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initiation of Vertebrate Limb Deveiopment Martin J. Cohn Thesis submitted for the degree of Ph. D. Department of Anatomy and Developmental Biology University College London University of London 1997 ProQuest Number: 10045536 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 10045536 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 Development of paired appendages at appropriate levels along the body axis characterizes the jawed vertebrate body plan. Molecular networks that operate within limb buds have received much attention, although very little is known about how limb budding is initiated. Here I show that beads soaked in Fibroblast growth factors (FGFs) and placed in prospective flank of chick embryos induce formation of ectopic limb buds, which contain their own signaling regions and develop into complete limbs. Application of FGF to anterior flank induces ectopic wings, and FGF applied to posterior flank induces ectopic legs. Hox genes are good candidates for encoding position in lateral plate mesoderm along the body axis, and thus determining where limbs form. If particular combinations of Hox gene expression determine where wings and legs develop, then formation of additional limbs from flank should involve changes in Hox gene expression which reflect the type of limb induced. Here I show that the same population of flank cells can be induced to form wing or leg. Induction of ectopic limbs is accompanied by specific changes in expression of 3 Hox genes in lateral plate mesoderm which reproduce, in the flank, expression patterns found at normal limb levels. Together, these results suggest that local production of FGF may initiate limb development, and that Hox gene expression may specify limb position. Pythons lack forelimbs, have vestigial hindlimbs, and regional specialization of the axial skeleton has been lost. I have examined patterns of Hox gene expression in python embryos, and show that the uniform pattern of vertebrae is associated with uniform Hox gene expression. Transplantation experiments and analysis of gene expression in python hindlimbs suggest that limb development is arrested due to failed apical ridge formation. Absence of a ridge in pythons may result from failure of limb ectoderm to respond to mesenchymal signals. ACKNOWLEDGEMENTS I would like to thank Professor Cheryll Tickle, my supervisor, for accepting me as her student and taking me under her extra wing. I am grateful to Cheryll for teaching, inspiring and encouraging me during my Ph.D. in her laboratory. I would also like to thank Professor Lewis Wolpert for many stimulating discussions and enthusiastic support for the project. Many thanks to Dr. Ketan Patel for being an excellent collaborator, teacher and friend. I am grateful to Dr. Ronald Nittenberg for tremendous friendship, for translating papers, and for many great discussions. I would also like to thank Prof. John Heath, Dr. Helen Abud, Dr. Juan Carlos Izpisüa-Belmonte, Dr. Jon Clarke, Dr. Robb Krumlauf and Dr. David Wilkinson, with whom I collaborated on various aspects of this project. I am grateful to Dr. Mike Coates for sharing his wealth of knowledge, for a terrific and what I hope will be a long-lasting collaboration, and for many "Dead" discussions. Thanks to Dr. Susan Evans for sharing her knowledge of reptiles. Many thanks to Esther Wenman of London Zoo, John Foden of Drayton Manor Zoo, Nick Jackson of The Welsh Mountain Zoo, and Miranda Stevenson of Edinburgh Zoo for generously providing me with fertile python eggs. Thanks also to past and present members of the lab, for creating a wonderful working environment. I would like to thank Prof. Owen Lovejoy for continuous encouragement, support and friendship. I would also like to thank Adam Levine, the Cosmic Charlies, the Levine family and the Bright family for making London feel like home. Special thanks to my parents and brothers for their endless support and encouragement. Finally, I thank Philippa for supporting and tolerating me. This work was supported by the BBSRC and Action Research. CONTENTS ABSTRACT 1 ACKNOWLEDGEMENTS 3 CHAPTER ONE: General Introduction 1. Structure of the limb 1 0 2. Patterning the anteroposterior axis of the limb 1 1 2.1 Polarizing activity in urodele amphibians 1 8 2.2 Molecular basis of polarizing activity 2 0 2.2.1 Retinoic acid 2 0 2.2.2 Sonic hedgehog 2 1 2.2.3 Hox genes in limbs 2 4 3. Proximodistal outgrowth and patterning 2 8 3.1 The apical ectodermal ridge 2 8 3.2 Molecular basis of ridge signaling 3 2 3.2.1 Fibroblast growth factors 3 2 3.3 The progress zone 3 4 3.4 Candidate molecules expressed in the progress zone 3 4 4. Dorsoventral polarity 3 5 5. Evolutionary conservation of signaling networks 3 8 6. Patterning the primary body axis 4 0 6.1 Hox genes 4 0 6.2. Hox genes and axial patterning. 44 6.3 Retinoids, limbs and the body plan 4 5 7. How are limbs induced? 4 6 7.1 Ectopic limb development 4 9 7.2 Initiation of limb budding 4 9 CHAPTER TWO: Materials and Methods 1. Application of FGF beads to chick embryos 5 2 2. Application of SHH beads to chick embryos 5 2 3. Application of retinoic acid beads to chick embryos 5 3 4. Whoie-mount skeletal preparations: Aldan green staining 5 3 5. Whoie-mount skeletal preparations: Alcian blue and Alizarin red staining 5 4 6. Scanning Electron Microscopy 5 4 7. Whoie-mount in situ hybridization 5 5 8. Tissue transplantations 5 5 9. lontophoretic application of Dil and DiAsp 5 6 10. Dig-FGF2 5 7 11. Microinjection of Dig-FGF2 5 7 12. Antibody staining 5 8 12a. Whoie-mount antibody staining 5 8 12b. Antibody staining frozen sections 6 0 13. Nile Blue Sulphate staining 61 CHAPTER THREE: FGF and Limb initiation 1. Background 6 2 2. Results 6 3 2.1 FGF beads induce additional limbs in chick embryos 6 3 2.2 Identity and morphological pattern of additional limbs 6 4 2.3 Early development of additional buds 7 0 2.4 FGF acts within two hours 7 3 2.5 Molecular polarity of early buds 7 7 2.6 Competence of flank cells to express Shh 8 0 2.7 Application of SHH to the flank 8 4 2.8 Application of FGF2 and SHH to the flank 8 5 3. Discussion 92 3.1 initiation of a Limb by Application of FGF 9 2 3.2 Reversed Polarity of the Additional Limbs 9 3 3.3 Establishment of the polarizing region 9 4 3.4 Which FGF member initiates limb development in the normal embryo? 9 7 3.5 Control of limb position 9 9 3.6 Molecular basis of limb induction 9 9 CHAPTER FOUR: Hox Genes and Specification Of Limbs 1 . Background 101 2 . Results 102 2.1 Cell lineage in ectopic wings and legs 1 02 2.2 Dynamics of Hoxb9 expression in lateral plate mesoderm 106 2.3 Comparative analysis of Hoxb9, Hoxc9 and Hoxd9 in lateral plate mesoderm 107 2.4 Hox9 gene expression and induction of ectopic limbs 108 2.5 Application of FGF to paraxial mesoderm 117 2.6 Effects of retinoic acid on Hoxb9 expression at wing level 121 2.7 Hoxb9 boundaries and cell lineage in the wing bud 121 2.6 Loss of Hoxb9 expression in posterior limb buds 125 3. scussion 129 3.1 Hox gene expression specifies limb position 129 3.2 The polarizing region pathway is independent of Hoxb9 130 3.3 Hoxb9 is reduced by local signals in the limb bud 130 3.4 Hox genes and the evolution of paired appendages 131 CHAPTER FIVE: Developmental Analysis of Limblessness and Axial Patterning in Python Embryos 1. Background 133 2. Results 135 2.1 Segmental identity in the python axial skeleton 136 2.2 Morphology of python limbs 142 2.3 Early development of the python hindlimb bud 145 2.4 Gene expression in the python limb ectoderm 151 2.5 Polarizing activity in python limbs and flank 155 2.6 Python SHH expression can be rescued by a chick apical ridge 156 2.7 Failure of apical ridge formation in python limb buds 160 3. Discussion 3.1 Hox gene expression and regionalization 164 of the python body axis 164 3.2 Dorsoventral position of the hindlimb 166 3.3 Failure of hindlimb development 166 3.4 Rescuing snake limb development 169 3.5 Evolution of the snake body plan 170 CHAPTER SIX: General Discussion 1. FGF as limb initiation signal 174 2. Positioning FGF along the body axis 174 3. Reprograming Hox expression in the flank 176 4. Implications for co-ordinating axial patterning 177 5. Evolution of body plans 178 References iso 8 LIST OF FIGURES AND TABLES CHAPTER ONE Figure 1. The Zone of Polarizing Activity (ZPA) patterns the anteroposterior axis of the limb 17 Figure 2. The Apical Ectodermal Ridge (AER) maintains proximodistal outrowth of the limb 3 1 Figure 3. Hox gene cluster diversity and evolution 4 3 CHAPTER THREE Table 1. Effects of beads soaked in FGF2 and implanted into the lateral plate mesoderm of chick embryos 6 7 Figure 4.
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