Journal: NPJ Regenerative Medicine

Article Title: GDNF promotes hair formation and cutaneous wound healing by targeting bulge stem cells

doi: 10.1038/s41536-020-0098-z

Figure Lengend Snippet: a GDNF overexpression in transgenic mice leads to increased hair formation. Right panels depict Masson’s trichrome staining of skin sections (blue = dermal collagens, red = muscle and keratin). Scale bars = 200 µm. b Quantification of the number of anagen HFs per 2 mm skin section at P50. n = 5, male control + / +; n = 5, male Tg/Tg(Ctsl-GDNF), p ≤ 0.001; error bars = SEM, two-way ANOVA with Bonferroni post-hoc test. c Immunofluorescence analysis of GDNF expression in skin. Hair follicle (HF). See also Figs. S1– . d GDNF promotes hair formation after depilation. Dorsal skins were wax depilated (outlined in white) and analyzed 4 days later. Top views of the depilated skins (upper panels), and H&E-stained tissue sections (lower panels). Hair shaft (arrow). Scale bars = 200 µm. e GDNF promotes the proliferative expansion of K15 + BSCs within HFs. Immunofluorescence analysis depicting representative HFs (outlined in white) at start (non-depilated; upper panels) and 1-day post depilation (lower panels). Magnifications of Ki67 + proliferating K15 + BSCs (bracket). Hair follicles (HF), hair germ (Hg). Scale bars = 20 µm. f qPCR analysis of GDNF-mediated hair formation 1 day after depilation. Two-tailed Student’s t -test ( p ≤ 0.05*≤0.001**≤0.001***), n = 3 skin samples per group; error bars = SEM. g BSCs purified from hair follicles using FACS. CD34 + /CD49f + purification of BSCs (top). Quantification of BSC colonies treated with either vehicle (PBS) or GDNF (100 ng/ml) at 3 or 6 days after plating (middle). BSC colonies (bottom, arrows). Error bars = SEM, two-way ANOVA, n = 3, p < 0.001. Scale bar = 0.1 mm.

Article Snippet: Intrawound injections of recombinant Rat GDNF protein (50–125 μg/wound in 50 μl of vehicle solution; R&D Systems, 512-GF/CF) or 50 μl of vehicle (PBS, 1 mM KH 2 PO 4 , 155 mM NaCl, 3 mM Na 2 HPO 4 -7H 2 O, pH 7.4) was administered using a 26-gauge needle and syringe.

Techniques: Over Expression, Transgenic Assay, Staining, Control, Immunofluorescence, Expressing, Two Tailed Test, Purification

Journal: NPJ Regenerative Medicine

Article Title: GDNF promotes hair formation and cutaneous wound healing by targeting bulge stem cells

doi: 10.1038/s41536-020-0098-z

Figure Lengend Snippet: Dorsal skins of P50 C57BL/6J mice were injected with a single dose of 10–50 µg recGDNF or vehicle (PBS). Asterisk represents the HF in the expanded inset. Surface views of the vehicle and recGDNF-treated animals reveal prominent hair growth after rec-GDNF treatment only (lower panels). Panniculus carnosus (PC). Scale bars = 200 µm.

Article Snippet: Intrawound injections of recombinant Rat GDNF protein (50–125 μg/wound in 50 μl of vehicle solution; R&D Systems, 512-GF/CF) or 50 μl of vehicle (PBS, 1 mM KH 2 PO 4 , 155 mM NaCl, 3 mM Na 2 HPO 4 -7H 2 O, pH 7.4) was administered using a 26-gauge needle and syringe.

Techniques: Injection

Journal: NPJ Regenerative Medicine

Article Title: GDNF promotes hair formation and cutaneous wound healing by targeting bulge stem cells

doi: 10.1038/s41536-020-0098-z

Figure Lengend Snippet: a Schema depicting the morphogenic and post-morphogenic stages of the murine hair cycle. b GFRA1–EGFP is expressed within cells of the bulge (Bg) and dermal papillary (Dp) compartments of anagen HFs from Gfra1 EGFP/+ mice. Scale bar = 200 µm (left panel). Scale bar = 50 µm (bulge, shaft, Dp panels). Sebaceous gland (Sg). See also Fig. . c GFRA1–EGFP is expressed within cells of the bulge (Bg) compartment but not within the dermal papillary (Dp) compartment of telogen HFs from Gfra1 EGFP/+ mice. Bars = 20 µm. Sebaceous gland (Sg). d GDNF targets bulge (Bg) cells but not dermal papillary (Dp) cells of hair follicles during the early onset of hair growth. Time-course depilation analysis reveals that GDNF-responding cells are restricted to the bulge (Bg) compartment during the early stages of anagen induction (i.e., 1–2 days post depilation). However, by 4-days post depilation, GFRA1–EGFP becomes expressed within both Bg cells, and Dp cells that have become encased by matrix cells. At higher magnification, GDNF-responding cells also include dermal cap (Dc) cells at the base of Dp cells. Scale bars = 20 µm. Sebaceous gland (Sg). e Distribution of GFRA1–EGFP + cells within hair follicles during telogen and anagen. Fisher’s exact test, two-sided, p = 0.0005. Dermal papilla (DP). f Model depicting GDNF targeting of BSCs during the early stages of hair formation.

