Jagged1 is necessary for postnatal and adult neurogenesis in the dentate gyrus

Abstract

Understanding the mechanisms that control the maintenance of neural stem cells is crucial for the study of neurogenesis. In the brain, granule cell neurogenesis occurs during development and adulthood, and the generation of new neurons in the adult subgranular zone of the dentate gyrus contributes to learning. Notch signaling plays an important role during postnatal and adult subgranular zone neurogenesis, and it has been suggested as a potential candidate to couple cell proliferation with stem cell maintenance. Here we show that conditional inactivation of Jagged1 affects neural stem cell maintenance and proliferation during postnatal and adult neurogenesis of the subgranular zone. As a result, granule cell production is severely impaired. Our results provide additional support to the proposal that Notch/Jagged1 activity is required for neural stem cell maintenance during granule cell neurogenesis and suggest a link between maintenance and proliferation of these cells during the early stages of neurogenesis.

Keywords: Dentate gyrus; Jagged1; Progenitors.

FIGURE 1. Jagged1 is necessary for postnatal-DG development

Prox1 staining of coronal sections of the hippocampus of P30 control (A), Nestin-CreER^T2;Jagged1^F/F (B), and Nestin-CreER^T2;Prox1^F/F (C) mice. The DG was smaller in Nestin-CreER^T2;Jagged1^F/F mice (B, D) and Nestin-CreER^T2;Prox1^F/F mice (C, D) than in controls. Fewer Nestin⁺ cells (E–H), Tbr2⁺ cells (I–L), and Calretinin⁺ cells (M–P) were observed in the DG of Nestin-CreER^T2;Jagged1^F/F mice (F, J, N) and Nestin-CreER^T2;Prox1^F/F mice (G, K, O) than in controls (E, I, M). Data in D, H, L, and P represent the number of positive cells ± s.d. per section. N=3 pups. Paired t test. (*) p<0.1; (**) p<0.01; (***) p<0.001; (****) p<0.0001.

FIGURE 2. Jagged1 is expressed in Gfap⁺ and Dcx⁺ cells during postnatal dentate gryus development

Double Jagged1/Gfap (A), Jagged1/Dcx (B) and Jagged1/Calretinin (CR) (C) IHCs of P10 postnatal dentate gyrus with corresponding orthogonal views. Jagged1 is expressed in Gfap⁺ (A) and Dcx⁺ (B) cells at P10. However, no Jagged1⁺/CR⁺ (C) cells were found at this stage.

FIGURE 3. Loss of Jagged1 function affects postnatal-DG neurogenesis without increasing cell death

TM-induction protocols were used to generate the TM0-5, TM0-10, and TM5-10 Nestin-CreER^T2;Jagged1^F/F P10 pups (A). Prox1 staining of coronal sections of the hippocampus of control (B), TM0-5 (C), TM0-10 (D), and TM5-10 (E) Nestin-CreER^T2;Jagged1^F/F pups shows various reductions in DG size (F). No increase in TUNEL⁺ cells was observed in any of the Nestin-CreER^T2;Jagged1^F/F pups (G). Fewer Nestin⁺ radial glia-like cells were observed in all of the conditional-mutant Nestin-CreER^T2;Jagged1^F/F (red bars) pups (H) compared to controls (blue). Fewer Sox2⁺ cells (I), Tbr2⁺ cells (J), and Calretinin⁺ cells (K) were observed in TM0-5 and TM0-10 Nestin-CreER^T2;Jagged1^F/F pups at P10. The number of Sox2⁺ and Tbr2⁺ observed in TM5-10 mice was comparable to that in controls (I, J), while the number in Calretinin⁺ cells was increased (K). CldU/IdU double-labeling (L) was combined with immunohistochemical analysis to study the maintenance and proliferation of the Nestin⁺ (M, N, O) and Sox2⁺ (P, Q, R) cell populations (see A for details). Nestin⁺ and Sox2⁺ cells are less maintained and re-enter S-phase less frequently in the Nestin-CreER^T2;Jagged1^F/F pups (M–R). Data in F–K represent the number of positive cells ± s.d. per section. Data are represented as the number of Nestin⁺ or Sox2⁺ cells that are IdU⁺, CldU⁺ or CldU⁺/IdU⁺ in the Nestin⁺ or Sox2⁺ population ± s.d. per section. N=3 pups. t test (*) p<0.1; (**) p<0.01; (***) p<0.001.

FIGURE 4. Jagged1 is expressed in Gfap⁺ and Dcx⁺ cells in the adult dentate gyrus

Double Jagged1/Gfap (A), Jagged1/Dcx (B) and Jagged1/Calretinin (CR) (C) IHCs of adult SGZ with corresponding orthogonal views. Jagged1 is expressed in Gfap⁺ (A) and Dcx⁺ (B) cells ; however, no Jagged1⁺/CR⁺ (C) cells were found.

