Hematoxylin and eosin (HE) staining of coronal sections of adult hippocampus of (A) control and (B) Nestin-Cre;Prox1^F/F adult brains shows the absence of a dentate gyrus (DG) in the mutant mice. (C) Mature granule cells are C-Prox1⁺ and NeuN⁺. (D) In the Nestin-Cre;Prox1^F/F brain, the few remaining Prox1⁺ cells present in the hippocampal region are C-Prox1⁺ NeuN⁺ mature granule cells that escaped deletion (insert in D). (E, F) Only a few wild-type C-Prox1⁺ granule cells remain in the Nestin-Cre;Prox1^F/F brain at E16.5. (G, H) There is a reduction in the number of N-Prox1⁺ cells in the Nestin-Cre;Prox1^F/F brain at this stage, although these cells are still able to migrate outside the dentate neuroepithelium (DNE) (arrows). Although reduced, Notch1 (I, J) and Ngn2 (K, L) expression remains in the DNE and DG of Nestin-Cre;Prox1^F/F embryos as shown by ISH. Moreover, Notch1 and Ngn2 progenitors (arrows) migrate outside of the mutant DNE. (M, N) The NeuroD1 expression domain, although reduced, remains in the migratory stream (MS) and the DG of Nestin-Cre;Prox1^F/F embryos as shown by ISH. Scale bar: 100 µm. CA, Pyramidal cell layer; T, Thalamus. Scale bar: 100 µm.

(A) Prox1 is expressed in the dentate gyrus neuroepithelium (DNE) at E14.5. (B) Nestin-Cre successfully removed most Prox1 expression from the DNE at this stage. However, the expression pattern of Notch1 (C, D) and Ngn2 (E, F) is not changed in the DNE of Nestin-Cre;Prox1^F/F littermates. (G, H) Lhx2 is expressed in the developing dentate gyrus (DG) but not in the fimbria neuroepithelium (FNE, arrows) of control and Nestin-Cre;Prox1^F/F brains. (I, J) Wnt3a expression is detected in the DG and FNE of control and conditional mutant brains at this stage (arrows). (K, L) Calretinin expression in the marginal zone (MZ) is also normal in the Nestin-Cre;Prox1^F/F brain. Cx, cortex; T, thalamus; GE, ganglionic eminence. Scale bar in (A, B): 50 µm. Scale bar in (C–L): 100 µm.

(A, D) BrdU (after 1-h pulse) and Ki67 (B, E) immunostaining revealed reduced proliferation in the dentate gyrus neuroepithelium (DNE) of Nestin-Cre;Prox1^F/F brains starting at E16.5. Similar results were obtained at E18.5 (C, F, G, H). (I) Cells in the DNE of Nestin-Cre;Prox1^F/F embryos have a longer cell cycle as shown by double BrdU/Ki67 immunostaining. (J, K) Cyclin E⁺ cells are absent from the DNE of E16.5 Nestin-Cre;Prox1^F/F brains. (L) C-Prox1⁺ cells are normally found in the DNE and migratory stream (MS) of control brains at E18.5. Following TM administration at E16.5, there are fewer C-Prox1⁺ cells in the DNE and MS (M) of Nestin-CreER^T2;Prox1^F/F brains. (N) As indicated by Ki67 counting, the number of cycling cells is reduced in the DNE of Nestin-CreER^T2;Prox1^F/F embryos. Data represent the mean number of positive cells per DG section ± SD (N = 3 embryos). Paired t test. * p<0.1; ** p<0.01; **** p<0.0001. Scale bar in (A–M): 50 µm.

(A–E) As shown by double-staining with anti-N-Prox1 (red) and anti-C-Prox1 (green), the number of Δ-Prox1 cells (red bars in E) is reduced in the Nestin-Cre;Prox1^F/F dentate gyrus (DG) at postnatal stages of development (blue bars in E represent double N-Prox1/C-Prox1⁺ wild-type cells). (F) There is an increase in the number of TUNEL⁺ cells in the mutant DG as granule cell maturation progresses. IHC using anti-N-Prox1 and anti-NeuroD1 antibodies revealed that all granule cells are NeuroD1⁺ both in P10 wild-type (G) and conditional mutant (H) DG. However, in the conditional mutant hippocampal region most of the N-Prox1⁺ cells are Dcx⁻ (J) and Calretinin⁻ (L). The only NeuN⁺ cells left in the Nestin-Cre;Prox1^F/F hippocampal region are the C-Prox1⁺ cells that have escaped Cre deletion (N). Panels I, K, and M correspond to controls. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. ** p<0.01; *** p<0.001; **** p<0.0001. CA, Pyramidal cell layer. Scale bar: 100 µm.

