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THY1-mediated mechanisms converge to drive YAP activation in skin homeostasis and repair

Nature Cell Biology volume 24, pages 1049–1063 (2022)Cite this article

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Abstract

Anchored cells of the basal epidermis constantly undergo proliferation in an overcrowded environment. An important regulator of epidermal proliferation is YAP, which can be controlled by both cell–matrix and cell–cell interactions. Here, we report that THY1, a GPI-anchored protein, inhibits epidermal YAP activity through converging molecular mechanisms. THY1 deficiency leads to increased adhesion by activating the integrin-β1–SRC module. Notably, regardless of high cellular densities, the absence of THY1 leads to the dissociation of an adherens junction complex that enables the release and translocation of YAP. Due to increased YAP-dependent proliferation, Thy1–/– mice display enhanced wound repair and hair follicle regeneration. Taken together, our work reveals THY1 as a crucial regulator of cell–matrix and cell–cell interactions that controls YAP activity in skin homeostasis and regeneration.

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Fig. 1: Deletion of Thy1 increases epidermal proliferation in adult mice.
Fig. 2: Loss of Thy1 enhances epidermal YAP activation.
Fig. 3: THY1 regulates ITGβ1 activation and cellular adhesion.
Fig. 4: THY1 controls the release of YAP from AJs.
Fig. 5: Loss of Thy1 enhances wound repair and regeneration.
Fig. 6: Increased migration during wound healing of Thy1–/– mice.
Fig. 7: Inhibition of YAP abrogates Thy1–/–-dependent phenotypes.
Fig. 8: Thy1–/––dependent phenotypes are predominant in the epidermis.

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Further data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We apologize to colleagues in the field whose important works we could not cite owing to space constraints. We thank all members of the Fuchs Lab as well as V. Sedov and O. Sedova for helpful comments; V. Zlobin at the PCRA for providing animal care; E. Barak at the Technion Flow Cytometry Unit for assisting with FACS experiments; and M. Allabi and K. Nafe for their warm support. Y.F. is the Deloro Chair and is funded by ICRF (2028184) and ISF (2027619) grants. Images were generated using BioRender software.

Author information

Author notes

  1. Egor Sedov

    Present address: Program in Epithelial Biology and Department of Dermatology, Stanford University School of Medicine, Stanford, CA, USA

  2. Elle Koren

    Present address: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA

Authors and Affiliations

  1. Laboratory of Stem Cell Biology and Regenerative Medicine, Department of Biology, Technion Israel Institute of Technology, Haifa, Israel

    Egor Sedov, Elle Koren, Sucheta Chopra, Roi Ankawa, Yahav Yosefzon, Marianna Yusupova, Alona Feldman & Yaron Fuchs

  2. Lorry Lokey Interdisciplinary Center for Life Sciences and Engineering, Technion Israel Institute of Technology, Haifa, Israel

    Egor Sedov, Elle Koren, Sucheta Chopra, Roi Ankawa, Yahav Yosefzon, Marianna Yusupova, Lucien E. Weiss, Alona Feldman, Yoav Shechtman & Yaron Fuchs

  3. Department of Biomedical Engineering, Technion Israel Institute of Technology, Haifa, Israel

    Lucien E. Weiss & Yoav Shechtman

  4. Department of Engineering Physics, Polytechnique Montreal, Montreal, Quebec, Canada

    Lucien E. Weiss

  5. Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Adnan Mahly, Arad Soffer & Chen Luxenburg

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Contributions

E.S., E.K. and Y.F. designed and analysed the experiments. E.S., E.K. and S.C. performed the experiments. Y.Y. provided technical assistance throughout the project. L.E.W. and Y.S. performed TIRF imaging. R.A., A.M., A.S. and C.L. provided assistance and performed in utero microinjections. M.Y. and A.F. assisted in lentiviral construct designs. Y.F. conceived and supervised the project. E.S., E.K. and Y.F. wrote the manuscript.

