Hair Follicle Specific ACVR1/ALK2 Critically Affects Skin

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between this versionandtheVersionrecord. Pleasecitethis articleasdoi:10.1111/wrr.12549. through thecopyediting, typesetting, paginationandproofreadingprocess, whichmayleadtodifferences This istheauthormanuscript acceptedforpublicationandhasundergone full peerreviewbuthasnotbeen National Endowment Award, the Association for Acade for Association the Award, Endowment National grants. B.L. received funding from NIH/NIGMS -K08GM109105-0 NIH/NIGMS from funding B.L. received [email protected] E-mail: 4 3 2 1 Surgery Foundation, American College of Surgeons. A Surgeons. of American College Foundation, Surgery Ann Arbor, MI 48109-0219 Arbor, Ann 48109-0219 MI 5340 SPC Center Taubman 2130 Drive Center Medical East 1500 System Health Michigan Universityof Departmentof Surgery Levi, MD Benjamin Association for the Surgery of Trauma Research & Ed & Research Trauma for of Surgery the Association David Fireman David Albert Einstein College of Medicine, Department of Department Medicine, of College Einstein Albert Schoo Sciences, Material and Biologic of Department Dep Surgery, Reconstructive and Plastic of Division CorrespondingAuthor: Michael SorkinMichael Hair Follicle Specific ACVR1/ALK2 Critically Affect Critically ACVR1/ALK2 Specific Follicle Hair

Accepted Article Healing Wound Attenuates and Morphogenesis 1

, John Li, John ,

1 , Shailesh Agarwal Shailesh ,

1 , Bin ZhaoBin , 1 , Kavitha Ranganathan Kavitha , artment of Surgery, University of Michigan, Ann Arb Ann Michigan, of University Surgery, of artment Genetics, Bronx, NY NY Bronx, Genetics, l of Dentistry, University of Michigan, Ann Arbor, Arbor, Ann Michigan, of University Dentistry, of l 3 , Yuji Mishina Yuji ,

ucation Foundation Scholarship, Plastic Plastic Scholarship, Foundation ucation .S and K.R. received NIF F32 Fellowship F32Fellowship NIF received and K.R. .S mic Surgery Roslyn Award American Roslyn Surgery mic , Plastic Surgery Foundation Foundation Surgery Plastic , 1 , Shawn Loder Shawn , 2 , , Cederna Paul 1 , David Cholok David , 1 , Benjamin LeviBenjamin , s Skin Skin s MI or, MI MI or, 1 , , 1,4 1,4

The bone morphogenic protein signaling (BMP) is int is (BMP) signaling protein Themorphogenic bone Abstract

healing. wound cellniche and regulation of stem cells through activation of BMP BMP of activation through cells of stem regulation demonstrate that hair follicle specific ALK2 is int is ALK2 specific follicle hair that demonstrate with targeted overexpression of the BMP receptor AL receptor BMP of the targeted overexpression with overexpression of ALK2 resulted in attenuation of c of attenuation in resulted of ALK2 overexpression critical role in cutaneous homeostasis and regenera homeostasis cutaneous criticalrole in evaluation demonstrated significant dysregulation i significantdysregulation demonstrated evaluation increased prevalence of CD34 and ITGA6fol and positive CD34 of prevalence increased characterize its role in skin development and postn development skin role in its characterize localization. Mutant follicles were found to exhibi follicles to found were Mutant localization. These demonstrated increased numbers of individual of individual numbers increased Thesedemonstrated

Accepted Article

This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration ricately involved in maintenance of the stem stem of the maintenance in ricatelyinvolved t elevated proliferative activity as well as as well activity elevated t proliferative atal wound healing. Initial histologic histologic Initial healing. atalwound tion. Here, we utilize a novel mouse model model mouse a novel Here, utilize we tion. n hair follicle morphogenesis in mutant mice. mice. mutant in morphogenesis hair follicle n receptors. Hair follicle stem cells play a play cells stem follicle receptors. Hair utaneous wound healing. These findings These findings healing. wound utaneous hair follicles with altered morphology and morphology altered with hairfollicles ricately involved in the quiescence and quiescence ricatelythe in involved K2/ACVR1 in hair follicle stem cells, to to cells, stem hair follicle in K2/ACVR1 licle stem cells. Interestingly, constitutive constitutive Interestingly, cells. stem licle 2

