Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: (A) Representative images of mock- or ZIKV-infected BCOs stained with neuronal markers (CTIP2 and NeuN), a neural progenitor cell marker (SOX2), and DAPI. Scale bars, 100 μm. (B) Quantification of BCO size p.i. with ZIKV. Significance was assessed by two-tailed Student’s t test, and experiments were performed in two batches with 12 organoids per group per batch. (C) BCO size fold change of ZIKV- and mock-treated groups over a period of 1 month. (D) Quantification of SOX2+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (E) Quantification of CC3+ cells in ZIKV- versus mock-infected groups. *p < 0.05 by two-tailed Student’s t test. (F) Quantification of SATB2+ cells within MAP2+ cells in ZIKV- versus mock-infected groups. **p < 0.01 by two-tailed Student’s t test. (G) Quantification of GFAP+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (H) Quantification of NeuN+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (I) Quantification of CTIP2+ cells in ZIKV- versus mock-infected groups. N.S., not significant by two-tailed Student’s t test. (J) Bright-field images of engraftment of two patient-derived GSCs (387 and 3565) transduced with GFP into human BCOs over a time course. Scale bars, 1 mm. (K) Engrafted GSCs (GFP+) with normal BCO immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 200 μm. (L) Quantification of integrin αvβ5+ cells in normal BCOs or GSC-BCOs. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (M) Representative images of GFP-labeled GSC-BCOs immunostained for integrin αvβ5 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (N) Representative images of GFP-labeled GSC-BCOs immunostained for SOX2 (red), GFP (green), and DAPI (blue). Scale bars, 100 μm. (O) Images of GFP-labeled GSC-GFP BCOs 13 days p.i. with ZIKV. Scale bars, 1 mm. (P) Representative images of residual GSCs (green) and DAPI staining (blue) of GFP-labeled GSC-GFP BCOs cultured under mock conditions or with ZIKV for 2–4 weeks. Scale bars, 200 μm. The percentage of GFP+ cells among DAPI+ cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-way ANOVA. (Q) Representative immunostaining for integrin αvβ5 (red), GFP (green), ZIKV-E (white), and DAPI (blue) of GFP-labeled GSC-GFP BCOs mock- or ZIKV-infected for 2–4 weeks. Scale bars, 200 μm (left) and 100 μm (center). The percentage of ZIKV-E+ cells among integrin αvβ5 cells was quantified. Values represent mean ± SEM. n = 6. ****p < 0.0001 by two-tailed Student’s t test. (R) Representative images of 387 and 3565 GSC-BCOs with or without ZIKV, respectively, stained with SOX2, ZIKV-E, and DAPI. GFP shows the presence of GSCs (scale bars, 50 μm). ZIKV-E+, GFP+, and ZIKV-E+ cells among GFP+ cells were quantified by counting (two GSCs cell lines, two repeats, n = 12 organoids/group); *p < 0.05 by two-tailed Student’s t test. (S) Schematic of the experiment design. (T) Volcano plot showing differences between GSC-BCO ZIKV versus GSC-BCO mock. 113 genes were differentially expressed (greater than 1.5-fold) between these two groups (*p < 0.05). (U) Network analysis of genes differentially expressed upon ZIKV infection, represented as a bubble plot.

Article Snippet: Rabbit polyclonal antibody to CTIP2 , US Biological , Cat# B0807-13E2; RRID:AB_2064140.

Techniques: Infection, Staining, Marker, Two Tailed Test, Derivative Assay, Transduction, Labeling, Cell Culture, Immunostaining

Journal: Cell stem cell

Article Title: Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin α v β 5 Axis

doi: 10.1016/j.stem.2019.11.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal antibody to CTIP2 , US Biological , Cat# B0807-13E2; RRID:AB_2064140.

