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The Role of Cell-Surface Neutral Metalloendopeptidases In THE ROLE OF CELL-SURFACE NEUTRAL METALLOENDOPEPTH)ASES IN CRANIOFACIAL DEVELOPMENT Submitted in fulfilment of the degree of Doctor of Philosophy, University of London. BRADLEY SPENCER-DENE BSc. 1995 Joint Department of Maxillofacial Surgery, Eastman Dental Institute for Oral Health Care Sciences and University College Hospitals, University of London and Developmental Biology Unit, Institute of Child Health, University of London, ProQuest Number: 10105162 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 10105162 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 It is proposed that cell-surface zinc-dependent metalloendopeptidases, by virtue of their capacity to cleave and inactivate a wide range of peptide morphogens, represent a hitherto unrecognised level of control during growth and differentiation of the mammalian head and face. The spatio-temporal distributions of two of these enzymes, neutral endopeptidase (NEP) and endopeptidase-2 (Endo-2), have been demonstrated immunohistochemically. Their presence in a wide range of craniofacial tissues in the rat, during a gestational period when these tissues are undergoing active morphogenesis, suggests that these enzymes play key roles in the development of the craniofacial region. In addition, the patterns of expression of NEP mRNA have been described using in situ hybridization. In order to investigate the roles played by NEP, the activity of the endogenous enzyme was blocked using two chemically distinct, highly selective NEP inhibitors, during whole embryo culture. At the end of the culture period, the treated embryos exhibited a characteristic asymmetric craniofacial dysmorphogenesis. Histological examination revealed a distension of the left internal carotid and first branchial arch arteries. The predominantly prosencephalic swelling was considerably exacerbated by an overgrowth of the overlying neuroepithelium. In addition, there was often incomplete closure of the cranial neural folds, and the branchial arches were of a dysmorphic appearance on the affected side. From these studies it can be concluded that both NEP and Endo-2 are present during development of the embryonic rat head and face, and that NEP appears to be essential for normal morphogenesis of the craniofacial region. ACKNOWLEDGEMENTS First and foremost, I feel I must pay tribute to my mother, Avril, for her unwavering motivation, encouragement and ceaseless drive. She has helped to keep me focused on my work, lifted me when my spirits faltered, and nurtured my ambition and determination to succeed. Both my girlfriend, now fiancée, Tracey, and my brother, Alastair have brought and continue to bring warmth, humour and support throughout my studies. On a professional level I must thank both of my Ph.D supervisors. Professor Brian Henderson and Professor Peter Thorogood for their advice, patience, enthusiasm, guidance, and for having the foresight to propose the original hypothesis which has laid the foundations of this study. I could not have completed this thesis without the unstinting encouragement and financial support of Professor Malcolm Harris, an extremely kind and generous man. Also I wish to thank the MRC for providing the initial funding for this project. To name all of the people who have helped me over the past three and a half years would run to several pages. However, I would especially like to thank Dr John Kenny, Dr Nick Lench and all of the staff of the Maxillofacial Surgery Research Unit (IDS) and the Developmental Biology Unit (ICH). A special mention must also be made to all of my collaborators in the UK and around the world. Their generous gifts, advice and invitations to meetings have helped to make this project so successful. ABBREVIATIONS ANP Atrial natriuretic peptide APES 3-Aminopropyltriethoxysilane ATP Adenosine triphosphate BLP Bombesin-like peptide BMP Bone Morphogenetic Protein BSA Bovine Serum Albumin CALLA Common Acute Lymphoblastic Leukemia Antigen CDIO Cluster of Differentiation 10 cDNA complementary Deoxyribonucleic acid CGRP Calcitonin Gene Related Peptide CTP Cytosine triphosphate DAB 3,3’ -Diaminobenzid ine dd dideoxy DEPC Diethylpyrocarbonate DTT Dithiothreitol ElO Embryonic/gestational day 10 EC-24.11 E.C.3.4.24.11 EC-24.18 E.C.3.4.24.18 EDTA Ethylenediaminetetraacetic acid EGF Epidermal Growth Factor Endo-2 Endopeptidase-2/E.C.3.4.24.18 FMLP f-Met-Leu-Phe GTP Guanosine triphosphate HACBO-Gly N-(2RS)-3-hydroxylaminocarbonyl-2-benzyl-l-oxopropyl-glycine