Generation and genotyping of mice
To generate littermate G3/G4 Terc+/−*
and G3/G4 Terc−/−
mice, G2/G3 Terc−/−
males were crossed with Terc+/−
females (Blasco et al., 1997
). Genotyping was performed as described in Blasco et al. (1997)
. Note that littermate G3/G4 Terc+/−*
and G3/G4 Terc−/−
mice are of an exact genetic background (C57BL6) as if they were derived from the same parents.
Mouse colonies were generated in a pure C57BL6 background and maintained at the Spanish National Cancer Center under specific pathogen-free conditions in accordance with the recommendations of the Federation of European Laboratory Animal Science Associations.
Telomeric repeat amplification protocol
Primary mouse embryonic fibroblasts (MEFs) were trypsinized and washed in PBS, and S-100 extracts were prepared as described in Blasco et al. (1997)
. Three protein concentrations were used for each sample (5, 2, and 1 μg). Extension and amplification reactions and electrophoresis were performed as described in Blasco et al. (1997)
. A negative control was included by preincubating each MEF extract with RNase for 10 min at 30°C before the extension reaction. An internal control for PCR efficiency was included (TRAPeze kit; Oncor).
To induce LRC mobilization, IFE hyperplasia, and anagen entry, tail skin from 71-d-old mice in the telogen (resting) phase of the hair cycle was topically treated every 48 h with TPA (20 nM in acetone) for a total of four doses. The control mice were treated with acetone only. 24 h after the last TPA treatment, mice were killed and the tail skin was analyzed. To induce anagen by physical stimulation, dorsal HFs in the telogen phase of the hair cycle were plucked from the back skin of 60-d-old G3 Terc+/−* mice and the corresponding G3 Terc−/− controls. 10 d after plucking, dorsal skins were harvested and prepared for histology.
Labeling of LRCs
LRCs were obtained as described in Bickenbach et al. (1986)
, Cotsarelis et al. (1990)
, and Braun et al. (2003)
, with some modifications. In brief, litters of neonatal mice were injected with 50 mg/kg of bodyweight BrdU (Sigma-Aldrich) diluted in PBS. Each animal received a daily injection beginning at day 4 of life for a total of 5 d. After the labeling period, mice were allowed to grow for 60 d before the initiation of any treatment. Cells retaining the label at the end of the treatment were identified as LRCs.
Preparation of whole mounts
Whole mounts of mouse tail epidermis were prepared as previously described in Braun et al. (2003)
. In brief, after mice were killed with CO2
and their tails were amputated, skin was peeled from the tails and incubated in 5 mM EDTA in PBS at 37°C for 4 h. Using forceps, intact sheets of epidermis were separated from the dermis and fixed in neutral-buffered formalin for 2 h at room temperature. Fixed epidermal sheets were maintained in PBS containing 0.2% sodium azide at 4°C before labeling.
Immunofluorescence of epidermal sheets
To detect LRCs in whole mounts of the tail skin, fixed epidermal sheets were blocked and permeabilized by incubation for 30 min in a modified phosphate buffer (Braun et al., 2003
) containing 0.5% BSA and 0.5% Triton X-100 in TBS. Subsequently, epidermal sheets were immersed for 30 min in 2 M HCl at 37°C, incubated overnight with a mouse anti-BrdU antibody conjugated with fluorescein (Roche) at 1:50 in modified PB buffer, washed four times in PBS containing 0.2% Tween 20, and mounted in Vectashield (Vector Laboratories).
A laser scanning confocal microscope (TCS-SP2-AOBS; Leica) was used to obtain fluorescence images. Image stacks of 60–80 μm were obtained through the z dimension at steps 1.0 μm apart, using a PL APO 20×/0.70 PH2 (Leica) as lens. Maximum intensity projections of the image stacks were then generated using LCS Software (Leica).
Mice were killed when they showed signs of poor health, such as reduced activity or weight loss, and subjected to exhaustive histopathological analysis. The organs we analyzed for age-related degenerative pathologies were the intestine (atrophy of the small and large intestine), kidney (glomerulonephritis and tubular degeneration), spleen (atrophy, hemosiderosis, and myeloid and lymphoid hyperplasia), liver (congestion, vacuolar degeneration, microgranuloma, and steatosis), testis (atrophy and ectasis of seminal vesicles), ovary (atrophy), uterus (cystic endometrial hyperplasia), skin (benign hyperplasia), lung (congestion), heart (congestion and cardiomyopathy), and brain (calcification).
Histology and immunohistochemistry of skin
Tail or back skin samples were harvested from mice and fixed overnight in neutral-buffered formalin at 4°C, dehydrated through graded alcohols and xylene, and embedded in paraffin. For determination of IFE thickness, dermis thickness, and HF length, dissected skin was cut parallel to the spine and sections were cut perpendicular to the skin surface to obtain longitudinal HF sections. 5-μM sections were used for hematoxylin-eosin staining.
For immunohistochemistry, tail skin samples were sectioned at 2–3 μm and processed with 10 mM sodium citrate, pH 6.5, cooked under pressure for 2 min. Slides were washed in water, and then in TBS Tween 20 0.5%, blocked with peroxidase, washed with TBS Tween 20 0.5% again, and blocked with FBS followed by another wash. The slides were incubated with the primary antibodies: rabbit monoclonal to Ki-67 antibody (prediluted; SP6; Master Diagnostica), rabbit polyclonal active caspase 3 at 1:200 (R&D Systems), mouse monoclonal p63 at 1:100 (clone A48, Neomarker), or rabbit polyclonal K14 at 1:100 (Neomarker). Slides were then incubated with secondary antibodies conjugated with peroxidase (DakoCytomation), goat anti-rabbit (1:50) in the case of Ki-67, active caspase 3, K14, and mouse on mouse (Vector Laboratories) in the case of p63. For signal development, DAB (DakoCytomation) was used as a substrate. Sections were lightly counterstained with hematoxylin and analyzed by light microscopy.
