycle dependent manner. To test this hypothesis, I examined Mto2 throughout the cell cycle and found that Mto2 is hyperphosphorylated during mitosis by Cdk1. Mutation of these sites to nonphosphorylatable alanine residues eliminates the mitotic phosphorylation but does not alter function. We therefore hypothesize that the mitotic phosphorylation inhibits Mto2 from activating the ?-TuC.

The process of implantation absolutely depends on synchronized development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state and a reciprocal dialogue between the blastocyst and the uterus. The uterus is comprised of heterogeneous cell types that respond differentially to ovarian steroid hormones, estrogen and progesterone (P4). P4 is commonly known as the hormone of pregnancy, acting through progesterone receptor (PR) to activate transcription of genes involved in ovulation, uterine receptivity, implantation, decidualization and pregnancy maintenance. However, various aspects of its roles throughout pregnancy are not well understood. Female mice missing Pgr, the gene that encodes PR, are completely infertile with failure of ovulation, fertilization and implantation. This severe phenotype precludes using these null mice to study potential new aspects of P4 function during pregnancy. In contrast, deletion of Fkbp52, a cochaperone for PR, results in uterine-specific P4 resistance, allowing us to address unique aspects of uterine P4-PR signaling during pregnancy. Using Fkbp52 null mice, we first show that while implantation completely fails in these null mice, ovulation, another P4-mediated event, is normal. These results suggest tissue-specific dependence and differential sensitivity of the ovary and uterus to FKBP52-PR mediated P4 action. This study, therefore, provides the first evidence for an in vivo role for FKBP52 in regulating tissue-specific PR and its critical role in uterine receptivity and implantation. We also present evidence that P4-PR-FKBP52 signaling is a function of genetic makeup of mice and is pregnancy stage specific. Collectively, our findings show that FKBP52 deficiency causes uterine P4 resistance during pregnancy, since null females have normal uterine PR and serum P4 levels with reduced PR activity. This work is clinically relevant to genetically diverse populations of women with P4-resistant recurrent pregnancy failure or various gynecological disorders, since there is a correlation between P4 supplementation and decreased risks of recurrent miscarriages and remission of endometriosis.

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.