Taken together we here show that the

Taken together, we here show that the recently identified marker SPOT14 labels low-proliferating NSPCs in the hippocampus that react dynamically to positive and negative neurogenic stimuli in vivo. These data further validate the usefulness of this restrictive and functionally relevant marker to analyze the behavior of NSPCs to various extrinsic stimuli. The availability of functional SPOT14 antibodies, a SPOT14 reporter line, as well as an inducible SPOT14-CreERT2 line (Knobloch et al., 2013) extend the current toolbox to study adult neurogenesis and will allow further dissection and better understanding of the complex regulation of NSPCs within the adult brain.
Experimental Procedures
Author Contributions
Acknowledgments
Introduction Human embryonic stem cells (hESCs) originate from the jak inhibitor inner cell mass (Thomson et al., 1998), which is in a hypoxic microenvironment estimated at 1.5%–5.3% O2 in the mammalian reproductive tract (Dunwoodie, 2009; Mohyeldin et al., 2010; Simon and Keith, 2008). hESCs grown in physiological O2 (∼5% or less O2) self-renew with reduced levels of spontaneous differentiation compared with hESCs grown in atmospheric O2 (∼21% O2) (Ezashi et al., 2005; Westfall et al., 2008). hESCs isolated and passed exclusively in physiological O2 contain two active X chromosomes (XaXa), marking a less differentiated state than that in atmospheric O2, which typically contains one inactive X chromosome (Lengner et al., 2010). Physiological O2 also improves the efficiency of defined factor-induced cellular reprogramming to a pluripotent-like state (Yoshida et al., 2009). Combined, these studies show the importance of physiological O2 in supporting stem cell self-renewal and in suppressing spontaneous, usually unwanted hESC differentiation. Studies on the role of O2 tension in promoting pluripotency have indicated hypoxia-inducible factor 2α (HIF2α) (also called endothelial PAS domain protein 1) and HIF3α in the transcriptional upregulation of OCT4 in hESCs (Forristal et al., 2010). These findings are also consistent with the role of HIF2α in transactivating Oct4 expression in mouse ESCs (Covello et al., 2006). Since the activation of HIF pathway appears to favor self-renewal, it might be expected that HIF activity would also inhibit purposeful hESC differentiation. Four studies have examined the effects of hypoxia on early hESC differentiation, but none has specifically examined the role of HIF. In these studies, hESCs in physiological O2 showed enhanced embryoid body (EB) formation or endothelial and cardiomyocyte differentiation (Ezashi et al., 2005; Lim et al., 2011; Ng et al., 2010; Prado-Lopez et al., 2010). However, physiological O2 induces pleiotropic cellular and molecular effects, and the underlying cause(s) for paradoxically enhanced EB or lineage formation in physiological O2 is unclear. For example, the O2 concentration is known to affect (1) oxidative stress and hESC growth (Ezashi et al., 2005); (2) the activity of O2-dependent enzymes, such as Jumonji domain-containing dioxygenases (Xia et al., 2009), which have broad roles in the epigenetic regulation of gene expression; (3) multiple O2-sensing signal transduction pathways, including the mechanistic target of rapamycin (mTOR) pathway (Wouters and Koritzinsky, 2008) and the unfolded protein response-activated endoplasmic reticulum stress pathway (Wouters and Koritzinsky, 2008); and (4) the HIF-controlled gene transcription network (Lendahl et al., 2009). Therefore, it remains unclear whether the enhancement of EB or lineage specific differentiation in physiological O2 occurs mainly through HIF transactivation or other molecular mechanisms. HIFs are major regulators of the cellular response to O2 tension (Denko, 2008; Lendahl et al., 2009; Majmundar et al., 2010). HIFs form a heterodimer composed of HIFα and HIF1β (also called aryl hydrocarbon receptor nuclear translocator) to transactivate hypoxia-responsive genes. They are regulated at the level of α-subunit protein stability in an O2-dependent manner. When oxygen is abundant, HIFα subunits are hydroxylated by prolyl hydroxylase domain (PHD) proteins (in the presence of Fe2+) and recognized by an E3 ubiquitin ligase, VHL (Von Hippel-Lindau), leading to degradation in the proteasome. In hypoxic conditions, decreased O2 diminishes enzymatic activity of PHDs. As a result, HIF1α and HIF2α proteins are stabilized and dimerize with HIF1β in the nucleus to transactivate specific target genes. In Hif1α, Hif2α, and Hif1β knockout mice, deficient HIF activity impaired placental development (Adelman et al., 2000; Cowden Dahl et al., 2005; Kozak et al., 1997), heart development (Krishnan et al., 2008; Licht et al., 2006), and endochondrial bone formation during early embryogenesis (Amarilio et al., 2007; Dunwoodie, 2009; Provot et al., 2007). Furthermore, conditional knockout mice of Hif1α in the central nervous system exhibit hydrocephalus accompanied by a reduction in neural cells and an impairment of spatial memory (Tomita et al., 2003). Those studies demonstrated the importance of Hifs in normal brain development. It is clear from the vast majority of studies on human pluripotent cell differentiation that typical protocols generate cells more akin to those found during the earliest stages of tissue formation, prior to significant tissue vascularization. What is less understood is whether methods to culture cells that more accurately replicate in vivo conditions can affect the developmental potential of pluripotent progeny.