Hippocampal Neurogenesis Figure 3. Malformation of the hippocampal DG structure in Ngn2 mutant animals. (A1–A3) Hematoxylin/Eosin staining performed on coronal sections from two-weeks old WT, Ngn2+/GFP and Ngn2GFP/GFP mutant animals, shows that the loss of Ngn2 results in the formation of a smaller hippocampus and primitive and undeveloped DG. (B1–B4) The malformation of the DG in Ngn2GFP/GFP mutant animals can already be observed two days after birth, throughout the hippocampal structure, along the rostro-caudal axis (see also supplementary figure 2). The inlets of rectangular panels in bottom right corners show a schematic drawing of the shape of the DG for Ngn2+/GFP and Ngn2GFP/GFP mutant animals; black arrowheads point to the undeveloped and missing dorsal blade and ventral blade of the DG, respectively, in Ngn2GFP/GFP mutant animal. Scale bars: 200 mm (A1–B4). doi:10.1371/journal.pone.0004779.g003 not shown) littermates (Figure 5B–C). This data confirm the importance of Ngn2 during the amplification of granule progenitor.s. To further explore whether Ngn2+ cells become post-mitotic or still proliferate after Ngn2 is downregulated, we performed immunohistochemistry on sections through the DG of Ngn2+/ GFP mice. We stained them with the mitosis marker Phospho- histone 3 (PH3) and NeuroD1, which is downstream of Ngn2 in the transcriptional cascade controlling neurogenesis. Thus, in the same sections we could identify whether cells that had initiated Ngn2 expression (GFP labeled), continued to divide (PH3+) or committed to neuronal differentiation (NeuroD1+). We observed some GFP+ cells that co-expressed PH3. They all exhibited morphological characteristics of one of the five mitotic phases: prophase, metaphase, anaphase, telophase and cytokinesis (Figure 5D and data not shown). Unexpectedly, cells that colabeled for GFP, PH3 and NeuroD1 (Figure 5E and figure S4A and S4B) were rare. This indicates that Ngn2+ cells undergo division/amplification and that they mature into post-mitotic neurons upon NeuroD1 expression. In mice lacking Ngn2, we found the cells expressing GFP localized to the malformed ventral blade of the DG (Figure 5F and G). All of these cells were Ki67+ and they only very rarely expressed PH3 (Figure 5F and G, and figure S4C) in the absence of Ngn2 most of the cells are arrested in the cell cycle prior to entering the M phase and their mitosis is impaired. These data suggest that the mechanism of action of Ngn2 is conserved from development of the DG to postnatal hippocampal neurogenesis. Our findings suggest that Ngn2 regulates amplification and cell cycle exit of DG granule progenitors. To examine this hypothesis, we compared the effects of Ngn2 and NeuroD1 on mitotic activity in embryonic cortico-hippocampal neurosphere-derived progeni- tors, 5 days upon transduction, in vitro. In cultures transduced with Ngn2 retrovirus, we found that 27.2% (64.1%) of the PH3+ cells had been transduced (Figure I1–J). These cells were immunopositive for MAP2 (Figure 5I1 and I2) and therefore represented dividing neuroblasts. By contrast, in NeuroD1 transduced cultures, only 7.3% of PH3+ cells were GFP+ (Figure 5H1, H2 and J). All of the NeuroD1-transduced cells became MAP2+ (Figure 5H). To confirm this data we pulse- labeled transduced cultures with chlorodeoxyuridine (CldU) for 48 hours, five days after differentiation. In contrast to NeuroD1- transduced cultures, we observed dividing cells transduced with Ngn2 retrovirus that were positive for PH3 and that had incorporated CldU (Figure 5K–L2). As the number of cells PH3+/CldU+/GFP+ we observed was low, we did not quantify this finding. Collectively, we have shown that Ngn2 is required for granule neuroblast production and amplification and that in the absence of Ngn2 the progenitors arrest in the cell cycle. NeuroD1, on the other hand, induces cell cycle exit and promotes rapid neuronal maturation. NeuroD1 directs neuronal differentiation and maturation Based on our previous observations we proposed that Ngn2 primarily controls amplification of granule neuroblasts and NeuroD1 directs neuronal differentiation. To test this hypothesis, we overexpressed Ngn2 or NeuroD1 in E14.5 cortico-hippocam- pal neurospheres and compared their effects after 5 days of differentiation of the progenitors. All cortico-hippocampal pro- genitors expressed Pax6 prior to differentiation (Figure S5). After 5 days, both factors suppressed Pax6 and Sox2 (Figure S5, figure 6C) and induced expression of Tbr1, Map2, NeuroD1 and PSA- NCAM (Figure 6D and E). Interestingly, all Ngn2-overexpressing cells co-expressed Neu- roD1. Based on these findings, we next explored whether Ngn2 overexpression induces NeuroD1 expression, and if NeuroD1 in turn directs neuronal differentiation. We compared the effects of both transcription factors in progenitors that either do or do not normally express them. Thus, we expressed Ngn2 in E14.5 neural progenitors isolated from three different brain regions: cortex/ hippocampus, lateral ganglionic eminence (LGE) and ventral mesencephalon (VM). Ngn2 expression in the developing forebrain is normally limited to the neocortex, and it does not PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4779