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The M3 Muscarinic Acetylcholine Receptor Promotes Epidermal Differentiation

  • Junyan Duan
    Affiliations
    Center for Complex Biological Systems, University of California, Irvine, Irvine, California, USA

    NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine, Irvine, California, USA
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  • Charles Grando
    Affiliations
    Department of Dermatology, School of Medicine, University of California, Irvine, Irvine, California, USA
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  • Shuman Liu
    Affiliations
    Division of Endocrinology, Department of Medicine, School of Medicine, University of California, Irvine, Irvine, California, USA
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  • Alex Chernyavsky
    Affiliations
    Department of Dermatology, School of Medicine, University of California, Irvine, Irvine, California, USA
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  • Jefferson K. Chen
    Affiliations
    Division of Endocrinology, Department of Medicine, School of Medicine, University of California, Irvine, Irvine, California, USA
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  • Author Footnotes
    7 These authors contributed equally to this work.
    Bogi Andersen
    Footnotes
    7 These authors contributed equally to this work.
    Affiliations
    Center for Complex Biological Systems, University of California, Irvine, Irvine, California, USA

    NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine, Irvine, California, USA

    Division of Endocrinology, Department of Medicine, School of Medicine, University of California, Irvine, Irvine, California, USA

    Department of Biological Chemistry, School of Medicine, University of California, Irvine, Irvine, California, USA
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  • Author Footnotes
    7 These authors contributed equally to this work.
    Sergei A. Grando
    Correspondence
    Correspondence: Sergei A. Grando, Department of Dermatology, University of California, Irvine, 362 Medical Surge II, Irvine, California 92697, USA.
    Footnotes
    7 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, School of Medicine, University of California, Irvine, Irvine, California, USA

    Department of Biological Chemistry, School of Medicine, University of California, Irvine, Irvine, California, USA

    Institute for Immunology, University of California, Irvine, Irvine, California, USA
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  • Author Footnotes
    7 These authors contributed equally to this work.
Open AccessPublished:July 20, 2022DOI:https://doi.org/10.1016/j.jid.2022.06.013
      The M3 muscarinic acetylcholine receptor is predominantly expressed in the basal epidermal layer where it mediates the effects of the autocrine/paracrine cytotransmitter acetylcholine. Patients with the autoimmune blistering disease pemphigus develop autoantibodies to M3 muscarinic acetylcholine receptor and show alterations in keratinocyte adhesion, proliferation, and differentiation, suggesting that M3 muscarinic acetylcholine receptor controls these cellular functions. Chmr3-/- mice display altered epidermal morphology resembling that seen in patients with pemphigus vulgaris. In this study, we characterized the cellular and molecular mechanisms through which M3 muscarinic acetylcholine receptor controls epidermal structure and function. We used single-cell RNA sequencing to evaluate keratinocyte heterogeneity and identify differentially expressed genes in specific subpopulations of epidermal cells in Chmr3-/- neonatal mice. We found that Chmr3-/- mice feature abnormal epidermal morphology characterized by accumulation of nucleated basal cells, shrinkage of basal keratinocytes, and enlargement of intercellular spaces. These morphologic changes were associated with upregulation of cell proliferation genes and downregulation of genes contributing to epidermal differentiation, extracellular matrix formation, intercellular adhesion, and cell arrangement. These findings provide, to our knowledge, previously unreported insights into how acetylcholine controls epidermal differentiation and lay a groundwork for future translational studies evaluating the therapeutic potential of cholinergic drugs in dermatology.

      Abbreviations:

      ACh (acetylcholine), GO (Gene Ontology), IFE (interfollicular epidermis), K (keratin), KC (keratinocyte), KO (knockout), mAChR (muscarinic acetylcholine receptor), P (postnatal day), scRNA-seq (single-cell RNA-sequencing), UMAP (Uniform Manifold Approximation and Projection), WT (wild-type)

