Figure 3. Ablation of Tbr2 results in loss of INPs and impairs postnatal and adult neurogenesis. A, TAM was administered to mice on P5 and P6, and animals were collected on P19. In control mice (Nestin–CreERT2;Tbr2flox/+;Z/EG), Tbr2 protein is present in the SGZ as expected (B, B1), whereas in Tbr2 icKO mice (Nestin–CreERT2;Tbr2flox/flox;Z/EG) it is essentially absent (C, C1). Highlighted regions in dashed boxes in B and C are shown in higher magnification in B1 and C1, respectively. Markers of INPs (NeuroD1) and neuroblasts (Prox1) are almost completely absent in Tbr2 icKO mice (E, G), whereas they are abundant in controls (D, F). Expression of calretinin (CalR) in new neuroblasts in the SGZ is strongly decreased in mutant mice (G, blue), as is expression of Prox1 (G, red). Use of a reporter strain to monitor the fate of recombined cells (Z/EG, GFP+ cells) shows reduced GFP+ cells with neuronal morphology in Tbr2 icKO mice (C, E, G, I), and fewer GFP+ cells are NeuroD1+ (E) or Prox1+ (G) in mutant mice. At P19, most GFP+ cells are NeuN+ new neurons in control mice (H; region highlighted in dashed box is shown in high magnification in H1), whereas very few GFP+ cells colocalize with NeuN in mutant mice (I, I1, arrows). High-magnification image of cells in Tbr2 icKO mice shows that most GFP+ cells do not have typical neuronal morphology (I1), whereas many GFP+ cells have typical granule neuron morphology in controls (H, H1, arrows). J, Schematic diagram of TAM and BrdU administration to adult mice. After administration of TAM, adult animals were collected at P12W, P14W, and P18W. K–L1, Tbr2 protein expression is present in the SGZ of control animals (Nestin–CreERT2;Tbr2flox/+) at P12W (K, K1, arrows) but is essentially absent from Tbr2 icKO mice (Nestin–CreERT2;Tbr2flox/flox) at this time (L, L1). M–O1, DCX staining of type-3 INPs and new neuroblasts in the SGZ remains consistent between P12W and P18W in control mice, with only a slight apparent reduction attributable to an age-related decline in neurogenesis (M–O, arrows). In Tbr2 icKO mice, DCX+ INPs and neuroblasts are reduced by P12W and continue to decline through P14W (M1–N1, arrows). By P18W, very few DCX+ INPs and neuroblasts remain in Tbr2 icKO mice (O1). P, P1, By P14W, NeuroD1+ INPs and neuroblasts are depleted from the SGZ in Tbr2 icKO mice. Q, Quantification of the total number of NeuroD1+ cells in control and mutant animals confirms decreased numbers of INPs and neuroblasts in Tbr2 icKO mice at P12W and P14W (n = 3, ANOVA, p < 0.01). R–T, BrdU pulse-chase (administered according to the schedule in J) shows that, in control mice, most BrdU+ cells colocalize with NeuN at P14W (R), whereas the proportion of BrdU+ cells coexpressing NeuN is reduced in Tbr2 icKO mice (S, T, orange bars; t test, p < 0.01, n = 4). U, Quantification of the total number of AC3 shows that the number of cells undergoing apoptosis in the SGZ does not differ between control and Tbr2 icKO mice at any stage examined (P19, P12W, P18W; ANOVA, p > 0.05, n = 3). Graphs represent the mean ± SEM for each group. **p < 0.01. Scale bars: B, D, H, K, M, P, 50 μm; B1, H1, K1, 20 μm; R, 100 μm.