mice were generated through Recombineering Technology (http://web.ncifcrf.gov/research/brb/recombineeringInformation.aspx
). Briefly, a tamoxifen-inducible CreERT2 gene (Feil et al., 1997
) was inserted through homologous recombination into the first exon of the Tlx
locus in a BAC clone (RP24-344A4). The correctly recombined BAC clones were confirmed by restriction digestion and sequencing. The genomic DNA was released by sequential digestion with BsiWI and AscI and separated from the vector backbone through a CL-4B sepharose column. Transgenic animals were then produced by pronuclear injection of fertilized mouse eggs by The Transgenic Core Facility at UT Southwestern. Twenty-three founders were identified after genotyping and were further screened for inducible expression of EYFP after crossing into Rosa-EYFP reporter mice and treatment with Tamoxifen. The pTlx-CreERT2
mice were kept in a mixed background of FVB, C57BL/6J and 129S1/SvImJ.
The strategies and methods for generating TlxLacZ/LacZ
mice or mice with conditional alleles of Tlx
have been described (Yu et al., 2000
; Zhang et al., 2008
). In short, TlxLacZ/LacZ
mice were generated by replacing exons 3–5 with LacZ
genes. Thus, expression of LacZ
is under the direct regulation of the endogenous Tlx
promoter and enhancers. To create a conditional allele of Tlx
, exon 2 was flanked by two loxP
sites through homologous recombination. Cre-mediated recombination resulted in a null allele of Tlx
by creating stop codons after reading frame shifts. Detailed information has also been provided for the generation and characterization of transgenic Nes-GFP
mice (Yamaguchi et al., 2000
) and conditional allele of Trp53
(Marino et al., 2000
). All mice were housed under a 12-h light/dark cycle and had free access to food and water in a controlled animal facility. No significant phenotypic differences were observed between male and female mice; thus, both genders were included in the analysis. Experimental protocols were approved by the Institutional Animal Care and Use Committee at UT Southwestern.
Administration of tamoxifen, bromodeoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU)
Tamoxifen (Sigma) was dissolved through bursts of sonication in sesame oil with a final stock concentration of 20 mg/ml. Mice were injected intraperitoneally once daily with 4 mg tamoxifen per 20 g body weight or sterile sesame oil (vehicle) for 5 consecutive days. The mice were sacrificed 24 hr after the last injection or at the indicated time points. Dividing cells in vivo were labeled by intraperitoneal injection of BrdU, CldU or IdU (in PBS) at the indicated dose for the specified duration.
Lentivirus production and stereotactic brain injections
cDNA was subcloned into either CMV-ires-GFP
lentiviral vector in which the expression of the transgene was driven by the CMV
promoter (Lee et al., 2008
). HEK293T cells were cotransfected with lentiviral and packaging plasmids (pMDL, VSV-G
) by CaPO4
method. Virus-containing supernatants were collected at 24, 48 and 72 hr post transfection, pooled and filtered through a 0.22 μm filter to remove cellular debris. Viral particles were then concentrated by centrifugation at 25,000 rpm for 2 hr at 4°C. Lentiviral titers were measured in either HEK293 cells (CMV
promoter) or primary astrocytes (hGfap
promoter). One μl of viruses (1 × 108
cfu/ml) were stereotactically injected into the DG. We used the following coordinates from bregma for Tlx
-null mice: anterior/posterior (AP), −1.3 mm; medial/lateral (ML), ±2 mm; and dorsal/ventral from skull (DV), −1.5 mm.
Tissue preparation and immunohistochemistry
The adult mice were sacrificed via CO2
overdose and perfused with 1xPBS followed by ice-cold 4% paraformaldehyde (PFA) in PBS. Brains were dissected and post-fixed overnight with 4% PFA at 4°C, followed by cryoprotection with 30% sucrose solution in PBS for another 24 hr. Frozen brains were sectioned at 40 μm with a sliding microtome (Leica) and free-floating sections were collected and stored in antifreeze solution at −20°C. For immunostaining, sections were washed 3 times with PBS and blocked for 1 hr at RT with blocking solution (3% BSA/0.2% Triton X-100 in PBS). The sections were then incubated overnight at 4°C with primary antibodies diluted in blocking solution. After 3 rinses with PBST buffer (0.2% Triton X-100 in PBS), the sections were further incubated with the corresponding secondary antibodies in blocking solution for 2 hr at RT. When necessary, nuclei were stained with Hoechst 33342 (Hst, Sigma) (1 μg/ml in PBS). Sections were washed and mounted onto Superfrost glass slides (Fisher Scientific) with mounting medium containing diazabicyclo-octane (DABCO, Sigma). For BrdU, CldU and IdU detection, prior to incubation with primary antibodies, sections were treated with 50% formamide in 2xSSC buffer for 2 hr at 65°C, followed by further treatment with 2 M HCl for 30 min at 37°C and equilibration with 0.1 M boric acid (pH8.5). Sequential detection of CldU and IdU were conducted essentially as described (Tuttle et al., 2011
). Briefly, HCl-treated brain sections were first incubated overnight with antibody against IdU/BrdU (mouse clone 3D4) at 4°C, followed by high stringency wash with fresh TBST buffer (36mM Tris, 50mM NaCl, 0.5% tween-20; pH 8.0) at 37°C. The sections were then incubated overnight with antibody against CldU/BrdU (rat BU1/75) at 4°C, washed with PBST and detected with corresponding secondary antibodies.
