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First published online 29 March 2005
doi: 10.1242/jcs.02298

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Endogenous Myc controls mammalian epidermal cell size, hyperproliferation, endoreplication and stem cell amplification

Jennifer Zanet^1,*, Sophie Pibre^1,*, Chantal Jacquet¹, Angel Ramirez², Ignacio Moreno de Alborán³ and Alberto Gandarillas^1,*,

¹ Institut de Génétique Moléculaire de Montpellier, CNRS/UMII, Montpellier, France
² Epithelial Damage, Repair and Tissue Engineering Program, CIEMAT, Madrid, Spain
³ Centro Nacional de Biotecnologia, CSIC, Madrid, Spain

View larger version (49K): [in a new window]   Fig. 4. Cell size and cell cycle analyses. (A-F) Representative flow cytometry analyses of keratinocytes isolated from control (Ct) or KO epidermis of skin as indicated. (A) Light scatter dot plot of whole populations. R1, region gating smaller, blunt, basal cells; R2, region gating larger and more complex, suprabasal (suprab) cells. (B) Keratin 1 (K1; FL-1) expression of cells in R2 or R1; numbers in brackets are percent of K1-negative cells (left histograms) or K1-positive cells (right histograms) within the total population; K1-positive cells are in green and K1-negative cells in red in the light scatter dot plots. (C) Overlays of cell size (forward scatter, FSC) of the populations indicated. (D) DNA content (FL2) profiles of basal (R1) or suprabasal (R2) keratinocytes. (E) DNA content profiles of proliferative (K1–) or differentiating (K1+) cells. M2 for the G1 phase; M1 for polyploid cells. (F) Cell size (FSC) versus DNA content (FL2); a line has been drawn to help comparing FSC values. 10,000 keratinocytes were counted for every sample (see Materials and Methods). Numbers are percent of cells in each gate within each plot.

View larger version (81K): [in a new window]   Fig. 1. Specific ablation of Myc in keratinocytes. (A-C) Southern blotting analysis of EcoRI digested genomic DNA; the probe detects the three alleles: Myc WT (20 kb), Myclox (10 kb) and deleted Myclox (KO; 16 kb). Genomic DNA is from embryos (E; A) or (B,C) from whole tail, tongue, liver, heart, skin (S), epidermis (Ep) or dermis (D) of adult mice bearing or not Cre as indicated (+ or –). Animals were aged 4 months to 1 year. (D) RPA on RNA from epidermis of control (Ct) or KO mice to detect Myc-related mRNAs. KO underlined indicates the presence of a severe phenotype, mth (month) indicates the age of the mouse. The experiment was performed on ten different pairs of littermates from one and a half months to one and a half years of age.

View larger version (73K): [in a new window]   Fig. 2. Macroscopic phenotype of epidermal Myc KO mice. (A) One-week-old male littermates. (B) 3-month-old female littermates. (C-E) Examples of ripped-off skin on neck (C), face (D) or abdomen (E). (F) Epidermal KO mice have difficulty stretching. (G) One-and-a-half year-old epidermal KO mouse with complete loss of hair and spontaneous lesions all over the body. Ct, control littermates with neither Cre nor Myclox alleles (wt), with just Cre, or with just myclox.

View larger version (82K): [in a new window]   Fig. 3. Microscopic phenotype of Myc KO epidermis. (A,B) Hematoxylin and eosin staining of control (A) or KO (B) epidermis. Note that KO epidermis is thinner and the nuclei smaller, with loss of cellularity in the basal layer (black arrows) and heterogenous mild hyperkeratosis (thickening of squamous layers: black arrowhead). (C,D) Skin sections stained for keratin 10 (green), keratin 5 (red), keratin 1 (green), filaggrin (red), involucrin (red), BrdU (red), Ki67 (green) or for nuclei with DAPI (DNA, either in blue or red) as indicated. Arrows: areas of terminal differentiation in the basal layer. White arrowhead, mild hyperkeratosis. DN, dermal nuclei. Note the smaller size of KO epidermal nuclei. (E,F) Percent of BrdU (E) or Ki67 (F) positive cells with respect to total basal nuclei. Bars are s.e.m. Bar, 20 µm. Ct, control. The dotted line indicates the basal membrane.

