Journal: Nature Communications

Article Title: Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture

doi: 10.1038/s41467-021-24624-5

Figure Lengend Snippet: a Doxycycline-inducible expression of V5-tagged transcription factors (TFs) or EGFP as a control, in astrocyte cultures to assess the contribution of Rorb , Dbx2 , Lhx2 and Fezf2 to astrocyte maturation. Cells are infected with the lentiviral vectors at day 6 of differentiation, followed by transgene induction with doxycycline from day 7 to day 14. b Immunolabeling for the transgene (EGFP or V5 tag) and the mature astrocyte enzyme glutamine synthetase (GS, gene Glul ). c Expression of mature astrocyte-specific genes (from Fig. ) in cultured astrocytes and cortical astrocytes. Heatmap of selected genes. Barplot showing that subsets of mature genes with low expression in control cultures are induced by the transcription factors analysed. d Calcium responses induced by mechanical stimulation in Rorb- or Fezf2-overexpressing and control (rtTA expressing only) astrocytes, measured by time-lapse imaging with the calcium-sensitive dye OGB. e , f Metabolic changes induced by Rorb or Fezf2 in vitro, measured by gas chromatography coupled with mass spectrometry (GC-MS). Relative changes of intracellular metabolite levels ( e ), and glutamine release into the culture medium ( f ). Statistical analysis and data presentation: b scale bar 50 μm; shown: means ± SD; n = 3 biological replicates, one-way ANOVA ( F = 117.4, p < 0.0001) with Tukey’s multiple comparisons test vs EGFP. c Differential genes: DESeq2 analysis, each n = 3; two-sided Wald test with Benjamini–Hochberg correction; significance threshold: adjusted p -value ≤ 0.05, absolute log2(fold change) ≥ 1. Shown are log2-transformed mean centred normalized expression values; d representative time-lapse series (pseudocoloured, scale bar 20 μm) and individual cell traces (displaying the change in fluorescence vs background (dF/F)), and barplots showing the fraction of responsive and oscillating cells (means ± SD, n = 3 biological replicates, repeated measures one-way ANOVA with Tukey’s multiple comparisons test; %responding: F = 7.711, p = 0.042; %oscillating: F = 7.878, p = 0.041); e Heatmap showing log2-normalised changes in metabolite levels relative to mean of each experiment ( n = 5 independent experiments), Two-sided t -tests vs control with Benjamini–Hochberg correction for multiple testing; significant changes (padj < 0.05) are highlighted); f quantification of glutamine in samples of originally glutamine-free medium (means ± SD shown, n = 5 independent experiments, repeated measures one-way ANOVA with Tukey’s multiple comparisons test; F = 32.87, p = 0.0021); See also Supplementary Fig. , Supplementary Data 7, Supplementary Movies – .

Article Snippet: Rorb , Dbx2 , Lhx2 , and Fezf2 open reading frame (ORF) constructs from Origene (MR225771, MR215327, MR206390, MR207270) were re-cloned into a modified version of the TetO-FUW-EGFP lentiviral expression plasmid (Addgene #30130) .

Techniques: Expressing, Control, Infection, Immunolabeling, Cell Culture, Imaging, In Vitro, Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry, Transformation Assay, Fluorescence

Journal: Nature Communications

Article Title: Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture

doi: 10.1038/s41467-021-24624-5

Figure Lengend Snippet: a ATAC-Seq genome tracks showing chromatin accessibility peaks around the transcriptional start site (TSS) of the mature gene Clmn in cultured astrocytes expressing transcription factors and in brain astrocytes (at postnatal day 4 (Astr_P4), and 2 months of age (Astr_2m)). Note the highlighted peaks that are present in cultured astrocytes only when Rorb or Lhx2 are expressed. b Heatmaps showing chromatin accessibility in cultured astrocytes around Rorb - and Lhx2 -induced peaks and in brain astrocytes. c Transcription factor motifs enriched in Rorb - and Lhx2 -induced peaks (de novo motif enrichment analysis). d Expression of mature astrocyte-specific genes induced by the combined expression of Rorb and Fezf2 . Astrocytes co-infected with Rorb- and Fezf2-inducing viruses were analysed along with samples from Fig. . Statistical analysis and data presentation: a ATAC tracks represent merged reads of 3 replicates (4 for Astr_2m, 6 for Control). b Heatmaps show normalized ATAC read count of merged samples in 20 bp windows ±1 kb of peak centres. Differential peaks: DESeq2 analysis, each n = 3 ( n = 4 for Astr_2m, n = 6 for Control); two-sided Wald test with Benjamini–Hochberg correction; significance threshold: adjusted p -value ≤ 0.05, absolute log2(fold change) ≥ 1. Shown are log2-transformed normalized expression values. d Differential genes: DESeq2 analysis, each n = 3 ( n = 6 for Control); two-sided Wald test with Benjamini–Hochberg correction; significance threshold: adjusted p -value (FDR) ≤ 0.001, absolute log2(fold change) ≥ 1. Shown are log2-transformed mean centred, normalized expression values. See also Supplementary Fig. , Supplementary Data .

Article Snippet: Rorb , Dbx2 , Lhx2 , and Fezf2 open reading frame (ORF) constructs from Origene (MR225771, MR215327, MR206390, MR207270) were re-cloned into a modified version of the TetO-FUW-EGFP lentiviral expression plasmid (Addgene #30130) .

Techniques: Cell Culture, Expressing, Infection, Control, Transformation Assay

Journal: The Journal of Comparative Neurology

Article Title: Mapping the distribution of language related genes FoxP1 , FoxP2 , and CntnaP2 in the brains of vocal learning bat species

doi: 10.1002/cne.24385

Figure Lengend Snippet: Details of antibodies used for immunohistochemistry

Article Snippet: RORBorigene , TA806996 , W001 , Origene , mouse , 9 , All species , 1:100 , 10% normalgoat serum , BiotinylatedGoat anti‐mouse , BA‐9200 , Vector Labs , 1:1000 , ABC.

Techniques: Blocking Assay, Plasmid Preparation