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Dissecting Wnt Signaling for Melanocyte Regulation during Wound Healing

  • Qi Sun
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA

    The Department of Cell Biology, New York University School of Medicine, New York, New York, USA
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  • Piul Rabbani
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA

    The Department of Cell Biology, New York University School of Medicine, New York, New York, USA
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  • Makoto Takeo
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA

    The Department of Cell Biology, New York University School of Medicine, New York, New York, USA
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  • Soung-Hoon Lee
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA

    The Department of Cell Biology, New York University School of Medicine, New York, New York, USA
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  • Chae Ho Lim
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA

    The Department of Cell Biology, New York University School of Medicine, New York, New York, USA
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  • EN-Nekema Shandi Noel
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA

    The Department of Cell Biology, New York University School of Medicine, New York, New York, USA
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  • M. Mark Taketo
    Affiliations
    Department of Pharmacology, Kyoto University, Sakyo, Kyoto, Japan
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  • Peggy Myung
    Affiliations
    Department of Dermatology, Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut, USA
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  • Sarah Millar
    Affiliations
    Departments of Dermatology and Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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  • Mayumi Ito
    Correspondence
    Correspondence: Mayumi Ito, 550 1st Avenue, Smilow 410, New York, New York 10016, USA.
    Affiliations
    The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA

    The Department of Cell Biology, New York University School of Medicine, New York, New York, USA
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Open ArchivePublished:February 08, 2018DOI:https://doi.org/10.1016/j.jid.2018.01.030
      Abnormal pigmentation is commonly seen in the wound scar. Despite advancements in the research of wound healing, little is known about the repopulation of melanocytes in the healed skin. Previous studies have shown the capacity of melanocyte stem cells in the hair follicle to contribute skin epidermal melanocytes after injury in mice and humans. Here, we focused on the Wnt pathway, known to be a vital regulator of melanocyte stem cells in efforts to better understand the regulation of follicle-derived epidermal melanocytes during wound healing. We showed that transgenic expression of Wnt inhibitor Dkk1 in melanocytes reduced epidermal melanocytes in the wound scar. Conversely, forced activation of Wnt signaling by genetically stabilizing β-catenin in melanocytes increases epidermal melanocytes. Furthermore, we show that deletion of Wntless (Wls), a gene required for Wnt ligand secretion, within epithelial cells results in failure in activating Wnt signaling in adjacent epidermal melanocytes. These results show the essential function of extrinsic Wnt ligands in initiating Wnt signaling in follicle-derived epidermal melanocytes during wound healing. Collectively, our results suggest the potential for Wnt signal regulation to promote melanocyte regeneration and provide a potential molecular window to promote proper melanocyte regeneration after wounding and in conditions such as vitiligo.

      Abbreviations:

      cKO (conditional knockout), McSC (melanocyte stem cell), TAM (tamoxifen), Tyr (tyrosinase)