      Introduction

      The muscarinic acetylcholine (ACh) receptors are single-subunit transmembrane glycoproteins of five subtypes (M1‒M5). On ACh binding, muscarinic ACh receptors (mAChRs) stimulate interactions of G proteins with signal transducing enzymes, leading to changes in second messengers, ion concentrations, and the modulations of protein kinase activities. Within the epidermis, ACh is released in an autocrine and paracrine manner, and the different mAChR subtypes are combinatorically expressed in a differentiation-specific manner, suggesting complex epidermal regulation (reviewed in
      • Grando S.A.
      Muscarinic receptor agonists and antagonists: effects on keratinocyte functions.
      ,
      • Grando S.A.
      Cholinergic control of epidermal cohesion.
      ).
      Previous research, primarily using in vitro models, has implicated mAChRs in multiple keratinocyte (KC) functions, including cell‒cell and cell‒substrate adhesion, migration, proliferation, and differentiation (reviewed in
      • Grando S.A.
      Muscarinic receptor agonists and antagonists: effects on keratinocyte functions.
      ,
      • Grando S.A.
      Cholinergic control of epidermal cohesion.
      ). Successful use of topical muscarinic agonist pilocarpine to heal skin erosions in patients with the autoimmune blistering disease pemphigus and the disappearance of psoriatic lesions due to systemic therapy with the muscarinic antagonist atropine highlight the importance of understanding the in vivo effects of the mAChR pathway (reviewed in
      • Grando S.A.
      Muscarinic receptor agonists and antagonists: effects on keratinocyte functions.
      ).
      Blocking mAChRs in undifferentiated human KC monolayers significantly increased cell numbers and inhibited differentiation when KCs were exposed to differentiation-inducing agents (reviewed in
      • Grando S.A.
      Muscarinic receptor agonists and antagonists: effects on keratinocyte functions.
      ). Blocking of mAChRs in organotypic cultures of human epidermis also altered epidermal differentiation as evidenced by increased expression of injury markers keratin (K)6 gene K6/K16 and decreased expression of the differentiation markers K10 and FLG as well as cell adhesion proteins (
      • Kurzen H.
      • Henrich C.
      • Booken D.
      • Poenitz N.
      • Gratchev A.
      • Klemke C.D.
      • et al.
      Functional characterization of the epidermal cholinergic system in vitro.
      ). The outcome was cell‒cell separation (acantholysis) in the basal and lower suprabasal layers with defective epidermal barrier and cell death through intrinsic activation of apoptosis (
      • Kurzen H.
      • Henrich C.
      • Booken D.
      • Poenitz N.
      • Gratchev A.
      • Klemke C.D.
      • et al.
      Functional characterization of the epidermal cholinergic system in vitro.
      ). Acantholysis caused by blockade of KC mAChRs was associated with increased phosphorylation of the adhesion molecules E-cadherin, desmoglein 3, and β- and γ-catenins, suggesting that regulation of KC cell‒cell adhesion through the mAChR class predominantly involves changes in the phosphorylation of intercellular adhesion molecules (
      • Nguyen V.T.
      • Chernyavsky A.I.
      • Arredondo J.
      • Bercovich D.
      • Orr-Urtreger A.
      • Vetter D.E.
      • et al.
      Synergistic control of keratinocyte adhesion through muscarinic and nicotinic acetylcholine receptor subtypes.
      ), but the effects of specific mAChRs subtypes on in vivo epidermal differentiation are incompletely understood.
      Previous studies of the role of cholinergic autocrine and paracrine regulation of non-neuronal cells through mAChRs have given contradictory results about the role of specific mAChR subtypes in cell proliferation, suggesting that mAChRs induce either cell cycle arrest (
      • Chang M.C.
      Areca nut extract and arecoline induced the cell cycle arrest but not apoptosis of cultured oral KB epithelial cells: association of glutathione, reactive oxygen species and mitochondrial membrane potential.
      ;
      • Jeng J.H.
      • Hahn L.J.
      • Lin B.R.
      • Hsieh C.C.
      • Chan C.P.
      • Chang M.C.
      Effects of areca nut, inflorescence piper betle extracts and arecoline on cytotoxicity, total and unscheduled DNA synthesis in cultured gingival keratinocytes.
      ;
      • Kurzen H.
      • Henrich C.
      • Booken D.
      • Poenitz N.
      • Gratchev A.
      • Klemke C.D.
      • et al.
      Functional characterization of the epidermal cholinergic system in vitro.
      ;
      • Thangjam G.S.
      • Kondaiah P.
      Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes.
      ) or promote cell proliferation (
      • Arredondo J.
      • Hall L.L.
      • Ndoye A.
      • Chernyavsky A.I.
      • Jolkovsky D.L.
      • Grando S.A.
      Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.
      ). Most of these experiments were performed with cultured human KCs rather than in an animal model and employed muscarinic ligands that lack selectively to specific mAChR subtypes. Therefore, the precise effects of specific mAChR subtypes on in vivo epidermal cell proliferation and molecular mechanisms mediating the physiologic regulation by ACh remain to be identified.
      To identify the cell types controlled by non-neuronal ACh in the murine epidermis, discern major cell functions, and identify the principal regulatory mechanisms in this study, we employed single-cell RNA-sequencing (scRNA-seq) technology and the receptor-knockout (KO) mice. We focused on the M3 mAChR subtype (Chrm3), the major mAChR mediating ACh signals in basal KCs. CHRM3 is preferentially coupled to the activation of pertussis toxin–insensitive G proteins of the G α q/11 family, which activate phospholipase C and produce inositol 1,4,5-triphosphate and diacylglycerol. These second messengers elicit the activation of protein kinase C and trigger the release of calcium ion from intracellular stores. CHRM3 is one of the major antigens targeted by autoantibodies in severe pemphigus (
      • Chernyavsky A.
      • Patel K.G.
      • Grando S.A.
      Mechanisms of synergy of autoantibodies to M3 muscarinic acetylcholine receptor and secretory pathway ca 2+/Mn 2+-ATPase isoform 1 in patients with non-desmoglein pemphigus vulgaris.
      ,
      • Chernyavsky A.
      • Amber K.T.
      • Agnoletti A.F.
      • Wang C.
      • Grando S.A.
      Synergy among non-desmoglein antibodies contributes to the immunopathology of desmoglein antibody-negative pemphigus vulgaris.
      ;
      • Kalantari-Dehaghi M.
      • Anhalt G.J.
      • Camilleri M.J.
      • Chernyavsky A.I.
      • Chun S.
      • Felgner P.L.
      • et al.
      Pemphigus vulgaris autoantibody profiling by proteomic technique.
      ;
      • Lakshmi M.J.D.
      • Jaisankar T.J.
      • Rajappa M.
      • Thappa D.M.
      • Chandrashekar L.
      • Divyapriya D.
      • et al.
      Correlation of antimuscarinic acetylcholine receptor antibody titers and antidesmoglein antibody titers with the severity of disease in patients with pemphigus.
      ;
      • Sinha A.A.
      Constructing immunoprofiles to deconstruct disease complexity in pemphigus.
      ). Patients with pemphigus develop intraepidermal cell‒cell detachment (acantholysis) above the basal cell layer, blisters, and nonhealing erosions (reviewed in
      • Grando S.A.
      Cholinergic control of epidermal cohesion.
      ).
      We found that Chrm3-/- neonatal mice feature abnormal epidermal morphology characterized by an increased number of basal cells and epidermal thickness. Intercellular spaces in the basal cell layer are increased, consistent with decreased cell‒cell adhesion. These morphologic changes were associated with upregulation of cell proliferation genes and downregulation of epidermal differentiation genes as well as downregulation of the expression of genes contributing to extracellular matrix formation, intercellular adhesion, and cell arrangement. These findings provide new insights into the molecular mechanisms by which ACh regulates epidermal development and lay the groundwork for translational studies on cholinergic drugs in dermatology.

