myc, CDK11, Fas ligand, GADD153, JunB, mdm2, p16, p21 and p53) [17,18,19,20,21,22,23,24,25,26,27,28] and genes involved in responses to a hypoxic environment [29]. Furthermore, Ets1 has been reported to directly interact with the tumor suppressor protein p53 and modulate its transcriptional activity [30,31,32,33,34]. Because Ets1 regulates the expression of a large cohort of target genes that influence cellular proliferation and survival, Ets1 might play an important role in early stages of the carcinogenic process in addition to its better known role in tumor invasion and metastasis. To investigate the ability of Ets1 to drive neoplastic or pre-neoplastic changes in stratified squamous epithelial cells, we developed a transgenic mouse model that allows us to inducibly express high levels of Ets1 protein in skin epithelium. Using this inducible model system, we have explored the role of Ets1 in SCC. Results Ets1 is expressed predominantly in the proliferative basal layer of the stratified squamous epithelium Although Ets1 has previously been reported to be expressed in embryonic mouse skin [35], the exact expression pattern of this transcription factor during epidermal keratinocyte differentiation Ets1 in Squamous Neoplasia has not been determined. We performed immunostaining for Ets1 protein in the skin epithelium of newborn and adult mice, which showed that expression of Ets1 is predominantly restricted to the nuclei of proliferative basal layer keratinocytes of the epidermis and hair follicles of newborn mice (Fig. 1A). In contrast, more differentiated suprabasal cells expressed little or no Ets1 protein. A similar pattern of expression was detected in adult mice, with the majority of Ets1 positive cells being localized to the basal layer and to the hair follicles (Fig. 1A). We conclude that in mice Ets1 is mainly expressed in undifferentiated keratinocytes of the skin and its expression is downregulated as cells commit to the terminal differentiation program. A similar pattern of expression of Ets1 in basal cells, but not suprabasal differentiated cells, has been detected in human skin samples [3]. Development of an inducible Ets1 transgenic model As discussed in the Introduction, high levels of Ets1 expression have been detected in SCC in humans and rodents. These observations along with the expression pattern of Ets1 in the proliferative compartment of the skin suggest that Ets1 may play a role in keratinocyte proliferation. However, Ets1 knockout mice do not display any obvious defects in the proliferation or differenti- ation of skin keratinocytes, although they are characterized by a Figure 1. Development of an inducible transgenic model to express Ets1 in differentiated epidermal keratinocytes. (A) Immuno- histochemical localization of Ets1 in the nuclei of epidermal keratinocytes in newborn and adult mice. (B) Schematic representation of the tetracycline-inducible Ets1 transgenic system. The system consists of two separate transgenes- the Tet driver transgene in which the tetracycline- regulated transactivator protein (tTA) is under the control of the involucrin promoter and enhancer elements and the Tet responder transgene in which an HA-tagged version of murine Ets1 is under the control of the pTight promoter containing tet-responsive elements (TRE, which are tTA binding sites). When Tet driver and Tet responder mice are crossed, BT offspring are produced. Maintaining these BT animals on Dox supplementation inhibits transgene expression, whereas withdrawal of Dox supplementation causes induced expression of the Ets1 transgene in cells that normally express involucrin (upper spinous and granular layers). (C) Western blot analysis of transgene expression in skin extracts from newborn wild-type and BT mice using anti-Ets1 and anti-HA antibodies. GAPDH serves as a loading control. (D) Immuno-histochemical localization of transgene expression using antibody against the HA epitope tag. doi:10.1371/journal.pone.0004179.g001 PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4179