Figure 1.
Ablation of Dicer by the K5-Cre transgene induces fur loss and epidermal dysmorphology in older mice.
A) Indistinguishable appearance of Dicer-conditional littermates with (DicerΔ/Δ) or without (Dicerc/c) the K5-Cre transgene at four weeks of age. B) Normal morphology of skin and hair follicles in four-week old control mice (Dicerc/c mice, left two panels) and age-matched DicerΔ/Δ mice (Dicerc/c, K5Cre+, right two panels). Scale bars equal 100 microns. C) Appearance of litter of K5-Cre negative (Dicerc/c) and K5-Cre+ (DicerΔ/Δ) mice at 10 weeks of age. D) Appearance of 8-month old Dicer heterozygous mouse (top) and Dicer-ablated mouse (bottom) Haplo-insufficieny for Dicer does not alter the fur of mice. E) PCR analysis of tissue-derived genomic DNA samples from representative K5Cre+, Dicerc/c (DicerΔ/Δ) mouse indicates presence of Dicer-ablated allele (283 bp product) in the tail and skin of the mouse, but not in the liver or spleen. PCR product of Dicer conditional allele and Dicer wildtype allele = 476 bp and 396 bp, respectively. Three control genomic DNA samples (Dicerc/c; K5Cre+, Dicerc/c; and wildtype) were used as controls. F) Kaplan-Meier survival curves for control (wildytype or Dicerc/c) cohorts and DicerΔ/Δ mouse cohorts. G) Hematoxylin and eosin staining of skin tissue sections of 7-month old mice. Deletion of Dicer in the epidermis (DicerΔ/Δ mice-bottom panels) results in defective follicle morphology. Wildtype (wt) and K5-Cre+ skin samples are used as controls (top panels). Scale bars equal 100 microns.
Figure 2.
Co-ablation of p53 does not alter fur loss in DicerΔ/Δ mice.
A) Appearance of mice at 5-months of age, with genotypes of the mice given beneath. As the two middle mice lack the K5-Cre transgene, not all conditional (c) alleles have undergone deletion. B) Fur loss was scored in Dicer-ablated mice bearing one (purple), two (red), or no (black) functional alleles of p53. Regardless of genotype, all DicerΔ/Δ mice began to lose fur between ages 5 and 6 weeks of age, and almost all fur is lost by week 12. C) Hematoxylin and eosin staining of skin tissue sections of two 8-week old K5-Cre transgenic control mice. D) Hematoxylin and eosin staining of skin tissue sections of two 8-week old Dicer skin-ablated mice. E) Hematoxylin and eosin staining of skin tissue sections of two 8-week old mice co-ablated in skin for Dicer and p53. Scale bars for 2C, 2D, and 2E equal 100 microns.
Figure 3.
Co-ablation of p53 decreases apoptosis and the survival of DicerΔ/Δ mice.
A) Hematoxylin and eosin staining of skin tissue sections of 5-month old mice. Control (K5-Cre+ transgene) was similar in appearance to wildtype control samples, whereas ablation of Dicer showed reduced and misshapen follicle morphology, with increased epithelial thickening, mitotic figures (arrow), and incidence of apoptosis (arrowheads). Ablation of p53 did not alter follicle dysmorphology seen in DicerΔ/Δ mice (upper right panel), though epithelial cellularity was more pronounced in some regions (see star on bottom right side panel) and fewer apoptotic events were noted. Scale bars equal 100 microns. B) Measurements of epidermal thickness in microns for control skin (white bar), Dicer-ablated skin (gray bar), and Dicer, p53 co-ablated skin (black bar). Bars represent average thickness, with standard deviation shown. Asterisks indicate p values<0.01 between control and Dicer-ablated skin or control and Dicer, p53 co-ablated skin. C) Average number of apoptotic cells observed in 40X field. No apoptotic figures were seen in control tissues (white bar) whereas apoptosis was increases in Dicer-ablated skin (gray bar) and in Dicer, p53 co-ablated skin (black bar) relative to controls. Apoptosis was decreased in Dicer-ablated skin co-deleted for functional p53. Asterisks indicate p values<0.01. D) Average number of mitotic figures seen in 40X field. Mitosis was increased in Dicer-ablated skin (gray bar) and in Dicer, p53 co-ablated skin (black bar) relative to controls (white bar). Asterisks indicate p values<0.01 between control and Dicer-ablated skin or control and Dicer, p53 co-ablated skin. E) Immuno-staining for the proliferation antigen Ki-67 confirms increased cell proliferation in the epidermis of Dicer-ablated mice in the presence or absence of functional p53. Scale bars equal 100 microns. F) PCR-based genotyping of tissues harvested from Dicer conditional, p53 conditional mice in the absence (Dicerc/c, p53c/c) or presence (DicerΔ/Δ p53Δ/Δ) of the K5-Cre transgene. Deletion of functional Dicer and p53 alleles is detected specifically in the skin and tumor tissues of K5-Cre+ mice. G) Kaplan-Meier survival curves for Dicer-ablated mice bearing one (purple; n = 17), two (red; n = 14), or no (black; n = 45) functional alleles of p53. Ablation of both Dicer and p53 in the skin greatly reduced viability of these mice relative to mice ablated for Dicer in skin (p<0.01) or mice ablated for Dicer and one allele of p53 in skin (p<0.01).
