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Studies of a Site-Specific Recombination System and Analysis of New Modulators of Notch Signaling in Caenorhabditis Elegans

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Studies of a Site-Specific Recombination System and Analysis of New Modulators of Notch Signaling in Caenorhabditis Elegans Studies of a site-specific recombination system and analysis of new modulators of Notch signaling in Caenorhabditis elegans. Marcus L. Vargas Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2012 © 2012 Marcus L. Vargas All Rights Reserved Abstract Studies of a site-specific recombination system and analysis of new modulators of Notch signaling in C. elegans. Marcus L. Vargas The ability to make transgenic animals has been a great tool for biologists to study living organisms. In C. elegans, the way transgenes are generated makes them problematic in many circumstances, and there is no single, simple, reliable approach that circumvents all of the problems with current methods of introducing transgenes into C. elegans. In Chapter 2, I discuss my attempt to develop a transgenic system in C. elegans using the bacteriophage φC31 integrase system. I show evidence that φC31 integrase is active in C. elegans somatic tissue. I have successfully integrated a transgene into the C. elegans genome in single-copy using φC31 dependent recombination-mediated cassette exchange. However, attempts to repeat φC31-mediated integration has been unsuccessful. In Chapter 3, I use genetic analysis to test many genes that were reported to be associated with the γ-secretase complex in a mammalian tissue culture system. The γ- secretase complex is an important component in the Notch signaling pathway. Not only is the γ-secretase complex essential in the Notch pathway, it is also implicated in the pathology of familial Alzeheimer’s disease (FAD). As γ-secretase complex components show a Notch loss-of-function phenotype in C. elegans, a reverse genetic approach, using genes encoding proteins that associate with Presenilin was used to identify putative new Notch modulators. Several genes were identified that suppress a glp-1(gf) allele and one gene that suppress a gfp-1(lf) allele. These genes are unlikely to be core components of the Notch signaling pathway. Table of Contents: Table of Contents ......................................................................................................... i List of Figures ............................................................................................................ iv List of Tables ................................................................................................................v Acknowledgements .................................................................................................... vi Chapter 1. General Introduction ..........................................................................................1 Notch signaling ..............................................................................................................2 Notch signaling mechanism .....................................................................................3 Notch signaling in C. elegans development ..................................................................5 Germline development and Notch ...........................................................................5 Embryogenesis and inductive signaling by Notch ...................................................7 The somatic gonad and lateral inhibition by Notch .................................................9 Vulval precursor cells and Notch mediated cell-fate decision ...............................11 Genetic approaches for Notch components .................................................................13 Suppressors and enhancers of lin-12 (sel) .............................................................14 Enhancers of glp-1(lf) (ego) ...................................................................................14 Suppressors of glp-1(lf) (sog) ................................................................................15 Tumurous enhancers of glp-1(gf) (teg) ..................................................................16 γ-secretase complex in Notch transduction and APP processing ................................16 Summary of results ......................................................................................................20 Chapter 1. Figures and Tables ...........................................................................................22 i Chapter 2. φC31 integration in C. elegans .........................................................................30 Abstract ........................................................................................................................31 Introduction ..................................................................................................................31 Materials and methods .................................................................................................37 Results and discussion .................................................................................................55 φC31 can mediate recombination in C. elegans ....................................................55 φC31 can mediate recombination in the C. elegans germline ...............................56 Constructing suitable landing platforms in the C. elegans genome .......................57 RMCE in the germline of C. elegans .....................................................................58 Conclusion ...................................................................................................................61 Chapter 2. Figures and Tables ...........................................................................................63 Chapter 3. Modulation of Notch signaling pathway by Presenilin associated proteins .....80 Abstract ........................................................................................................................81 Introduction ..................................................................................................................81 Material and methods ...................................................................................................85 Results ..........................................................................................................................92 RNAi screen for positive modulators of Notch in C. elegans ...............................93 Positive modulators of GLP-1/Notch .....................................................................94 Negative regulators of GLP-1/Notch .....................................................................96 Discussion ....................................................................................................................97 G0alpha ...................................................................................................................99 CDC-48/p97 .........................................................................................................100 ii ERLIN ..................................................................................................................102 SEC-22 .................................................................................................................102 Glucose Transporters ...........................................................................................103 RAB-11 ................................................................................................................104 Chapter 3. Figures and Tables .........................................................................................106 Chapter 4: General Discussion .........................................................................................119 Summary of Results ...................................................................................................120 Discussion ..................................................................................................................120 φC31 potential uses and future prospects ............................................................120 Genetics and potential issues ...............................................................................121 Difference in assay types .....................................................................................122 Difference in alleles .............................................................................................123 Soma-to-germline interaction ..............................................................................125 Assessing γ-secretase activity ..............................................................................127 References ........................................................................................................................131 iii List of Figures: Chapter 1. Figures and Tables ...........................................................................................22 Figure 1: Domain organization of Notch receptors from C. elegans, D. melanogaster and H. sapiens ..............................................................................................................24 Figure 2: Overview of LIN-12 signaling in C. elegans ...............................................25 Figure 3: Germline development and proliferation maintenance ................................26 Figure 4: Embryogenesis and inductive signaling .......................................................27 Figure 5: AC specification and lateral inhibition .........................................................28 Figure 6: VPC fate specification and LIN-12 ..............................................................29
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