Browsing by Subject "Wnt Proteins"
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Item Beta-Catenin and Development of the Urogenital System(2009-09-04) Merkel, Calli E.; Carroll, Thomas J.The urogenital system is composed of the kidneys, gonads, urinary and reproductive tracts. Components of the urogenital system play many important roles in the body; kidneys function by regulating the body's fluid volume, acidity and mineral composition, while the reproductive tract is necessary for propagation of our species. Therefore, the study of urogenital system development is important in the understanding of disorders associated with both the kidneys and the reproductive tract and their treatment. Urogenital system development begins with the formation of an epithelial tube, called the Wolffian duct. From the Wolffian duct forms a ureteric bud, which, along with the metanephric mesenchyme, will undergo a series of morphogenetic changes, eventually giving rise to the adult kidney. The Wolffian or M��rian ducts, along with the bipotential gonads, will develop into the male or female reproductive tracts,respectively. Although many signals are involved in development of the urogenital system, canonical Wnt/beta-catenin signaling is known to play a significant role. To better understand the role Wnt signaling plays in reproductive tract development, we conditionally removed beta-catenin from the Wolffian duct using a HoxB7cre line of mice. We determined that beta-catenin is necessary for M��rian duct formation. Additionally, removal of beta-catenin from the Wolffian duct leads to premature differentiation, preventing degradation of the Wolffian duct in females and inhibiting proper formation of the Wolffian duct into components of the male reproductive tract. In addition to our mouse model, we validated the efficacy of small molecule inhibitors of Wnt signaling in embryonic kidney culture. Functional small molecule Wnt inhibitors will provide an important tool for the continued study of urogenital system development along with the potential treatment of diseases associated defective Wnt signaling.Item Discovery and Characterization of Novel Chemical Inhibitors of Wnt Ligand Production with Implications for Anti-Cancer Therapy(2012-07-20) Dodge, Michael E.; Lum, LawrenceThe widespread and indispensable nature of Wnt morphogens during early development has long been a focal point of embryogenesis research. More recently, pathway activation has been uncovered in adult stem cell niches and cancers arising from associated tissues. Consequently, interest has shifted towards achieving chemically based control over aberrant pathway responses, with the ultimate goal of creating therapeutic utility. To this end our laboratory screened a diverse 200K compound library using cell based transcriptional reporters and uncovered several classes of small molecules that modulate distinct Wnt regulatory nodes. One class in particular, termed IWP (Inhibitor of Wnt Production), represents the first specific chemical inhibitors of a Membrane Bound O-Acyltransferase family member: Porcupine. Herein we postulate that IWPs function by binding to and disrupting Porcupine?s active site, preventing subsequent acylation of Wnt ligands necessary for downstream pathway activation. Further SAR studies reveal first generation IWP lead structures to be superior at this task in vitro, with even modest structural changes negatively impacting compound activity. Finally, we propose that due to Porcupine?s upstream position within the Wnt pathway, IWP administration has the potential to disrupt oncogenic contributions made by recently uncovered non-canonical Wnt signaling events. Together these findings advance our understanding of Wnt chemical tractability with long-term implications for targeted therapies.Item Feedback Regulation of Wnt Signaling by Naked Cuticle (Nkd) During Drosophila Embryogenesis(2008-05-12) Chan, Chih-Chiang; Wharton, Keith A.Wnt/beta -catenin signals are essential for many developmental and physiological processes in animals. Deregulation of the Wnt signaling pathway in mammals can cause diseases such as birth defects, cancer, osteoporosis, and diabetes. In Drosophila, the naked cuticle (nkd) gene antagonizes the Wnt/beta -catenin signaling in every segment of the embryo. Nkd is a modular, evolutionarily conserved protein that uses an EF-hand motif and adjacent sequences to target the cytoplasmic Wnt signal transducer Disheveled (Dsh). The mechanism by which Nkd antagonizes Wnt signaling in Drosophila embryos is not well understood. The abundance and bulk distribution of Dsh is not altered in nkd mutants as compared to wild type embryos, and overexpression of Nkd transgenes in nkd mutants did not alter Dsh distribution or abundance by confocal microscopy. Nkd transgenes lacking Dsh-binding regions were mostly able to rescue nkd mutants, suggesting that the Dsh-binding regions of Nkd contribute little to Nkd activity, at least when the transgenes were overexpressed. In this thesis, I have investigated non-Dsh binding regions that are critical for Nkd function. Our lab's findings indicate that a conserved 30 amino acid motif is essential for Nkd nuclear localization and function. Substitution of the 30aa motif with a heterologous nuclear localization sequence (NLS) rescued some nkd mutants to adulthood. In support of Nkd's role in the nucleus, Nkd binds to Importin-alpha 3, an adaptor for the canonical nuclear import apparatus. I identified that Nkd associates with Importin-alpha 3 via a motif ("D6") that is conserved between D. melanogaster and D. pseudoobscura. NkdΔD6, lacking the Importin-alpha 3-binding motif, was defective in nuclear localization and in rescuing nkd mutants. RNAi knockdown of importin-alpha 3 prevented the nuclear localization of Nkd. The findings that Nkd possesses two NLSs, each of which is required for function, and that Nkd associates with a component of the nuclear import apparatus, suggest that Nkd antagonizes the Wnt/beta -catenin signaling in the nucleus. Furthermore, I also addressed the function of the N-terminus of Drosophila Nkd. Unlike mammalian Nkd homologs that have N-terminal myristoylation consensus sequences responsible for membrane association, the N-terminus of Drosophila Nkd, also conserved in mosquito Nkd, lack such a sequence. Nonetheless, Nkd's N-terminus was required for function and membrane association. Substitution of the N-terminus with heterologous myristoylation sequences did not restore nkd function, indicating that the mechanism by which Drosophila Nkd associates with the membrane is different than mammalian Nkds. Therefore, Nkd appears to function in the membrane, in the cytoplasm to target Dsh, and in the nucleus to antagonize Wg signaling.Item Functional Genomics Based Interrogation of Cell-Fate Determination Pathways(2011-08-10) Jacob, Leni Susan; Lum, LawrenceThe Hedgehog (Hh) and Wnt signal transduction pathways are master regulators of embryogenesis and tissue renewal and represent anticancer therapeutic targets. Using genome-wide RNA interference screening in murine cultured cells, I established previously unknown associations between these signaling pathways and genes linked to developmental malformations, diseases of premature tissue degeneration, and cancer. I identified functions in both pathways for the multitasking kinase Stk11 (also known as Lkb1), a tumor suppressor implicated in lung and cervical cancers. Stk11 loss resulted in disassembly of the primary cilium, a cellular organizing center for Hh pathway components, thus dampening Hh signaling. Loss of Stk11 also induced aberrant signaling through the Wnt pathway. Chemicals that targeted the Wnt acyltransferase Porcupine or that restored primary cilia length by inhibiting the tubulin deacetylase HDAC6 (histone deacetylase 6) countered deviant pathway activities driven by Stk11 loss. My study demonstrates that Stk11 is a critical mediator in both the Hh and the Wnt pathways, and that functional genomics based approaches to dissecting cell-fate determination pathways may support the development of targeted therapeutic strategies.Item Mechanisms of Genome Buffering and Cell Fate Coordination in Adult Tissue Homeostasis(2016-07-26) Tuladhar, Rubina; Amatruda, James F.; Scherer, Philipp; DeBerardinis, Ralph J.; Lum, LawrenceSelf-renewal competency of adult stem cells is essential for tissue homeostasis. The corruption of genes essential for genome preservation or for niche-stem cell interactions frequently results in loss of stem cell viability and disease. The two components of my thesis focus on understanding adult stem cell preservation - the integration of metabolism and intercellular communication mediated by the Wnt family of secreted signaling molecules, and epigenetic mechanisms that buffer the proteome against insertion/deletion (INDEL)-type genetic mutations. Wnt-mediated signaling is essential for embryogenesis and the maintenance of adult tissues. Lipidation of Wnt proteins by the acyltransferase Porcupine (Porcn) is crucial for secretory pathway exiting. Using chemically based approaches, I have demonstrated that Porcn active site features conserved across animals enforce ω-7 cis fatty acylation of Wnt proteins. Deviant acylation of a Wnt protein using an exogenously supplied trans fatty acid cripples its ability to traverse the secretory pathway due to a previously unappreciated stereoselectivity of the Wnt chaperone Wntless (WLS) for fatty acids. My findings provide a mechanistic account of chemical specificity observed in Porcn inhibitors, and delineate a universal mechanism for integrating communal cell fate decision-making with metabolic fitness. As part of my efforts to generate isogenic cells for the expression of LKB1, a tumor suppressor that regulates Wnt protein production, I encountered the emergence of foreign LKB1 proteins subsequent to the introduction of INDELs by the DNA editing enzyme CRISPR-Cas9. I demonstrate that these novel proteins are the products of: a) the installation of internal ribosomal entry sites (IRES), b) the induction of exon skipping due to compromised exon splicing enhancers (ESEs), and c) the conversion of pseudo-mRNAs to protein-coding mRNAs due to the unwanted elimination of premature termination codons. I propose that these molecular events serve as compensatory mechanisms employed by cells to restore proteome integrity in the face of INDEL-type challenges to the genome posed by pathogens and environmental mutagens. Taken together, these two projects will: a) delineate intervention strategies premised upon the attack of an universally conserved point of intersection between metabolism and cell-to-cell communication, b) facilitate the personalization of medicine, and c) accelerate tissue engineering initiatives.Item Quantitative Single-Cell Imaging Reveals Insulation of Morphogenic Signal Transduction(2014-07-24) Coster, Adam D.; Alto, Neal; Altschuler, Steven J.; Wu, Lani; Ranganathan, Rama; Amatruda, James F.How cells integrate external cues in order to make behavioral decisions is a central problem of cell biology. In development and in tissue-homeostasis, cell-fate decisions are made by the integration of multiple morphogenic signals, but how cells convert such combinations of signals into distinct behaviors is not well understood. A major complication is our incomplete knowledge of which signal properties encode the information that cells use for decision-making. A further complication is that the static networks we use to describe cellular signaling pathways are likely to be overly-complex; the true signaling network, in a given cellular context and at a particular point in time, may be much simpler. Using a rigorous and quantitative single-cell imaging approach, I find that such simplicity is present in the integration between Wnt and Transforming Growth Factor Beta (TGFB), which are key developmental pathways. Surprisingly, this insulation extends to the integration of signals within the TGFB superfamily, which are expected to compete for shared components and so interfere with one another during signal transduction. My results thus add clarity to and simplify our understanding of how cells integrate information from the Wnt and TGFB pathways, and further suggest that insulation of signal transduction may be a common feature of morphogenic pathways.Item Wnt9b Signals Through Two Distinct Pathways to Regulate Progenitor Maintenance, Differentiation and Morphogenesis During Kidney Development(2010-01-12) Karner, Courtney Michael; Carroll, Thomas J.The development of the kidney is dependent upon reciprocal interactions between the ureteric bud epithelium and a mesenchymal progenitor population known as the metanephric mesenchyme. Signals from the ureteric bud cause a subset of the progenitors to differentiate into tubules while the remainder of the mesenchyme undergoes proliferation/self renewal. This process of progenitor expansion and differentiation reiterates itself during the course of development, ultimately resulting in the formation of approximately 20,000 tubules per mouse kidney. Establishment of the correct number of tubules depends on a careful balance between the induction of new tubules and the expansion of the existing tubule progenitor cells. The molecular mechanisms by which this balance is achieved are poorly understood. Previous studies suggested that Wnt/_-catenin signaling was necessary and sufficient to induce tubule formation. Further, it was hypothesized that the pathway must be repressed in the progenitors to keep them in an undifferentiated state. However, using a combination of microarray analysis and genetics, I have found that Wnt/_-catenin signaling is active in the progenitor compartments. My data indicate that rather than inhibiting _-catenin activity, factors in the progenitor cells (primarily the transcription factor Six2) alter the transcriptional output of _-catenin signaling. Wnt/_-catenin signaling is required for stem cell renewal/proliferation of the progenitor cells. These data reveal a mechanism through which cells receiving the same Wnt9b signal can respond in distinct ways (differentiation vs. proliferation). Utilizing this simple mechanism, the kidney balances the induction of new tubules and the maintenance of tubule progenitors, thereby insuring formation of the proper number of tubules. While the mechanisms regulating tubule formation have been well studied, little is known of the mechanisms that establish the length or diameter of these tubules. In the kidney, defects in the establishment or maintenance of tubule diameter are associated with one of the most common inherited human disorders, polycystic kidney disease. Here, I show that attenuation of Wnt9b signaling during kidney morphogenesis affects the planar cell polarity of the epithelium and leads to tubules with significantly increased diameter. Although previous studies showed that polarized cell divisions played an important role in the maintenance of postnatal kidney tubule diameter, we find that during early embryonic development, cell divisions are randomly oriented. Therefore the orientation of cell division plays little role in kidney tubule diameter establishment. Instead, our data suggest that diameter is established during early morphogenetic stages by convergent extension processes and maintained by polarized cell divisions. Wnt9b, signaling through the non-canonical Rho/Jnk branch of the Wnt pathway, is necessary for both of these processes. These findings provide novel insights into the molecular mechanisms that regulate the kidney tubule development and morphogenesis and greatly enhance our ability to generate kidney stem cells and engineer tubules for damaged organs.