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Hedgehog-Wnt Interactions During Pathologic Epithelial Bud Development and Skin Tumorigenesis

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HEDGEHOG-WNT INTERACTIONS DURING PATHOLOGIC EPITHELIAL BUD DEVELOPMENT AND SKIN TUMORIGENESIS by Hoseong Yang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in The University of Michigan 2008 Doctoral Committee: Professor Andrzej A. Dlugosz, Chair Professor Eric R. Fearon Professor Deborah L. Gumucio Associate Professor Thomas M. Glaser Associate Professor Theodora S. Ross © Hoseong Yang 2008 Acknowledgements I am grateful to have had a wonderful mentor and committee chair, Anj Dlugosz. I thank him for all his endless support, training, ideas, guidance, friendship, and instilling his tireless love and passion for science. Anj gave me the freedom and autonomy to explore and develop my own ways to approach a research problem, but yet his door was always open and he was always there to provide incredible amounts of guidance and advice. He truly is a wonderful ‘boss’ to work for, and a colleague for years to come. Research is a life-long learning experience, and I have gained a strong foundation in the scientific method and the way to approach a research problem under his tutelage. I also have to thank my thesis committee members, Drs. Eric Fearon, Tom Glaser, Deb Gumucio, and Theo Ross. They have been incredibly supportive and I have appreciated their advice and guidance throughout my Ph.D. training. I am also incredibly indebted to all the wonderful members of the Dlugosz laboratory, both past and present: Marina Grachtchouk, Jenny Ferris, Evan Michael, Anna Wang, Jianhong Liu, Alex Ermilov, Lijyun Syu, Jim Diener, Monique Verhagen, and Mark Hutchin for all their support and wonderful memories. I also send out my special thanks to Karen Myers, Penny Morris, and Ellen Elkin for their assistance. I also would like to thank Dr. Sewon Kang for his generosity and especially for setting up a meeting with Anj during my M2 year in medical school. Special thanks to Drs. Elia Duh and Don Zack, my mentors at the ii Wilmer Eye Institute at Johns Hopkins School of Medicine, for providing mentorship and guidance years after I have left Hopkins. I also would like to thank Drs. Sarah Millar and Thomas Andl at U. Penn., who have been incredibly generous and helpful throughout my graduate career, and also for providing TRE-Dkk1 transgenic mice. I thank the Center for Organogenesis Training Grant and Deb Gumucio; the Department of Dermatology; and MSTP for the funding support. I am especially grateful for my parents, Seungjin and Jeongok Yang, for their support and endless love throughout my life and during my graduate career. They have sacrificed so much for my well-being, taught me the value of tirelessly working hard to achieve my goals, and led by example how to live life and always strive to be a better person. Thank you. iii Table of Contents Acknowledgements ...........................................................................................................ii List of Figures..................................................................................................................vii Abstract...................................................................................................................... ……x Chapter I Introduction Introduction………………………………………………………………………..1 Morphogenesis of the Hair Follicle: Wnt and Hh Pathways Play Sequential and Distinct Roles……………………………………………………………………...3 The Hair Cycle…………………………………………………………………...10 Hair Follicle Bulge: The Stem Cell Compartment………………………………14 Canonical Wnt Signaling………………………………………………………...16 Hedgehog Signaling……………………………………………………………...22 Wnt Signaling in Disease………………………………………………………...31 Hh Signaling in Disease………………………………………………………….33 Unanswered Questions…………………………………………………………...39 Summary…………………………………………………………………………40 Reference List……………………………………………………………………43 Chapter II Pathological responses to oncogenic Hedgehog signaling in skin are dependent on canonical Wnt/β-catenin signaling Summary…………………………………………………………………………56 Introduction………………………………………………………………………56 Materials and Methods…………………………………………………………...60 Transgenic mice and conditional transgene activation………………….60 Tissue harvesting and whole-mount reparation……...………………….61 Immunostaining…………………….…………………………………….62 Immunoblotting…………………………………………………………..63 RNA isolation and semi-quantitative RT-PCR…………………………...64 Results…………………………………………………………………………....66 Expression of similar lineage markers in human superficial BCCs and embryonic hair buds……………………………………………………..66 Ectopic Hh signaling in mouse epidermis produces superficial BCC-like downgrowths resembling embryonic hair buds………………………….68 Canonical Wnt signaling in human and murine Hh pathway-driven epithelial buds……………………………………………………………75 M2SMO-induced hair buds form follicular hamartomas instead of hair follicles…………………………………………………………………...79 Development of M2SMO-induced epithelial buds and hamartomas is blocked by Dkk1…………………………………………………….