The Role of Hair Follicles and Edar Signalling in Cutaneous Wound Healing A thesis submitted to the University of Manchester for the degree of PhD in the Faculty of Life Sciences 2015 Clare Garcin Contents List of Figures .............................................................................................. 5 List of Supplementary Figures ................................................................... 6 List of Tables ................................................................................................ 7 Abbreviations ............................................................................................... 8 Abstract ...................................................................................................... 10 Alternative Format Thesis ......................................................................... 11 Declaration ................................................................................................. 11 Copyright Statements ................................................................................ 11 Acknowledgements ................................................................................... 12 1 Chapter 1. Introduction ...................................................................... 13 1.1 The importance of an intact integument .................................................. 13 1.2. Skin structure and function ...................................................................... 13 1.2.1 Epidermis .............................................................................................. 14 1.2.2 The Basement Membrane ..................................................................... 18 1.2.3 Dermis .................................................................................................. 19 1.3. Skin Development ..................................................................................... 21 1.4 Skin appendage development ................................................................... 22 1.5. The Adult Hair Follicle .............................................................................. 26 1.5.1 The bulge and hair follicle stem cells ..................................................... 27 1.5.2 The isthmus and junctional zone ........................................................... 29 1.5.3 The hair cycle ........................................................................................ 30 1.6. Wound healing .......................................................................................... 34 1.6.1 Haemostasis and inflammation ............................................................. 35 1.6.2 Re-epithelialisation ................................................................................ 36 1.6.3 Fibroplasia ............................................................................................ 38 1.6.4 Contraction and remodelling .................................................................. 39 1.7. Involvement of the hair follicle in epidermal maintenance and repair .. 40 1.7.1 Hair follicle contribution to epidermal maintenance ................................ 40 1.7.2 Hair follicle contribution to wound healing .............................................. 40 1.7.3 Hair cycle influence on wound healing .................................................. 45 1.7.4 Skin appendages and regeneration following wounding ........................ 46 1.8. Ectodysplasin ............................................................................................ 47 1.8.1 The Ectodysplasin pathway ................................................................... 47 1.8.2 Ectodysplasin ligands ............................................................................ 48 1.8.3 Ectodysplasin Receptors ....................................................................... 49 1.8.4 Ligand-receptor interactions .................................................................. 50 1.8.5 Expression of Eda/Edar during development ......................................... 52 1.8.6 Potential Eda/Edar target/regulated genes ............................................ 54 1.9 Clues from X-linked Hypohidrotic Ectodermal Dysplasia ....................... 56 1.9.1 X-linked Hypohidrotic Ectodermal Dysplasia ......................................... 56 1.9.2 Mouse models of X-linked Hypohidrotic Ectodermal Dysplasia ............. 56 1.9.3 Edar in hair follicle development and adult hair biology ......................... 58 1.9.4 A role for EDAR in wound healing? ....................................................... 60 1 1.10 Specific project aims ............................................................................... 64 2 Chapter 2 ............................................................................................. 66 Hair follicle bulge stem cells appear dispensable for the acute phase of wound re-epithelialisation ......................................................................... 66 Abstract ............................................................................................................ 67 Introduction ...................................................................................................... 68 Materials and methods .................................................................................... 70 Animal experimentation. .............................................................................. 70 Generation and treatment of transgenic mice. .......................................... 70 Wounding procedure. .................................................................................. 70 Tissue harvesting......................................................................................... 71 Immunohistochemistry. ............................................................................... 71 Quantitative immunohistomorphometry of proliferating HF cells. ........... 71 Co-localisation immunofluorescence studies. .......................................... 72 Results .............................................................................................................. 73 Injury rapidly induces peri-wound hair follicle proliferation that propagates radially from the wound edge. ................................................. 73 Anagen hair follicles proliferate more rapidly in response to injury. ....... 73 Peri-wound hair follicles do not display bulge proliferation. .................... 73 Bulge cells in peri-wound hair follicles do not appear to migrate from the niche to contribute to healing. .................................................................... 74 Bulge cells appear ‘hyper-adhesive’ at the wound edge. .......................... 74 Bulge stem cell apoptosis can be selectively induced using a K15CrePR;DTA transgenic line. .................................................................... 79 Partial bulge stem cell depletion does not delay wound repair. ............... 79 Discussion........................................................................................................ 82 Acknowledgements ......................................................................................... 85 Conflict of interest ........................................................................................... 85 References ....................................................................................................... 89 3 Chapter 3 ............................................................................................. 92 The Ectodysplasin A pathway contributes to human and murine skin repair. .......................................................................................................... 92 Abstract ............................................................................................................ 93 Introduction ...................................................................................................... 94 Results .............................................................................................................. 96 Edar expression is elevated at the wound edge. ....................................... 96 Eda null (Tabby) mice exhibit delayed wound healing, which can be restored by acute pathway activation. ........................................................ 96 Activation of EDAR signalling accelerates healing in wild type mice. ..... 99 EDAR signalling activation restores healing in ovariectomised mice ... 100 Activation of EDAR signalling promotes human healing. ....................... 103 Discussion...................................................................................................... 105 Materials and Methods .................................................................................. 108 Wounding ................................................................................................... 108 Tissue processing, histology and analysis .............................................. 108 In vitro scratch wounding .......................................................................... 109 Ex vivo human skin wounding .................................................................. 109 Statistical analysis ..................................................................................... 109 Supplemental Methods .................................................................................. 109 In utero rescue of Tabby phenotype ......................................................... 109 Analysis of cell