      Results

      Altered intercellular cohesion of basal cells and epidermal hyperplasia in neonatal Chrm3-/- mice

      We studied postnatal day (P) 0 wild-type (WT) and Chrm3-/- mice to define the epidermal role of ACh acting through CHRM3 (Figure 1a). In the epidermis of WT mice, basal layer KCs form a single row of polygonal epithelial cells with indistinct cell borders; the intercellular spaces are invisible. By contrast, the epidermis of Chrm3-/- mice contains an increased number of basal cells, giving the impression of crowding of basal KCs (n = 3‒3, P = 0.013) (Figure 1b). The overall epidermal thickness is also increased in Chrm3-/- mice (n = 7‒13, P = 0.018) (Figure 1b). Basal layer cells in Chrm3-/- mice also appear smaller with distinct cell borders, revealing intercellular spaces (Figure 1a, arrows). At a higher magnification, cellular bridges crossing the intercellular spaces can be seen in the Chrm3-/- epidermis (Figure 1a). Taken together, these results suggest that lack of Chrm3 signaling is associated with decreased cell‒cell adhesion in the basal layer and increased cell proliferation leading to epidermal hyperplasia.
      Figure thumbnail gr1
      Figure 1Chrm3-/- thickens mouse epidermis. (a) The differences between epidermal morphology between WT versus Chrm3(-/-) mice were manifested by an increased thickness of the basal layer comprised of crowded and slightly shrunk keratinocytes in KO mice. Bar = 10μm. (b) Quantitative analysis of the differences in epidermal morphology between WT and Chrm3(-/-) mice showed an increase in both the thickness of epidermis and the number of nucleated basal cells within IFE in KO mice. P - value was calculated from two-tailed t-test. (c) UMAP showing the clusters of seven major epidermal cell types, including IFE, HF, melanocytes, Langerhans cells, Merkel cells, sebaceous gland, and T cells. The marker gene list is shown in . (d) All the seven epidermal cell types are present in both WT and Chrm3(-/-) epidermis. (e) WT and Chrm3(-/-) Cellular composition of epidermis. The black box highlights the basal populations, which are increased in the Chrm3(-/-) epidermis. HF, hair follicle; IFE, interfollicular epidermis; KO, knockout; UMAP, Uniform Manifold Approximation and Projection; WT, wild-type.

      Expansion of stem and progenitor cells in the Chrm3-/- mouse neonatal epidermis

      To understand the cellular and transcriptomic changes underlying the epidermal abnormalities described earlier, we collected dorsal skin from Chrm3-/- P0 C57BL/6J mice (n = 2), isolated epidermal cells, and performed scRNA-seq. In total, 19,486 cells passed the quality control and were included in the data analysis. To allow for comparison with normal neonatal epidermis, we integrated the Chrm3-/- dataset with our previously published scRNA-seq data of 13,353 epidermal cells similarly collected from WT P0 C57BL/6J mouse epidermis (n = 2) (
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ). The integrated dataset contains 32,839 cells (Figure 1c).
      Seven major epidermal cell types were identified in the dataset: interfollicular epidermis (IFE) KCs, hair follicle KCs, sebaceous gland cells, Merkel cells, melanocytes, Langerhans cells, and T cells (Figure 1c). The IFE readily forms five subpopulations. Using canonical marker genes (
      • Joost S.
      • Annusver K.
      • Jacob T.
      • Sun X.
      • Dalessandri T.
      • Sivan U.
      • et al.
      The molecular anatomy of mouse skin during hair growth and rest.
      ,
      • Joost S.
      • Zeisel A.
      • Jacob T.
      • Sun X.
      • La Manno G.L.
      • Lönnerberg P.
      • et al.
      Single-cell transcriptomics reveals that differentiation and spatial signatures shape epidermal and hair follicle heterogeneity.
      ;
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ), we identified three of them as basal cell clusters (basal IFE 1, n = 2,971; basal IFE 2, n = 3,041; basal IFE 3, n = 3,319), identified one as a transitional cell cluster (transition IFE, n = 1,895), and identified one differentiated cell cluster (differentiated IFE, n = 1,188). These IFE cluster assignments correspond well to our previous study on the role of GRHL3 in epidermal differentiation, showing several basal subtypes and a prominent cluster of cells transitioning between the basal and suprabasal layers (
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ).
      Chrm3 KO does not result in the loss of epidermal cell types or the formation of new cell types. The Chrm3-/- cells do not form a unique cluster, and their positions in the Uniform Manifold Approximation and Projection (UMAP) generally overlap with those of the WT cells (Figure 1d). However, the cellular composition of the Chrm3-/- epidermis is different from that of the WT. The proportion of IFE KCs in the skin and the total proportion of the basal IFE KC subpopulations almost doubled and tripled, respectively, in the Chrm3-/- mice (Figure 1e). These data indicate that the increased epidermal thickness and cellularity of the Chrm3-/- (Figure 1a) is due to the expansion of IFE stem and progenitor cells.
      We also noted significant decreases in the proportion of Merkel cells (WT 1 = 0.14%, WT 2 = 0.19%, KO 1 = 0.06%, KO 2 = 0.06%) and Langerhans cells (WT 1 = 1.28%, WT 2 = 1.50%, KO 1 = 0.45%, KO 2 = 0.60%) in the Chrm3-/- epidermis (Figure 1e), indicating that Chrm3 may play a role in sensory and epidermal immune development.

      Expansion of stem cells, including cycling cells, and contraction of differentiated cells in the Chrm3-/- IFE