Techniques: Control, Negative Control, Virus, Recombinant, In Vitro, Transfection, cDNA Synthesis, Plasmid Preparation, Imaging, TUNEL Assay, SYBR Green Assay, Reverse Transcription, Bradford Assay, shRNA, Software, Gene Expression, Flow Cytometry, Microscopy

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet: Rbp4-Cre neurons have L5-PN identity (A) Top: Single-cell RNA sequencing workflow ( Figure S1 ). Bottom: Expression profile of layer-specific genes , , in Rbp4-Cre neurons. (B) Top: Rbp4-Cre neurons (green), Bcl11b (magenta), Hoechst (blue). Bottom: fraction of Rbp4-Cre neurons expressing Bcl11b. n = number of Rbp4-Cre neurons. (C) Correlation of cortical layer-specific neuronal genes’ expression between Rbp4-Cre neurons and adult cortical layers for up to 150 genes. , (D) UMAP embedding of Rbp4-Cre neurons’ single cell transcriptomes. Color: Leiden clusters. (E) Top: Rbp4-Cre types (colored as in D) embedded in a triangle representing the similarity between each cell’s expression profile from the three adult L5-PN types (NP, IT, and PT) ( Figure S1 ). Bottom: For each adult type, percent of Rbp4-Cre neurons of each embryonic type associated with that adult type. (F) Percent of neurons from each type on each embryonic day (colored as in D and labels derived from E). Scale bars: 20 transcripts (A), 10 μm (B). See also Figure S1 .

Article Snippet: Rat monoclonal anti CTIP2/BCL11B , Merck , Cat# MABE1045.

Techniques: RNA Sequencing Assay, Expressing, Derivative Assay

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet: Rbp4-Cre neurons are located in the region from the subplate to the surface of cortex, are distinct from Cajal-Retzius cells and subplate neurons, and express layer 5 markers, related to Figure 2 (A) Within the UMAP embedding of all 25681 sequenced excitatory neuron transcriptomes (gray), cells expressing a number of genes previously associated with Cajal-Retzius cells , , overlap in a location (green outline) distinct from positively identified Rbp4-Cre neurons (as in Figure S1 ) (red outline; Rbp4-Cre neurons) (labeled in bottom right). Clusters of Rbp4-Cre neurons are additionally labeled based on the cluster identification from Figure 1 E. (B) Within the UMAP embedding of all 25681 sequenced excitatory neuron transcriptomes (gray), cells expressing genes previously associated with subplate neurons overlap in a location (green outline) distinct from positively-identified Rbp4-Cre neurons (as in Figure S1 ) (red outline; Cre neurons) (labeled in the bottom right). Clusters of Rbp4-Cre neurons are additionally labeled based on the cluster identification from Figure 1 E. (C) Rbp4-Cre neurons (green), counterstained with Hoechst (blue), show that neurons in the deep layer lie physically within the subplate (SP; dotted line), as localized by the expression of a common subplate marker, Nr4a2 (Nurr1) (magenta). (D) Only a small fraction of Rbp4-Cre neurons express Nr4a2 from E13.5 to E18.5. (E) Rbp4-Cre neurons are located in the region from the subplate to the surface of cortex. Rbp4-Cre neurons (stained using GFP antibody labeling GCaMP6s, green) in both spatial configurations (on E14.5, E16.5, and E18.5) co-labeled with antibodies labeling different zones in the developing cortical wall (Pax6 (first row), Tbr2 (second row), Tbr1 (third row), Satb2 (fourth row), red), counterstained with Hoechst (blue). Dotted line outlines the area from subplate to surface of cortex. (F) Rbp4-Cre neurons (stained using GFP antibody, green), counterstained with Hoechst (blue), show that neurons within both the superficial and deep layers at E14.5, as well as neurons within both the intermediate and deep layers at E18.5, all colocalize with Bcl11b (red), the expression of which is restricted in the adult cortex to layer 5. Arrows: example Rbp4-Cre neurons. (G) Genes selective for each embryonic layer 5 type. Transcript counts for each gene shown as colored circles in all types at all ages. Radius of circle: fraction of cells expressing the gene; color of circle: mean normalized transcripts per cell (log 2 ). Bold text: genes used for in situ hybridization, shown in Figure 2 C. (H) Spatial distribution of Rbp4-Cre neurons into layers is indistinguishable when expressing GCaMP6s compared to tdTomato on both E14.5 and E18.5, and Rbp4-Cre neurons. Quantifying the distribution of Rbp4-Cre neurons into layers at E14.5 (top) and E15.5 (bottom) in embryos generated by using the GCaMP6s-tTA2 reporter line, or a tdTomato reporter line. Probability: χ 2 test comparing the fraction of Rbp4-Cre neurons in each layer for the two different reporter lines, p = 0.05. Scale bars: 20 μm (C, E, F).

