Cortical Neurogenesis Requires Bcl6-Mediated Transcriptional Repression of Multiple Self-Renewal-Promoting Extrinsic Pathways

Abstract

During neurogenesis, progenitors switch from self-renewal to differentiation through the interplay of intrinsic and extrinsic cues, but how these are integrated remains poorly understood. Here, we combine whole-genome transcriptional and epigenetic analyses with in vivo functional studies to demonstrate that Bcl6, a transcriptional repressor previously reported to promote cortical neurogenesis, acts as a driver of the neurogenic transition through direct silencing of a selective repertoire of genes belonging to multiple extrinsic pathways promoting self-renewal, most strikingly the Wnt pathway. At the molecular level, Bcl6 represses its targets through Sirt1 recruitment followed by histone deacetylation. Our data identify a molecular logic by which a single cell-intrinsic factor represses multiple extrinsic pathways that favor self-renewal, thereby ensuring robustness of neuronal fate transition.

Keywords: Bcl6; FGF signaling; Notch signaling; SHH signaling; Wnt signaling; brain development; cyclins; neurogenesis; stemness; transcription.

Graphical abstract

Figure 1

Bcl6 Promotes a Neurogenic Transcription Program and Represses Selective Genes of the Main Proliferative Pathways (A) Scheme representing the differentiation protocol of Bcl6-inducible A2 lox.Cre mouse embryonic stem cells into cortical progenitors. Bcl6 expression was induced at day 12 using a single doxycycline pulse for 24 h. (B) Gene Ontology analysis showing statistically significant enrichment for some categories of up- and downregulated genes following Bcl6 induction (see also Table S2 for complete lists). (C) Histograms representing the log-fold change of a series of significantly up- or downregulated genes, respectively indicated in red or blue, selected upon their expression and/or function during cortical differentiation. IP, intermediate progenitors; NE, neuroepithelial cells; RGC, radial glial cells (see also Table S1 for complete lists). (D) Histograms representing the log-fold change of significantly up- or downregulated genes, respectively indicated in red or blue, belonging to the main proliferative pathways in cortical progenitors. Only the potential target genes with known expression in embryonic cortical progenitors are indicated. For the complete list of genes taken into consideration for the analysis, see also Table S3. Genes marked with an asterisk also are target genes of the pathway itself. SVZ, subventricular zone; VZ, ventricular zone. (E) Scheme of the canonical Wnt pathway depicting the role in the cascade of the ensemble of Wnt/β-catenin-related genes bound and/or altered by Bcl6 investigated in this study. (F) qRT-PCR analysis of the Notch target Hes5, Wnt-related genes Ctnnb1 and Ccnd2, and the cell fate markers Pax6, Eomes, and Tubb3 in day 12 in vitro cortical progenitor cells treated with DMSO (control) or doxycycline for 6, 12, or 24 h. Data are presented as mean + SEM of Dox over control (Ctrl) absolute levels (n = 7–9 [6 h], 27–30 [12 h], and 9 [24 h] from at least 3 independent differentiations for each group). ^∗p < 0.05; ^∗∗p < 0.01 using Student’s t test. See also Figure S1.