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NFATc1 through cyclosporine A as well as geneticde as Aas well cyclosporine through NFATc1 been implicated to play an important role in this p this role in important an play to beenimplicated follicle morphogenesis proceeds through a sequence through proceeds morphogenesis follicle follicle. These bulge stem cells are marked by exp are by cells marked stem These bulge follicle. maintained through self-renewal anddifferentiation through self-renewal maintained which reside within the hair bulge, a morphological bulge, hair the reside which within factors can also affect wound healing (9). (9). healing wound affect also factors can adult stem cell populations (1). Hair follicles rep follicles (1). Hair cell populations stem adult (catagen) and stasis (telogen) (3, 4). The hair cyc hair (3,4). The (telogen) (catagen)and stasis dysfunctional hair follicle morphogenesis as well a as well morphogenesis hair follicle dysfunctional hair stem cell regulation and quiescence are still still are and quiescence cellregulation hairstem (5). (5). Introduction Introduction

follicle morphogenesis, and that deletion of Smad1/ deletion that and morphogenesis, follicle activation and precocious entry into the anagen pha entry the into and precocious activation regulation, and wound healing, we utilized a transg utilized we healing, and wound regulation, and Smad 5, both key effectors of the canonical BMP canonical key effectorsof the both 5, andSmad maintenance of hair bulge stem cells that are predo are that cells stem hair bulge maintenanceof constitutive expression of the BMP receptor ALK2/AC of receptor BMP the expression constitutive hair bulge cell specific promoter NFATc1. A better A better NFATc1. promoter cellspecific hair bulge NFATc1, a calcium sensitive transcription factor, h factor, transcription sensitive a calcium NFATc1, To more clearly define the role of BMP on hair foll on BMP role the of define moreclearly To

Accepted Article of an microenviron intricate composed is skin Human

This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration resent a unique source of stem cells that are that cells stem of source a unique resent unknown, multiple signaling pathways have have signaling pathways multiple unknown, le itself is predominantly regulated by cells, regulatedcells, by predominantly is itself le rocess. Kandyba et al have shown that Smad 1 1 Smad that shown have al et rocess.Kandyba ression of CD34, Itga6, Lhx2, Tcf3, and Sox9 Tcf3, and Sox9 Lhx2, Itga6, of CD34, ression s postnatal hair shaft differentiation (8). These (8). hair differentiation shaft postnatal s minantly quiescent. In fact, inhibition of Ininhibition fact, quiescent. minantly enic mouse model that is characterized by by characterized is that model mouse enic se (6, 7). While the exact mechanisms driving mechanisms exact the se(6,7). While during the hair cycle (2). In this process, hair In process,hair this (2). cycle during hair the ly distinct compartment of the mature hair matureof hair the compartment lydistinct 5 or the BMPR1a receptor results in in results receptor BMPR1a or the 5 understanding of these processes may of these understanding of proliferation (anagen), destruction destruction (anagen), of proliferation pathway, are required for undisturbed for undisturbed required are pathway, letion causes premature cell cycle cellcycle premature causes letion VR1 under the conditional control of the control under conditional the VR1 icle stem cell morphology, cycle cycle cellmorphology, iclestem as previously been implicated in the the in beenimplicated as previously ment that provides niches to several several to niches provides that ment 3

provide the basis for developing therapeutic approa therapeutic for developing basis the provide healing. wound

Accepted Article

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photometric analysis with ImageJ software (NIH, BetImageJsoftware (NIH, with analysis photometric Animals Animals promoter (10). Eight-week old female mice were used female were mice old (10). Eight-week promoter in overexpression of ALK2 in the setting of Cre rec setting ofthe Cre in of ALK2 overexpression in Methods Methods

mutant littermate controls that did not carry not N the did that controls littermate mutant dressing was removed each time and then replaced. W replaced. and then time each wasremoved dressing evaluated every other day obtaining standardized ph standardized day obtaining everyother evaluated were performed in accordance with the guidelines in guidelines the with accordance in were performed subcutaneous injection every 12 hours for the first the for hours 12 every injection subcutaneous Buprenex; Reckitt Benckiser Pharmaceuticals Inc,Ri BenckiserPharmaceuticals Reckitt Buprenex; and littermate control mice (n=8) underwent the sam the underwent (n=8) mice control andlittermate Laboratory Animals from the InstituteLaborator for the from LaboratoryAnimals an occlusive Tegaderm dressing (3M, Maplewood, MN). Maplewood, (3M, dressing anTegaderm occlusive suture to prevent skin contraction from the pannicu the from contraction preventskin to suture approved by the Institutional Animal Care and use C Care Institutionaland use Animal approved the by midline. Round shaped silicone rings were applied t applied were rings shaped silicone Round midline. removed, and two 6 mm full thickness circular wound circular thickness full mm 6 removed,and two Mice were anesthetized according to protocol using using according protocol to anesthetized Micewere Injury Skin (PRO0001553).

