Figure 3. Co-expression analysis of PAX3-positive epidermal melanocytes of normal skin. Epidermal melanocytes of normal skin show a variable differentiation status: less differentiated melanocytes co-expressing PAX3 (mouse monoclonal antibody, DSHB) and HES1 (A); more differentiated co-expressing PAX3 (rabbit polyclonal, Invitrogen) and MLANA (B); and mature melanocytes expressing only MLANA (B). Single HES1-labelled cells in the epidermis are keratinocytes (A). C) PAX3 (mouse monoclonal antibody, DSHB) and Ki67 co-expressing melanocytes are also observed in the epidermis of sun-exposed skin. Lines in (A), (B) and (C) demarcate the epidermal-dermal border (EDB) or epidermal surface (ES). D) Graph shows the distribution of differenti- ation marker expression in normal skin melanocytes with respect to melanocyte location (in epidermis, outer root sheath (ORS), or hair follicle bulb). doi:10.1371/journal.pone.0009977.g003 compared to the ORS (23.7%) and the epidermis (27.9%) (Figure 3D). By contrast, MLANA was expressed in more that 60% of both epidermal and ORS PAX3-positive melanocytes, compared to 36% of those in the matrix of the hair bulb (Figure 3D). We have also observed a significant number of single MLANA-labelled melanocytes (Figure 3B) (30.5% in the epider- mis, 3.9% in ORS, and 40% in the hair bulb). This indicates that melanocytes, both follicular and epidermal, have a variable differentiation status: from less differentiated PAX3, MITF and HES1-positive to more differentiated PAX3, PAX3 in Melanocytes MITF and MLANA-positive. The observed PAX3-negative, MLANA-positive epidermal melanocytes may represent mature terminally differentiated melanocytes. In addition, Yang and colleagues [28] have recently proposed that in UV-treated skin TGF-b signalling from keratinocytes is downregulated, which increases PAX3 expression in epidermal melanocytes and stimulates their proliferation. In order to check if PAX3-positive melanocytes observed here are proliferating we co- stained PAX3 with a marker of proliferation, Ki67, and found 18.1% of epidermal melanocytes in samples of sun-exposed skin (scalp) to be double-labelled (Figure 3C). In contrast to this, in samples that had relatively little sun exposure (breast) one single double labelled cell was observed in the epidermis (accounting for 1.4% of PAX3-positive cells). PAX3 in cell survival: Co-expression with BCL2L1 To further characterise the phenotype of PAX3-positive melanocytes relative to melanoma cells, expression of an antiapoptotic factor BCL2L1 was assessed in PAX3-positive cells (Figure 5A). We have observed that a similar proportion of PAX3- positive cells were also BCL2L1-positive in melanocytes of normal skin, in naevi and in melanoma cells in all of the samples analysed (Figure 5D). The exception is melanoma metastases, in which BCL2L1 was detected in only one sample (out of four) and both PAX3 and BCL2L1 were weakly stained and very rarely co-stain. BCL2L1 expression in epidermal keratinocytes, as reported previously [33], served as an internal positive control for the staining. BCL2L1 expression in normal PAX3-positive melanocytes indicates that they might utilise the same cell survival regulatory mechanism as melanoma cells, in order to sustain continuous renewal in the ever-changing environment of the epidermis. It is possible that one of the roles PAX3 plays in adult melanocytes (similar to that in development) is to ensure their maintenance and survival, while regulating their specification and proliferation. PAX3 in cell migration: Co-expression with MCAM Expression of the melanoma progression marker MCAM was also analysed relative to PAX3. MCAM expression was observed in most naevi and melanoma samples analysed; 80% (4/5) naevi samples, 66.7% (4/6) primary melanomas and 100% (5/5) of melanoma metastases. There was no MCAM expression in epidermal melanocytes of normal skin (Figure 5B). Notably we frequently observed MCAM expression in hair follicle cells of normal skin [34–36], mainly in the ORS and not exclusively in melanocytic cells (Figure 5C). MCAM expression in the endothelial cells of the blood vessels served as an internal positive control [36,37]. In naevi samples MCAM expression was observed in specific areas, mainly in nests of cells in the dermal component of the naevus and only a small proportion (14.7%) of PAX3-positive cells were MCAM-positive (Figure 5B, E). Compared to naevi, primary melanomas showed MCAM expression in a larger proportion of PAX3-positive cells, 56.4% (Figure 5B, E), which are again primarily located in the dermal component of the lesion. Samples of melanoma metastases showed MCAM expression in the majority (93.2%) of PAX3-positive cells (Figure 5B, E). In general, MCAM co-expressed with PAX3 in all MCAM- positive samples. In accordance with previous reports [34], the number of MCAM expressing PAX3-positive cells increased with malignant and particularly metastatic transformation. The expres- sion of MCAM in follicular melanocytes, in contrast to the absence of expression in epidermal melanocytes, highlights again the difference between these two melanocyte populations. PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e9977