Expression of Vertebrate Hox Genes During Craniofacial Morphogenesis
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Expression of Vertebrate Hox Genes During Craniofacial Morphogenesis Paul Nicholas Hunt Thesis for the Degree of Doctor of Philosophy August 1991 MRC Division of Eukaryotic Molecular Genetics, National Institute of Medical Research, The Ridgeway, Mill Hill, London NW7 1AA University College London, Gower Street, London WC1E 6BT 1 ProQuest Number: 10609329 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 10609329 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 Acknowledgements I would like to thank my external supervisor, Dr. Robb Krumlauf, for his excellent help, support and enthusiasm throughout my Ph.D, for many stimulating comments and discussions and for the opportunities for travel to meetings he provided. I want also to thank Dr. Cheryl Tickle for fulfilling the role of internal supervisor and introducing me to craniofacial development. I would like to thank the other members of EKG/K over the years for many interesting discussions and proofreading; Chitrita Chaudhury, Martyn Cook, Heather Marshall, Ian Muchamore, Angela Nieto, Stefan Nonchev, Nancy Papalopulu, Melissa Rubock, and Jenny Whiting. A particular thank you to Mai-Har Sham, for her patient help with practical techniques over the years. The comments and suggestions of Drs. Nigel Brown, Jonathan Cooke, Andrew Lumsden, Roger Morris and Peter Thorogood are greatly appreciated, as were the efforts of the NIMR photographic section. This research and my studentship were funded by the UK MRC. I would like to thank the people who wrote the papyrus package, which has saved many hours of tedium. Finally I would like to thank Harry Lamb and Romita Das Gupta for their support and constant friendship over the years, and for providing the incentive for many a great meal. 2 Abstract Previous work has implicated Hox genes in the development of the branchial region. At SV2 days I have shown Hox 2 expression in neural crest emerging from the neural plate, while at 9 days of mouse development, after neural crest migration is complete, each branchial arch has a distinct code of Hox 2 expression. Arch 1 has no expression, arch 2 expresses Hox 2.8 alone, and arch 3 expresses Hox 2.8 and Hox 2.7. Given the role of the homologous genes in insects, and the suggestion that neural crest is specified before migration, it is possible that a step in the development of this level of the body is a specification of the hindbrain and branchial arches by a code of Hox expression. Retinoic acid is known to affect craniofacial development, and is able to alter the expression of Hox genes in vitro in a coordinated way. I have analysed gene expression in rat embryos treated with a drug unrelated to retinoic add that nevertheless causes similar fusion of first and second branchial arches, and show that in addition rhombomere compression and fusion of cranial ganglia occur. The implications of this on the signalling events occuring during development of the branchial region are discussed. There are three other Hox clusters in vertebrates as well as Hox 2, thought to be derived from a single ancestral cluster of genes present in the common ancestor of vertebrates and insects. I have shown that equivalent genes in other clusters show identical expression domains in hindbrain and neural crest to their Hox 2 equivalents at W 2 d.p.c. In contrast, the expression limits of subfamily members are offset from one another in the trunk somites. The implications of this on head versus trunk development are discussed. 3 4 Contents Acknowledgements 2 Abstract 3 List of Figures 8 List of Data Tables 9 Chapter 1 - Introduction 11 1.1 Introduction to Head Development 11 1.2 The Neural Crest 15 13 Genes Involved in Patterning of the Head 17 1.4 The Antennapedia Class Homeobox Genes 19 Chapter 2 - Materials and Methods 25 2.1 Buffers and Stock-Solutions! ^ ^ a > 25 2.1.1 General Molecular Biology ............................ 25 2.13 In situ reagents .V. — . 29 2.13 PCR reagents ............................................................................ 30 ■ ■ " k ' f , j'. '. y . - i i J . .■ : y ' 4 :' 2.2 Bacterial Media, and Antibiotics 30 2.3 Animals 31 2.4 Cloning and DNA Methods 31 2.4.1 Bacterial strain ......................................................................... 31 2.4.2 The production of cells competent for Transformation . 31 2.43 Bacterial transformation ......................................................... 