Accepted Articlemod mouse a transgenic developed have previously We

This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration FATc1 cre transgene. All animal procedures procedures animal All creFATc1transgene. 72 hours after injury. Wound healing was healing after Wound hours 72 injury. lus carnosus. Wounds were then covered with with covered were then carnosus. Wounds lus ombinase expression driven by the NFATc1 NFATc1 bythe driven expression ombinase o the wounds and secured with 6-0 Nylon 6-0 Nylon with and secured wounds the o otographs. For this purpose, the occlusive occlusive the purpose, For otographs. this y Animal Research (ILAR, 2011) and were andwere 2011) (ILAR, yResearch Animal inhaled isofluorane. Dorsal hair was Dorsal isofluorane. inhaled the Guide for the Use andCare of for Guide Use the the e treatment. Buprenorphine (0.01 mg/kg, (0.01 Buprenorphine e treatment. ommittee of the University of Michigan Universityof the of Michigan ommittee chmond, VA) was administered by VA) was administered chmond, hesda, MD). MD). hesda, s were created equidistant from the the from createdequidistant were s for all aspects of this study with non- study with of this foraspects all ound healing curves were generated using using generated were healing ound curves caALK2 NFATc1 mutant mice (n=8) mice mutant NFATc1 caALK2 el that carries a mutation resulting acarries mutation that el 5

Histology Histology Flow cytometry cytometry Flow FACS Aria (BD Biosciences) and subsequently analyze and subsequently Biosciences) Aria (BD FACS Statistical significance was determined using the s the using determined significance was Statistical analysis Statistical OR). significant. consideredstatistically (eBioscience) and Itga6 (eBioscience) antibodies. F antibodies. Itga6(eBioscience) and (eBioscience) described, including H&E and immunohistochemistry f and immunohistochemistry H&E described,including type 1 collagenase solution (Sigma-Aldrich, St. Lou St. (Sigma-Aldrich, solution collagenase type 1 Images were taken with a Nikon E-800 upright micros upright E-800 Nikon a with Imagestaken were

Dorsal skin was harvested from mutant and littermat mutant from was harvested Dorsalskin brightmicroscopy. field and40x 24 hours, and embedded in paraffin prior to section to paraffinin prior and embedded hours, 24 described. Dorsal skin from mutant and littermate control mice control and littermate mutant from Dorsalskin

Acceptedand enzy minced, finely dissected, were Thetissues Article

This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration tudent’s unpaired tudent’s ing. Histology was performed as previously as previously was performed ing. Histology low cytometry was performed on a BD a on performed was cytometry low is, MO) and the stained using CD34 using CD34 stained MO) and the is, e control mice (n=3 per group) as previously group)as previously per (n=3 mice econtrol cope (Nikon, Melville, NY) utilizing 10x 10x NY)utilizing Melville, (Nikon, cope or pSMAD and Ki67 (Santa Cruz, CA). CA). Cruz, (Santa andKi67 or pSMAD d using FloJo software (Tristar, Ashland, (Tristar,Ashland, software FloJo using d was harvested, fixed in formalin for formalin in fixed was harvested, matically digested in a 0.075% a 0.075% in digested matically t -test. A -test. p -value < 0.05 was < 0.05 -value 6

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proliferative anagen phase of the hair cycle andma cycle hairof the anagen phase proliferative phenotypic differences when compared to littermate littermate to compared when phenotypicdifferences arrested in this phase and cannot transition furthe transition cannot and phase arrestedthis in in color and more disoriented in alignment. Further alignment. in colorand moredisoriented in skin, we utilized a double fluorescent reporter mou a reporter double fluorescent we utilized skin, H&E staining, we found that the number of hair foll of hair number the that found we H&Estaining, when treated with depilatory cream indicating deepe indicating cream depilatory with whentreated ( cre mice. In the resulting mice, all cells that exp that cells all mice, In resulting the cre mice. significantly increased ( increased significantly all other cells are red. Interestingly, we were abl we were Interestingly, red. are othercells all Constitutive expression of caALK2 affects hair foll hair caALK2 affects of expression Constitutive Results