The following primary antibodies were used: GFP (rabbit, 1:500, Molecular Probes; chick, 1:1000, Aves Labs); GFAP (mouse, 1:500, Sigma; guinea pig, 1:1000, Advanced Immunochemicals); BrdU/CldU (rat BU1/75, 1:500, Accurate); BrdU/IdU (mouse clone 3D4, 1:1000, BD Pharmingen); NeuN (rabbit, 1:500, Millipore); Sox2 (rabbit, 1:500, Millipore); BLBP (rabbit, 1:500, Millipore); Nestin (mouse, 1:200, Pharmingen); Ki67 (rabbit, 1:500, Novocastra); MCM2 (rabbit, 1:500, Cell Signaling); DCX (goat, 1:150, Santa Cruz); Olig2 (rabbit, 1:500, Millipore), pSMAD1/5/8 (rabbit, 1:500, Cell Signaling); GSTπ (mouse, 1:200, BD Sciences); S100β (rabbit, 1:1000, Swant); RIP (mouse, 1:250, Hybridoma Bank, Iowa); and Histone H3 (rabbit, 1:500, Cell Signaling). Alexa Fluor 488-, 594-, or 647-conjugated secondary antibodies produced in goat or donkey (Invitrogen) were used for indirect fluorescence. Images were taken using a Zeiss LSM510 confocal microscope. A Cell Counter software plugin in the ImageJ program was used to count cells. Data were obtained from 12 random sections from 3–5 mice in each group.
NSC culture, immunocytochemistry and flow cytometry
NSCs from 6-to-8-week-old Tlx+/LacZ;CreERTM
(CZ) or Tlxflox/LacZ;CreERTM
(FCZ) mice were isolated and cultured in growth medium (DMEM/F12 medium supplemented with N2 (Invitrogen), heparin (5ug/ml, Sigma), EGF (20ng/ml, Peprotech) and bFGF (20ng/ml, Peprotech)), as previously described (Zhang et al., 2008
NSCs were sorted based on LacZ
expression using FluoReporter lacZ Flow Cytometry Kits, according to the user’s manual (Invitrogen). To acutely delete Tlx
, FCZ cells were treated with 10 nM 4-hydroxytamoxifen (Sigma) for the indicated duration. Similarly, Tlx
-null NSCs were isolated from E18.5 cortices and cultured in growth medium. These cells were labeled the following day with 1 μM 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, Molecular Probes) for 15 min at RT, followed by lentiviral transduction. Four hr later, the cells were washed with growth medium and continuously cultured for another 14 days with medium change in every other day. DiI and GFP were used for gating in flow cytometry. For immunocytochemistry, cultured cells on chamber slides (BD Sciences) were fixed with 4% PFA, washed with PBS, blocked for 30 min at RT, followed by overnight incubation with primary antibodies in blocking solution at 4°C. For detection of BrdU-labeled cells, the fixed cells on slides were treated with 2 M HCl at 37°C for 30 min, followed by washing with PBS and incubation with primary antibodies.
FACS of Nes-GFP cells and RNA-Seq
Three-week-old mice (n = 5–9 for each genotype) were used. Brains were cut into 1 mm coronal sections with a brain matrix, and fresh tissues surrounding the lateral ventricles were microdissected on ice. Tissues were enzymatically digested for 30 min at 37°C with PPD solution, which consists of papain (2.5 u/ml), DNase I (250 u/ml) and dispase II (1.0 u/ml) in DMEM culture medium supplemented with 4.5 g/L glucose. After trituration with a 5-ml pipette, cells were sequentially washed 3 times with DMEM/10% FBS and once with PBS/4% FBS. They were then filtered through a 40 μm cell strainer for live cell sorting based on GFP expression. GFP+ cells were directly sorted into TRIzol® LS reagent (Invitrogen) and the total RNAs were isolated using RNeasy Mini Kit (Qiagen). RNA quality was determined by Bioanalyzer (Agilent). cDNAs were synthesized from 50 ng of total RNA and were further amplified with an RNA-seq Ovation system (NuGen). A cDNA library was prepared and subjected to parallel sequencing using Genome Analyzer II (Illumina). Sequence tags were analyzed as described (Masui et al.). A total of 20.8 million unique tags were obtained for wild-type cells and 19.7 million with RNAs from Tlx−/− cells. Based on the rpkm (reads per kilobase of exon model per million mapped reads) value for Mcm2, which is an essential gene for licensing DNA replication, a cut-off value of 2.0 was used for either wild-type or Tlx−/−. Gene expression changes were calculated as the ratio of the rpkm for wild-type to that of Tlx−/− and further analyzed with DAVID program for KEGG signaling pathways.
Quantitative RT-PCR (qRT-PCR) analysis
Total RNAs from cultured or FAC-sorted cells were isolated using TRIzol reagent (Invitrogen) and RNeasy Mini Kit (Qiagen). The Superscript III system (Invitrogen) and random primers were used to synthesize cDNA from 1.0 μg total RNA from cultured cells. For sorted cells, 20 ng of total RNA was used to make the first strand cDNAs, which were further amplified using the RNA-seq Ovation system, according to manufacturer’s protocol (NuGEN). Gene expression was analyzed using the SYBR Greener system (Invitrogen) on a 384-well ABI 7900HT thermocycler (Applied Biosystems). Primer sequences for PCR reactions are available upon request.
Differences between groups were determined for significance using the two-tailed Student’s t test with equal variance or ANOVA. A p value of < 0.05 was considered significant.