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Screen to identify the novel pancreatic gene Synaptotagmin 13 (Syt13)

Title: Screen to identify the novel pancreatic gene Synaptotagmin 13 (Syt13)

Doctoral Thesis / Dissertation , 2016 , 242 Pages , Grade: bestanden

Autor:in: Stefanie Willmann (Author)

Biology - Developmental Biology
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The pancreas is a compound gland which regulates nutrition homeostasis in mammals. Impairment in the Insulin-producing β-cells of the pancreas will lead to either type 1 or 2 diabetes. We focused on pancreas development, as the different lineages of the pancreas segregate. In the mRNA screening approach during the so-called secondary transition of the pancreas, we identified known and novel pancreatic genes. Thus, faciliating the understanding of pancreatic signaling and lineage determing factors. Furthermore, the novel pancreatic-related candidate gene Syt13 was analyzed for localization and function in the pancreatic gland, highlighting endocrine lineage commitment and ß-cell development.

Der Pankreas ist ein komplexes Organ dass für das Ernährungsgleichgewicht im Körper wichtig ist. Beeinträchtigungen in den Insulin produzierenden β-Zellen des Pankreas führt entweder zu Typ1 oder Typ2 Diabetes. In der Entwicklung des Pankreas entstehen die unterschiedlichen Verzweigungen des Pankreas wie azinare, duktale und endokrine Zellen. In einem mRNA Screening Ansatz haben wir in der sekundären Transition, in der sich die unterschiedlichen Linien des Pankreas aufteilen, bekannte und unbekannte Gene identifiziert. Dabei haben wir das Gen Syt13 weiter analysiert in Bezug auf Lokalisation und Funktion im Pankreas. Die bisherigen Experimente zeigten einen Zusammenhang zwischen Syt13 in der Aufspaltung in endokrine Zellen und in der Reifung zu ß-Zellen [...]

Excerpt


Inhaltsverzeichnis (Table of Contents)

  • Danksagung
  • Contents
  • Abstract
  • Introduction
    • The early embryonic development
    • Development of endodermal derived organs
    • Development of the pancreas
    • Regulatory networks of pancreas development
    • The model of endocrine formation
    • Establishment of epithelial asymmetry
    • The family of Synaptotagmins (Syt)
    • Aim of this thesis
  • Results
    • Generation of the Foxa2Venus mouse line
      • Design and generation of the Foxa2Venus (FVF) targeting vector
      • Analysis of the Foxa2Venus mouse line in the pancreas
    • Genome-wide expression profile of the pancreas in the secondary transition
      • Bioinformatic analysis of pathways in the secondary transition
      • Bioinformatic analysis of genes in the secondary transition
    • Identification and characterization of pancreatic genes
      • Temporal and spatial progression of pancreatic genes
      • Temporal and spatial progression of unknown pancreatic genes
    • Analysis of the novel pancreas gene Synaptotagmin 13 (Syt13)
      • Bioinformatic analysis of Syt13
        • The family of Synaptotagmins
        • Interaction partner of SYT13
        • Target gene prediction of SYT13
      • Functional analyses of Syt13
        • The gene Syt13
        • The amino acid (aa) sequence of Syt13
      • Generation of the genetically modified mouse line Syt13
        • Design, generation and verification of the Syt13GT targeting vector
      • Syt13 reporter gene expression in the early embryo
        • Characterization of Syt13 reporter gene expression in the crown
      • Syt13 mutants present defects in the adult pancreas
      • Syt13 expression in pancreas organogenese
      • Syt13 associated SNP reveal T2D susceptibility
      • Delamination of endocrine precursors in Syt13 mutants is impaired
      • Syt13 mutants show polarity defects
    • Discussion
      • FVF marks the multipotent progenitors in the pancreas
      • The FVF mouse line is a valuable tool for genome wide expression profiles
      • Molecular pathways guiding pancreas organogenesis
      • The pancreas gene selection for known and unfamiliar genes
      • Generation of different mouse lines
      • Generation of the Syt13GT/GT mouse line
      • Syt13 expression in a distinct subset of tissue
      • Pancreatic multipotent progenitors and endocrine cells marked by Syt13
      • Syt13 initiates morphogenesis in the pancreatic epithelium
      • The subcellular localization of Syt13 suggests a role in polarity membrane complexes along with BB positioning
      • Potential mechanism of Syt13 in endocrine lineage formation
      • Hypothetical molecular function of Syt13
    • Material and Methods
      • Material
        • Equipment
        • Consumables
        • Kits
        • Chemicals
        • Buffer and solutions
        • Enzymes
        • Sera and Antibodies
        • Oligonucleotides
        • Cell lines
        • Culture media
        • Molecular weigth markers
        • Mouse lines
      • Methods
        • Bioinformatics methods
          • Affymetrix®Gene 1.0 ST Array
          • Affymetrix®Gene 1.0 ST Array card
          • Affymetrix®Gene 1.0 ST Array card quality control
          • Affymetrix®Gene 1.0 ST Array card analysis
          • Pancreas gene selection using the digital database Genepaint.org
        • Cell culture
          • Embryonic stem cell culture and spheres culture
          • Culture of primary murine embryonic fibroblasts
          • Treatment of MEF with mytomycin
          • Freezing –Thawing of MEFs
          • Freezing -Thawing of ES cells
          • Passaging of ES cells
          • Electroporation of ES cells
          • Picking of ES cell clones
        • Molecular biology
          • DNA extraction
          • RNA preparation
          • DNA/RNA concentration
          • Reverse transcription
          • Gelelectrophorese
          • DNA sequencing
        • Protein biochemistry
          • Protein extraction from tissue
          • Bradford assay for determining protein concentration
          • Western blot
          • Western blot immunostaining
          • Immunohistochemistry
        • Embryology
          • Genotyping of mice and embryos
          • PCR Programs for genotyping
          • Isolation of embryos and organs
          • Tissue clearing with BABB
          • X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) staining
        • Histology
          • Paraffin sections
          • Counterstaining with Nuclear Fast Red (NFR)
          • Cryosections
      • Supplement
        • Abbreviations
        • Figures and tables
        • Literature
        • Curriculum Vitae
        • Congresses and Publications
        • Additional Figures
        • Alternative Discussion

      Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

      The main objective of this thesis is to identify novel pancreatic genes involved in the formation of the endocrine lineage during pancreas development, with a focus on the secondary transition phase (E12.5- 15.5). The research utilizes the Foxa2-Venus fusion reporter mouse line to gain insight into epithelial-mesenchymal interactions and to identify specific genes involved in the differentiation process. The goal is to generate a comprehensive gene regulatory network of the pancreas during this critical developmental stage.

      • Pancreas organogenesis and lineage commitment
      • Global gene expression profiling during the secondary transition
      • Identification and characterization of novel pancreatic genes
      • Role of Synaptotagmin 13 (Syt13) in endocrine lineage formation and development
      • Epithelial-mesenchymal interactions and signaling pathways involved in pancreatic development

      Zusammenfassung der Kapitel (Chapter Summaries)

      The Introduction provides a comprehensive overview of pancreas development, outlining the critical stages, signaling pathways, and key transcription factors involved in the differentiation of the different pancreatic lineages. The focus is on the secondary transition phase, where the multipotent pancreatic progenitors segregate into the endocrine, exocrine, and ductal lineages. The chapter also describes the role of epithelial asymmetry and the Synaptotagmin family of proteins in cellular processes.

      The Results section details the research conducted, starting with the generation of the Foxa2Venus reporter mouse line. The chapter outlines the bioinformatic analysis of the global gene expression profile of the pancreas during the secondary transition, highlighting significant pathways and genes involved in the process. The identification and characterization of novel pancreatic genes, particularly Synaptotagmin 13 (Syt13), are presented. Functional analyses of Syt13, including its expression pattern, protein interactions, and potential role in endocrine lineage formation are discussed.

      The Discussion section interprets the research findings, highlighting the significance of the Foxa2Venus mouse line as a valuable tool for studying pancreas development. The chapter analyzes the implications of the identified gene regulatory network, emphasizing the role of signaling pathways like Wnt, FGF, and Notch in orchestrating the differentiation process. The discussion focuses on the novel pancreatic gene candidate Syt13 and its potential involvement in polarity establishment, vesicle trafficking, and asymmetric cell division. The chapter also discusses the implications of Syt13 in the context of diabetes and other pancreatic-related diseases.

      Schlüsselwörter (Keywords)

      The main keywords and focus topics of the thesis are pancreas development, secondary transition, endocrine lineage, global gene expression profiling, Synaptotagmin 13, epithelial-mesenchymal interactions, signaling pathways, Wnt, FGF, Notch, epithelial asymmetry, polarity establishment, vesicle trafficking, asymmetric cell division, diabetes, and pancreatic-related diseases.

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Details

Title
Screen to identify the novel pancreatic gene Synaptotagmin 13 (Syt13)
College
Technical University of Munich  (IDR)
Grade
bestanden
Author
Stefanie Willmann (Author)
Publication Year
2016
Pages
242
Catalog Number
V338115
ISBN (eBook)
9783668296107
ISBN (Book)
9783668296114
Language
English
Tags
sreen synaptotagmin
Product Safety
GRIN Publishing GmbH
Quote paper
Stefanie Willmann (Author), 2016, Screen to identify the novel pancreatic gene Synaptotagmin 13 (Syt13), Munich, GRIN Verlag, https://www.grin.com/document/338115
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