      To further examine the cellular abnormality in the neonatal Chrm3-/- IFE, we computationally isolated the IFE KCs and performed subcluster analysis. Eight IFE clusters formed: four basal populations (basal 1 = 1,641, basal 2 = 1,524, basal proliferating = 813, and basal aberrant = 1,533), two transitional populations (transition 1: n = 1,574 and transition 2: n = 381), one differentiated population (differentiated = 1,034), and one terminally differentiated population (terminally differentiated = 249) (Figure 2a). The expression pattern of KC differentiation marker genes is consistent with that in previous studies establishing canonical markers for these populations (
      • Joost S.
      • Zeisel A.
      • Jacob T.
      • Sun X.
      • La Manno G.L.
      • Lönnerberg P.
      • et al.
      Single-cell transcriptomics reveals that differentiation and spatial signatures shape epidermal and hair follicle heterogeneity.
      ;
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ) (Figure 2b). K14, K5, Col17a1, and Itga6 are expressed at high levels in the basal populations, with the proliferating basal cells expressing additional proliferation marker genes such as Mki67 and Top2. K10, K1, Klf4, and Tgm3 are expressed in the differentiated populations. Loricrin gene Lor specifically marks the terminally differentiated population. The transition populations express marker genes for both basal and differentiated KCs but at lower levels (Figure 2b and c).
      Figure thumbnail gr2
      Figure 2Chrm3-/- expands the population of basal cells and cycling cells. (a) Upper: UMAP showing the eight clusters of the IFE, including four basal populations, two transition populations, one differentiated population, and a terminally differentiated population. Lower: Bar graph presenting the cellular composition of the WT and Chrm3-/- IFE. (b) Expression of marker genes projected onto the UMAP to identify the basal cells (K14, K5, Col17a1, Itga6), differentiated cells (K10, K1, Klf4, Tgm3), and terminally differentiated cells (Lor). Red denotes high expression, and blue denotes low expression. (c) Heatmap showing the top 10 marker genes for each of the clusters defined in a. Blue boxes highlight some of the canonical marker genes. The complete marker gene list is shown in . (d) Upper: cell cycle assignment projected onto the UMAP. Lower: bar graph showing the percentage of each cell cycle stage in the WT and Chrm3-/- IFE. (e) A representative image of the epidermis WT and Chrm3-/- mice staining with anti‒Ki-67 antibody (bar = 20 μ m) as well as direct count of Ki-67‒postive cells showing more positive cells in the basal layer of KO mice. P-value was calculated from two-tailed t-test. IFE, interfollicular epidermis; K, keratin; KO, knockout; Lor, loricrin; UMAP, Uniform Manifold Approximation and Projection; WT, wild-type.
      The increase in the proportion of basal cells in the Chrm3-/- IFE is further confirmed in the subclustering analysis. All the four basal subpopulations are increased in the Chrm3-/- epidermis, with the most significant increase in the basal 2 (WT 1 = 5.6%, WT 2 = 5.2%, KO 1 = 21.9%, and KO 2 =22.6%) and the basal aberrant clusters (WT 1 = 9.3%, WT 2 = 7.5%, KO 1 = 19.1%, and KO 2 =10.5%) (Figure 2a). Consistent with the pattern found in WT (
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ), Chrm3-/- basal 1 expresses high levels of H19, Igf2, and Wnt4, whereas Chrm3-/- basal 2 cluster expresses the marker genes of basal 1 but higher levels of decorin gene Dcn, a gene that encodes the extracellular matrix protein decorin, and Sox4 (Figure 2c). Similar to the aberrant basal population found in the Grhl3-/- epidermis (
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ), the basal aberrant cluster in Chrm3-/- mice expresses the marker genes of both basal 1 and basal 2 but also some cell cycle marker genes such as Ccnd1 and Nasp.
      Cell cycle scoring using Seurat suggests that the basal aberrant cluster consists of cells in the S-phase. In fact, the proportion of cells expressing S-phase markers is twice higher in the Chrm3-/- than in the WT IFE (WT 1 = 16.4%, WT 2 = 14.3%, KO 1 = 27.7%, KO 2 = 32.7%) (Figure 2d). Consistent with scRNA-seq findings, immunofluorescent staining of the epidermis from Chrm3-/- and WT mice with anti‒Ki-67 antibody revealed more positive cells (n = 3‒3, P = 0.013) in the basal layer of KO mice than in the WT mice (Figure 2e).
      In addition, the population of differentiated KCs is decreased in the Chrm3-/- epidermis (WT 1 = 26.3%, WT 2 = 28.5%, KO 1 = 6.7%, KO 2 = 4.7%), whereas the terminally differentiated population is unchanged (WT 1 = 2.5%, WT2 = 2.1%, KO 1 = 2.0%, KO 2 = 2.3%) (Figure 2a). In summary, these data show the expansion of stem cells and a decrease in differentiated cells in the Chrm3-/- IFE, indicating that Chrm3 promotes differentiation of IFE stem cells.

      Downregulation of epidermal differentiation genes and upregulation of cell proliferation genes in the Chrm3-/- epidermis

      Having established changes in epidermal differentiation and cellular composition of the Chrm3-/- IFE, we next studied changes in gene expression. Differential gene expression analysis performed on all IFE populations collectively reveals downregulation of genes related to KC differentiation in the Chrm3-/- IFE (Figure 3a and Supplementary Table S3). Gene Ontology (GO) analysis shows that the downregulated genes are enriched for biological processes such as epidermis development, regulation of water loss through the skin, and establishment of skin barrier (Figure 3b). These gene expression changes are not caused solely by the decrease in the proportion of differentiated KCs in the Chrm3-/- IFE. GO analysis performed on downregulated genes in Chrm3-/- differentiated and terminally differentiated IFE clusters alone gives similar results (Supplementary Figure S1 and Supplementary Table S4). Taken together, the decreased IFE differentiation in Chrm3-/- neonatal mice is evident at both cellular and molecular levels.
      Figure thumbnail gr3
      Figure 3Differentiation is downregulated in the Chrm3-/- IFE. (a) Heatmap showing 120 of the differentially expressed genes between the WT and Chrm3-/- IFE. The blue boxes highlight the genes related to keratinocyte differentiation. The complete gene list is shown in . (b) GO analysis reveals that keratinocyte differentiation is downregulated in the Chrm3-/- IFE. (c) GO analysis reveals that terms related to cell proliferation are upregulated in the Chrm3-/- IFE. (d) Distribution patterns of K5- and K10-positive keratinocytes in the epidermis of WT and Chrm3-/- mice visualized by confocal microscopy of skin samples stained with anti-K5 (red) and anti-K10 (green) antibodies (bar = 20 μ m) and quantitative analysis of the intensity of fluorescence produced by each antibody within IFE showing an increase in K5 and decrease in K10 expression in KO mice. p− value was calculated from two-tailed t-test. (e) Expression patterns of the six groups of genes defined in Lin et al. (2020) shown on the left and enrichment score and corresponding false discovery rate q-value obtained from GSEA shown on the right. GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; IFE, interfollicular epidermis; K, keratin; KO, knockout; WT, wild-type.
      By contrast, several marker genes of basal KCs such as K14, K5, and Itgb1 are upregulated in the Chrm3-/- IFE. The upregulated genes are overrepresented in GO biological processes such as RNA splicing, covalent chromatin modification, and regulation of chromosome organization (Figure 3c). These results point to the upregulation of cell division in the Chrm3-/- IFE, which is consistent with our finding of higher proportions of S-phase and basal cells in the Chrm3-/- IFE (Figure 2a and d).
      The analysis of epidermal cytokeratin expression at the protein level using fluorescence staining revealed differences in the distribution of K5- and K10-positive KCs between the epidermis of WT and Chrm3-/- mice. Confocal microscopic images of the epidermis double stained for K5 and K10 revealed increased staining for K5 and decreased staining for K10 in Chrm3-/-mice (Figure 3d). The number of K5-positive cells within the IFE of Chrm3-/- mice was significantly (n = 2‒2, P = 0.028) higher than that in WT mice. By contrast, the number of K10-positive cells within the IFE in Chrm3-/- mice was significantly lower than in WT mice (n = 2‒2, P = 0.007). These results support the findings of the scRNA-seq that Chrm3 promotes epidermal differentiation.

