Fig 1.
Age-associated structural changes in the tail epidermis.
(A) Schematic diagram of the mouse tail skin. (B) Hematoxylin-eosin stained tail skin sections of young (2-month-old) and old (2-year-old) mice. Arrowheads indicate the micro-undulation in the basal region. Degenerating hair follicle is observed (asterisk). Scale bars: 50 μm. (C) Quantification of the thickness of the scale and interscale IFE. The epidermal thickness is quantified from ≥6 epidermal units per mouse. N = 3. Error bars show S.E.M. Student’s t-test. *; P < 0.05, ns: not significant; P = 0.22 (C, the interscale IFE). (D) The proportion of epidermal units harboring micro-undulations. ≥30 epidermal units are counted in each mouse to score micro-undulations of basal regions of the IFE. N = 3. Student’s t-test is used. **; P < 0.01. (E) Immunostaining of tail skin sections of young (2-month-old) and old (2-year-old) mice with K14 (basal cell marker, green) and PDGFRα (dermal cell marker, red) antibodies. Hoechst is used to stain nucleus (blue). Arrowheads indicate the micro-undulation. Scale bars: 100 μm. (F, G) Quantification of the mean signal intensity of K14 (F) or PDGFRα (G). The intensity of immunostaining signals in skin section is measured and averaged from ≥50 individual cells per mouse. N = 3. Student’s t-test. ***; P < 0.001. ns: not significant; P = 0.46 (F). (H) Immunostaining of tail skin sections of young (2-month-old) and old (2-year-old) mice with α6 integrin (basal cell marker, green) and Collagen IV (basement membrane marker, red). Scale bars: 100 μm. The area within the yellow box is shown with higher magnification. Scale bars: 20 μm. (I) Quantification of the mean signal intensity of α6 integrin. The intensity of immunostaining signals in skin section is measured and averaged from ≥50 individual cells per mouse. N = 3. Student’s t-test. ns: not significant; P = 0.35 (I).
Fig 2.
Epidermal structural changes in the SAM mouse model.
(A) Hematoxylin-eosin stained tail skin sections of senescence-resistant SAMR1 or senescence-prone SAMP1 and SAMP8 strains at 6 months of age. Scale bars: 50 μm. (B, C) Quantification of the epidermal thickness. IFE, interfollicular epidermis. The epidermal thickness is quantified from ≥6 epidermal units per mouse. N = 3. One-way ANOVA. Error bars show S.E.M. ns: not significant. (B) SAMR1 vs. SAMP1; P = 0.13. SAMR1 vs. SAMP8; P = 0.53. (C) SAMR1 vs. SAMP1; P = 0.40. SAMR1 vs. SAMP8; P = 0.79. (D) The proportion of epidermal unit harboring the micro-undulation. ≥30 epidermal units are counted in each mouse to score micro-undulations of basal regions of the IFE. N = 3. One-way ANOVA. ns: not significant. SAMR1 vs. SAMP1; P = 0.87. SAMR1 vs. SAMP8; P = 0.61. (E) Hematoxylin-eosin stained tail skin sections of senescence-resistant SAMR1, senescence-prone SAMP1 and SAMP8 stains at 1 year of age. Arrowheads indicate the micro-undulations in the basal region. Scale bars: 50 μm. (F, G) Quantification of the IFE thickness. The epidermal thickness is quantified from ≥6 epidermal units per mouse. N = 3. One-way ANOVA. ns: not significant. (F) SAMR1 vs. SAMP1; P = 0.17. SAMR1 vs. SAMP8; **; P < 0.01. (G) SAMR1 vs. SAMP1; P = 0.56. SAMR1 vs. SAMP8; P = 0.94. (H) The proportion of epidermal unit harboring the micro-undulation. ≥30 epidermal units are counted in each mouse to score micro-undulations of basal regions of the IFE. N = 3. One-way ANOVA. ns: not significant. SAMR1 vs. SAMP1; P > 0.99. SAMR1 vs. SAMP8; *; P < 0.05.
Fig 3.
The apoptotosis in the basal layer remains largely unchanged with age.
