Mitochondrial Function in Murine Skin Epithelium Is Crucial for Hair Follicle Morphogenesis and Epithelial–Mesenchymal Interactions  Jennifer E. Kloepper, Olivier R. Baris, Karen Reuter, Ken Kobayashi, Daniela Weiland, Silvia Vidali, Desmond J. Tobin, Catherin Niemann, Rudolf J. Wiesner, Ralf Paus  Journal of Investigative Dermatology  Volume 135, Issue 3, Pages 679-689 (March 2015) DOI: 10.1038/jid.2014.475 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 TfamEKO retards hair follicle (HF) morphogenesis, reduces HF density, and induces HF dystrophy. (a) Photos of mouse back skin (hematoxylin and eosin). (b) Thickness of the subcutis of TfamEKO mouse skin is significantly thinner, n=3, seven visual fields (VFs)/mouse. (c) HFs in late morphogenesis stages (6–8) are reduced in TfamEKO mice, n=2–4 (bars = 50 μm). (d) The amount of HFs in TfamEKO mice is significantly lower, n=5–6, 10 VFs/mouse. (e) Hair shafts of TfamEKO mice show no melanin banding pattern; melanin is ectopic, and dermal papillae (DPs) are condensed and look telogen-like. Black arrows indicate points of interest (bars=50 μm). (f, g) TfamEKO mice have condensed DPs (white asterisk: condensed DP), n=3, 5 VFs/mouse (bars = 13 μm). Mean±SEM, *P<0.05, **P<0.01, using the Mann–Whitney test (b–d) or the unpaired t-test (g). Journal of Investigative Dermatology 2015 135, 679-689DOI: (10.1038/jid.2014.475) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 TfamEKO hair follicles (HFs) show increased apoptosis, reduced proliferation, and pigmentation abnormalities. (a) Photos showing matrix keratinocytes in situ in high-resolution light microscopy; red arrows, proliferation; black arrows, apoptosis; n=3 (bars = 13 μm). (b) TfamEKO mice show HFs with melanin clumping and melanin disruption. (c, d) Proliferating cells (BrdU+: purple, keratin14: green) are decreased in TfamEKO animals. (c) Representative photos (bars=75 μm, left panels; 25 μm, right panels). (d) ***P<0.001, n=10 HFs/mouse from two mice per time point, Student‘s t-test for unpaired samples. (e) TUNEL+ cells in TfamEKO animals and littermates (bars = 20 μm). (f) Loss of Tfam results in an increased TUNEL signal in TfamEKO animals, ***P<0.001, n=5 mice per time point, Student‘s t-test for unpaired samples. Tfam, mitochondrial transcription factor A. Journal of Investigative Dermatology 2015 135, 679-689DOI: (10.1038/jid.2014.475) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Epithelial progenitor markers are partially abnormal in TfamEKO mice. (a) Representative photos show keratin15 (green) and 4,6-diamidino-2-phenylindole (DAPI; purple) immunoreactivity (bars = 25 μm). (b) Significant decrease in keratin15 immunoreactivity, n=20 hair follicles (HFs), from two mice per time point. (c) Lrig1 expression (green) is not affected; DAPI (purple; bars = 100 μm). (d) Representative photos show Plet1 (green, propidium iodide nuclear counterstaining purple) immunoreactivity (bars = 50 μm). (e) Only on day 5 after birth Plet1 immunoreactivity is significantly decreased, n=25–50 HFs/mouse, from 4 mice per time point. Mean±SEM, **P<0.01, ***P<0.001, analyzed by Student‘s t-test for unpaired samples. Journal of Investigative Dermatology 2015 135, 679-689DOI: (10.1038/jid.2014.475) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Loss of Tfam severely impairs hair follicle differentiation and sebaceous gland development. (a, b) Reduced expression of Lef1 in TfamEKO mice (green, nuclear counterstaining purple, bars = 50 μm), n=50–68 HFs/mouse, from four mice. (c) Reduced expression of membranal β-catenin (green) in TfamEKO HFs (counterstaining purple, bars = 100 μm). (d, e) Adipophillin (green, bars = 50 μm) as % of positive HFs, significant reduction in TfamEKO mice, n=50–68 HFs/mouse, from three mice. (f, g) Scd1 (purple) immunoreactivity (keratin14: green), significantly decreased in TfamEKO mice, n=42–98 HFs/mouse, from 4 mice (bars = 25 μm). (h) Oil red (red), nuclear staining (blue), 3 mice (bars = 100 μm). Mean±SEM, *P<0.05, ***P<0.001, Student‘s t-test for unpaired samples. TFAM, mitochondrial transcription factor A. Journal of Investigative Dermatology 2015 135, 679-689DOI: (10.1038/jid.2014.475) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 TfamEKO changes the distribution of immune cells. (a) MHC II immunostaining (red). (b) MHC II+ cells in different compartments (n=7–14 HFs, three mice) and (c) in the epidermis (n=11–16 visual fields, three mice). (d) Immunoreactivity of MHC II (n=6–118 HFs, three mice), (e) area size of compartment B and C (n=9–15 HFs, three mice). (f) CD11b immunostaining (brown). (g) Number of CD11b+ cells in different compartments (n=15–20 HFs, three mice) and (h) in the epidermis (n=15–20 visual fields, three mice). (i) Total number of CD11b+ cells per HF. Mean±SEM, *P<0.05, **P<0.01, Student‘s t-test for unpaired samples (bars = 50 μm). A.U., arbitrary units; A, connective tissue sheath 50 μm around the HF; B, upper HF compartment, 10 nuclei away from epidermis; C, lower HF compartment, below B; HF, hair follicles; MHC, major histocompatability complex. Journal of Investigative Dermatology 2015 135, 679-689DOI: (10.1038/jid.2014.475) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 TfamEKO disturbs the epithelial–mesenchymal cross-talk. (a, b) Alkaline phosphatase (AP) active dermal papillae (DPs) in blue (n=19–32 hair follicles (HFs), three mice). (c–e) Versican immunostaining, white lines demarcate the versican+ DP areas and immunoreactivity (IR) was measured (n=9–30 of HFs, 2–3 mice). (f, g) NCAM immunostaining, white lines demarcate the NCAM+ DP areas (1) and the dermal sheath cap (2). (f) Areas of NCAM+ DPs, (g) dermal sheath cap, and (i) immunoreactivity of DPs and dermal sheath cap (j). (n=9–30 HFs, 2–3 mice). Mean±SEM, *P<0.05, **P<0.01, unpaired Student‘s t-test, bars = 50 μm. Journal of Investigative Dermatology 2015 135, 679-689DOI: (10.1038/jid.2014.475) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions