The study of cytokinesis in fission yeast S.pombe has revealed a signaling network, the septation initiation network (SIN) that serves to coordinate cytokinesis with mitotic exit in the fission yeast Schizosaccharomyces pombe. The timing of cytokinesis and septum formation hinges upon the activation of the GTPase Spg1. Given the central role of Spg1 GTPase in regulating this pathway we investigated the role of Cdc7 its effector, and Byr4 the GAP component that serves to inactivate it. In order to elucidate Cdc7 regulation I have identified various functional domains within Cdc7. I show that a region adjacent to the kinase domain is responsible for association with Spg1 but insufficient for SPB localization. In addition I find that Cdc7 self associates which is required for binding to Spg1. Our findings indicate that there are potentially multiple modes of regulating Cdc7 function and hence the SIN. I have investigated the regulation of the GAP component Byr4 by phosphorylation as a means of regulating Spg1activation and further characterized its interaction with Spg1 and Cdc16. Together these results point to modes of regulating the activation of Spg1 and its effector as a means of controlling septum formation and cytokinesis.

The work contained in this document provides the first description and characterization of Bves, a gene product identified by our laboratory, in epithelial cell types. Studies addressed a central hypothesis that Bves plays a role in epithelial cell-cell interaction and integrity, which is supported by an initial protein distribution analysis at cell membranes in both cells and tissues. Localization, interaction, and functional assays establish that Bves is an essential component of the tight junction complex and interacts with the protein complex containing ZO-1 in epithelial cells. Furthermore, in vivo experiments demonstrate that Bves is clearly important for epithelial morphogenesis during X. laevis development, and establish an excellent model system to perform future studies. This dissertation provides an important contribution to the body of literature on the Bves by defining the essential nature of Bves in epithelial integrity and by providing the groundwork for further examination of how Bves participates in epithelial cell interaction.

Dynamic spatiotemporal expression of the nodal gene and its orthologs is involved in the dose-dependent induction and patterning of mesendoderm during early vertebrate embryogenesis. In loss of function studies, a strong knockdown of Xenopus antivin/lefty (Xatv/Xlefty) function was achieved by coinjecting translation- and splicing-blocking morpholino oligonucleotides that target both the XatvA and XatvB alloalleles. A greater expansion of the Organizer and mesendoderm tissues in my studies than noted in previous Xlefty/Xatv knockdown experiments in Xenopus, with long-term maintenance of expanded axial tissues strongly suggests that the regulation of Xenopus nodal-related (Xnr) signaling by Xlefty/Xatv is essential for proper cell layer specification during early embryogenesis, and tissue patterning at late stages. Inhibitors specific to Xnr signaling were used to provide evidence that Xnr-mediated induction was inherently long-range in the Xlefty/Xatv-deficient embryo, essentially being capable of spreading over the entire animal hemisphere. While the expansion of Xnr1 and Xnr2 expression is limited to the marginal zone by Xlefty/Xatv deficiency, inhibition of Xbra function using Xbra-EnR in Xlefty/Xatv-deficient embryos caused a much larger increase in the level and spatial extent of Xnr expression. However, Xnr2 expression was constrained to the superficial cell layer in any experimental conditions, suggesting a fundamental tissue-specific competence to express Xnrs. These studies reveal a two-level suppressive mechanism for restricting the strength, range and duration of Xnr signaling via both Xlefty/Xatv-mediated extracellular inhibition and Xbra-mediated indirect transcriptional repression. Therefore, the tight regulation of Xnr signaling and expression by multiple influences is essential for precisely refining cell layer specification and patterning during gastrulation. Conserved Nodal/Lefty/Pitx2 cassette in left LPM has been implicated in left-right (L-R) axis patterning during post-gastrulation and later asymmetric morphogenesis. Detailed genetic pathways for these processes, however, are still elusive because of lack of focused studies on L-R development. Under this rationale, I tried to identify new genes involved in L-R pathways using several screening methods such as PCR-based subtractive cDNA screening and microarrays. Though any candidates were not isolated in these studies, results suggest that an unbiased and rapid screening with Affymetrix GeneChipsË seems to be an appropriate method for isolating new molecules involved in L-R patterning because of its reliable detection of controls, such as Xnr1 and Xlefty/Xatv.

