terminal differentiation [26]. Once Mitf levels reach a certain threshold this repression is removed, allowing Mitf, in the presence of b-catenin, to activate Dct expression and melanocyte matura- tion. This model of regulation of melanocyte differentiation suggests that Pax3 expression is either negligible or significantly reduced in adult differentiated melanocytes. Several studies report PAX3 expression in naevi and melanomas [2,4], and show no expression in melanocytes of normal skin [5,6], thus concluding that PAX3 re-expression might be involved in melanocyte transformation. In contrast to this widely accepted assumption, other studies note PAX3 expression in melanocytes of normal skin [22,27], and more specifically its up-regulation as a result of the UV-induced loss of TGFb signalling from keratinocytes [28]. Additionally, PAX3 expression has been described in cultured melanocytes [3,29] and melanoblasts with no significant change in levels of either mRNA or protein between these two cell types [29]. Here we confirm that PAX3 is present in the majority of naevus and melanoma cells. We also show conclusively that PAX3 is expressed in melanocytes of normal skin where it is co-expressed with known melanocyte markers. Since the precise role played by PAX3 in melanomas is not clear, we have looked into potential PAX3 regulated pathways within normal melanocytes. We have chosen pathway-representative markers that are also downstream targets of PAX3 and analysed their co-expression with PAX3 in normal skin melanocytes, naevi and melanomas. Interestingly, while the majority of PAX3-positive melanocytes in the epidermis of normal skin show a mature phenotype, a portion (around 20%) show a less differentiated and proliferative phenotype. Moreover, PAX3-positive melanocytes frequently show expression of BCL2- like 1 (BCL2L1, also known as Bcl-X, bcl-xL or BCL-XL/S), an antiapoptotic marker. Taken together these results indicate that melanocytes of normal skin display a phenotype that is predisposed to malignant transformation (antiapoptotic, undiffer- entiated and proliferative). We also show that PAX3- positive epidermal melanocytes of normal skin are molecularly distinct from those of the hair follicle, which like melanoma cells express Melanoma cell adhesion molecule (MCAM, also known as MUC18/CD146), associated with cell migration and melanoma progression and metastasis. Results PAX3 expression in normal skin, naevi and melanoma PAX3 expression was analysed by immunohistochemistry in tissue sections from paraffin embedded samples of normal skin, naevus, primary melanoma and melanoma metastases. Positive staining for PAX3 was obtained after performing heat-induced antigen retrieval in an EDTA/Tris buffer, pH 8.0 and incubating the primary antibody (anti-PAX3, DSHB) at room temperature (Figure 1A, top panel). Antigen retrieval using the most common 10mM citrate buffer, pH 6.0 with the same staining protocol, failed to produce a positive signal in several of these samples, and in those that were positive after citrate antigen retrieval, the intensity of staining was dramatically reduced. Negative controls included omitting the primary antibody from the procedure resulting in no immunohistochemical signal (not shown). Mouse monoclonal anti-PAX3 antibody (DSHB) has been previously confirmed by immunohistochemistry to be specific for the PAX3 protein [30]. PAX3-specific staining was also confirmed using a different commercially available anti-PAX3 antibody (Invitrogen) (Figure 2, A–C and E). However, we have observed less intensive staining overall with this polyclonal antibody (Invitrogen), compared to that observed with the monoclonal (DSHB). PAX3 in Melanocytes All of the samples analysed showed positive PAX3 staining in more than 10% of cells (Figure 1A, top panel). In normal skin PAX3-positive cells showed a pattern of distribution that is characteristic of melanocytes; i.e. they were found at the epidermal-dermal boundary and along the hair follicle (Figure 1A–D). When adjacent sections were stained with MITF, a known marker of melanocytes, a comparable pattern of distribution was observed (Figure 1A, bottom panel). The intensity of PAX3 staining varied between cells within the same lesion and between different samples. There was no obvious correlation between either intensity or number of PAX3-positive cells and lesion type. However, PAX3-positive cells of normal skin samples generally showed weaker staining and far fewer cells were positive, compared to those in naevi and primary melanoma samples. In contrast, melanoma metastases have an overall low level of PAX3 expression and these cells were mainly located at the periphery of the lesion. Additional analysis by quantitative RT-PCR was performed on a separate set of samples, to confirm PAX3 expression in normal skin, naevi, primary melanomas, and melanoma metastases. PAX3 expression was detected in all naevi (5/5), primary melanomas (5/ 5) and melanoma metastases analysed (5/5), and in one out of two (1/2) normal skin samples. The highest levels of PAX3 expression were found in melanoma metastases (mean fold change of 258361198), followed by naevi and primary melanomas with 101639 and 73655- fold change, respectively (Figure 1E). Even though these results suggest that PAX3 might be up-regulated in advanced melanomas (melanoma metastases vs. primary melano- mas) comparison with immunohistochemistry results show that expression levels are not due to increased amounts of PAX3 per individual cell, but rather they reflect the number of cells expressing PAX3 (Figure 1A). Nevertheless RT-PCR results confirm PAX3 expression in melanocytes of normal skin. Characterisation of PAX3-positive cells: Co-expression of PAX3 and MITF In order to validate PAX3 expression in naevi and melanomas as well as to confirm that PAX3-positive cells in normal skin are in fact melanocytes, co-staining of PAX3 and MITF was assessed by immunofluorescence. Indeed, in all of the samples analysed, including normal skin, PAX3-positive cells co-express MITF (Figure 2, A–C and E). However, in normal skin (as in other samples) a small number of cells showed only MITF and not PAX3 expression, highlighting the variation in normal melanocyte cell phenotype. In normal skin samples 81.6% of all MITF-positive melanocytes were also PAX3-positive (Figure 2D). Both naevi and primary melanoma samples show similar numbers of MITF-positive cells that also express PAX3 (97.2% and 94.5%, respectively) (Figure 2D). Interestingly, melanoma metastases show significantly less PAX3-positive than MITF-positive cells (Figure 2D), and generally these were weakly stained for PAX3. However, this was observed primarily when using rabbit polyclonal antibody to PAX3 (Invitrogen) for immunofluorescent staining. When the mouse monoclonal anti-PAX3 antibody (DSHB) was used for immunohistochemistry staining, many more PAX3-positive cells were observed on the same sample. The discrepancy between these two results is probably due to the sensitivity of the two antibodies. Nevertheless, the intensity of PAX3 staining in individual cells within the same tissue sample varied when either antibody was used, whereas MITF levels were uniform across each sample (Figure 2, A–C). This is shown clearly in cells of the A2058 metastatic cell line (Figure 2C) and may reflect differential cell status/phenotype. PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e9977