Acute ablation of either C/EBPa or C/EBPb alone in adult mouse skin does not produce a major skin phenotype To ablate C/EBPa and C/EBPb in postnatal adult mouse skin we used a conditional inducible approach utilizing K14-CreERtam mice [36]. The K14 promoter directs the expression CreERtam to the basal layer of the epidermis, ORS follicular keratinocytes of the hair follicle as well as the progenitor and proliferative populations of sebocytes of the sebaceous gland of mouse skin. The activity of CreERtam is regulated through topical treatment with the synthetic ER antagonist, 4-hydroxytamoxifen (4OHT). While the overall goal was to simultaneously acutely ablate C/ EBPa and C/EBPb in adult mouse skin, it was first necessary to characterize the epidermis of adult mice in which C/EBPa or C/ EBPb were individually acutely ablated. While the phenotypes of the epidermal specific germline knockouts of C/EBPa and C/ EBPb have been previously characterized [25,26], it could not be assumed that acute ablation in fully-developed adult skin would mimic the germline embryonic knockouts where developmental compensatory events could have occurred in the embryo to mask a postnatal phenotype. Moreover, in order to accurately define and interpret the phenotype of the double knockout it was important to determine the phenotype of single C/EBP knockouts in the same experimental model system. K14-CreERtam;C/EBPaff mice and K14-CreERtam;C/EBPbff mice were generated and we first analyzed K14-CreERtam;C/ EBPbff mice (hereafter referred to as (inducible knock out) IKOb mice). Before treating mice with 4OHT, the levels of C/EBPb protein in the epidermal homogenates of untreated IKOb and untreated C/EBPbff mice (bff) were examined. Immunoblot analysis demonstrated epidermal C/EBPb levels were similar in the two genotypes indicating the CreERtam fusion protein was not significantly active in the absence of 4OHT treatment (Fig. 1C). 4OHT treatment (1.0 mg 4OHT/day for days 1–5 and 13–17; skin was collected on day 21), had no significant effect on C/EBPa or C/EBPb levels in floxed-b mice (Fig. 1C), however, 4OHT treatment resulted in near complete ablation of C/EBPb in IKOb epidermis and this was consistently accompanied by a modest increase in C/EBPa levels suggesting possible compensatory upregulation (Fig. 1C). C/EBPb is expressed in three forms (LAP* (39 kD), LAP (36 kD) and LIP (20 kDa) due to the use of alternative translation initiation sites and all forms of C/EBPb were decreased in 4OHT-treated IKOb mice (data not shown). Immunohistochemical (IHC) staining for C/EBPb confirmed the loss of C/EBPb in the IFE and revealed the loss of C/EBPb in the ORS follicular keratinocytes of the hair follicle and sebocytes of sebaceous gland (Fig. 1D). The 4OHT-treated IKOb epidermis displayed a moderate increase in: epidermal thickness (Fig. 1E right), the number of nucleated cell layers (Fig. 1F) and the number of BrdU positive basal keratinocytes following a 1 hour pulse was increased (Fig. 1G first panel). Overall, with the exception of the epidermal thickness and the increase in nucleated cell layers, the morphology of the IFE, ORS of the hair follicle and sebaceous glands of the 4OHT-treated IKOb epidermis were indistinguishable from 4OHT-treated bff mice (Fig. 1E). IHC staining for K5, K10, loricrin and involucrin in C/EBPbff and IKOb epidermis treated with 4OHT revealed the correct spatial expression patterns, however, similar to germline C/EBPb knockout mice [26], we observed a modest decreased staining intensity for K10 (Fig. 1H). Next, K14-CreERtam;C/EBPaff (IKOa) were examined. Base- line levels of C/EBPa protein in the epidermis of untreated IKOa mice and control C/EBPaff (aff) mice were similar and the topical application of 4OHT to IKOa mice resulted in greatly reduced levels of C/EBPa and a modest increase in C/EBPb (Fig. 2A). Roles of C/EBPs in Adult Skin Both isoforms of C/EBPa ((p42 (42 kD) and p30 (30 kD)) were similarly decreased after treatment with 4OHT (data not shown). IHC staining for C/EBPa demonstrated the loss of C/EBPa in the IFE, ORS cells of the hair follicle and sebocytes in 4OHT treated IKOa animals (Fig. 2B). Epidermal thickness and basal cell proliferation as measured by BrdU incorporation was moderately increased in 4OHT-treated IKOa vs. 4OHT-treated C/EBPaff (Fig. 2D). With the exception of a slight increase epidermal thickness proliferation, the morphology of the IFE, ORS follicular epidermis and sebaceous glands of the 4OHT-treated IKOa epidermis were indistinguishable from 4OHT-treated C/EBPaff mice (Fig. 2C) as was the IHC staining for K5, K10, loricrin and involucrin in epidermis (data not shown). In summary, with the exception of modest increases in epidermal proliferation in 4OHT-treated IKOa mice, the acute single ablation of C/EBPa or C/EBPb in adult mouse epidermis results in a phenotype similar to the germline knockout phenotype [25,26], indicating developmental compensatory events do not have a major role in masking the postnatal germline knockout phenotype. Figure 2. Acute ablation C/EBPa does not produce a major skin phenotype. (A) Immunoblot analysis of epidermal lysates from untreated and 4OHT-treated C/EBPaff and IKOa mice (n = 3 mice/ genotype/treatment). (B) IHC staining for C/EBPa in 4OHT-treated C/ EBPaff and 4OHT-treated IKOa mouse skin. (C) H&E staining of 4OHT- treated C/EBPaff and 4OHT-treated IKOa mouse skin. (D) Quantification of nucleated cell layers and number of basal BrdU positive cells in 4OHT-treated C/EBPaff and IKOa mice (n = 3 mice/genotype/treatment). All scale bars represent 30 microns. *indicates significantly different from controls p,.05, Student’s t test. doi:10.1371/journal.pone.0009837.g002 PLoS ONE | www.plosone.org 4 March 2010 | Volume 5 | Issue 3 | e9837