Proper function of the brain requires that neurons adopt different morphologies and connections. In the nematode <i>C. elegans</i>, VA and VB motor neurons arise from a common precursor cell but adopt different morphologies and accept input from separate sets of command interneurons. In <i>unc-4</i> mutants, VA motor neurons are miswired with VB-type inputs. We have proposed that miswiring results when VB genes are ectopically expressed in the VAs in <i>unc-4</i> mutants. Previous work revealed that UNC-4 functions with the UNC-37/Groucho co-repressor protein to repress the VB-specific genes <i>acr-5, del-1, glr-4</i>. However, our genetic data rule out roles for these VB genes in synaptic choice. To identify the missing <i>unc-4</i> target genes, a microarray-based strategy for profiling VA motor neurons was adopted. A comparison of VA-specific transcripts isolated by mRNA-tagging from wildtype and <i>unc-37</i> mutant animals revealed ~250 upregulated transcripts in <i>unc-37</i> animals. One of these genes, <i>ceh-12</i>, is the <i>C. elegans</i> homolog of HB9, a homeodomain transcription factor with conserved roles in motor neuron fate in flies and vertebrates (Arber et al 1999, Broihier and Skeath 2002). In <i>C. elegans, ceh-12</i>::GFP is exclusively expressed in VB motor neurons in wildtype animals. In <i>unc-4</i> and <i>unc-37</i> mutants, <i>ceh-12</i>::GFP is also expressed in VA motor neurons as suggested by the microarray data. Thus, CEH-12 is a strong candidate for an UNC-4 target gene that regulates synaptic choice. To test this idea, the <i>unc-4</i> promoter was used to drive CEH-12 expression in wildype VA motor neurons. These animals exhibit an Unc-4 like backward movement defect, as expected for a model in which ectopic CEH-12 is sufficient to impose VB type inputs. In addition, we also showed that <i>ceh-12</i> deletion mutants are partial suppressors of Unc-4 movement, thereby confirming that CEH-12 is also required for the Unc-4 miswiring defect. We conclude the VB-specific gene, <i>ceh-12</i>, is normally repressed in VA motor neurons to prevent the imposition of VB-type inputs. The incomplete suppression of <i>unc-4</i>, however, suggests that UNC-4 also controls other downstream target genes that function in parallel pathways to regulate synaptic choice.

Prostaglandin G/H synthases (PGHS), commonly referred to as cyclooxygenases (COX-1 and COX-2), catalyze a key step in the synthesis of biologically active prostaglandins (PGs), the conversion of arachidonic acid (AA) into prostaglandin H₂ (PGH₂). PGs have important functions in a variety of physiologic and pathologic settings, including inflammation, cardiovascular homeostasis, reproduction, and carcinogenesis. However an evaluation of prostaglandin function in early mammalian development has been difficult due to the maternal contribution of prostaglandins from the uterus. The emergence of zebrafish as a model system has begun to provide some insights into the roles of this signaling cascade during vertebrate development. In zebrafish, COX-1 derived prostaglandins are required for two distinct stages of development, namely during gastrulation and segmentation. During gastrulation, PGE₂ signaling promotes cell motility, without altering the cell shape or directional migration of gastrulating mesodermal cells via the G-protein coupled prostaglandin E₂ receptor (EP4). During segmentation, COX-1 signaling is also required for posterior mesoderm development, including the formation of vascular tube structures, angiogenesis of intersomitic vessels, and pronephros morphogenesis. We propose that deciphering the role for prostaglandin signaling in zebrafish development could delineate mechanistic details underlying various disease processes that result from perturbation of this pathway and uncover novel potential therapeutic targets.

