Many diverse cell types differentiate from the mammalian neural crest during embryogenesis. Two such cell types are the cells responsible for pigmentation, the melanocytes, and the mechanoreceptive Merkel cells. Both cell types reside in the basal layer of the epidermis of the skin and are associated with particularly aggressive forms of cancer, namely melanoma and Merkel cell carcinoma. The differentiation of any cell type is a tightly regulated process, requiring the correct temporal co-ordination of extracellular ligands and transcriptional regulators. In some instances, dysregulation of these control mechanisms in differentiated cell populations can contribute to oncogenesis. This thesis has been concerned with an analysis of transcription factors implicated in the proper development of these two epidermal cell populations, and has related the findings to cancer cell lines derived from these malignancies.
In vitro culture systems are useful model systems for molecular analysis of both differentiation and malignancy. Here, the establishment of unpigmented human melanoblast cell strains from neonatal epidermis using a low calcium medium supplemented with Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2 is described. By comparison with cultures of human melanocytes, the ability of these cells to differentiate in vitro upon growth factor removal to pigmented melanocytes was demonstrated by induction of DOPA reactivity, melanosome maturation and pigmented cell pellets after harvesting. These findings are consistent with those reported by other laboratories for the differentiation of cultured murine melanoblasts. Immunoblot and immunohistochemical analysis of the expression of the melanogenic enzymes tyrosinase and tyrosinase related protein-1 suggest that acquisition of the pigmented phenotype by human melanoblasts in vitro was due to a maturation of these proteins, rather than de novo synthesis.
Having established an in vitro system to study melanocytic differentiation, the expression, localisation and/or DNA binding activity of a number of transcription factors that are implicated in melanocyte development was examined. Thus, it was demonstrated that the POU domain factor BRN2 and the SOX family member SOX 10 are highly expressed in melanoblasts, but are down-regulated upon differentiation. In contrast, expression of MITF, which directly regulates several pigmentation-associated genes, and the neural crest developmental regulator PAX3, remain relatively constant during the melanoblast to melanocyte transition. Furthermore, evidence for expression of an additional SOX family protein(s) in this cell lineage was found.
Culture of melanocytes in melanoblast growth medium induced both the expression and DNA binding activity of BRN2. Additional experiments demonstrated that synergism between any two of Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2 increased BRN2 expression and DNA binding activity in cultured melanocytes. Furthermore, as growth factor mediated BRN2 induction precedes that of SOX 10, and BRN2 ablated melanoma cell lines lack SOX 10 expression, BRN2 may regulate the expression and/or DNA binding activity of SOX 10 in melanocytic cells. Due to the highly conserved manner in which POU and SOX proteins interact to co-regulate transcription, and the similar expression profiles of BRN2 and SOX 10 during in vitro melanoblast differentiation, it appears likely that the combinatorial action of these proteins regulates a pivotal step in melanocytic development.
To date, no method for the long-term in vitro propagation of Merkel cells has been described, preventing a detailed molecular analysis of this cell. However, cell lines established from Merkel cell carcinoma biopsies have been classified into two groups; Classic lines that retain several neuroendocrine markers of differentiated Merkel cells, and Variant cell lines, thought to represent a more undifferentiated phenotype and arise from more aggressive tumours with poorer clinical outcomes. Using these cell lines, expression of the POU domain factor BRN3c and the bHLH transcription factor HATH1 was examined. Both of these factors are required for development of mechanosensory cells of the inner ear, and furthermore, a requirement for MATH1 in murine Merkel cell development has been demonstrated. BRN3c and HATH1 correlated with the differentiated phenotype of Classic cells lines, whereas expression of these factors was not detected in Variant cell lines. Co-immunofluorescent staining of human neonatal epidermal sheets using chromogranin immunoreactivity to identify Merkel cells demonstrated that BRN3c and HATH1 was expressed by these cells. Thus, BRN3c and HATH1 are candidates for regulation and/or acquisition of the Merkel cells neuroendocrine phenotype in vivo.