Hippocampal Neurogenesis Figure 2. Ngn2+ cells become hippocampal granule neurons. (A1–J2) Indirect immunofluorescence performed on coronal sections from two- weeks old WT and Ngn2+/GFP mice. (A1–A2) Ngn2-expressing cells co-express Pax6 (.90%), but not NeuN. Ngn2+ cells are often observed by doublet of cells. (B1–B2) Almost all Ngn2+ cells express Tbr2, however only half of Tbr2+ cells co-express Ngn2. (C–F) Mash1 expression precedes that of Ngn2. (C) All Mash1+ cells express Pax6, and exhibit high mitotic activity (See also supplementary Figure 1J). Mash1 expression ceased at the onset of Tbr2 (D) or Ngn2 expression (E). (F) Mash1 cells never co-expressed NeuroD1. (G1–G2) GFP (driven by the Ngn2 promoter) starts upon Mash1 downregulation. Mash1 expression and GFP expression do not co-localize. Ngn2 GFP-expressing progenies become DG granule neurons, the latter expresses NeuroD2 during their maturation. Ngn2 GFP-expressing progenies sequentially express Tbr2 and NeuroD1 (H1–H2), the cellular membrane markers PSA-NCAM and Calretinin (I1–I2) and Prox1, an identity marker of mature hippocampal granule neurons (I1–J2). (K) Expression patterns of cellular markers and transcription factors during hippocampal DG granule neurogenesis. The type-2a stage has been divided into early and late type- 2a stages, based on the sequential expression of Mash1 and Ngn2. Panels A2, B2, G2–J2 represent a higher magnification of framed areas in corresponding panels A1, B1, G1–J1. The white crosshairs in these panels were positioned to show co-expression of the markers of interest in single cells. Rectangular images on the bottom and right of the panels A2, B2, D–F and J2–G2 represent a projection of 14-Z stacks images (total of 10– 20 mm thick). Scale bars: 50 mm (A1, B1, C, G1, H1, I1 and J1), 25 mm (A2, B2, D, E, F, G2, H2, I2 and J2). doi:10.1371/journal.pone.0004779.g002 PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4779