      Introduction

      Upon injury, migration and proliferation of epithelial cells result in re-epithelialization of the wound area, a process that occurs in association with the formation of underlying granulation tissues. However, the new skin tissue replenished in the wound area typically leaves an appearance distinct from the original skin, partly because of imperfect skin pigmentation. Skin hypopigmention is a common complication, particularly after excisional surgical procedures or burn injuries (
      • Massaki A.B.
      • Fabi S.G.
      • Fitzpatrick R.
      Repigmentation of hypopigmented scars using an erbium-doped 1,550-nm fractionated laser and topical bimatoprost.
      ). On the other hand, wound injuries or inflammatory processes can also lead to skin hyperpigmentation (
      • Nordlund J.J.
      • Abdel-Malek Z.A.
      Mechanisms for post-inflammatory hyperpigmentation and hypopigmentation.
      ). In fact, abnormal pigmentation is one of the most visible changes caused by wounding or inflammation and, therefore, a common source of cosmetic and psychosocial concerns to patients (
      • Wisely J.A.
      • Hoyle E.
      • Tarrier N.
      • Edwards J.
      Where to start? Attempting to meet the psychological needs of burned patients.
      ). Currently, there are no treatment options to permanently and completely overcome this complication (
      • Chadwick S.
      • Heath R.
      • Shah M.
      Abnormal pigmentation within cutaneous scars: a complication of wound healing.
      ). This is partly rooted in our incomplete understanding of how melanocytes are recruited to the wound site and how they modulate pigment production during wound healing.
      Stem cells are responsible for replenishing cells that are lost to homeostatic cellular turnover, injury, or diseases. In humans, melanocytes are located in the basal layer of the epidermis and in the hair follicles. Stem cells in the melanocytic lineage residing within the skin epidermis have not yet been identified. Therefore, the presence, identity, and function of epidermal melanocyte stem cells (McSCs) remain elusive. In adult mice, melanocytes are not detectable in the interfollicular epidermis but are located in the bulge/secondary hair germ (also referred to as the sub-bulge) area and the bulb area of the hair follicle (
      • Nishimura E.K.
      Melanocyte stem cells: a melanocyte reservoir in hair follicles for hair and skin pigmentation.
      ,
      • Nishimura E.K.
      • Jordan S.A.
      • Oshima H.
      • Yoshida H.
      • Osawa M.
      • Moriyama M.
      • et al.
      Dominant role of the niche in melanocyte stem-cell fate determination.
      ). The bulge/secondary hair germ area of the hair follicle harbors McSCs throughout the distinct regenerative phases of the hair follicle cycle (
      • Muller-Rover S.
      • Handjiski B.
      • van der Veen C.
      • Eichmuller S.
      • Foitzik K.
      • McKay I.A.
      • et al.
      A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.
      ,
      • Nishimura E.K.
      • Jordan S.A.
      • Oshima H.
      • Yoshida H.
      • Osawa M.
      • Moriyama M.
      • et al.
      Dominant role of the niche in melanocyte stem-cell fate determination.
      ). Hair bulb melanocytes that produce pigment for the hair during the hair follicle cycle are progeny of McSCs and are present only during the growth phase of the hair cycle. Previous studies established that McSCs in the hair follicle also have the potential to migrate to the skin epidermis to give rise to functional epidermal melanocytes. This function can be clearly observed in the recovery process of vitiligo in humans, a disease characterized by loss of epidermal melanocytes (
      • Birlea S.A.
      • Costin G.E.
      • Roop D.R.
      • Norris D.A.
      Trends in regenerative medicine: repigmentation in vitiligo through melanocyte stem cell mobilization.
      ,
      • Picardo M.
      • Dell’Anna M.L.
      • Ezzedine K.
      • Hamzavi I.
      • Harris J.E.
      • Parsad D.
      • et al.
      Vitiligo.
      ). Clinical observations and experimental data with vitiligo skin suggest that repigmentation of the de-pigmented skin occurs from hair follicle melanocytes either spontaneously or after narrow band UVB treatment (
      • Cui J.
      • Shen L.Y.
      • Wang G.C.
      Role of hair follicles in the repigmentation of vitiligo.
      ,
      • Goldstein N.B.
      • Koster M.I.
      • Hoaglin L.G.
      • Spoelstra N.S.
      • Kechris K.J.
      • Robinson S.E.
      • et al.
      Narrow band ultraviolet B treatment for human vitiligo is associated with proliferation, migration, and differentiation of melanocyte precursors.
      ). Furthermore, lineage tracing of McSCs with a label retaining technique definitively showed the ability of follicular McSCs to undergo epidermal melanocyte fate in mice after injuries or UVB treatment (
      • Chou W.C.
      • Takeo M.
      • Rabbani P.
      • Hu H.
      • Lee W.
      • Chung Y.R.
      • et al.
      Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.
      ). When epidermal melanocytes are present, they may be primary contributors to the regeneration of melanocytes after injury; however, a recent study showed that follicular McSCs can populate epidermal melanocytes in human skin after epidermal ablation, regulated by endothelin signaling (
      • Takeo M.
      • Lee W.
      • Rabbani P.
      • Sun Q.
      • Hu H.
      • Lim C.H.
      • et al.
      EdnrB governs regenerative response of melanocyte stem cells by crosstalk with Wnt signaling.
      ). These studies suggest that follicular McSCs serve as additional reservoirs for melanocytes that are kept in a relatively protected area away from the skin surface.
      Wnt/β-catenin signaling is a central pathway in melanocyte biology. Binding of Wnt ligand(s) to the cell surface receptors frizzled and their co-receptor, LRP, leads to the stabilization of cytosolic β-catenin and its subsequent translocation to the nucleus, where it interacts with LEF/TCF transcription factors to regulate the transcription of downstream genes (
      • Barker N.
      The canonical Wnt/beta-catenin signalling pathway.
      ,
      • van Amerongen R.
      • Nusse R.
      Towards an integrated view of Wnt signaling in development.
      ). Genes targeted by Wnt signaling include those vital for pigmentation such as MITF, Dct, and tyrosinase (Tyr) (
      • Dorsky R.I.
      • Raible D.W.
      • Moon R.T.
      Direct regulation of nacre, a zebrafish MITF homolog required for pigment cell formation, by the Wnt pathway.
      ,
      • Takeda K.
      • Yasumoto K.
      • Takada R.
      • Takada S.
      • Watanabe K.
      • Udono T.
      • et al.
      Induction of melanocyte-specific microphthalmia-associated transcription factor by Wnt-3a.
      ,
      • Wang X.
      • Liu Y.
      • Chen H.
      • Mei L.
      • He C.
      • Jiang L.
      • et al.
      LEF-1 regulates tyrosinase gene transcription in vitro.
      ,
      • Yasumoto K.
      • Takeda K.
      • Saito H.
      • Watanabe K.
      • Takahashi K.
      • Shibahara S.
      Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling.
      ). Consistently, this pathway plays a critical role in regulating McSC behavior. Forced activation of this pathway in McSCs leads to their premature differentiation, whereas loss of β-catenin in melanocytes results in defective differentiation of McSCs into hair bulb melanocytes (
      • Rabbani P.
      • Takeo M.
      • Chou W.
      • Myung P.
      • Bosenberg M.
      • Chin L.
      • et al.
      Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.
      ). Moreover, UV irradiation induces McSCs to activate Wnt signaling and abrogation of Wnt leads to defects in melanocyte migration from the hair follicle to the epidermis (
      • Yamada T.
      • Hasegawa S.
      • Inoue Y.
      • Date Y.
      • Yamamoto N.
      • Mizutani H.
      • et al.
      Wnt/beta-catenin and kit signaling sequentially regulate melanocyte stem cell differentiation in UVB-induced epidermal pigmentation.
      ). These studies prompted us to dissect the function and regulation of this pathway in McSCs and their progeny during skin wound healing.
      In this study, we used multiple genetic mouse models to investigate the impact of the gain and loss of function of Wnt/β-catenin signaling in hair follicle-derived melanocytes after skin excisional wounds in mice. Taking advantage of the tissue-specific promoters that target melanocytes or epithelial cells, we show (i) the vital function of Wnt signaling in recruitment of follicle-derived epidermal melanocytes to the wound area and (ii) the essential role for epithelium-derived Wnt ligands in the Wnt activation of follicle-derived epidermal melanocytes in the healed area of the adult skin.