The lining of the gastrointestinal tract, a single layer of epithelial cells, forms a dynamic barrier that protects the organism from harmful substances present in the lumen. Maintenance of a healthy gastrointestinal tract requires a delicate balance of proliferation, migration, and apoptosis. In response to damage of the intestinal epithelium, cells immediately surrounding the wounded area rapidly migrate into the denuded area to close the wound and restore the integrity of the epithelium in a process called restitution. A number of growth factors found in the gastrointestinal tract accelerate restitution in in vitro models of intestinal injury. Several of these factors are currently being tested in clinical trials as therapeutic treatments for inflammatory bowel diseases, of which the most promising so far is epidermal growth factor (EGF). However, the molecular mechanisms by which these factors activate intestinal epithelial cell migration are poorly defined. Using an in vitro model of intestinal epithelial cell wound closure developed in our laboratory, we have previously shown that EGF treatment increases intestinal epithelial cell migration to accelerate wound closure. In this dissertation work I have explored the molecular mechanism by which EGF stimulates intestinal epithelial cell migration. The experimental data presented here demonstrate that the small GTPase Rac is required for EGF stimulated cell migration. Treatment of intestinal epithelial cells with EGF rapidly activates Rac and enhances lamellipodia formation at the wound margin. EGF receptor (EGFR) kinase activity is required for increased cell migration and Rac activation in response to EGF. Activation of Src family kinases and phosphoinositide 3-kinase by EGF are required for Rac activation, demonstrating a novel signaling pathway downstream of EGFR that contributes to intestinal epithelial cell migration. These data contribute to our knowledge of the complex molecular mechanism that control intestinal epithelial repair with implications for understanding the role of growth factor regulation of this small GTPase in intestinal cellular migration and differentiation programs in vivo.

Constitutively activating mutations of members of the RAS family of small G proteins provide an important oncogenic contribution to a significant percentage of human cancers. Despite nearly 25 years of investigation, important questions about the function of these proteins and their relationships to one another remain unresolved. We have undertaken a series of studies to address some of these questions and herein report novel findings regarding the relationship of these family members to one another and their respective contributions to the progression of human disease. Briefly, we examine the uniquely potent oncogenic contribution of K-RAS to cell lines derived from human tumors and identify some previously unappreciated findings regarding the mechanisms by which this protein promotes tumorigenesis. Interestingly, we present data to support that K-RAS requires the activity of another family member, NRAS to realize its full oncogenic potential in certain contexts, and that represents the first biochemical evidence of a relationship between RAS family members. We additionally challenge earlier notions about effector pathways long thought to be essential for RAS-driven transformation, as well as disprove an accepted and widespread assay for measuring the activity of RAS proteins.

The microtubule cytoskeleton of most animal oocytes differs from that of somatic cells in that the centrioles are lost during oogenesis. In most animals, female meiotic spindles assemble in the absence of centrosomes; instead, microtubule nucleation by chromatin, motor activity, and microtubule dynamics drive the self-organization of a bipolar meiotic spindle. Meiotic spindle assembly commences when microtubules gain access to chromatin after nuclear envelope breakdown (NEBD) during meiotic maturation. While many studies have addressed the chromatin-based mechanism of female meiotic spindle assembly, it is less clear how signaling influences microtubule localization and dynamics prior to NEBD. This thesis work analyzes microtubule behavior in response to hormonal signaling in Caenorhabditis elegans oocytes at the early stages of the meiotic maturation process. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase I and is accompanied by MAP kinase activation, NEBD, and meiotic spindle assembly. In C. elegans, sperm trigger oocyte meiotic maturation and ovulation using the major sperm protein (MSP) as an extracellular signaling molecule. To examine the role of MSP in regulating the oocyte microtubule cytoskeleton, we investigated microtubule organization in oocytes in the presence and absence of sperm. When sperm are present, microtubules are dispersed evenly throughout the cytoplasm. In contrast, when sperm are absent, microtubules are enriched at the cortical edges between oocytes. Females injected with purified MSP demonstrate that MSP is sufficient to reorganize oocyte microtubules. Using confocal microscopy and live-cell imaging, we show that MSP signaling reorganizes oocyte microtubules prior to NEBD and fertilization by affecting their localization and dynamics. By analyzing regulators of meiotic maturation, we have discovered several genes involved in organizing oocyte microtubules in response to MSP. We present evidence that MSP reorganizes oocyte microtubules through a signaling network involving antagonistic GÑo/i and GÑs pathways and gap-junctional communication with somatic cells of the gonad. We propose several biological functions for microtubule reorganization including a role in promoting meiotic spindle assembly through facilitating the search and capture of microtubules by meiotic chromatin following NEBD.