……85 Discussion………………………………………………………………………..95 iv Reference List………………………………………………………..…………100 Chapter III Forced activation of canonical Wnt/β-catenin signaling is sufficient to drive advanced hair follicle differentiation in normal hairless volar and M2SMO skin Introduction…………………………………………………………………..…107 Materials and Methods………………………………………………………….110 Generation of transgenic mice and transgene activation…………...….110 Tissue harvesting, whole-mount preparation and whole-mount Oil Red O staining………………………………………………………………….111 Semi-quantitative RT-PCR…...………….………………………...……112 Immunostaining and whole-mount immunoflourescence staining……………………………………………………………….…112 Results…………………………………………………………………………..113 Stabilized β-catenin induced in adult epidermis causes formation of functional ectopic hair follicles in multiple regions of skin…………….113 Normal hairless volar skin is competent to form mature hair follicle lineages following expression of stabilized β-catenin (βcat*)……...….117 βcat* expression results in preputial gland keratinization and differentiation………………………………………………………..…121 M2SMO-induced ectopic buds and hamartomas in volar skin retain the capacity to produce late-stage follicle lineages with expression of stabilized β-catenin……………………………………………………..123 Discussion………………………………………………………………………126 Reference List………………………………………………………..…………132 Chapter IV Epithelium-specific activation of Hh signaling is sufficient to reactivate growth of resting hair follicles Introduction…………………………………………………………………..…134 Materials and Methods………………………………………………………….137 Transgenic mice and conditional transgene activation…………...……137 Tissue harvesting……………………………………………………….138 Immunostaining…………………………………………………………138 Results…………………………………………………………………………..138 Epithelial-specific Hh activation by SmoA1 in telogen skin is sufficient to reactivate growth of resting hair follicles……………………………....138 Preferential expansion of outer root sheath cells near follicle bulge, the stem cell compartment, in SmoA1-induced growing hair follicles……..143 SmoA1 transgene is expressed in both ORS and matrix compartments, but distinct cell populations show differing responsiveness to SmoA1…….145 Expression of stem cell markers K15 and CD34 is lost over time in follicles reactivated to grow by SmoA1…………………….…………..149 Activation of Hh Signaling in the stem cell compartment does not induce anagen…………………………………………………………….…….155 Discussion………………………………………………………………………159 Reference List………………………………………………………..…………165 v Chapter V Heightened Hh signaling in epidermis stimulates ectopic pigment accumulation and melanogenesis Introduction…………………………………………………………………..…167 Materials and Methods………………………………………………………….172 Generation of transgenic mice………………………………………….172 Tissue harvesting, whole-mount preparation and whole-mount X-gal staining………………………………………………………….………172 Semi-quantitative RT-PCR……...………………………….……..…….173 Results…………………………………………………………………………..173 Heightened Hh signaling in epidermis stimulates melanogenesis……...174 Summary...……………………………………………………………………...178 Reference List………………………………………………………..…………181 Chapter VI Summary and Future Directions Hh-Wnt crosstalk in Hh-driven skin pathology………………………………...184 β-catenin drives advanced stages of hair follicle differentiation…………...…..195 Epithelial Hh signaling reactivates growth of resting hair follicles…………….196 Pathologic Hh signaling and melanogenesis……………………………………199 Reference List………………………………………………………..…………202 vi List of Figures Figure 1-1. Model for hair follicle morphogenesis……………………………………….9 Figure 1-2. The hair cycle…………………………………………………………….....13 Figure 1-3. The canonical Wnt signaling pathway……………………………………...20 Figure 1-4. Dkk1 and Kremen inhibit canonical Wnt/β-catenin signaling by removing LRP6 from the cell surface………………………………………………………………21 Figure 1-5. The Hedgehog signaling pathway…………………………………………..30 Figure 1-6. Models of Hh pathway activation in human cancers……………………….38 Figure 2-1. Human superficial basal cell carcinoma expresses hair bud lineage markers………………………………………………………………………………….. 68 Figure 2-2. Hairless region of mouse volar skin………………………………………...71 Figure 2-3. Ectopic Hh signaling in M2SMO-expressing hairless mouse skin drives superficial BCC-like downgrowths resembling hair buds……………………………….72 Figure 2-4. Ectopic Hh-induced buds in hairless volar skin express hair bud markers K17 and Epcam………………………………………………………………………………..73 Figure 2-5. Mouse hair bud cellular compartments and epithelial lineage markers. …...74 Figure 2-6. M2SMO-induced ectopic epithelial buds express hair bud lineage markers…………………………………………………………………………………...75
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