Figure 4.
Deletion of Dicer in the epidermis induces DNA damage.
A) Representative hematoxylin and eosin staining of skin sections of 7-8 month old K5-Cre+ control mice, and of 7-8 month old Dicer-ablated mice with (DicerΔ/Δ) or without functional p53 (DicerΔ/Δ, p53Δ/Δ). B) Phospho-H2A.X staining of representative skin samples reveals DNA damage in Dicer-ablated skin. C) Staining of sections for phospho-ATM indicates activation of DNA damage signaling (ATM auto-phosphorylation) specifically in Dicer-ablated skin, regardless of p53 status. Scale bars equal 100 microns. D) Quantification of DNA damage levels in the epidermis of Dicer-ablated mice. Average percentage of phospho-H2A.X positive nuclei shown (bars), with standard deviation. Little phospho-H2A.X staining was observed in the epidermis of wildtype mice or of K5-Cre control mice (white bars), whereas increased staining was seen in Dicer-ablated skin (gray bar) and in Dicer, p53 co-ablated skin (black bar) relative to controls. Asterisks indicate p values<0.01 between control and Dicer-ablated skin or control and Dicer, p53 co-ablated skin.
Figure 5.
Spontaneous skin tumorigenesis in p53-deficient mice.
A) Kaplan-Meier survival curve for K5-Cre+ mice bearing one (n = 14), two (n = 14), or no (n = 18) ablated alleles of p53 (blue, orange and black lines, respectively). Survival of p53-wildtype mice was not compromised, whereas loss of one or both alleles of p53 reduced survival. Log-rank test indicates p53 skin-ablated mice die significantly faster than p53 skin-haploinsufficient mice or p53 wildtype mice (P<0.01). B) Tumor formation in mice deleted for one or both alleles of p53. Left panel is cystic tumor formed on ear of p53wt/Δ mouse, middle panel is basal cell carcinoma formed on the face of a p53Δ/Δ mouse, right panel is a representative well-differentiated squamous cell carcinoma formed on the flank of a p53Δ/Δ mouse. Scale bars equal 100 microns.
Table 1.
Tumor spectrum of mice deleted for p53 and/or Dicer in skin.
Figure 6.
Dicer and p53 cooperate to suppress skin carcinogenesis.
A) Kaplan-Meier tumor curves for various mouse cohorts. K5-Cre+ mice that bear Dicer and p53 wildtype alleles (n = 18) do not present with spontaneous tumors (black curve). Tumorigenesis in K5-Cre+ cohorts that contain one ablated allele of p53 (blue curves), with (n = 13) and without (n = 12) Dicer co-ablation, or in K5-Cre+ cohorts that bear two ablated alleles of p53 (orange curves), with (n = 25) or without (n = 12) Dicer co-ablation reveals that loss of Dicer (DicerΔ/Δ) accelerates cancer formation in p53-haploinsufficient mice (p<0.0001) and p53-ablated mice (p<0.0001). B) Representative DicerΔ/Δ, p53Δ/Δ mouse (on right) presenting with multiple incidence of tumor formation. DicerΔ/Δ mouse (on left) is an age-matched, 12-month old mouse with wildtype p53 alleles. C) PCR analysis of tumor-derived and tissue-derived genomic DNA samples from representative DicerΔ/Δ, p53Δ/Δ mice reveals the presence of the Dicer-ablated allele and p53 ablated allele in the skin and tumor of the mouse, but not in control mice (right panels). D) Hematoxylin and eosin staining of skin tumors arising in Dicer-ablated mice deleted for one (p53Δ/wt) or both copies (p53Δ/Δ) of p53. Top left panel is a BCC. Mice co-deleted for both Dicer and p53 form moderately or poorly differentiated SCC (top right panel) and poorly differentiated and invasive carcinomas (bottom two panels). Scale bars equal 100 microns.