Fig. 1.

Mice overexpressing Plk4 have extra centrosomes in cells of the interfollicular epidermis (IFE). (A) Transgene expression cassette for the induction of Plk4, and epidermal-specific activation of the transgene. Note that Plk4 contains a YFP tag added to its C terminus, which proved useful in vitro and also for Western blotting, but not epifluorescence imaging, of frozen skin sections. (B) Genotype percentages of F1 generation mice at time of birth when Plk4^fl+ animals are mated to mice expressing Cre driven by the epidermal-specific K14 promoter. (C) Immunoblot of transgene reporter H2B-mRFP expression in isolated E17.5 epidermis from corresponding genotypes, with β-actin as a loading control. (D) Quantification by qRT-PCR of Plk4 expression levels in isolated epidermis of eight control and three Plk4 cOE embryos. Error bars indicate SEM. (E) Immunoblot of YFP-tagged Plk4 expressed in cultured keratinocytes and immunoprecipitated using an anti-GFP antibody. H2B-GFP was transfected into keratinocytes as a control for the immunoprecipitation scheme. UB, unbound fraction; In, input fraction; IP, immunoprecipitated fraction. (F) Immunofluorescence images of IFE sections demonstrating expression of the H2B-mRFP reporter in the dermis and epidermis. DAPI is in blue. (Scale bar: 25 μm.) (G) Immunofluorescence images of Plk4-overexpressing skin demonstrating the presence of cells with extra centrosomes in both the basal layer (yellow arrow) and the suprabasal layer (red arrow) of the epidermis, exemplified by the presence of more than two foci marked by pericentrin, a centrosomal marker. The white dashed line represents the basement membrane. The blue dashed line demarcates the cell periphery. DAPI is in blue. (Scale bar: 5 μm.) (H and I) Images (H) and quantification (I) of centrosome number in isolated cells of the basal (K5⁺) and suprabasal (K10⁺) layers of the IFE, after dissociation, cytospinning, and immunostaining. Basal cells: eight control and four Plk4 cOE embryos; suprabasal cells: six control and four Plk4 cOE embryos. DAPI is in blue. Error bars represent SEM. (Scale bar: 5 μm.) (J) Immunofluorescence images of primary keratinocytes (MK) cultured from Plk4^fl+ mice and infected with lentivirus expressing Cre. These cells express Plk4-YFP and have extra centrosomes, as numbered and depicted in the enlarged panels (top to bottom, 1–3). (Scale bar: 10 μm.)