FIGURE 5. Jagged1 inactivation results in a transient increase in adult-SGZ neurogenesis

TM-induction protocol used to generate 12-week-old Nestin-CreER^T2;Jagged1^F/F mice (A). Jagged1 expression is reduced in the treated Nestin-CreER^T2;Jagged1^F/F mice (B). The number of Tbr2⁺ cells (C), Dcx⁺ cells (D), and Calretinin⁺ cells (E) was higher in the 12-week-old Nestin-CreER^T2;Jagged1^F/F mice (red bars) than in controls (blue bars). CldU/IdU double-labeling was performed as shown in (A). The number of IdU⁺ cells (F) was higher, and that of CldU⁺ cells (G) was comparable in the 12-week-old Nestin-CreER^T2;Jagged1^F/F mice compared to that in controls. Furthermore, the percentage of Tbr2⁺/IdU⁺ cells (grey bars, H), Dcx⁺/CldU⁺/IdU⁺ cells (light grey bars, I), Dcx⁺/IdU⁺ cells (grey bars, I), and Calretinin⁺/IdU⁺ cells (grey bars, J) was higher in the 12-week-old Nestin-CreER^T2;Jagged1^F/F mice (right bars) than in controls (left bars). The percentage of Dcx⁺/CldU⁺ cells (black bars, I) and Calretinin⁺/CldU⁺ cells (black bars, J) in the 12-week-old Nestin-CreER^T2;Jagged1^F/F mice was also higher. TM-induction protocol used to generate 16-week-old Nestin-CreER^T2;Jagged1^F/F mice (K). Fewer Tbr2⁺ cells (L) were detected in the 16-week-old Nestin-CreER^T2;Jagged1^F/F mice, but the number of Dcx⁺ cells (M) was similar to that in controls. Data in C–G represent the number of positive cells ± s.d. per 1000 DAPI-labeled cells. Data in H–J represent the percentage of Tbr2⁺ cells (H), Dcx⁺ cells (I), and Calretinin⁺ cells (J) that are also CldU⁺/IdU⁺ (light grey bars), IdU⁺ (grey bars), or CldU⁺ (black bars) ± s.d. per section. White bars represent the single-labeled cell population in H–J. N=3 mice. t test. In the multiple-bar graphs, asterisks represent the significance of the black bars; daggers, the grey bars; and hash marks, the light grey bars. (*, †, or #) p<0.1; (**) p<0.01; (***) p<0.001.

FIGURE 6. The transient increase in neurogenesis in the Nestin-CreER^T2;Jagged1^F/F mice is accompanied by a reduction in the Nestin⁺ and Sox2⁺ cell populations

TM-induction protocol used to generate 12-week-old Nestin-CreER^T2;Jagged1^F/F mice (A). Fewer Nestin⁺ cells were observed in the 12-week-old Nestin-CreER^T2;Jagged1^F/F mice (B, red bars) than in controls (B, blue bars). However, the number of Sox2⁺ cells was comparable (C). CldU/IdU double-labeling was performed, as shown in (A). The percentage of Nestin⁺/IdU⁺ cells was lower in the 12-week-old Nestin-CreER^T2;Jagged1^F/F mice (right) than in controls (left) (grey bars, D); furthermore, that of Sox2⁺/CldU⁺/IdU⁺ cells (light grey bars, E) and Sox2⁺/IdU⁺ cells (grey bars, E) was higher, and that of Sox2⁺/CldU⁺ cells (black bars, E) was lower. TM-induction protocol used to generate 16-week-old Nestin-CreER^T2;Jagged1^F/F mice (F). Fewer Nestin⁺ cells (G) and Sox2⁺ cells (H) were detected in the 16-week-old Nestin-CreER^T2;Jagged1^F/F mice than in controls. Data in B, C, G, H represent the number of positive cells ± s.d. per 1000 DAPI-labeled cells. Data in D and E represent the percentage of Nestin⁺ cells (D) and Sox2⁺ cells (E) that are also CldU⁺/IdU⁺ (light grey bars), IdU⁺ (grey bars), or CldU⁺ (black bars) ± s.d. per section. White bars represent the single-labeled population in H and I. N=3 mice. t test. In the multiple-bar graphs, the asterisks represent the significance of the black bars; daggers, the grey bars; and hash marks, the light grey bars. (* or #) p<0.1; (** or ††) p<0.01; (***) p<0.001.

FIGURE 7. RbpJ and Jagged1 functional inactivation affects neurogenesis in a similar way

Prox1 staining of coronal sections of the hippocampus of control (A) and TM0-10 Nestin-CreER^T2;RbpJ^F/F pups (B) shows a reduction in DG size (C) without an increase in TUNEL⁺ cells (D) at P10. Fewer Nestin⁺ cells (E), Sox2⁺ cells (F), Tbr2⁺ cells (G), and Calretinin⁺ cells (H) were observed in the Nestin-CreER^T2;RbpJ^F/F pups (red bars) than in controls (blue bars). CldU/IdU double-labeling, with CldU at P5 and IdU 1h before collection at P10, was combined with immunohistochemical analysis to study the proliferation (I) and maintenance (J, K) of the Nestin⁺ radial glia-like cell population. Less Nestin⁺/ldU⁺ (I), Nestin⁺/CldU⁺ (J) and Nestin⁺/CldU⁺/IdU⁺ (K) cells were observed in the Nestin⁺ population of Nestin-CreER^T2;RbpJ^F/F pups than in controls. After TM adult induction, less Nestin⁺ (L), Sox2⁺ (M), or Dcx⁺ (N) cells were observed in the Nestin-CreER^T2;RbpJ^F/F pups than in controls Data in panels C-N represent the number of positive cells ± s.d. per section. N=3 pups. t test. (*) p<0.1; (**) p<0.01; (***) p<0.001; (****) p<0.0001.