      Results

      Dkk1 expression inhibits the generation of epidermal melanocytes after wounding

      To determine the impact of loss of function of Wnt signaling in melanocytes during wound healing, we sought to overexpress a potent Wnt inhibitor, Dkk1, in melanocytes. Members of the Dkk family of secreted Wnt inhibitors specifically inhibit Wnt/LRP signaling by forming a complex with LRP that is internalized, removing LRP from the cell surface (
      • Bafico A.
      • Liu G.
      • Yaniv A.
      • Gazit A.
      • Aaronson S.A.
      Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow.
      ,
      • Mao B.
      • Wu W.
      • Li Y.
      • Hoppe D.
      • Stannek P.
      • Glinka A.
      • et al.
      LDL-receptor-related protein 6 is a receptor for Dickkopf proteins.
      ,
      • Semenov M.V.
      • Tamai K.
      • Brott B.K.
      • Kuhl M.
      • Sokol S.
      • He X.
      Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.
      ). This approach specifically inhibits Wnt signaling, unlike the deletion of β-catenin, which has a Wnt-independent function in cell-cell adhesion (
      • Rabbani P.
      • Takeo M.
      • Chou W.
      • Myung P.
      • Bosenberg M.
      • Chin L.
      • et al.
      Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.
      ). We generated Dct-rtTA; tetO-Dkk1; tetO-H2B-GFP transgenic mice (Dct:Dkk1:GFP mice) (
      • Chu E.Y.
      • Hens J.
      • Andl T.
      • Kairo A.
      • Yamaguchi T.P.
      • Brisken C.
      • et al.
      Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis.
      ,
      • Zaidi M.R.
      • Davis S.
      • Noonan F.P.
      • Graff-Cherry C.
      • Hawley T.S.
      • Walker R.L.
      • et al.
      Interferon-gamma links ultraviolet radiation to melanomagenesis in mice.
      ) in which Dkk1 and GFP reporter can be inducibly expressed in Dct+ melanocytes upon doxycycline treatment. Dct is expressed by undifferentiated McSCs and their differentiated melanocyte progeny (
      • Nishimura E.K.
      • Jordan S.A.
      • Oshima H.
      • Yoshida H.
      • Osawa M.
      • Moriyama M.
      • et al.
      Dominant role of the niche in melanocyte stem-cell fate determination.
      ). We created 1-cm2 excisional wounds (
      • Chou W.C.
      • Takeo M.
      • Rabbani P.
      • Hu H.
      • Lee W.
      • Chung Y.R.
      • et al.
      Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.
      ) on the back skin of 8-week-old Dct:Dkk1:GFP mice and fed them with a doxycycline-containing diet until harvesting the wound tissues (Figure 1a). As a control experiment, we used Dct-rtTA; tetO-H2B-GFP mice in which only GFP reporter is expressed in Dct+ melanocytes upon doxycycline treatment. At 8 weeks old, the only population of Dct+ melanocytes in the back skin is McSCs that are in the bulge/secondary hair germ areas of hair follicles (
      • Nishimura E.K.
      • Jordan S.A.
      • Oshima H.
      • Yoshida H.
      • Osawa M.
      • Moriyama M.
      • et al.
      Dominant role of the niche in melanocyte stem-cell fate determination.
      ) (Figure 1b). Previous studies showed that McSCs of the hair follicles in the wound periphery migrate to the epidermis and establish epidermal melanocytes in the wound (
      • Chou W.C.
      • Takeo M.
      • Rabbani P.
      • Hu H.
      • Lee W.
      • Chung Y.R.
      • et al.
      Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.
      ). Consistent with past observations, examination of whole mount skin epidermis of control wound tissues showed GFP+ melanocytes in the wound epidermis by 44 days after wounding, and these melanocytes were sporadically located without being uniformly localized in the wound area (Figure 1c). In contrast, mice with Dkk1 expression in melanocytes generated significantly reduced numbers of GFP+ melanocytes in the wound epidermis compared with the control mice, suggesting that Wnt signaling is required for the generation of epidermal melanocytes (Figure 1d and e). These results show that Wnt signaling is required for follicular McSCs to generate epidermal melanocytes during skin wound healing (Figure 1p).
      Figure 1
      Figure 1Overexpression of Dkk1 in melanocytes inhibits the wound-induced generation of epidermal melanocytes. (a–e) Dct-rtTA; tetO-Dkk1; tetO-H2B-GFP (Dct:Dkk1:GFP) and Dct-rtTA; tetO-H2B-GFP (Dct:GFP) mice were wounded and treated with doxycycline for 44 days. (a) Experimental scheme. (b) Immunofluorescence of Dct before wounding. (c, d) Dct:GFP signals in whole mount wound epidermis at postwound day 44. (e) Quantification of c and d. (f–o) Dct:Dkk1:GFP and Dct:GFP mice were treated with doxycycline from 3 weeks old and depilated at 7 weeks old. Tissues were harvested at 12 days after depilation. (f) Experimental scheme. (g) Image of mouse at 12 days after depilation. (h–m) Immunofluorescence of indicated markers and corresponding brightfield images. (n–o) Quantification of jm. S100+/Dct cells in k and m are dermal papilla cells. (p) Schematic summary. Dashed lines in c and d indicate boundary between wound and intact areas. Dashed lines in b and hm indicate boundary of epithelium and dermis. Arrowheads indicate melanocyte stem cells. Data are represented as mean ± standard error of the mean. P < 0.05, ∗∗∗P < 0.001. Scale bar = 50 μm in b and hm, 500 μm in c and d, and 1 cm in g. d, day; HF, hair follicle; MC, melanocyte; McSC, melanocyte stem cell; P, post-depilation; PW, postwound; sHG, secondary hair germ.
      The intact area of Dkk1-expressing mice verified an apparent defect in hair pigmentation (Figure 1fûp) that is known to rely on Wnt/β-catenin signaling (
      • Rabbani P.
      • Takeo M.
      • Chou W.
      • Myung P.
      • Bosenberg M.
      • Chin L.
      • et al.
      Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.
      ). In normal skin without wounding, McSCs regenerate differentiated melanocytes in the hair bulb that are responsible for hair pigmentation in association with the hair follicle cycle. We thus examined hair bulb melanocytes in these mice. Compared with control (Figure 1h), Dct:Dkk1:GFP mice had a significantly reduced number of hair bulb melanocytes (Figure 1i, left hair follicle). Even when bulb melanocytes were detected in Dkk1-expressing mice, they often lacked pigment (Figure 1i, right hair follicle) and failed to display immunoreactivity for melanocyte differentiation markers MITF and S100, which are typically up-regulated in hair bulb melanocytes (Figure 1j–o) (
      • Rabbani P.
      • Takeo M.
      • Chou W.
      • Myung P.
      • Bosenberg M.
      • Chin L.
      • et al.
      Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.
      ). The Dkk1-expressing melanocytes maintained expression of Sox10 (
      • Harris M.L.
      • Buac K.
      • Shakhova O.
      • Hakami R.M.
      • Wegner M.
      • Sommer L.
      • et al.
      A dual role for SOX10 in the maintenance of the postnatal melanocyte lineage and the differentiation of melanocyte stem cell progenitors.
      ), similar to control mice (see Supplementary Figure S1 online). The overall phenotype seen in Dkk1-expressing hair melanocytes (Figure 1p) is very similar to those reported in β-catenin–deficient mice (
      • Rabbani P.
      • Takeo M.
      • Chou W.
      • Myung P.
      • Bosenberg M.
      • Chin L.
      • et al.
      Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.
      ), verifying that the role of β-catenin in melanocyte differentiation is mainly mediated by Wnt signaling, instead of the cell adhesion role of β-catenin.