View larger version (91K): [in a new window]   Fig. 5. Wound healing and cell migration. (A-J,M,N) Two 3 mm punch biopsies were made on the back of each control (Ct) or KO mouse as indicated. Wounds 2 days (A,E) or 4 days (B,F) after wounding. (C,G) Hematoxylin eosin staining of wound sections after 2 days. (D,H) Higher magnification details of C and G. (I,M) ß1 integrin (green) and involucrine (red) staining on wound-healing fronts. J,N) Representative BrdU staining on wound-healing fronts (green). Green cornified layer in M is nonspecific staining. Nuclei were stained with DAPI (DNA either red or blue). (K) Percent of BrdU incorporation in wound-healing fronts after 2 or 4 days (D). (L,P) Representative tracks (16) of elongated-moving keratinocytes (without mitomycine C) from control mouse (L, green) or KO epidermis (P, red). (O) Length of tracks followed by control (Ct) or KO keratinocytes without or after treatment with mitomycine C (mitoC). A dash (–) locates the mean, and bars the s.e.m. (20 cells). Bar, 40 µm in C,D,G,H, 20 µm in I,J,M,N. Dotted line for basal membrane.

View larger version (80K): [in a new window]   Fig. 6. Proliferation in hair follicles and sebaceous function. (A) Hair follicle sections from control (Ct) or KO skin stained in red for keratin 5 (K5) or keratin 6 (K6) as indicated; nuclei were stained with DAPI (blue). Arrows indicate areas of loss of expression. (B,C) Oil red O staining on sections from back (B) or tail (C) skin to detect sebaceous secretion. Arrowheads point at sebaceous glands. Every study was carried out with mice of the same litter. Bar, 20 µm.

View larger version (47K): [in a new window]   Fig. 7. Effects on stem cells in vivo. (A) Label-retaining analyses; percent of BrdU-positive cells with respect to total number of nuclei 2 months after the last injection. all, all skin; Ct, control, F, hair follicles; IF, interfollicular epidermis. S.e.m. is shown. (B) Skin sections stained for ß1 integrin (white) or p63 (green); nuclei stained with DAPI (DNA, blue). Arrows indicate p63 positive cells. Bars are s.e.m. Bar, 20 µm.

View larger version (20K): [in a new window]   Fig. 9. Model for Myc function in epidermis. In the presence of Myc, daughters of stem cells undergo clonal expansion (TAC). A blue square represents the stem cell decision to enter the (TAC) compartment before terminal differentiation; Myc would behave as an `amplifier'. In the absence of Myc, stem cells are able to divide, but their daughters cannot amplify and directly undergo terminal differentiation. Stem cells would be driven to divide and differentiate more frequently to replenish the epidermis and their compartment might be depleted over time. SC, stem cells; TD, terminal differentiation.

View larger version (81K): [in a new window]   Fig. 8. Stem cell amplification and fate in culture. (A) Clonogenicity assay with 2.5 x105 total keratinocytes from 2-month-old control (Ct) or KO mice. (B) Clonogenicity assay with 105 basal keratinocytes from Ct or KO newborns (see Materials and Methods). Keratinocyte colonies are red, fibroblast-feeder layer is blue. (C) Microscopic view of Ct or KO (arrows) colonies. Note that KO colonies are constituted by one or three flattened cells only. (D) Immunofluorescence on Ct or KO keratinocytes in culture. Ks stands for keratins K5 and K6 (mix of two antibodies to detect keratinocytes stained in green), nuclei in blue (DNA, DAPI). A 2 hour BrdU incubation was done after 5 and 21 days (stained in red). Bar, 20 µm. D, days.