      Basal genes are prominently upregulated in the terminally differentiated cells of the Chrm3-/- epidermis

      Previously, we have comprehensively characterized the P0 murine epidermal differentiation program by defining six groups of genes with distinct pseudotemporal expression patterns during IFE KC differentiation (
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ) (Figure 3e, left). Groups 1 and 2 genes are highly expressed in basal cells with decreasing expression as differentiation progresses. Group 3 genes are lowly expressed in basal cells, peak in the middle of differentiation, and decrease in expression toward terminal differentiation. Groups 4, 5, and 6 genes are lowly expressed in basal cells with rising expression as differentiation progresses. Using these six groups of genes as gene sets, we performed Gene Set Enrichment Analysis (
      • Mootha V.K.
      • Lindgren C.M.
      • Eriksson K.-F.
      • Subramanian A.
      • Sihag S.
      • Lehar J.
      • et al.
      PGC-1 alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes.
      ;
      • Subramanian A.
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      • Ebert B.L.
      • Gillette M.A.
      • et al.
      Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles.
      ) using genes differentially expressed in the Chrm3-/- IFE as input (Supplementary Table S3). We found that group 1 and group 2 genes are overrepresented in upregulated genes and that groups 3, 4, 5, and 6 are overrepresented in downregulated genes in the Chrm3-/- IFE (Figure 3e, right). This analysis indicates that Chrm3 broadly suppresses progenitor genes and activates differentiation genes.
      We also used the six groups of differentiation genes to define more precisely the differentiation defect in the Chrm3-/- IFE; we scored the expression of each group in each differentiation stage of the WT and Chrm3-/- IFE (Figure 4a). This analysis shows that the most striking defect in the Chrm3-/- IFE is in the terminally differentiated KCs where groups 1 and 2 genes are highly upregulated and groups 4, 5, and 6 genes are highly downregulated.
      Figure thumbnail gr4
      Figure 4Epidermal differentiation is downregulated in suprabasal cells and adhesion molecules are downregulated in Chrm3-/- basal cells. (a) Scoring of the cells in each differentiation stage on the basis of their expression level of six groups of genes defined in
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      shows that the disturbance in the differentiation profile is most evident in the differentiated cells. P-value was calculated from t-test. ns: p > 0.05, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗ P ≤ 0.001, and ∗∗∗∗ P ≤ 0.0001. (b) Gene Ontology analysis on genes downregulated in Chrm3-/- basal cells show enrichment for skin development and keratinocyte differentiation. Genes contributing to the enrichment of some of the terms are listed. The complete list of differentially expressed genes in Chrm3-/- versus in WT basal cells is provided in . (c) Adhesion molecules are downregulated in the Chrm3-/- basal cells. P-value was calculated from t-test. ns: p > 0.05, ∗p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001. ns, not significant; WT, wild-type.
      GRHL3 is a transcription factor that promotes differentiation (
      • Ting S.B.
      • Caddy J.
      • Hislop N.
      • Wilanowski T.
      • Auden A.
      • Zhao L.
      • et al.
      A homolog of drosophila grainy head is essential for epidermal integrity in mice.
      ;
      • Yu Z.
      • Lin K.K.
      • Bhandari A.
      • Spencer J.A.
      • Xu X.
      • Wang N.
      • et al.
      The grainyhead-like epithelial transactivator Get-1/Grhl3 regulates epidermal terminal differentiation and interacts functionally with LMO4.
      ) and suppresses Wnt signaling in and expansion of epidermal stem cells (
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ). Although the epidermal differentiation defect in Chrm3-/- bears similarity to that of the Grhl3-/- epidermis (
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ) because both mutants show accumulation of stem and progenitor cells and reduction in differentiated KCs, the marked upregulation of basal genes in the terminally differentiated KCs is unique to the Chrm3-/- mutant (Supplementary Figure S2).

      Adhesion molecules are downregulated in Chrm3-/- basal KCs

      Because Chrm3 is most highly expressed in the basal cell compartment (
      • Kurzen H.
      • Berger H.
      • Jäger C.
      • Hartschuh W.
      • Näher H.
      • Gratchev A.
      • et al.
      Phenotypical and molecular profiling of the extraneuronal cholinergic system of the skin.
      ;
      • Ndoye A.
      • Buchli R.
      • Greenberg B.
      • Nguyen V.T.
      • Zia S.
      • Lawry M.A.
      • et al.
      Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human Epidermis.
      ), we isolated the four basal clusters and identified genes that are differentially expressed in the Chrm3-/- basal cells (Supplementary Table S5). The GO terms enriched for the upregulated and downregulated genes in the Chrm3-/- basal cells are similar to the ones found in differentially expressed genes and GO analysis done for the whole IFE, with processes related to cell division being upregulated (not shown) and processes related to epidermis development being downregulated (Figure 4b). A closer look at the downregulated genes contributing to GO terms such as skin development, epidermis development, KC development, and epithelial cell development reveals the downregulation of genes contributing to extracellular matrix formation, intercellular adhesion, and cell arrangement, including Jup, Evpl, Barx2, Arrdc3, and Gja1 (Figure 4c). The downregulations of adhesion molecules can explain, in part, the increase in intercellular space between Chrm3-/- basal cells (Figure 1a).

