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  • To define the transcriptome of

    2018-11-12

    To define the transcriptome of the ExE lineage in the present study, we analyzed primitive endoderm and trophectoderm markers using qRT-PCR. Interestingly, we found that markers of the primitive endoderm, such as DDAH2, LAMB1, DAB2, AFP and NR2F1 (COUP-TF1), and markers of the trophectoderm, such as GATA3, MSX1 and KRT7, were upregulated upon RA treatment. The result of our study corroborates previous investigation reporting that over-expression of NR2F1 in murine embryonic stem order A-366 leads to RA-induced ExE endoderm gene expression (Zhuang and Gudas, 2008). The DAB2 and GATA factors play a major role in the primitive endoderm during early embryogenesis. Through studies with knockout mice, it was established that GATA4 and GATA6 are required for ExE lineage formation (Fujikura et al., 2002). DAB2 is expressed in the ExE visceral endoderm during early mouse development and is required for growth of the inner cell mass (Morris et al., 2002; Yang et al., 2002). A selective expression of GATA3 is observed in the trophectoderm and is also responsible for trophoblast development (Home et al., 2009; Ralston et al., 2010b). To further characterize the ExE lineage, we analyzed the amount of placental hormone hCG produced by hESCs treated with RA in the presence or absence of exogenous FGF-2. An elevation of hCG production was observed in a time dependent manner during ExE differentiation. The ExE lineage develops into multiple placental cell types. Trophoblast cell lineage differentiation and development is restricted to ExE tissues. In this context, epigenetic modifications are involved in normal trophoblast differentiation and ExE tissue functions (Hemberger, 2010). The transcription factor GATA3 is capable of the induction of trophoblast differentiation in parallel to CDX2 (Ralston et al., 2010). In addition, the expression of the DDAH2 and EOMES genes is required for the establishment of trophoblast lineage (Ayling et al., 2006; Russ et al., 2000; Tanaka et al., 1998). The hypermethylation of DDAH2 has been previously reported in trophoblast stem cells (Tomikawa et al., 2006) and is highly consistent with our observed hypermothylation of TDGF1 and GATA3 and, to a lesser extent, EOMES. The microarray expression patterns of these genes correspond to the methylation status, with no expression of DDAH2, 42.8-fold downregulation of TDGF1, 7.5-fold upregulation of GATA3 and 1.2-fold downregulation of EOMES. The growth factor TDGF1 is required for the anterior–posterior axis positioning and is restricted to the ExE region (Ding et al., 1998; Rodriguez et al., 2005). The expression of KRT7, GATA3 and TFAP2α was assessed order A-366 with immunocytochemistry and western blotting. ExE markers appear to be expressed prominently during RA treatment, and the presence of FGF-2 did not affect their expression. In conclusion, our study shows that treatment with RA in the presence of FGF-2 induces the phosphorylation of SMAD1/5 and facilitates the translocation of β-catenin to the nucleus, thus further affecting the downstream targets of the WNT, BMP and TGF-β signaling pathways. TGF-β signaling is associated with trophectoderm differentiation, and stimulation of BMP4 leads to the activation of GATA4 and GATA6 and consequently to GATA3 mediated trophoblast differentiation (Fig. 5C). We propose that RA influences cell fate changes by modulating the WNT, BMP and TGF-β signaling pathways during embryogenesis and directs ExE differentiation in hESCs. The findings from this study reveal the dynamics of gene regulation through RA-driven ExE lineage formation.
    Author disclosure statement
    Acknowledgments This work was supported by the “Embryonic Stem Cell-based Novel Alternative Testing Strategies” (ESNATS) EU project (grant agreement no.: FP7 — 201619). In addition, we thank Mrs. Böttinger for her technical assistance.
    Introduction Radial glia cells play important roles in neuronal migration and neurogenesis during development (Kriegstein and Alvarez-Buylla, 2009; Malatesta et al., 2003; Noctor et al., 2001). These cells have their cell bodies in the ventricular zone, and radial processes extending to the pial surface. By the end of the developmental period, most of the radial glia cells differentiate into astrocytes (Alves et al., 2002; Levitt and Rakic, 1980; Mission et al., 1991; Voigt, 1989). During development, radial glia cells correspond to the neural stem cells (NSCs), since they are able to proliferate, self-renew, and also differentiate into neurons, astrocytes and oligodendrocytes (Anthony et al., 2004; Costa et al., 2010; Merkle et al., 2004; Noctor et al., 2001, 2002; Spassky et al., 2005). It has also been shown that radial glia cells originate resident NSCs in the subventricular zone (SVZ) in the adult brain (Merkle et al., 2004). There are two regions in the mammalian brain where neurogenesis persists during adulthood: the SVZ, around the lateral ventricles (LV); and the subgranular layer (SGL) of the hippocampal dentate gyrus (DG) (Gage, 2000; Gage et al., 1998; Kaplan and Bell, 1984; Kriegstein and Alvarez-Buylla, 2009; Lois and Alvarez-Buylla, 1993).