      Constitutive activation of Wnt signaling promotes the generation of epidermal melanocytes after wounding

      Next, we asked whether constitutive activation of Wnt/β-catenin signaling in melanocytes affects the generation of epidermal melanocytes. For this, we generated Tyr-CreER; β-catenin fl(ex3)/+; Dct-LacZ mice (β-catenin-STA mice) (
      • Bosenberg M.
      • Muthusamy V.
      • Curley D.P.
      • Wang Z.
      • Hobbs C.
      • Nelson B.
      • et al.
      Characterization of melanocyte-specific inducible Cre recombinase transgenic mice.
      ,
      • Harada N.
      • Tamai Y.
      • Ishikawa T.
      • Sauer B.
      • Takaku K.
      • Oshima M.
      • et al.
      Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene.
      ,
      • Zhao S.
      • Overbeek P.A.
      Tyrosinase-related protein 2 promoter targets transgene expression to ocular and neural crest-derived tissues.
      ) that specifically express a stabilized mutant form of β-catenin in melanocytes upon tamoxifen (TAM) treatment. These mice contain a Dct-LacZ reporter that allows us to detect Dct+ melanocytes by LacZ expression. We created 1-cm2 excisional wounds (
      • Chou W.C.
      • Takeo M.
      • Rabbani P.
      • Hu H.
      • Lee W.
      • Chung Y.R.
      • et al.
      Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.
      ) as described using β-catenin-STA mice and control mice (Dct-LacZ mice) and then treated them with TAM for 21 days (Figure 2a). By 44 days after wounding, forced Wnt activation in melanocytes resulted in epidermal melanocytes that were distributed over the entire wound scar area (Figure 2c). This was in clear contrast to the control samples in which melanocytes were sparsely distributed and largely concentrated in the peripheral area of the wound scar (Figure 2b). In quantifications, we observed a considerably higher number of melanocytes in the wound area of β-catenin–STA mice compared with control mice (Figure 2d). Tissue section analysis showed the presence of Dct+ melanocyte in the wound epidermis but not the dermis of control and β-catenin–STA mice (Figure 2e and f). There was significant increase of the percentage of nuclear β-catenin+ epidermal melanocytes in β-catenin–STA mice (90%) compared with control (55%) and increased expression of nuclear β-catenin in McSCs in the hair follicles of β-catenin–STA mice, confirming the successful forced Wnt activation in melanocytes of these mice (Figure 2e–g). These results show that forced activation of Wnt signaling via β-catenin stabilization enhances the number and distribution of melanocytes within the wound epidermis.
      Figure 2
      Figure 2Overexpression of β-catenin promotes the generation of epidermal melanocytes after wounding. Tyr-CreER; β-catenin fl(ex3)/+; Dct-LacZ (β-catenin-STA), and Dct-LacZ control mice were wounded and treated with tamoxifen for 21 days. Wound tissues were harvested at postwound day 44. (a) Experimental scheme. (b, c) β-galactosidase staining of whole mount wound epidermis. (d) Quantification of b and c. (e, f) Immunofluorescence of indicated markers in wound center and hair follicles in wound periphery. (g) Quantification of the percentage of nuclear β-catenin+ melanocytes in wound epidermis of e and f. Dashed lines outline the boundary between wound and intact areas. Dotted boxes outline magnified regions in separate fluorescent channels. Data are represented as mean ± standard error of the mean. P < 0.05. Scale bar = 1,000 μm in b and c, 25 μm in e and f, and 10 μm in magnified images in e and f. PW, postwound.

