ANATOMY AND DEVELOPMENT OF TENDONS IN VERTEBRATE LIMBS Deana D’Souza A thesis submitted for the degree of Ph.D Department of Anatomy and Developmental Biology University College London University of London July 1999 1 ProQuest Number: 10630946 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 com plete 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 10630946 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 To my family 2 ABSTRACT The gross and microscopical anatomy of developing tendons in chick limbs is described. Expression patterns of genes encoding EphA4, a tyrosine kinase receptor involved in direct cell-cell signalling, Six-1, a transcription factor, and Follistatin, a TGF-(3 antagonist, are documented in developing chick tendons. EphA4 expression is compared with tenascin and collagen I expression. Follistatin applied ectopically to chick limbs inhibits tendon development suggesting a role for TGF-P signalling. Manipulations on chick limbs are carried out to examine co-ordination of tendon and cartilage development. Manipulations that invoke ectopic cartilage lead to ectopic tendons expressing both Follistatin and EphA4 while manipulations that invoke cartilage truncation lead to loss of tendons and expression. Ectodermal signalling is known to control dorso-ventral limb pattern including tendons. EphA4 is expressed in both dorsal and ventral tendons but the former are flattened while the latter round. In limbs of transgenic mice in which Wnt-7a, a dorsalising signal produced by dorsal ectoderm, had been functionally inactivated, the EphA4 expression pattern is ventralised early in dorsal tendon development. In chickens in which Lmx-1, a transcription factor expressed in dorsal mesenchyme in response to Wnt-7a signalling, is ectopically expressed ventrally, no early changes in EphA4 expression pattern in ventral tendons could be detected. However, later, established tendons in ventral regions come to resemble dorsal tendon and double nails form. Spatial and temporal expression patterns of Wnt-7a and Lmx-1 were examined and compared with EphA4 expression in tendons. These analyses suggest that dorso-ventral patterning and specification of tendons involves complex series of parallel interactions between tendon-forming cells and both ectoderm and mesenchyme. A polydactylous human foot with double nails is dissected and the toes identified by the tendons. The anatomy is interpreted from a developmental viewpoint. 3 ACKNOWLEDGEMENTS I wish to extend my gratitude to my supervisor Prof Cheryll Tickle who guided and encouraged me throughout this work and for her understanding nature. She dared to allow a static anatomist into the world of dynamic anatomy. It would be remiss of me not to acknowledge the amount of free time Cheryll spent critical reading this thesis instead of walking on the hills and vales of Scotland and I thank her for that. I would like to thank Dr K Patel for collaboration through out this work. I am also grateful to Drs Jon Clarke and Paul Martin for harbouring me in time of need and for helpful suggestions. I am grateful to many members in the lab and department, Anne, Ronald, Litsa, Monica, Paris, Aris, Mel, Neil, Muriel, Alison, Juan and many others for useful discussion and technical guidance . My special thanks to Litsa for being there when no one else was. Though not directly associated with this work, I wish to thank Drs Paul O Higgins, Chris Dean and Breda and Pia who gave me enormous moral support. Last but not the least, I would like to thank my family Godfrey, Andrea and Adrian and my brother Dal for their support and understanding and very specially Godfrey for taking on more domestic chores during the writing up of this thesis. 4 CONTENTS ABSTRACT 3 ACKNOWLEDGEMENTS 4 CONTENTS 5 LIST OF FIGURES 11 LIST OF TABLES 14 LIST OF ABBREVIATIONS 15 CHAPTER ONE: GENERAL INTRODUCTION 16 1.1 Development of the vertebrate Limb 17 1.2 Outline of Limb development 17 1.3 Initiation of limb development 19 1.4 Signalling during limb bud development 19 1.4.1 Proximo-distal development of the limb 20 1.4.2 Patterning along the anterior-posterior axis 23 Hox genes 24 1.4.3 Patterning along dorso-ventral aixs 27 1.4.3.1 Role of ectoderm 27 1.4.3.2 Models for dorso-ventral patterning 27 1.4.3.3 Molecules involved in dorsal patterning 28 1.5 Differentiation of tissues in the limb 32 1.6 Tendons 33 1.6.1 Development of tendon 34 1. 6. 1.1 Formation of digital tendons 36 1.6.1.2 How do tendons attach appropriately to the skeleton? 