Hippocampal Neurogenesis Figure 1. Characterization of the molecular signatures defining each phases of cell maturation during hippocampal granule neuronal differentiation, in two-weeks old newborn mice. (A1–J2) Indirect immunofluorescence performed on hippocampal coronal sections from 2 weeks old newborn mice. (A1–B2) Type-1 radial glia stem cells are identified by the co-expression of Nestin, Glast, GFAP and Pax6. Radial glia cells locate in both dorsal and ventral blades of the DG. (C1–C2) Type-2 amplifying progenitors expressing Tbr2 rarely co-express PSA-NCAM or Dcx. (D1–D2) Type-3 maturing granule neurons co-express Tbr1, PSA-NCAM and Dcx. (E1–E2) Type-3 maturing granule neurons co-express NeuroD1 but not Pax6. Pax6 progenitors co-expressing NeuroD1 were rarely found and located in the hilus. (F1–F2) Granule neurons maturation occurs through the expression of NeuroD1, PSA-NCAM and Calretinin. (G1–G2) The transition from type-2 amplifying progenitors to mature neurons takes place when the type 2 cells express Tbr2 followed by expression of NeuroD1 and thereafter NeuN. (H1–H2) The transition from type-3 amplifying progenitors to mature granule neurons occurs through the expression of NeuroD1, Tbr1 and NeuN. (I1–I2) The expression of Prox1 starts soon after that of NeuroD1. (J1–J2) NeuroD2 expression starts soon after that of NeuroD1 and persists in mature granule neurons, in contrast to NeuroD1. Panels A2–J2 represent a high magnification of framed areas in corresponding panels A1–J1. Rectangular images on the bottom and right of the panels A2– J2 represent projected images of 14-Z stacks (total of 10–14 mm thick). The white crosshairs in these panels were positioned to show co-expression of markers of interest in single cells. See each panel label. SGZ = subgranular zone, GL = granule layer, DB = dorsal blade and VB = ventral blade. Scale bars: 50 mm (A1, B1, C1, D1, E1, F1, G1, H1, I1 and J1), 25 mm (A2, B2, C2, D2, E2, F2, G2, H2, I2 and J2). doi:10.1371/journal.pone.0004779.g001 data from Ngn2+/GFP mice are entirely consistent with our earlier observations using immunocytochemistry to label Ngn2-expressing cells and with GFP undergoing slow degradation after the expression of Ngn2 has ceased. Importantly, none of the GFP- expressing cells, including those only weakly fluorescent, residing in the SGL expressed the astrocytic marker GFAP or the oligodendrocytic markers CNPase (data not shown). Taken together, the data show that Ngn2 progenies become neurons and never generate astrocytes or oligodendrocytes. Marked reduction of hippocampal granule neurons in absence of Ngn2 To determine the role of Ngn2 during the initiation of hippocampal granule neurogenesis, we analyzed the hippocampus of mice lacking Ngn2 (Ngn2GFP/GFP). In contrast to the mice use by Galichet and coworkers, our mice have a lifespan of only 2 weeks after birth. Therefore, we analyzed 2 week-old and younger mice. Consistent with previous observations [20], the hippocam- pus in Ngn2GFP/GFP mutants is clearly malformed (Figure 3A). In two week-old Ngn2GFP/GFP mice, the ventral blade of the DG is completely absent. Already on postnatal day 1–2, the ventral blade of the DG is malformed along its whole rostro-caudal axis (Figure 3B). As neither migration defects nor cell death cause the reduced number of hippocampal neurons in Ngn2 mice [20], we asked if Ngn2 is necessary for production of hippocampal granule neuroblasts. We first injected 2 day-old mouse pups with BrdU 2 hours prior to sacrifice, allowing us to evaluate cell proliferation and ongoing neurogenesis. We also injected BrdU into female mice on their final day of pregnancy and examined the brains of their pups 48 hours later (corresponding to P1–P2 old pups). This allowed us to assess the number of neurons derived from