Acute co-ablation of C/EBPa and C/EBPb results in severe morphological defects in epidermis involving hyperplasia, dysplasia and hyperkeratosis To address potential functional redundancies between C/EBPa and C/EBPb and unmask their function in epidermis and sebaceous glands, K14-CreERtam;C/EBPaff;C/EBPbff (IKOab) and C/EBPaff;C/EBPbff (affbff) mice were generated and characterized. Immunoblot analysis demonstrated the levels of C/EBPa and C/EBPb in the untreated epidermis of IKOab mice ff ff mice were similar (Fig. 3A), and the topical application and a b of 4OHT to IKOab and affbff mice resulted in dramatic decreases in both C/EBPa and C/EBPb in IKOab but not 4OHT-treated ff ff mice (Fig. 3A). All forms of C/EBPb (LAP*, LAP and LIP) a b and C/EBPa (p42 and p30) were similarly decreased (data not shown). Loss of C/EBPa and C/EBPb in the IFE, ORS follicular keratinocytes and sebocytes was demonstrated by co-IF staining for C/EBPa and C/EBPb in 4OHT-treated IKOab and 4OHT- treated affbff mouse skin (Fig. 3B). In normal IFE, basal keratinocytes progress upwards through morphologically distinct spinous and then granular suprabasal layers, eventually ending in the production of a nonviable stratum corneum or cornified layer. Histological analysis of dorsal skin sections from 4OHT-treated IKOab and 4OHT-treated a ff b ff mice revealed a ff b ff IFE was normal (Fig. 3D). In contrast, the 4OHT-treated IKOab IFE and infundibular ORS keratinocytes were hyperplastic (Fig. 3E&F) and Roles of C/EBPs in Adult Skin the IFE displayed a significant increase in the number of nucleated epidermal cell layers (Fig. 3C). In addition, the basal and spinous layers of the 4OHT-treated IKOab epidermis were dysplastic as characterized by the disorganization of the layers and by highly abnormal variations in cell and nucleus size (Fig. 3G&H). There were also focal and regional areas of moderate to severe hyperplasia of the follicular infundibular epithelium and ORS keratinocytes (Fig. 3F) in the 4OHT-treated IKOab epidermis. In contrast to the disorganized basal and spinous layers, the granular layer of keratinocytes in 4OHT-treated IKOab mice appeared less affected and keratohyalin granules, a hallmark of granular layer, were present at levels similar to 4OHT-treated control mice (Fig. 3I-J). The stratum corneum in 4OHT-treated IKOab epidermis was significantly thickened (hyperkeratosis) (Fig. 3E-G) and displayed low levels of parakeratosis. Collectively, these morphological and histological alterations in the 4OHT-treated IKOab epidermis indicate the acute loss of C/EBPa and C/EBPb disrupts epidermal homeostasis, and within the viable epidermis, the basal and spinous layers appear to be most severely affected. We also examined mice at different times after the start of 4OHT treatment to determine when epidermal abnormalities appeared. We observed focal areas of hyperplasia in as early as four days from start of treatment and these areas became more exten- sive and severe at 8, 12, and 18 days from start of treatment (Fig. 3K). Figure 3. Acute co-ablation of C/EBPa/b results in epidermal morphological defects. (A) Immunoblot analysis of epidermal lysates from untreated and 4OHT-treated a ff b ff and IKOab mice (n = 3 mice/genotype/treatment). (B) Co-IF staining for C/EBPa and C/EBPb in 4OHT-treated a ff b ff and 4OHT-treated IKOab mouse skin. (C) Quantification of epidermal nucleated cell layers in 4OHT-treated mice. (D) H&E staining of 4OHT-treated ff ff mice. (E) H&E staining of 4OHT-treated IKOab mice. (F) Characteristic H&E staining of lesion in 4OHT-treated IKOab mouse. (G) H&E staining of a b 4OHT-treated IKOab epidermis with (H) region enlarged displaying dysplasia. (I) H&E staining and TEM micrograph of keratohyalin granules in 4OHT- ff ff and 4OHT-treated (J) IKOab epidermis. (K) H&E staining of lesions from 4OHT-treated animals from different days after start of 4OHT treated (I) a b treatment. Scale bars represent 30 microns unless otherwise notated. *indicates significantly different from controls p,.05, Student’s t test. doi:10.1371/journal.pone.0009837.g003 PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9837