mice. Taken together, these findings suggest adverse effect on epidermal homeostasis and hair-cycle regulation caused by disruption of the RASSF9 gene. We also examined the expression of keratin 6 (K6), a marker of hyperproliferative epidermis, whose expression is highly restricted to a single cell layer in the hair follicle that surrounds the innermost layer of the outer root sheath, but otherwise not typically expressed in epidermis [16,17,18]. In RASSF92/2 mice, K6 was highly expressed throughout the epidermal layers and entire root sheath of hair follicles in abnormally thick layers of cells (Figure 5). Keratin 14 (K14), a marker of epidermal proliferating compart- ment, was confined to the basal cells in WT skin as expected, whereas it was also detected in the suprabasal layer of RASSF92/ 2 mice, also abnormally thick, together with K6 (Figure 5). The striking co-expression of K6 and K14 in abnormally thickened epidermal tissues suggests hyperproliferation in the epidermis of two-week-old RASSF92/2 mice (Figure 5). Aberrant differentiation in skin of RASSF92/2 mice Epidermal homeostasis of the skin requires a coordinated regulation of cell proliferation and differentiation. Thus we investigated the status of epidermal differentiation in RASSF92/2 mice. We performed immunofluorescence staining for keratin 5/ keratin 14 and keratin 1/keratin 10 (K5/K14 and K1/K10), markers for the basal and suprabasal layers of the cutaneous skin, respectively [19]. In RASSF92/2 mice at 4 days post-partum (dpp), RASSF9 Regulated Epidermal Homeostasis K5 and K14 were detected not only in the basal layer but also the suprabasal layers of epidermis, abnormal patterns that echoed the epidermal hyperplasia observed of two-week-old RASSF92/2 mice (K5: Figure 6; K14: Figure 7). Moreover, detection of K1/K10 expression revealed drastic thickening of suprabasal layers in RASSF92/2 mice, in contrast to the WT control (K1: Figure 8; K10: Figure 9). Furthermore, aberrant expression of K1 and K10 was observed in RASSF92/2 follicles, an anomaly strongly suggesting possible epidermalization of the hair follicles in the skin of RASSF92/2 mice (Figures 8–9; detailed images of hair follicles in 8B and 9B). Finally, immunostaining of filaggrin, a marker expressed by cells of granular layer, revealed abnormal expression of filaggrin in multiple layers of granular cells beneath the stratum corneum of RASSF92/2 mice at two weeks of age, in contrast to the thin-layer pattern typically maintained in the granular layer of WT skin (Figure 10; detailed images in 10B). Taken together, these findings showed that RASSF92/2 mice suffered from a severe defect in epidermal homeostasis characterized by abnormal thickening of epidermis, dysregulated cellular proliferation, and disruption of keratinocyte maturation as revealed by the altered patterns of keratin and filaggrin expressions. RASSF9 expression profiles in WT mouse To further evaluate the significance of RASSF9 gene expression for normal mouse development, we determined the expression Figure 6. Aberrant differentiation in the skin of RASSF92/2 mice—K5 (red). (A) Frozen sections of dorsal skins of WT (+/+) and RASSF92/2 (2/2) mice were immunostained with red fluorescence for K5 in 4-dpp mice. Scale bar = 100 mm. (B) Images at higher magnification. Scale bar = 40 mm. Blue, DAPI staining of cell nuclei. doi:10.1371/journal.pone.0017867.g006 PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e17867