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Isolation and Functional Analysis of Xenopus Ephrin-A3 Isolation and Functional Analysis of Xenopus Ephrin-A3 Taslima Khan A Thesis for the Degree of Doctor of Philosophy 1999 Division of Developmental Biology and Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 lAA. University College, London, Gower Street, London, WCIE 6BT. ProQuest Number: 10014866 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10014866 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ''We shall not cease from exploration And the end of all our exploring Will be to arrive where we started And know the place for the first time. Through the unknown, remembered gate When the last o f the earth left to discover Is that which was the beginning; ” Little Gidding, T. S. Eliot. To My Family For Encouraging Me, Entertaining Me and For Trying, So Often, To Understand This PhD ABSTRACT ABSTRACT Segmentation is a primary requirement for the establishment of the vertebrate body plan and it is therefore of great interest to identify proteins involved in these patterning events. The Eph family is the largest sub-group of the Receptor Tyrosine Kinases (RTKs), and several Eph family members have been shown to have important roles in development, including maintenance of segmental boundaries and as guidance cues within the nervous system. Ligands of the Eph family, known as ephrins, have been identified in many vertebrate species where they have been shown to be expressed in many tissues during development and in the adult. A PCR based strategy was used to isolate members of the ephrin-A class in Xenopus. RNAase protection analysis indicated that at two of the ephrins are expressed during gastrula and/or neurula stages. A library screen isolated XLIG4, one of these putative ephrins. Sequence analysis shows that XLIG4 is an ephrin-A3 homologue. Whole mount in situ hybridisation and RNAase protection in Xenopus embryos, revealed that Xephrin- A3 expression occurs throughout gastrulation, neurulation and tailbud stages, with dynamic expression in the migrating crest. Functional analysis was carried out by overexpression of soluble and full length forms of Xephrin-A3 in the Xenopus embryo. Since Xephrin-A3 has expression within specific neural crest streams, it was possible Xephrin-A3 had a role in these cells. Utilising whole mount in situ analysis with molecular markers, it was found that both ephrin forms disrupted the migration pattern of neural crest, the severity of which depended on the concentration of ectopic Xephrin-A3 expressed. Ephrin-A class ligands interact EphA class receptors of which EphA4 is expressed in the third arch neural crest and EphA2 in the second arch neural crest in Xenopus. It is proposed that complementary and overlapping expression of ephrin-A3 and EphA receptors is involved in the targeted migration of branchial neural crest. TABLE OF CONTENTS TABLE OF CONTENTS PAGE Title 1 Dedication 2 Abstract 3 Table of Contents 4 List of Figures 10 List of Tables 13 Abbreviations 14 CHAPTER ONE: INTRODUCTION 1.1 Introduction 17 1.2 Mesoderm Induction and Dorsalisation 18 1.3 Signalling Proteins and Neural Induction 18 1.4 TGF-p Superfamily 20 1.4.1 Activin 20 1.4.2 BMP4 21 1.4.3 Noggin, Follistatin and Chordin 21 1.5 Receptor Tyrosine Kinases 23 1.5.1 Structure 24 1.5.2 Signalling 27 1.5.3. SH2 and SH3 Domains 28 1.6 The Eph Receptor Family 28 1.6.1 Structure of Eph Receptors 29 1.6.2 Ligands of the Eph Receptor Tyrosine Kinase Family 33 1.6.3 Ephrin Structure and Binding Specificity Classes 33 1.6.4 Receptor and Ephrin Binding Analysis 33 1.6.5 Biochemistry of Eph Receptors 34 TABLE OF CONTENTS 1.6.6 Cytoskeletal Associations 38 1.6.7 Signalling Through Ephrins 42 1.6.8 Eph Family Developmental Expression Patterns 45 1.6.9 Roles in Axonal Pathfinding in the Retinotectal System 46 1.6.10 Axonal Repulsion and Axon Fasiculation 48 1.6.11 Signal Transduction Through an Ephrin 49 1.7 Hindbrain Segmentation 50 1.7.1//ojc genes 51 1.1.2 Krox20 51 1.1.3 Kreisler 54 1.8 Compartments and Boundaries 55 1.8.1 A Role for an Eph Receptor in Hindbrain Segmentation 57 1.9 Neural Crest 60 1.9.1 Segmentation of Branchial Neural Crest 61 1.9.2 Neural Crest Migration 62 1.10 Cell Adhesion Molecules 63 1.11 Expression of Eph Receptors and Ligands in Neural Crest 64 1.11.1 Roles of Eph Receptors in Neural Crest Pathfinding 65 1.12 Roles in Angiogenesis 66 1.13 Roles in Adhesion 71 1.14 Aims of the Project 68 1.14.1 Hypothesis 72 CHAPTER TWO: MATERIALS AND METHODS 2.1 Standard Solutions: 