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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Dev Biol. 2014 Feb 13;388(1):11–21. doi: 10.1016/j.ydbio.2014.02.004

JAGGED1 IS NECESSARY FOR POSTNATAL AND ADULT NEUROGENESIS IN THE DENTATE GYRUS

Alfonso Lavado 1,*, Guillermo Oliver 1,*
PMCID: PMC4009513  NIHMSID: NIHMS569127  PMID: 24530424

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: Jagged1, progenitors, dentate gyrus

INTRODUCTION

The formation of the dentate gyrus (DG) is a complex process that involves cell migration and neurogenesis during prenatal and postnatal stages of development (Pleasure et al., 2000; Li and Pleasure, 2005). Neurogenesis also occurs in the subgranular zone (SGZ) of the DG throughout adulthood (Altman and Das, 1965; Altman and Das, 1967; Kuhn et al., 1996; Eriksson et al., 1998). Molecules that regulate the development of this brain region are thought to have a similar function during adult-SGZ neurogenesis. Multiple signaling molecules, including Wnt, Noggin/BMP, Shh, and Notch, regulate neural stem cell maintenance, proliferation, and differentiation in the adult SGZ (Alvarez-Buylla and Lim, 2004; Doe, 2008; Fuentealba et al., 2012).

Notch signaling has recently emerged as a key player during postnatal- and adult-SGZ neurogenesis (Ables et al., 2011). It is well established that the Notch pathway regulates neural progenitor maintenance during embryonic development (Louvi and Artavanis-Tsakonas, 2006; Pierfelice et al., 2011), is necessary for proper proliferation during postnatal-DG neurogenesis (Breunig et al., 2007), and is required for neural stem cell maintenance during adult-SGZ neurogenesis (Ables et al., 2010; Ehm et al., 2010; Lugert et al., 2010). Furthermore, Notch signaling modulates dendrite morphology in newborn neurons (Breunig et al., 2007) and is required for synaptic plasticity in the hippocampus (Alberi et al.). In the adult subventricular zone (SVZ), canonical Notch signaling maintains both neural stem cells (Imayoshi et al., 2010) and ependymal cell quiescence (Carlen et al., 2009).

Notch signaling also has been considered a candidate for coupling neural stem cell maintenance and cell production during adult neurogenesis (Alvarez-Buylla and Lim, 2004; Pierfelice et al., 2011). In the adult SVZ, a non–cell autonomous EGFR/Notch mechanism may control the balance between neural stem cells and intermediate progenitor cells (Aguirre et al., 2010). Recently, we reported that the lack of Prox1 during adult-SGZ neurogenesis reduces the survival of Jagged1-expressing cells and eventually leads to the depletion of the stem cell population (Lavado et al., 2010). This result suggests that a Notch/Jagged1 mechanism is necessary for neural stem cell maintenance in the adult SGZ. Jagged1 is a canonical Notch ligand widely expressed during brain development and in the adult brain (Lindsell et al., 1996; Stump et al., 2002; Irvin et al., 2004; Breunig et al., 2007). Jagged1 is expressed in the SVZ (Nyfeler et al., 2005; Carlen et al., 2009) and promotes neural stem cell maintenance in SVZ cultures (Nyfeler et al., 2005). However, no data are yet available on the functional role(s) of Jagged1 during DG development and adult-SGZ neurogenesis.

Here we have analyzed the role of Jagged1 during DG development and adult-SGZ neurogenesis. We determined that functional inactivation of Jagged1 during postnatal-DG development results in a smaller DG, consequence of defective neural stem cell maintenance and proliferation. We also report that conditional inactivation of Jagged1 during adult-SGZ neurogenesis depletes the neural stem cell population and ultimately hinders neurogenesis. We identified Jagged1 as a critical ligand for Notch-dependent neural stem cell maintenance during postnatal- and adult-SGZ neurogenesis. Together, our results reinforce the idea of an important role for Notch/Jagged1 in the maintenance of neural stem cells during adult-SGZ neurogenesis. It also suggests of a link between neural stem cell maintenance and proliferation during early stages of neurogenesis.

MATERIALS AND METHODS

Mice and tamoxifen treatment

Jagged1F/F (Mancini et al., 2005), Prox1F/F (Harvey et al., 2005), RbpJF/F (Han et al., 2002), and Nestin-CreERT2 (Cicero et al., 2009) mice have been previously described. Mice were kept in the NMRI background. TM (Sigma, St. Louis, MO) was dissolved in safflower oil at 20 mg/ml. To induce Cre recombination during embryonic development, pregnant dams were administered orally TM (2mg/20g body weight) at E12.5. To induce Cre recombination postnatally, pups were fed TM (4 mg/20 g body weight) daily from P0 until P10. In adults (8-week-old), TM (4 mg/20 g body weight) was administered by gavage 3 times/week for 4 weeks. Genotypes were determined by PCR analysis. Nestin-CreERT2;Jagged1F/+, Nestin-CreERT2;Prox1F/+ and Nestin-CreERT2;RbpJF/+ mice were indistinguishable from wild-type mice and thus were used as controls.

Immunohistochemistry

Immunohistochemical analysis of Jagged1 and the other proteins was performed as described (Lavado et al., 2010). The following antibodies and dilutions were used: rabbit anti-Prox1 (1:1000; Millipore, Billerica, MA), goat anti-Prox1 (1:100; R&D, Minneapolis, MN), goat anti-Nestin (1:100; R&D), rabbit anti-Sox2 (1:500; Invitrogen, Carlsbad, CA), rabbit anti-GFAP (1:1000, Dako), rabbit anti-Hes1 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Tbr2 (1:250; Abcam), rabbit anti-Dcx (1:250; Abcam), rabbit anti-Calretinin (1:5000; Millipore), goat anti-Jagged1 (1:100; R&D), and goat anti-Jagged1 (1:50, Santa Cruz Biotechnology). The following secondary antibodies were used: anti-rabbit, anti-mouse, or anti-goat Alexa 488, Alexa 594 (Invitrogen), Cy3 or Cy5 (Jackson Immunoresearch, West Grove, PA). Low-magnification images were obtained with a Leica MZFLIII stereomicroscope equipped with a ProgRes C14 camera and a Zeiss Axiovert 1.0 microscope equipped with an Axiocam MRm. The remaining images were obtained with a Zeiss LSM 510 NLO Meta confocal microscope.

TUNEL and proliferation assays

TUNEL assays were performed on tissue sections, as previously described (Lavado et al., 2008). For proliferation assays at early postnatal stages, P5 pups were injected with CldU (50 μg/g body weight, subcutaneous) (Sigma, St Louis, MO). P10 pups were injected with IdU (50 μg/g body weight, subcutaneous) (Sigma) 1 h before harvest. Brains were perfused with 4% paraformaldehyde (PFA) and cryoprotected in 30% sucrose. For proliferation assays at adult stages, animals were injected with IdU or CldU (50 μg/g body weight, subcutaneous) at different time points. Brains were perfused in 4% PFA and cryoprotected in 30% sucrose. IdU/CldU incorporation was exposed after 15-min treatment in 2N HCl. Mouse anti-BrdU (1:10; BD Pharmigen, San Diego, CA) antibody was used to reveal IdU, and rat anti-BrdU (1:500; Accurate Chemical & Scientific Corporation, Westbury, NY) antibody was used to detect CldU. Sections were counterstained with DAPI to label cell nuclei.

RESULTS

Conditional deletion of Jagged1 during postnatal neurogenesis results in a smaller dentate gyrus

Standard Jagged1-null embryos die at embryonic day (E) 10.5 (Xue et al., 1999); therefore, to evaluate the possible functional roles of Jagged1 during DG development we used a conditional-inactivation approach. Jagged1-floxed mice (Mancini et al., 2005) were bred with Nestin-CreERT2 mice (Cicero et al., 2009), and tamoxifen (TM) was administered to pregnant dams at embryonic (E) day E12.5 and embryos were collected at E14.5 and E16.5. However, no abnormalities in DG development were observed at those stages in the absence of Jagged1 (Fig. S1). Next, we analyzed the role of Jagged1 during postnatal stages of DG development by administering TM daily to controls and Nestin-CreERT2;Jagged1F/F pups from postnatal day (P) 0 to P10. Pups analyzed at P30 revealed that TM-treated Nestin-CreERT2;Jagged1F/F mice had a smaller DG (Fig. 1B, D) than control littermates (Fig. 1A, D). We have previously shown that postnatal deletion of Prox1 reduces the number of Jagged1+ cells and results in a smaller DG (Lavado et al., 2010) (Fig. 1C, D). Interestingly, at P30 both TM-treated Nestin-CreERT2;Jagged1F/F and Nestin-CreERT2;Prox1F/F had comparable small DGs (Fig. 1D). Therefore, to identify a possible functional correlation between Prox1 and Jagged1, we next analyzed whether Nestin-CreERT2;Jagged1F/F pups exhibited defects similar to those identified in Nestin-CreERT2;Prox1F/F pups at P30. This analysis revealed that the number of Nestin+ radial glia-like cells was reduced in both mutant strains (Fig. 1E–H). Similar results were obtained with the intermediate progenitor marker Tbr2 (Fig. 1I–L) and the immature neuron marker Calretinin (Fig. 1M–P). These results demonstrated that postnatal deletion of Jagged1 or Prox1 leads to a similar phenotype at P30.