Figure 2

Bcl6 Alters β-Catenin Signaling In Vivo to Promote Neurogenesis (A) In situ hybridization of Axin2 Wnt reporter gene on coronal sections of E12.5 wild-type and Bcl6^−/− frontal and parietal telencephalon. Scale bars, 500 μm. (B) Normalized gray scale quantifications of Axin2 levels were performed using ImageJ software. Data are presented as mean + SEM. ^∗p < 0.05 using Student’s t test. (C–E) In utero electroporation of pCIG, pCIG+pCAG-Δ(1-90)Ctnnb1, pCIG-Bcl6+pCIG or pCIG-Bcl6+pCAG-Δ(1-90)Ctnnb1 at E13.5. (C) Hoechst and GFP immunofluorescence was performed on coronal sections of E15.5 brains. Dashed lines mark the basal and apical margins of the VZ, SVZ, intermediate zone (IZ), and cortical plate (CP). Scale bar, 50 μm. (D) Histograms show the percentage of GFP+ cells in VZ, SVZ, IZ, and CP. ^∗∗p < 0.01 pCIG-Bcl6+pCIG versus pCIG and ^#p < 0.05, ^###p < 0.001 pCIG-Bcl6+pCAG-Δ(1-90)Ctnnb1 versus pCIG-Bcl6+pCIG using two-way ANOVA followed by Tukey post hoc test. (E) Histograms show the percentage of Pax6+ and Tuj1+ cells among the GFP+ cells. ^∗∗p < 0.01, ^∗∗∗p < 0.001 pCIG-Bcl6+pCIG versus pCIG+pCIG and ^###p < 0.001 pCIG-Bcl6+pCAG-Δ(1-90)Ctnnb1 versus pCIG-Bcl6+pCIG using one-way ANOVA followed by Tukey post hoc test. Data are presented as mean + SEM of n = 6 control embryos (1,856 cells), n = 10 embryos for stabilized β-catenin gain of function (2,727 cells), n = 9 embryos for Bcl6 gain of function (1,922 cells), and n = 9 embryos for Bcl6 and stabilized β-catenin double gain of function (1,588 cells). (F–H) In utero electroporation of scramble (control), scramble+Ctnnb1, scramble+Hes5, scramble+Ctnnb1+Hes5, scramble+Bcl6, scramble+Ctnnb1+Bcl6, scramble+Hes5+Bcl6, and Ctnnb1+Hes5+Bcl6 shRNAs at E13.5. (F) Representative images of Hoechst and GFP immunofluorescence performed on coronal sections of E16.5 brains. Dashed lines mark the basal and apical margins of the VZ, SVZ, IZ, and CP. Scale bar, 50 μm. (G) Histograms show the percentage of GFP+ cells in VZ, SVZ, IZ, and CP. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 versus control and ^#p < 0.05, ^###p < 0.001 versus Bcl6+control shRNAs using two-way ANOVA followed by Tukey post hoc test. (H) Histograms show the percentage of Pax6+ and β3-tubulin+ cells among the GFP+ cells. ^∗p < 0.05, ^∗∗p < 0.01 versus control and ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 versus Bcl6+control shRNAs using one-way ANOVA followed by Tukey post hoc test. Data are presented as mean + SEM of n = 15 control embryos (4,476 cells), n = 7 embryos for Ctnnb1 shRNA (1,851 cells), n = 13 embryos for Hes5 shRNA (3,893 cells), n = 7 embryos for Ctnnb1+Hes5 shRNA (1,932 cells), n = 6 embryos for Bcl6 shRNA (2,093 cells), n = 9 embryos for Bcl6+Ctnnb1 shRNA (2,642 cells), n = 10 embryos for Bcl6+Hes5 shRNA (2,614 cells), and n = 9 embryos for Bcl6+Ctnnb1+Hes5 shRNA (2,993 cells). See also Figures S2, S3, and S4.

Figure 3

Bcl6 Directly Affects Ccnd1/Ccnd2 Expression In Vivo to Promote Neurogenesis (A–D) In situ hybridization of (A) Ccnd1 and (C) Ccnd2 genes on coronal sections of E12.5 wild-type and Bcl6^−/− frontal and parietal telencephalon. Normalized gray scale quantifications of (B) Ccnd1 and (D) Ccnd2 levels were performed using ImageJ software. Data are presented as mean + SEM. ^∗p < 0.05, ^∗∗p < 0.01 using Student’s t test. Black scale bars, 500 μm. Red scale bar, 100 μm. (E–G) In utero electroporation of scramble (control), scramble+Ccnd1+Ccnd2, scramble+Bcl6, and Ccnd1+Ccnd2+Bcl6 shRNAs at E13.5. (E) Representative images of Hoechst and GFP immunofluorescence performed on coronal sections of E16.5 brains. Dashed lines mark the basal and apical margins of the VZ, SVZ, IZ, and CP. Scale bar, 50 μm. (F) Histograms show the percentage of GFP+ cells in VZ, SVZ, IZ, and CP. ^∗p < 0.01, ^∗∗p < 0.01 versus control and ^###p < 0.001 versus Bcl6+control shRNAs using two-way ANOVA followed by Tukey post hoc test. (G) Histograms show the percentage of Pax6+ and β3-tubulin+ cells among the GFP+ cells. ^∗∗p < 0.01, ^∗∗∗p < 0.001 versus control and ^###p < 0.001 versus Bcl6+control shRNAs using one-way ANOVA followed by Tukey post hoc test. Data are presented as mean + SEM of n = 12 control embryos (3,627 cells), n = 7 embryos for Ccnd1+Ccnd2 shRNA (1,519 cells), n = 8 embryos for Bcl6 shRNA (1,998 cells), and n = 12 embryos for Bcl6+Ccnd1+Ccnd2 shRNA (3,475 cells). See also Figures S3 and S4.

