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

Contents

1 Abstract

2 Introduction

2.1 The early embryonic development

2.2 Development of endodermal derived organs

2.3 Development of the pancreas

2.4 Regulatory networks of pancreas development

2.5 The model of endocrine formation

2.6 Establishment of epithelial asymmetry

2.7 The family of Synaptotagmins (Syt)

2.8 Aim of this thesis

3 Results

3.1 Generation of the Foxa2Venus mouse line

3.1.1 Design and generation of the Foxa2Venus (FVF) targeting vector

3.1.2 Analysis of the Foxa2Venus mouse line in the pancreas

3.2 Genome-wide expression profile of the pancreas in the secondary transition

3.2.1 Bioinformatic analysis of pathways in the secondary transition

3.2.2 Bioinformatic analysis of genes in the secondary transition

3.3 Identification and characterization of pancreatic genes

3.3.1 Temporal and spatial progression of pancreatic genes

3.3.2 Temporal and spatial progression of unknown pancreatic genes

3.4 Analysis of the novel pancreas gene Synaptotagmin 13 (Syt13)

3.4.1 Bioinformatic analysis of Syt13

3.4.1.1 The family of Synaptotagmins

3.4.1.2 Interaction partner of SYT13

3.4.1.3 Target gene prediction of SYT13

3.4.2 Functional analyses of Syt13

3.4.2.1 The gene Syt13

3.4.2.2 The amino acid (aa) sequence of Syt13

3.4.3 Generation of the genetically modified mouse line Syt13

3.4.3.1 Design, generation and verification of the Syt13GT targeting vector

3.4.4. Syt13 reporter gene expression in the early embryo

3.4.4.1 Characterization of Syt13 reporter gene expression in the crown

3.4.5 Syt13 mutants present defects in the adult pancreas

3.4.6 Syt13 expression in pancreas organogenese

3.4.7 Syt13 associated SNP reveal T2D susceptibility

3.4.8 Delamination of endocrine precursors in Syt13 mutants is impaired

3.4.9 Syt13 mutants show polarity defects

4. Discussion

4.1 FVF marks the multipotent progenitors in the pancreas

4.2 The FVF mouse line is a valuable tool for genome wide expression profiles

4.3 Molecular pathways guiding pancreas organogenesis

4.4 The pancreas gene selection for known and unfamiliar genes

4.5 Generation of different mouse lines

4.6 Generation of the Syt13GT/GT mouse line

4.7 Syt13 expression in a distinct subset of tissue

4.8 Pancreatic multipotent progenitors and endocrine cells marked by Syt13

4.9 Syt13 initiates morphogenesis in the pancreatic epithelium

4.10 The subcellular localization of Syt13 suggests a role in polarity membrane complexes along with BB positioning

4.11 Potential mechanism of Syt13 in endocrine lineage formation

4.12 Hypothetical molecular function of Syt13

5 Material and Methods

5.1 Material

5.1.1 Equipment

5.1.2 Consumables

5.1.3 Kits

5.1.4 Chemicals

5.1.5 Buffer and solutions

5.1.6 Enzymes

5.1.7 Sera and Antibodies

5.1.8 Oligonucleotides

5.1.9 Cell lines

5.1.10 Culture media

5.1.11 Molecular weigth markers

5.1.12 Mouse lines

5.2 Methods

5.2.1 Bioinformatics methods

5.2.1.1 Affymetrix®Gene 1.0 ST Array

5.2.1.2 Affymetrix®Gene 1.0 ST Array card

5.2.1.3 Affymetrix®Gene 1.0 ST Array card quality control

5.2.1.4 Affymetrix®Gene 1.0 ST Array card analysis

5.2.1.5 Pancreas gene selection using the digital database Genepaint.org

5.2.2 Cell culture

5.2.2.1 Embryonic stem cell culture and spheres culture

5.2.2.2 Culture of primary murine embryonic fibroblasts

5.2.2.3 Treatment of MEF with mytomycin

5.2.2.4 Freezing –Thawing of MEFs

5.2.2.5 Freezing -Thawing of ES cells

5.2.2.6 Passaging of ES cells

5.2.2.7 Electroporation of ES cells

5.2.2.8 Picking of ES cell clones

5.3 Molecular biology

5.3.1 DNA extraction

5.3.2 RNA preparation

5.3.3 DNA/RNA concentration

5.3.4 Reverse transcription

5.3.5 Gelelectrophorese

5.3.6 DNA sequencing

5.4 Protein biochemistry

5.4.1 Protein extraction from tissue

5.4.2 Bradford assay for determining protein concentration

5.4.3 Western blot

5.4.4 Western blot immunostaining

5.4.5 Immunohistochemistry

5.5 Embryology

5.5.1 Genotyping of mice and embryos

5.5.2 PCR Programs for genotyping

5.5.3 Isolation of embryos and organs

5.5.4 Tissue clearing with BABB

5.5.5 X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) staining

5.6 Histology

5.6.1 Paraffin sections

5.6.2 Counterstaining with Nuclear Fast Red (NFR)

5.6.3 Cryosections

6 Supplement

6.1 Abbreviations

6.2 Figures and tables

6.3 Literature

6.4 Curriculum Vitae

6. Congresses and Publications