FIGURE 1. Jagged1 is necessary for postnatal-DG development.

FIGURE 1

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Prox1 staining of coronal sections of the hippocampus of P30 control (A), Nestin-CreERT2;Jagged1F/F (B), and Nestin-CreERT2;Prox1F/F (C) mice. The DG was smaller in Nestin-CreERT2;Jagged1F/F mice (B, D) and Nestin-CreERT2;Prox1F/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-CreERT2;Jagged1F/F mice (F, J, N) and Nestin-CreERT2;Prox1F/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.

In the postnatal SGZ, Jagged1 is detected in Gfap+ radial glia-like cells (Fig. 2A) As neurogenesis progresses, Jagged1 is also expressed in Dcx+ (Fig. 2B), but not in Calretinin+ (Fig. 2C), cells. Because Notch signaling is involved in neural stem cell maintenance and proliferation (Louvi and Artavanis-Tsakonas, 2006; Pierfelice et al., 2011; Fuentealba et al., 2012), we reasoned that the lack of Jagged1 might also affect any of those processes. To analyze in more detail the role of Jagged1 during postnatal stages of brain development, we generated 3 distinct Jagged1-conditional mutant mice by administering TM daily to Nestin-CreERT2;Jagged1F/F pups at different time windows: in the first group TM was administered from P0 to P5 (TM0-5), in the second one from P0 to P10 (TM0-10), and in the third one from P5 to P10 (TM5-10) (see Fig. 3A for details) (Fig. S2). All 3 different groups of Jagged1-conditional mutant mice were analyzed at P10 and all exhibited a smaller DG (Fig. 3B–F) in the absence of an increase in cell death (Fig. 3G).

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

FIGURE 2

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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.

FIGURE 3

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TM-induction protocols were used to generate the TM0-5, TM0-10, and TM5-10 Nestin-CreERT2;Jagged1F/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-CreERT2;Jagged1F/F pups shows various reductions in DG size (F). No increase in TUNEL+ cells was observed in any of the Nestin-CreERT2;Jagged1F/F pups (G). Fewer Nestin+ radial glia-like cells were observed in all of the conditional-mutant Nestin-CreERT2;Jagged1F/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-CreERT2;Jagged1F/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-CreERT2;Jagged1F/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.

Next, we used the same 3 groups of Nestin-CreERT2;Jagged1F/F pups to analyze the number of Nestin+ radial glia-like cells, of Sox2+ progenitors, of Tbr2+ intermediate progenitors, and of Calretinin+ immature neurons at P10. We found that the number of Nestin+ cells was reduced in the 3 groups of TM-induced Nestin-CreERT2;Jagged1F/F pups (Fig. 3H). However, the number of Sox2+ cells (Fig. 3I) and Tbr2+ cells (Fig. 3J) was comparable to that of controls in the TM5-10 pups. The number of Calretinin+ cells was reduced in the TM0-5 and TM0-10 Nestin-CreERT2;Jagged1F/F pups, but it was increased in the TM5-10 mice (Fig. 3K). These results indicated that the early deletion of Jagged1 resulted in an overall defect in neurogenesis and DG size (Fig. 3B–K); instead, deletion at later stages although reduced the number of Nestin+ cells and increased that of Calretinin+ cells, did not have a profound effect on DG size (Fig. 3E, F, H–K). These results show that the lack of Jagged1 during postnatal DG development results in neurogenesis defects in the absence of cell death, suggesting a role for Jagged1 in neural stem cell maintenance and/or progenitor proliferation during DG formation.

Next, we analyzed the maintenance and proliferation profile of Nestin+ radial glia-like and Sox2+ progenitors cells by double labeling with chlorodeoxyuridine (CldU) and iodo-deoxyuridine (IdU) (Burns and Kuan, 2005; Vega and Peterson, 2005) at different time points (Fig. 3L). To analyze cell maintenance, we administered CldU for long-term labeling, and to analyze proliferation, we administered IdU 1 h before collection (see Fig. 3A for details). First, we analyzed the Nestin+ population and found that the percentage of Nestin+/IdU+ (Fig. 3M) and Nestin+/CIdU+ (Fig. 3N) cells was reduced in the 3 groups of TM-induced Nestin-CreERT2;Jagged1F/F pups. Further, an analysis of the Nestin+/CIdU+ that re-entered S-phase and acquired IdU revealed a marked reduction of these population in the Nestin-CreERT2;Jagged1F/F pups (Fig. 3O). Then, we studied the Sox2+ population and found that the percentage of Sox2+/ldU+ cells was reduced in TM0-5 Nestin-CreERT2;Jagged1F/F pups, but increased in the TM0-10 and TM5-10 ones (Fig. 3P). We also found that Sox2+ cells maintenance was reduced in all the 3 different Nestin-CreERT2;Jagged1F/F pups (Fig. 3Q) and that more Sox2+/CldU+ re-entry S-phase (Fig. 3R). These results argue that conditional deletion of Jagged1 reduces the maintenance of Nestin+ and Sox2+ cells, but increases the percentage of long-term–labeled Sox2+ cells that re-enter S-phase before sample collection.

A reduction in Nestin+ and Sox2+ cell maintenance in the absence of an increase in cell death might imply an increase in cell production. In order to analyze this possibility, we quantified the percentage of Calretinin+/CldU+ cells and found that it was increased in all the 3 different groups of Nestin-CreERT2;Jagged1F/F pups (Fig. S3), a result implying an increase in Calretinin+ cell production.

Removal of Jagged1 from the adult SGZ results in a transient increase in neurogenesis

Similarly to postnatal development, Jagged1 is initially expressed in Gfap+ radial glia-like cells during adult-SGZ neurogenesis (Fig. 4A). Later, Jagged1 is detected in Dcx+ (Fig. 4B), but not in Calretinin+ cells (Fig. 4C). Furthermore, we have previously demonstrated that in the absence of Prox1, intermediate progenitors fail to survive, Jagged1 expression is absent, neural stem cells lack active Notch signaling, and neural stem cell maintenance is affected in the SGZ (Lavado et al., 2010). Therefore, we analyzed whether the lack of Jagged1 in the adult SGZ alters neurogenesis through a defect in neural stem cell maintenance without affecting cell survival.

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

FIGURE 4

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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.

To inactivate Jagged1 in the adult SGZ, we administered TM to 8-week-old Nestin-CreERT2;Jagged1F/F mice. Four weeks after induction (see Fig. 5A for details), Jagged1 expression was greatly reduced (Fig. 5B) and the mice were analyzed. In the absence of Jagged1, the number of Tbr2+ cells (Fig. 5C), of Dcx+ cells (Fig. 5D), and the ratio of non-radial to radial Dcx+ cells increased (Fig. S4). The number of Calretinin+ cells also increased in the 12-week-old Nestin-CreERT2;Jagged1F/F mice (Fig. 5E). These results suggest that the lack of Jagged1 in the adult SGZ stimulates neurogenesis.

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

FIGURE 5

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TM-induction protocol used to generate 12-week-old Nestin-CreERT2;Jagged1F/F mice (A). Jagged1 expression is reduced in the treated Nestin-CreERT2;Jagged1F/F mice (B). The number of Tbr2+ cells (C), Dcx+ cells (D), and Calretinin+ cells (E) was higher in the 12-week-old Nestin-CreERT2;Jagged1F/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-CreERT2;Jagged1F/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-CreERT2;Jagged1F/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-CreERT2;Jagged1F/F mice was also higher. TM-induction protocol used to generate 16-week-old Nestin-CreERT2;Jagged1F/F mice (K). Fewer Tbr2+ cells (L) were detected in the 16-week-old Nestin-CreERT2;Jagged1F/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.

