Hippocampal Neurogenesis Figure 6. Ngn2 induces NeuroD1 expression, in vitro. (C1–G2) Indirect immunofluorescence performed on cortico-hippocampal, ventral mesencephalic and lateral ganglionic eminence neurospheres derived from tissue of E14.5 rat embryos. (A) Schematic representation of the retroviral constructs used in this study. (B) Schematic representation of the experimental design. (C1–C4) Upon five days of differentiation, Ngn2- and NeuroD1- transduced cells (GFP+) downregulate the immature neural marker Sox2 (red), as compared to control transduced cells. (D1–D4) Ngn2 and NeuroD1 overexpression induce Tbr1 expression and MAP2. Data in C4 and D4 are expressed as 6SEM; *** P,0.0001 (E1–F) Overexpression of Ngn2 in cortico- hippocampal and ventral mesencephalic neurospheres induces the expression of NeuroD1 and leads to neuronal differentiation, as marked by the expression of PSA-NCAM (red). (G1–G2) The ectopic overexpression of Ngn2 induces ectopic expression of NeuroD1 and neuronal differentiation in LGE neurospheres. (H) Schematic illustration of molecular events upon Ngn2 induction, in vitro. Scale bars: 50 mm (C1–3, D1–3, E1–G2). doi:10.1371/journal.pone.0004779.g006 Sequential expression of different transcription factors and cellular markers during hippocampal neurogenesis We clarified the pattern of expression of various markers expressed during postnatal and adult hippocampal granule neurogenesis in detail. We found that the transcription factor Pax6 is initially expressed by both radial glia stem cells (type-1) and early amplifying progenitors (type-2a). The next transcription factor to appear in chronology is Mash1. Mash1 expression characterizes the early stage of hippocampal progenitor amplifi- cation (early type-2a). The role of Mash1 during hippocampal granule neurogenesis is still unclear. Mash1 mutant mice do not display any clear malformation of the DG [20]. However, overexpression of Mash1 alone in the DG leads to the generation of oligodendrocytes [36]. Based on these observations, we hypothesize that Mash1-expressing cells are not yet committed towards a granule cell fate. They may still have the potential to generate both neurons and oligodendrocytes, as is the case in the SVZ [37]. In agreement with different developing brain regions, we found that Ngn2 starts to be expressed in Mash1-positive transiently amplifying progenitors, i.e. ‘‘late type-2a cells’’ according to the classification we propose. We observed that Pax6 expression persisted longer than that of Mash1, in Ngn2-expressing cells (late type-2a cells), in juvenile but not adult DG (Fig. 1 and figure S1). Later on, Ngn2 is downregulated, whilst Tbr2 expression persists (type-2b cells). The transition from amplifying progenitor to neuroblast is defined by the expression of NeuroD1. Thus, Ngn2 is expressed at the beginning of the transiently amplifying progenitor phase while NeuroD1 marks the end of that period. This applies both to the juvenile and adult rat brain [21,25]. After NeuroD1 is turned on the progenitors leave the cell cycle, gradually mature, express PSA-NCAM, NeuroD2, Calretinin, Prox1, Tbr1 and, finally, NeuN. NeuroD1 expression persists at low levels in mature neurons (Figure 9; [10]). We found the sequential expression of these markers conserved in juvenile (P2 and 2 weeks) and adult rodent brains. Importantly, we observed that the same transcrip- tion factors and neuronal markers are also present in the adult human hippocampus and arranged spatially in a manner reminiscent with what we saw in rodents. It is possible, however, that some of these markers are expressed for longer or shorter periods at postnatal and adult stages of hippocampal progenitor maturation. Moreover, one can ask to which extent the number of divisions occurring during neuroblasts maturation is conserved at postnatal and adult stages of neurogenesis. A new function for Ngn2 in maintenance of a progenitor state for granule neuron production/amplification during embryonic and postnatal hippocampal neurogenesis In agreement with earlier work, we confirm that Ngn2 is indispensable for hippocampal development and plays a vital role in postnatal hippocampal granule neurogenesis. Ngn2 mutant animals display a reduced size of the cornu ammonis and a malformed DG (Figure 3 and 4; [20]). We show that the number of newborn neurons (incorporated BrdU) is decreased in absence of Ngn2, but hippocampal granule neuron subtype specification is not affected, post-nataly. Likewise, neurons from other brain regions, e.g. ventral midbrain dopamine neurons, can be generated in the absence of Ngn2 [30,31]. The reduced numbers of mature neurons in the hippocampus and ventral midbrain of Ngn2 mutant animals does not appear to be due to cell death or migration defects [20,31]. Instead it may be due to a defect in the generation and/or amplification of neuronal progenitors. Indeed, in the hippocampus of postnatal animals, we observed that cells lacking Ngn2 arrest in the cell cycle, maintain Pax6 expression and cease to proliferate. When overexpressed in neural progenitors in vitro, Ngn2 lead to up-regulation of NeuroD1 and caused neuronal differentiation (Figure 6), which was not always the case when we overexpressed the same factor in vivo (Figure 7L, N and O). We also saw that Ngn2 does not always efficiently induce cell cycle exit. Typically some mitotically active eGFP+ cells were present in the DG of rats injected with the Ngn2 retrovirus (Figure 7P), and we observed that cultured Ngn2-transduced cells are still capable of dividing. Thus, Ngn2 does not always promote cell cycle exit and neuronal commitment, but depending on the state of the cells, Ngn2 may instead promote alternate cellular fates [38–40]. Indeed, an earlier study has shown that Ngn2-overexpressing progenitors generate oligodendrocytes when grafted to the adult spinal cord [40]. We have also seen that Ngn2-overexpressing neural progenitors from the embryonic midbrain form astrocytes when grafted to the striatum (unpublished observations). Probably, Ngn2 cannot influence already committed cells, but rather would control neuroblasts production. One could speculate that Ngn2 oscillates during granule neuron formation, as recently demonstrated during neocorticogenesis [41,42]. NeuroD1, but not Ngn2, is obligatory for granule neuron progenitor differentiation Hippocampal granule progenitors can mature and express Tbr1 and Prox1, both during the development and postnatally, even in the absence of Ngn2. Because neuronal differentiation still occurs in Ngn2 mice, it is clear that compensatory, Ngn2- independent mechanisms induce NeuroD1 expression. One candidate is Ngn1, which is expressed during neocorticogenesis, the specification of olfactory sensory neurons and during embryonic rat hippocampal development [43–47]. Both Ngn1 and Ngn2 regulate NeuroD1 [19,48]. The introduction of two mutant forms of Ngn1, a deletion of the basic region of Ngn1 and a substitution of two amino acids in the C-terminal basic region, prevents NeuroD1 expression and neuronal differentiation [48]. The double Ngn1/Ngn2 mutant displays a more severe phenotype than single gene (Ngn1 or Ngn2) mutant mice. For example, the total brain size of the double mutants is much smaller [10,32]. If the role of Ngn1 during hippocampal neurogenesis is to activate NeuroD1 and that of Ngn2 is to control granule neuroblasts production/amplification, the DG of double Ngn1/ Ngn2 knockout mice should resemble that of NeuroD1 mutant mice. PLoS ONE | www.plosone.org 11 March 2009 | Volume 4 | Issue 3 | e4779