Article Snippet: Intrawound injections of recombinant Rat GDNF protein (50–125 μg/wound in 50 μl of vehicle solution; R&D Systems, 512-GF/CF) or 50 μl of vehicle (PBS, 1 mM KH 2 PO 4 , 155 mM NaCl, 3 mM Na 2 HPO 4 -7H 2 O, pH 7.4) was administered using a 26-gauge needle and syringe.

Techniques:

Journal: NPJ Regenerative Medicine

Article Title: GDNF promotes hair formation and cutaneous wound healing by targeting bulge stem cells

doi: 10.1038/s41536-020-0098-z

Figure Lengend Snippet: a Wound repair in P19 non-transgenic (+/+) mice at 4 DPI. Staining was done with Masson’s trichrome stain (Blue = collagen types, red = keratin and muscle). Scale bar = 200 µm. b Wound repair in P19 hemizygous Tg(Ctsl-Gdnf)/0 mice at 4 DPI. Masson’s trichrome. Scale bar = 200 µm. c , c’ Wound repair and healing in P50 non-transgenic (+/+) mice at 4–7 DPI. Masson’s trichrome. Scale bar = 200 µm. d , d’ Wound repair and healing in P50 hemizygous Tg(Ctsl-Gdnf)/0 mice at 4–7 DPI. Masson’s trichrome. Scale bar = 200 µm. e Quantification of wound closure in non-transgenic and hemizygous Tg(Ctsl-Gdnf)/0 mice. Wound size measured at 0–7 DPI: P19 set, non-transgenic, n = 4; hemizygous Tg(Ctsl-Gdnf)/0, n = 7. P50 set, non-transgenic, n = 6; hemizygous Tg(Ctsl-Gdnf)/0, n = 8. * p ≤ 0.05; error bars = SD, two-way ANOVA with Bonferroni post-hoc test. f Hair follicle stage within uninjured skin. Masson’s trichrome. Scale bar = 100µm. g Peri-wound hair formation assessed using the uDISCO clearing method in P50 mice. Wound (W) blood vessel formation depicted at 2 DPI (upper panel). Peri-wound hair formation assed at 18 DPI (lower panels). h Enhanced Gdnf and Vegfa expression in injured Tg(Ctsl-Gdnf)/0 wounds. Real-time qPCR was performed at 1 DPI using non-transgenic and Tg(Ctsl-Gdnf)/0 wounds. Non-transgenic and hemizygous Tg(Ctsl-Gdnf)/0, n = 5. * p ≤ 0.05, *** p ≤ 0.001; error bars = SD, Student’s t -test.

Article Snippet: Intrawound injections of recombinant Rat GDNF protein (50–125 μg/wound in 50 μl of vehicle solution; R&D Systems, 512-GF/CF) or 50 μl of vehicle (PBS, 1 mM KH 2 PO 4 , 155 mM NaCl, 3 mM Na 2 HPO 4 -7H 2 O, pH 7.4) was administered using a 26-gauge needle and syringe.

Techniques: Transgenic Assay, Staining, Expressing

Journal: NPJ Regenerative Medicine

Article Title: GDNF promotes hair formation and cutaneous wound healing by targeting bulge stem cells