Hematoxylin and eosin staining (A, B) of coronal sections of the hippocampus shows a reduced dentate gyrus (DG) in 2-mo-old Nestin-CreER^T2;Prox1^F/F mice treated with TM from P0 to P15 (B). There are no changes in the neural stem cell population at P10, as indicated by Nestin (C–E), Sox2 (F–H), and Id1 (I–K) IHC in the Nestin-CreER^T2;Prox1^F/F DG. BrdU staining after a 1-h pulse shows a similar level of proliferation in the DG of wild-type (L, N) and Nestin CreER^T2;Prox1^F/F brains (M, N). The number of Tbr2⁺ intermediate progenitors (O–Q) and Dcx⁺ (R, S) cells is reduced in the Nestin-CreER^T2;Prox1^F/F DG at P10. We also observed an increase in the number of TUNEL⁺ cells (T–V) in the DG area of the Nestin-CreER^T2;Prox1^F/F brains at P10. Data represent the mean number of positive cells per DG section ± SD (N = 3 mice). Blue bars are controls. Red bars are TM-treated Nestin CreER^T2;Prox1^F/F brains. Paired t test. * p<0.1; ** p<0.01. CA, pyramidal cell layer; T, thalamus; H, Hilus. Scale bar: 100 µm.

During adult neurogenesis, Prox1 is not expressed in Nestin⁺ (A), Sox2⁺ (B), or Id1⁺ (C) adult neural stem cells or in Acsl⁺ Type-IIa intermediate progenitors (D). Instead, Prox1 is expressed in Tbr2⁺ (E) and Dcx⁺ Type-IIb intermediate progenitors (F), Type-III intermediate progenitors (G), and Calretinin⁺ immature neurons. Graphs compare the number of Tbr2⁺ (K) Dcx⁺ (N) and Calretinin⁺ (Q) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER^T2;Prox1^F/F (red) mice. A reduced number of Tbr2⁺ (J, K), Dcx⁺ (M, N), and Calretinin⁺ (P, Q) cells was observed in the SGZ of 4-mo-old Nestin-CreER^T2;Prox1^F/F mice. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. * p<0.1; ** p<0.01; *** p<0.001; **** p<0.0001. GCL, granule cell layer; IML, inner molecular layer; M, month. Scale bar in (A–H): 25 µm. Scale bar in (I, J, L, M, O, P): 50 µm.

A reduced number of Tbr2⁺ intermediate progenitors (A–C) and of Dcx⁺ (D–F) and Calretinin⁺ (G–I) cells was observed in the SGZ of Nestin-CreER^T2;Prox1^F/F mice. An increase in the number of TUNEL⁺/Tbr2⁺ (J, M) and TUNEL⁺/Dcx⁺ (J, L) cells was observed in 12-wk-old and 16-wk-old (K) Nestin-CreER^T2;Prox1^F/F mice. As a consequence of the reduction in the number of intermediate progenitors, fewer BrdU⁺ cells are found in the DG of Nestin-CreER^T2;Prox1^F/F mice 4 (N–P) and 8 (Q–S) wk after the beginning of TM treatment. Data represent the mean number of positive cells per DG section ± SD. (N = 4 mice). Blue bars are controls. Red bars are TM-treated Nestin CreER^T2;Prox1^F/F brains. Paired t test. * p<0.1; ** p<0.01; *** p<0.001; **** p<0.0001. W, weeks. Scale bar: 100 µm. Scale bar in (L, M): 10 µm.