6. Additional Figures

6. Alternative Discussion

Objectives and Topics

This thesis aims to characterize the gene regulatory network during the secondary transition of pancreas organogenesis, specifically investigating the role of the novel pancreatic gene Synaptotagmin 13 (Syt13) in endocrine lineage commitment and vesicle trafficking.

• Characterization of pancreatic endodermal epithelium (FVF+) using Foxa2-based reporter models.
• Global transcriptional expression profiling to identify genes regulated during pancreatic lineage segregation.
• Functional analysis of Syt13 through the generation of genetically modified mouse models (knock-in/knock-out).
• Investigation of polarity defects and delamination processes in Syt13-deficient mice.
• Analysis of the correlation between SYT13 variants and susceptibility to type 2 diabetes.

Excerpt from the Book

Summary of Chapters

Abstract: Provides a high-level overview of the thesis, summarizing the identification of Syt13 and its role in pancreatic lineage segregation and diabetes susceptibility.

Introduction: Covers the developmental stages of the mouse embryo, the transcriptional hierarchy governing pancreatic development, and the family of Synaptotagmin proteins.

Results: Details the generation of the Foxa2Venus mouse line, global gene expression profiling, the characterization of novel pancreatic genes, and the functional analysis of the Syt13 gene.

Discussion: Interprets the findings regarding FVF usage, gene expression profiles, signaling pathways, and the hypothetical molecular function of Syt13 in endocrine lineage formation and polarity.

Material and Methods: Lists the specific equipment, reagents, mouse lines, and experimental protocols (genotyping, IHC, bioinformatics) used throughout the study.

Supplement: Provides detailed lists of abbreviations, additional experimental figures, supplemental tables, and the comprehensive literature reference list.

Keywords

Pancreas organogenesis, secondary transition, Synaptotagmin 13, Syt13, Foxa2, endocrine lineage, Ngn3, Pdx1, gene regulatory network, ciliogenesis, type 2 diabetes, polarity, epithelial-to-mesenchymal transition, mouse models, tubulogenesis.

Frequently Asked Questions

What is the core focus of this research?

This doctoral thesis investigates the gene regulatory networks active during the "secondary transition" of pancreatic development in mice, with a specific focus on identifying and characterizing novel pancreatic genes.

Which gene did the researcher identify as a key candidate?

The study highlights Synaptotagmin 13 (Syt13) as a novel pancreatic gene involved in the segregation of endocrine lineages and potential diabetes susceptibility.

What is the main objective of the thesis?

The primary goal is to decipher the regulatory mechanisms of pancreatic lineage commitment, identify potential disease genes, and evaluate these in the context of diabetes therapy and organogenesis.

Which scientific methods were utilized?

The researcher employed a broad spectrum of methods, including the generation of novel transgenic mouse lines (knock-in/knock-out), global transcriptomic profiling (Affymetrix Gene Arrays), bioinformatic pathway analysis, immunohistochemistry (IHC), and secondary raster electron microscopy (SEM).

What is covered in the main body of the work?

The main body (Results section) details the development of the Foxa2-Venus reporter mouse, the generation of a gene regulatory network, and extensive characterization of Syt13 expression, function, and its impact on cell polarity.

How is this research relevant to diabetes?

The study connects Syt13 to endocrine progenitor function, vesicle trafficking, and insulin secretion, and provides clinical relevance by identifying single nucleotide polymorphisms (SNPs) in SYT13 that correlate with insulin secretion defects in humans.

How does Syt13 affect pancreas development?

Syt13 is shown to be involved in the delamination of endocrine progenitors; deficiency leads to structural defects, such as atypical Islets of Langerhans, cystic structures, and impairment in endocrine cell differentiation.

Is Syt13 involved in exocytosis like other Synaptotagmins?

Unlike Syt1, Syt13 lacks traditional calcium-binding residues in its C2 domains, suggesting it operates via a non-canonical mechanism, potentially involving vesicular docking and transport on the microtubule network rather than traditional calcium-triggered fusion.