Persistence of postnatal NSCs in Tlx-null brains. A, Existence of Tlx-expressing cells, indicated by blue staining for β-galactosidase in the neurogenic niches of Tlx-null mice. Deletion of Tlx leads to a hypomorphic DG and enlarged ventricles. B, Although lacking proliferating cells (indicated by BrdU staining), Tlx-null mice continue to express stem cell markers (Nestin, Sox2, and GFAP) in the neurogenic niche. Images were taken from the lateral ventricle (LV). C, Inducible deletion of Tlx does not result in down-regulation of stem cell markers (Sox2 or Nestin) in culture. Tlx⁺ stem cells were isolated based on LacZ expression. The Tlx gene was then deleted through Cre/loxP-mediated recombination after tamoxifen (TM) treatment. Vehicle (ethanol) was used as a control. Gene expression was determined by qRT-PCR at the indicated time points (n = 3; *p = 0.014, and **p = 0.0001). D, Persistence of Nes-GFP-expressing cells in Tlx-null mice. Expression of Nes-GFP in the dentate gyrus of 30-day-old mice was detected by confocal analysis. Enlarged views of the boxed regions are also shown. Arrows denote mis-positioned GFP⁺ cells. Hoechst 33342 (Hst)-staining was used to reveal the nuclei. Scale, 50 μm. E, NSCs in adult Tlx-null brains. Enlarged views are taken from the boxed regions. NSCs have long, radial processes and co-express GFAP, BLBP and Sox2. Arrows indicate colocalization. Please also note the displacement of Tlx-null NSCs from the subgranule zone of the dentate gyrus. Scales, 20 μm.

Age-dependent inactivation of postnatal NSCs due to loss of TLX function. A–D, Endogenous marker Ki67 (A–B) or PCNA (C–D) was used to label actively proliferating NSCs in the DG during early postnatal stages (postnatal day 7, 14, and 21). Stem cells were identified by expression of Nes-GFP. E–F, Activated stem cells were examined by expression of MCM2. Please note that only a fraction of MCM2⁺GFP⁺ cells were undergoing active cell cycling (Ki67⁺GFP⁺ or PCNA⁺GFP⁺). n = 12 sections from 3 mice for each genotype. (*p < 0.005 and **p < 0.002; Scale, 20 μm).

Activation of label-retaining Tlx-null NSCs by reintroducing exogenous TLX. A, Ectopic TLX rescued proliferation of Tlx-null cells, indicated by a reduction of DiI label. Isolated Tlx-null NSCs were transduced by the indicated lentiviruses. Flow cytometry was performed after 14 days in culture. B, Inactive adult type-1 NSCs induced by Tlx deletion. Proliferating type-1 cells in the DG of 9 weeks old mice were labeled by BrdU-pulse (100 mg/kg, twice a day for 5 days) and staining for Nes-GFP and GFAP. A BrdU-labeled type-1 cell with radial morphology and an adjacent one with semi-tangential orientation in wild type mice are indicated by an arrow and an asterisk, respectively. C, Enhancing proliferation of Tlx-null NSCs by exogenous TLX in vivo. Six-week-old Tlx-null;Nes-GFP mice were injected with control lentiviruses or viruses expressing TLX. Three weeks later, proliferating cells were labeled by BrdU-pulse. A proliferating type-1 cell induced by re-expression of TLX is indicated by an arrow. D, Reactivation of label-retaining cells with exogenous TLX. Tlx-null mice were pulsed with CldU (50 mg/kg) at E18.5 and E19.5, chased for 6 weeks, stereotactically injected with lentiviruses expressing GFP or GFP-T2A-TLX, and then pulsed again with IdU (100 mg/kg, twice a day for 7 or 10 days). A few examples of TLX-induced GFP⁺CldU⁺IdU⁺ cells are shown. Scales for images at lower magnification, 50 μm; scales for the rest of images: 20 μm.

Role of p53 signaling in TLX-dependent regulation of NSC proliferation. A, Increased p21 expression upon inducible deletion of Tlx by 4-hydroxytamoxifen (TM)-treatment of cultured adult Tlx^flox/LacZ NSCs. The expression of Tlx and p21 were determined by qRT-PCR. Veh, ethanol as vehicle control. B, Transient ectopic expression of p21 in cultured NSCs inhibited their proliferation (n=4; *p = 0.019). C, p53-dependent regulation of p21 expression by TLX. Cultured NSCs with the indicated genotypes were transduced with adenoviruses expressing either GFP or GFP-Cre. D, Confirmation of altered p53 signaling. Gene expression was determined by qRT-PCR using sorted Nes-GFP⁺ NSCs from 3-week-old wild-type or Tlx-null mice. E, Efficiency of Cre-mediated deletion of either Tlx or p53 in cultured NSCs determined by qRT-PCR. F, Concomitant deletion of p53 in cultured NSCs rescued their proliferation defects that was induced by Cre-mediated deletion of Tlx (n = 4; *p = 0.023, **p < 0.0001). G, A model showing the role of TLX in postnatal NSCs. Tlx-expressing cells generate both activated and inactive postnatal NSCs, which are identified by marker expression. TLX is required for inactive NSCs to proliferate by modulating p21 expression in a p53-dependent manner. Besides p53 signaling, TLX also modulates many other signaling pathways, which may contribute to the regulation of NSC activation.

Tracing TLX lineage in inactive and activated adult NSCs. A, The CreER^T2 gene was knocked into the first exon of the Tlx gene in a BAC clone through homologous recombination. Transgenic mice were produced through pronuclear injection of fertilized eggs. B, Tamoxifen-induced expression of GFP in adult neurogenic niches. LV, lateral ventricle. DG, dentate gyrus. C, Induced expression of GFP in type-1 (C1–C5) or type B (C6–C9) stem cells (marked by co-localization with GFAP expression and long processes, arrows) in the DG or the LV, respectively. Asterisks, tangentially oriented type-2 cells. C2 is a projection view of C1. C2–C5 and C7–C9 are enlarged views of boxed regions in C1 and C6, respectively. D, Induced GFP expression in inactive (arrow, GFAP⁺MCM2⁻) and activated (arrow head, GFAP⁺MCM2⁺) stem cells. Transient amplifying cells (asterisk, GFP⁺GFAP⁻MCM2⁺) were also observed. Scale in B, 100 μm. Scales in C and D, 20 μm.

Deletion of Tlx does not lead to spontaneous differentiation of postnatal Nes-GFP⁺ stem cells. The identity of Nes-GFP+ cells in 30-day-old mice was analyzed by co-labeling with markers for astrocytes (A, B), oligodentrocytes (C, D) and neurons (E, F). Some of the glial cells are indicated by arrows. GS, glutamine synthetase. Scale, 50 μm.

Mis-positioning of label-retaining Nes-GFP cells in Tlx-null mice. A, NSCs are indicated by Nes-GFP expression in the DG during the early postnatal and adult stages. To quantify the position of NSCs, the DG was divided into the outer and the inner halves. Scale, 20 μm. B, Quantification of the positions of NSCs in the DG. Values are presented as percentage of GFP⁺ cells in either the inner or the outer half of dentate blade (n = 3; *p < 0.005 and **p < 0.002). C, Confocal images of BrdU-retaining cells co-labeled with a mature neuronal marker (NeuN) or a radial glia marker (GFAP). Cells were pulse-labeled with BrdU at P0 and examined at P24. Scale, 20 μm. D, Quantification of BrdU-retaining Nes-GFP cells in the outer half of the granule cell layer. Cells were pulse-labeled with BrdU at E15.5, P0, or P7 and examined at postnatal day P21, P24 or P43, respectively (n = 3; *p<0.002). E, Schematic representation of BrdU-retaining Nes-GFP⁺ cells (indicated by red dots) in the DG of either wild-type or Tlx-null mice.

TLX-dependent signaling in postnatal NSCs. A, Whole-genome expression was examined by sequencing of the RNAs isolated from sorted Nes-GFP⁺ cells from 3-week-old wild-type or Tlx-null brains. Representative genes for 4 categories: no expression (Myod1), no change on expression (Nes, Sox2, and Blbp), up-regulated (p21) and down-regulated (Ccnd2). RPKM, reads per kilobase of exon model per million mapped reads. B, KEGG pathway analysis. Please note the significant changes on genes involved in cancer/glioma, cell cycle, Wnt signaling, DNA replication and p53 signaling. C, D, Expression changes on genes in DNA replication. C, Sequencing data. D, Heatmap representation of fold changes on gene expression, which was determined by sequencing or qRT-PCR analysis of RNA from Tlx-null NSCs. Data from microarray analysis of adult NSCs after inducible deletion of Tlx (Zhang et al., 2008) was also included. E, Inducible deletion of Tlx leads to inactive stem cells. Tlx⁺/LacZ adult NSCs with a CreER^TM transgene and/or a floxed allele of Tlx (FCZ and CZ, respectively) were treated with 4-hydroxytamoxifen (TM) or vehicle (ethanol). Genomic DNA was isolated and measured at the indicated time points after treatment (n = 3. *p = 0.02 and **p = 0.001).