from those we obtained when we transduced the embryonic brain with a viral vector expressing Ngn2. In this latter case, Ngn2 did not induce neurons and the transduced cells did not express NeuroD1. Therefore, Ngn2 appears to direct non-neuronal cell type specification in vivo [38–40]. Taken together, we have confirmed that NeuroD1 plays a key role in neuronal differenti- ation in the hippocampus, both during development and in the adult brain. Concluding remark We present a detailed classification of different stages in hippocampal neurogenesis. Our detailed molecular mapping of hippocampal neurogenesis allows for a more accurate analysis of how new factors stimulate neurogenesis at different steps in the development of granule neurons. Thereby we hope to facilitate the development of new agents, which stimulate endogenous progen- itors in the treatment of diseases. Materials and Methods Animal tissue preparation The creation of the Ngn2 transgenic mice was reported elsewhere (ref guillemot). Heterozygote male and female mice were crossed to obtain WT, heterozygote (Ngn2+/GFP) and mutant (Ngn2GFP/GFP) animals. Tails of the Ngn2 offspring were used to obtain DNA for determination of the genotype using a polymerase chain reaction (PCR) assay as previously reported [31]. As mutant Ngn2GFP/GFP do not survive longer than 2.5– 3 weeks after birth, Ngn2GFP/GFP and their littermates were sacrificed at the postnatal ages of two days (P2) or two weeks for this study. Neurogenesis was assessed in two weeks or two months old WT mice. Sprague Dawley pregnant rats were ordered from B&K Universal Ltd, Sollentuna, Sweden (hppt://www.bku.com). All animals were housed in groups with ad libitum access to food and water at a 12-h light/dark cycle. All experimental procedures conducted in this study had been approved by the Ethical Committee at Lund University. For immunohistochemical analysis, mice (from two weeks old and adult stage) and juvenile rats were sacrificed by transcardial perfusion with saline for 5–10 minutes, followed by 4% parafor- maldehyde (PFA) for 10 minutes. Brains were kept in PFA overnight at 4uC and subsequently cryopreserved in a 20–30% sucrose/0.1 M phosphate buffer solution until sectioning on a microtome apparatus (30 mm thickness sections, Microm Zeiss). Seven series of coronal sections were cut throughout the brain. Free-floating sections were preserved in antifreeze solution until immunohistochemistry was performed. Heads of postanatal two days old mice were decapitated and soaked in PFA 4% for 24 hours, at 4uC and transferred into sucrose solution until sectioning on cryostat apparatus (16 mm thickness; Leica CM3000). Sections were mounted on Superfrost glass slides and stored at 280uC until immunohistochemistry was performed. Cloning, subcloning, virus production and titer measurement The Moloney leukemia-derived retroviral vectors used in this study, pCMMP-IRES2eGFP-WPRE and pCMMP-Ngn2-IRE- S2eGFP-WPRE were previously described [40,49]. To generate the construct pCMMP-NeuroD1-IRES2eGFP-WPRE, mouse NeuroD1 cDNA was amplified from a pCS2+mtNeuroD1 plasmid (kindly provided by Professor Jackie Lee, Denver university, Boulder, USA) by PCR to introduce the restriction sites PmeI in 59 and XhoI in 39. Amplification of cDNA was performed as previously described [49]. The construction was verified by Hippocampal Neurogenesis enzymatic restriction and by DNA sequencing using BigDye 3.1 (ABI). All infectious particles were produced using the producer cell line 293VSV-G and as previously described [49]. The titer of each retrovirus was measured by flow-cytometry based on eGFP expression, four days following infection of HT1080 cells and ranged from 0.5610922.16109 TU/ml (All details on how to produce infectious particles can be provided upon request). Neurosphere generation, transduction and differentiation Pregnant female Sprague-Dawley rats (B & K Universal, Sollentuna, Sweden) were terminally anesthetized by an overdose of sodium pentobarbital (i.p., 60 ng/ml). Embryos at stage embryonic day E14.5 (Plug day as day 0) were collected and cortical-hippocampal neurospheres were generated following dis- section of the dorso-posterior part of the cortical tissue, and generated as previously described [49]. In this study, second passage (P2) neurospheres were used to study the effect of the overexpression of Ngn2 and NeuroD1 on neuronal differentiation. Each well, containing an equal starting population of 200,000 cells/ml, corresponding to 15–25 neurospheres, was transduced indepen- dently with each retrovirus at a multiplicity of infection (MOI) of 1, in proliferation medium supplemented by protamine-sulfate (4 mg/ ml, Sigma). To induce differentiation, the medium was replaced with normal basic differentiation medium two days post-transduc- tion and subsequently changed every other day until fixation. Immunocytochemistry, immunohistochemistry and microscopy The antibodies used in this study are: rabbit anti-GFAP (1:1000; DAKO), mouse anti-Nestin (1:100; BD PharMingen), guinea pig anti-Glast (1:500; Chemicon), rabbit anti-Prox1 (1:1000; Cov- ance), goat anti-Ngn2 (1:20; Santa Cruz), goat anti-NeuroD1 (1:200; Santa Cruz), rabbit anti-Pax6 (1:150; Covance), rabbit anti-Trb2 (1:500; Chemicon), rabbit anti-Trb1: (1:1000; Chemi- con), goat anti-Dcx: (1:500; Santa Cruz), mouse anti-PSA-NCAM: (1:500; Chemicon), mouse anti-NeuN (1:300; Chemicon), rabbit anti-Calretinin (1:500; Swant); mouse anti-Sox2 (1:100; R&D systems), mouse anti-Mash1 (1:100; BD PharMingen), rabbit anti- NeuroD2 (1:300; ABCAM), mouse anti-MAP2 (1:500; Sigma), rabbit anti-Ki67 (1:150; NovaCastra) and rabbit anti-phospho- Histone H3. The secondary antibodies (1:200) Cy2, FITC, Cy3 and Cy5 were from Jackson IR laboratories, Alexa-fluor 488, 568, 595 and 647 from Invitrogen-Molecular Probes. DAPI (1:1000) was purchased from Sigma. For immunocytochemistry, cultures were fixed in 4% parafor- maldehyde at day five, rinsed with PBS three times prior to pre- incubation with a blocking solution (10% donkey serum, 0.25% TritonX100 in PBS) for 1 hour. The remainder of the procedure was performed as previously described [49]. Specimen analyses were performed using a Leica confocal microscope (Leica software, equipped with a GreNe and a HeNe laser, using the following lines of excitation: 488 nm, 594 nm and 647 nm). Samples were analyzed using 206, 406 and 636 objectives, sometimes zoomed. Figures were composed in CANVAS-X software. Bromo-deoxyuridine (BrdU) and Cloro-deoxyuridine (CldU) pulse labeling and immunohisto- and immunocyto-chemistry To assess cell proliferation and ongoing neurogenesis in vivo, animals were injected with BrdU (100 mg/kg, Sigma), two hours prior to sacrifice (for both P2 and two weeks old Ngn2 mice). To assess neurogenesis, BrdU (100 mg/kg) was injected 48 hours PLoS ONE | www.plosone.org 15 March 2009 | Volume 4 | Issue 3 | e4779