previously-unreported white-spotted coat color, suggesting a defect in melanocyte proliferation, survival or migration (Fig S1A–C). The lack of an obvious keratinocyte phenotype in Ets1 knockout mice may be due to functional compensation by other Ets family members, a number of which are expressed in skin. Indeed, the closely related Ets2 protein is highly expressed in skin and has been shown to play a role in hair follicle morphogenesis [36]. Although Ets1 is not essential for epidermal homeostasis under physiological conditions, it may contribute to oncogenic transfor- mation of these cells by promoting proliferation, epithelio- mesenchymal transition (EMT) and/or invasion and metastasis. In order to study the function of the Ets1 oncogene in skin carcinogenesis, we developed a Tet-inducible Ets1 transgenic mouse model. The full-length murine Ets1 cDNA was cloned in- frame with an N-terminal HA epitope tag and inserted downstream of the doxycycline-regulated pTight promoter in the vector pTRE-Tight to generate the construct pTight-Ets1 (Figure 1B). The pTight-Ets1 construct was micro-injected into fertilized mouse oocytes and six transgenic founders were obtained. To test the ability of the six transgenic lines to inducibly express Ets1, we crossed each founder with previously-described transgenic mice expressing the tetracycline-transactivator (tTA) under the control of the human involucrin promoter and enhancer elements (INV-tTA mice) [37]. In the resulting bitransgenic (BT) offspring (pTight-Ets1/ INV-tTA), HA-Ets1 can be induced in the upper spinous and granular layers of skin and other stratified squamous epithelial tissues when doxycyline (Dox) is withheld. Note that this induced Ets1 expression pattern is opposite to the normal pattern of Ets1 gene expression in the basal layer of the epidermis. When Ets1 expression was induced during embryonic develop- ment by withholding Dox, BT newborn animals derived from two of the six pTight-Ets1 founder animals, line C and E exhibited a dramatic skin phenotype leading to a defect in epidermal barrier formation and neonatal lethality (data not shown). In agreement with the skin phenotype, line C and E were shown to express the HA-Ets1 transgene by Western blotting (Figure 1C and data not shown). The majority of the studies reported herein were performed using the transgenic line C, but similar results were obtained with line E. To determine whether the transgene was faithfully expressed in the differentiated layers of the epidermis, immunohistochemical analysis using anti-HA antibodies was carried out. As shown in Figure 1D, HA staining was evident in the differentiated layers of the skin epithelium, but not in the undifferentiated basal layers. The Ets1 transgene would also be expressed in differentiated cells of the oral mucosa and other internal, stratified epithelia (i.e., esophagus, forestomach and anogenital linings) since the involu- crin promoter has been shown to be active in these sites [37]. Transgene expression could be suppressed by supplementation with Dox as expected and mice maintained on Dox during embryonic development and in the post-natal period were normal at birth, did not have an epidermal barrier defect and survived to adulthood. Thus, we have developed a Doxycycline–regulated, inducible transgenic model system that allows us to turn Ets1 expression on or off in the stratified squamous epithelia of mice at different ages and for different periods of time. Expression of the Ets1 transgene in adult mice leads to conspicuous changes in the skin As noted above, induction of Ets1 expression during embryonic development leads to neonatal lethality due to a failure to establish a normal skin barrier function. To overcome this problem, we suppressed expression of the Ets1 transgene during the embryonic Ets1 in Squamous Neoplasia and postnatal period by maintaining the mice on Dox supple- mentation. After weaning, Dox was withdrawn allowing transgene expression (Figure 2A). In adult BT mice, obvious epidermal alterations became evident within a three- to six-week period after Dox withdrawal (Figure 2B–D). In particular, BT mice exhibited patchy hair loss, crusting, scaly skin and open sores covering the body surface. The skin of the paws and tail was frequently affected. In addition, BT mice were often very thin and wasted compared to control littermates, potentially due to difficulties in feeding caused by changes to the oral cavity, esophagus or forestomach. We noted some variability in the severity of the phenotype of induced mice, this might reflect differences in genetic background modifiers as the pTight-Ets1 transgenic mice were initially derived from a mixed C3H6C57BL/10 genetic background and subsequently crossed to FVB/N mice. Alternatively, the differences in phenotype might reflect variegation in transgene expression or in the precise timing of transgene induction. The studies described below assessing histological changes in the skin epidermis were performed using samples from mice that displayed a visible phenotype after 3–6 weeks of Ets1 induction. Histological examination of BT mouse skin revealed striking hyperplastic and dysplastic alterations when compared to single transgenic or non-transgenic control mice. The epidermal changes were most prominent in sections of the affected dorsal trunk, ear and tail skin (Figure 3A–C). The BT epidermis was dramatically thickened and hyperkeratotic when compared to control mice and was composed of predominantly large, pleomorphic cells with eosinophilic cytoplasm, suggesting that the expanded cell popu- lation was spinous in nature (acanthosis). Some keratinocytes of the stratum corneum, though cornified, retained nuclei (paraker- atosis) indicating a partial block to terminal differentiation. The alteration of the normal pattern of keratinocyte differentiation was also apparent from the presence of keratin pearls scattered throughout the skin. In addition, there was an extensive invasion of the underlying dermis by the hyperplastic epidermal cells. The keratinocytes invaded the dermis in the form of large cohesive masses or thick downgrowths (10–20 cells across) often enclosing islands of dermal tissues within them. In some cases, enlarged sebaceous glands and the proximal end of hair follicles formed the leading edge of the invasive front. As a secondary response to the epidermal alterations, inflammatory reaction typified by leukocytic infiltration was also observed. Closer examination showed an increase in nuclear-to-cytoplasmic ratio, hyperchromatic nuclei and prominent nucleoli in the keratinocytes (Figure 3D). The proliferative capacity of the affected epidermis was augmented as evidenced by increased number of mitotic cells. Interestingly, these actively dividing cells were not restricted to the proliferative basal compartment of the epidermis, but were present in the suprabasal layers too. This suggests that some partially-differentiated keratinocytes retained or acquired proliferative capability in the supra-basal layers. A number of these dividing cells had atypical (tripolar) mitotic spindles, a feature often associated with transformation (Fig. 3E). Therefore, induction of Ets1 in differentiating keratinocytes leads to extensive hyperplasia associ- ated with dysplastic differentiation and downgrowths of chords of keratinocytes into the dermal compartment. The keratinocyte differentiation program is disrupted in epithelia expressing Ets1 The histological analyses described above suggested that the normal differentiation program of epidermal keratinocytes was disrupted by expression of Ets1 in the differentiating layers of the skin. To confirm this result, we stained sections of the dorsal skin of BT and control mice with antibodies specific for marker proteins PLoS ONE | www.plosone.org 3 January 2009 | Volume 4 | Issue 1 | e4179