Fig 1.
Nuclear β-catenin is detected in the DP during anagen.
Skin biopsies of normal C57BL/6 mice were collected at the indicated age and processed for paraffin sections. (A-F) Expression of β-catenin (green color) was visualized by immunofluorescence staining and counterstained with DAPI (blue nuclei). The DP was circled by white dashed lines in each hair follicle. (J-L) Same β-catenin immunostaining as in (A-F) (green color) without DAPI counterstaining. (M-R) DAPI staining of skin tissue sections used in (A-F) (blue nuclei). Scale bar: 100 μm
Fig 2.
Endogenous CD133 expression in postnatal murine hair follicles and lineage tracing using ZsGreen1 reporter mice.
(A-F) Frozen sections of back skin biopsies of normal C57BL/6 mice at each indicated age were immunostained with an anti-CD133 antibody. CD133 was expressed in a subpopulation of cells in the DP (circled by white dashed line) of P28 to P38 anagen hair follicles (A-E) but not in hair follicles that in catagen or later (F). (G-L) Lineage tracing of CD133+ DP cells using CD133-CreERT2; ZsGreen1 reporter mice. The DP is co-stained for the expression of alkaline phosphatase (red), which is a specific DP marker. GFP+ cells were first detected in DPs at P28 (G) and present throughout the entire anagen (H-K). An average of 20% of telogen hair follicles contained a small population of GFP+ cells in the DP (L). (M-R) Images of ZsGreen1 expression (green) in CD133+ DP cells during the hair cycle without staining for alkaline phosphatase expression. Green fluorescent DP cells were indicated with red arrows. For every indicated mouse age, at least three mice were analyzed. Scale bar: (A-F), 100 μm; (G-R), 200 μm
Fig 3.
Expression of ΔN-β-catenin in CD133+ DP cells accelerates spontaneous hair anagen (A) Illustration of the triple transgenic mouse model. Expression of ΔN-β-catenin in CD133+ DP cells requires administration of both tamoxifen, which induces rtTA expression, and doxycycline, which forms a complex with rtTA to bind to the tetO promoter. (B) The time scheme for mouse induction. (C) Detection of ∆N-β-catenin expression in CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mutant mice by RT-PCR. Back skin biopsies from CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mutant mice. (H-K) and control littermates (D-G) were stained with H&E and photographed at indicated stages. Scale bars: 100 μm. (L) The numbers of hair follicles at P35 were counted and compared between CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mutant mice and control littermates (mean ± s.d.). A minimum of three skin biopsies from three pairs of mutant and control mice was analyzed. Two-tailed paired Students t-test was employed to calculate statistical significance.
Fig 4.
Expression of ΔN-β-catenin in CD133+ DP cells accelerates hair follicle differentiation.
5-μm-thick paraffin sections from P35 CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mutant mice and control littermates were processed for immunofluorescence staining of following markers: Sox9 for outer root sheath (control: A; mutant: E); Gata3 for inner root sheath (control: B; mutant: F); AE13 and AE15 for hair keratins (control: C, D; mutant: G, H); versican for anagen DP (control: I; mutant: J). Sections were nuclear counterstained with DAPI (blue). K. The numbers of versican+ DP cells in each hair follicle were counted and compared between CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mutant mice and control littermates (mean ± s.d). A minimum of three skin biopsies from three pairs of mutant and control mice was analyzed. Two-tailed paired Student’s t-test was employed to calculate statistical significance. (L-M) β-catenin expression in hair follicle was examined by immunohistochemistry. Images shown are representative of at least three replicates at each indicated age. Scale bars: 200 μm for A-J; 100 μm for L-M.
Fig 5.
Expression of ΔN-β-catenin in CD133+ DP cells does not block hair follicle regression.
Back skin biopsies from CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mutant mice (H-K) and control littermates (D-G) were stained with H&E and photographed at indicated stages. Scale bars: 100 μm. (L) The numbers of hair follicles at P35 were counted and compared between CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mutant mice and control littermates (mean ± s.d.). A minimum of three skin biopsies from three pairs of mutant and control mice was analyzed. Two-tailed paired Student’s t-test was employed to calculate statistical significance.
Fig 6.
Increased proliferation in both matrix keratinocytes and DP cells in CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN hair follicles.
Co-expression of Ki67 (green) and Lef1 (red) was examined in skin samples collected from CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mice and control littermates at P32 (control: A; mutant: E) and P35 (control: B; mutant: F). Paraffin slides from BrdU-incorporated skin biopsies co-stained with anti-versican antibody showing enlarged DP compartment in hair follicles of from CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mice with increased numbers of proliferating DP cells and matrix cells (G) than in littermate controls (C). Cyclin D1 staining indicated accelerated cell cycle progression in hair matrix cells when ΔN-β-catenin was expressed in CD133+ DP cells (H). Images shown are representative of at least three replicates at each indicated age. Total cell number (I) and number of Lef1+ DP cells (J) in each hair follicle at P28 and P35 were counted and compared between CD133-CreERT2; Rosa-rtTA; tetO-Ctnnb1ΔN mice and control littermates. 20 hair follicles were counted for each mouse (mean ± s.d.). For each indicated age, at least three pairs of mutant and control mice were used. Scale bars: 200 μm for A-C and E-G; 100 μm for D and H.