      Wnt ligands secreted by epithelial cells are essential for the activation of Wnt/β-catenin signaling in epidermal melanocytes

      Next, we investigated the source of Wnt ligands that drive Wnt activation in epidermal melanocytes during wound healing. For this, we isolated epidermal melanocytes (
      • Kawaguchi A.
      • Chiba K.
      • Tanimura Y.
      • Motohashi T.
      • Aoki H.
      • Takeda T.
      • et al.
      Isolation and characterization of Kit-independent melanocyte precursors induced in the skin of steel factor transgenic mice.
      ) and epithelial cells from healed wound at 17 days after wounding (see Supplementary Figure S2 online) and determined the expression of Wnt ligands in both populations. We found that epithelial cells significantly up-regulate several Wnt ligands, including Wnt3, 4, 10a, and 16 compared with epidermal melanocytes, suggesting that epithelial cells may be the major source of Wnt ligands in the wound area (Figure 3a).
      Figure 3
      Figure 3Wnt ligands provided by epithelial cells are required for Wnt activation and differentiation of epidermal melanocytes. (a) Wnt ligand quantitative PCR of epidermal melanocytes and keratinocytes isolated from wound epidermis 17 days after wounding. (b–j) K14-CreER; Wls fl/fl (K14-Wls cKO) or K14-CreER; Wls fl/fl; Dct-LacZ (K14-Wls cKO-Dct-LacZ) mice and control littermates were wounded and immediately treated with tamoxifen for 7 days. Wound tissues were harvested at indicated time points. (b) Experimental scheme. Immunofluorescence of indicated markers and corresponding brightfield images at (c, d) postwound day 7 and (f, g) postwound day 21. (h–j) Quantification of f and g. (e) β-galactosidase staining of whole mount wound tissue at postwound day 44. Dashed lines outline the boundary of epithelium and dermis. Dotted boxes outline magnified regions in separate fluorescent channels. Arrowheads point to epidermal melanocytes. Data are represented as mean ± standard error of the mean. P < 0.05. Scale bar = 50 μm in ce, 25 μm in f and g, and 10 μm in magnified images in c, d, and f. cKO, conditional knockout; PW, postwound; sHG, secondary hair germ; UD, undetected.
      Although several studies suggest the potential function for epithelial Wnt ligands to influence McSC behavior (
      • Rabbani P.
      • Takeo M.
      • Chou W.
      • Myung P.
      • Bosenberg M.
      • Chin L.
      • et al.
      Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.
      ,
      • Yamada T.
      • Hasegawa S.
      • Inoue Y.
      • Date Y.
      • Yamamoto N.
      • Mizutani H.
      • et al.
      Wnt/beta-catenin and kit signaling sequentially regulate melanocyte stem cell differentiation in UVB-induced epidermal pigmentation.
      ), the necessity for these extrinsic ligands has never been addressed. To determine whether Wnt ligands derived from epithelial cells are required for Wnt signaling in melanocytes during wound healing, we generated K14-CreER; Wls fl/fl; Dct-LacZ mice (K14-Wls conditional knockout [cKO] mice) in which Wls, which is essential for Wnt ligand secretion (
      • Banziger C.
      • Soldini D.
      • Schutt C.
      • Zipperlen P.
      • Hausmann G.
      • Basler K.
      Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells.
      ,
      • Bartscherer K.
      • Pelte N.
      • Ingelfinger D.
      • Boutros M.
      Secretion of Wnt ligands requires Evi, a conserved transmembrane protein.
      ,
      • Goodman R.M.
      • Thombre S.
      • Firtina Z.
      • Gray D.
      • Betts D.
      • Roebuck J.
      • et al.
      Sprinter: a novel transmembrane protein required for Wg secretion and signaling.
      ), was ablated in keratin 14+ basal epithelial cells after TAM treatment (
      • Carpenter A.C.
      • Rao S.
      • Wells J.M.
      • Campbell K.
      • Lang R.A.
      Generation of mice with a conditional null allele for Wntless.
      ,
      • Vasioukhin V.
      • Degenstein L.
      • Wise B.
      • Fuchs E.
      The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin.
      ). These mice contain the Dct-LacZ reporter to visualize Dct+ melanocytes. We created 1-cm2 excisional wounds as described, using K14-Wls cKO mice and control mice (Dct-LacZ) and treated them with TAM for 7 days (Figure 3b). At 7 days after wounding, we observed substantial expansion of Dct+ McSCs in the hair follicle of the wound periphery area, and they migrate upward into the epidermis, consistent with a previous study (
      • Chou W.C.
      • Takeo M.
      • Rabbani P.
      • Hu H.
      • Lee W.
      • Chung Y.R.
      • et al.
      Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.
      ); some of these Dct+ melanocytes are Wnt active, as evidenced by nuclear β-catenin signals (Figure 3c). In contrast, loss of epithelial Wnt ligands in K14-Wls cKO mice inhibited Wnt activation in Dct+ McSCs and their expansion into the epidermis (Figure 3d). As a result, at 44 days after wounding, the examination of whole mount wound samples showed a significant reduction of epidermal melanocytes in the wound area of K14-Wls KO mice compared with control mice (Figure 3e). This phenotype was reminiscent of what was seen upon Wnt inhibition in melanocytes in Dkk1 mice (Figure 1a–e). Indeed, the analyses of wound tissue sections from K14-Wls cKO mice showed a clear defect in Wnt signal activation in epidermal melanocytes, shown by the lack of nuclear β-catenin signals (Figure 3f). They also down-regulated another marker of Wnt activation, Tcf1, compared with epidermal melanocytes of control mice (see Supplementary Figure S3 online). In control wounds, about 55–60% of epidermal melanocytes showed Wnt activation with nuclear β-catenin expression (Figure 3h). Moreover, epidermal melanocytes in K14-Wls cKO mice showed defect in pigment production, and they failed to express melanocyte differentiation marker MITF, which is expressed by normal epidermal melanocytes (Figure 3f, g, i, and j). These results showed that epithelium-derived Wnt ligands are essential for Wnt activation of McSCs and epidermal melanocytes, which is required to recruit functional melanocytes to the wound area.