Over-activation of the BMP pathway results in aberr in results BMPthe pathway of Over-activation cells within the hair follicle and specifically the and specifically hair the follicle within cells cell rich clusters localizing in the subcuticular f subcuticular the in localizing cellrich clusters stem cell maintenance and wound healing healing wound maintenanceand stemcell in the mutant mice throughout the hair follicle and follicle hair the throughout mice mutant the in subcuticular layer as compared to the controls controls the to as compared layer subcuticular Figure 1d Figure Mutant mice overexpressing ALK2 within NFATc1 posit NFATc1 within ALK2 overexpressing mice Mutant

Immunofluorescence performed on the skin demonstrat skin the on performed Immunofluorescence

Acceptedlaye this in of hair follicles ).The localization Article Figure 1c Figure This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration ) and, the hair follicles appeared highlydysmorphi follicles )hair and,the (Figure 2a) (Figure at just superficial to the panniculus carnosus panniculus the superficial to just at hair bulb bulb hair ress the NFTAc1 gene were labeled green, while labeled genewere NFTAc1 the ress e to directly localize the NFATc1 expressing expressing NFATc1 the directly to localize e r. In order to identify NFATc1 expression in the the in expression In NFATc1 identify to r. order within the hair bulb located in the deeper deeper the located in hair the bulb within se model that was cross-bred with NFATc1- with was cross-bred that semodel y indicate that the mutant hair follicles are are hair follicles ymutant the that indicate more, mutant mice did not fully lose their hair fullyhair their lose not did mice mutant more, icles observed in mutant skin was skin mutant observed icles in r-rooted hair follicles ( follicles r-rootedhair controls. Their hair was noted to belighter to noted was Their hair controls. icle morphology. iclemorphology. ant regulation of cell proliferation, cellproliferation, of regulation ant r is commonly observed during the the during observed commonly ris (Figure 1e) (Figure . . ed increased pSMAD expression expression pSMAD edincreased ive cells demonstrated distinct distinct demonstrated cells ive . . Figure1a c with large large cwith ). Based on ).on Based 7

by these markers was increased in the mutant group, mutant the in was increased bythesemarkers NFATc1 cells cells NFATc1 contraction. We observed a significant attenuation attenuation a significant observed We contraction. mice compared to the controls, which resulted in de in resulted which controls, the to compared mice

region of the anagen hair follicles in the mutant m mutant the in follicles hair anagen of region the After isolating cells from the skin of mutant and c of mutant skin the from cells After isolating

healing, we utilized a well-established mouse model mouse a well-established wehealing,utilized evaluated the expression of Integrin 6 and CD34 wit Integrin and CD34 6 of expression evaluated the 2c) markers have been shown to represent hair follicle follicle represent to hair shown been markershave . Furthermore, expression of proliferation marker K marker of proliferation expression Furthermore, .

Accepted Article2b) (Figure

This article isprotected by copyright. All rights reserved. . To assess the effect of caALK2 constitutive expre constitutive caALK2 effect of the assess To . Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration ontrol mice using flow cytometry, we we cytometry, using mice flow ontrol ice and co-localized with GFP positive GFP positive with iceand co-localized of the wound healing process in the mutant mutant the in process healing wound of the stem cells. The number of cells as identified as ofidentified cells The number cells. stem layed wound healing ( healing layedwound that prevents healing by primary primary by preventshealing that hin these cell populations, sincethese thesecell populations, hin although not statistically significant significant statistically although not i67 was also localized to the hair bulb hair the bulb to localized also was i67 Figure2d ssion on wound wound on ssion ). ). (Figure (Figure 8