(A, B) Tail whole-mount epidermal sheets are stained with cleaved caspase-3 (apoptosis marker, red), β4 integrin (basal cell marker, green) and Hoechst (blue). (A) and (B) represent the immunostaining of wild-type young (2-month-old) and old (2-year-old) mice. The area within the red box is shown with higher magnification. Arrowhead represents apoptotic cells in the basal layer (B). Asterisks represent hair follicles. White dotted lines represent the boundary of scale and interscale. Scale bars: 100 μm (left) or 20 μm (right). (C) Quantification of the number of caspase3+/β4 integrin+ cells per an interscale or scale structure. The caspase3+ basal cells are scored from ≥50 interscale or scale IFE structures per mouse. N = 4. Error bars show S.E.M. Mann-Whitney U test. ns: non-significant; P = 0.22 in interscale IFE; P = 0.85 in scale IFE. (D) Quantification of the mean signal intensity of β4 integrin. Signal from whole-mount staining shown in (A, B) is measured. N = 4. Student t-test. ns: not significant. P = 0.93. (E) FACS dot plot shows gates to define basal cells in the interfollicular epidermis (α6 integrin+/CD34−/Sca1+), followed by the analysis for 7-AAD and Annexin V signals. (F, G) The mean signal intensity of α6 integrin (F) and Sca1 (G) measured by FACS. N = 3. Student’s t-test. ns: not significant; P = 0.65 (F); P = 0.66 (G). (H) Graph shows the percentage of apoptotic cells (Annexin V+/7-AAD−) in the basal IFE. N = 3. Student’s t-test. ns: not significant; P = 0.77. (I) Quantification of the number of caspase3+/β4 integrin+ cells in SAM mice at 1-year-old. The caspase3+ basal cells are scored from ≥50 interscale or scale IFE structures per mouse. N = 3. Kruskal-Wallis test for multiple comparison is used. ns: non-significant; SAMR1 vs. SAMP1; P > 0.99. SAMR1 vs. SAMP8; P > 0.99 in the interscale IFE and SAMR1 vs. SAMP1; P > 0.99. SAMR1 vs. SAMP8; P > 0.99 in the scale IFE. (J) Quantification of the mean signal intensity of β4 integrin. Signal from whole-mount staining (S2B Fig) is measured. N = 3. One-way ANOVA. ns: not significant; SAMR1 vs. SAMP1; P = 0.57. SAMR1 vs. SAMP8; P = 0.99.
Fig 4.
Age-dependent decrease in proliferation in the scale epidermis.
(A) Schematic view of the interscale and scale structures in the tail whole-mount epidermis. (B) Tail whole-mount epidermal sheets are stained with BrdU (proliferation marker, green) and Hoechst (blue). White dotted lines represent the boundary of scale and interscale. Asterisks represent hair follicles. Scale bars: 100 μm. (C, D) Quantification of BrdU-positive cells within each interscale and scale structure. The number of BrdU+ cells is scored from ≥8 interscale or scale IFE structures per mouse. N = 3. Error bars show S.E.M. Student’s t-test is used. **; P < 0.01 (C). ns: not significant; P = 0.22 (D). (E) BrdU (proliferation marker, green) and Hoechst (blue) staining in the tail skin sections. (F, G) Proportion of BrdU+ cells per basal cells in the scale (F) or interscale IFE (G). The number of BrdU+ cells is scored from ≥100 basal cells per mouse. N = 3. Student’s t-test. ns: not significant; **; P < 0.01 (F), and P = 0.31 (G).
Fig 5.
Decrease in scale proliferation in senescence prone SAMP8 mice.
