sebaceous glands, demonstrating the presence of terminally differentiated sebum producing sebocytes associated with nearly every hair follicle (Fig. 6G). In contrast, the sebaceous gland lobules associated with hair follicles in 4OHT-treated IKOab mice did not stain with Oil Red O (Fig. 6G right and bottom right panels) indicating the absence of sebum and terminally differen- tiated sebocytes. To further characterize the sebaceous gland defect in 4OHT-treated IKOab mice, we examined K14, fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD3) and melanocortin 5 receptor (MC5R). K14 is expressed in the basal IFE keratinocytes and is also expressed in the undifferentiated peripheral sebocytes in sebaceous glands, while SCD3 and MC5R have been characterized as specific markers of sebocyte differen- tiation. Within the sebaceous glands of 4OHT-treated affbff mice, K14 is appropriately expressed in the undifferentiated sebocytes around the periphery of sebaceous gland (Fig. 6I). In contrast, the entire 4OHT-treated IKOab sebocyte lobule uniformly expressed K14 suggesting that the lobule may be composed of a population of undifferentiated sebocytes. To further identify these cells within the lobule as sebocytes we conducted IHC staining for FASN. ff ff mice expressed FASN as Sebaceous glands in 4OHT-treated a b did 4OHT-treated IKOab sebocyte lobules indicating that the lobules are comprised of sebocytes (Fig. 6J). The intense FASN staining of 4OHT-treated IKOab sebocyte lobules compared to control glands is likely due to reduced cytoplasmic volume of IKOab sebocytes. FASN mRNA levels were similar in skin between the two genotypes (Fig. 6H). SCD3 and MC5R are markers of terminally differentiated sebocytes and real time quantitative PCR revealed mRNA of both were greatly reduced in 4OHT-treated IKOab. Collectively these morphological, biochemical and molecular results indicate that acute co-ablation of C/EBPa and C/EBPb in adult mice results in a significant sebaceous gland defect in which sebocyte terminal differentiation and lipid production is impaired. Discussion The co-ablation of C/EBPa and C/EBPb in adult mouse skin revealed critical biological functions for these transcription factors in keratinocytes and sebocytes that are not present in germ line single knockout mice [25,26] as well as the inducible conditional single knockout mice described in this study. For example, sebaceous glands of C/EBPa or C/EBPb knockout mice were indistinguishable from wild type sebaceous glands. In contrast, the co-ablation of C/EBPa and C/EBPb resulted in severe sebaceous gland morphological defects and a failure of sebocyte differenti- ation. Likewise, acute co-deletion of C/EBPa and C/EBPb produces profound changes in epidermal homeostasis not seen in single KOs that involves the disruption of stratified squamous differentiation, disorganization of the epidermis and the hyper- proliferation of the IFE keratinocytes as well as ORS follicular keratinocytes. Thus the co-ablation of C/EBPa and C/EBPb has revealed previously unknown functional roles for these transcrip- tion factors in sebocyte biology and has confirmed and eclipsed earlier studies in C/EBPb knockout mice that indicated a role for C/EBPb in early events of keratinocyte differentiation involving K1/K10 expression and growth arrest [26]. Our results indicate that functional redundancies exist between C/EBPa and C/EBPb and/or these two transcription factors cooperate to regulate basal keratinocyte cell cycle withdrawal and early differentiation in keratinocytes and sebocytes. Sebaceous glands are formed from a sebocyte progenitor that expresses Blimp 1, keratin 14 (K14) and K5 [29], and these cells produce a proliferative population of sebocytes [30,32] which in Roles of C/EBPs in Adult Skin turn differentiate into the lipid/sebum producing cells. Through the inducible conditional co-ablation of C/EBPa and C/EBPb we unmasked novel roles for these transcription factors in sebaceous gland differentiation. Sebocytes lacking C/EBPa and C/EBPb were morphologically altered and sebum production was blocked. IKOab mice retained distinct sebaceous gland lobules, but these cells did not produce sebum, a hallmark of sebocyte differentia- tion. It appears the proliferating cells located on the periphery of the gland continued dividing but their progeny were unable to initiate terminal differentiation. Our observation that the cells in the lobules of IKOab mice express K14 and FASN is consistent with this idea, providing compelling evidence that the cells within the lobule are sebocytes. In addition, we observed decreased levels of SCD3 and MC5R, two markers of differentiated sebocytes. Our results indicate the adipogenic transcription factors, C/EBPa and C/EBPb, also have critical roles in sebocyte differentiation through the regulation of lipogenesis and sebum production. The critical importance of these transcription factors in sebocyte homeostasis was further strengthened by the profound effect of their co-ablation on the specialized sebocytes of the Meibomian and preputial glands. Meibomian glands of mice lacking C/EBPa and C/EBPb were severely disorganized, dysplastic and there was evidence of bacterial infection. Posterior blepharitus is a human disease often characterized by improper sebum production by Meibomian glands, lending an environment favorable for bacterial growth, and accompanied by inflammation [41]. Acne, seborrheic dermatitis, blepharitis, and sebaceous cancer are among several human health conditions involving altered sebocyte homeostasis. It seems plausible the transcription factors C/EBPa and C/EBPb could have involvement in these disease conditions. Molecular analysis of IKOab epidermis revealed that K5 and K14, markers of basal keratinocytes, were expressed throughout the basal and suprabasal layers, while suprabasal markers K1 and K10 were delayed or weakly expressed. A concomitant increase in hyperproliferative basal and suprabasal IFE keratinocytes was observed in IKOab epidermis. These results indicate an expansion of the basal compartment and reduced ability of these basal keratinocytes to initiate the early events of stratified squamous differentiation involving K1 and K10 expression and cell cycle withdrawal. Both C/EBPa and C/EBPb can bind to and activate the K10 promoter and C/EBP binding sites [42] have been identified in both the K1 and K10 promoters [26] suggesting differences in K1 and K10 expression could be regulated directly by C/EBPs at the promoter level. Chromatin immuno-precipita- tion experiments will be required to resolve these issues. In general, the granular layers of IKOab epidermis appeared less affected; keratohyalin granules were present and involucrin and filaggrin were expressed, albeit at increased levels. These changes in increased involucrin and filaggrin expression could be a response to the disruption of epidermal homeostasis due to the defective basal to spinous transition or it is possible that C/EBPa and C/EBPb contribute to the suppression of granular differen- tiation as has been reported for Hes1 in epidermal development [19]. Mechanisms of epidermal development and postnatal differen- tiation are not necessarily synonymous. For example, ablation of Notch in epidermal development produces the loss of spinous and granular layers and a hypoproliferative phenotype [18], while postnatal ablation of Notch1 produce an opposite effect involving a hyperproliferative phenotype and an increased granular layer [20]. Hes1 ablation dramatically alters epidermal development involving the spinous to granular transition, but appears to have a minimal role in adult skin [19]. Recently the co-deletion of C/ EBPa and C/EBPb in developing mouse skin produced mice that PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9837