Expression patterns of the glial cell line–derived neurotrophic factor, neurturin, their cognate receptors GFRα-1, GFRα-2, and a common signal transduction element c-Ret in the human skin hair follicles  Mohamed A. Adly, PhD, Hanan A. Assaf, MD, Paolo Pertile, Mahmoud R. Hussein, MSc, MD, PhD, MRCPath, EBP, Ralf Paus, MD  Journal of the American Academy of Dermatology  Volume 58, Issue 2, Pages 238-250 (February 2008) DOI: 10.1016/j.jaad.2007.10.014 Copyright © 2008 American Academy of Dermatology, Inc. Terms and Conditions

Fig 1 Immunoreactivity (IR) for glial cell line–derived neurotrophic factor (GDNF) in human scalp hair follicle (HF), shown in red with avidin-biotin complex (ABC) and tyramide signal amplification (TSA) techniques. A to H, Anagen VI HF. A and E, Distal region of follicle. Upper (B and F) and lower (C and D) central regions of follicle. D and H, Bulb region of proximal follicle. I and J, Part of early catagen HF. K, Telogen HF. L, Strong GDNF expression in correct regions of mouse brain (cerebellum) as positive control. M, Negative control for A to D. N, Schematic summary for anagen VI HF showing GDNF expression in red. CTS, Connective tissue sheath; DP, dermal papilla; HCo, hair cortex; HMC, hair matrix cells; HMe, hair medulla; HS, hair shaft; INF, infundibulum; IRS, inner root sheath; ORS, outer root sheath; SG, sebaceous gland. Journal of the American Academy of Dermatology 2008 58, 238-250DOI: (10.1016/j.jaad.2007.10.014) Copyright © 2008 American Academy of Dermatology, Inc. Terms and Conditions

Fig 2 Reverse transcription (RT)-polymerase chain reaction (PCR) analysis of glial cell line–derived neurotrophic factor (GDNF) messenger RNA (mRNA) expression in human scalp skin and anagen VI hair follicle (HF) using primers for GDNF (A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (B). a, Whole thickness of frontal skin; b, whole thickness of scalp skin after HF extraction; c, isolated anagen VI HF; d, microdissected epidermis with papillary dermis. bp, Base pair. Journal of the American Academy of Dermatology 2008 58, 238-250DOI: (10.1016/j.jaad.2007.10.014) Copyright © 2008 American Academy of Dermatology, Inc. Terms and Conditions

Fig 3 Immunoreactivity (IR) for neurturin (NTN) in human scalp hair follicle (HF), shown in red with avidin-biotin complex (ABC) and tyramide signal amplification (TSA) techniques. A to F, Anagen VI HF by ABC and TSA stainings, respectively. A and D, Parts of distal region of follicle. B and E, Central region of follicle. C and F, Bulb region of proximal follicle. G and H, Parts of early catagen HF. Positive control (I) (mouse brain: caudatus) and negative control (J) for A to C. K, Schematic summary for anagen VI HF showing NTN expression in red. CTS, Connective tissue sheath; DP, dermal papilla; HCo, hair cortex; HMC, hair matrix cells; HMe, hair medulla; HS, hair shaft; INF, infundibulum; IRS, inner root sheath; ORS, outer root sheath; SG, sebaceous gland. Journal of the American Academy of Dermatology 2008 58, 238-250DOI: (10.1016/j.jaad.2007.10.014) Copyright © 2008 American Academy of Dermatology, Inc. Terms and Conditions

