Fig. 5: Different transcription factors control distinct aspects of astrocyte maturation.
From: Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture
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. 2c) 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. 7, Supplementary Data 7, Supplementary Movies 1–3.