Article Snippet: Rat monoclonal anti CTIP2/BCL11B , Merck , Cat# MABE1045.

Techniques: Expressing, Labeling, Marker, Staining, Antibody Labeling, In Situ Hybridization, Generated

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet: Rbp4-Cre neurons show two phases of increased spontaneous activity (A) Schematic diagram of in vivo para-uterine two-photon calcium imaging ( Figure S3 ). Top right: single embryonic neuron; arrowhead: soma activity; arrow: neurite activity; color: normalized calcium activity. (B) Mating strategy to drive GCaMP6s expression in Rbp4-Cre neurons. (C and D) Two-photon imaging of somas (red, C) and neurites (blue, D) of Rbp4-Cre neurons. Two regions of interest (ROIs) (left) and their recorded activity traces (right). (E and F) Activity of individual somas (E) and neurites (F) of Rbp4-Cre neurons. Circles: activity of each ROI; box (25–75 percentile) and whisker (5–95 percentile); white line: median; n = number of somas or neurites. Recordings from 3 (E13.5), 9 (E14.5), 5 (E15.5), 4 (E16.5), 5 (E17.5), and 6 (E18.5) embryos ( Figures S4 and ). (G) Activity (mean ± SEM) (data from E [soma, red] and F [neurite, blue]). Dotted line: separation of active phases and transition phase. (H) Distribution of activity (data from E [soma, red] and F [neurite, blue]) in log-scale. Horizontal lines: median (black: soma; white: neurite). (I) Activity in the two active phases across the three layers. (J) Schematic of Rbp4-Cre neuron development, highlighting the two circuit motifs and phases of activity. (K) Left: schematic of electroporations. Right: Immunostaining of electroporated Rbp4-Cre neurons (red), Bcl11b (white), Hoechst (blue). (L) Distribution of mKir2.1- or Kir2.1-positive Rbp4-Cre neurons’ normalized depths (as in Figure 2 A) (10 mKir2.1-tdTomato and 10 Kir2.1-tdTomato electroporated embryos). Colored lines: medians. n = number of neurons. (E, F, H, L) Wilcoxon rank-sum test. Scale bars: 10 μm (inset, A), 40 μm (left, C, D), 25 s and 25 %ΔF/F (right, C), 25 s and 50 %ΔF/F (right, D), 2 ave. %ΔF/F (G), 20 μm (K), 10% (L). See also Figures S3 , , and .

Article Snippet: Rat monoclonal anti CTIP2/BCL11B , Merck , Cat# MABE1045.

Techniques: Activity Assay, In Vivo, Imaging, Expressing, Whisker Assay, Immunostaining