IL-ljg Interleukin-1 beta IPTG Isopropyl jS-D-Thiogalactopyranoside NEP Neutral endopeptidase/E.C.3.4.24.11 NTE NaCl (sodium chloride)/Tris/EDTA PABA N-benzoyl-L-tyrosyl-p-aminobenzoic acid PBS Phosphate Buffered Saline PCR Polymerase chain reaction PFA Paraformaldehyde mRNA messenger Ribonucleic Acid RT-PCR Reverse transcriptase-PCR SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis SEM Scanning Electron Microscopy SSC NaCl/sodium citrate Taq Thermus aquaticus TE Tris-EDTA TAE T r is-Acetate-EDT A TBE Tris-Borate-EDT A TEMED N,N, N ’, N’-Tetramethylethylenediamine TGFa Transforming Growth Factor alpha TGFjg Transforming Growth Factor beta Tris/Trizma Tris(hydroxymethyl)aminomethane UTP Uridine triphosphate VIP Vasoactive Intestinal Peptide X-gal 5-Bromo-4-chloro-3-indolyl-j8-D-galactoside TABLE OF CONTENTS Abstract pg 2 Acknowledgements pg 3 Abbreviations pg 4 List of Tables pg 8 List of Figures pg 9 CHAPTER 1, INTRODUCTION pp 11-39 1.1 Background pg 12 1.2 Substrate specificity and mechanism of action of pg 12 the neutral metalloendopeptidases 1.3 Protein structures and molecular genetics pg 18 1.3.1 NEP pg 18 1.3.2 Endo-2 pg 19 1.3.3 Regulation of NEP pg 22 1.4 Distribution of NEP pg 23 1.4.1 Expression in adult tissues pg 23 Central nervous system pg 23 Peripheral organs pg 23 1.4.2 Developmental expression pg 24 1.5 Distribution of Endo-2 pg 28 1.6 Inhibition of the metalloendopeptidases pg 29 1.6.1 Inhibitors of NEP pg 29 1.6.2 Inhibitors of Endo-2 pg 31 1.7 The role of biologically active peptides and growth factors pg 31 during mammalian craniofacial development 1.8 The role played by growth factors in abnormal human pg 37 craniofacial development 1.9 HYPOTHESIS pg 39 1.10 Objectives of this study pg 39 CHAPTER 2, MATERIALS AND METHODS pp 40-83 2.1 Animals used in this study pg 40 2.1.1 Determination of embryonic stage pg 40 2.1.2 Sacrificing of animals and dissection of embryo pg 40 2.1.3 Dissection of adult rat kidneys pg 42 2.2 Preparation of microvillar membranes pg 42 2.2.1 Extraction of a membrane fraction from rat embryos pg 43 2.3 Fixation of tissues pg 44 2.3.1 Fixation of frozen sections for immunohistochemistry pg 44 2.3.2 Fixation of embryos for wax histology pg 45 2.3.3 Fixation of cultured embryos for scanning electron pg 45 microscopy 2.4 Tissue processing pg 46 2.4.1 Freezing tissue and cryosectioning pg 46 2.4.2 Wax embedding and sectioning pg 47 2.4.2.1 Black and white photography pg48 2.4.3 Scanning electron microscopy pg48 2.4.3.1 Black and white photography pg 49 2.5 Preparation of APES-coated slides pg 49 2.6 Immunohistochemistry pg 49 2.6.1 Western blotting pg 50 2.6.1.1 Controls pg 51 2.6.2 Immunohistochemical localization of NEP pg 51 2.6.3 Immunohistochemical localization of Endo-2 pg 52 2.6.4 Enhancement of DAB pg 53 2.6.5 Controls used for immunohistochemistry Pg54 2.6.6 Counterstaining Pg55 2.6.7 Colour photography Pg55 2.7 In situ hybridization Pg55 2.7.1 Preparation of p^S]-radiolabelled probes pg56 2.7.1.1 Transcription pg56 2.7.1.2 In situ riboprobe preparation using a G-50 drip column Pg57 2.7.2 Pre-treatment of frozen sections Pg58 2.7.3 Hybridization Pg59 2.7.4 Post-hybridization washes pg 60 2.7.5 Autoradiography pg 60 2.7.5.1 Preparation of ILFORD K5 emulsion pg61 2.7.5.2 Developing emulsion pg61 2.7.5.3 Colour photography pg62 2.7.6 Digestion of plasmid DNA with restriction endonucleases Pg62 2.8 Nucleic acid extraction pg63 2.8.1 Extraction protocols for plasmid DNA; standard mini-prep pg63 2.8.2 Ethidium bromide/high salt midi-prep pg 64 2.8.3 Extraction of messenger RNA pg65 2.8.4 Preparation of genomic DNA from rat kidney pg67 2.9 Synthesis of cDNA using reverse transcriptase (RT) Pg68 2.10 Polymerase chain reaction (PCR) pg 70 2.10.1 Primers pg 70 2.10.2 PCR conditions pg 71 2.10.3 PCR reaction mixture pg 71 2.10.4 Quantitative determination pg 72 2.11 Ligation of RT-PCR products into pGEM-T vector pg 73 2.12 Transformation of high efficiency competent cells pg 74 2.13 Blue/white colour screening for recombinants pg 74 2.13.1 Preparation of LB-ampicillin-XGAL-IPTG plates pg 75 2.14 Extraction of DNA from an agarose slice pg 76 2.15 Sequencing pg 77 2.15.1 Alkaline dénaturation of double stranded plasmid DNA Pg77 2.15.2 Annealing template and primer pg 78 2.15.3 Labelling reaction Pg78 2.15.4 Termination reactions Pg79 2.16 Denaturing gel electrophoresis Pg79 2.17 Gel drying and autoradiography Pg81 2.18 Whole embryo culture pg 81 2.18.1 Inhibitor treatment pg 82 2.18.2 Assessment of normality pg 83
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