RASSF9 Regulated Epidermal Homeostasis Figure 3. Histological abnormalities in RASSF92/2 skin. (A) H&E staining of skin sections from two-week-old mice. The dashed yellow lines denote the epidermis-dermis and dermis-adipose borders. Similar results were obtained from three independent pairs of mice. The low and high magnification images were shown in the left and right panels, respectively. Left panel, scale bar = 200 mm; Right panel, scale bar = 20 mm. Epi, epidermis; Der, dermis; Adi, adipose; HF, hair follicle. (B, C) Quantitative analysis of the thickness and cellular layers of the epidermis, dermis and adipose tissues of histological skin sections from ten individual regions of each genotyping mouse at two weeks after birth. Values were measured by thickness and numbers of cellular layer in panel (B) and (C), respectively. Mean 6 SD (n = 3, per genotype); * P,0.001, ** P,0.005, *** P,0.05. Panel A–C: +/+, wild type; +/2, heterozygotes; 2/2, homozygotes. doi:10.1371/journal.pone.0017867.g003 multiple organs, as confirmed by reverse-transcription PCR (RT-PCR; Figure 2C; Figure S2A, the top second panel). Perturbation of skin architecture in RASSF92/2 mice Since homozygous mutant mice showed phenotypes typical of the alopecia syndromes, we next sought to characterize its skin pathology. Hematoxylin and eosin (H&E) staining of fixed skin tissue sections revealed thickening of the epidermis and dermis of RASSF92/2 skin by ,3-fold and 1.7-fold, respectively, versus that of the WT mice at two weeks old (Figure 3A and B). The histological features of dermal and epidermal thickening of RASSF92/2 skin could also be observed with Masson’s trichrome staining, which stained collagen blue for better contrast of collagen-rich dermis with collagen-free epidermis (Figure S3). On the other hand, the subcutaneous adipose tissue of the mutant mouse was only 60% of that of the WT mouse (Figure 3A and B; Figure S3). In the mouse, the hair cycle consists three stages: anagen (hair growth), catagen (apoptosis-driven regression), and telogen (resting maintenance). Strikingly, in contrast to anagenic WT hair follicles, the majority of the mutant hair follicles were at PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e17867