known genes in the neurogenesis process; including Six6, FGF3, POU3F1, NPAS1, CYP26A1 and FGF8 (Fig. 6D). The higher expression levels of genes that modulate post mitotic neuron maintenance, such as FBXO2, may be required for the regionalization of neurons [28]. Several genes that were up regulated in NI are among a set of genes that are already known to be highly expressed in the developing CNS, including the Six6 gene, which is expressed abundantly in the brain, cerebellum and specific precursors of neural retina cells [29], and the FGF3 gene, which is expressed in the hindbrain and whose expression is required for hindbrain patterning [30]. POU3F1 also known as OCT6 is a member of the pou domain family of proteins and is involved in neural ectoderm formation; its expression is down regulated upon ESC differentiation and increases again during brain development [31]. The protein encoded by the NPAS1 gene is a member of the basic helix-loop-helix (bHLH)-PAS family of transcription factors and is specifically expressed in neural tissue [32]. NPAS1 in mice modulates the transcription of erythropoietin by binding to its enhancer region in vivo; thus, it indirectly controls oxygen- responsive elements during late embryogenesis and central nervous system development [33]. CYP26A1 plays a key role in retinoic acid (RA) metabolism. Many isoforms of this gene and other subunits of the P450 cytochrome are overrepresented at the NI stage, highlighting the importance of the retinoic acid (RA) metabolic pathway in the neural initiation stage [34]. Another gene, CPZ, modulates the WNT signaling pathway by cleaving some undefined protein or by binding to the WNT molecule [35]. FGF8 is also overexpressed in NI, and its expression is downregulated as differentiation progresses (Fig. 6). FGF8 is a paracrine factor that appears to have a function during dopaminergic neuron specification and proliferation [36]; it works cooperatively with SHH in the specification of midbrain neurons. Neural rosette cells comprise neural progenitors from the neural crest and CNS neurons. Neural crest progenitors express S100A11, MSX1, TFAP2A, TFAP2B and ERBB3 abundantly in the rosette stage (Fig. 6B). During the early development of neural cells, the MEIS1 and MEIS2 homeobox genes can positively control PAX6 transcription and induce hESCs toward neuralization [37]. The extrinsic factor RA may trigger neural specific genes and induce the neural fate [38], and RA may also affect anterior-posterior pattern formation by inhibiting BMP signaling with Noggin, which induces the formation of neural ectoderm. Shh protein can induce Foxa2 and ventralize neural progenitors and, in a positive regulatory loop, FOXA2 can induce endogenous SHH and inhibit NKX2.2 [39] and also the serotonergic phenotype. Endogenous transcription of FGF8 resulting from RA exposure [40] can induce WNT1 expression that cooperatively with FGF8 can induce neural progenitors to differentiate into TH-producing cells. A major group of differentially expressed genes includes transcripts that were down-regulated during differentiation (Fig. 5). This group includes genes involved in the cell cycle, mitosis and metabolism as well as genes involved in the development of other germ layer clusters (Fig. 6E). Several major ESC markers, including OCT4, NANOG, PRDM14, and GAL, were grouped in this cluster (Fig. 6E). Because OTX2, a gene involved in forebrain-hindbrain patterning, was also down- regulated during differentiation, the results suggest that poster- iorized neurons were generated during differentiation in this study [41]. Matrix associated genes, including MMP1, THBS1, and ITGB1BP3, were also among the hESC enriched genes, suggesting that expression of these genes provides an environment conducive to the proliferation of stem cells. DNA methyltransfer- Gene Expression Analysis of hESC Differentiation ase genes and noncoding genes homologous to OCT4 control the epigenetic state of hESCs and are important classes of genes for stem cell maintenance. Some antagonists of FGF signaling, such as SPRY1, were also overrepresented in hESCs. SPRY1 is involved in cortical neuron pattern formation and inhibits caudal cell fates [42]; its role in hESCs with a high concentration of FGF is not clear and may be important for the fine tuning of FGF signaling in ESCs (Fig. 6E). Overall, our study found that the transcriptome is extremely complex and dynamic during early neural differentiation of hESCs. All transcripts that were modulated during differentiation were placed in upregulated and downregulated clusters and were analyzed using the KEGG pathway database and ClueGO software. With a two-sided hypergeometric test, genes were assigned to pathways. The pathway information for each cluster was analyzed, and pathways represented in each cluster were identified and compared. Our results showed that some transcripts that were involved in metabolic pathways were downregulated during differentiation. Pentose and glucuronate interconversions, fatty acid turnover (for membrane biogenesis), DNA replication, mismatch repair, recombination and immune response pathways were hyper expressed in hESCs, but not in neurons (Fig. 7, Table S4). Nucleotide metabolism and cell cycle pathway members were also highly expressed in hESCs. Genes involved in nitrogen metabolism, apoptosis, NOTCH signaling, axon guidance, neurotrophin signaling, Parkinson’s disease and prion disease are highly abundant molecular pathways that were overrepresented in differentiated neural cells (Fig. 7, Table S4). In ESCs, mTOR signaling can stabilize OCT4, SOX2 and NANOG expression and can negatively control the induction of endoderm and mesoderm from ESCs. Inhibition of mTOR with rapamycin enhanced the expression of endoderm and mesoderm markers in treated EB and impaired the pluripotency of hESCs, but this effect was not observed in neural differentiation [43]. According to our results, mTOR signaling functions in neural induction. Transcripts associated with mTOR (S6K1) were upregulated during neuro- genesis (Table S4). NOTCH signaling is also active in hESC- derived neural progenitors and has an important function in the proliferation and differentiation of NPCs. Inhibition of NOTCH can disrupt the maintenance of stem cell characteristics of NPCs [44]; [45], by suppressing the HES1 and HES5 genes, which negatively control the expression of the proneural genes MASH1 and NGN1 [46,47,48]. The NOTCH signaling members NOTCH1, DTX1, DTX3, DLL1, DLL3, HES5 and JAG2 were expressed more strongly at the NE stage compared to hESCs, and this stronger expression continued as the cells differentiated into final differentiated neurons. As shown previously, NOTCH signaling negatively controls neurogenesis in a stepwise process; in the first step, its activation leads to gliogenesis as opposed to neurogenesis, and in the second step, its activation promotes the production of astrocytes and inhibits the production of oligoden- drocytes and neural fates [48]. Another pathway that confers one of the characteristics of NPs is the Toll like receptor (TLR) pathway. The TLR pathway is involved in the development of CNS and the innate immune system and functions in cell proliferation and NPC differentiation [49,50]. The TLR pathway has an immunomodulatory effect on NPCs via the activation of TNF-alpha [51]. It seems that members of the TLR pathway (AKT1, CD14, FOS, IFNAR1, IL8, JUN, MAPK10, MAPK11, MAPK3, PIK3R1, PIK3R2, RELA, SPP1) are more highly expressed in NEs and DNs (Table S4). WNT signaling, MAPK signaling, Jak-STAT signaling, Hedgehog signaling, and TGF-beta signaling are active pathways in NEs and DNs, but these pathways do not share common proteins. The PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22856