Figure 4. Co-expression analysis of PAX3-positive follicular melanocytes. A) The transverse section of the hair follicle shows both PAX3 and HES1 co-expressing (enlarged in B), and single PAX3- expressing (enlarged in C), melanocytes in the outer root sheath (ORS). The line circumscribes the hair follicle. PAX3 was labelled with mouse monoclonal antibody (DSHB). D) The longitudinal section of the hair follicle shows PAX3 and MLANA co-expressing (enlarged in the insert on the left) and single PAX3-expressing (enlarged in the insert on the right) PAX3 in Melanocytes melanocytes in the outer root sheath (ORS). E) Single PAX3-expresing (arrows) and PAX3 and MLANA co-expressing (arrowheads) melano- cytes in the matrix of the hair bulb. PAX3 was labelled with rabbit polyclonal antibody (Invitrogen). doi:10.1371/journal.pone.0009977.g004 Discussion PAX3 expression in normal skin, naevi and melanoma In agreement with previous reports, we confirm here that PAX3 is expressed in all melanoma and naevi samples analysed. There is however a paucity of studies analysing PAX3 expression in differentiated melanocytes in human skin. Traditionally consid- ered a critical embryonic regulator, PAX3 has been suggested to maintain melanocyte stem cells in the bulge area of the hair follicles in mice [26], but was thought to be down-regulated upon terminal differentiation of melanocytes. This belief has been supported by reports detailing the absence of PAX3 expression in melanocytes of normal human skin in contrast to naevi and melanomas [5,6]. Our results show conclusively that PAX3 is expressed in both epidermal and follicular melanocytes of human skin. Furthermore, co-expression with MITF confirms the melanocytic origin of these PAX3-positive cells. We also show here, for the first time, the variable phenotype of epidermal melanocytes of normal skin. The observed frequency of PAX3 expression in naevi, melanomas and specifically in melanocytes in normal skin is much higher than reported elsewhere [5,6]. One possible explanation could be the methodology we used, namely EDTA/ Tris antigen retrieval buffer, rather than the citrate buffer commonly used by other researchers, which was, in our experience, less efficient. It might also reflect the influence of environmental factors, namely sun exposure and solar radiation. It has recently been suggested that in UV-treated skin TGF-b signalling from keratinocytes is down-regulated, increasing PAX3 expression in epidermal melanocytes thus stimulating their proliferation [28]. All the samples used in this study are collected from people living in Australia, where the solar radiation levels are in the high to extreme range for much of the year. A recent report from New Zealand [27] shows PAX3 expression in all naevi, melanomas and normal skin samples, confirming our findings. Further studies are required to investigate differences in PAX3 expression in skin samples derived from the Southern vs. the Northern hemispheres. Normal skin samples used in this study, mainly originating from anatomic locations frequently exposed to the sun (such as limbs and face), were free of any melanocytic lesion having been used for other diagnostic purposes. We have included here two normal skin samples from breast reductions which would have lower sun exposure levels and these samples also show PAX3- positive melanocytes in the epidermis. We have looked to see if PAX3-positive melanocytes in our samples were proliferative, by co-staining with Ki67. In our samples of sun-exposed skin, 18.1% of epidermal PAX3-positive melanocytes were proliferative, but the majority were not. This was in contrast to samples subjected to low levels of sun exposure, where we observed one single PAX3, Ki67-positive cell. However, to accurately address the question whether the high frequency of PAX3 expression in normal melanocytes observed in this study, relative to previously published papers, is caused in any way by the extent of sun exposure, a more thorough analysis of skin subjected to chronic and/or acute UV exposure relative to sun-protected skin, would need to be performed. PLoS ONE | www.plosone.org 6 April 2010 | Volume 5 | Issue 4 | e9977