Development of Reaggregated Chick Hindlimb A thesis submitted for the degree of Doctor of Philosophy at the University of London By Adrian Paul Hardy Department of Anatomy and Developmental Biology University College London January 1996 ProQuest Number: 10106026 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 10106026 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 Abstract The developing chick limb has two major signalling centres; the apical ectodermal ridge maintains expression of several important genes and outgrowth of the limb, and the polarising region specifies the pattern of skeletal elements along the anteroposterior axis. In this study, reaggregated leg grafts (mesenchyme dissociated into single cells, placed in an ectodermal jacket and grafted to a host) were used to study patterning in a system where the developmental axes were severely disrupted. Reaggregates from different regions of leg mesenchyme developed correspondingly different digits, giving a system in which skeletal phenotype could be compared with the expression of genes thought to be important in patterning. Posterior third and whole leg reaggregates gave rise to different digits, yet expressed the same combination of HoxD, Bmp-2 and shh genes throughout their development. Anterior thirds initially only express the 3' end of the HoxD cluster but activate the more 5' members of the cluster sequentially over a period of 48 hours, a period during which Bmp-2 is activated but no shh or Fgf-4 expression, or polarising activity, could be detected. These results suggest that there are at least two independent mechanisms for activating the HoxD complex, one polarising region-dependent and one independent, and that shh expression may not be necessary to maintain outgrowth and patterning once a ridge has been established. The study also examined over-expression of a number of gene products in reaggregates, using avian retroviruses. Over­ expression of BMP-2 resulted in the grafts forming large amounts of cartilage, almost to the exclusion of most other tissues. Conversely, over-expression of FGF-4 resulted in the grafts almost completely failing to differentiate into tissues typical of the chick limb, instead forming bags of undifferentiated mesenchyme. CONTENTS Page Title Page 1 Abstract 2 Contents 3 List of Figures 10 List of Tables 13 Abbreviations used in text 14 Acknowledgements 16 Chapter One - General Introduction to Limb Development 1.1 Prologue 18 1.2 A brief review of limb embryology 20 1.2.1 Early development of the limb 20 1.2.2 The proximodistal axis 22 1.2.2.1 The apical ectodermal ridge 22 Effects of AER removal and addition 22 Mutants with affected AERs 24 Activities of the AER 24 1.2.2.2 The progress zone model 25 1.2.3 The dorsoventral axis 26 1.2.4 The anteroposterior axis 27 1.2.4.1 The zone of polarising activity 27 Effects of polarising region grafts 27 Maps of the polarising region 28 Is the polarising region necessary? 28 Responding cell populations 29 Commonality of polarising region signalling 29 1.2.4.2 The morphogen model 30 A graded signal 30 Effects of attenuating signalling 30 1.2.4.3 The polar co-ordinate model 32 1.2.4.4 Prepattern 33 1.2.5 Summary 33 1.3 The molecular basis of limb development 35 1.3.1 Candidates for the polarising region signal 35 1.3.1.1 Retinoids 35 Presence of associated molecules 36 Retinoids may not be the polarising region signal 36 1.3.1.2 Sonic hedgehog 36 Vertebrate Shh 37 Cleavage of Shh 37 1.3.2 Homeobox genes 38 1.3.2.1 HoxD genes 40 HoxD genes may be controlled by the polarising region 41 Activation of HoxD genes requires a signal from the AER 41 Evidence from mouse mutants 43 A combinatorial model of skeletal specification 43 Possible late roles of HoxD genes 44 1.3.2.2 HoxA genes 45 1.3.2.3 msh-class homeobox genes 47 Roles of Msx-1 48 Roles of Msx-2 49 1.3.3 Fibroblast growth factors 49 1.3.3.1 Roles in limb development 50 FGF-2 50 FGF-4 an d -8 51 1.3.3.2 Possible roles in limb initiation 52 A model of limb initiation 53 1.3.3.3 Fibroblast growth factor receptors 54 FGFRs in the developing limb 54 1.3.4 Bone morphogenetic proteins 55 Bmp-2 55 Bmp-4 57 Bmp-7 57 1.3.5 Summary: A molecular model of limb development 58 1.4 The aims of this thesis 58 Chapter Two - General Materials and Methods 2.1 Handling and Preparing Eggs 62 2.1.1 Chicken (Gallus domesticus) 62 2.1.2 Quail {Coturnix coturnixjaponica) 62 2.2 Grafting Procedures 63 2.2.1 General 63 2.2.2 Preparation of reaggregates 63 2.3 Processing and staining procedures 67 2.3.1 Wholemount staining for cartilage 68 2.3.2 Histology 68 2.3.2.1 Haematoxylin and Eosin (H&E) 69 2.3.2.2 Mallory's triple stain 69 2.3.2.3 Giemsa stain 70 2.3.2.4 Harris’s haematoxylin stain 10 Chapter Three - Characterisation of the Development of Reaggregates made with Leg Mesenchyme 3.1 Introduction 72 3.2 Materials and Methods 73 3.2.1 Cell tracing experiments 73 3.2.1.1 Quail-chick chimaeras 73 3.2.1.2 DU 75 3.2.2 Wholemount staining for cell death 75 3.2.3 Wholemount staining for nerve supply 75 3.2.4 Scanning electron microscopy 76 3.3 Results 77 3.3.1 Early appearance and histology 77 3.3.1.1 General observations 11 3.3.1.2 Early histology 11 3.3.2 A description of tissues present 79 3.3.2.1 The development of the skeleton 79 Normal leg bud 82 Reaggregated leg bud mesenchyme 83 3.3.2.2 Musculature 83 3.3.2.3 Tendon 87 3.3.2.4 Nerves 90 3.3.3 Cell sorting experiments 90 3.3.3.1 Quail-chick chimaeras 90 3.3.3.2 DU 92 3.3.4 Cell death 95 3.4 Discussion 96 3.4.1 Tissues present in reaggregates 96 3.4.1.1 Cartilage 96 3.4.1.2 Muscle and tendon 99 3.4.1.3 Nerves 100 3.4.2 Cell sorting and death in reaggregates 101 3.4.2.1 Cell sorting 101 3.4.2.2 Cell death 103 Chapter Four - The Development of Reaggregates Made from Different Regions of Leg Mesenchyme 4.1 Introduction 105 4.2 Materials and methods 106 4.2.1 Making reaggregates from different regions of leg mesenchyme 106 4.2.2 Assigning digit identity 108 4.2.3 Morphometric analysis of digit identity 110 4.2.4 Assessing the polarising activity of reaggregated mesenchyme 112 4.2.5 Measuring the height of reaggregate AERs 114 4.2.6 Dil labelling of anterior third leg 116 4.3 Results 116 4.3.1 Reaggregates made from different regions of leg mesenchyme give rise to different digits 116 4.3.1.1 Digits obtained from different types of reaggregate 116 4.3.1.2 A morphometric assessment of digit identity 120 4.3.2 Polarising activity in reaggregated mesenchyme 124 4.3.3 The AER in reaggregates made from different regions of mesenchyme 127 4.3.4 Morphogenetic potential of anterior third mesenchyme 130 4.3.4.1 DU labelling of anterior third leg 130 4.3.4.2 Intact anterior third leg 133 4.3.4.3 Anterior third recombinants 133 4.3.4.4 Fragmented anterior third recombinants 133 100 pm fragments 138 50-75 pm fragments 138 4.4 Discussion 138 4.4.1 Different digits from different regions of reaggregated leg mesenchyme 138 4.4.2 Polarising activity in reaggregates 141 4.4.3 The morphogenetic potential of anterior third mesenchyme 142 Chapter Five - Gene Expression in Reaggregates 5.1 Introduction 146 5.2 Materials and Methods 147 5.2.1 hybridisation 147 5.2.1.1 Wholemount in-siiu hybridisation 147 Riboprobe synthesis 147 Embryo processing 149 Preparation of chicken embryo powder 149 Pre-hybridisation treatment and hybridisation 150 Post-hybridisation treatment 150 Post-antibody washes 151 Sectioning wholemount in-situ hybridisation stained embryos 151 Solutions and buffers for wholemount hybridisation 152 5.2.1.2 In-situ hybridisation to tissue sections 153 Riboprobe synthesis 153 Embryo processing 154 Pre-hybridisation treatment and hybridisation 155 Post-hybridisation treatment 156 Autoradiography 156 Solutions and buffers for in-situ hybridisation to tissue sections 157 The use of a retroviral vector to effect gene over­ expression in reaggregates 158 5.2.2.1 Background 158 5.2.2.2 Concentrating viral supernatant by ultracentrifugation 158 5.2.2.3 Infecting mesenchyme for reaggregation 160 5.2.2.4 Control studies 160 5.3 ts 161 Gene expression in reaggregated leg mesenchyme 161 5.3.1.1 Expression of HoxD genes in reaggregates 161 The normal leg bud 161 Whole leg reaggregates 161 Posterior third reaggregates 163 Anterior third reaggregates 163 5.3.1.2 Expression o/Bmp-2 and Bmp-4 in reaggregates 170 Bmp-2 170 Bmp-4 171 5.3.1.3 Expression of Shh in reaggregates 175 5.3.1.4 Expression o/Fgf-4 in reaggregates 178 5.3.1.5 Expression of Msx-1 and Msx-2 in reaggregates 178 Over-expression of selected genes using an avian retrovirus 181 5.3.2.1 Over-expression of BMP-2 in reaggregates using RCAS BP(A) 187 Cartilage pattern 187 Histology 190 5.3.2.2 Over-expression of FGF-4 in reaggregates using RCASBP(A) 190 Cartilage pattern 190 Histology 194 5.3.2.3 Over-expression ofHOXD-11 in reaggregates using RCASBP(A) 199 5.3.2.4 Control studies with RCAS BP(A) encoding alkaline phosphatase 200 5.4 Discussion 200 5.4.1 HoxD genes in reaggregated leg mesenchyme 200 5.4.2 HoxD and shh expression in reaggregates