Isolation of newborn keratinocytes
2-d-old mice were killed and soaked in betadine (5 min), in a PBS antibiotic solution (5 min), in 70% ethanol (5 min), and again in a PBS antibiotic solution (5 min). Limbs and tail were amputated and the skin was peeled off using forceps. Skins were then soaked in PBS (2 min), PBS antibiotic solution (2 min), 70% ethanol (1 min), and again in PBS antibiotic solution (2 min). Using forceps, each skin was floated on the surface of 1× trypsin solution (4 ml on a 60-mm cell culture plate; Sigma-Aldrich) for 16 h at 4°C. Skins were transferred to a sterile surface. The epidermis was separated from the dermis using forceps, minced, and stirred at 37°C for 30 min in serum-free Cnt-02 medium (CELLnTEC Advanced Cell Systems AG). The cell suspension was filtered through a sterile Teflon mesh (Cell Strainer 0.7 m; BD Biosciences) to remove cornified sheets. Keratinocytes were then collected by centrifugation (160 g) for 10 min and seeded on collagen I–precoated cell culture plates (BD Biosciences).
Colony-forming assay and culture conditions
1,000 mouse keratinocytes per genotype were seeded onto 10 μg/ml mitomycin C (2 h), treated with J2-3T3 fibroblasts (105 per well, 6-well dishes), and grown at 37°C/5% CO2 in Cnt-02 medium. After 10 d of cultivation, dishes were rinsed twice with PBS, fixed in 10% formaldehyde, and then stained with 1% Rhodamine B to visualize colony formation. Colony size and number were measured using three dishes per experiment.
Telomere length analysis by Q-FISH
Freshly isolated splenocytes were obtained by squeezing the spleen through a cell strainer (70 μm; Nylon; BD Biosciences). Red cells were lysed by osmotic shock, and the splenocytes were resuspended in RPMI 1640 containing 10% FBS and 0.55 μM β-mercaptoethanol. Concanavalin A (Sigma-Aldrich) was added to a concentration of 5 μg/ml and splenocytes were grown for 48 h. The cells were incubated with 0.1 μg/ml colcemide (Invitrogen) for 2 h and fixed in methanol/acetic acid (3:1). Q-FISH was performed as described in Herrera et al. (1999)
and Samper et al. (2000)
. To correct for lamp intensity and alignment, images from FluoroSpheres (fluorescent beads; Invitrogen) were analyzed using the TFL-Telo software (provided by P. Lansdorp, Terry Fox Laboratory, Vancouver, Canada). Telomere fluorescence values were extrapolated from the telomere fluorescence of lymphoma cell lines LY-R (R cells) and LY-S (S cells) with known telomere lengths of 80 and 10 kb, respectively. There was a linear correlation (r2
= 0.999) between the fluorescence intensity of the R and S telomeres. We recorded the images using a camera (CCK; COHU) on a fluorescence microscope (DMRb; Leica). A mercury vapor lamp (CS 100 W-2; Philips) was used as a source. We captured the images using the Q-FISH software (Leica) in a linear acquisition mode to prevent oversaturation of fluorescence intensity. We used the TFL-Telo software (Zijlmans et al., 1997
) to quantify the fluorescence intensity of telomeres from at least 10 metaphases for each data point.
Exponentially growing primary keratinocytes were fixed in methanol/acetic acid, and Q-FISH of interphase nucleus was performed. For Q- FISH in tail skin, paraffin-embedded tail sections were deparaffinated. Both keratinocytes and deparaffinated sections of tail skin were hybridized with a PNA-telomeric probe and telomere fluorescence was determined as described in Gonzalez-Suarez et al. (2000)
and Muñoz et al. (2005)
. More than 60 nuclei from each mouse and condition were captured at 100 magnification using a microscope (CTR MIC; Leica) and a camera (High Performance CCD; COHU). Telomere fluorescence was integrated using spot IOD analysis in the TFL-TELO program (Zijlmans et al., 1997
Cytogenetic analysis using telomere Q-FISH on metaphases
Metaphases from keratinocytes of the indicated genotypes were obtained by adding 1 μg/ml colcemide (Invitrogen) to primary keratinocytes during 5 h and then fixing in methanol/acetic acid (3:1). Q-FISH was performed as described in Herrera et al. (1999)
and Samper et al. (2000)
. For analysis of chromosomal aberrations, 50 metaphases per genotype were analyzed by superimposing the telomere image on the DAPI image using the TFL-telo software.
Unless otherwise indicated, data are given as mean values ± SEM of n and have been analyzed for statistically significant differences using t test.
Online supplemental material
Fig. S1 shows rescue of HF stem cell mobilization defects in late generation telomerase-reconstituted G4 Terc+/−*
mice. Fig. S2 shows similar proliferation rates in G3 Terc−/−
and G3 Terc+/−*
tail skin. Fig. S3 shows no detectable apoptosis in the skin of G3 Terc+/−*
mice and G3 Terc−/−
siblings. Fig. S4 shows no differences in p63 expression between Terc−/−
and G3 Terc+/−*
tail skin. Fig. S5 shows no differences in keratin 14 expression between Terc−/−
and G3 Terc+/−*
tail skin. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200704141/DC1