Gene Expression Analysis of hESC Differentiation Figure 7. Pathway analysis of enriched transcripts in hESC (light blue) and differentiated cells (dark blue). Enriched transcripts searched against KEGG pathway database for finding the members involved in the signaling pathways, mostly transcribed members in each stage (up to 60%) in undifferentiated or neural differentiated cells compared and depicted in the figure. Common active pathways in both stages have not been shown. doi:10.1371/journal.pone.0022856.g007 expression of WNT1 and most WNT receptors decreased, although the expression of WNT4, WNT7A, WNT7B and FZD9 increased with neural differentiation (Table S4). Neural progenitors (NPs) have some overlap with ESCs in the expression patterns of WNT pathway molecules. The DKK1 gene, an inhibitor of WNT signaling, was suppressed during neurogenesis. However, another inhibitor, DKK3, had an increased expression level, which may explain how different members of the WNT gene family may control differentiation of different cell types. Metabolism associated genes are found in both ESCs and differentiated cells. Genes involved in the pentose phosphate pathway, galactose metabolism, ascorbate and aldarate metabo- lism and fatty acid elongation and metabolism have increased expression levels in stem cells. The beta-1,4-galactosyltransferase (B4GALT1) protein is an enzyme that participates in glycocon- jugation, lactose biosynthesis, and galactose metabolism in liver cells. This enzyme is targeted to the plasma membrane of intestinal cells and absorbs galactose from the lumen; it may also function in adhesion [52]. Previous studies have shown that the B4GALT1 promoter has a binding site for E1AF, and induction of B4GALT1 expression by E1AF can lead to lung cancer [53]. Genes in the glycosphingolipid biosynthetic pathway are overrepresented in neural cells compared with ESCs. Glyco- sphingolipids (GSLs) are a group of bioactive glycolipids that includes cerebrosides, globosides, and gangliosides and are involved in cell adhesion, modulation of growth factor/hormone receptors, antigen recognition, and protein trafficking. GSLs are more active in neural tissues compared to hESCs, and their dysfunction may lead to the accumulation of GSLs and lysosomal storage diseases (LSDs) [54]. NOTCH signaling in neural system development has multiple functions. Not only can NOTCH switch a neural cell fate decision [55], but NOTCH signaling also plays an important role in the maintenance of neural stem cells [56]. NOTCH molecules are also needed for cell fate determination in hESCs as they differentiate into the three germ layers [57]. In this study expressed transcripts in this pathway were delta-like ligands (DLL1, DLL3), jagged (JAG2) and the receptor, NOTCH1. Activation of NOTCH1 can activate the transcription of HES5 and determine neural fate. Regulatory network of NPs at the NE stage We established an interaction network of modulated genes at the NE stage using the STRING database. The full regulatory interaction network was composed of 96 nodes that were up- or down regulated more than 1.5-fold in rosette cells compared with hESCs. The data are presented in Figure S3. We then constructed a subnetwork of 44 nodes of NE-enriched genes (Fig. 8). Our results highlight the role of the TGFß family in the proliferation of neural progenitors. Genes in the TGFß superfamily are major players in the differentiation and maintenance of NPs. ID1 is a key gene in the proliferation of NPs [58] and is under the control of LEFTY2, another helix-loop-helix transcription regulator. The genes ID3 and ID2 are also activated by LEFTY2 and are important for the survival, proliferation [59] and specification [60] of neural crest progenitor cells. It seems that the LEFTY 2 gene is involved in NP proliferation (Fig. 8). MSX1 can activate BMP4, PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e22856