the last day of intrauterine development. Regardless of whether the mice were WT or hetero/homozygous mutant pups, we found cells that had incorporated BrdU in the hippocampal subventricular zone (hSVZ), fimbria and the DG (Figure 4A and B; [28]), suggesting that progenitors originating in the hSVZ divide while migrating towards the DG. Most of them were organized in chains typical of migrating cells (Figure 4A and B and figure S2). In Ngn2GFP/GFP mutant mice injected with BrdU 2 hours prior to sacrifice, we observed a decrease in the number of BrdU+ cells compared to heterozygous littermates. Thus, the numbers of newborn cells were reduced in the hSVZ/fimbria (133.367.5 for Ngn2+/GFP vs 69.765.0 for Ngn2GFP/GFP) and DG (132.069.1 for Ngn2+/GFP vs 42.662.2 for Ngn2GFP/GFP)(Figure 4C and D). We also examined the number of newborn cells differentiating into neurons, and identified them by the co-expression of BrdU and NeuroD1. As expected, they were relatively few in numbers because the short delay (2 hours) between BrdU administration and sacrifice in this first experimental paradigm. In Ngn2GFP/GFP mutants, we found the number of cells differentiating into neurons to be reduced by 60% and 71% in the hSVZ/fimbria (8.661.4 for Ngn2+/GFP vs 3.460.5 for Ngn2GFP/GFP) and DG (9.761.1 for Ngn2+/GFP vs 2.860.6 for Ngn2GFP/GFP), respectively, (Figure 4E and F). As a result, the number of NeuroD1+ cells in the DG was reduced by 80% (580.3627.7 for Ngn2+/GFP vs 116.267.3 for Ngn2GFP/GFP; Figure 4G). We obtained similar results in the second paradigm, i.e. in mice that we sacrificed 48 hours after BrdU administration and in which a larger number of the newborn cells had time to mature into neurons. In this case, the number of BrdU+/NeuroD1+ cells in the DG was reduced by 73% of Ngn2GFP/GFP mutant animals (91.5624.1 for Ngn2+/GFP vs 26.463.3 for Ngn2GFP/GFP) (Figure 4J). We found no differences in the proportion of BrdU+ cells that expressed NeuroD1 in Ngn2+/GFP and Ngn2GFP/GFP mice (14.261.0% and 17.461.8% respectively; Figure 4K). This shows that the few cells that manage to proliferate in the Ngn2 mice have the same ability to differentiate into neurons as those in mice with one Ngn2 allele. Interestingly, the absence of Ngn2 did not alter the identity of neurons in the DG granule layer in 2 week-old Ngn2 mice. The maturing granule progenitors sequentially expressed Pax6, Tbr2 (Figure S3A and S3B), NeuroD1 and Calretinin (Figure 4L), PSA-NCAM (Figure 4N), Tbr1 (data not shown) and Prox1 (Figure 4M). We confirmed that the same transcriptional cascade is active in the 20% (compared to mice with one Ngn2 allele) residual granule neurons that are formed two day-old in Ngn2GFP/ GFP mice (Figure S3D–G and data not shown). We next investigated if the cells that failed to develop into neurons in Ngn2GFP/GFP mutant mice, became glial cells. We found that cells expressing GFP never co-labeled with the astrocyte marker GFAP (Figure 4N) or the oligodendrocyte marker CNPase (data not shown). Altogether, our results show that Ngn2 plays an important role during the production/amplification of hippocampal granule neuroblasts, but not the acquisition of the granule neuron identity (Figure S7). Ngn2 controls the amplification of granule neuron progenitors We next monitored the mitogenic activity of Ngn2+ cells in the DG of 2 week-old WT mice. We observed that over 50% of Ngn2+ cells in the DG of WT mice co-expressed the mitosis marker Ki67 (Figure 5A). Inspired by this finding, we injected BrdU into 2 week-old WT and Ngn2GFP/GFP mice and compared cell proliferation in the DG. We observed 92% reduction in number of proliferating cells in the SGZ of Ngn2 mutant mice (6.760.5) compared to WT (83.661.2) and heterozygotic (data PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4779