Proliferating neuronal progenitors in the postnatal hippocampus transiently express the proneural gene Ngn2 Ilknur Ozen, 1, * Christophe Galichet, 2, * Colin Watts, 1 Carlos Parras, 3 Franc¸ois Guillemot 2 and Olivier Raineteau 1 1 Department of Clinical Neurosciences, University of Cambridge, Robinson Way, Cambridge CB2 2PY, UK 2 Division of Molecular Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK 3 Unite Mixte de Recherche INSERM U-711, Hoˆ pital de la SalPetriere, 75651 Paris cedex 13, France Keywords: neurogenesis, neurogenic niche, proneural transcription factor, transgenic mice Abstract Little is known of the transcription factors expressed by adult neural progenitors produced in the hippocampal neurogenic niche. Here, we study the expression of the proneural basic helix-loop-helix (bHLH) transcription factor Neurogenin-2 (Ngn2) in the adult hippocampus. We have characterized the pattern of expression of Ngn2 in the adult hippocampus using immunostaining for Ngn2 protein and a Ngn2-green fluorescent protein (GFP) reporter mouse strain. A significant proportion of Ngn2-expressing cells were mitotically active. Ngn2-GFP expression was restricted to the subgranular zone and declined with age. Neuronal markers were used to determine the phenotype of Ngn2-expressing cells. The vast majority of Ngn2-GFP-positive cells expressed the immature neuronal markers, doublecortin (DCX) and polysialic acid-neural cell adhesion molecule (PSA-NCAM). Finally, the pattern of Ngn2 expression was studied following seizure induction. Our data show an increase in neurogenesis, detected in these animals by bromodeoxy- uridine (BrdU) and DCX staining that was contemporaneous with a marked increase in Ngn2-GFP-expression. Taken together, our results show that Ngn2-GFP represents a specific marker for neurogenesis and its modulation in the adult hippocampus. Ngn2 transient expression in proliferating neuronal progenitors supports the idea that it plays a significant role in adult neurogenesis. Introduction The dentate gyrus (DG) is a major component of the hippocampal formation and is essential for normal hippocampal function. The birth of neurons persists in this brain region long into adulthood (Altman & Das, 1965; Bayer et al., 1982). Neural progenitor cells in the DG are generated in the subgranular zone (SGZ), a discrete region directly underlying the dentate granule cell layer. These immature neurons migrate from the SGZ into the granule cell layer and where they differentiate into functional neurons (Jones et al., 2003; Abrous et al., 2005; Esposito et al., 2005; Ge et al., 2006; Zhao et al., 2006). The neurogenic niche in which progenitor cells proliferate and initiate differentiation is composed of different cell types that have been classified on the basis of morphological criteria and immunoh- istochemical properties (Seri et al., 2004). Little is known about the transcription factors and signalling molecules that these cells express. During development, the migrating precursors of granule cells have been shown to express the basic helix-loop-helix (bHLH) gene Mash1 and the receptor Notch1 and at least some of these cells additionally express the ligand Delta1, the HLH gene Id3 and the bHLH gene Hes5 (Pleasure et al., 2000). Previous work with mutant mice has shown that different transcription factors are implicated in distinct steps of dentate gyrus formation. Thus, mice lacking Emx2 exhibit early deficits in the migration of SGZ precursors (Oldekamp et al., 2004). In contrast, mice lacking the bHLH genes NEX ⁄ Math2 and NeuroD1 show normal granule cell birth but a failure of these cells to mature (Schwab et al., 2000). In the adult hippocampus, only the expression of Pax6 has been described and it is restricted to GFAP-positive (GFAP+) early progenitor cells (Maekawa et al., 2005; Nacher et al., 2005). The bHLH transcription factor Neurogenin-2 (Ngn2) regulates neurogenesis in vertebrate embryos by coupling neuronal determin- ation with the specification of glutamatergic neuronal phenotypes in various regions of the developing central nervous system (Fode et al., 2000; Parras et al., 2002). Ngn2 acts downstream of Pax6 in the developing cerebral cortex (Heins et al., 2002; Scardigli et al., 2003), where it is involved in neuronal commitment of multipotent progen- itors, specification of cortical neuron identity and radial migration of postmitotic cortical neurons (Farah et al., 2000; Nieto et al., 2001; Schuurmans et al., 2004). Our previous observations suggest that Ngn2 expression is normally maintained postnatally in the hippocam- pal subgranular zone (Raineteau et al., 2004). To develop these observations further, we combined both immunostaining for Ngn2 with transgenic mice expressing a green fluorescent protein (GFP) reporter gene under the Ngn2 genomic regulatory sequences (Ngn2 + ⁄ GFP mice; Seibt et al., 2003) to detect Ngn2-expressing cells in the postnatal hippocampus. Our results demonstrate that Ngn2-GFP is expressed in proliferating neuronal progenitors and that its expression faithfully follows neurogenic modulation. The expression of this proneural gene in neuronal progenitors of the adult dentate gyrus supports the idea that Ngn2 may have a significant role in postnatal neurogenesis. Correspondence: Dr Olivier Raineteau, as above. E-mail: olr21@cam.ac.uk *I.O. and C.G. contributed equally to this work. Received 21 July 2006, revised 13 March 2007, accepted 15 March 2007 European Journal of Neuroscience, Vol. 25, pp. 2591–2603, 2007 doi:10.1111/j.1460-9568.2007.05541.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd