Fig. 5. The hair follicle stem cells (SCs) and their lineage. Schematics depict the mature hair follicle (Left) and the lineages that arise upon transition from the resting (telogen) to growing (anagen) phase of the hair cycle (Right). Growth is accompanied by SC activation, self-renewal, and conversion to proliferating progeny that subsequently progress to differentiate along distinct lineages to produce the hair shaft, and the ORS and IRS.

Fig. 6. Postnatal ablation of Bmpr1a affects quiescence within the SC niche in adult hair follicles. (A) Status of active canonical BMP signaling in WT follicles, as depicted by anti-P-Smad1/5/8 immunofluorescence (Upper), anti-BMPR1A immunofluorescence (Lower Insets), and Bmpr1a in situ hybridization (ISH) (Lower) with digoxygenin cRNA probes specific for exon 2 of BMPR1A. Analyses were performed over the period encompassing the first telogen (P19) to anagen (P20 and P24) transition. Note lack of labeling in anti-P-Smad1/5/8 immunofluorescence in new germ (hg). Note that Bmpr1a ISH correlates well with anti-P-Smad1 staining at anagen P24, suggesting that its pattern of expression may be more faithful than immunofluorescence. (B) H&E-stained sections of skins from Bmpr1a(fl/fl)/K14-Cre^TM and Bmpr1a(fl/fl) mice before TM treatment began at P44. Initiation of TM treatment coincided with the onset of the second telogen phase of the hair cycle. (C) Genotyping of DNAs isolated from Bmpr1a CON^TM and cKO^TM skins. PCR analysis confirmed deletion of exon2 of Bmpr1a (Bottom) only after TM treatment and only in mice carrying the double-floxed allele (Top) and K14-Cre^TM (Middle). (D) Lack of anti-P-Smad-1/5/8 immunofluorescence shows that the Bmpr1a gene was efficiently targeted within 16d of topical TM treatment. (E) ISH with a cRNA probe specific for the deleted exon 2. Note the absence of hybridization in Bmpr1a cKO^TM skin at P59. (F) Prior to TM treatment of mice, the CD34 monoclonal antibody marks the bulge SCs of the P44 resting telogen-phase follicles in both Bmpr1a(fl/fl)/K14-Cre^TM and Bmpr1a(fl/fl) mice. Note that there is autofluorescence of the hair shaft and background fluorescence within the dermis. The anti-CD34 staining of the epithelium, however, faithfully recapitulates the natural expression and specificity of CD34. (G and H) Increased BrdU incorporation to bulge and to the cells migrating out of the niche in Bmpr1a cKO^TM mice after receiving a 4-h pulse at P59 and P77, respectively. In contrast, no anti-BrdU antibody labeling was detected in the bulge of CON^TM animals at P59, and only a few cells were marked in the hair germ at the onset of anagen at P77. Broken lines denote dermal-epidermal boundaries, and arrows denote labeling. Bu, bulge; hg, hair germ; Mx, matrix.

SI Figure 7

Fig. 7. Expansion of the proliferating populations of hair follicles after inhibition of BMP signaling postnatally. cKO^TM and CON^TM mice were targeted for Bmpr1a ablation at P44 and analyzed just after the first morphological changes were observed at P59. At this stage, the CON^TM follicles were still in telogen, but the cKO^TM follicles had all precociously entered anagen and had formed an early hair germ. Frozen skin sections (10 mm) were subjected to indirect immunofluorescence and ISH. (A) Anti-Sox9 staining was detected in the cKO^TM and CON^TM bulge, and anti-Sox9 immunofluorescence was very strong in the early hair germ of the cKO^TM follicles at P59. (B) Anit-Lhx2 staining was detected in the cKO^TM and CON^TM bulge but was consistently slightly stronger in the CON^TM bulge. Anti-Lhx2 immunofluorescence was very strong in the early hair germ of the cKO^TM follicles at P59. Note that intense Lhx2 staining overlaps with up-regulated P-cad staining in the cKO^TM germ. (C) ISH of Sox4 shows expression in the hair germ and the bulge of the cKO^TM, but not CON^TM, P59 follicle. (D) Shh ISH shows expression in the distal portion of the hair germ close to the DP of P59 cKO^TM follicles. No expression in the niche was detected. Broken lines mark dermo-epithelial borders, and brackets denote bulge (Bu). hg, hair germ.

Fig. 8. Regeneration of epidermal wounds by hair follicle cells following targeted removal of BMP receptor 1A expression. Two different experiments were conducted to examine this issue. (A) Bmpr1a (fl/fl)/K14-Cre and control mice were bred on the background of K14-H2BGFP. At birth, epidermis was removed enzymatically by dispase treatment (Upper), discarded, and then the dermis harboring BMPR1A-deficient or WT hair follicles was grafted to the backs of nude mice. Reepithelialization was judged by the number of GFP-positive cells (arrowheads) in the BMPR1A-deficient and WT hair follicle regenerated epidermis. (B) Bmpr1a cKO^TM mice were mated to Rosa26-fl-stop-fl-lacZ mice; Bmpr1a ablation was induced during the extended second telogen phase of adult mice, and marked by permanent β-galactosidase expression. Following Bmpr1a ablation, skins were scraped to remove the epidermis, and wound repair was monitored for the periods indicated by X-gal staining to detect β-galactosidase activity and counterstain with eosin. Signs of reepithelialization were judged by the presence of β-gal-expressing cells within the regenerated epidermis (arrows). Epi, epidermis; Der, Dermis; HF, hair follicle.

Fig. 9. Precocious and sustained activation of Lef1/β-catenin and P-PTEN/PI3K/AKT signaling in hair follicles lacking BMPR1A. cKO^TM and CON^TM mice were analyzed at P77 after TM treatment. Frozen skin sections (10 mm) were subjected to indirect immunofluorescence or immunohistochemistry by using the antibodies indicated in the Lower Right of each frame. Color coding is according to the fluorescent tags of the secondary antibodies. (A) Immunofluorescence shows up-regulated nuclear Lef1 staining in the SC niche and cells migrating out of the niche (arrowheads) in cKO^TM P77 follicles. In CON^TM follicles, only a few cells in the new hair germ show nuclear Lef1 labeling. (B) Nuclear b-catenin staining is markedly increased in P77 cKO^TM follicles in both the SC niche and in the cell masses migrating down out of the niche. In the control CON^TM anagen follicle, nuclear b-catenin staining is only seen in the hair germ at P77. (C) Immunofluorescence reveals that anti-P-GSK3b staining is enhanced in the P77 cKO^TM niche and in the proliferating progeny emanating from the bulge. In control CON^TM follicles, anti-P-GSK3b staining is seen within the P77 bulge and is increased at the telogen-to-anagen transition (see Fig. 3C for comparison with telogen bulge). (D) Anti-P-PTEN immunohistochemistry shows that P-PTEN is normally only seen in the emerging hair germs (hg) of WT follicles at the telogen-to-anagen transition (P21 and P23, first transition; and P77, second transition). In the P77 cKO^TM bulge and emerging cell mass, P-PTEN is up-regulated. (E) As judged by immunofluorescence, 14-3-3z was detected in the P77 cKO^TM SC niche but was up-regulated in the proliferating progeny migrating out of the niche. In the control CON^TM skin, 14-3-3z was also detected in the bulge and elevated in the hair germ at the start of the new anagen. Lam5, laminin5; β-cat, β-catenin; β4, β4-integrin; Bu, bulge; hg, hair germs. Arrowheads denote aberrant expression.

Fig. 10. Mice expressing a dominant active BMPR1A transgene display precocious hair follicle differentiation. dTg mice for the K14-Tet^on doxycycline-regulatable transcription factor (rtTA), and for a doxycycline-regulatable enhancer driving the expression of BMPR1A-CA, an HA-epitope tagged, constitutively active form of the BMPR1A, were engineered as described in the main text. (A) PCR analysis of DNAs isolated from dTg^Dox and CON^Dox mice confirmed the presence of transgenes. (B) Elevated anti-P-Smad-1/5/8 immunofluorescence in dTg^Dox skin after doxycycline induction. (C) Absence of anti-CD34 staining in the SCs of the dTg^Dox niche at P47. Note that a cyst emerges from the dTg^Dox hair follicle and that the hair shaft displays autofluorescence, as do hair-shaft cells, present within the P47 follicle. (D) Monoclonal antibody AE13 staining marks the hair-specific keratins. In CON^Dox follicles, it is present within the cortical cells that form the hair shaft; in dTg^Dox skin, AE13 marks some cells within the cyst. (E) Filaggrin staining (Fil) marks the granular layer of epidermis. This marker is excluded from the cysts of dTg^Dox. (F-H) Immunofluorescence and ISH show highly reduced levels of Sox9, Sox4 and Shh, respectively, in dTg^Dox ,whereas in CON^Dox, expression was observed in the expected pattern. Mx, matrix; Bu, bulge.