Hippocampal Neurogenesis Figure 5. Ngn2 and NeuroD1 mark the initiation and end of amplification of hippocampal DG granule neuroblasts, respectively. (A1–C3 and D1–K2) Indirect immunofluorescence performed on hippocampal coronal sections from postnatal two-weeks old WT, Ngn2+/GFP and Ngn2GFP/GFP mutant animals and Ngn2 and NeuroD1 retrovirally transduced E14.5 cortico-hippocampal neurospheres. (A1 and A2) Fifty percent of Ngn2+ cells co-label with Ki67. (B1 and B2) One BrdU injection with a two-hour chase labels 50% of the Ngn2-expressing cell population, representing half of the total number of proliferating cells in the DG. (C1–C4) In the DG of two-weeks old Ngn2GFP/GFP mutant animals, the number of dividing cells is substantially decreased compared to WT (data expressed as 6SEM; *** P,0.0001). (D1 and D2) In Ngn2+/GFP mice, GFP/Ngn2-expressing cells undergo division and give rise to GFP+/NeuroD12 cells. These cells have a baso-apical orientation. (E1 and E2) GFP/Ngn2-expressing cells stop dividing when NeuroD1 expression starts. Dividing cells expressing GFP and NeuroD1 have a planar orientation (see also supplementary figure 4). (F1 and G2) All cells brightly GFP present in the ventral blade of the DG of Ngn2GFP/GFP mutant animals co-express Ki67; however, only very few divide, indicating that in absence of Ngn2, Ngn2 progenies stay arrested in the cell cycle. (H1–J) Presence of GFP+/MAP2+/PH3+ dividing neuroblasts in Ngn2-transduced E14.5 hippocampo-cortical neurospheres compared to NeuroD1-transduced cultures. (K–L2) Five days differentiated Ngn2 transduced cells (GFP+) that have been cultures for two more additional days in medium supplemented with CldU, are still capable of dividing after 7 days, as opposed to NeuroD1 transduced cells. Rectangular images on the bottom and right of the panels A2, B2, C3, D2, F2, G2, I1 and J1 represent a projection of 14-Z stacks images (total of 10–14 mm thick) from framed areas or pointing arrows in panels A1, B2, C3, D2, F2, G2, I1 and J1, respectively. The white crosshairs in these panels were positioned to show co-expression in single cells of markers of interest, as labeled above each panel. Arrows and arrowheads point at cells of interest. Scale bars: 100 mm (B1, C1 and C2), 50 mm (A1, D1, E1, F1, G1, H1, I1, K and L1), 25 mm (A2, B2, and F2), 10 mm (L2), 5 mm (C3, E2, G2, H2 and I2). doi:10.1371/journal.pone.0004779.g005 appear in LGE tissue [29]. In the developing VM, both Ngn2 and NeuroD1 are expressed, but not when neural progenitors from this region are cultured in vitro [30,31]. In our experiments, cortico- hippocampal progenitors could differentiate into neurons, al- though they did not normally express NeuroD1 (Figure 6E). When we overexpressed Ngn2 in VM and LGE progenitors, the cells started to express both NeuroD1 and PSA-NCAM (Figure 6F and G). We then overexpressed NeuroD1 in the same types of cultured progenitors and found that the cells became immunoreactive for PSA-NCAM (Figure 6G and data not shown). Thus, NeuroD1 is sufficient to direct neuronal differentiation in cortico-hippocam- pal-, LGE- and VM-derived progenitors. Our results from in vitro cultures and the analysis of the DG of Ngn2 mutant animals collectively show the neuron-inducing effect of NeuroD1 in hippocampal granule cell progenitors [32]. NeuroD1 directs exclusive neuronal differentiation of hippocampal granule neuron progenitors As the next step, we tested the effects of NeuroD1 in vivo and compared them with those of Ngn2. We injected retroviruses carrying the gene for either Ngn2 [33] or NeuroD1, and the reporter gene eGFP into the ventricles E15.5 rat embryos, in utero (Figure 7A). Three weeks later, when the rats were about two weeks old, we examined their hippocampi and analyzed the eGFP+ cells. The rats injected with control vector exhibited eGFP+ cells within the hippocampus that were