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hair follicle resulted in an increased number and s number increased an in resulted hairfollicle morphology and function. Our results are consistent are Our results function. and morphology the importance of homeostasis within the hair the cycle within of homeostasis importance the hair follicle. In this study, we demonstrate that A we that demonstrate Instudy, this hairfollicle. function and hair loss in mice (12). The adult hair adult (12). mice The in and hair loss function demonstrate that BMP and Wnt signaling important is Wnt and BMP that demonstrate segment, and a lower basal layer that undergoes int layerundergoes that basal anda lower segment, follicle stem cells located within the bulge.the While within located cells stem follicle skin in that an imbalance of these factors results results of thesefactors an imbalance that in skin cycle. The anagen phase requires local activation o activation requireslocal phase anagen cycle.The A balance of numerous signaling pathways determines pathways signaling of numerous A balance Discussion

follicle was increased in our mutant model, this do this model, mutant our in was increased follicle follicle (11). A disturbance in homeostasis within within homeostasis in (11). Adisturbance follicle al showed a similar indirect relationship between h between relationship indirect a showed al similar follicle, and BMP promotes quiescence of the stem c stem of the quiescence promotes and BMP follicle, the regenerative function and ability to transition andto ability function regenerative the of ceramide synthase 4 inhibition; loss of ceramide loss inhibition; 4 synthase ofceramide result of overexpression of Alk2. The mechanism by The mechanism of Alk2. of overexpression result BMP and Wnt signaling, resulted in inappropriate ac inappropriate in resulted signaling, and Wnt BMP likely involves a direct interaction between Alk2 a Alk2 between interaction a direct likelyinvolves human hair follicle stem cells with enlargement of of enlargement with cells stem hair follicle human

Acceptedre of Alk2 stabilization specifically, Inmodel our Article

This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration in a prolonged anagen phase within the hair the within phase anagen a prolonged in through the hair cycle is likely inhibited as a likely inhibited is hair cycle through the LK2 overexpression within NFATc1 cells of the cells NFATc1 within overexpression LK2 follicle consists of an upper permanent permanent of an upper consists follicle the number and size of each individual hair individual of each and size number the ize of individual follicles with an aberrant with follicles of individual ize these pathways results in loss of stem cell of stem loss in results these pathways nd the bulge stem cells specifically. Peters et. et. Peters specifically. cells bulgethe nd stem es not directly correlate with function. In fact,function. with correlate directly esnot air follicle number and function in the setting the in andfunction number airfollicle f hair follicle cells within the bulge of the hair the bulgethe of within cells fhair follicle the sebaceous glands (12). Gnedeva et al. al. et Gnedeva glands(12). sebaceous the synthase 4, a factor that is also affected also by is that afactor synthase 4, ermittent growth and regression with each with andregression growth ermittent and tissue regeneration. Rishikaysh et al. al. et Rishikaysh regeneration. and tissue with those of previous studies that highlight that studies of previous those with tivation and subsequent exhaustion of exhaustion and subsequent tivation ells within the hair follicle niche (8, 13). niche (8,13). hair the follicle within ells which this occurs is unclear to date, but but date, to unclear is occurs this which for maintaining the barrier function of function barrier the for maintaining the architecture and organization of the organization and architecture the sulted in quiescence of the local hair local the quiescence in of sulted 9

populations within the hair follicle niche, we can niche,we hair the follicle within populations cell populations within the U itself. hair within follicle cell populations expression of Integrin 6 and CD34 was increased in in was increased CD34 Integrinand 6 of expression mice. Such differences may explain why the mutant why mutant the explain may differences Such mice. maintenance. Furthermore, modulation of the hair fo hairof the modulation Furthermore, maintenance. bala an important Thus, (14). origin dermal papilla eliminate completely dorsomorphin inhibitor BMP the fashion a homeostatic in signaling BMP that showed

hair follicle cycle maintenance and tissue regenera andtissue maintenance cycle hairfollicle link between hair follicle morphogenesis, function, morphogenesis, follicle betweenhair link While our findings suggest the importance of the BM of the importance the findingsour suggest While in depth histologic and biomolecular analysis of th analysis and biomolecular histologic depth in overexpression of Alk2, additional studies are requ studies additional of Alk2, overexpression regeneration. to understand the underlying mechanism. Further stu Further mechanism. underlying the understand to relationship between these variables. Byunderstand variables. betweenthese relationship

Acceptedof hair follicle apparent function is that it While Article

This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration nder the influence of stabilized Alk2, the the Alk2, of stabilized influence the nder harness these findings to optimize tissue optimize findingsto these harness tion in comparison to the control mice (6). (6). mice control the to comparison in tion e phases of wound healing is required in order order in required is healing wound eof phases nce is required for proper hair cycle hair proper for required nce is and tissue regeneration. regeneration. and tissue ired to more clearly delineate a direct a direct delineate moreclearly iredto ing the role of Alk2 on local stem cell stem local on Alk2 ingrole the of the mutant mice compared to the control control the to compared mice mutant the llicle cycle is affected by the local niche and niche bylocal the cycleaffected is llicle dies will therefore be needed to establish a establish to therefore needed will dies be stem cells have altered function in terms of terms in function altered have cells stem is critical for hair growth, and that use ofuse and that growth, criticalfor hair is P pathway on gross wound healing, a more healing, wound gross pathwayon P d hair-inducing activity from cells of activitycells from hair-inducing d stem cells is affected by the affectedthe by is cells stem 10 Page 10of15 Page 11of15

photographs demonstrate attenuated wound closure in closure attenuated wound demonstrate photographs Figure 1: NFATc1 regulated caALK2 overexpression re caALK2 overexpression regulated Figure NFATc1 1: Legends: Figure

< p 0.05. * SEM. asmean ± Photographs of NFATc1/caALK2 mutant mice and litter and mice mutant NFATc1/caALK2 ofPhotographs cells expressing CD34 and Itga6. healing (D) Wound and CD34 expressing cells staining of adult skin with quantification of h (C) quantification with skin ofstaining adult wound healing. (A) Immunofluorescence for pSmad 1/5 for pSmad Immunofluorescence (A) healing. wound Immunofluorescence of skin and hair follicle in dou in and hair follicle of skin Immunofluorescence (B) Immunofluorescence for proliferation marker Ki6 marker for proliferation Immunofluorescence (B) Cre/ROSA26mTmG. Values are expressed as mean ± SEM. ± as mean expressed Values are Cre/ROSA26mTmG. Figure 2: Hair follicle ALK2 critically regulates c regulates critically ALK2 follicle FigureHair 2:

Accepted Article

This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration air follicle number and (D) hair follicle size. (E) size. hair follicle (D) and number follicle air ell proliferation, stem cell maintanence and maintanence and cell stem proliferation, ell ble transgenic reporter mouse NFATc1- mouse transgenic reporter ble curve and corresponding representative representative corresponding and curve 7. (C) Flow cytometry quantification of quantification Flow(C) cytometry7. mutant mice (n=8). Values are expressed expressed Valuesare (n=8). mice mutant sults in aberrant hair morphology. (A) (A) morphology. hair aberrant in sults /8 in adult mutant and wild-type skin. skin. mutant andwild-type adult in /8 mate controls after depilation. (B) HE(B) depilation. after controls mate * p < 0.05. < p 0.05. * 11

9. Lewis CJ, Mardaryev AN, Poterlowicz K, Sharova T Sharova K, Poterlowicz Mardaryev AN, CJ, Lewis 9. 2007;104(24):10063-8. A. S U Sci morphogenetic protein signaling suppresses wound-in suppresses signaling protein morphogenetic keratinocyte proliferation and migration. J Invest Invest J migration. and proliferation keratinocyte Lhx2 differentially regulates Sox9, Tcf4 and Lgr5 i Tcf4 Lgr5 Sox9, and regulates Lhx2differentially follicle stem cells in the absence of bone morphoge of bone absence the in cells stem follicle 1. Gurtner GC, Werner S, Barrandon Y, Longaker MT. MT. Longaker Y, Barrandon S, Werner GC, Gurtner 1. References:

epidermal regeneration after injury. Development. 2 Development. injury. after regeneration epidermal 4. Hsu YC, Li L, Fuchs E. Emerging interactions bet EmergingE. interactions L,Fuchs Li YC, Hsu 4. 2001;117(1):3-15. Dermatol. Invest J cyclestages. quiescence and proliferation of skin stem cells. cells. Ce stem of skin proliferation and quiescence 2. Alonso L, Fuchs E. The hair cycle. J Cell Sci. 2 CellSci. The J cycle. hair E. Fuchs L, Alonso 2. 2008;453(7193):314-21. Nature. 6. Keyes BE, Segal JP, Heller E, Lien WH, Chang CY, Chang WH, Lien Heller E, Segal JP, BE, Keyes 6. 5. Mardaryev AN, Meier N, Poterlowicz K, Sharov AA, Sharov K, Poterlowicz N, Meier MardaryevAN, 5. 2014;20(8):847-56. Nat Med. niches. 8. Kobielak K, Stokes N, de la Cruz J, Polak L, Fuc de L, N, Polak Cruz la Stokes J, K, Kobielak 8. 3. Muller-Rover S, Handjiski B, van der Veen C, Eic C, der van Veen B, Handjiski Muller-RoverS, 3. aging in hair follicle stem cells. Proc Natl Acad S Acad Natl Proc cells. stem follicle hair in aging A comprehensive guide for the accurate classificati accurate forthe guide Acomprehensive 7. Horsley V, Aliprantis AO, Polak L, Glimcher LH, LH, Glimcher L, Polak AO, Aliprantis V, Horsley 7.