Corresponding author

Correspondence to Yaron Fuchs.

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Nature Cell Biology and the authors thank Valerie Horsley, Barry Thompson, and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Thy1-/- mice display increased numbers of hair follicles, but no difference in hair follicle cycling.

a,b, Quantifications for number (a) and distance (b) between adult (P56) dorsal hair follicles (HFs) (n = 5 mice). c, Immunofluorescence for KRT15 on tail skin wholemounts from adult (P56) wild type (WT) and Thy1-/- mice. d,e, Quantifications for average number (d) and distance (e) of tail skin HFs (n = 5 mice). f, Schematic of HF cycling after waxing of adult telogenic (P56) mice. Ana, anagen. g-i, Photographs of depilated WT and Thy1-/- mice at day 1 (g), day 5 (h), and day 9 (i) post waxing (n = 3 mice). j, Immunofluorescence for KRT5 on dorsal skin sections from WT and Thy1-/- mice at day 9 post waxing when mice are in late anagen (Ana-VI). k, Brightfield images of WT and Thy1-/- anagenic HFs. Dashed lines demarcate HFs. l, HF length at day 9 post waxing (n = 7 mice). All images and quantifications are representative of at least n = 3 mice per genotype, unless otherwise indicated. Quantifications were performed on fields of ≥ 60 samples ranging from 100-200μm, unless otherwise specified. Error bars indicate mean ± s.e.m. Scale bars, 50 μm (k), 100 μm (j) or 200 μm (c).

Source data

Extended Data Fig. 2 Loss of THY1 leads to enhanced YAP activation.

a, Representative confocal images of dorsal skin sections from wild type (WT; shown in inset) and Thy1-/- mice stained for pan-YAP and ITGβ4 (CD104). White arrows indicate nuclear YAP. b, YAPactive signal was normalized using β-actin as a loading control (relating to Fig. 2c) (n = 3 independent blots). c., Signal of pYAPS127 level normalized to α-tubulin (relating to Fig. 2e) (n = 3 independent blots). d, Immunostaining against active YAP in scrambled (shSCR) control mice and uninfected littermates (of shTHY1). e. Number of active YAP+ epidermal cells (n = 3 independent cultures). f, Scramble control (Ctrl) and THY1-silenced (shTHY1) keratinocytes (HaCaT) positive for GFP, demonstrating high lentiviral transduction efficiency (n ≥ 3 independent cultures). g, qPCR analysis of THY1 mRNA post infection (n = 3 independent cultures, analyzed in triplicate). Values were normalized to RPLP0. h, Whole lysate proteins isolated from Ctrl and shTHY1 HaCaT cells immunoblotted against THY1 and β-actin. i, Number of keratinocytes per colony in Ctrl and shTHY1 cultures (n = 5 independent cultures of each treatmen)]. j, Ctrl and shTHY1 keratinocytes immunostained against MCM2. k, Relative number of MCM2+ cells per colony (n = 15 colonies, from three independent cultures). l, Relative number of active YAP+ cells per colony (n = 15 colonies in three independent cultures). m, Whole lysate proteins isolated from Ctrl and shTHY1 non-confluent keratinocytes immunoblotted against YAPactive and β-actin. n, RNA isolated from Ctrl and THY1-OE (THY1-overexpression) subjected to qPCR analysis of THY1 (n = 3 independent cultures analyzed in triplicates). Values were normalized to RPLP0. o, Whole lysate proteins isolated from Ctrl and THY1-OE keratinocytes immunoblotted against THY1 and GAPDH. p, Densitometry of pYAPS127 signal normalized to β-actin protein levels. q, Percentage nuclear active YAP+ cells per field in confluent cultures (n = 10 colonies in three independent cultures). All images are representative of at least n = 3 mice of each genotype, unless otherwise stated. Error bars indicate the mean ± s.e.m. Scale bars: 20 μm (d,j) or 50 μm (a,f).

Source data

Extended Data Fig. 3 Lack of THY1 results in increased adhesion.

a, Quantification of relative cell size of Ctrl and shTHY1 keratinocytes (n = three independent cultures). b, Equal numbers of Ctrl and shTHY1 keratinocytes passaged and seeded, then monitored for adhesion (n = three independent cultures). c, Quantifications of cell adhesion of Ctrl and shTHY1 keratinocytes after seeding. d, Confluent Ctrl and shTHY1 keratinocytes were incubated with Trypsin/EDTA (0.25%) and monitored for detachment. e, Confluent ITGα6+/SCAI+ keratinocytes from WT and Thy1-/- mice were incubated with Trypsin/EDTA (0.25%) and monitored for detachment. f, Representative image of remaining (adhered) shSCR (Ctrl) and shTHY1 cells. g, Quantification of remaining (adhered) shSCR (Ctrl) and shTHY1 cells (n = four independent cultures). h, TIRF microcopy image of THY1-OE (THY1-overexpressed) keratinocytes immunostained for ITGβ1 and THY1. i, Ctrl, shTHY1 and THY-OE HaCaT cells immunoblotted against ITGβ1 and β-actin (n = 3 independent cultures of each). j, Dorsal skin cells immunoblotted against ITGβ1 and β-actin (n = 3 pooled mice each). k, Signal of pSRCY418 level normalized to β-actin (relating to Fig. 3k). l, Whole lysate proteins isolated from non-confluent and confluent Ctrl and shTHY1 HaCaT cells immunoblotted against pYAPY357 and β-actin. m, Immunostaining of active pYAPY357 in shSCR (control) and shTHY1 HaCaT cells. n, Primary dorsal keratinocytes treated with SRC inhibitor dasatinib immunostained for active ITGβ1 and active YAP. o, Relative percentage of active YAP+ primary keratinocytes. p, Whole lysate proteins isolated from Ctrl and shTHY1 HaCaT cells, treated with ROCK inhibitor (Y27632) and dasatinib, immunoblotted against active YAP and β-actin. All images are representative of at least n = 3 independent cultures per genotype, unless otherwise indicated. Scale bars, 5 μm (h), 20 μm (m) or 50 μm (f,n).

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Extended Data Fig. 4 Co-localization of cell-cell and cell-matrix proteins.

a, Schematic of Förster resonance energy transfer (FRET) using the secondary antibodies Alexa Fluor-488 and Alexa Fluor-594 as donor/acceptor pairs. b-e, FRET experiments on HaCaT cells utilizing antibodies against (b) ITGβ1and THY1 (b), ITGβ1 and α-catenin (c), α-catenin and ITGβ1 (d), and α-catenin and THY1 (e). f, Electron donor control for FRET experiments utilizing single antibodies (donor) and secondary antibodies alone (acceptor). g, Secondary antibody controls show no fluorescence in the absence of primary antibodies. h, Control for FRET utilizing distant electron donor/acceptor pairs ITGβ1 and Ki-67. i. High-magnification confocal image of non-confluent THY1-OE (THY1-overexpressed) keratinocytes immunostained against THY1 and α-catenin (n = 3 independent cultures per treatment). Panels on the right show single fluorescent channels. j, Whole lysate proteins isolated from WT and Thy1-/- dorsal skins immunoblotted against α-catenin and β-actin (n = 3 pooled mice of each). Scale bars, 10 μm (b,h) or 20 μm (i).

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Extended Data Fig. 5 Dissecting cell-cell vs. cell-matrix mechanisms of THY1.

a, Immunostaining of active YAP in single HaCaT cells silenced for THY1 and co-treated with ITGβ1 blocking antibody, YAP inhibitor verteporfin or SRC inhibitor dasatinib. b, Percentage active YAP+ single cells (n = three independent cultures). c, qPCR for YAP targets in confluent cells: shSCR (Ctrl), shTHY1 and shTHY1 treated with either ITGβ1 blocking antibody, YAP inhibitor verteporfin or SRC inhibitor dasatinib. Values were normalized to RPLP0 (n = 3 independent cultures, analyzed in triplicates). d, Immunostaining of proliferation marker MCM2 in single HaCaT cells silenced for THY1 and co-treated with ITGβ1 blocking antibody, verteporfin or dasatinib. e, Percentage of MCM2+ single cells (n = three independent cultures). f, Relative number active YAP+ cells in the suprabasal layer of dorsal skins from WT and Thy1-/- mice (n = 5 mice). g, Percentage of active ITGβ1+ cells in the suprabasal layer of dorsal skins from WT and Thy1-/- mice (n = 3 mice). All images and quantifications are representative of at least n = 3 mice per genotype or 3 independent cultures per group, unless indicated otherwise. Error bars indicate mean ± s.e.m. Scale bars, 20 μm (a,d).

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Extended Data Fig. 6 Thy1-/- mice display enhanced repair and regeneration.

a, Wound contraction at day 1 post wound infliction (PWI) (n = 7 mice). b,c, WT and Thy1-/- wounds imaged at day 7 (b) and day 18 (c) PWI. Dotted lines demarcate wound border. Asterisks denote areas of mechanical damage from epidermis/dermis separation. d, Immunostaining against active YAP and Ki-67 in the WT and Thy1-/- wound site at 18 days PWI. e-h, Active YAP expression in dorsal wounds after 3 days (e) and 7 days (f) PWI, or MCM2 expression after 3 days (g) and 7 days (h) PWI. i-k, Quantifications for percentages of active YAP+ (i) and MCM2+ (j) cells in the suprabasal layer, and number of cells (k) in the basal layer (n = 5 mice). l, Immunoblot of active YAP and β-actin in separated epidermal and dermal fractions from adult (P56) WT and Thy1-/- dorsal skins. m, Number of active YAP+ dermal cells throughout the wound healing process (n = 5 mice). n, Immunoblot of active YAP and β-actin in separated epidermal and dermal fractions from WT and Thy1-/- dorsal skins at 7 days PWI. o, Densitometry of active YAP signal (relating to panel n) (n = 3 pooled mice per group). p, qPCR for TGFβ1, SMAD2, SSMAD7 and p21 mRNA in WT and Thy1-/- dorsal skins at 7 days PWI. Values were normalized to RPLP0 (n = 3 mice, analyzed in triplicates). q, H&E images of WT and Thy1-/- dorsal wounds at 7 days PWI. Dashed black lines demarcate epidermis/dermis border. r, Regenerated Thy1-/- HFs (white arrowheads) immunostained against MCM2 and KRT15. s. Maximum projection images of WT and Thy1-/- wounds at 30 days PWI. t. Immunostaining against non-phosphorylated (active) β-catenin at day 7 PWI. u, qPCR against CTGF, SHH, WNT10a, WNT10b, WNT4 and WNT3 at day 12 PWI. Values were normalized to RPLP0 (n = 3 mice, analyzed in triplicates). All images are representative of at least n = 6 mice of each genotype, unless otherwise indicated. Error bars indicate mean ± s.e.m. Scale bars, 20 μm (d), 50 μm (e,f,g,h,t), 100 μm (r), 200 μm (q,s), 500 μm (b,c).