Figure 4

Bcl6 Binds to Core Genes of the Wnt/β-Catenin Pathway to Alter Their Expression (A) qRT-PCR analysis of Wnt-related genes from DMSO- and doxycycline-treated cells at day 12. Data are presented as mean + SEM of absolute levels (n = 21 from 8 differentiations). ^∗p < 0.05 and ^∗∗p < 0.01 using Student’s t test. (B) ChIP-qPCR validation of screened Bcl6-binding sites on regulatory regions and negative control sites of significantly downregulated Wnt-related genes in E12.5 wild-type and Bcl6^−/− telencephalon using a Bcl6 antibody. Data are presented as mean + SEM of input enrichment (n = 4). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 using Student’s t test. Genes are represented using 5′ to 3′ orientation with light blue showing 5′ UTR and 3′ UTR exons and thicker dark blue showing the exons of the coding sequence and the introns according to RefSeq gene sequences from mouse genome mm10 assembly (https://genome.ucsc.edu). Black boxes indicate amplified regions (BS, region comprising the Bcl6-predicted binding site inside the ChIP-seq significant peaks; NEG, region with no predicted Bcl6 matrix using the Jaspar software [http://jaspar.genereg.net]). See also Figures S5 and S6.

Figure 5

Bcl6 Binding to Significantly Downregulated Wnt-Related Genes Induces Chromatin Remodeling and Sirt1 Recruitment (A) ChIP-qPCR of histone acetylation marks on Bcl6 binding sites of significantly downregulated Wnt-related genes in E12.5 wild-type and Bcl6^−/− telencephalon using control (rabbit immunoglobulin G [IgG]) and H1.4K26ac and H4K16ac antibodies. Data are presented as mean + SEM of input enrichment (n = 6). ^∗p < 0.05 and ^∗∗p < 0.01 using Student’s t test. (B) ChIP-qPCR on validated Bcl6 binding sites of significantly downregulated Wnt-related genes in E12.5 wild-type and Bcl6^−/− telencephalon using a Sirt1 antibody. Data are presented as mean + SEM of input enrichment (n = 6). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 using Student’s t test. See also Figure S6.

Figure 6

Bcl6 and Sirt1 Bind to Ccnd1 and Ccnd2 Regulatory Regions, Leading to the Removal of the β-Catenin Effector Tcf7l1 (A) Schematic representation of the genomic region 2 kb upstream of Ccnd1 and Ccnd2 transcription starting sites showing validated Bcl6 (red) as well as putative Tcf7l1 (blue) binding sites and negative regions for either transcription factor (green) as predicted by the Jaspar software (http://jaspar.genereg.net). The arrows represent the amplified regions by qPCR used to measure the enrichment following ChIP. (B) ChIP-qPCR analysis of the Bcl6, Sirt1, and Tcf7l1 binding sites on the Ccnd1 and Ccnd2 regulatory regions in cortical progenitors derived from Bcl6 A2 lox.Cre mouse ESCs (differentiation day 12, 24 h DMSO [Ctrl] or Dox treatment). Data are presented as mean + SEM of input enrichment (n = 3 differentiations). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 using Student’s t test. (C) ChIP-qPCR analysis of the H4K16ac histone acetylation mark on Bcl6 binding sites of the Ccnd1 and Ccnd2 regulatory regions in cortical progenitors derived from Bcl6 A2 lox.Cre mouse ESCs (differentiation day 12, 24 h DMSO [Ctrl] or Dox treatment). Data are presented as mean + SEM of input enrichment (n = 3 differentiations). ^∗p < 0.05 using Student’s t test. See also Figure S7.