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet: Perturbing autism-associated genes selectively in Rbp4-Cre neurons disrupts circuit organization and activity during embryonic development (A) Expression (circles) of selected genes associated with autism spectrum disorder in the three Rbp4-Cre neuron types and adult L5-PN types. Radius of circles: fraction of cells expressing the gene; color of circles: mean normalized transcripts per cell (log 2 ). (B) Fraction of genes with a mean transcript count greater than the number of transcripts shown on the x axis, for all genes (black), and genes associated with autism spectrum disorder (magenta) in Rbp4-Cre neurons (top) and adult L5-PNs (bottom). Inset: Fold change of autism-associated gene expression compared to all genes in embryos and adult. (C) Immunostaining of cortex of Rbp4-tdTomato-Chd8 +/− (top) and Rbp4-tdTomato-Grin2b +/− (bottom) mice ( Figure S8 ). Rbp4-Cre neurons (red), Bcl11b, (white), Hoechst (blue). (D) Normalized depths of Rbp4-Cre neurons (as in Figure 2 A) in Rbp4-tdTomato (WT) and the two mutant (top, Chd8 +/− ; bottom, Grin2b +/− ) embryos ( Figure S9 ). 125 neurons from each mouse line, sampled at random. χ 2 test. (E) Mating strategy to generate Rbp4-GCaMP6s-tTA2-Chd8 +/− (Chd8 +/− ) and Rbp4-GCaMP6s-tTA2-Grin2b +/− (Grin2b +/− ) embryos. (F) Example recordings from Rbp4-Cre neurons’ dendrites in Rbp4-GCaMP6s-tTA2 (WT), Grin2b +/− , and Chd8 +/− embryos at E16.5 using 3D acousto-optic two-photon microscope. (G) Distribution of activity in E16.5 embryos, shown in log-scale, for WT and two mutant genotypes. Circles: activity of each neurite; red line: median; shading: distribution. Wilcoxon rank-sum test. n = number of neurites. (H) Immunostaining of local patches of cortical disorganization in Rbp4-tdTomato-Chd8 +/− and Rbp4-tdTomato-Grin2b +/− mice, at E18.5. Rbp4-Cre neurons (red), Bcl11b, (white), Hoechst (blue). (I) Fraction of mutant mice, of each genotype, showing at least one patch, summed across E16.5 to E18.5. Red line: Average across all four genotypes. Data from 14 (Rbp4-tdTomato-Chd8 +/− ), 11 (Rbp4-tdTomato-Chd8 −/− ), 9 (Rbp4-tdTomato-Grin2b +/− ), and 6 (Rbp4-tdTomato-Grin2b −/− ) embryos. Fisher’s exact test (p = 0.05, prior to Bonferroni correction). (J) Fraction of neurons within the superficial layer that are located within patches of disorganization, in embryos with at least one patch. n = number of superficial layer neurons on each embryonic day. Scale bars: 20 μm (C), 25s and 25 %ΔF/F (F), 50 μm (H). See also Figures S8 and .

Article Snippet: Rat monoclonal anti CTIP2/BCL11B , Merck , Cat# MABE1045.

Techniques: Activity Assay, Expressing, Immunostaining, Mutagenesis, Microscopy

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet: Perturbing autism-associated genes selectively in Rbp4-Cre neurons disrupts organization of layer 5 during embryonic development, related to Figure 7 (A) Rbp4-Cre neurons (stained using tdTomato antibody, red) in Rbp4-tdTomato-Chd8 −/− (top) and Rbp4-tdTomato-Grin2b −/− (blue) mice, from E14.5 to E18.5, within the cortical plate (magenta), subplate (cyan), and intermediate zone (yellow), counterstained with Hoechst (blue). (B) Distribution of Rbp4-Cre neuronal locations as a fraction of the cortical plate and subplate thickness, from E14.5 to E18.5, in control (WT, black), Rbp4-tdTomato-Chd8 −/− (top, green), and Rbp4-tdTomato-Grin2b −/− (bottom, orange) mice. 85 neurons from each mouse line, sampled at random, displayed on each embryonic day. Layer boundaries derived from Figure 2 A (blue: superficial layer; gray: intermediate layer; beige: deep layer). Probability: χ 2 test comparing the fraction of Rbp4-Cre neurons in each layer between the conditional knockout mouse and control mice, on each embryonic day; p = 0.05. (C) Local patches of disorganization in Rbp4-tdTomato-Chd8 and Rbp4-tdTomato-Grin2b conditional knockout (cKO) mice (examples from each embryonic day from E16.5 to E18.5) show Rbp4-Cre neurons (stained using tdTomato antibody, red) at the surface and disrupted intermediate layer, including both neurons expressing Cre (red) and not expressing Cre (stained using Bcl11b antibody, white), counterstained with Hoechst (blue). (D) Superficial layer Rbp4-Cre neurons in E18.5 control mouse, without disorganization of the underlying intermediate layer. Scale bar: Scale bar: 20 μm (A, C, D).