Transient asymmetric Nodal signaling in the left lateral plate mesoderm (L LPM) during tailbud/early somitogenesis stages is associated in all vertebrates examined with the development of stereotypical left-right (L-R) organ asymmetry. In Xenopus, asymmetric expression of Nodal-related 1 (Xnr1) begins in the posterior L LPM shortly after the initiation of bilateral perinotochordal expression in the posterior tailbud. The L LPM expression domain rapidly shifts forward to cover much of the flank of the embryo before being progressively downregulated, also in a posterior-to-anterior (P-to-A) direction. The mechanisms underlying the initiation and propagation of Nodal/Xnr1 expression in the L LPM, and its transient nature, are not well understood. Removing the posterior tailbud domain prevents Xnr1 expression in the L LPM, consistent with the idea that normal embryos respond to a posteriorly derived asymmetrically acting positive inductive signal. The forward propagation of asymmetric Xnr1 expression occurs LPM-autonomously via planar tissue communication. The shifting is prevented by Nodal signaling inhibitors, implicating an underlying requirement for Xnr1-to-Xnr1 induction. It is also unclear how asymmetric Nodal signals are modulated during L-R patterning. Small LPM grafts overexpressing Xnr1 placed into the R LPM of tailbud embryos induced the expression of the normally L-sided genes Xnr1, Xlefty, and XPitx2, and inverted body situs, demonstrating the late-stage plasticity of the LPM. Orthogonal Xnr1 signaling from the LPM strongly induced Xlefty expression in the midline, consistent with findings in the mouse and demonstrating for the first time in another species conservation in the mechanism that induces and maintains midline barrier function. My studies suggest that there is long range contralateral communication between L and R LPM, involving Xlefty in the midline, over a substantial period of tailbud embryogenesis, and therefore lend further insight into how, and for how long, the midline maintains a L versus R status in the LPM. My results directly support very recent findings in mouse that were gathered concurrently and that led to a SELI (Self-Enhancement and Lateral Inhibition) model for pan-embryonic integration of L-R asymmetry information by communication across the midline. The consistency in findings between mouse and Xenopus demonstrate further conservation in the L-R specification program. The unidirectional P-to-A shifting of Xnr1 expression in the L LPM during tailbud stages occurs rapidly within ~6-8 hours. It is uncertain whether the time that is required for the biochemical processes involved in signal receipt, intracellular signal transduction, ligand production and secretion between individual cells can occur fast enough to be accommodated during the period of observed Xnr1 expression shifting. I used a pharmacological approach to block Xnr1 signaling specifically at the level of the receptor to further investigate the role of Xnr1 autoregulation in maintaining and propagating its own expression within the L LPM. Preliminarily, I found that Xnr1 and Xlefty transcripts are rapidly degraded upon inhibiting the Xnr1 autoregulatory loop, providing novel insight into the stability of these mRNAs.

In eukaryotes, all nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs). These giant proteinaceous structures are embedded in the nuclear envelope where the outer nuclear membrane and inner nuclear membrane join to form a pore. A functional NPC is composed of approximately 30 proteins, termed nucleoporins (Nups). However, the precise mechanism of how the NPC assembles is still unknown. Based on previous studies, we hypothesized that integral membrane proteins play a crucial role in NPC biogenesis and function. To test this hypothesis, I analyzed Pom34p and Pom152p, two integral membrane Nups in S. cerevisiae. The first part of my studies characterized Pom34p membrane orientation and its role in NPC structure organization. The results indicated that POM34 encodes a double pass transmembrane protein with two cytoplasmic domains. It has broad genetic interactions with other NUPs, including NUP170, NUP188, NUP59, GLE2, NUP159 and NUP82. Lack of the Pom34p N-terminal domain in a nup188 null (∆) background leads to mislocalization of a subset of Nups and nucleocytoplasmic transport defect. These data indicated that Pom34p is important for the maintenance of NPC structure. The second part of my studies utilized genetic approaches to identify potential NPC assembly factors. To this end, I conducted a synthetic lethal screen with a pom34∆ pom152∆ double mutant, which shows no apparent defects in growth, NPC structure and nuclear transport. The screen revealed several mutants allelic to NUP188, NUP170, and NUP192, whose gene products comprise the core framework of the NPC. The synthetic lethal phenotype further illustrates the close relationship between pore membrane proteins (Poms) and structural Nups. Particularly, NUP192 is an essential gene encoding the largest Nup. This is the first report to link the function of Nup192p to Poms. In addition, I performed a split ubiquitin yeast two hybrid screen aimed at identifying potential interaction partners of Pom152p. The combination of these genetic studies shed light into the understanding of NPC structure organization, and the functional defects associated with structure perturbations.