(A) Schematic representation of tissues and cell lines used for microarray hybridization experiments and Northern analysis. Embryos, tissue types, and cell lines in the rectangle were used in this study. (B) Scatter-plot analysis of ES versus TS6.5 cells microarray experiment. Average expression levels (arbitrary units) of each gene were calculated from three independent hybridizations. Genes that show significantly different expression levels between ES and TS6.5 cells at the 5% significance level (P < 0.05) with expression levels above the background are displayed as colored spots, and the other genes are displayed as dark blue spots. For genes that are expressed higher in ES cells than in TS6.5 cells, blue indicates genes expressed greater than 10-fold, green indicates those expressed between two- and 10-fold, and yellow indicates those expressed less than twofold. For genes that are expressed higher in TS6.5 cells than in ES cells, red indicates genes expressed greater than 10-fold, pink indicates those expressed between two- and 10-fold, and orange indicates those expressed less than twofold. Several examples of genes are indicated. (C) k-means cluster-analysis of differentially expressed genes among ES, TS3.5, TS6.5, and MEF at a 5% significance level (P < 0.05). By using the k-means algorithm, 2,969 genes were grouped into 15 distinct clusters based on their similarities of expression patterns among ES, TS3.5, TS6.5, and MEF. The name of individual clusters is indicated, followed in parentheses with the number of genes in each cluster. Only statistically significant, sequence-validated genes are represented here. (D) Functional classifications of ES-, TS-, MEF-, and stem cell-specific genes. The number of genes identified as specific to the cell type is indicated within parentheses. Annotated genes were classified by their function (Kargul et al. 2001), and are indicated on the right with its color co-ordinate. The unknown category includes sequences that are in the database but have yet to be characterized, as well as completely novel sequences.
