The present study indicated that induction of glutamatergic

The present study indicated that induction of glutamatergic markers by nucleotides involved the P2Y4-Gq signaling axis. Expression of the P2Y4 nucleotide receptor was transiently augmented in the course of neuronal differentiation of ESCs, which is coincident with the window of nucleotide-induced vGluT expression. P2Y4 expression was observed in type 2 NSCs of the adult mouse hippocampus, mainly in type 2b. In vitro experiments indicated that activation of the P2Y4 expressed in the type 2 NSCs was sufficient for induction of glutamatergic markers in their descendant neurons. Since the decision regarding neuronal cell fate is thought to occur in the early stage of type 2 NSCs (Kempermann et al., 2004), the differentiation stage sensitive to the nucleotide-induced glutamatergic subtype selection appears to follow the decision of the neuronal lineage, but precedes terminal differentiation of neurons. Thus far, some in vitro methods for induction of glutamatergic neuron subtype have been reported (Reyes et al., 2008; Gaspard et al., 2008; Shi et al., 2012); however, it remains uncertain which stage of differentiating NSCs is sensitive to these induction methods. The present study indicated a differentiation stage in which expression of neuronal subtype markers can be affected by extracellular nucleotides.
Experimental Procedures
Author Contributions
Acknowledgments This work was supported by the Grant-In Aid for Scientific Research (C) (JSPS KAKENHI number 23500392) from the Japan Society for the Promotion of Science, and by the Seamless Technology Transfer Program through Target-Driven R&D (AS232Z00373F) of the Japan Science and Technology Agency. This work was also partially supported by the Shimizu Foundation for Immunology and Neuroscience.
Introduction Zinc finger and SCAN domain containing 4 (ZSCAN4) is a DNA-binding protein that is specifically expressed in two-cell stage embryos during mouse development (Falco et al., 2007). In vitro, interestingly, Zscan4 is transiently expressed in a minor population of embryonic stem colorimetric (ESCs) at one time (Carter et al., 2008) but is eventually expressed in all (Zalzman et al., 2010). It functions for telomere elongation and genomic stability (Zalzman et al., 2010) and thus is considered as a rejuvenation factor. ESCs are a heterogeneous population. If cultured in conventional serum-containing medium supplemented with leukemia inhibitory factor (LIF), they remain undifferentiated but closer studies show they are actually a mixture of cells with higher and lower potential of differentiation (reviewed in Nakai-Futatsugi and Niwa, 2013). Recently even a minor population of two-cell-stage-like ESCs that are not only pluripotent but also capable of differentiating into extra-embryonic lineages was found in the heterogeneous ESC population (Macfarlan et al., 2012). The heterogeneity of ESCs is accompanied by fluctuation of the expression of pluripotency-associated genes such as Rex1 (also known as Zfp42) (Toyooka et al., 2008), Nanog (Chambers et al., 2007; Singh et al., 2007), Klf4 (Niwa et al., 2009), Tbx3 (Niwa et al., 2009), Stella (Hayashi et al., 2008), and so on. However, among the pluripotency-associated genes, Oct3/4 (also known as Pou5f1), whose expression does not fluctuate, is an exception. It is the master gene of pluripotency (Nichols et al., 1998). A constant expression level of Oct3/4 is crucial for the maintenance of pluripotency, as a slight increase leads to differentiation into primitive endoderm and mesoderm while a slight decrease leads to differentiation into trophoectoderm (Niwa et al., 2000). The expression level of Oct3/4 is maintained at a constant level downstream of a robust transcription factor network in mouse ESCs (Niwa et al., 2009). Rex1, although not essential for ESC self-renewal and pluripotency (Masui et al., 2008), decreases in its mRNA-expression level when the master gene Oct3/4 is either hyper-expressed or hypo-expressed (Niwa et al., 2000). Thus we consider the promoter activity of Rex1, which is high only when the expression of Oct3/4 is maintained at an optimal range, as a good indicator of pluripotency.