Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells

Abstract

Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest 'immortal' DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.

Figure 1

Strategy to detect divisions during hair follicle cycle. (A) The hair cycle. After morphogenesis (∼PD17), HFs enter the adult phase of development characterized by cycles of breakdown (catagen), rest (telogen), and growth (anagen). HFSCs are located in the bulge area. The bulge cells are quiescent during telogen, but proliferate in anagen, when they contribute to differentiated follicle cell lineages and the generation of a new hair shaft (red). APM, arrector pili muscle; DP, dermal papillae; SG, sebaceous gland. (B) Top: tetracycline-inducible (tet-off) double transgenic mouse system drives expression of histone H2B–GFP from the epithelial keratin 5 (K5) promoter (pulse). Doxy administration in mouse diet turns off H2B–GFP expression (chase) when proliferating epithelial cells dilute the label between daughter cells by divisions. Bottom: when doxy is added to diet at time points indicated, the proliferation history of bulge cells during the first and second hair cycle can be assessed based on H2B–GFP dilution.

Figure 2

H2B–GFP system counts cell divisions in bulge cells. (A) Confocal images collected side by side from skin sections of pTRE–H2B–GFP/K5tTA mice at PD21 (no chase; Telogen I); PD49 (4-weeks chase; Telogen II), and PD77 (8-weeks chase; Telogen III). Top panels: note decrease of overall H2B–GFP signal, with brightest signal retained in the bulge area. Bottom panels: TOPRO-3 is a DNA stain. (B) Skin cells stained for surface expression of CD34 and α6-integrin analysed for fluorescence (FL) by FACS: dot plot of all live PI negative skin cells (left); GFP histograms of double positive CD34+/α6-integrin+ cells at time points indicated (right). PD21 (no chase, blue); PD49 (4-weeks chase, red); and PD77 (8-weeks chase, green). (C) Average frequency for each GFP peak sub-population as defined in (B, right) in CD34+/α6-integrin+ cells with standard deviation among mice at time points indicated (PD49, N=7; PD77, N=6; PD21, N=2). (D) Linear regression analysis of GFP intensity (Int) in bulge sub-populations at PD49 shows decrease of the GFP level by two-fold. (E) FACS analyses of single cells isolated from mice treated on the back skin with TPA (mouse back left of midline) or acetone (mouse back right of midline) for 3 weeks during the doxy chase. (F) Projections through confocal stack images show hair follicles from 3-weeks acetone-treated (left) versus TPA-treated (right) skin section (100 μm). TOPRO-3 DNA stain is shown in red. Note lack of bright GFP signal in TPA-treated skin. Hair follicles were in telogen in acetone treatment area and in anagen in TPA treatment area of skin, as expected from the known effect of TPA in promoting anagen. (G) Projection through fluorescence image optical stack collected from skin section (40 μm) shows BrdU incorporation (red) by bulge cells after 1 week of both TPA and BrdU treatment, and 5 weeks of doxy chase. Bu, bulge. (H) Quantification of BrdU+ cells in bulge cells shown in (F), represented as three arbitrary classes of GFP brightness, after acetone versus TPA treatment. (I) Normal log of GFP^Peak1 median intensity (Int) relative to time of chase allows derivation of H2B–GFP degradation rate from the slope. (J) GFP^Peak1- (top panels) and GFP^Peak2 (bottom panels)-sorted cells from PD49 mice treated with BrdU during the entire 4-weeks doxy chase period. Note rare BrdU+ cells (red) in GFP^Peak1 population and nearly 100% BrdU+ cells in GFP^Peak2. DAPI is DNA staining. (K) Quantification of BrdU+ cells in GFP sub-population from mice in (J). Data are shown as average of cells counted from three mice at PD49, with standard deviations. Total number of counted cells is indicated at top of the graph. Designation of GFP cell population as defined in Figure 2B is shown at the bottom with presumed divisions in each population. Scale bars, 50 μm.

Figure 3

H2B–GFP system counts cell divisions in individual bulges in situ. (A) Confocal optical Z-stack is shown as tiled images. Numbers indicate actual optical slice and arrows point to a bright H2B–GFP cell throughout the stack. (B) Stack in (A) is shown as maximal projection through the slices on XY, ZY, and XZ planes. Arrow points to the same cells as in (A). (C) Total intensity after background subtraction (Int) in each optical Z-section for cell indicated by arrows in (A, B) used to obtain total 3D intensity. (D) Average 3D intensity (Int) of bulge cells measured at PD21 (no chase, black) and six sub-populations with average intensities decreasing by two-fold at PD49 (4-weeks chase, grey) is shown with standard errors of the mean (s.e.m.) bars. (E) Linear regression analysis to verify the ranking of bulge cells in classes differing by two-fold in GFP intensity. The slope measured experimentally did not differ significantly from a −1 slope, as predicted by two-fold dilution (P=0.97, Student's t-test). (F) Cross comparison of average intensity of cells from each bulge (no chase) is shown with error bars (s.e.m.). Accurate definition of nuclei was difficult in unchased follicles, which contained dense clusters of bright cells. This resulted in higher H2B–GFP signal variation than expected from previous FACS analyses. Eight out of ten follicles showed no significant differences in intensity (P=0.3147, Wilcoxon test). HFs 5 and 6 were significantly different from the eight follicles (P=0.001, Wilcoxon test). (G) Distribution of the six GFP populations defined in (D) with distinct divisions (0 × –5 ×) detected at PD49 (4 weeks of chase) among 10 HFs (hf01–hf10). Average intensities per HF for each of the six GFP populations detected at PD49 (4 weeks of chase) are shown with error bars (s.e.m.). Wilcoxon test showed no significant difference in the GFP signal among each population in all HFs (P=0.1997), with the exception of one population (1 × division) in two HFs (hf01& hf06; P=0.06). HFs 5 and 10 marked with asterisks were partially truncated by our skin and/or optical sectioning, and were also missing the undivided (0 ×) cell population.