      Inhibition of Wnt ligand secretion from melanocytes does not affect their Wnt activation

      If Wnt signaling in epidermal melanocytes is controlled by epithelial Wnt ligands, Wnt ligand secretion from melanocytes themselves may not influence their regenerative behavior. To verify this, we generated Tyr-CreER; Wls fl/fl (Tyr-Wls cKO) mice in which Wls can be deleted in melanocytes (
      • Bosenberg M.
      • Muthusamy V.
      • Curley D.P.
      • Wang Z.
      • Hobbs C.
      • Nelson B.
      • et al.
      Characterization of melanocyte-specific inducible Cre recombinase transgenic mice.
      ,
      • Carpenter A.C.
      • Rao S.
      • Wells J.M.
      • Campbell K.
      • Lang R.A.
      Generation of mice with a conditional null allele for Wntless.
      ) after TAM treatment. After wounding and subsequent TAM treatment on Tyr-Wls cKO and control mice (Figure 4a), we found no difference in the pigmentation and number of epidermal melanocytes between these mice (Figure 4b, d, and e). Additionally, we did not detect a difference in Wnt activation of epidermal melanocytes between melanocytes of control and Tyr-Wls cKO mice, assessed by expression of nuclear β-catenin, between the melanocytes of control and Tyr-Wls cKO mice (Figure 4b and c). These results show that although melanocytes have the potential to express Wnt ligands in some conditions (Figure 3a) (
      • Ye J.
      • Yang T.
      • Guo H.
      • Tang Y.
      • Deng F.
      • Li Y.
      • et al.
      Wnt10b promotes differentiation of mouse hair follicle melanocytes.
      ), such intrinsic Wnt ligands are dispensable for Wnt signal activation of epidermal melanocytes during skin wound healing.
      Figure 4
      Figure 4Melanocytes do not produce Wnt ligands to drive their Wnt activation and differentiation. (a–e) Tyr-CreER; Wls fl/fl (Tyr-Wls cKO) mice and control littermates were wounded and immediately treated with tamoxifen for 21 days. Wound tissues were harvested 21 days after wounding. (a) Experimental scheme. (b) Immunofluorescence of indicated markers and corresponding brightfield images in wound center and hair follicles in wound periphery. (c-e) Quantification of b. (f) Schematic model: upon wounding, epithelial cells generate Wnt ligands to drive the Wnt activation and upward migration of quiescent McSCs. Once in the epidermis, Wnt ligands from epithelial cells continue to induce Wnt activation in epidermal melanocytes, which differentiate and produce pigment. Wnt activation is also required for McSCs to generate hair bulb melanocytes. Dashed lines outline the boundary of epithelium and dermis. Dotted boxes outline magnified regions in separate fluorescent channels. Arrowheads indicate epidermal melanocytes. Data are represented as mean ± standard error of the mean. Scale bar = 25 μm and 10 μm in magnified images. cKO, conditional knockout; McSC, melanocyte stem cell; n.s., not significant; PW, postwound; sHG, secondary hair germ.

      Discussion

      Previous studies have noted the distinct behaviors in melanocytic lineage after global modification of Wnt signaling in adult skin through various approaches, including injection/topical application of Wnt inhibitors, agonists, or small interfering RNA. For example, intradermal injection of Wnt3a and Wnt10b in mouse skin promoted melanocyte differentiation in the hair follicle, and injection of the Wnt inhibitor sFRP4 inhibited this process (
      • Guo H.
      • Xing Y.
      • Liu Y.
      • Luo Y.
      • Deng F.
      • Yang T.
      • et al.
      Wnt/beta-catenin signaling pathway activates melanocyte stem cells in vitro and in vivo.
      ,
      • Guo H.
      • Lei M.
      • Li Y.
      • Liu Y.
      • Tang Y.
      • Xing Y.
      • et al.
      Paracrine secreted frizzled-related protein 4 inhibits melanocytes differentiation in hair follicle.
      ,
      • Ye J.
      • Yang T.
      • Guo H.
      • Tang Y.
      • Deng F.
      • Li Y.
      • et al.
      Wnt10b promotes differentiation of mouse hair follicle melanocytes.
      ). Topical application of the GSK3β-inhibitor LiCl promoted the regeneration of pigmented hair in the wound scar (
      • Yuriguchi M.
      • Aoki H.
      • Taguchi N.
      • Kunisada T.
      Pigmentation of regenerated hairs after wounding.
      ). Intradermal injection of the Wnt inhibitor IWR-1 and small interfering RNA against Wnt7a in mouse skin inhibited Wnt activation in McSCs and their migration into the epidermis after UVB irradiation (
      • Yamada T.
      • Hasegawa S.
      • Inoue Y.
      • Date Y.
      • Yamamoto N.
      • Mizutani H.
      • et al.
      Wnt/beta-catenin and kit signaling sequentially regulate melanocyte stem cell differentiation in UVB-induced epidermal pigmentation.
      ). This study specifically inhibited and activated Wnt signaling in melanocytes using genetic mouse models during wound healing to definitively show the vital role for Wnt signaling in the recruitment of melanocytes to the injured skin (Figure 4f).
      Our results suggest that modulation of Wnt signaling in melanocytes may promote the recovery phase of vitiligo or, conversely, prevent undesirable hyperpigmentation caused by UV irradiation, a theory previously proposed by other studies that used non–tissue-specific approaches (
      • Yamada T.
      • Hasegawa S.
      • Inoue Y.
      • Date Y.
      • Yamamoto N.
      • Mizutani H.
      • et al.
      Wnt/beta-catenin and kit signaling sequentially regulate melanocyte stem cell differentiation in UVB-induced epidermal pigmentation.
      ). Further, our study extended these findings to also show that Wnt signaling is vital for regeneration of hair melanocytes through analyses of transgenic overexpression of the Wnt inhibitor Dkk1 in melanocytes. Thus, the differential fate choice for McSCs to become epidermal melanocytes versus hair melanocytes may not be necessarily determined by Wnt signaling. Instead, we propose that Wnt signaling may function to enhance proliferation and differentiation of McSCs, thereby reinforcing recruitment of melanocytes to the wound site, which is similar to the effect of endothelin signaling (
      • Takeo M.
      • Lee W.
      • Rabbani P.
      • Sun Q.
      • Hu H.
      • Lim C.H.
      • et al.
      EdnrB governs regenerative response of melanocyte stem cells by crosstalk with Wnt signaling.
      ). Such an effect of Wnt signaling is in contrast to that of Mc1R, whose inhibition specifically blocks epidermal melanocyte fate without affecting hair melanocyte production (
      • Chou W.C.
      • Takeo M.
      • Rabbani P.
      • Hu H.
      • Lee W.
      • Chung Y.R.
      • et al.
      Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.
      ,
      • Takeo M.
      • Lee W.
      • Rabbani P.
      • Sun Q.
      • Hu H.
      • Lim C.H.
      • et al.
      EdnrB governs regenerative response of melanocyte stem cells by crosstalk with Wnt signaling.
      ). How the Wnt and Mc1R receptor pathways cooperatively regulate epidermal melanocyte generation during wound healing or during the recovery process of vitiligo will be an important subject for future investigation. More broadly, understanding the crosstalk between Wnt-driven melanocyte regeneration and mechanisms of wound healing (e.g., inflammatory signals) may illuminate the mechanism behind the fate decision of McSCs.
      Our results definitively show the essential role for epithelial Wnt ligands in the activation of Wnt signaling and subsequent melanocyte functions in the wound epidermis (Figure 4f). Supporting this, previous studies detected Wnt10b protein in regenerating epithelial cells up to 3 days and Wnt4 protein up to 5 days after wounding (
      • Okuse T.
      • Chiba T.
      • Katsuumi I.
      • Imai K.
      Differential expression and localization of WNTs in an animal model of skin wound healing.
      ), and Wnt7a is up-regulated in epidermal cells and hair follicle stem cells upon UV irradiation, which promotes generation of epidermal melanocytes (
      • Yamada T.
      • Hasegawa S.
      • Inoue Y.
      • Date Y.
      • Yamamoto N.
      • Mizutani H.
      • et al.
      Wnt/beta-catenin and kit signaling sequentially regulate melanocyte stem cell differentiation in UVB-induced epidermal pigmentation.
      ). It is intriguing that epithelial Wnt ligands are the primary source for Wnt activation in melanocytes and that the lack of epithelial Wnt ligands cannot be compensated for by other sources. Interactions between epithelial cells and melanocytes have been well delineated in melanocyte biology. Ligands of multiple signaling pathways including endothelins (
      • Hara M.
      • Yaar M.
      • Gilchrest B.A.
      Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.
      ,
      • Imokawa G.
      • Yada Y.
      • Miyagishi M.
      Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes.
      ), α-melanocyte-stimulating hormone (
      • De Luca M.
      • Siegrist W.
      • Bondanza S.
      • Mathor M.
      • Cancedda R.
      • Eberle A.N.
      Alpha melanocyte stimulating hormone (alpha MSH) stimulates normal human melanocyte growth by binding to high-affinity receptors.
      ,
      • Schauer E.
      • Trautinger F.
      • Köck A.
      • Schwarz A.
      • Bhardwaj R.
      • Simon M.
      • et al.
      Proopiomelanocortin-derived peptides are synthesized and released by human keratinocytes.
      ), and TGF-β (
      • Lee H.S.
      • Kooshesh F.
      • Sauder D.N.
      • Kondo S.
      Modulation of TGF-beta 1 production from human keratinocytes by UVB.
      ,
      • Nishimura E.K.
      • Suzuki M.
      • Igras V.
      • Du J.
      • Lonning S.
      • Miyachi Y.
      • et al.
      Key roles for transforming growth factor beta in melanocyte stem cell maintenance.
      ) are secreted from epithelial cells, which are thought to influence adjacent melanocytes in the skin through paracrine signaling. Our experiments exemplify the strikingly passive nature of melanocytes, and this knowledge will help direct and formulate strategies to manipulate melanocytes in efforts to enhance their regeneration or modulate their pigment production.

      Materials and Methods

      Mice

      All animal protocols were approved by the Institutional Animal Care and Use Committee at New York University School of Medicine. Tyr-CreER, K14-CreER, Wntless fl/fl mice were obtained from Jackson Laboratories (Bar Harbor, ME). The tetO-Dkk1 mice were from Sarah Millar at University of Pennsylvania (Philadelphia, PA). β-catenin fl(ex3)/+ mice were from M. Mark Taketo. Dct-lacZ mice were from Paul Overbeek. Dct-rtTA and tetO-H2B-GFP mice were from NCI Mouse Repository (Frederick, MD). Both male and female mice were used in all experiments. To induce Cre recombination, tamoxifen (Sigma-Aldrich, St. Louis, MO) treatment was performed by intraperitoneal injection (0.1 mg/g body weight) of a 20-mg/ml solution in corn oil per day (
      • Ito M.
      • Yang Z.
      • Andl T.
      • Cui C.
      • Kim N.
      • Millar S.E.
      • et al.
      Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.
      ). For tetracycline-inducible mouse models, mice were given doxycycline-containing chow (20g/kg) (Bio-Serv, Flemington, NJ) for the indicated time period.

      Wound experiment

      Wound experiments were performed as described with minor modification (
      • Chou W.C.
      • Takeo M.
      • Rabbani P.
      • Hu H.
      • Lee W.
      • Chung Y.R.
      • et al.
      Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.
      ). Briefly, mice were anesthetized with isoflurane. The dorsal fur was clipped, and a 1-cm2 area of full-thickness skin was excised with scissors.
      For whole-mount wound epidermis analysis, wound scar tissues were harvested at indicated time points. The connective tissues were carefully removed with forceps. The wound tissue was incubated in 20 mmol/L EDTA for 4 hours to separate the wound epidermis from dermis. The epidermal tissue was carefully removed from the dermal tissue with forceps and then fixed in 4% paraformaldehyde (PFA) for 10 minutes at room temperature.