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Extended Data Fig. 7 Effects of Thy1 loss on various cell types during wounding.

a, Confocal images of the WT and Thy1-/- wound beds at 3 days post wound infliction (PWI) immunostained against T cell marker CD3. White dotted lines demarcate epidermis/dermis. b, Flow cytometry on wounds resected at day 3 PWI gated for CD45-APC+ cells (n = 3 pooled mice of each genotype). c,d., Immunoblots against NFκB p65 (c) and IκBα (d) in epidermal and dermal cells of WT and Thy1-/- telogenic (P56) dorsal skins. e, Dermis of adult WT and Thy1-/- mice after 7 days PWI stained for MCM2 and the dermal fibroblast marker PDGFRα. f., Percentage of proliferating papillary and reticular dermal cells. g,h, Immunostaining against Vimentin and Ki-67 (g), and CD31 and Ki-67 (h) in wound site dermis at day 3 PWI. i, Immunostaining against CD31 and Ki-67 in wound site dermis at 7 days PWI. j, Relative numbers of proliferating CD31+ cells at 3 and 7 days PWI in WT and Thy1-/- mice (n = 3 mice). All images and quantifications are representative of at least n = 3 mice of each genotype, unless otherwise indicated. Quantifications were performed on ≥ 60 fields of view ranging from 100-200μm. Western blots were performed on pooled proteins from n = 3 mice per genotype. Error bars indicate mean ± s.e.m. Scale bars, 50 μm (a,e,g,h,i).

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Extended Data Fig. 8 Inhibition of YAP abrogates Thy1-/–dependent phenotypes.

a, WT and Thy1-/- dorsal skins treated with 2.5% DMSO, and Thy1-/- treated with verteporfin (vert) at 36 h post BrdU (n = 9 mice per treatment). b, Number of BrdU+ cells from WT (DMSO-treated), Thy1-/- (DMSO-treated) and Thy1-/- (Vert-treated) mice (n = 9 mice per treatment). c, Schematic representation of YAP inhibition and dorsal wound infliction. d, Photographs of WT dorsal wounds treated with DMSO or Vert at day 7 post wound infliction (PWI). e, Re-epithelialization dynamics of WT skins. Percentage of wound coverage was calculated versus original wound size (n = 4 mice). f, Immunostaining against MCM2 and ITGβ4 (CD104) in dorsal wounds from WT (DMSO), Thy1-/- (DMSO) and Thy1-/- (Vert) mice. g, Percentage of MCM2+ suprabasal cells in treated mice (n = 4 mice). h, WT (DMSO), Thy1-/- (DMSO) and Thy1-/- (Vert) treated dorsal skins immunostained for MCM2 at day 7 PWI. i. Number of de novo hair follicles in the dorsal wound bed from WT (DMSO), Thy1-/- (DMSO) and Thy1-/- (Vert) mice at 7 days PWI (n = 4 mice). j. Schematic representation of ITGβ1 inhibition and dorsal wound infliction. k, WT dorsal skin treated with IgG or ITGβ1 antibody (ITGβ1 Ab) immunostained for active YAP and Ki-67 at day 7 PWI. l, Relative percentage active YAP+ cells in suprabasal layer from WT and Thy1-/- mice treated with IgG or ITGβ1 Ab at day 0 PWI (n = 4 mice). m. Dorsal skin sections from WT and Thy1-/- mice treated with IgG or ITGβ1 Ab immunostained for active YAP and Ki-67 at day 7 PWI. n-o. Quantifications of active YAP+ (n) and Ki-67+ (o) basal cells from WT and Thy1-/- mice treated with IgG or ITGβ1 Ab at day 7 PWI (n = 3 mice). p-q, Quantifications of active YAP+ (p) and Ki-67+ (q) suprabasal cells from WT and Thy1-/- mice treated with IgG or ITGβ1 antibodies at day 7 PWI (n = 3 mice). All images and quantifications are representative of at least n = 6 mice per genotype and treatment, unless otherwise stated. Error bars indicate mean ± s.e.m. Scale bars, 50 μm (a,f,h,k,m).

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Sedov, E., Koren, E., Chopra, S. et al. THY1-mediated mechanisms converge to drive YAP activation in skin homeostasis and repair. Nat Cell Biol 24, 1049–1063 (2022). https://doi.org/10.1038/s41556-022-00944-6

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