Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: (A1-A3) Experimental strategy to target/manipulate PyN gene expression and label ChCs in the same neocortical layer. (A1) Schematic drawing of E15.5 in utero electroporation (IUE) targeting nascent layer II/III (LII/III) PyNs in embryos from Swiss Webster (SW) females that were bred with Nkx2.1-CreER+/−;Rosa26-loxpSTOPloxp-tdTomato (Ai9)+/+ males. The position of the positive (+) and negative (−) electrodes used to target neocortical progenitors in the ventricular zone (VZ) is depicted. (A2) Tamoxifen (TMX) administration at E18.5 induces Cre activity and excision of a STOP cassette resulting in tdTomato red fluorescent protein (RFP) expression in ChC progenitors. (A3) Representative 200 μm × 200 μm confocal image of a single RFP+ ChC and neighboring electroporated GFP+ PyNs in LII of somatosensory cortex. Scale bar, 20 μm. Enlarged view of the boxed area showing a GFP+ PyN innervated at its AIS by an RFP+ ChC cartridge (arrow) is depicted on the right. Scale bar, 5 μm. AISs are visualized by immunostaining for ankyrin-G (AnkG) (blue).

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques: Gene Expression, In Utero, Electroporation, Activity Assay, Expressing, Immunostaining

Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: (A) Temporal profile of PyN AIS innervation by ChCs in LII of somatosensory cortex from Nkx2.1-CreER;Ai9 mice. Representative images of RFP+ ChC cartridges, AISs of neighboring PyNs, and gephyrin puncta at time points spanning P8 to P28. Scale bar, 5 μm.

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques:

Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: (A) Representative images of PyNs innervated by ChC cartridges in LII of somatosensory cortex from Nkx2.1-CreER;Ai9 mice electroporated at E15.5 with plasmids expressing EGFP and shCtrl, shL1CAM#1, shL1CAM#2, or shL1CAM#1 + human L1CAM (hL1CAM) and sacrificed at P28. Scale bar, 10 μm.

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques: Expressing

Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: (A) Representative images of PyNs innervated by ChC cartridges in LII of somatosensory cortex from Nkx2.1-CreER;Ai9 mice electroporated at E15.5 with plasmids expressing EGFP and shCtrl, shL1CAM#1, shL 1 CAM# 1 +hL 1CAM-WT, or shL1CAM#1+hL1CAM-Y1229H and sacrificed at P28. Scale bar, 10 μm.

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques: Expressing

Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques: Virus, Control, Recombinant, Protease Inhibitor, DNA Ligation, Bicinchoninic Acid Protein Assay, Western Blot, Sequencing, shRNA, Plasmid Preparation, Software, Imaging

Journal: iScience

Article Title: In vivo reprogramming of NG2 glia improves bladder function after spinal cord injury

doi: 10.1016/j.isci.2026.114850

Figure Lengend Snippet: SOX2-mediated glial reprogramming in the adult mouse spinal cord (A) Experimental scheme. SOX2-expressing lentiviruses were injected into the adult spinal cord, and tissues were analyzed 4 weeks (wk) later using immunohistochemistry (IHC). (B) Quantification of SOX2-induced DCX + immature neurons (mean ± SEM, n = 3–4 mice per group). Notably, DCX + cells were nearly absent in the p75-2-only condition. (C) Confocal images of DCX + cells induced by either wild-type SOX2 (SOX2 WT ) or the phospho-mimetic mutant SOX2 (SOX2 S251E ). Scale bars, 20 μm (D) Experimental scheme for tracing the cellular origin of mutant SOX2-reprogrammed cells. Adult transgenic mice were treated with tamoxifen (Tam), followed by intraspinal viral injections, and analyzed 4 weeks later. (E) Confocal images and quantifications (mean ± SEM, n = 3 mice per group) showing that NG2 glia are the cellular origin of mutant SOX2-reprogrammed cells in the adult spinal cord. NG2 glia and astrocytes were lineage-traced using Pdgfra-CreER T2 ;R26R-tdT and Aldh1l1-CreER T2 ;R26R-tdT mice, respectively. Scale bar, 20 μm (F) Experimental scheme for a time-course analysis of mutant SOX2-mediated reprogramming. dpv, days post virus injection. (G) Quantification of mutant SOX2-induced ASCL1 + neural progenitors (mean ± SEM, n = 3 mice per group). (H) Confocal images showing induction of ASCL1 + neural progenitors by mutant SOX2. Scale bar, 20 μm (I) Experimental scheme for tracing the progeny of mutant SOX2-induced ASCL1 + progenitors in the adult spinal cord. wpv, weeks post virus injection. (J) Quantification of mutant SOX2-induced tdT + cells at 4 wpv (mean ± SEM, n = 5 mice). (K) Confocal images showing robust detection of DCX + tdT + cells in mutant SOX2-expressing spinal cord. Scale bar, 20 μm.