      Discussion

      In this study, we used Chrm3-/- mice and scRNA-seq to characterize the role of non-neuronal ACh in epidermal gene expression and differentiation. We found that Chrm3 promotes the expression of cell‒cell adhesion molecules in the basal cell layer and differentiation throughout the epidermis. These findings support the idea that the cytotransmitter ACh—produced and released by epidermal KCs and signaling through the CHRM3 subtype expressed in epidermal stem cells—has an important epidermal developmental role. This study also sheds new light on the pathophysiology of pemphigus vulgaris where some patients develop autoantibodies inactivating CHRM3 through receptor desensitization (
      • Chernyavsky A.
      • Khylynskyi M.M.
      • Patel K.G.
      • Grando S.A.
      Chronic exposure to the anti-M3 muscarinic acetylcholine receptor autoantibody in pemphigus vulgaris contributes to disease pathophysiology.
      ). Patients with pemphigus vulgaris feature some of the same alterations in adhesion, epidermal stem cell proliferation, and differentiation as observed in the experimental model of Chrm3 inactivation. Similar to the epidermal changes in Chrm3-/- mice, anti-CHRM3 pemphigus autoantibodies upregulate K5 and downregulate K10 (
      • Chernyavsky A.
      • Khylynskyi M.M.
      • Patel K.G.
      • Grando S.A.
      Chronic exposure to the anti-M3 muscarinic acetylcholine receptor autoantibody in pemphigus vulgaris contributes to disease pathophysiology.
      ) in mice—findings that are mirrored in the epidermis of patients with pemphigus vulgaris (
      • Williamson L.
      • Raess N.A.
      • Caldelari R.
      • Zakher A.
      • Bruin A. de
      • Posthaus H.
      • et al.
      Pemphigus vulgaris identifies plakoglobin as key suppressor of c-myc in the skin.
      ).
      Changes in the cellular composition of the Chrm3-/- epidermis support the prodifferentiation role of Chrm3 in epidermal stem cells. The proportion of undifferentiated basal cells, including proliferating basal cells, increases. By contrast, the proportion of differentiated epidermal cells decreases (Figures 1e and 2a and d). Morphologic analyses support these scRNA-seq findings. The number of basal epidermal cells (Figure 1b), the epidermal thickness (Figure 1a and b), and the basal cell proliferation (Figure 2e) are increased, whereas the expression of K10 differentiation marker is decreased (Figure 3d). Our gene expression analysis showed that downregulated genes in the differentiated cells of the Chrm3-/- IFE play a role in epidermal differentiation (Figure 3a and b and Supplementary Figure S1). By contrast, upregulated genes in the Chrm3-/- IFE facilitate the proliferation of basal cells (Figure 3a and c and Supplementary Figure S1).
      The mechanisms underlying the prodifferentiation effects of Chrm3 remain unknown, but given that Chrm3 is expressed to the highest levels in basal cells, we believe that it acts at the level of epidermal stem cells. Possibly, the primary function of Chrm3 is to promote the expression of cell‒cell and cell‒matrix adhesion molecules. Consistent with this idea, increased intercellular spaces between basal KCs are a prominent feature of the Chrm3-/- epidermis (Figure 1a). Also consistent with this finding, we noted decreased expression of a number of important cell‒cell and cell‒matrix adhesion molecules in the Chrm3-/- basal cells (Figure 4a and b). Loss of cell‒cell adhesion may impair the ability of basal KCs to undergo differentiation and promote proliferation. In other cell types, it has been shown that Chrm3 controls the assembly of the cytoskeleton, stimulates the formation of cell‒cell and cell‒substrate attachments, and inhibits cell proliferation (
      • Chernyavsky A.I.
      • Arredondo J.
      • Wess J.
      • Karlsson E.
      • Grando S.A.
      Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors.
      ;
      • Shafer S.H.
      • Puhl H.L.
      • Phelps S.H.
      • Williams C.L.
      Activation of transfected M1or M3Muscarinic acetylcholine receptors induces cell-cell adhesion of Chinese hamster ovary cells expressing endogenous cadherins.
      ;
      • Shafer S.H.
      • Williams C.L.
      Elevated Rac1 activity changes the M3Muscarinic acetylcholine receptor-mediated inhibition of proliferation to induction of cell death.
      ;
      • Strassheim D.
      • May L.G.
      • Varker K.A.
      • Puhl H.L.
      • Phelps S.H.
      • Porter R.A.
      • et al.
      M3 muscarinic acetylcholine receptors regulate cytoplasmic myosin by a process involving RhoA and requiring conventional protein kinase C isoforms.
      ;
      • Williams C.L.
      • Hayes V.Y.
      • Hummel A.M.
      • Tarara J.E.
      • Halsey T.J.
      Regulation of E-cadherin-mediated adhesion by muscarinic acetylcholine receptors in small cell lung carcinoma.
      ).
      Many of the downregulated genes in the Chrm3-/- basal cells play important roles in extracellular matrix formation, intercellular adhesion, and cell arrangement. Jup encodes plakoglobin, which forms adherence junctions and facilitates the formation of desmosomes. Itga6 participates in the production of integrin, which is important for cell‒substrate adhesion. Evpl encodes a hemidesmosome component. Autoimmunity to envoplakin is associated with paraneoplastic pemphigus (also known as paraneoplastic autoimmune multiorgan syndrome) (
      • Nguyen V.T.
      • Ndoye A.
      • Bassler K.D.
      • Shultz L.D.
      • Shields M.C.
      • Ruben B.S.
      • et al.