Article Snippet: Rat monoclonal anti CTIP2/BCL11B , Merck , Cat# MABE1045.

Techniques: Staining, Derivative Assay, Knock-Out, Expressing

Journal: Cell

Article Title: Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex

doi: 10.1016/j.cell.2023.03.025

Figure Lengend Snippet:

Article Snippet: Rat monoclonal anti CTIP2/BCL11B , Merck , Cat# MABE1045.

Techniques: Recombinant, Electron Microscopy, Multiplex Assay, Expressing, Plasmid Preparation, Sequencing, Positive Control, Negative Control, Software, RNA Sequencing Assay, Transmission Assay, Microscopy, Laser-Scanning Microscopy, Imaging

Journal: The Journal of Cell Biology

Article Title: δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte–neuron cadherin interactions

doi: 10.1083/jcb.202303138

Figure Lengend Snippet: Neuronal δ-catenin is required for astrocyte complexity. (a) Schematic of experimental design. Ctnnd2 was silenced in upper-layer neurons by IUE at E15.5. At P0, wild-type astrocytes were labeled by mCherry-CAAX (cyan) by PALE. Brains were collected at P21 for analysis of astrocyte morphology and territory size. SVZ, subventricular zone. (b) Representative images of the primary visual cortex after IUE and PALE. Upper-layer neurons (green) are transfected with shControl or shCtnnd2, lower-layer neurons (magenta) are labeled with Ctip2, and wild-type astrocytes (cyan) are labeled with mCherry-CAAX. (c) Representative images of P21 astrocytes after upper-layer neurons were transfected with shControl or shCtnnd2. Whole astrocytes were reconstructed using the Imaris filament tracing tool. Inset shows a confocal image of the astrocyte with a convex hull denoting astrocyte territory volume. (d) Silencing δ-catenin expression in upper-layer neurons resulted in a significant decrease in upper-layer astrocyte complexity (P = 5.99 × 10 −4 ) but not in lower-layer astrocyte complexity (P = 0.96). Quantification of in vivo astrocyte complexity with 3D Sholl analysis. n = 13–17 astrocytes from six to nine mice per condition. ANOVA, linear mixed model with Tukey HSD. ns, not significant. (e) Silencing δ-catenin expression in upper-layer neurons resulted in a significant decrease in upper-layer astrocyte territory volume (P = 0.0005) but not in lower-layer astrocyte complexity (P = 0.45). Quantification of in vivo astrocyte territory volumes by convex hull analysis. Astrocytes from the upper (green) and lower purple) layers of the V1 cortex were imaged and analyzed. Average astrocyte territory volume of individual mice is plotted in black. n = 13–17 astrocytes from six to nine mice. Nested t test for each layer. ns, not significant. All data are presented as mean ± SEM. The scale bar in b is 100 μm, while the scale bar in c is 10 μm. *** P < 0.001.

Article Snippet: For immunostaining in PALE experiments, 100 μM floating sections were washed and permeabilized in 1X TBS containing 0.4% Triton X-100 (0.4 % TBST), blocked in 10% NGS diluted in 0.4% TBST, and incubated shaking in the following primary antibodies for three nights at 4°C: rat anti-Ctip2 (1:500, #Ab00616-7.4; Absolute Antibody), rabbit anti-RFP (1:2,000), and mouse anti-Satb2 (1:500, #ab51502; Abcam).

Techniques: Labeling, Transfection, Expressing, In Vivo