PAX3 in Melanocytes Figure 1. PAX3 expression in melanocytic and melanoma cells. A) Immunohistochemistry shows PAX3 expression (top panel) in representative samples of normal skin, naevus, primary melanoma and melanoma metastasis, compared to MITF expression in adjacent sections (bottom panel). B–D) Show the distribution of PAX3-positive melanocytes (arrowheads) in normal skin: along the hair follicle (early (B) and late (C) anagen); and in the epidermis (D). Arrows in (D) point to the cytoplasmic melanin deposit distinguishable from the nuclear PAX3 staining (arrowheads). Asterisk in (B) and (C) marks a dermal papilla. PAX3 was labelled with mouse monoclonal antibody (DSHB). E) PAX3 expression was analysed by RT-PCR and the graph shows the mean fold increase of PAX3 expression in naevi, primary melanomas and lymph node metastases normalised to the expression in normal skin. doi:10.1371/journal.pone.0009977.g001 PAX3 in melanocyte differentiation: Co-expression of PAX3 with MITF, HES1 and MLANA Even though the overall percentage of MITF-positive melano- cytes that were also PAX3-positive in normal skin was not significantly different in either benign or malignant melanocytic lesions (Figure 2D), the distribution of cells that co-expressed PAX3 and MITF differed between the epidermis and hair follicles of normal skin. Around 90% of MITF-positive melanocytes in the epidermis and the outer root sheath (ORS) of the hair follicle were PAX3-positive, compared to only 65% of those in the bulbar area (Figure 3D). Interestingly there was a distinct location in the hair bulb, at the very base of the hair, populated exclusively by MITF- positive melanocytes (Figure 2E). Given this difference between epidermal and follicular melano- cytes, we thought it necessary to further characterise PAX3- positive melanocytes in the normal skin. We therefore co-stained PAX3 cells with Hairy and enhancer of split 1 (HES1) (Figure 3A and 4A–C) or Melan-A (MLANA, also known as MART-1) (Figure 3B and 4D, E), markers of a less or more differentiated cell PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e9977