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Characterization of Primary Cilia and Intraflagellar Transport 20 in the Epidermis Characterization of Primary Cilia and Intraflagellar Transport 20 in the Epidermis Steven H. Su Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2020 © 2020 Steven H. Su All Rights Reserved Abstract Characterization of Primary Cilia and Intraflagellar Transport 20 in the Epidermis Steven H. Su Mammalian skin is a dynamic organ that constantly undergoes self-renewal during homeostasis and regenerates in response to injury. Crucial for the skin’s self-renewal and regenerative capabilities is the epidermis and its stem cell populations. Here we have interrogated the role of primary cilia and Intraflagellar Transport 20 (Ift20) in epidermal development as well as during homeostasis and wound healing in postnatal, adult skin. Using a transgenic mouse model with fluorescent markers for primary cilia and basal bodies, we characterized epidermal primary cilia during embryonic development as well as in postnatal and adult skin and find that both the Interfollicular Epidermis (IFE) and hair follicles (HFs) are highly ciliated throughout development as well as in postnatal and adult skin. Leveraging this transgenic mouse, we also developed a technique for live imaging of epidermal primary cilia in ex vivo mouse embryos and discovered that epidermal primary cilia undergo ectocytosis, a ciliary mechanism previously only observed in vitro. We also generated a mouse model for targeted ablation of Ift20 in the hair follicle stem cells (HF-SCs) of adult mice. We find that loss of Ift20 in HF-SCs inhibits ciliogenesis, as expected, but strikingly it also inhibits hair regrowth. Closer examination of these mice reveals that Ift20 is crucial in maintaining HF-SC identity. Specifically, ablation of Ift20 in HF-SCs results in loss of SOX9 expression in HF-SCs and results in ectopic expression of the IFE marker KLF5 in HF-SCs. Additionally, ectopic differentiation is observed in HF-SCs following loss of Ift20. Finally, using both in vitro and in vivo models, we also characterize the role of primary cilia and Ift20 in epidermal wound healing. We find that loss of Ift20 slows collective keratinocyte migration in vitro and also slows HF-SC migration in vivo during wound repair. Interestingly our data suggests that Ift20 regulates keratinocyte migration in a primary cilia-independent manner. Instead, we find that Ift20 mediates focal adhesion (FA) turnover during keratinocyte migration. Specifically, Ift20 together with Rab5, regulates recycling of FA integrins and loss of Ift20 inhibits proper return of integrins to the keratinocyte surface. Overall, we demonstrate that the epidermis is highly ciliated throughout development and in postnatal skin. We show that Ift20 is crucial in maintaining HF- SC identity and the telogen to anagen transition in HFs. We finally demonstrate that Ift20 regulates keratinocyte migration independent of its function in ciliogenesis and instead regulates recycling of FA integrins through a Rab5 dependent mechanism. Table of Contents List of Charts, Graphs, Illustrations ............................................................................................... iv Acknowledgments.......................................................................................................................... vi Chapter 1: Introduction ................................................................................................................... 1 1.1 Mammalian skin: architecture and cell populations ......................................................... 1 1.2 The hair cycle ................................................................................................................... 5 1.3 Homeostatic self-renewal of epidermal stem cell populations......................................... 9 1.4 The dynamics of epidermal wound healing ................................................................... 11 1.5 Cell migration and focal adhesions ................................................................................ 15 1.6 Epidermal development during embryogenesis ............................................................. 16 1.7 Primary cilia: function and pathophysiology ................................................................. 20 1.8 Ciliogenesis and cilia disassembly ................................................................................. 24 1.9 Ift20 has both ciliary and non-ciliary functions ............................................................. 28 Chapter 2: Characterization of primary cilia in embryonic and postnatal epidermis in a transgenic mouse model ................................................................................................................................. 30 2.1 Introduction .................................................................................................................... 30 2.2 Characterization of Arl13b-mCherry; Centrin 2-GFP developing epidermis ................ 30 2.3 Characterization of Arl13b-mCherry; Centrin 2-GFP postnatal and adult epidermis ... 35 2.4 Cilia length, morphology, and live ex-vivo imaging ..................................................... 39 2.5 Discussion ...................................................................................................................... 44 Chapter 3: Ift20 and hair follicle homeostasis .............................................................................. 47 i 3.1 Introduction ......................................................................................................................... 47 3.2 Postnatal loss of Ift20 and primary cilia results in gross hair regrowth defect in mice ...... 48 3.3 Wnt signaling is absent in IFT20 cKO hair follicles .......................................................... 52 3.4 Loss of Ift20 in HF-SCs results in loss of SOX9 expression, stem cell identity and ectopic expression of differentiation markers ....................................................................................... 56 3.5 Discussion ........................................................................................................................... 62 Chapter 4: Ift20 in Cell Migration and Wound Healing ............................................................... 65 4.1 Introduction and background .............................................................................................. 65 4.2 Ift20 is required for polarized migration of cultured primary keratinocytes ...................... 65 4.3 Ablation of Ift20 does not affect Golgi polarization or overall structure in cultured keratinocytes ............................................................................................................................. 68 4.4 Loss of Ift20 leads to defects in mechano-chemical signal transduction that is independent of the primary cilium ................................................................................................................ 71 4.5 Ift20 is required for focal adhesion reformation after microtubule-induced FA disassembly. ................................................................................................................................................... 75 4.6 Integrin surface expression is altered when Ift20 function is ablated ................................. 79 4.7 Ift20 maintains its association with the Golgi apparatus during focal adhesion reformation after MT-induced FA disassembly............................................................................................ 82 4.8 Ift20-dependent FA reformation does not require Golgi function ...................................... 86 4.9 Ift20 is required to traffic β1 integrin through Rab5 endosomes ........................................ 86 4.10 Ift20 is required for the in vivo mobilization of epidermal stem cells in response to epidermal injury ........................................................................................................................ 91 4.11 Ift20 is required for stem cell migration from skin explants taken from wound biopsies 97 ii 4.12 Discussion ......................................................................................................................... 97 Conclusion .................................................................................................................................. 103 References ................................................................................................................................... 109 Appendix A: Methods and Materials .......................................................................................... 116 Appendix B: Abbreviations ........................................................................................................ 128 iii List of Charts, Graphs, Illustrations Figure 1.1 Schematic of sagittal section of skin…………………………………………………..4 Figure 1.2 Illustration of the hair cycle……………………………………………………………8 Figure 1.3 Schematic of sagittal section of skin during wound healing…………………………14 Figure 1.4 Schematic of epidermal development………………………………………………..19 Figure 1.5 Schematic of intraflagellar transport…………………………………………………27 Figure 2.1 Characterization of E14.5 Arl13b-mCherry; Centrin 2-GFP epidermis……………..33 Figure 2.2 Characterization of E15.5 Arl13b-mCherry; Centrin 2-GFP epidermis……………..34 Figure 2.3 Characterization of P1 Arl13b-mCherry; Centrin 2-GFP epidermis…………………36 Figure 2.4
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