Functional genomic analysis of neural crest development

Human Genome Research

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Abstract

Neural crest stem cells give rise to multiple lineages including the peripheral nervous system and melanocytes. Many birth defects, diseases and cancers including melanoma are caused by defects in neural crest expressed genes. The expression of neural crest genes is controlled by a complex transcriptional regulation. One of the transcription factors needed for appropriate expression is SOX10. The target genes of SOX10 may themselves be essential for proper neural crest development. To identify potential SOX10 target genes, we have analyzed the effects of altered SOX10 expression occurring in melanoma samples on the expression of multiple genes simultaneously using cDNA expression microarrays. Successful microarray experiments require: 1) appropriate mRNA populations to compare, 2) appropriate cDNA populations on the array, 3) critical secondary screens, and 4) defined functional assays. 1) We have and are continuing to develop sets of tools to discover and analyze genes that are involved in neural crest-derived melanocyte development and disease. We used cDNA microarray cluster analysis comparing RNA from twenty cell lines from varying stages of neural crest development and melanoma. The cDNA microarray contained over 4500 human melanocyte expressed ESTs; over 2000 mouse ESTs with similarity to known genes; and control genes with important functions in NC-M development and melanoma. Analysis of the clustered expression profiles identified two blocks of cDNAs; each containing neural crest control cDNAs as well as cDNAs that have not been attributed to development or disease. We have also developed an efficient method for functional analysis of these genes by over-expression in immortalized NC-Ms in vitro and melanoblasts in vivo. This involves using a retrovirus system to direct overexpression of genes into defined lineaegs in mice and in cell culture.