Astrogenesis persists until adult stages of the DG.Figure 3A Experimental scheme of adult BrdU birth-dating paradigm used in B-E. Adult mice were administered with BrdU via drinking water for 12 consecutive days. For all experiments shown in B-H adult hGFAPeGFP mice were used. B Confocal image of an adult DG populated by distinct cells identified by cell type-specific protein expression and morphology. All cells newly-generated within the 12 days incorporated BrdU (magenta); rNSCs were identified by expression of SOX2 (cyan), localization of their cell bodies to the SGZ, and a GFAP+ radial process (green); IPCs were located within the SGZ and express SOX2 only (cyan); neuroblasts in the SGZ and GZ were labelled by DCX (white; arrows); astrocytes in the hilus, GZ, and ML (arrowheads) expressed SOX2 (cyan). C Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs + neuroblasts, astrocytes) in the DG. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C n = 5 ; dotted lines border SGZ and GZ; scale bars = 50 µm (B D Representative picture of astrocytes located to distinct DG layers (hilus GZ, ML). Co-expression of BrdU/SOX2/GFAP revealed that all DG compartments harboured astrocytes which were newly generated during adulthood (arrowheads). E Quantification of the proportions of generated astrocytes per DG layer out of all generated DG astrocytes. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): E) n = 5, dotted lines border GZ; scale bars = 50 µm D, F Representative image of proliferating astrocytes (SOX2+/MCM2+; cyan and magenta, respectively; arrowheads) of all DG layers. G Quantification of the percentages of proliferating astrocytes in distinct DG layers over all proliferating DG astrocytes. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (G) n = 4, dotted lines border GZ; scale bars = 50 µm F, H Confocal images showing GFP+/GFAP+/SOX2+ astrocytes of distinct DG layers co-expressing the proliferation marker MCM2 Data information: scale bars = 10 µm (H I Scheme illustrating adult fate-mapping experiment using NestinCreERT2; GFP mice to monitor rNSCs and their generated astrocytic progeny. Two potential modes of adult astrogenesis and their lineage relation are depicted: (1) rNSC-derived astrogenesis, (2) astrocyte-derived astrogenesis. J Tamoxifen induction protocol used in K-N. Adult NestinCreERT2; GFP mice were intraperitoneally injected with tamoxifen for 10 times at 5 consecutive days (12 h apart) and killed 12 dpi. K Representative image of recombined cells (GFP+, green) of different cellular identity: rNSCs [SOX2+ (cyan) with a NESTIN+ radial process (white)], IPCs (SOX2+ nuclei in the SGZ; cyan), DCX+ neuroblasts in the SGZ and GZ (magenta), and astrocytes (SOX2+ cells in the hilus, GZ, and ML; cyan). L Quantification of the proportions of recombined cell types over all recombined DG cells (rNSCs, IPCs + neuroblasts, astrocytes). Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (L n = 3; SGZ and GZ; scale bars = 50 µm K, M Quantification of the percentages of rNSC-derived astrocytes in distinct DG layers over all recombined DG astrocytes. N Confocal images of proliferating rNSC-derived astrocytes (GFP+/SOX2+/MCM2+; arrowheads) in hilus and GZ. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): M) n = 3 scale bars = 10 µm N) O Experimental timeline of in vivo two-photon imaging hGFAP-RFP. AAV2/9-hsyn-jGCaMP7 virus served to provide neuronal landmarks and was injected immediately before the implantation of the hippocampal imaging window. Repetitive imaging was performed 28 days post injection/implantation. P Scheme of hippocampal imaging window facilitating optical access to the DG and even hilus. Q Representative examples of field views captured within the hippocampal fissure (indicated by the abundant large vessels), the GZ with its densely packed granule neurons and the hilus containing much larger neuronal somata; expression of GCaMP7 virus (green), labelling of astrocytes by hGFAP-RFP (red). Data information: scale bars = 50 µm Q, R Examples of locally dividing astrocytes (arrows) derived from two experimental animals. Arrowheads depict landmarks that served for identification. Data information: scale bars = 50 µm R). dpi = days post injection.