74 2.2 Cloning and DNA Manipulation 75 2.2.1 Preparation of Competent Cells for Plasmid Transformation 76 2.2.2 Subcloning and Ligation 76 2.2.3 Bacterial Transformation 76 2.2.4 Isolation of Plasmid DNA in a Mini-Preparation 76 2.2.5 Maxi-Preparation of Plasmid DNA 77 2.2.6 Restriction Endonuclease Digests of DNA 77 2.2.7 Electrophoretic Separation of DNA and RNA 78 TABLE OF CONTENTS 2.3 Embryo Culture and Manipulations 78 2.3.1 Microinjection of Xenopus Embryos 78 2.4 RNA Isolation 78 2.4.1 RT-PCR Cloning of Ephrins 79 2.4.2 Gel-Purification of DNA 80 2.5 DNA Sequence Analysis 80 2.5.1 Sequence Comparisons 80 2.6 Library Screening 81 2.6.1 Obtaining DNA from XgtlO Phage Library 81 2.6.2 Southern Blot 82 2.7 RNAase Protection 82 2.7.1 Preparation of Probe 82 2.7.2 Probe Purification 83 2.7.3 Hybridisation 83 2.7.4 Degradation of Non-Protected RNA 84 2.8 Whole Mount in situ Hybridisation 84 2.8.1 Preparation of Probe 84 2.8.2 Preparation of Embryos and Hybridisation 85 2.8.3 Post-Hybridisation Washes 85 2.8.4 Antibody Washes and RNA Detection 86 2.8.5 Fluorescein Dextran Detection 86 2.8.6 Clearing Embryos 87 2.8.7 Sectioning Embryos 87 2.9 In vitro Transcription and Translation 87 2.9.1 Creation of Truncated Xephrin-A3 87 2.9.2 In vitro Translation Reaction 88 2.9.3 In vitro Transcription Reaction 89 2.10 Northern Blotting Analysis 89 2.10.1 Preparation of Gel and RNA Samples 89 2.10.2 Preparation of Probe and Hybridisation 90 TABLE OF CONTENTS CHAPTER THREE: THE CLONING OF XENOPUS EPHRINS 3.1 Introduction 91 3.2 Results 91 3.2.1 RT-PCR Procedure 91 3.2.2 PCR Clone Analysis 97 3.2.3 PCR Class Categorisation 100 3.2.4 Sequence Comparisons of XLIG Class Clones 100 3.2.5 Analysis of XLIG 1-5 Classes 105 3.2.6 Isolation and Sequence Analysis of an XLIG4 cDNA Clone 108 3.2.7 Analysis of XLIG4 cDNA Sequence 113 3.3 Discussion 113 3.3.1 XLIG Classes 123 3.3.2 The XLIG4 cDNA Analysis 124 3.3.3 Pseudotetraploidy 128 CHAPTER FOUR: THE EXPRESSION PATTERN OF XENOPUS EPHRINS 4.1 Introduction 129 4.2 Results 129 4.2.1 Temporal Profile of XLIG 1-5 Classes 129 4.2.2 Expression Analysis of Xephrin-A3 132 4.2.3 Northern Blot Analysis 132 4.2.4 Whole Mount in situ Hybridisation 137 4.2.4.1 Early Neurula Stage Expression 137 4.2.4.2 Late Neurula to Tailbud Stage Expression 142 4.2.4.2.1 Summary 145 4.2.4.3 Late Tailbud Stage Expression 145 4.3 Discussion 145 4.3.1 Xephrin-A3 Expression in the Neural Crest 148 4.3.2 Role of the Eph Family in Neural Crest Migration 149 4.3.3 Xephrin-A3 in other species 152 4.3.4 Non-neuronal Xephrin-A3 expression 152 TABLE OF CONTENTS CHAPTER FIVE: FUNCTIONAL ANALYSIS OF XEPHRIN-A3 5.1 Introduction 153 5.2 Results 153 5.2.1 Subcloning the full length and soluble forms of Xephrin-A3 153 5.2.2 Microinjection of Full Length and Soluble Xephrin-A3 156 5.2.2.1 Microinjection of Full Length Xephrin-A3 156 5.2.2.1.1 XKrox20 Analysis: Overexpression of 3-2ng Xephrin-A3 161 5.2.2.1.2 XKroxlO Analysis: Overexpression of 2-lng Xephrin-A3 164 5.2.2.1.3 XKrox20 Analysis: Overexpression of 0.5-0. Ing Xephrin-A3 171 5.2.2.1.4 XAP2 and EphA2 Analysis 171 5.2.2.2 Microinjection of Soluble Xephrin-A3 176 5.2.2.3 Targeted Blastomere Injections of Full Length and Soluble Xephrin-A3 182 5.2.2.4 Observations of Gastrulation 182 5.3 Discussion 186 5.3.1 The Effect of Ectopic Expression of Xephrin-A3 on Neural Crest Cell Migration 186 5.3.2 The Role Of Overlapping and Complementary Expression in Xephrin-A3 Function 190 5.3.2.1 The Role of Overlapping Expression in the Eph Family 190 5.3.2.2 The Potential Role of Xephrin-A3 Expression in the First Arch Neural Crest Cells 192 5.3.2.3 The Role of Complementary Expression in the Eph Family 193 5.3.3 The Effects of Lower Ectopic Xephrin-A3 Expression 194 5.3.4 Soluble Xephrin-A3 Ectopic Expression 195 5.3.5 Gastrulation Defects 196 5.3.5.1 Xephrin-A3 Ectopic Expression 196 5.3.5.2 Targeted Injections of Xephrin-A3 197 5.3.5.3 Fate change of Injected Blastomeres 197 TABLE OF CONTENTS 5.3.6 Similarity of Full Length and Soluble Xephrin-A3 Overexpression Phenotypes 198 5.3.7 Comparing and Contrasting the Effect of Dominant-Negative EphA Receptors in Xenopus Neural Crest Migration with Xephrin-A3 198 CHAPTER SIX: DISCUSSION 6.1 Neural Crest Cell Migration 200 6.1.1 Generating Neural Crest Free Zones 200 6.1.2 Neural Crest Cell Guidance 200 6.1.3 The Role of the Eph Family 201 6.2 General Roles of Eph Receptors and Ephrins 202 6.3 Xephrin-A3 Expression Patterns 203 6.4 General Summary 204 6.5 Future Directions
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