Nestin (B, C), Sox2 (E, F), and Id1/PECAM (H, I) immunostaining of the subgranular zone (SGZ) of 4-mo-old controls (B, E, H) and Nestin-CreER^T2;Prox1^F/F (C, F, I) mice treated with TM from P0 to P15. Graphs compare the number of Nestin⁺ (A), Sox2⁺ (D), and Id1⁺PECAM⁻ (G) cells in P20, 2-mo-old, 4-mo-old, and 8-mo-old control (blue) and Nestin-CreER^T2;Prox1^F/F (red) mice treated with TM from P0 to P15. At P20, the number of Nestin⁺, Sox2⁺, or Id1⁺PECAM⁻ NSCs is similar in controls and Nestin-CreER^T2;Prox1^F/F brains (A, D, G). However, after P20, the number of Nestin⁺ (A–C), Sox2⁺ (D–F), or Id1⁺PECAM⁻ (G–I) cells is reduced in Nestin-CreER^T2;Prox1^F/F mice. Graphs compare the number of Nestin⁺ (J), Sox2⁺ (M), and Id1⁺ PECAM⁻ (P) cells in 12-wk-old and 16-wk-old control (blue) and Nestin-CreER^T2;Prox1^F/F (red) mice treated with TM for 4 wk starting at 8 wk of age. The number of Nestin⁺, Sox2⁺, and Id1⁺ PECAM⁻ adult NSCs was increased in 12-wk-old conditional mutant brains (J, M, P). At 16 wk, the number of Nestin⁺ (J, L), Sox2⁺ (M, O), and Id1⁺ PECAM^- (P, R) cells is reduced in the SGZ of conditional-mutant mice. The percentage of Nestin⁺/b-Gal⁺ and Sox2⁺/b-Gal⁺ cells is similar in 12-wk-old (S) and 16-wk-old (T) control (blue) and Nestin-CreER^T2;Prox1^F/F (red) brains that carry the ROSA allele. However, the percentage of Tbr2⁺/b-Gal⁺ and Dcx⁺/b-Gal⁺ cells is reduced at both stages in the Nestin-CreER^T2;Prox1^F/F;ROSA SGZ (S, T). Data represent the mean number of positive cells per DG section ± SD. N = 4 brains. Paired t test. * p<0.1; ** p<0.01; *** p<0.001; **** p<0.0001. GCL, Ganglion cell layer. Scale bar in (B, C, E, F, H, I): 25 µm. Scale bar in (K, L, N, O): 100 µm. M, month; W, weeks. Scale bar in (Q, R): 50 µm.

(A) Prox1⁺/Jagged1⁺ cells in the SGZ of adult mice. (B) TM administration schedule. (C, D) Jagged1⁺ cells were not detected in 12-wk-old Nestin-CreER^T2;Prox1^F/F mice after 4 wk of TM treatment. (E) Nestin⁺Hes1⁺ cells are present in the SGZ of control and Nestin-CreER^T2;Prox1^F/F mice. However, the number of double positive cells is reduced in TM-treated Nestin-CreER^T2;Prox1^F/F in 12-wk-old (F) and 16-wk-old mice (G). Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. *** p<0.001. Scale bar in (A, E): 25 µm. Scale bar in (C, D): 100 µm.

Anti-C-Prox1 staining (A, control) shows a reduced dentate gyrus (DG) in P30 Nestin-Cre;JoJo(Prox1) mice (B, V). There is no difference in the number of Prox1⁺ cells in the DG region of control (C, E) and Nestin-Cre;JoJo(Prox1) (D, E) at E16.5. There are no differences either in number of Sox2⁺ (F) or Ki67⁺ (G) cells in the dentate neuroepithelium (DNE) and the DG at E16.5. At P8, there is an increase in the number of Dcx⁺ (H, I) and NeuN⁺ (J, L) cells in TM-treated Nestin-CreER^T2;Prox1^F/F brains. However, the number of Nestin⁺ (M, N) cells is reduced at this stage. (O) Cell fate analysis of the Prox1 ectopic-expressing cells, showing that they predominantly differentiate towards a neuronal lineage. The number of Nestin⁺ adult NSCs (P, Q, T) and Dcx⁺ intermediate progenitors (R, S, U) is reduced in the SGZ of P30 Nestin-Cre;JoJo-Prox1 animals. Data represent the mean number of positive cells per DG section ± SD. N = 3 brains. Paired t test. *** p<0.001. Blue bars are controls. Red bars are Nestin-Cre;JoJo-Prox1. MS, Migratory stream; HF, Hippocampal field. Scale bar: 100 µm.