Fig 4 Immunoreactivity (IR) for GFRα-1 in human scalp hair follicle (HF), shown in red with avidin-biotin complex (ABC) and tyramide signal amplification (TSA) techniques. A to E, GFRα-1 IR in anagen VI HF by ABC and TSA staining, respectively. A, B, and D, Parts of central region of follicle. C and E, Bulb region of proximal follicle. F and G, Parts of early catagen HFs. Positive control from mouse brain (striatum) (H) and negative control (I). J, Schematic summary for anagen VI HF showing GFRα-1 IR in red. CTS, Connective tissue sheath; DP, dermal papilla; HCo, hair cortex; HMC, hair matrix cells; HMe, hair medulla; HS, hair shaft; INF, infundibulum; IRS, inner root sheath; ORS, outer root sheath; SG, sebaceous gland. Journal of the American Academy of Dermatology 2008 58, 238-250DOI: (10.1016/j.jaad.2007.10.014) Copyright © 2008 American Academy of Dermatology, Inc. Terms and Conditions

Fig 5 Immunoreactivity (IR) for GFRα-2 in human scalp hair follicle (HF), shown in red with avidin-biotin complex (ABC) and tyramide signal amplification (TSA) techniques. A to F, GFRα-2 IR in anagen VI HF by ABC and TSA techniques, respectively. A and D, Parts of distal region of follicle. B and E, Central region of follicle. C and F, Bulb region of proximal follicle. G to I, Parts of early catagen HF. J, Positive control from mouse cerebral cortex. K, Negative control. L, Schematic summary for anagen VI HF showing GFRα-2 IR in red. CTS, Connective tissue sheath; DP, dermal papilla; HCo, hair cortex; HMC, hair matrix cells; HMe, hair medulla; HS, hair shaft; INF, infundibulum; IRS, inner root sheath; ORS, outer root sheath; SG, sebaceous gland. Journal of the American Academy of Dermatology 2008 58, 238-250DOI: (10.1016/j.jaad.2007.10.014) Copyright © 2008 American Academy of Dermatology, Inc. Terms and Conditions

Fig 6 Immunoreactivity (IR) for Ret in human scalp hair follicle (HF), shown in red with avidin-biotin complex (ABC) and tyramide signal amplification (TSA) techniques. A to F, Ret IR in anagen VI HF by ABC and TSA stainings, respectively. A and D, Infundibulum and parts of distal region of follicle. B and E, Central region of follicle. C and F, Bulb and lower central regions of proximal follicle. G, Part of early catagen HF. H, Telogen HF. I, Negative control. J, Positive control from mouse cerebral cortex. K, Schematic summary for anagen VI HF showing Ret IR in red. CTS, Connective tissue sheath; DP, dermal papilla; HCo, hair cortex; HMC, hair matrix cells; HMe, hair medulla; HS, hair shaft; INF, infundibulum; IRS, inner root sheath; ORS, outer root sheath; SG, sebaceous gland. Journal of the American Academy of Dermatology 2008 58, 238-250DOI: (10.1016/j.jaad.2007.10.014) Copyright © 2008 American Academy of Dermatology, Inc. Terms and Conditions

Fig 7 Quantitative analysis of Tunel+ and Ki67+ cells in human anagen VI hair follicles incubated with glial cell line–derived neurotrophic factor (GDNF) and neurturin proteins for 8 days in culture. Indicated are mean values and SEM. Statistical analysis was carried out using Mann-Whitney test. Total of 100 ng/mL of GDNF increased number of Ki67+ keratinocytes significantly versus that estimated under mere incubation with transforming growth factor (TGF)β2 (∗P < .05). Journal of the American Academy of Dermatology 2008 58, 238-250DOI: (10.1016/j.jaad.2007.10.014) Copyright © 2008 American Academy of Dermatology, Inc. Terms and Conditions

Fig 8 Effect of glial cell line–derived neurotrophic factor (GDNF) on spontaneous and transforming growth factor (TGF)β2-induced catagen development of organ-cultured human anagen VI hair follicle (HF). Total of 100 ng/mL of GDNF retarded premature, TGFβ2-induced catagen development. Hair cycle staging was performed by quantitative histomorphometry of hematoxylin-eosin–stained paraffin sections after 8 days of HF organ culture. Journal of the American Academy of Dermatology 2008 58, 238-250DOI: (10.1016/j.jaad.2007.10.014) Copyright © 2008 American Academy of Dermatology, Inc. Terms and Conditions