      Classification, clinical manifestations, and immunopathological mechanisms of the epithelial variant of paraneoplastic autoimmune multiorgan syndrome: a reappraisal of paraneoplastic pemphigus.
      ). Barx2 plays a critical role in cell adhesion and cytoskeleton remodeling, and its knockdown stimulates cell proliferation in human bronchial epithelial cells (
      • Chen H.
      • Zhang M.
      • Zhang W.
      • Li Y.
      • Zhu J.
      • Zhang X.
      • et al.
      Downregulation of BarH-like homeobox 2 promotes cell proliferation, migration and aerobic glycolysis through wnt/ β-catenin signaling, and predicts a poor prognosis in non-small cell lung carcinoma: Barx2 and non-small cell lung carcinoma.
      ); Arrdc3 negatively regulates integrin β4. Gja1 encodes connexin 43, which is important for gap junctions between KCs and is targeted by pemphigus autoimmunity (
      • Abreu-Velez A.M.
      • Howard M.S.
      • Jiao Z.
      • Gao W.
      • Yi H.
      • Grossniklaus H.E.
      • et al.
      Cardiac autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in Colombia, South America.
      ).
      We also observed a significant decrease in the proportion of Langerhans cells in the Chrm3-/- epidermis. It has been previously noted that activation of the CHRM3 is essential for optimal immune responses, including both T helper 1 and T helper 2 cytokine production. Chrm3 deficiency in mice significantly abrogates the ability to launch an effective adaptive immune response to helminth and bacterial infections (
      • Darby M.
      • Schnoeller C.
      • Vira A.
      • Culley F.J.
      • Bobat S.
      • Logan E.
      • et al.
      The M3 muscarinic receptor is required for optimal adaptive immunity to helminth and bacterial infection.
      ;
      • McLean L.P.
      • Smith A.
      • Cheung L.
      • Urban J.F.
      • Sun R.
      • Grinchuk V.
      • et al.
      Type 3 muscarinic receptors contribute to intestinal mucosal homeostasis and clearance of nippostrongylus brasiliensis through induction of TH2 cytokines.
      ).
      In addition to pemphigus, altered signaling of autocrine/paracrine ACh through CHMR3 has been shown in breast, colorectal, and several other cancers (
      • Chen J.
      • Cheuk I.W.Y.
      • Shin V.Y.
      • Kwong A.
      Acetylcholine receptors: key players in cancer development.
      ;
      • Lin G.
      • Sun L.
      • Wang R.
      • Guo Y.
      • Xie C.
      Overexpression of muscarinic receptor 3 promotes metastasis and predicts poor prognosis in non-small-cell lung cancer.
      ;
      • Tolaymat M.
      • Larabee S.M.
      • Hu S.
      • Xie G.
      • Raufman J.P.
      The role of M3 muscarinic receptor ligand-induced kinase signaling in colon cancer progression.
      ), asthma and chronic obstructive pulmonary disease (
      • Yamada M.
      • Ichinose M.
      The cholinergic pathways in inflammation: a potential pharmacotherapeutic target for COPD.
      ), immune-mediated inflammation (
      • Kawashima K.
      • Fujii T.
      Expression of non-neuronal acetylcholine in lymphocytes and its contribution to the regulation of immune function.
      ), Sjögren syndrome (
      • Fox R.I.
      • Stern M.
      • Michelson P.
      Update in Sjögren syndrome.
      ), cholinergic urticaria (
      • Tokura Y.
      New etiology of cholinergic urticaria.
      ), wound reepithelialization (
      • Chernyavsky A.I.
      • Arredondo J.
      • Wess J.
      • Karlsson E.
      • Grando S.A.
      Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors.
      ), and diabetes (
      • Kruse A.C.
      • Li J.
      • Hu J.
      • Kobilka B.K.
      • Wess J.
      Novel insights into M3 muscarinic acetylcholine receptor physiology and structure.
      ) as well as gastrointestinal and urinary bladder functions (
      • Ehlert F.J.
      • Pak K.J.
      • Griffin M.T.
      Muscarinic agonists and antagonists: effects on gastrointestinal function.
      ;
      • Yamanishi T.
      • Chapple C.R.
      • Chess-Williams R.
      Which muscarinic receptor is important in the bladder?.
      ). These and some other human diseases may therefore benefit from pharmacologic modulation of CHRM3.
      We previously studied the role of GRHL3 in epidermal differentiation (
      • Kudryavtseva E.I.
      • Sugihara T.M.
      • Wang N.
      • Lasso R.J.
      • Gudnason J.F.
      • Lipkin S.M.
      • et al.
      Identification and characterization of grainyhead-like epithelial transactivator (GET-1), a novel mammalian grainyhead-like factor.
      ;
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ;
      • Yu Z.
      • Lin K.K.
      • Bhandari A.
      • Spencer J.A.
      • Xu X.
      • Wang N.
      • et al.
      The grainyhead-like epithelial transactivator Get-1/Grhl3 regulates epidermal terminal differentiation and interacts functionally with LMO4.
      ) and observed the expansion of basal cells and decreased number of differentiated cells in the Grhl3-/- epidermis. Although we also observed these features in the Chrm3-/- epidermis, we noticed striking differences between the differentiation defects in these two mutants. In particular, the gene expression programs associated with basal cells were uniquely upregulated in the terminally differentiated KCs of the Chrm3-/- epidermis (Supplementary Figure S2). We speculate that the combined deletion of Ghrl3 and Chrm3 would result in an additive effect with increased expansion of basal KCs and a reduced number of differentiated cells.
      In conclusion, Chrm3 plays an important role in epidermal development, promoting adhesion of basal cells, suppressing basal cell proliferation, and promoting epidermal differentiation.

