IDENTIFICATION OF PROTEINS CONTROLLING GASTRULATION MOVEMENTS BY A PROTEOMIC APPROACH IN ZEBRAFISH DISSERTATION zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Dipl.-Biochem. VINZENZ LINK Geboren am 12. Januar 1974 in Frankfurt/Main, Deutschland Gutachter: Professor Dr. Bernard Hoflack, Technische Universität Dresden Dr. Carl-Philipp Heisenberg, Max Planck Institut für Molekulare Zellbiologie und Genetik, Dresden Professor Dr. Herman P. Spaink, Institute of Biology, Leiden University Eingereicht am 20.1.2006 Verteidigt am 19.4.2006 The Lord had said to Abram: “Go from your country, your people and your father's household to the land I will show you! I will make you into a great nation, and I will bless you; I will make your name great, and you will be a blessing.” Mose 12, 1-2 1 SUMMARY During vertebrate gastrulation, a well-orchestrated series of cell movements leads to the formation of the three germ layers: ectoderm, mesoderm and endoderm. In zebrafish, a model organism for vertebrate development, the mesendodermal progenitor cells separate from the ectodermal cells and migrate towards the animal pole. To identify proteins controlling these processes, I used a comparative proteomic approach following two alternative strategies: (1) Based on the notion that Wnt11 regulates cell movement and morphology during gastrulation independent of transcriptional regulation, I performed a screen aimed at the identification of proteins phosphorylated upon Wnt11 signalling. To regulate Wnt11 expression tightly, I engineered a transgenic slb/wnt11-/- fish line expressing wnt11 under the control of a heat shock promoter. Using this line, I performed a quantitative comparison of protein phosphorylation with or without Wnt11 pathway activation by analysing 32P- labelled embryo extracts on 2D gels. (2) Since these experiments did not reveal any Wnt11 targets, I addressed, in the second approach, proteomic differences causal for the changes in cell adhesion and motility observed in mesendodermal cells upon involution. Quantitative 2D gel analysis comparing ectodermal and mesendodermal cells revealed 37 significantly regulated spots, 36 of which I identified by mass spectrometry. Interestingly, the majority of these proteins were not regulated on a transcriptional level as determined by an accompanying microarray analysis confirming the complementary nature of proteomics and transcriptomics. Among the identified targets, several proteins, including Ezrin2, had previously been assigned a cytoskeleton-related function. I characterised Ezrin2 in more detail showing that Ezrin2 is specifically activated by phosphorylation in mesendodermal cells and that it is required for proper gastrulation movements. In the course of this study, I developed techniques for proteomic analysis of early zebrafish embryos, including a protocol to remove the yolk. I identified several cytoskeleton-related proteins in a comparative proteomic screen for regulators of gastrulation movements. The subsequent characterisation of Ezrin2 confirmed the power of proteomics for the analysis of developmental processes. In conclusion, this work provides a foundation to study developmental and cell biological questions in early zebrafish embryos using proteomics. 11 TABLE OF CONTENTS INDEX OF FIGURES .........................................................................V INDEX OF TABLES......................................................................... VI ABBREVIATIONS..........................................................................VII 1 INTRODUCTION .........................................................................1 1.1 Zebrafish as a model system .......................................................... 1 1.1.1 Genetics............................................................................................................1 1.1.2 Development and cell biology ......................................................................2 1.1.3 Limitations .......................................................................................................3 1.2 Gastrulation movements................................................................ 3 1.2.1 Morphological processes ...............................................................................4 1.2.2 Signals and signalling......................................................................................5 1.2.3 Effector proteins.............................................................................................7 1.2.4 Targeting effector proteins............................................................................7 1.3 Proteomics ..................................................................................... 8 1.3.1 2D gel electrophoresis....................................................................................8 1.3.2 Why proteomics?..........................................................................................10 1.3.3 Limitations of 2D gel-based proteomics...................................................11 1.3.4 Alternative proteomic approaches .............................................................12 1.3.5 Protein identification by mass spectrometry.............................................13 1.4 Aims of this thesis.........................................................................14 2 RESULTS ..................................................................................16 Development of techniques......................................................................16 2.1 Deyolking of embryos for proteomics and western blotting........16 2.1.1 Deyolking of embryos..................................................................................16 2.1.2 Improvement of western blotting ..............................................................19 2.2 2D gel electrophoresis of embryonic zebrafish extracts...............21 2.3 Application of mass spectrometry for the identification of zebrafish proteins......................................................................... 23 2.3.1 Comparison of databases.............................................................................23 Phosphoproteomics of Wnt11 signalling................................................. 27 2.4 Conditional activation of Wnt11 expression................................. 27 111 2.4.1 Generation of the transgenic wnt11 line...................................................28 2.4.2 Wnt11 induction ...........................................................................................28 2.5 Functional proteomics – phosphorylation................................... 29 2.5.1 32P-γ-ATP labelling .......................................................................................30 32 2.5.2 Pi labelling....................................................................................................33 2.5.3 Phosphorylation specific staining...............................................................35 Comparative proteomics of the germ layers ........................................... 37 2.6 Proteomic analysis of ectodermal versus mesendodermal cells............................................................................................... 37 2.7 Comparison of proteomic and genomic approaches....................41 2.7.1 Genechip analysis..........................................................................................41 2.7.2 Comparison of proteomic and genomic results.......................................43 2.8 Characterisation of Ezrin2 ........................................................... 44 2.8.1 Gene expression pattern..............................................................................45 2.8.2 Regulation of Ezrin2 ....................................................................................46 2.8.3 Functional analysis........................................................................................50 3 DISCUSSION .............................................................................53 3.1 Deyolking and proteomics in zebrafish....................................... 53 3.2 Wnt11 signalling targets ............................................................... 54 3.2.1 Limited number of responsive cells...........................................................54 3.2.2 Timing of phosphorylation .........................................................................56 3.2.3 Perturbation by the preparation procedure...............................................57 3.2.4 Properties of potential targets.....................................................................57 3.3 Proteins identified comparing mesendodermal and ectodermal cells............................................................................ 58 3.4 Functions of Ezrin....................................................................... 60 3.5 Conclusions.................................................................................. 62 4 MATERIALS AND METHODS .....................................................63 4.1 Materials....................................................................................... 63 4.1.1 Antibodies......................................................................................................63 4.1.2 Morpholino oligonucleotides......................................................................63 4.1.3 mRNA generation and injection.................................................................64