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First published online May 17, 2004
doi: 10.1242/10.1242/dev.01148

Development 131, 2737-2748 (2004)
Published by The Company of Biologists 2004
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Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis

Sergio Ruiz1, Mirentxu Santos1, Carmen Segrelles1, Hugo Leis1, José Luis Jorcano1, Anton Berns2,{dagger}, Jesús M. Paramio1,{dagger} and Marc Vooijs2,*

1 Department of Cell and Molecular Biology and Gene Therapy, CIEMAT, Madrid E28040, Spain
2 Division of Molecular Genetics and Centre of Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan, 121 Amsterdam, 1086 CX, The Netherlands



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  		Fig. 1. Keratinocyte-specific Cre expression leads to pRb ablation in vivo. (A) Schematic representation of the wild-type, the floxed (F19) and the inactivated Rb allele ({Delta}19). Exons are indicated as numbered boxes and loxP sites as triangles. Rb18 and Rb19 indicate the primers used to characterize the different Rb alleles. (B-E) ß-Galactosidase staining (blue) identifies Cre activity in X-Gal stained tissue sections from epithelia of double transgenic R26R:K14cre animals in epidermis (B), tongue (C), thymic epithelium (D) and hard palate (E). (F) Cre-mediated recombination detected by PCR analysis on tails RbF19/F19;K14cre mice. (G) Immunoblot analysis for pRb in RbF19/F19 keratinocytes 48 hours post-infection with Ad-GFP or Cre-coding adenovirus. (H) Immunoblot analysis for pRb family members on primary keratinocytes derived from newborn mice with the indicated genotypes. Scale bars: 150 µm.

 


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  		Fig. 2. Consequences of epidermal Rb ablation in vivo. (A) External appearance of P8 mice with epidermal-specific Rb loss. (B-G) Haematoxylyn-Eosin (H/E) stained skin sections from RbF19/F19 (B), RbF19/F19;p107–/– (C), RbF19/+;p107–/–;K14cre (D), RbF19/F19;K14cre (E), RbF19/F19;p107+/–;K14cre (F) and RbF19/F19;p107–/–;K14cre (G) mice at P8. There are no alterations in mice bearing one functional copy of Rb and the hyperplasia and hyperkeratosis promoted by epidermal pRb loss is evident, which becomes more progressive with the concomitant loss of p107. (H) Quantitative analysis of epidermal hyperplasia measured by epidermal thickness (in µm) taken from three different aged-matched mice of each genotype counting 2-4 sections in each (mean±s.d.). Scale bars: 50 µm.

 


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  		Fig. 3. Proliferation defects in epidermal Rb-deficient mice. (A-D) Representative sections showing BrdU incorporation in the epidermis of RbF19/F19 (A), RbF19/F19;K14cre (B), RbF19/F19;p107+/–;K14cre (C) and RbF19/F19;p107–/–;K14cre (D). (E-E') Quantitative analysis of the percentage of BrdU-positive nuclei per mm of epidermis in the basal layer (E) and number of BrdU-positive nuclei per mm in suprabasal layer (E') of mice with the indicated genotypes. Three 10-day-old mice of each genotype were analyzed, scoring 2-4 sections in each (mean±s.d.). (F-I) Representative sections showing BrdU incorporation in hair follicles of 10 days old mice of each genotype. (J-M) Apoptosis detection (TUNEL) in hair follicles of 10-day-old mice of each genotype. Scale bars: 100 µm. (N,N') Label-retaining population at 30 (N) and 75 (N') days after BrdU labeling in mice of the quoted genotypes. Data in N and N' come from the study of five different mice analyzing 2-4 independent sections from each animal (mean±s.d.).

 


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  		Fig. 4. Altered epidermal differentiation in Rb-deficient skin. (A-D) Double-immunofluorescence on P10 skin sections showing K5 (green) and K10 (red) or (E-H) K6 (green) and K10 (red) of RbF19/F19 (A,E), RbF19/F19;K14cre (B,F), RbF19/F19;p107+/–;K14cre (C,G) and RbF19/F19;p107–/–;K14cre (D,H). Yellow staining indicates co-expression. DAPI (blue) was used to stain nuclei. Scale bars: 50 µm.

 


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  		Fig. 5. Ectopic proliferation of differentiated Rb-deficient keratinocytes. (A-C,E,F) Double immunofluorescence against BrdU (green) and K10 (red) in the epidermis of adult RbF19/F19 (A), RbF19/F19;K14cre (B) and RbF19/F19;p107+/–;K14cre mice (C); and in RbF19/F19 (E) RbF19/F19;K14cre (F) primary keratinocytes upon 24 hours of Ca2+-induced differentiation. Arrows indicate BrdU incorporation in K10-expressing cells. (D) Triple immunofluorescence against K5 (red), K10 (blue) and BrdU (green) showing BrdU incorporation in cells expressing K10, but not K5 (arrows). (G) Summary of the BrdU incorporation in K10-positive primary keratinocytes of the indicated genotypes. Data are from the analysis of three independent experiments scoring at least 500 cells. White lines in A-C indicate the dermal epidermal junction. Scale bars: 50 µm.

 


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  		Fig. 6. Consequences of pRb and p107 loss in primary keratinocytes. (A) Doubling times of cultured primary keratinocytes showing a reduction in RbF19/F19;p107+/–;K14cre and RbF19/F19;p107–/–;K14cre with respect to wild-type and pRb-deficient cells. (B) FACS analysis of cell cycle profiles of asynchronous growing keratinocytes showing no significant differences among the different genotypes. Data are from the analysis of four independent experiments. In B, at least 105 cells were scored on each experiment (mean±s.d.). (C) Percentage of BrdU incorporation in keratinocytes growing under low (0.05 mM) and high (1.2 mM) Ca2+ medium for the indicated times and re-stimulated with low Ca2+ medium. (D) Percentage of BrdU incorporation in primary keratinocytes of the indicated genotypes after adeno cre or adeno GFP infection and before culture under the conditions indicated. Data come from the analysis of three independent experiments scoring at least 1000 cells on each (mean±s.d.). (E) Luciferase reporter activity of E2F of primary keratinocytes of the indicated genotypes cultured in low Ca2+ or upon Ca2+-induced differentiation for 24 and 48 hours. Transfections were performed in triplicate, and the mean and standard error were calculated for each condition. Two independent transfection experiments were performed and luciferase activity was normalized to the values obtained with control, RbF19/F19, cells in low Ca2+.

 

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© The Company of Biologists Ltd 2004