of Ngn2 expression result in a markedly smaller hippocampus and an almost complete absence of the DG. We show that Ngn2 is required for granule neuroblasts production/amplification. Gain- of-function of NeuroD1 during development of the DG results in an efficient generation of granule neuroblasts, an effect that we do not observe when we overexpress Ngn2. Finally, we demonstrate that the same transcription factors and cellular markers seen in mouse and rat tissue are also present in the human hippocampus. Results Expression of transcription factors and cellular markers define different phases of postnatal hippocampal granule neuron maturation We first analyzed in detail the chronology of expression of different transcription factors and cell-specific markers during DG granule neuron formation and correlated our findings to the previously established classification of adult hippocampal neuro- genesis [3]. The current classification describes hippocampal stem cells and progenies into three categories (type-1, -2 and -3 cells) depending on the markers they express as they mature. In the adult mouse DG, only a very small proportion of cells undergo mitosis at any one given time [22]. Consequently, in adult animals it is difficult to analyze which transcription factors are involved in the transition phase from one cell category to the next. Therefore, we studied 2 week-old rodents which have higher numbers of maturing granule neurons in the DG. We performed triple immunohistochemistry for all markers of interest and compared results from 2 week- and 2 month-old mice (Figure 1 and figure S1). We first confirmed that stem cells in the SGZ exhibit characteristics of radial glia [3,11,16]. They extend radial processes from the SGZ to the apex of the subgranular layer (SGL) and the basal molecular layer. Moreover, they express the intermediate filament protein nestin, glial fibrillary acidic protein (GFAP) and glial glutamate transporter (GLAST) [16] (Figure 1A and B and figure S1A and S1B). The GFAP- and GLAST+ cells are defined as type-1 cells [3]. They also express the paired- homeodomain transcription factor Pax6 (Figure 1B). These radial glia-like stem cells divide relatively infrequently, and are believed to undergo symmetric division (giving rise to two identical stem cells) under some conditions. They can also divide asymmetrically and give rise to a new stem cell and one neuronal progenitor, which usually is defined as a type-2 cell (Figure 2B) [3]. Type-2 cells are divided into two different populations: type-2a expressing nestin and type-2b co-expressing nestin and double- cortin (Dcx) [23]. Based on a previous report [24], we examined the expression pattern of the T-domain transcription factors Tbr1 and Tbr2. We found that Tbr2 is expressed in type-2a cells, in agreement with a recent report [25]. In the SGZ of 2 week-old mice, we found an average of 50–70 Tbr2+ cells per 30 mm-thick section throughout the rostro-caudal axis of the dorsal hippocam- pus. This number was reduced by 35% in 2 month-old mice (Figure 1C with figure S1C and data not shown). As opposed to Tbr1 (Figure 1D and figure S1D), Tbr2+ cells did not co-express Dcx or PSA-NCAM (Figure 1C and figure S1C). We observed that Tbr1-immuoreactivity gradually decreased as neurons matured and started to express NeuN (Figure 1H and figure S1H). Thus, Tbr2 labels type-2 cells, while Tbr1 is expressed by immature granule neurons. We then examined the molecular phenotype of type-3 cells, a cell type that transiently expresses Calretinin [26]. We hypothe- sized that hippocampal type-3 cells might also express the bHLH transcription factor NeuroD1, known to partially overlap with Tbr2 and Tbr1 in different brain regions [24]. We found that Hippocampal Neurogenesis NeuroD1 expression in the hippocampus starts in Tbr2+ cells and extends to post-mitotic Tbr1+ cells, which also express the hippocampal granule identity transcription factor Prox1 (Figure 1G and 1I and figure S1G and S1I). While Tbr1 expression ceases during granule maturation, that of NeuroD1 is weakly maintained when NeuN expression starts (Figure 1G and 1H and figure S1G and S1H). We found that calretinin expression decreased during granule neuron maturation, before NeuroD1 was reduced (Figure 1F). Thus NeuroD1 is expressed in type-2b and type-3 cells, as well as immature granule neurons. Finally, we found that NeuroD2 starts to be expressed just after NeuroD1 and, unlike NeuroD1, continues to be highly expressed in mature neurons (Figure 1J and data not shown). Mash1 and Ngn2 define the early versus late type-2a stage in 2 weeks old mice Having established the hierarchy of transcription factors and cellular markers appearing during hippocampal neurogenesis, we set out to clarify the position of the two bHLH transcription factors Ngn2 and Mash1, in this process. We found that more than 90% of Ngn2-immunoreactive cells are Pax6 positive (Figure 2A), and almost all co-express Tbr2 (Figure 2B). Thus, Ngn2 appears to be expressed by type-2a cells. As our antibodies against Ngn2 and NeuroD1 were made in the same species, we could not determine if there was an overlap between these two proteins. However, by comparing their overlapping expression pattern with that of Tbr2, we propose that some Ngn2+ cells co-express NeuroD1, and that they appear at the onset of NeuroD1 expression and downregu- lation of Ngn2. We base this assumption on the facts that more than 50% of the Tbr2+ cells are Ngn2+ and more than 50% of Tbr2+ cells also express NeuroD1. The expression of Ngn2 in adult hippocampus was hard to detect using immunohistochem- istry due to the low expression of the protein (data not shown). However, it is present, as previously described using reporter mice and as revealed by in situ hybridization [21]. We next examined the expression of Mash1 and compared it with that of Ngn2, Pax6 and Tbr2. We found some Mash1+ cells (10–20 cells per section) in the dorsal hippocampus. Almost all of them co-expressed Pax6 (Figure 2C). Only few cells co-expressed Tbr2 (Figure 2D) or Ngn2 (Figure 2E), suggesting that Mash1 is downregulated when these two proteins are expressed. None of the Mash1-expressing cells were positive for NeuroD1 (Figure 2F). Mash1+ cells were still undergoing mitosis (Figure 2C) and consequently over 90% of them co-labeled with the cell-cycle marker Ki67 (Figure S1J). Taken together, we show that Mash1 and Ngn2 are expressed in early and late stages of maturation of type2a progenitors, respectively, and that both transcription factors are co-expressed briefly when cells transit from early to late phase type2a cells. Ngn2 progenies become hippocampal granule neurons Previous work has shown that in Mash1 mutant mice hippocampal neurogenesis is not reduced and the hippocampus is not malformed [20]. Therefore we focused our initial analysis on the role of Ngn2 in hippocampal neurogenesis. We characterized the fate of Ngn2-expressing cells using Ngn2 Knock-in green fluorescent protein (GFP) mice (Ngn2+/GFP) [27]. In the SGZ of 2 week-old hippocampi, we found bright GFP-expressing cells that were positive for Tbr2 (Figure 2H), the immature neuronal marker PSA-NCAM (Figure 2I) and NeuroD1 (Figure 2J). Furthermore, Prox1+, NeuroD2+ and calretinin+ cells were also weakly positive for GFP. We made similar observations in 2 month-old mice (data not shown) confirming earlier published results [21]). We did not observe Mash1+/GFP+ and NeuN+/GFP+ cells (Figure 2G). These PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4779