RASSF9 Regulated Epidermal Homeostasis Figure 4. Aberrant proliferation in RASSF92/2 skin. (A) BrdU incorporation (green) and K14 (red) were co-stained by immunofluorescence in the skin cross-sections of two-week-old RASSF92/2 (2/2; right panel) and WT mice (+/+; left panel). Areas marked by yellow dashed line are enlarged in the adjacent column (‘‘Enlarge’’). Scale bar = 100 mm. ORS, outer root sheath; HB, hair bulb. The abbreviations Epi, Derm, Adi were defined as described in Figure 3A. (B) High-magnification images of BrdU incorporation in dermal tissues. Abnormal patterns of proliferating cells in Epi, ORS and HB of 2/2 skin are consistent with those observed in (A). The dashed white lines denote the epidermis-dermis border. Similar results were obtained from three independent pairs of mice. Scale bar = 20 mm. (C) Skin proliferation was evaluated by quantitation of BrdU incorporation using ImageJ software. Analyses were performed for the number of BrdU-positive nuclei in basal layer (top panel; per mm of epidermis); the number of BrdU-positive nuclei in dermal hair follicles (middle panel; per mm2 of dermis layer); and the percentage of HB with more than five BrdU-positive nuclei (bottom panel). Data represent scoring of at least 20 hair bulbs from each mouse and three mice for each genotype. *, P,0.05; **, P,0.001. doi:10.1371/journal.pone.0017867.g004 the catagen and telogen phases, with many of them devoid of hair shafts [Figure 3A and Figure S3, lower left panels; contrast with upper left (WT) panels]. These observations strongly suggest that RASSF9, the gene disrupted by the transgene insertion in this mutant line, is crucial for epidermal development. Aberrant proliferation in skin of RASSF92/2 mice The skin of RASSF92/2 mouse exhibited a significant epidermal thickening. We therefore investigated whether RAFF9 affected the epidermal proliferation by determining bromode- oxyuridine (BrdU) incorporation in the skin of two-week-old PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17867