Hippocampal Neurogenesis Figure 7. NeuroD1 directs neuronal differentiation of hippocampal progenitors in vivo. (B1–P2) Indirect immunofluorescence performed on hippocampal coronal sections of two-weeks old rats that received a retroviral injection with Ngn2, NeuroD1 or control retroviruses, at the intra- uterine age of E15.5. (A) Schematic representation of the experimental design for in utero retroviral transgene delivery procedure. (B1–B3) Presence of GFP-expressing cells in the hippocampus, three weeks after retroviral injection with Ngn2, NeuroD1 or control vectors. (B4) The proportion of GFP- granule neuron-like cells (blue) and GFP-progenitors/glial-like cells (red) in animals injected with each retrovirus (expressed in percentage of GFP cells) indicate a complete differentiation of NeuroD1-transduced hippocampal progenitors into granule neurons-like cells. (C1–F) Ngn2 overexpression in vivo results in the generation of GFAP-expressing astrocytes, also observed in control group. NeuroD1 overexpression did not induce astrocytic differentiation. (G1–J) In utero injections of control and Ngn2 retroviruses result in the generation of Tbr1+ cells. Only a few Tbr1+/ eGFP+ cells were observed in the hippocampal DG of the animals injected with NeuroD1 retrovirus. (K–O) NeuroD1 overexpression induces exclusive neuronal differentiation, in contrast to Ngn2 overexpression. (P1 and P2) A few GFP+ cells that were transduced with Ngn2 retrovirus are found still dividing. Data are expressed as 6SEM; *** P,0.0001 and ** P,0.01. Scale bars: 50 mm (B1–3, C1, D1, E, G1, H1, I, K, L, M1 and P1), 25 mm (C2, D2, G2, H2 and M2), 10 mm (P2). doi:10.1371/journal.pone.0004779.g007 We demonstrated that NeuroD1 directs neuronal differentiation both in vitro and in vivo. In progenitors isolated from different embryonic brain regions and cultured in vitro, we found that overexpression of NeuroD1 induced neuronal differentiation. The neurons generated expressed PSA-NCAM, Dcx and MAP2. After in utero retroviral vector-mediated gene delivery, virtually all cells transduced with NeuroD1 became neurons. In contrast, progen- itors transduced with Ngn2 or control retroviruses adopted a neuron-like morphology in only 70–85% of cases. Under in vitro cell culture conditions, when we overexpressed Ngn2 in progen- itors derived from the LGE we observed robust expression of NeuroD1 and neuronal differentiation. These in vitro results differ PLoS ONE | www.plosone.org 12 March 2009 | Volume 4 | Issue 3 | e4779