32 2.4.4 Standard DNA methods ......................................................... 32 2.4.5 Restriction Digests ....................... 32 2.4.6 Agarose gel electrophoresis.................................................... 33 2.4.7 Isolation of DNA fragments from g e ls.................................. 33 2.4.8 Sub-cloning and ligations......................................................... 34 2.4.9 Isolation of plasmid DNA - Mini-prep.................................. 34 2.4.10 Isolation of plasmid DNA - Maxi-prep ............................... 35 2.4.11 Southern blot............................................................................ 36 2.4.12 Colony screen......................................................................... 36 5 2.5 Generation of Probes by PCR 37 2.5.1 PCR reactions.......................................................................... 37 2.5.2 Fragment preparation ............................................................ 38 2.6 DNA sequencing 38 2.6.1 Labelled primer preparation ................................................. 38 2.6.2 Enzymatic sequencing reactions ............................................ 39 2.6.3 Preparation and running of 50cm sequencing gels .............. 39 2.7 In situ hybridisation 40 2.7.1 In vitro Transcription ............................................................ 40 2.7.2 Preparation of subbed slides ................................................. 41 2.7.3 Embedding and Sectioning ................................................... 41 2.7.4 Slide pretreatment ................................................................... 42 2.7.5 Hybridisation and washing ..................................................... 43 2.7.6 Autoradiography ..................................................................... 43 2.7.7 Developing, histology and serial reconstruction ................. 44 2.8 Northern Analysis 44 2.8.1 Agarose-formaldehyde gels .................... .............................. 44 2.8.2 Northern blot .......................................................................... 44 2.83 Filter hybridisation ................................................................. 45 'i~; Chapter 3 - Expression of Hox 2 Genes in. Branchial Neural Crest ui ; i 5 47 ^ 3.1 Expression of Hox 2.8 in Crest at Time of {Emergence 47 .................................................................................... ?■ P/:r:Li.r> 3.2 Expression of Hox 2.8 After Cranial Crest Migration is Complete 50 3.3 Establishment of Hox 2.8 Expression in the Surface Ectoderm 52 3.4 Other Genes of Hox 2 are Expressed in Specific Branchial Arches 54 3.5 Discussion 56 3.5.1 The Role of Mesoderm in Neural Plate Regionalisation . 56 3.5.2 The Transmission of Spatial Specification to Other Parts of the Head........................................................................................ 60 3.5.3 Areas of reduced crest emigration........................................ 62 3.5.4 The Mechanism of Head Segmentation ............................... 63 3.5.5 Differences in Extent of Specification in Cranial Crest . 63 Chapter 4 - Effects of a Triazole Drug on Craniofacial Development 65 4.1 Introduction 65 6 4.2 Effects of a Fungicide on Craniofacial Development 67 4.3 Gene Expression in a 23 Somite Rat Embryo Showing Branchial Arch Fusion 70 4.4 Gene Expression in Other Treated Rat Embryos 78 4.4.1 Embryo # 2................................................................................ 78 4.4.2 Embryo # 4................................................................................ 78 4.4.3 Embryo # 7................................................................................ 79 4.5 Expression of Hox 2.8 in Treated Mouse Embryos 79 4.6 Discussion 81 4.6.1 The Genesis of the Phenotype of Treated Anim als 82 4.6.2 Implications for the Neural Crest ........................................... 84 4.73 Implications for the Role of Retinoic Add in the Head. 85 Chapter 5 - Expression of Genes of Hox 1 and Hox 4 in the Head 89 5.1 The Problem of Four Vertebrate Hox Ousters 89 5.2 Comparison of Subfamily Expression Domains in the Hindbrain 90 5 3 Expression of Subfamily Members in the Branchial Arches s i 91 5.4 The Labial Subfamily 96 53 Discussion - The Hox Code of the Head 96 . .. .” •' p ‘ , : Chapter 6 - Discussion 105 6.1 Hox Genes and the Evolution of the Neural Crest 106 6.2 The Labial Family 108 6.3 Hox Genes in the Head and Trunk 108 6.4 Future Directions 111 References 115 Publications Arising From this Work 125 7 List of Figures Chapter 1 - Introduction Figure 1.1: