Yasushi Hanakawa, Hong Li, Chenyan Lin, John R

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Desmogleins 1 and 3 in the Companion Layer Anchor Mouse Anagen Hair to the Follicle Yasushi Hanakawa, Hong Li, Chenyan Lin, John R. Stanley, George Cotsarelis Journal of Investigative Dermatology Volume 123, Issue 5, Pages 817-822 (November 2004) DOI: 10.1111/j.0022-202X.2004.23479.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Companion layer expresses desmoglein (Dsg)1 and Dsg3 in normal anagen hair follicles. Longitudinal (A, E) and transverse (C, D) sections through the suprabulbar (A–D) and bulbar (E, F) portions of anagen hair follicles immunostained with antibodies against Dsg1 and Dsg3. Dsg1 was present in the outer root sheath (ORS), inner root sheath (IRS) and companion layer (c). Dsg3 was detected in the precortical cells (PC) and companion layer (B, D, F). The dermal papilla (p) stains weakly for DSG1 and not for DSG3 (scale bar=50 μm). Journal of Investigative Dermatology 2004 123, 817-822DOI: (10.1111/j.0022-202X.2004.23479.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Desmoglein (Dsg)3-/-mice lose hair after injection with exfoliative toxin A (ETA) during anagen. (A) Mice as labeled were injected with ETA or phosphate-buffered saline (PBS), and 4 h later were subjected to an adhesive tape test. Only Dsg3-/- mice treated with ETA showed hair loss (seen on tape in bottom row). (B) Semiquantitation of hair loss on adhesive tape by weight. Dsg3-/- mice treated with ETA showed statistically significant loss of hair compared to Dsg3+/- mice treated with ETA (p<0.01). Journal of Investigative Dermatology 2004 123, 817-822DOI: (10.1111/j.0022-202X.2004.23479.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Desmoglein (Dsg)3-/-mice injected with exfoliative toxin A (ETA) develop a split between the outer root sheath (ORS) and inner root sheath (IRS). Histology of skin from Dsg3-/-mouse treated with ETA (B, D, F, H) demonstrates a split between the IRS and ORS (arrow), compared to skin from control Dsg3+/- mouse treated with ETA (A, C, E, G). Hair matrix cells in the bulb remain in both the treated (G) and control (H) follicles (scale bars=50 μm). Journal of Investigative Dermatology 2004 123, 817-822DOI: (10.1111/j.0022-202X.2004.23479.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The hair shaft, inner root sheath (IRS). and companion layer are removed after exfoliative toxin A (ETA) treatment of Dsg3-/-mice. (A) Toluidine blue staining of hair and surrounding cells removed from a Dsg3-/- mouse after injection with ETA shows a smooth surface and fractured base. (B) Transverse cryosection of removed hair and surrounding cells stained with toluidine blue shows deep blue staining of the hair shaft (center), blue staining of the IRS (middle), and a peripheral clear companion layer. Double staining of removed hairs with AE13 (stains hard keratins in the hair shaft) and anti-K6 (stains companion layer and medulla) antibodies shows presence of hair shaft (green; C, E) and companion layer and medulla (red; D, G). Double staining with AE15 (detects trichohyalin in IRS and medulla) and anti-K6 antibodies shows IRS ([arrowhead] green; F, H) encircled by companion layer ([arrow] red; G, H) (scale bars=50 μm). Journal of Investigative Dermatology 2004 123, 817-822DOI: (10.1111/j.0022-202X.2004.23479.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The outer root sheath (ORS) and bulb matrix remain in the skin of desmoglein (Dsg)3-/-mice after exfoliative toxin A (ETA) treatment. Immunofluorescence staining using anti-K14 and AE13 antibodies on Dsg3-/- mouse skin after ETA treatment and gentle hair removal (C, D). K14 antibody staining detects the basal epidermis and ORS in both control (A) and treated (C) mice. Lack of AE13 staining in depilated Dsg3-/- mice treated with ETA is consistent with the loss of the hair shaft (D). Pulse labeling of bromodeoxyuridine (BrdU) detects matrix keratinocytes in the hair bulb. Control (Dsg3-/- with phosphate-buffered saline (PBS) and BrdU) showed positive incorporation of BrdU in matrix cells (E). Depilated skin from Dsg3-/- mice treated with ETA showed staining for BrdU (F), while plucked hair was negative (G) suggesting a split above the matrix within the precortical cells in the ETA-treated Dsg3-/- mice. Scale bar= 50 μm Journal of Investigative Dermatology 2004 123, 817-822DOI: (10.1111/j.0022-202X.2004.23479.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions