Roles of C/EBPs in Adult Skin Figure 6. Co-ablation of C/EBPa/b results in morphological and molecular defects in sebaceous, Meibomian and preputial glands. ff ff and (B) 4OHT-treated IKOab mice. Co-IF staining in (C) Meibomian glands and (D) (A) H&E staining of sebaceous glands/lobules of 4OHT-treated a b ff ff and preputial glands in untreated controls. H&E staining of (E) Meibomian glands (circled) and (F) preputial glands (circled) of 4OHT-treated a b ff ff (arrows point to sebaceous glands) and 4OHT-treated IKOab mice 4OHT-treated IKOab mice. (G) Oil Red O staining for lipids in 4OHT-treated a b ff ff and 4OHT- (arrows point to sebaceous lobules). (H) FASN: Semi-quantitative reverse transcriptase PCR on whole skin mRNA from 4OHT-treated a b treated IKOab mice. SCD3 and MC5R: TaqManH Real Time PCR. (n = 5 mice/treatment/genotype; *p,0.05). IHC staining for (I) keratin 14 and (J) FASN ff ff and 4OHT-treated IKOab mouse epidermis (AD-Adipose Tissue). Scale bars represent 30 microns. in 4OHT-treated a b doi:10.1371/journal.pone.0009837.g006 expression and for alterations in 4OHT-treated IKOab mice. Meibomian glands are found at the rim of the eyelid of mammals and the Meibomian sebocytes produce and release a sebum-like substance onto the eye that functions to prevent the evaporation of the tear film on the eye [40]. The preputial gland, found in some male mammals and located in the subcutaneous tissue of the inguinal area, is involved in the production and secretion of pheromones and lipids. Co-IF staining revealed C/EBPa and C/ EBPb were co-expressed in sebocytes of both the Meibomian and preputial glands (Fig. 6C&D). Gross examination of 4OHT- treated IKOab mice revealed that eyes of these mice appeared dry, swollen, and partially closed. Histological analysis revealed atrophied Meibomian glands characterized by greatly reduced lobule size and diminished numbers of differentiated sebocytes with clear vacuolated cytoplasm (Fig. 6E). In addition, there was severe regional epidermal hyperplasia of the haired skin eyelid epidermis as well as hyperplasia of the Meibomian ducts and follicular infundibula, which was similar to skin epithelium (Fig. 6E ff ff mice were right panel). In contrast, eyes of 4OHT-treated a b normal and the mice contained normal appearing Meibomian glands and ducts (Fig. 6E). Preputial glands from 4OHT-treated IKOab mice were also abnormal. The 4OHT-treated affbff mice displayed preputial glandular cells with typical, abundant, clear, finely vacuolated, translucent cytoplasm and distinct round nuclei (Fig. 6F circle). In contrast, 4OHT-treated IKOab mice had marked atrophy of preputial gland lobules and decreased numbers of clear finely vacuolated sebocytes (Fig. 6F right). Thus, the acute lost of C/EBPa and C/EBPb severely impairs sebaceous, Meibomian, and preputial gland homeostasis and results in the loss of morphologically distinct differentiated sebocytes in all of these glands. To provide biochemical evidence for altered sebocyte ff ff differentiation, histological sections of skin of 4OHT-treated a b and 4OHT-treated IKOab mice were subjected to Oil Red O staining. Oil Red O stains lipid and sebum and positive Oil Red O staining of sebaceous glands is a hallmark of sebocyte differenti- ff ff mice, Oil Red O stained all ation. In 4OHT-treated a b PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9837