(A, B) Tail whole-mount staining of senescence-resistant SAMR1 and senescence-prone SAMP1 and SAMP8 mice at 6 months (A) and 1 year (B) with BrdU (proliferation marker, green) and Hoechst (blue). White dotted lines represent the boundary of scale and interscale. Asterisks represent hair follicles. Scale bars: 100 μm. (C-F) Quantification of BrdU-positive cells per scale or interscale units among SAM mice at 6 months (C, D) or 1 year (E, F) of age. The number of BrdU+ cells is scored from ≥8 interscale or scale IFE structures per mouse. N = 3. Error bars show S.E.M. One-way ANOVA. ns: not significant. (C) SAMR1 vs. SAMP1; P = 0.53. SAMR1 vs. SAMP8; P = 0.86. (D) SAMR1 vs. SAMP1; P = 0.91. SAMR1 vs. SAMP8; P = 0.30. (E) SAMR1 vs. SAMP1; P = 0.07. SAMR1 vs. SAMP8; *; P < 0.05. (F) SAMR1 vs. SAMP1; P = 0.77. SAMR1 vs. SAMP8; P = 0.35. (G) BrdU (proliferation marker, green) and Hoechst (blue) staining in the tail sections of SAMR, SAMP1 and SAMP8 at 1 year. (H, I) Proportion of BrdU+ cells per basal cells in the scale (H) or interscale IFE (I). The number of BrdU+ cells is scored from ≥100 basal cells per mouse. N = 3. One way ANOVA used. ns: not significant; (H) SAMR1 vs. SAMP1; P = 0.35. SAMR1 vs. SAMP8; *; P < 0.05. (I) SAMR1 vs. SAMP1; P = 0.31. SAMR1 vs. SAMP8; P = 0.26.
Fig 6.
Age-dependent changes of the interscale differentiation marker.
(A) Schematic view of the interscale and scale IFE differentiation in the tail whole-mount epidermis. (B) Young and old tail epidermis stained with Keratin (K) 10 (differentiation marker of interscale, red), K31 (differentiation marker of scale, green) and Hoechst (blue). The greyscale images show K10 staining. Scale bars: 100 μm. (C, D) Quantification of the mean signal intensity of K10 (C) and K31 (D) from young and old tail whole-mount epidermis. N = 4. Error bars show S.E.M. Student’s t-test. ***; P < 0.001 (C). ns: not significant; P = 0.38 (D). (E) Young and old tail epidermis sections stained with K10 (red), K31 (green) and Hoechst (blue). The greyscale images show the K10 staining. Scale bars: 100 μm. (F, G) Quantification of the mean signal intensity of K10 (F) and K31 (G). The intensity of immunostaining signals in skin section is measured and averaged from ≥50 individual cells per mouse. N = 3. Student’s t-test. *; P < 0.05 (F). ns: not significant; P = 0.54 (G).
Fig 7.
Decrease in the interscale differentiation marker in senescence prone SAMP8 mice.
(A) Whole-mount tail epidermis stained with K10 (differentiation marker of interscale, red), K31 (differentiation marker of scale, green) and Hoechst (blue) of 6-month-old SAM mice. The grey scale images show K10 staining. Scale bars: 100 μm. (B, C) Quantification of the mean fluorescence intensity of K10 (B) and K31 (C). N = 3. One-way ANOVA. Error bars show S.E.M. ns: not significant; (B) SAMR1 vs. SAMP1; P = 0.91. SAMR1 vs. SAMP8; P = 0.99. (C) SAMR1 vs. SAMP1; P = 0.90. SAMR1 vs. SAMP8; P = 0.95. (D) Whole-mount staining of K10, K31 and Hoechst in 1-year-old SAM mice. (E, F) Quantification of the mean fluorescence intensity of K10 (E) and K31 (F). Scale bars: 100 μm. (E) SAMR1 vs. SAMP1; P = 0.89. SAMR1 vs. SAMP8; *; P < 0.05. (F) SAMR1 vs. SAMP1; P = 0.80. SAMR1 vs. SAMP8; P = 0.62. (G) Tail sections of SAMR, SAMP1 and SAMP8 mice stained with K10 (red), K31 (green) and Hoechst (blue) at 1 year of age. The greyscale images show the K10 staining. Scale bars: 100 μm. (H, I) Quantification of the mean signal intensity of K10 (H) and K31 (I). The intensity of immunostaining signals in skin section is measured and averaged from ≥50 individual cells per mouse. N = 3. One-way ANOVA. (H) SAMR1 vs. SAMP1; P = 0.90. SAMR1 vs. SAMP8; **; P < 0.01. (I) SAMR1 vs. SAMP1; P = 0.93. SAMR1 vs. SAMP8; P = 0.92.