Identification and characterization of interacting partners of cytoplasmic domain of Xenopus paraxial protocadherin [Elektronische Ressource] / presented by Yingqun Wang

Identification and Characterization of
interacting partners of cytoplasmic domain of
Xenopus Paraxial Protocadherin























DISSERTATION

submitted to the
Combined Faculties of the Natural Sciences and Mathematics
of the Ruprechtr-Karls University of Heidelberg, Germany

for the degree of
Doctor of Natural Sciences







presented by
Master of Sciences Yingqun Wang
born in Wuhan, P. R. China



Oral examination: ……………………




Identification and Characterization of
interacting partners of cytoplasmic domain of
Xenopus Paraxial Protocadherin














Referees: Prof. Dr. Herbert Steinbeisser
Prof. Dr. Thomas Holstein





















DEDICATED TO THE MEMORY OF MY MOTHER

李竹生 ZHUSHENG LI









Table of Contents
Table of Contents

Table of Contents....................................................................................................................................i
1. SUMMARY ....................................................................................................................................... 1
2. INTRODUCTION............................................................................................................................. 3
2.1 Morphogenetic movements in gastrulation........................................................................... 4
2.1.1 CE movements.............................................................................................................. 6
2.1.2 Tissue seperation .......................................................................................................... 8
2.2 Molecular basis of gastrulation movements.......................................................................... 9
2.2.1 Wnt signaling pathway ................................................................................................ 9
2.2.1.1 Canonical Wnt/β-catenin pathway ................................................................ 11
2.2.1.2 Planar cell polarity pathway 11
2+
2.2.1.3 Wnt/Ca pathway........................................................................................... 12
2.2.1.4 Regulation of gastrulation by Wnt signaling ................................................ 13
2.2.2 FGF signaling ............................................................................................................. 14
2.2.3 BMP and Nodal signaling .......................................................................................... 16
2.2.4 Endocytosis ................................................................................................................. 17
2.2.5 Cytosketon remodeling .............................................................................................. 18
2.2.6 Extracelluar matrix.................................................................................................... 20
2.2.7 Cell adhesion molecules ............................................................................................. 21
2.2.7.1 Classic cadherins 22
2.2.7.1.1 Classical cadherins in morphogenesis ................................................ 22 2 Catenins in morphogenesis.................................................................. 22
2.2.7.2 Protocadherins................................................................................................. 23
2.2.7.2.1 Axial protocadherin ............................................................................. 23 2 NF-protocadherin................................................................................. 23
2.2.7.2.3 Paraxial protocadherin ........................................................................ 24 4 Protocadherin in Neural crest and Somites ....................................... 25
2.2.7.3 Atypical cadherins........................................................................................... 25
2.3 Aim of the study .................................................................................................................... 26
3. Materials and Methods................................................................................................................... 28
3.1 Materials ................................................................................................................................ 28
3.1.1 Chemicals 28
3.1.2 Enzyme and Kit systems............................................................................................ 28
3.1.3 Laboratory instruments and accessories.................................................................. 29
3.1.4 General buffers and media........................................................................................ 30
3.1.5 Stock solutions, media and buffers for yeast work.................................................. 31
3.1.6 Antibodies ................................................................................................................... 33
3.1.7 Bacterial, yeast strains and yeast two-hybrid cDNA library.................................. 33
3.1.8 Oligonucleotides ......................................................................................................... 33
3.1.9 Plasmids ...................................................................................................................... 35
3.1.9.1 Plasmids for yeast two-hybrid assay.............................................................. 35
3.1.9.2 Plasmids for GST pull-down assay ................................................................ 36
3.1.9.3 Plasmids for expression in Xenopus embryos ............................................... 36
3.2 Molecular biology methods .................................................................................................. 37
3.2.1 Maintenance of bacterial strains............................................................................... 37
3.2.2 Preparation of competent bacteria ........................................................................... 37
3.2.3 Transformation of E. coli .......................................................................................... 37
3.2.4 Plasmid Minipreparation 37
3.2.5 Plasmid Midipreparation 37
3.2.6 Enzymatic modification of DNA 38
i Table of Contents
3.2.6.1 Digestion of DNA by restriction endonucleases............................................ 38
3.2.6.2 Generation of blunt-end DNA fragments...................................................... 38
3.2.6.3 Dephosphorylation of plasmid DNA.............................................................. 38
3.2.6.4 Ligation of DNA fragments ............................................................................ 38
3.2.7 DNA electrophoresis .................................................................................................. 38
3.2.8 DNA purification........................................................................................................ 39
3.2.8.1 Purification of DNA fragments ...................................................................... 39
3.2.8.2 Extraction of DNA fragments from agarose gels.......................................... 39
3.2.9 In vitro transcription of CAP-mRNA ....................................................................... 39
3.2.10 Determination of DNA and RNA concentration.................................................... 39
3.2.11 Polymerase Chain Reaction..................................................................................... 39
3.2.12 Mutagenesis of plasmids .......................................................................................... 40
3.2.13 DNA Sequencing....................................................................................................... 40
3.3 Embryological methods ........................................................................................................ 41
3.3.1 Preparation of Xenopus laevis embryos.................................................................... 41
3.3.2 Microinjection and manipulation of Xenopus embryos.......................................... 41
3.3.2.1 Micorinjection ................................................................................................. 41
3.3.2.2 Explantation of animal caps........................................................................... 41
3.3.3 RT-PCR (Reverse transcription PCR)..................................................................... 42
3.3.3.1 Isolation of RNA from embryos or explants................................................. 42
3.3.3.2 Reverse Transcription (RT)............................................................................ 42
3.3.3.3 PCR .................................................................................................................. 42
3.4 Protein biochemical methods ............................