3 7 1.6.1.3 Muscle-tendon-cartilage relationship 37 1.6.1.4 Myotendinous junction 38 1.7 Molecules expressed in tendons 39 1.7.1 Eph receptors and ligands 39 1.7.1.1 Receptor Signalling 40 1.7.1.2 Ligand signalling 40 1.7.1.3 Eph signalling during development of Nervous System 40 1.7.2 TGF-13 family members involved in cartilage and tendon development 43 1.7.3 Transcription factors in tendon 45 1.7.3.1 Six genes 45 1.7.3.2 Eya genes 45 1.8 Work described in this thesis 46 CHAPTER TWO: GENERAL MATERIALS AND METHODS 47 2.1 Embryos for manipulations or analytical study 48 2.2 Preparation of fixatives 48 2.3 Fixing embryos 48 2.3.1 For paraffin wax sections 48 2.3.2 For in situ hybridisation or antibody staining 48 2.4 Staining with Mallory’s trichrome 49 2.5 Alcian green staining for cartilage 49 2.6 Whole mount in situ hybridisation 49 2.6.1 Preparation of Linear DNA from plasmid DNA 49 2.6.2 Synthesis of riboprobe for whole mount in situ hybridisation 50 2.6.3 Preparation of embryos for hybridisation 50 2.6.4 Hybridisation, post hybridisation washes and visualising the signal 51 2.7 Preparation of Template DNA 52 2.7.1 Bacterial Transformation 52 2.7.2 Mini (Zippy) Preparation of Plasmid DNA 52 2.8 Frozen sectioning of embryos 53 CHAPTER THREE: MACROSCOPIC AND MICROSCOPIC PATTERN OF TENDONS IN CHICK LIMBS 54 3.1 Introduction 55 3.2 Materials and methods 60 3.3 Results 60 3.3.1 Pattern of tendons in adult wing 60 6 3.3.1.1 Extensor tendons 60 3.3.1.2 Flexor tendons 64 3.3.1.3 Interrosseus tendons 64 3.3.2 Tendon pattern in 10-day embryonic wing reflects that seen in the adult. 64 3.3.2.1 Extensor tendons 64 3.3.2.2 Flexor tendons 66 3.3.2.3 Interosseus tendons 66 3.3.3 Gross and histological study of tendons in chick toes 66 3.3.3.1 Extensors 67 3.3.3.2 Flexors 67 3.3.4 Embryonic development of tendons in the wing 67 3.3.5 Formation of tendon sheath 72 3.4 Discussion 76 3.4.1 Development of individual digital tendons 76 3.4.2 Role of mesenchymal lamina in tendon-cartilage attachment 78 3.4.3 Tendon sheath 79 CHAPTER FOUR: MOLECULAR SIGNALLING IN TENDON DEVELOPMENT 80 4.1 Introduction 81 4.2 Materials and Methods 83 4.2.1 Antibody staining 83 4.2.2 In situ hybridisation 83 4.2.3 Preparation and application of beads 83 4.2.4 Removal of apical ectodermal ridge 83 4.2.5 Alcian green staining 83 4.3 Results 84 4.3.1 Characterisation of expression pattern of EphA4 in chick leg tendons 84 4.3.1.1 Early expression pattern of EphA4 in foot plate 84 4.3.1.2 Description of tendon development in relation to EphA4 85 4.3.1.3 Relation of EphA4 to the mesenchymal lamina 86 4.3.1.4 Expression of EphA4 in later stages (32-36) 86 4.3 .2 Comparison of expression of EphA4 with Tenascin and Collagen I in tendons 93 4.3.3 Expression of Six 1 in chick limb buds 98 4.3.4 Expression of Bmp-4 and Follistatin during chick tendon development 101 4.3.5 Relationship between cartilage and tendon development 104 4.3.5.1 TGF-131 induces molecules associated with early tendon development 104 4.3.5.2 Ectopic Follistatin results in loss of tendon-associated markers 104 4.3.5.3 Effects of ridge removal on EphA4 and Follistatin expression in tendons 107 4.4 Discussion 109 4.4.1 EphA4 is expressed in relation to tendon development 109 4.4.2 EphA4 is expressed at tendon insertion sites to cartilage elements 110 4.4.3 Tendons are fully formed by stage 35 111 4.4.4 Potential role of EphA4 during tendon development 111 4.4.5 Six 1 is expressed in proximal regions of developing tendons 113 4.4.6 Expression of Follistatin is associated with tendons 114 4.4.7 Co-ordination of skeletal and tendon development 115 4.4.8 Does Follistatin have a role in joint formation? 118 CHAPTER FIVE: PATTERNING OF TENDONS IN DORSO-VENTRAL AXIS OF VERTEBRATE LIMBS 120 5.1 Introduction 121 5.2 Materials and Methods 128 5.2.1 Infection with virus 128 5.2.2 Staining with antibody to Lmx-1 128 5.2.3 In situ hybridisation 129 5.2.4 Histology 129 5.3 Results 129 5.3.1 Expression profile of Wnt-7a and Lmx-1 in relation to tendon development in chick limbs 129 5.3.1.1 Expression of Wnt-7a in late stages of limb development 129 5.3.1.2 Spatial and temporal relation of expression of Lmx 1 to tendon development 133 5.3.2 Expression of EphA4 in tendons of wild type and Wnt-7a mutant mouse 139 8 5.3.2.1 Expression of EphA4 in wild type mouse limbs at day 14.5 139 Wild type forelimb 139 Wild type hindlimb 140 5.3.2.2 Wnt7a mutants have bi-ventral pattern of tendons at 14.5 dpc 140 Mutant Forelimb 140 Mutant Hindlimb 144 5.3.2.3 Early development of tendon in wild type and Wnt-7a mutant mouse limb 145 5.3.3 Misexpression of Lmx-1 in chick limb buds 150 5.3.3.1 Pattern of EphA4 expression 150 5.3.3.2 Dorso-ventral alterations after misexpression of Lmx-1 152 5.4 Discussion 158 5.4.1 Wnt-7a signal may have a proximo-distal gradient 159 5.4.2 Relation of Wnt-7a to Lmx-1 159 5.4.3 Relation of Wnt-7a and Lmx-1 expression to tendon development.