Hippocampal Neurogenesis Figure 4. Ngn2 directs the production of hippocampal DG granule neuroblasts. (A1–B5, H–I2 and L1–N1) Indirect immunofluorescence performed on hippocampal coronal sections from two-days old Ngn2+/GFP and Ngn2GFP/GFP mutant animals (A1–B5 and H–I2) and two-weeks old Ngn2+/GFP mutant animals (L1–N1). (A1–B5) Presence of BrdU+ cells (red) in the hSVZ, chains of migrating cells and DG of two-days old Ngn2+/GFP and Ngn2GFP/GFP mutant animals injected with BrdU two hours prior to sacrifice. A4, A5, B4 and B5 represent high magnification of the framed areas in A1 and B1, respectively. (C–I2) Cell proliferation and neurogenesis are severely decreased in Ngn2GFP/GFP mutant animals. The histograms C–G and J and K depict the average number or percentage (6SEM); P,0.0001 = *** and P,0.01 = **. (L1–M2) Granule neuron maturation is confirmed by indirect immunofluorescence on hippocampal sections from two-weeks old Ngn2GFP/GFP mutant animals, as revealed by the co-expression of Calretinin, NeuroD1, Prox1 and NeuN in GFP-expressing cells. (N) In the absence of the Ngn2 protein, GFP-expressing cells do not switch from a neuronal commitment to an astrocytic phenotype. Rectangular images on the bottom and right of the panels L2, M2 and N represent a projection of 14-Z stacks images (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, as labeled above each panel. Scale bars: 200 mm (A1–3 and B1–3), 100 mm (H and H1), 50 mm (A4, A5, B4, B5, L1 and M1), 25 mm (I2, L2, M2 and N). doi:10.1371/journal.pone.0004779.g004 PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4779