br Discussion We have described an approach

Discussion We have described an approach that achieves inducible, retrovirus-mediated gene expression by using the coding sequence of TFs fused to an ERT2 motif. Fusion of the ERT2 motif to two TFs that regulate fate-choice decisions in the course of adult neurogenesis (i.e., ASCL1 and NEUROD1) allowed tight temporal control of functional gene expression in cultured NSPCs as well as within a neurogenic niche of the adult brain. The approach described here extends the range of applications for retrovirus-mediated gene manipulation. This highly selective and extremely fast strategy to test gene function in the context of mammalian neurogenesis can be combined with an ERT2-mediated system for temporal control of gene expression, allowing one to switch gene expression on (and off) with the use of TAM. Thus, with this simple two-step cloning protocol, it is now possible to test the function of genes in distinct stages or for different durations during neural development both in vitro and in vivo. Clearly, the approach described here is not limited to studying genes in the context of adult neurogenesis. A large number of experiments aiming to manipulate TFs will benefit from an ERT2-based temporal control of gene expression (in retroviral or plasmid DNA format) in both adult and embryonic tissues (e.g., using in utero electroporation of plasmids encoding for TF-ERT2-fusion proteins). In addition, the approach described here will be useful for studying the temporal requirements of selected TFs in the context of cellular reprogramming of somatic p-Cresyl sulfate toward distinct lineages or pluripotency (Takahashi and Yamanaka, 2006; Vierbuchen et al., 2010).
Experimental Procedures
Acknowledgments
Introduction The relationship between cell-cycle control and regulation of differentiation is a major question in stem cell biology. Neural stem cells (NSCs) are among the best characterized mammalian stem cells; they generate the central nervous system during development and support adult neurogenesis throughout life in the subventricular zone (SVZ) and subgranular layer of the hippocampus (Bonfanti and Peretto, 2007; Doetsch, 2003). NSCs were the first somatic stem cell type shown to grow indefinitely in vitro under self-renewing conditions as neurospheres (Reynolds and Weiss, 1992). NSC cultures can be derived ex vivo from both the developing and adult brain or from embryonic stem (ES) cells and can differentiate into the three brain lineages: neurons, astrocytes, and oligodendrocytes (Conti et al., 2005; Pollard et al., 2006). This differentiation is governed by extracellular ligands and cytokines (Gangemi et al., 2004) and is associated with the downregulation of NSC markers such as Nestin, SOX2, and PAX6 (Conti et al., 2005; Gómez-López et al., 2011). Self-renewing cells with gene expression patterns similar to normal NSCs can also be found in glioblastoma multiforme (GBM), supporting the concept of cancer stem cells (Nicolis, 2007). We recently showed that the canonical DNA damage response (DDR) signaling pathways (Figure S1A available online) are functional in NSCs (Schneider et al., 2012). Generation of DNA double-strand breaks (DSBs), e.g., by ionizing radiation, leads to activation and focal recruitment of the apical PI3K-like serine/threonine kinase (ATM), which labels chromatin at DNA lesions through phosphorylation of the histone H2A variant H2AX (γH2AX). ATM also phosphorylates the serine/threonine-glutamine (S/TQ) motif of many downstream effectors, some of which are focally recruited at DSBs (e.g., 53BP1), whereas kinases and transcription factors like CHK2 and p53 further relay DDR signaling, causing transient cell-cycle arrest to allow DNA repair or, depending on the nature of the DNA damage, apoptosis or cellular senescence (d’Adda di Fagagna, 2008; Jackson and Bartek, 2009; Shiloh, 2006).
Results
Discussion Terminal differentiation of stem and progenitor cells is defined by an irreversible cell-cycle arrest, loss of expression of stem/progenitor cell markers, and upregulation of differentiation-associated genes. We observed this in both ES-derived and adult forebrain NSCs after irr. Moreover, we also observed loss of DDR signaling and DDR gene expression in irr NSCs, which is consistent with their differentiation toward the astrocytic lineage (Schneider et al., 2012). The differentiation bias of irr NSCs toward astrocytes may be explained by their glial nature (Doetsch, 2003). Indeed, NSCs sustaining mitochondrial DNA damage were reported to be more prone to astroglial fate when stimulated to differentiate (Wang et al., 2011).