doi: 10.1038/s41536-020-0098-z

Figure Lengend Snippet: a , b recGDNF accelerates wound re-epithelization and granulation tissue maturation by 4 DPI. Vehicle-treated wounds depicted by H&E staining (panels a , a” ). Epithelial tongue (Ep. t), granulation tissue (Gr), eschar (es). recGDNF-treated wounds (50 µg/wound) formed the neoepidermis (Neo-ep) by 4 DPI ( b ). Appearance of vascularized red blood cells (arrows) within granulation tissue (panel b’ ). Dermis (D). Scale bars = 200 µm ( a , b ), and 100 µm ( a’ – b’ ). c Recombinant GDNF improves dorsal skin wound closure by 7 DPI. Single intrawound injection of 33 µM recGDNF (50 µg recGDNF in 100 µl PBS). Vehicle (x injected site). d , e Improved skin healing after recGDNF treatment by 7 DPI. Masson’s trichrome stain (Blue = collagen types, red = keratin and muscle) of vehicle-treated wound sections (panel d ). Polarized light microscopy of picrosirius red stained control wound sections reveal large parallel bundles of collagen (arrows) analogous to scar formation ( d’ ). In recGDNF-treated wounds, reduction and maturation of the granulation tissue with hair follicle (hf) formation ( e ), coupled with resolution of scar tissue into interwoven collagen bundles ( e’ ). Scale bars = 200 µm ( d , e ). See also Fig. . f Restoration of COL4A1 expression in the reticular dermis at the wound site after recGDNF treatment. COL4A1 immunofluorescence staining at 7 DPI (upper panels). The dotted vertical lines encompass the wound region. The papillary (p) dermis is the uppermost layer of the dermis, while the reticular (r) layer is the lower layer of densely packed collagen fibers. The horizontal dotted line in the recGDNF-treated sample separates the papillary and reticular dermal layers based on COL4A1 expression. DAPI and COL4A1 merged images (lower panels). Epidermis (e), hair follicle (hf). COL4A1 expression of uninjured skin is depicted in Driskell et al. 2013 . Scale bars = 200 μm.

Article Snippet: Intrawound injections of recombinant Rat GDNF protein (50–125 μg/wound in 50 μl of vehicle solution; R&D Systems, 512-GF/CF) or 50 μl of vehicle (PBS, 1 mM KH 2 PO 4 , 155 mM NaCl, 3 mM Na 2 HPO 4 -7H 2 O, pH 7.4) was administered using a 26-gauge needle and syringe.

Techniques: Staining, Recombinant, Injection, Light Microscopy, Control, Expressing, Immunofluorescence

Journal: NPJ Regenerative Medicine

Article Title: GDNF promotes hair formation and cutaneous wound healing by targeting bulge stem cells

doi: 10.1038/s41536-020-0098-z

Figure Lengend Snippet: a Experimental design using the K15 -CrePR1:R26R-Confetti multi-label reporter line to study the effect of recGDNF on the fate of cells derived from BSCs after injury. b Commitment of BSC-derived keratinocytes to the injured neoepidermis in vehicle-treated mice. Neo-epidermis (dotted line) at the wound site marked off by the white vertical bars (left panel). Confetti-labeled keratinocytes (arrows) are present in the neoepidermis by 7 DPI. Quiescent peri-wound HFs (boxed) that show initial Confetti labeling of BSCs (magnification) by 7 DPI (middle and right panels). Bulge (Bg), sebaceous glands (Sg). Scale bars = 200 μm; scale bar (magnification) = 20 μm. c Recombinant GDNF promotes the recruitment of BSC progeny to the injured neoepidermis by 7 DPI. Increased migration and commitment of GDNF-stimulated BSC-derived progeny to the wound neoepidermis (left panel). Cohesive streaks of BSC-descendants recruited to the wound site (middle panel). GDNF-mediated activation of BSCs (arrow) that give rise to RFP + cells which define a HF (right panel). Es (eschar). Scale bars = 200 μm. d Quantification of Confetti-labeled cells in the neoepidermis after injury. Left graph depicts the individual fluorescent proteins (termed XFPs) observed within the neoepidermis by 7 DPI. Middle graph depicts the number of individual clones (i.e. represented by cohesive single XFPs) present in the injured neoepidermis. The right graph depicts the number of cells within a clone at the injured neoepidermis. For statistics, a total of four serial sections through the wound midline were averaged and assessed per mouse at 7 DPI. Vehicle-treated, n = 3 male mice; recGDNF-treated, n = 4 male mice. * p = 0.04, ** p = 0.003; error bars = SEM, two-tailed Student’s t -test. Days post depilation (DPI).

Article Snippet: Intrawound injections of recombinant Rat GDNF protein (50–125 μg/wound in 50 μl of vehicle solution; R&D Systems, 512-GF/CF) or 50 μl of vehicle (PBS, 1 mM KH 2 PO 4 , 155 mM NaCl, 3 mM Na 2 HPO 4 -7H 2 O, pH 7.4) was administered using a 26-gauge needle and syringe.

Techniques: Derivative Assay, Labeling, Recombinant, Migration, Activation Assay, Clone Assay, Two Tailed Test