PAX3 in Melanocytes Figure 5. Co-expression of PAX3 with markers of cell survival and migration in melanocytic and melanoma cells. A) Double immunofluorescent staining showing PAX3 (mouse monoclonal antibody, DSHB) and BCL2L1 co-expression in representative samples of normal skin, naevus, primary melanoma and melanoma metastasis. B) PAX3 (mouse monoclonal antibody, DSHB) and MCAM co-expression in normal skin (epidermal melanocytes), naevus, primary melanoma and melanoma metastasis. Lines in (A) and (B) demarcate the epidermal-dermal border (EDB) or epidermal surface (ES). C) In contrast to the epidermal melanocytes, some PAX3-positive melanocytes in the outer root sheath (ORS) co-express MCAM. D) Graph showing the overall number of PAX3, BCL2L1 double-labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of PAX3-positive cells that are also BCL2L1-positive, averaged across all samples. E) Graph showing the overall number of PAX3, MCAM double-labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of PAX3-positive cells that are also MCAM-positive, averaged across all samples. doi:10.1371/journal.pone.0009977.g005 PAX3 expression in melanocytes of normal adult skin: A role in regulating differentiation PAX3 and MITF are crucial regulators of melanocyte development and not surprisingly have overlapping expression. To some extent they lie on opposing sides of the differentiation pathway, PAX3 being upstream of MITF and MITF initiating the melanogenic cascade and differentiation. We were interested to see if there is an association between expression of PAX3 and MITF (indicated by their co-expression) and the stage of melanocyte differentiation (based on the cell location, mainly in the hair follicle), but found no correlation. Studies on mice and human skin both show the existence of skin stem cells in the lower permanent portion of the hair follicle, or bulge [35,38,39]. It is reported that mouse bulge melanocyte stem cells are PAX3- positive and MITF-negative [40]; however no reports confirm this to be the case in human skin. In this study we were not able to find such cells. Instead the majority of melanocytes we observed, both in the epidermis and the outer root sheath of the hair follicle, were PAX3 and MITF-positive. In contrast to this, the matrix of the hair bulb has far fewer PAX3 and MITF-positive melanocytes and the base of the hair comprises solely MITF-positive (PAX3- negative) melanocytes. Similarly, the number of PAX3, MLANA- double-labelled melanocytes in the epidermis and ORS were around 60%, but were much lower (around 36%) in the hair matrix. This would indicate that some epidermal melanocytes resemble more the undifferentiated transient amplifying cells of the growing hair rather than differentiated cells of the hair matrix. On the other side of differentiation is the transcription factor HES1, suggested to prevent premature differentiation of melano- cytes. Hes1 is reported to be a mediator of Notch signalling in skin development and in hair follicle maintenance where it regulates commitment of epidermal keratinocytes to terminal differentiation [32,41]. Hes1 is expressed in spinous layers and mediates spinous cell gene induction and thus terminal differentiation of epidermal keratinocytes [32]. In a mature hair follicle it is most prominent in the inner root sheath, in matrix cells committed to terminally differentiate to form the hair shaft [31,32]. On the other hand, in the melanocytic lineage, Notch signalling, acting through Hes1, plays a crucial role in survival of immature melanoblasts and melanocyte stem cells, by preventing initiation of apoptosis [42]. PLoS ONE | www.plosone.org 7 April 2010 | Volume 5 | Issue 4 | e9977