System for marking slow-cycling SCs in vivo and monitoring their fate. (A) Strategy. (B to D) Skin sections of mice before and after 4-week chase. Shown are epifluorescence of H2B-GFP (green) and 4′,6′-diamidino-2-phenylindole (DAPI) (blue), and indirect immunofluorescence with antibodies (Abs) indicated (Texas Red). The hair cycle stage is indicated on each set of “after chase” frames (see also fig. S1, B to D, and fig. S2). Arrows (B) denote Ki67⁺ sebaceous gland cells in telogen. Arrowheads [(B) and (C)] denote transition zone between bulge and newly generated follicle downgrowth. Late anagen (Ki67 in red): GFP-bright cells are retained in the bulge; their progeny rapidly divide, diluting H2B-GFP. (D) Early anagen II bulb overexposed for GFP and double-labeled (small arrowheads) with Abs against each differentiation cell type. (E) Mice “after chase” were scratch-wounded and analyzed by immunofluorescence. Arrows denote likely directions of movements of GFP-positive LRCs and progeny. Abbreviations: Bu, bulge; DP, dermal papilla; Mx, matrix; hg, hair germ; Ep, epidermis; asterisk, hair shaft (autofluorescent); hf, hair follicle; Cx, cortex; ORS/IRS, outer/inner root sheaths; BM, basement membrane; In, infundibulum; W, wound. Scale bars, 50 μm.

Transcriptional profiling of bulge LRCs relative to other SCs. Duplicate mRNAs of bulge LRCs and their two progeny populations were amplified and hybridized to Affymetrix oligonucleotide chips. Files were analyzed by Microarray Suite (MAS5.0) Affymetrix software followed by public database searches, functional annotation, and comparison with similar databases from embryonic (ESC), neural (NSC), and hematopoietic (HSC) SCs (15, 16). (A) Bar graphs: Percentage of mRNAs called present in skin LRCs (4839 total; left) and of mRNAs increased in LRCs versus BL/ORS (154 total; right) and also called present in three other SCs (black), two other SCs (red), one other SC (yellow), and no other SCs (white). (B) GFP epifluorescence and immunofluorescence of skin sections from 8-week-old mice after 4-week chase. Abs are against known SC markers found up-regulated in bulge LRCs. Abbreviations are as in Fig. 1 legend; scale bars, 50 μm. Lower magnification for tenascin-C illustrates marker-specificity. (C) Semiquantitative RT-PCR. Probes are for SC markers, up-regulated in skin LRCs. Mouse ESC mRNAs are shown for comparison. PCR was run for 29, 32, and 35 cycles.

Comparison of cellular localization of bulge LRC mRNAs increased relative to BL/ORS and mRNA present in LRCs. Left, increased in LRCs relative to BL/ORS (154 total); right, present in skin LRCs (three pools of 150 mRNAs each of the 4839 present were analyzed). Black, expressed sequence tags; red, intracellular/cytosolic; blue, nuclear; gray, integral to membrane; white, secreted.

Isolation and preliminary characterization of bulge LRCs and progeny. Animals were Tet-fed for 4 weeks beginning at t = 4 weeks. (A) FACS analyses of single-cell suspensions of skins. GFP fluorescence (FL) is in arbitrary units. (B) Two-color FACS analyses for GFP and five surface markers. α6/CD34 data illustrate that GFP^high LRCs represent only ∼30% of α6/CD34/K5-H2B-GFP–positive cells. White, without primary Abs; red, with Abs. Percentages of total cells scoring positive are indicated. (C) GFP^high FACS population analyzed by immunofluorescence to illustrate homogeneity. Figure S4B provides quantification of all three fractions screened for six markers. (D) Propidium iodide (PI)–FACS cell cycle profiles by DNA content: G₀/G₁ (n = 1), G₂/M (n = 2), and S (n = 1 or 2). Percentage of total cells in G₂/M is indicated.

Implementation of array analyses to examine characteristics and dynamics of the skin SC niche. GFP (green) and immunofluorescence (red) of skin sections from 8-week-old mice (4-week chase). Examples shown: (A) An mRNA up-regulated >2× in LRCs relative to epidermis/ORS. (B) Activated (nuclear phospho-Smad2; arrowheads) or up-regulated (LTBP-1) LRC factors involved in TGFβ signaling. Quantification is at right (graph). (C) Tissue polarity proteins expressed in the SC niche. EphA4, EphB4, and EfnB1 (right of EphB4); boxed bulge in frame is also shown with GFP colabeling. (D) Dynamics of the niche during cycles of SC activation (telogen/anagen transition). Abbreviations are as in Fig. 1 legend; APMu, arrector pili muscle; asterisk denotes hair shaft autofluorescence. Scale bars, 50 μm.