either star-shaped, progenitor/glial-like cells (14.863.4%) or neuron-like cells with long neurites (85.363.4%) (Figure 7B). In rats that we had injected with the vector encoding Ngn2, the eGFP-labeled, the transduced hippocampal cells were composed of 31.9% (65.7%) progenitor/ glia-like cells and 69.4% (66.1%) neuron-like cells (Figure 7B). The glia-like subpopulation was immunopositive for the astrocytic marker GFAP (Figure 7C–D). In contrast, virtually all of the transduced cells in rats injected with the NeuroD1 vector became neuron-like cells (99.9%60.1; Figure 7B). None of these cells stained for GFAP (Figure 7E and F). They were positioned in the external layers of both the ventral and dorsal blades of the DG (Figure 7B3, E and I and figure S6A). When we examined neuronal maturation of Ngn2- and NeuroD1-transduced cells, we observed that only a few of those transduced with the NeuroD1 vector expressed the early mature neuronal marker Tbr1 (3.161.4%; Figure 7I and J). They were located within the basal layers, near the SGL, of the ventral and dorsal blades of the DG. In contrast, a greater proportion of the cells transduced with Ngn2 still expressed Tbr1 (19.6%63.3%; Figure 7H and J), indicating that they were less mature than the vast majority of the NeuroD1-transduced cells. Immunohisto- chemistry for NeuN confirmed these data (Figure 7M and O, and figure S6A). Indeed, when we overexpressed Ngn2 the proportion of hippocampal progenitors that became NeuN+ was no greater than in rats transduced with the control virus (Figure 7F). As a whole, our in utero injection experiments confirm that NeuroD1 has a stronger neuron-inducing effect than Ngn2 when overexpressed in hippocampal progenitors. Involvement of Mash1, Ngn2, Tbr and NeuroD proteins during human hippocampal neurogenesis Finally, we examined whether the transcription factors and cellular markers that are expressed in rodents are also expressed in human hippocampus. We performed immunohistochemistry on aged human hippocampal DG and found GFAP-, Sox2-, Pax6-, Nestin-, Prox1- and NeuN-immunopositive cells. This indicates that radial glia-like stem cells, neural progenitors and mature granule neurons are present in the aged human hippocampus (Figure 8A–E). While Prox1 is found mainly in granule neurons, Sox2 and Pax6 are exclusively localized to cells in the SGZ (Figure 8D and E). As for aged rodents, we did not observe the presence of Mash1, Ngn2, Tbr2, Tbr1 and NeuroD1 proteins in aged human hippocampus, using immunohistochemistry. However, we could identify the presence of the transcripts, using RT-PCR (Figure 8F). In addition, we detected mRNA for Prox1, Calbindin1 and Calbindin2/Calretinin (Figure 8F). Interestingly, we also observed Sonic Hedgehog and Wnt-3A transcripts, indicating that they are present in the human DG and supporting previous claims that they are important for the regulation of adult hippocampal neurogenesis (Figure 8F; [34,35]). Finally, we also found mRNA for GLAST, Pax6 and GFAP which suggests that radial glia-like stem cells are present in the adult human hippocampus, and may play a role in adult human hippocampal neurogenesis (Figure 8F; [8]). Overall, our data show that different proteins known to play key roles in rodent hippocampal granule neurogenesis are present in the adult human hippocampus. Discussion In this study we determine the functions of Ngn2 and NeuroD1 during hippocampal neurogenesis. First, we map the hierarchy of molecular markers of neurogenesis in the DG. Second, we describe that Ngn2 is necessary for granule progenitor produc- tion/amplification. Third, we demonstrate that NeuroD1 directs neuronal differentiation of granule progenitors. Finally, we show that different cellular markers expressed during hippocampal neurogenesis in rodents are present in human. PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4779