Article Snippet: Mouse: Pdgfra-CreER T2 , The Jackson Laboratory , JAX: 032770; RRID: IMSR_JAX: 032770.

Techniques: Expressing, Injection, Immunohistochemistry, Mutagenesis, Transgenic Assay, Virus

Journal: The Journal of Experimental Medicine

Article Title: Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation

doi: 10.1084/jem.20171127

Figure Lengend Snippet: Bcl11b negatively regulates the production of Th2 cytokines in vitro . (a) Naive CD4 T cells were isolated from Bcl11b fl/fl -CreER T2 mice and cultured under Th1, Th2, or Th17 conditions with or without 4-OHT for 4 d (early). Primed cells were restimulated with PMA and ionomycin for 4 h. Cytokine production was assessed by intracellular staining. (b) Naive CD4 T cells were isolated from Bcl11b fl/fl -CreER T2 mice and cultured under Th2 conditions for 4 d. Then, after resting in IL-2–containing medium for 1 d, the primed cells were further cultured under Th2 conditions (late Th2) with or without 4-OHT for 4 d. Cytokine production was assessed by intracellular staining after restimulation with PMA and ionomycin. Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01. Error bars represent mean ± SD.

Article Snippet: Bcl11b fl/fl mice ( ) on C57BL/6 background were bred to CreER T2 (Taconic line 10471) to generate Bcl11b fl/fl -CreER T2 .

Techniques: In Vitro, Isolation, Cell Culture, Staining

Journal: The Journal of Experimental Medicine

Article Title: Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation

doi: 10.1084/jem.20171127

Figure Lengend Snippet: Bcl11b inhibits in vitro Th2 response through modulating GATA3 expression and functions. (a) Early Th2 cells prepared as described in . Cells were stained for intracellular GATA3 expression. Left peaks are fluorescence minus one (FMO) control. (b) UCSC Genome Browser view of Bcl11b and GATA3 binding to the Gata3 gene locus including the NK and T cell enhancer (280 kb downstream of the Gata3 gene body) in Th2 cells. (c) Late Th2 cells were prepared as in . Cells were stained for intracellular GATA3 expression. (d and e) Late Th2 cells from Bcl11b fl/fl -CreER T2 or Bcl11b fl/fl -Gata3 fl/fl -CreER T2 mice were restimulated with PMA and ionomycin for 4 h. IL-4, IL-5, and IL-13 expression were assessed by intracellular staining. Data are representative of one (b) and two (a and c–e) independent experiments. *, P < 0.05; **, P < 0.01; n.s., not significant. Error bars represent mean ± SD.

Article Snippet: Bcl11b fl/fl mice ( ) on C57BL/6 background were bred to CreER T2 (Taconic line 10471) to generate Bcl11b fl/fl -CreER T2 .

Techniques: In Vitro, Expressing, Staining, Fluorescence, Control, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation

doi: 10.1084/jem.20171127

Figure Lengend Snippet: Bcl11b is important for optimal Th cell proliferation. (a) The expression of Ki-67 in early Th2 cells was measured by intracellular staining. Left peaks are the FMO controls. (b and c) Naive CD4 T cells purified from Bcl11b fl/fl -CreER T2 mice were labeled with CFSE and cultured under Th2 conditions in vitro with or without 4-OHT. After 3 d, cells were restimulated with PMA and ionomycin. IL-4 expression was assessed by intracellular staining. Proliferation index was calculated using FlowJo software. The percentage of IL-4–expressing cells was calculated in cell populations that underwent different cycles of division based on CFSE dilution. (d) RNA-seq analysis was performed with the WT and Bcl11b KO early and late Th2 cells. Cell cycle–related genes regulated by Bcl11b are shown. The total cell cycle gene list (Table S1) is from Qiagen. (e) Early Th2 cells were processed as in b. The expression of p21 and CFSE dilution were assessed. Red, WT Th2 cells; blue, Bcl11b KO Th2 cells. Data are representative of three (a) and two (b, c, and e) independent experiments. *, P < 0.05. Error bars represent mean ± SD.

Article Snippet: Bcl11b fl/fl mice ( ) on C57BL/6 background were bred to CreER T2 (Taconic line 10471) to generate Bcl11b fl/fl -CreER T2 .

