Neurogenin2 Directs Granule Neuroblast Production and Amplification while NeuroD1 Specifies Neuronal Fate during Hippocampal Neurogenesis Laurent Roybon1*, Tord Hjalt1, Simon Stott2, Francois Guillemot3, Jia-Yi Li1., Patrik Brundin1. 1 Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund, Sweden, 2 Division of Developmental Neurobiology, MRC National Institute for Medical Research, The Ridgeway, London, United Kingdom, 3 Division of Molecular Neurobiology, MRC National Institute for Medical Research, The Ridgeway, London, United Kingdom Abstract The specification and differentiation of dentate gyrus granule neurons in the hippocampus require temporally and spatially coordinated actions of both intrinsic and extrinsic molecules. The basic helix-loop-helix transcription factor Neurogenin2 (Ngn2) and NeuroD1 are key regulators in these processes. Based on existing classification, we analyzed the molecular events occurring during hippocampal neurogenesis, primarily focusing on juvenile animals. We found that Ngn2 is transiently expressed by late type-2a amplifying progenitors. The Ngn2 progenies mature into hippocampal granule neurons. Interestingly, the loss of Ngn2 at early stages of development leads to a robust reduction in neurogenesis, but does not disturb granule neuron maturation per se. We found that the role of Ngn2 is to maintain progenitors in an undifferentiated state, allowing them to amplify prior to their maturation into granule neurons upon NeuroD1 induction. When we overexpressed Ngn2 and NeuroD1 in vivo, we found NeuroD1 to exhibit a more pronounced neuron-inductive effect, leading to granule neuron commitment, than that displayed by Ngn2. Finally, we observed that all markers expressed during the transcriptional control of hippocampal neurogenesis in rodents are also present in the human hippocampus. Taken together, we demonstrate a critical role of for Ngn2 and NeuroD1 in controlling neuronal commitment and hippocampal granule neuroblast formation, both during embryonic development and in post-natal hippocampal granule neurogenesis. Citation: Roybon L, Hjalt T, Stott S, Guillemot F, Li J-Y, et al. (2009) Neurogenin2 Directs Granule Neuroblast Production and Amplification while NeuroD1 Specifies Neuronal Fate during Hippocampal Neurogenesis. PLoS ONE 4(3): e4779. doi:10.1371/journal.pone.0004779 Editor: Thomas A. Reh, University of Washington, United States of America Received July 29, 2008; Accepted January 14, 2009; Published March 10, 2009 Copyright: 2009 Roybon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by the Swedish Research Council, Hjarnfonden, the Swedish Parkinson Foundation, the Syskonen Svenssons Foundation and the Konung den V:s och Drottning Victorias Stiftelse and PROMEMORIA (From cell-cell recognition to memory formation; new strategies for the treatment of dysfunctional plasticity, learning and memory LSHM-CT-2005-512012). L.R. was supported by a European grant (LHSB-CT-2006-037328; STEMS) and currently supported by a Hjarnfonden fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: laurent.roybon@med.lu.se . These authors contributed equally to this work. Introduction Neurons are born not only during development of the central nervous system, but neurogenesis also continues into adulthood. In both rodent and human adult brain, neurogenesis is active in two distinct zones of the forebrain: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) [1–5]. The molecular mecha- nism underlying neurogenesis in the DG is not fully understood. Clearly, a cascade of transcriptional events controls the specifica- tion of neuronal identity in the DG [3,6–8], but details of the expression pattern and function of each transcription factor remain elusive. The paired-box homeodomain transcription factor Pax6 and the bHLH transcription factors Ngn2 and NeuroD1 are important when cells acquire a pan-neuronal character and a specific neuronal subtype [9,10]. In the developing neocortex, Pax6 is expressed in dividing radial glial cells at the ventricular surface [9]. In the adult hippocampus, Pax6 is expressed in astrocytes in the SGZ and are considered to be the true stem cells [9,11]. Loss- and gain-of-function studies indicate that Pax6 is involved in regulating the proliferation of neocortical and hippocampal progenitors [9,12–16]. During neocorticogenesis, high concentrations of Pax6 induce the expression of the bHLH transcription factor Neuro- genin2 (Ngn2) [17]. In turn, Ngn2 causes cell cycle exit [18], an event that takes place when NeuroD1 starts to be expressed [19]. In the developing hippocampus, the absence of Ngn2 leads to a reduced number of granule neurons [20]. NeuroD1, on the other hand, is essential for the differentiation and survival of hippocam- pal granule neurons [10]. Interestingly, in the absence of Ngn2, NeuroD1 can still be activated and neuronal differentiation can still take place [20]. This has led to the idea that the primary role of Ngn2 is not to direct neuronal differentiation. Only a small number of studies have addressed the role of Ngn2 and NeuroD1 in hippocampal neurogenesis [10,20,21]. Therefore, we now re-examine their roles in hippocampal neurogenesis in detail, using gain- and loss-of-function experiments. We first establish a hierarchy of transcriptional events that occur during neurogen- esis in the DG and then define the place that Ngn2, NeuroD1 and other transcription factors have in this cascade. We find that a lack PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4779