Moreover, it prevents differentiation of melanoblasts and mela- nocyte stem cells before they reach the hair bulb and regulates their proper location in the ORS and in the hair matrix [43]. Therefore it is not surprising that we see a decrease in the number of PAX3 and HES1-positive cells in the hair bulb compared to the ORS (10.5% and 23.7%, respectively). What is surprising is that around 28% of PAX3-positive epidermal melanocytes are also HES1-positive, indicating they are not terminally differentiated. In summary, it seems that epidermal melanocytes are not all terminally differentiated and similar to those in the hair follicle, they exhibit variable differentiation status with persistent PAX3 expression. Melanocytes in the ORS of the hair follicle are mostly PAX3 and MITF-positive and not all are fully differentiated (23.7% HES1-positive, 61.5% MLANA-positive). In the matrix of the hair bulb the number of PAX3-positive melanocytes is lower (around 65%), with only a small proportion still showing a less differentiated phenotype (10.5% HES1-positive and 36% MLANA-positive). Epidermal PAX3-positive melanocytes show a similar profile to those in the ORS (27.9% HES1-positive, 62% MLANA-positive). Interestingly, there are a similar number of MLANA-positive (but PAX3-negative) melanocytes in the epider- mis (30.5%) and the hair matrix (40%), in contrast to the ORS where such cells are rare (3.9%). PAX3 expression seems to correlate with undifferentiated (co-expressing HES1 and MITF) and differentiating (co-expressing MLANA and MITF) cells, whereas it might be diminished in terminally differentiated cells (PAX3-negative but MLANA or MITF-positive) that are further along the continuum of differentiation. It is possible that PAX3 is involved in maintenance of melanocyte ‘‘stemness’’, at least while they are migrating towards their destination, either in the hair follicle or the epidermis. PAX3 in apoptosis It is also very likely that PAX3 is involved in keeping melanocytes alive, since three quarters of epidermal and more than half ORS and matrix PAX3-positive melanocytes show expression of the PAX3 target, antiapoptotic BCL2L1. PAX3 is previously reported as an antiapoptotic regulator in tumours, including melanoma [44–49]. Several known antiapoptotic factors, such as tumour suppressors p53, PTEN and BCL2L1, are involved in Pax3-induced cell survival [48,50,51]. BCL2L1 is directly transcriptionally regulated by PAX3, and in rhabdomyo- sarcoma treatment with PAX3 or BCL2L1 antisense oligonucleo- tides, individually or in combination, decreases cell viability to a similar extent, suggesting that they lie in the same antiapoptotic pathway [51]. Similarly, BCL2L1 is associated with melanoma cell survival, since its expression correlates with melanoma progression [52,53] and treatment with antisense oligonucleotides resulted in reduction of cell viability [54,55]. However, its expression is observed in naevi and normal skin [52,53] and in melanocytes it also affects cell viability [55]. As expected we have observed positive BCL2L2 staining in all normal skin, naevi and primary melanoma samples analysed. The majority of PAX3-positive cells in melanomas, naevi and normal skin co-express BCL2L2 at a similar frequency. These results support the potential role of PAX3 as one of many cell survival regulators. The results also indicate that normal melanocytes might utilise the same cell survival regulatory pathway as melanoma cells, both in the epidermis and in hair follicles. PAX3 in Melanocytes expression in melanomas shows significant correlation with poor disease free survival and mortality [36,56–58,60,61]. Up-regula- tion of MCAM, together with loss of keratinocyte-dependence, is one of the crucial events that allows melanoma cells to invade the dermis and progress to vertical growth phase (for review see [62]). MCAM can mediate both homotypic adhesion between melano- ma cells, promoting local tumour growth, and heterotypic adhesion between melanoma cells and endothelial cells of blood vessels, facilitating metastatic spread [59,63,64]. Pax3-transfected melanocyte cells show increased expression of MCAM, both at an mRNA and protein level, suggesting that MCAM is a downstream target of Pax3 [65,66]. Our results confirm co-expression of PAX3 and MCAM in melanomas and naevi, mainly in the cells that form nests located in the dermal component of the lesion, with melanoma samples showing larger numbers of PAX3, MCAM-positive cells compared to naevi. This indicates that some PAX3-positive cells in naevi and melanomas have the capability to undergo vertical spread/migration. It suggests that regulation of migration might be one of the roles of PAX3 in melanoma, as it is in development, where Pax3 regulates cMet [67]. The frequent observation of MCAM expression in cells of the hair follicle might be associated with downwards growth into the dermis and migration of follicular cells. The observation that some PAX3-positive melanocytes in the ORS show MCAM expression, in contrast to those in the epidermis, suggests that MCAM is associated with migration of normal melanocytes from the bulge area to the developing hair follicle and to the epidermis. Upon reaching the epidermis, MCAM expression is downregulated. Alternatively, epidermal melanocytes do not arise and migrate from the bulge, but rather arise from interfollicular melanocyte stem cells, similar to those believed to maintain interfollicular epidermal homeostasis [68–70]. Even though some epidermal melanocytes resemble the transient amplifying melanocytic cells of the hair follicle with respect to their differentiation status, they are not thought to have the same migratory propensity. In summary, in addition to detection in the majority of naevus and melanoma cells, we conclusively show PAX3 expression in melanocytes of normal skin. Epidermal PAX3-positive melano- cytes, like follicular cells, exhibit a variable differentiation status; the majority show a more mature phenotype, but a proportion (around 20%) show a less differentiated and proliferative phenotype. Moreover, these melanocytes frequently show expres- sion of a cell survival marker. Our results suggest that a previously identified role for PAX3, as a regulator of an undifferentiated plastic state, may operate in melanocytes of normal skin. This role, possibly required for cellular response to environmental stimuli, may predispose a proportion of these cells to a successful malignant transformation as they are antiapoptotic, undifferenti- ated and proliferative. In addition, our findings that PAX3 expression is specific for both the melanocytic lineage and melanocytic lesions, could have potential applications in the clinical setting, as an immunohisto- chemical assay for the differential diagnosis of melanoma. The effectiveness of the commercially available anti-PAX3 antibodies, and optimisation of staining protocols shown in this paper, might also prove useful in a clinical setting. Materials and Methods PAX3 in migration The cell adhesion molecule MCAM has been associated with melanoma progression and metastatic potential [56–59]. Even though MCAM is frequently expressed in naevi [36] its increased Sample collection De-identified archival tissue samples were obtained from several pathology laboratories in accordance with the Human Research Ethics Committee of Edith Cowan University (ethics approval PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e9977