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This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration ci U S A. 2013;110(51):E4950-9. 2013;110(51):E4950-9. A. S U ci Dermatol. 2014;134(3):827-37. 2014;134(3):827-37. Dermatol. ll. 2008;132(2):299-310. 2008;132(2):299-310. ll. n hair follicle stem cells to promote promote to cells stem follicle hair n netic protein signaling. Proc Natl Acad Natl Proc signaling. protein netic on of murine hair follicles in distinct hair distinct in follicles hair of murine on 006;119(Pt 3):391-3. 006;119(Pt3):391-3. 011;138(22):4843-52. 011;138(22):4843-52. hs E. Loss of a quiescent niche but not not niche but quiescent of a Loss E. hs Fuchs E. NFATc1 balances NFATc1 balances E. Fuchs hmuller S, Foitzik K, McKay IA, et al. al. McKay et IA, K, Foitzik hmullerS, ween skin stem cells and their their and cells stem skin ween duced skin repair by inhibiting inhibiting by repair ducedskin Guo X, et al. Nfatc1 orchestrates orchestrates Nfatc1 al. et Guo X, Y, Aziz A, Sharpe DT, et al. Bone Bone al. et DT, Aziz Sharpe Y, A, Sharova TY, Ahmed MI, et al. al. et MI, Ahmed TY, Sharova Wound repair and regeneration. regeneration. and Woundrepair 12 Page 12of15 Page 13of15

10. Agarwal S, Loder SJ, Brownley C, Eboda O, Peter O, Eboda Brownley C, SJ, Loder AgarwalS, 10.

signaling mediated by constitutively active Activin active constitutively by mediated signaling ectopic bone formation extremit formation distal localizedto bone ectopic 11. Rishikaysh P, Dev K, Diaz D, Qureshi WM, Filip Filip WM, Qureshi D, Diaz K, Dev P, Rishikaysh 11. follicle morphogenesis and development. Int J J S Int Mol development. and morphogenesis follicle 12. Peters F, Vorhagen S, Brodesser S, Jakobshagen Jakobshagen Brodesser S, S, Vorhagen F, Peters 12. Ceramide synthase 4 regulates stem cell homeostasis cell stem 4 regulates synthase Ceramide 13. Genander M, Cook PJ, Ramskold D, Keyes BE, Mert Ramskold BE, Cook Keyes PJ, D, M, Genander 13. 2015;135(6):1501-9. Dermatol. signaling and its pSMAD1/5 target genes differentia genes pSMAD1/5 target its and signaling 14. Gnedeva K, Vorotelyak E, Cimadamore F, Cattaros F, Cimadamore E, Vorotelyak K, Gnedeva 14. Cell. 2014;15(5):619-33. CellStem lineages. Derivation of hair-inducing cell from human pluripo from cell human of hair-inducing Derivation 2015;10(1):e0116892.

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This article isprotected by copyright. All rights reserved. Manuscript underreview-CONFIDENTIAL Wound Repair andRegeneration type 1 receptor (ACVR1) results in in (ACVR1) results 1 receptor type y joints. Dev Biol. 2015;400(2):202-9. 2015;400(2):202-9. Biol. Dev yjoints. ci. 2014;15(1):1647-70. 2014;15(1):1647-70. ci. lly regulate hair follicle stem cell cell stem follicle llyhair regulate tent stem cells. PLoS One. PLoS One. cells. stem tent and hair follicle cycling. J Invest Invest J cycling. follicle hair and S, Mokry J. Signaling involved hair in Signaling Mokry S, J. K, Bruning JC, Niessen CM, et al. et CM, Niessen JC, Bruning K, son JR, Hayano S, et et al. BMP S, Hayano JR, son si G, Giusto E, Terskikh VV, et al. al. Terskikh et VV, E, Giusto G, si z AF, Sandberg R, et al. BMP BMP et al. R, Sandberg zAF, 13

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