Techniques: Expressing, Staining, Purification, Labeling, Cell Culture, In Vitro, Software, RNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation

doi: 10.1084/jem.20171127

Figure Lengend Snippet: Bcl11b/GATA3 complex inhibits the expression of Th1-associated genes. (a) Overlap of genes that are negatively regulated by Bcl11b (B-N), positively regulated by Bcl11b (B-P), negatively regulated by GATA3 (G-N), or positively regulated by GATA3 (G-P) in late Th2 cells. (b) Overlap of genes that are negatively regulated by both Bcl11b and GATA3 (B-N, G-N), negatively regulated by Bcl11b but positively regulated by GATA3 (B-N, G-P), cobound by Bcl11b and GATA3 with at least one overlap peak, and cobound by Bcl11b and GATA3 without overlap peaks. (c) Overlap of Th1-associated genes and genes that are negatively regulated by Bcl11b and GATA3. (d) Total RNA was harvested from Bcl11b fl/fl -CreER T2 and Gata3 fl/fl -CreER T2 late Th2 cells with or without 4-OHT. Relative (Rel.) mRNA expression of Havcr2 , Ccl5 , Sell , Anxa1 , Serpinb6b , Igtp , Irgm2 , and Csf1 was measured by qRT-PCR. (e and f) Th1 and Th2 cells were prepared as . Tim3 (coded by Havcr2 gene) expression was measured by cell surface staining (e). Th1 and Th2 cells were stimulated with CD3/CD28 beads for 24 h, and CCL5 amounts in supernatant were measured by ELISA (f). Data (d–f) are representative of two independent experiments. **, P < 0.01. Error bars represent means ± SD.

Article Snippet: Bcl11b fl/fl mice ( ) on C57BL/6 background were bred to CreER T2 (Taconic line 10471) to generate Bcl11b fl/fl -CreER T2 .

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Staining, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation

doi: 10.1084/jem.20171127

Figure Lengend Snippet: Bcl11b regulates chromatin epigenetic modification. Early and late Th2 cells were prepared from Bcl11b fl/fl -CreER T2 mice with or without 4-OHT for DHSs and H3K27ac modification analyses. (a and b) UCSC Genome Browser view of Bcl11b and GATA3 binding and histone H3K27ac modification (in early and late Th2 cells) at the Havcr2 and Ccl5 gene loci. (c and d) UCSC Genome Browser view of Bcl11b and GATA3 binding and histone H3K27ac modification (in early Th2 cells) at the Il4/Il13 and Gata3 gene loci. (e) The ratio of observation and predication was calculated on genes with Bcl11b binding only, GATA3 binding only, and Bcl11b/GATA3 cobinding (with at least one overlapped peak) versus H3K27ac modification changed genes in Bcl11b -deficient early Th2 cells. (f) Scatter plot for the mean of H3K27ac level between Bcl11b KO cells and WT cells versus the fold-change in H3K27ac level for regions enriched with H3K27ac in early Th2 cells. (g) Scatter plot for the mean of accessibility between Bcl11b KO cells and control cells versus the fold-change in accessibility for DHSs in early Th2 cells. (h) Boxplot for the fold-change in H3K27ac level at nonpromoter DHSs sorted based on their change in accessibility induced by Bcl11b KO; similar results were obtained for promoter DHSs. H3K27ac data shown in the genome browser view (a–d) represent one of the two biological repeats.

Article Snippet: Bcl11b fl/fl mice ( ) on C57BL/6 background were bred to CreER T2 (Taconic line 10471) to generate Bcl11b fl/fl -CreER T2 .

Techniques: Modification, Binding Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation

doi: 10.1084/jem.20171127

Figure Lengend Snippet: Bcl11b inhibits Th2 responses in vivo. (a) Diagram of the mouse acute asthma model. (b) Bcl11b fl/fl , Bcl11b fl/fl -CreER T2 , or Bcl11b fl/fl -Gata3 fl/fl -CreER T2 bone marrow chimeric mice were treated as in panel a. CD4 T cells purified with CD4 (L3T4) microbeads were stimulated with plate-bound anti-CD3/CD28 antibodies for 2 h. Total RNAs were harvested, and Il4 , Il5 , and Il13 mRNA levels were assessed by qRT-PCR. Rel., relative. (c and d) BAL fluids (c) and sera (d) were collected from Bcl11b fl/fl and Bcl11b fl/fl -CreER T2 bone marrow chimeras treated as in panel a. IL-13 and IL-5 in BALF (c) and IgE (d) in serum were measured by ELISA. (e) CD4 T cells were purified from Bcl11b fl/fl and Bcl11b fl/fl -CreER T2 bone marrow chimeras treated as in panel a. Cells were further cultured with OVA peptide in the presence of antigen-presenting cells for 24 h. IL-5 was measured by ELISA. (f) Late Th2/OT-II cells were transferred into CD45.1/CD45.2 F1 recipients. Mice were then challenged with OVA for 4 d. The percentage and cell number of SiglecF + CD11c − eosinophils in the lung were measured through cell surface staining. Data are representative of two (b–f) independent experiments. *, P < 0.05; **, P < 0.01. Error bars represent means ± SD.

Article Snippet: Bcl11b fl/fl mice ( ) on C57BL/6 background were bred to CreER T2 (Taconic line 10471) to generate Bcl11b fl/fl -CreER T2 .

Techniques: In Vivo, Purification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining