Gene expression profiling in a model of human neuronal differentiation using oligonucleotide arrays

Abstract

In an effort to understand the process of human neuronal differentiation, we have monitored gene expression in a cell culture model, NTERA2/D1 (NT2). This pluripotent human teratinocarcinoma cell line was induced to differentiate with retinoic acid (RA) over a 21-day period. We monitored gene expression at four time points using a custom-fabricated oligonucleotide GenechipÒ. A panel of human genes that were expected to participate in the process of neurogenesis or act as differentiation markers were chosen and FASTA analysis determined regions of unique sequence. Oligonucleotide selection, masking and fabrication of GenechipsÒ were performed at Affymetrix. Arrays contained 50 probe pairs per gene and 589 genes per chip, as well as controls (probe pairs for housekeeping genes and spiked samples). We analysed data using GenechipÒ software and performed pairwise comparison between untreated NT2 at 0 days and RA-treated NT2 at 3, 7, 14 and 21 days. Increased and decreased expression of select genes was seen throughout the time course. The largest differences observed were in the order of a 50-fold difference from control. As expected, expression of the intermediate filament neuroepithelial marker nestin increased rapidly following RAtreatment and decreased toward the end of the time course. This regulation likely reflects the appearance and differentiation of neuronal precursors in the cultures. These data suggest that the experimental design and analysis techniques were valid. Expression of Hox genes A1, A4, B2, B3, B5 and C5 were greatly altered throughout the time course, as were expression of helix-loop-helix domain-containing genes important to the process of neuronal specification. The ability to study the coordinate regulation of such genes with oligonucleotide arrays may enable signal transduction pathways associated with human neurogenesis and differentiation to be deciphered.