To better characterize this phenotype, we next performed CldU/IdU double labeling (see Fig. 5A for details). The number of IdU+ (short-term–labeled) cells increased in the 12-week-old Nestin-CreERT2;Jagged1F/F mice (Fig. 5F), but the number of Cldu+ (longer-term–labeled) cells was comparable to that of controls (Fig. 5G). Moreover, the number of CldU+/IdU+ cells increased (Fig. S5), as did the percentage of Tbr2+/IdU+ cells (Fig. 5H), Dcx+/IdU+ cells (Fig. 5I), and Calretinin+/IdU+ cells (Fig. 5J) in 12-week-old Nestin-CreERT2;Jagged1F/F mice. We did not observe Tbr2+/CldU+ cells in controls or Nestin-CreERT2;Jagged1F/F mice, but we found a higher percentage of Dcx+/CldU+ cells (Fig. 5I) and Calretinin+/CldU+ cells (Fig. 5J) in the 12-week-old Nestin-CreERT2;Jagged1F/F mice. Moreover, the percentage of Dcx+/CldU+/IdU+ cells was increased in the conditional-mutant mice (Fig. 5I). No Calretinin+/CldU+/IdU+ cells were observed. These results further support the notion that the production of Tbr2+ cells, Dcx+ cells, and Calretinin+ cells is increased in 12-week-old Nestin-CreERT2;Jagged1F/F mice.

To determine whether this increase in neurogenesis is transient or permanent, we analyzed 16-week-old Nestin-CreERT2;Jagged1F/F mice at 8 weeks after TM induction (see Fig. 5K for details). Unlike the 12-week-old Nestin-CreERT2;Jagged1F/F mice, the 16-week-olds had fewer Tbr2+ cells than -controls (Fig. 5L), and the number of Dcx+ cells was similar (Fig. 5M). These results show that the lack of Jagged1 in the adult SGZ induces a transient increase in neurogenesis.

The transient increase in neurogenesis in the adult SGZ of Jagged1 conditional-mutant mice leads to the depletion of the neural stem cell population

The transient increase in neurogenesis that we observed in the TM-induced adult Nestin-CreERT2;Jagged1F/F mice (Fig. 6A) suggested the possibility of a defect in neural stem cell maintenance. An initial analysis revealed that the number of Nestin+ radial glia-like cells in 12-week-old Nestin-CreERT2;Jagged1F/F mice was lower than that in controls (Fig. 6B); however, we found no changes in the number of Sox2+ cells (Fig. 6C). Using CldU/IdU labeling (see Fig. 6A for details), we found that the percentage of Nestin+/IdU+ cells was lower in the Jagged1 conditional-mutant mice (Fig. 6D), and we rarely detected Nestin+/CldU+ cells in any of the genotypes. To facilitate the analysis of the Nestin+ radial glia-like population, we repeated the CldU/IdU double-labeling but with a shorter labeling (Fig. S6A). Under these conditions, Nestin+/CldU+ cells were easily identified in the TM-induced Nestin-CreERT2;Jagged1F/F mice. The percentage of Nestin+/CldU+/IdU+ cells increased, and that of Nestin+/IdU+ cells and Nestin+/CldU+ cells decreased (Fig. S6B). Furthermore, the percentage of Sox2+/IdU+ cells and Sox2+/CldU+/IdU+ cells increased, and that of Sox2+/CldU+ cells decreased (Fig. 6E) in the 12-week-old Nestin-CreERT2;Jagged1F/F mice when the CldU/IdU labeling was performed as described in Fig. 6A. These results indicate that the increased neurogenesis observed in 12-week-old Nestin-CreERT2;Jagged1F/F mice is accompanied by an increased number of proliferating Nestin+ radial glia-like cells and Sox2+ progenitor cells; however, fewer Nestin+ cells were found.

FIGURE 6. The transient increase in neurogenesis in the Nestin-CreERT2;Jagged1F/F mice is accompanied by a reduction in the Nestin+ and Sox2+ cell populations.

FIGURE 6

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TM-induction protocol used to generate 12-week-old Nestin-CreERT2;Jagged1F/F mice (A). Fewer Nestin+ cells were observed in the 12-week-old Nestin-CreERT2;Jagged1F/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-CreERT2;Jagged1F/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-CreERT2;Jagged1F/F mice (F). Fewer Nestin+ cells (G) and Sox2+ cells (H) were detected in the 16-week-old Nestin-CreERT2;Jagged1F/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.

To evaluate whether the lack of Jagged1 in the adult SGZ results in more Nestin+ radial glia-like cells without active Notch signaling, we analyzed Nestin+ radial glia-like cells from control (n=294) and 12-week-old Nestin-CreERT2;Jagged1F/F mice (n=245) by performing Nestin/Hes1 double-immunohistochemical analysis. In the 12-week-old Nestin-CreERT2;Jagged1F/F mice, the number of Nestin+/Hes1− cells increased (Fig. S7).

We then analyzed whether the Nestin+ and Sox2+ cell populations were further depleted in the 16-week-old Jagged1 conditional-mutant mice (Fig. 6F) and found that the number of Nestin+ cells (Fig. 6G) and Sox2+ cells (Fig. 6H) were reduced. Together, these results suggest that the transient increase in neurogenesis in Jagged1 conditional-mutant mice is produced by a defect in neural stem cell maintenance.

Conditional RbpJ deletion mimics the phenotype observed in Jagged1 conditional mutant animals

The genetic manipulation of Notch1 regulates the proliferation of neural stem cells at postnatal stages of DG development (Breunig et al., 2007), and Notch signaling is necessary for neural stem cell maintenance during adult-SGZ neurogenesis (Ehm et al., 2010). Therefore, we analyzed whether the absence of functional Notch signaling during postnatal-DG development resembles the phenotype observed in the Nestin-CreERT2;Jagged1F/F pups.

RbpJ-floxed mice (Han et al., 2002) were bred with Nestin-CreERT2 mice, and similar TM induction protocols were applied to Nestin-CreERT2;RbpJF/F pups from P0 to P10. Analysis of these P10 pups revealed a smaller DG (Fig. 7A–C) without an increase in cell death (Fig. 7D). Furthermore, the number of Nestin+, Sox2+, Tbr2+, and Calretinin+ cells was reduced in the Nestin-CreERT2;RbpJF/F pups (Fig. 7E–H). Moreover, the proliferation of Nestin+ cells (Fig. 7I) and the maintenance of Nestin+/CldU+ cells were reduced (Fig. 7J), and fewer Nestin+/CldU+ cells re-entered S-phase in the Nestin-CreERT2;RbpJF/F pups (Fig. 7K). Again, the maintenance of Sox2+ progenitor cells was reduced (Fig. S8), and more Sox2+/CldU+ re-entered S-phase (Fig. S8). These resulted in an increased Calretinin+ cell production (Fig. S8). These results are comparable to those obtained in the Nestin-CreERT2;Jagged1F/F, and suggest that Jagged1 is a key ligand for Notch signaling-mediated neural stem cell maintenance and proliferation during postnatal-DG development.

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

FIGURE 7

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Prox1 staining of coronal sections of the hippocampus of control (A) and TM0-10 Nestin-CreERT2;RbpJF/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-CreERT2;RbpJF/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-CreERT2;RbpJF/F pups than in controls. After TM adult induction, less Nestin+ (L), Sox2+ (M), or Dcx+ (N) cells were observed in the Nestin-CreERT2;RbpJF/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.

To determine whether the absence of functional Notch signaling during adult-SGZ neurogenesis resembles the phenotype observed in Nestin-CreERT2;Jagged1F/F mice, we took advantage of Nestin-CreERT2;RbpJF/F mice. Eight-week-old Nestin-CreERT2;RbpJF/F mice were induced with TM as described in Fig. 5A and analyzed 4 weeks later. The number of Nestin+ radial glia-like cells (Fig. 7L) and Sox2+ progenitor cells (Fig. 7M) was reduced, and that of Dcx+ cells increased (Fig. 7N). Occasionally, we found some Nestin+ radial glia-like cells that ectopically expressed Prox1 (Fig. S9). These results show that the conditional deletion of Jagged1 or RbpJ results in a similar reduction of the Nestin+ cell population and in an increase in neurogenesis and suggest that Jagged1 is also a critical Notch-signaling ligand during adult neurogenesis.

DISCUSSION

Here we report for the first time the function of Jagged1 during postnatal mammalian brain development. We determined that Jagged1 is necessary for neural stem cell maintenance and proliferation during postnatal stages of granule cell neurogenesis. Moreover, conditional deletion of RbpJ, the downstream effector of canonical Notch signaling, similarly affected neural stem cell maintenance and proliferation and resulted in a reduction in the size of the DG comparable to that of Nestin-CreERT2;Jagged1F/F mice. These data, together with results showing that conditional removal of Notch1 during postnatal-DG neurogenesis reduces proliferation (Breunig et al., 2007), support a key role for Notch1/Jagged1 signaling during this process by regulating the maintenance and proliferation of progenitor cells. A similar role has been described for Notch signaling during mammalian embryonic development (de la Pompa et al., 1997; Hitoshi et al., 2002; Lutolf et al., 2002).

We analyzed the role of Jagged1 during adult-SGZ neurogenesis and found that similar to postnatal development, Jagged1 is necessary for neural stem cell maintenance and proliferation. Jagged1 is expressed in Gfap+ radial glia-like cells and Dcx+ cells, but not in Calretinin+ post-mitotic cells (Fig. 4). We demonstrated that the lack of intermediate progenitors increases the number of Nestin+/Hes1− radial glia-like cells and eventually results in depletion of neural stem cells (Lavado et al., 2010). Consistently, TM-induced Nestin-CreERT2;Jagged1F/F mice had more Nestin+/Hes1− cells (Fig. S7). Furthermore, the conditional deletion of RbpJ by GLAST-CreERT2 (Ehm et al., 2010) or Nestin-CreERT2 (Fig. 7) depleted the radial glia cell population. The deletion of RbpJ reduced the neural stem cell population faster than deletion of Jagged1 (the Nestin+ and Sox2+ populations were both reduced in the 12-week-old Nestin-CreERT2;RbpJF/F mutants, but only Nestin+ radial glia-like cells were reduced) in these mice. This difference may be explained by the cell-autonomous effect of the lack of RbpJ in the neural stem cell population, while in Nestin-CreERT2;Jagged1F/F mice, compensatory effects may be instilled by the cells that escaped deletion due to the cell-to-cell nature of the Notch/Jagged1 interaction. A compensatory effect by other Notch ligands may also be possible, though Jagged1 is the highest-expressed ligand in the SGZ, and Delta-1 expression is not changed in the TM-treated Nestin-CreERT2;Jagged1F/F mice (data not shown). Other ligands appeared to be either expressed in only a few mature neurons (Breunig et al., 2007) or absent (Irvin et al., 2004; Brooker et al., 2006). Still, we cannot exclude the possibility of Notch activation by other cell types (e.g., vascular endothelial cells) (Phng and Gerhardt, 2009) that are not affected by the use of Nestin-CreERT2. In any case, the lack of Jagged1 eventually increases the number of Nestin+/Hes1− cells and, similar to what was seen in Nestin-CreERT2;RbpJF/F conditional mutants, promotes SGZ neurogenesis (Fig. 5, Fig. 7) (Ehm et al., 2010).

Prox1 directly inhibits Notch1 gene expression, and a mechanism by which Prox1 controls the balance between neural progenitor cell self-renewal and neuronal differentiation in the spinal cord has been suggested (Kaltezioti et al., 2010). Our previous detailed characterization of Prox1 conditional-mutant mice led us to propose that intermediate progenitors may be an important component of neural stem cell maintenance in the adult SGZ through Notch1/Jagged1 signaling (Lavado et al., 2010). We have also shown that ectopic expression of Prox1 induces premature neuronal differentiation and neural stem cell depletion during postnatal- and adult-SGZ neurogenesis (Lavado et al., 2010). In this paper, we now show that the lack of RbpJ results in ectopic Prox1 expression in the Nestin+ cell population (Fig. S9). Therefore, all of these results suggest that Prox1 activity during postnatal- and adult-SGZ neurogenesis is involved not only in controlling intermediate progenitor survival but also in regulating Notch signaling.

CONCLUSIONS

In summary, we have provided evidence that Jagged1 plays an important role during postnatal- and adult-SGZ neurogenesis by controlling neural stem cell maintenance and proliferation. In the absence of Jagged1, DG formation and adult-SGZ neurogenesis are affected.

Supplementary Material

01

HIGHLIGHTS.

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    Jagged1 is not necessary for embryonic dentate gyrus formation

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

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    Notch/Jagged1 is required for progenitor proliferation and maintenance

Acknowledgments

We thank Suzanne Baker for the Nestin-CreERT2 mice, Luisa Iruela-Arispe for the Jagged1F/F mice and Jane Johnson for the RbpjF/F mice. We also thank Xin Geng and Joshua Breunig for helpful discussion and comments and Angela McArthur for editing this manuscript. This work was supported, in part, by NIH grant EY12162 to G. Oliver, Cancer Center Support grant CA-21765, and the American Lebanese Syrian Associated Charities (ALSAC).

Footnotes

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