      Immunofluorescence

      Immunofluorescence was performed as reported (
      • Rabbani P.
      • Takeo M.
      • Chou W.
      • Myung P.
      • Bosenberg M.
      • Chin L.
      • et al.
      Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration.
      ). Briefly, skin tissues were embedded in paraffin and cut into 6-μm sections. Tissue sections were incubated with the primary antibodies listed for 2 hours at room temperature or overnight at 4°C, followed by incubation with Alexa 488/594-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Sections were counterstained with DAPI (Vector Laboratories, Burlingame, CA). The primary antibodies used were mouse anti-β-catenin (1:400; Sigma-Aldrich, c7207), mouse anti-MITF (1:100; Vector Laboratories, VP-M650) (used in Figures 3 and 4), mouse anti-MITF (1:100; Abcam, Cambridge, UK, ab12039) (used in Figure 1), goat anti-sox10 (1:100; Santa Cruz Biotechnology, Dallas, TX, sc-17342), goat anti-Dct (1:100; Santa Cruz Biotechnology, sc-10451), rabbit anti-S100 (1:100; Dako, Glostrup, Denmark, Z0311) and rabbit anti-Tcf1 (1:100, Cell Signaling Technology, Danvers, MA, C63D9). Images were taken with an inversed Eclipse Ti microscope (Nikon, Tokyo, Japan) or an upright Axioplan microscope (Carl Zeiss, Oberkochen, Germany).

      Whole mount β-galactosidase staining

      Whole mount β-galactosidase staining was performed as published (
      • Chou W.C.
      • Takeo M.
      • Rabbani P.
      • Hu H.
      • Lee W.
      • Chung Y.R.
      • et al.
      Direct migration of follicular melanocyte stem cells to the epidermis after wounding or UVB irradiation is dependent on Mc1r signaling.
      ,
      • Ito M.
      • Yang Z.
      • Andl T.
      • Cui C.
      • Kim N.
      • Millar S.E.
      • et al.
      Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding.
      ) using wound epidermis or whole wound tissue.

      FACS isolation of melanocytes and keratinocytes from wound epidermis

      Wound scar tissue was harvested at day 17 after wounding. Wound tissue (lacking hair follicles) was carefully cut out from the wound periphery with scissors. The connective tissues were carefully removed with forceps. The wound tissue was incubated in 0.25% trypsin (Invitrogen, Waltham, MA) for 2 hours at 37°C. Wound epidermis was separated from the dermis using forceps and scalpel blades, and the epidermis was chopped finely and transferred into Media A (DMEM, 10% fetal bovine serum, 1× penicillin/streptomycin). The epidermal melanocyte and keratinocyte mixture was stirred at room temperature for 20 minutes to generate single cells. The obtained single cell suspension was filtered through a 70-μm nylon filter and centrifuged at 200g for 7 minutes and resuspended in phosphate buffered saline plus 10% fetal bovine serum. The cell suspension was incubated with PE-Cy7-anti-mouse CD117 (1:300, BD Pharmingen, San Diego, CA) for 15 minutes at room temperature. Cells were pelleted and washed twice in phosphate buffered saline plus 10% fetal bovine serum, then incubated with APC-CY7–anti-mouse CD45 (1:300, BD Pharmingen) for 15 minutes at room temperature. Cells were washed and sorted on a SY3200 cell sorter (Sony Biotechnology, San Jose, CA). Epidermal melanocytes were collected as PE-Cy7–positive and APC-Cy7–negative population (
      • Kawaguchi A.
      • Chiba K.
      • Tanimura Y.
      • Motohashi T.
      • Aoki H.
      • Takeda T.
      • et al.
      Isolation and characterization of Kit-independent melanocyte precursors induced in the skin of steel factor transgenic mice.
      ). Epidermal keratinocytes were collected as PE-Cy7–negative and APC-Cy7–negative population.

      Quantitative real-time PCR

      RNA was obtained from FACS-sorted cells and prepared using RNeasy micro kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. RNA was reverse transcribed with Superscript III First Strand Synthesis System (Invitrogen) for cDNA synthesis. cDNA was pre-amplified using TaqMan PreAmp Master Mix Kit (Applied Biosystems, Foster City, CA). Real-time amplification was carried out using the StepOnePlus Real-Time PCR System (Applied Biosystems). The target gene transcripts were quantified relative to the housekeeping gene, GAPDH. Data were analyzed using ΔΔCT method. For each Wnt ligand, the expression level in keratinocytes is calculated relative to that of melanocytes. For Wnt ligands that were not detected in melanocytes, the relative expression level in keratinocytes was considered as 1. Supplementary Table S1 online shows the list of primers used.

      Quantification and statistical analyses

      For whole wound tissue (epidermis) analysis, whole wound tissues from at least three mice were analyzed. For tissue section analysis, 20 epidermal melanocytes or hair follicles were analyzed in tissues from at least two mice. Quantification was done in a blinded manner by at least two investigators. Data were analyzed using Student t test (two tailed). A value of P less than 0.05 was deemed significant. Statistical analyses were performed using Microsoft Excel (Microsoft, Redmond, WA).

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank the New York University Langone’s Cytometry and Cell Sorting Laboratory (supported in part by grant P30CA016087 from the National Institutes of Health/National Cancer Institute ) for cell sorting. MI is grateful for the supports from the National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases grants R01 AR059768 , R01 AR066022 , and the Arnold and Mabel Beckman Foundation. QS and MT were supported by a NYSTEM institutional training grant (contract no. C026880). CHL was supported by New York University Cutaneous Biology and Skin Disease Training Program ( National Institute of Arthritis and Musculoskeletal and Skin Diseases T32 AR064184 ).

      Supplementary Material

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