      Materials and Methods

      Mice and reagents

      The Chrm3-/- mice were a generous gift from Jurgen Wess (Laboratory of Bioorganic Chemistry, Molecular Signaling Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD). This KO mouse line has been used in our previous experiments (e.g.,
      • Chernyavsky A.I.
      • Arredondo J.
      • Wess J.
      • Karlsson E.
      • Grando S.A.
      Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors.
      ).
      The Ki-67 mouse antibody was purchased from GeneTex (dilution 1:500). The K5 rabbit antibody was purchased from Abcam (Cambridge, United Kingdom) (dilution 1:500). The K10 mouse antibody was from Covance (Princeton, NJ) (dilution 1:500).
      All protocols were approved by the University Laboratory Animal Resources at the University of California, Irvine (Irvine, CA).

      scRNA-seq experiments

      Sample collection and sequencing

      Sample collection was done as previously described (
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ). In brief, skin samples were collected from P0 Chrm3-/- mice and incubated overnight in epidermal separation buffer. The epidermis was then separated from the dermis, and the suspension of epidermal cells was passed through a 40-μ M filter. Dead cells were removed using Dead Cell Removal kit.
      University of California, Irvine Genomic High Throughput Facility prepared the Chromium Single Cell, version 3.1 (10x Genomics, Pleasanton, CA) libraries, which were sequenced with Illumina NovaSeq6000.

      scRNA-seq data analysis

      Raw sequencing files were processed using Cell Ranger 3.0.2 with the MM10 reference as stated in
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      .
      The Chrm3-/- dataset (two mice) and the previously published WT dataset (
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      ) (two mice) were processed in R using Seurat, version 3 (
      • Stuart T.
      • Butler A.
      • Hoffman P.
      • Hafemeister C.
      • Papalexi III, E.
      • Mauck W.M.
      • et al.
      WMM. Comprehensive integration of single-cell data.
      ) according to the vignette.
      For all samples, only cells with 900‒7,700 genes and <10% mitochondrial genes were kept for further analysis. Each of the four samples was individually log normalized, and 2,000 highly variable genes were identified before integration. Integration was performed according to the Seurat standard integration workflow, and the features.to.integrate variable was set to include all genes in the datasets. The integrated dataset was then scaled, and principal component analysis was done. Clustering analysis on the integrated dataset used the Louvain algorithm, and the output was visualized with UMAP.
      For marker genes for each cell type in the skin (or differentiation stages in the IFE), we only ran the differential gene expression test on genes that are expressed in >25% of the cell-type population, and only the genes with <0.05 adjusted and >0.25 log fold change were reported.
      For differential gene expression tests between the two genotypes, WT and Chrm3-/-, Wilcoxon rank sum tests were performed, and any genes with <0.05 adjusted P-values were used for GO analysis.
      GO analyses were performed using ClusterProfiler in R (
      • Yu G.
      • Wang L.-G.
      • Han Y.
      • He Q.-Y.
      clusterProfiler: an R package for comparing biological themes among gene clusters.
      ). Gene Set Enrichment Analysis was performed on the desktop software Gene Set Enrichment Analysis, version 4 (
      • Mootha V.K.
      • Lindgren C.M.
      • Eriksson K.-F.
      • Subramanian A.
      • Sihag S.
      • Lehar J.
      • et al.
      PGC-1 alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes.
      ;
      • Subramanian A.
      • Tamayo P.
      • Mootha V.K.
      • Mukherjee S.
      • Ebert B.L.
      • Gillette M.A.
      • et al.
      Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles.
      ). Gene set scores were added to scRNA-seq datasets using the AddModuleScore function provided in Seurat.

      Other methods

      The number of nucleated basal cells within the IFE was measured within the 12 × 100 μ m rectangle frame applied to the images of IFEs taken at magnification ×20. The number of Ki-67‒postive cells within the IFE was measured within the 70 × 100 μ m rectangle frame applied at ×20. The intensities of fluorescence produced by anti-K5 and anti-K10 antibodies were measured below the stratum corneum within IFE, the background nonspecific fluorescence was subtracted, and the resultant values were divided by the number of DAPI-positive cell numbers inside the analyzed area.

      Statistical analysis

      The data were analyzed using t-test with a significance cutoff value of 0.05 and presented as mean ± standard error. All statistical analyses and graphs were done in R.

      Data availability statement

      The single-cell RNA-sequencing data of the two wild-type epidermis samples were previously published in
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      and were deposited in the Gene Expression Omnibus database under GSE154579 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154579). The single-cell RNA-sequencing data of the two Chrm3-/- samples are available in the Gene Expression Omnibus database under GSE201885 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE201885).

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgment

      This work was supported by a research grant from the Dysimmune Diseases Foundation and internal funds of the Department of Dermatology, School of Medicine, University of California, Irvine (to SAG); National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases grants P30AR075047 and R01AR44882 and the Irving Weinstein Foundation (to BA); and an NSF grant DMS1763272 and a grant from the Simons Foundation (594598) (to JD).

      Author Contributions

      Conceptualization: SAG, BA; Data Curation: JD, CG; Formal Analysis: JD; Funding Acquisition: SAG, BA; Investigation: JD, CG, SL, AC, JKC; Methodology: SAG, BA; Project Administration: SAG, BA; Resources: SAG, BA; Software: JD; Supervision: SAG, BA; Validation: JD, CG, SL, AC, JKC, SAG, BA; Visualization: JD; Writing – Original Draft Preparation: JD, SAG, BA; Writing – Review and Editing: JD, CG, SAG, BA

      Supplementary Materials

      Figure thumbnail fx1
      Supplementary Figure S1Gene Ontology results obtained from genes that are downregulated in the Chrm3-/- differentiated and terminally differentiated cells alone show that the downregulation of differentiation we observe when comparing Chrm3 -/- and WT IFE is not solely due to the decrease in the proportion of differentiated cells in the KO. IFE, interfollicular epidermis; KO, knockout; WT, wild-type.
      Figure thumbnail fx2
      Supplementary Figure S2Scoring of the cells in each differentiation stage on the basis of their expression level of six groups of genes defined in
      • Lin Z.
      • Jin S.
      • Chen J.
      • Li Z.
      • Lin Z.
      • Tang L.
      • et al.
      Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states.
      shows that Chrm3-/- and Grhl3-/- cause similar differentiation disruptions (upregulation of genes associated with basal cells and downregulation of genes associated with suprabasal cells). The defects caused by Grhl3 -/- is not as prominent as the defects caused by Chrm3 -/-. ns: p > 0.05. ∗p≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001. KO, knockout; ns, not significant.

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