Figure 4

Bulge cells dilute the BrdU label in divisions counted by H2B–GFP system. (A) Diagram showing BrdU and doxy simultaneous pulse-chase scheme. Arrows represent six BrdU subcutaneous injections at PD3–PD5. Mice were killed at PD21 (2-weeks chase) and PD47 (6-weeks chase). (B) Mice not treated with BrdU were used to estimate the frequency of GFP sub-populations (as defined in Figure 2C) among CD34+/α6+ bulge cells (PD21, N=2; PD47, N=3) and α6+ cells (PD6, N=2), with standard deviation bars. (C) Sub-population of bulge cells from mice in (A) sorted on slides and immunofluorescence stained showed BrdU+ cells in all sorted bulge cell fractions. (D) Frequencies of BrdU+ cells in each bulge cell sub-population are shown as average among mice with standard deviation bars (N=2 mice at PD21 and 3 mice at PD47). (E) Total intensity of BrdU signal per cell normalized between two experiments is plotted as average for each GFP sub-population at PD21. Note decrease of BrdU signal from one population to another. (F) Same as (E) for mice killed at PD47. (G) Average BrdU intensity measured in GFP^Peak1−7-sorted bulge sub-population from (E, F) is plotted as log₂ and shows linear regression fit supporting two-fold dilution of BrdU with divisions.

Figure 5

Long-term BrdU LRCs divide infrequently and dilute the label over time. (A) Diagram showing BrdU and doxy pulse-chase scheme. Small arrows represent six subcutaneous BrdU injections at time points indicated. (B) Skin sections from mice in (A) were stained for BrdU with fluorescently labelled antibodies. Scale bar, 50 μm. Blue is DNA DAPI stain. (C) Quantification of BrdU signal in fluorescence images of skin sections at time points indicated: PD6 is no chase; PD21, 2-weeks BrdU chase; PD77, 10-weeks BrdU chase. Images of BrdU-stained follicles were acquired with the same exposures and the brightest cells in each follicle were surveyed for level of BrdU signal using the IP Lab Imaging software. Note progressive decrease of label over time. (D) Skin cells from mice in (A) stained for CD34 and α6-integrin surface expression, sorted in GFP bulge sub-populations on microscopy slides and immunostained for BrdU. Percent BrdU+ cells in each GFP^Peak1−7 was determined relative to negative control (secondary antibody or no BrdU-labelled cells); PD47, N=3 mice; PD77, N=4 mice. Total number of cells counted is indicated at top in appropriate colour. (E) Data obtained in (D) are transformed to illustrate the frequency of BrdU LRCs with defined numbers of presumed divisions among total bulge LRCs. (F) A random pool of sorted cells from (D) was measured for total BrdU signal in single wide-field fluorescence images (N=4 mice at PD77 (black)). For comparison of BrdU levels at PD21, the H2B–GFP cells corresponding to one division during morphogenesis (2 weeks of BrdU and doxy chase: PD6–PD21) were also sorted and stained for BrdU (white dots, N=2 mice). The BrdU signal is plotted as log₂. Presumed divisions indicated by H2B–GFP intensity at bottom. Under each column dot pattern in black represents cells from the four mice analysed. White circles underline 2 of 850 cells (found in 1 of 4 mice) that did not follow the decreasing trend of BrdU signal with increasing number of divisions. Number of cells analysed is shown at top. NC1 (negative control 1) are all live cells from mice not injected with BrdU. NC2 (negative control 2) are bulge cells from PD77 mice stained with secondary antibody only.

Figure 6

Labelled chromosomal foci were diluted in nuclei of dividing cells. CD34+/α6+ bulge cells with distinct H2B–GFP levels and presumed divisions were sorted as before from PD77 mice treated with BrdU and doxy as shown in Figure 5A. Cells were spotted on slides, fixed, and stained for BrdU. Each panel shows fluorescence images of a single nucleus as sum projections through 3D optical Z-stacks. Examples are of the brightest BrdU+ cells found in individual sorted bulge sub-population. Numbers in corner indicate presumed divisions during two consecutive hair cycles. Note only 1–2 BrdU nuclear foci representative of distinct chromosome labelling in nuclei from cells with >4 presumed divisions. Scale bar, 5 μm.