RA plays an instrumental role in neuronal differentiation
RA plays an instrumental role in neuronal differentiation and neuronal patterning as well as motor axon generation. In addition, RA is required for the maintenance of mature adult neurons (reviewed in Ferri et al., 2007). Various concentrations of RA (from 1 to 10μM) have been used to induce ESCs into neuronal progenitor buy 3X FLAG tag Peptide or to specific neuronal cell populations that represent early or late stages of differentiation (reviewed in Baharvand et al., 2007; Bel-Vialar et al., 2002; Dhara and Stice, 2008; Pankratz et al., 2007). However, the concentration of RA and the time of addition may determine the commitment of cells to specific differentiation programs. The current study reveals the differential regulation of 102 neuronal differentiation-related markers as a consequence of RA treatment (Table 1, GO:0030182~neuron differentiation). RA and FGF-2 treatment downregulates early neuronal markers such as NESTIN, SOX1, and PAX6 and, in contrast, PAX6 shows an upregulation in the presence of FGF-2. A previous study showed that early neuroectodermal cells express PAX6 but not SOX1, and these cells were posteriorized with RA and sonic hedgehog, which resulted in the development of spinal motor neurons (Li et al., 2005). In another study, it has been shown that hESC-derived neuroectodermal cells express the anterior transcription factor OTX2 but not HOXB4 without RA treatment (Hu et al., 2010). The addition of RA after days 10 to 24 of glial differentiation produces OTX2-negative and HOXB8-positive cells (Hu et al., 2010). The early addition of RA in the present study caused a negative expression of OTX2 (fore-mid brain) and LHX2 (forebrain) (Supplementary Fig. 3A) (Wigle and Eisenstat, 2008). By contrast, we observed over-expression of axon guidance molecules such as NTRK2, SLITRK6, ROBO2 and SEMA5A in the presence of FGF-2 (Supplementary Fig. 3B) (Dickson, 2002). Although the expression of these molecules commits cells to neuronal patterning, we did not find any specific neuronal pattern markers (neither early nor late); these markers may be sensitive to the concentration and/or time of addition of RA. Gene markers such as BMP4, DCN, PITX2, and TGFb2 are well-documented members of TGF-β signaling pathways and were upregulated upon RA treatment. We determined that genes involved in TGF-β signaling were upregulated following RA and FGF-2 treatment. SMAD1/5 are known to be vital for germ cell lineage commitment, and SMAD1 is necessary in inducing the growth of the ExE lineage (Hayashi et al., 2002; Tremblay et al., 2001). In this context, SMAD2/3 activation is associated with the pluripotency of hESCs, while SMAD1/5 activation is associated with differentiation (James et al., 2005). SMAD4 activation and phosphorylation of SMAD1/5 results in their translocation to the nucleus, where they collectively regulate target gene expression with other transcription factors (Shi and Massague, 2003). Receptor activation by BMPs leads to the phosphorylation of Smad1/5, which is translocated to the nucleus after forming a complex with Smad4. In condition I (with FGF-2), we found that Smad1/5 was not phosphorylated when cells were only treated with FGF-2, and a high phosphorylation was observed in the absence of FGF-2. This lack of phosphorylation in the presence of FGF-2 is due to the maintenance of the pluripotent state in hESCs. In condition II (with RA), an elevation of the phosphorylation (serine/threonine) was observed at 6h of RA and FGF-2 treatment. This result suggests that RA modulates TGF-β signaling in the presence of FGF-2. A number of transcripts involved in WNT signaling, such as WNT4, FZD, WIF1 and DKK1, were downregulated upon RA treatment. WNT activation plays a crucial role in directing cell fates during embryogenesis, and this signaling also maintains pluripotency via FGF-2 stimulation and phosphorylation of GSK3β followed by nuclear translocation of β-catenin. Immunocytochemistry analysis showed that β-catenin localizes to the nucleus in the presence of FGF-2 and thereby activates WNT target genes to maintain pluripotency. By contrast, RA treatment coupled with the absence of FGF-2 causes β-catenin to relocate to the plasma membrane, which demonstrates that RA overrides the effect of FGF-2 by potentially inhibiting WNT signaling and directing cells toward ExE differentiation.