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. 2023 Nov 17;9(46):eadd0676.
doi: 10.1126/sciadv.add0676. Epub 2023 Nov 15.

MKL/SRF and Bcl6 mutual transcriptional repression safeguards the fate and positioning of neocortical progenitor cells mediated by RhoA

Alexia Cossard  1 , Kirsten Stam  1 , Aurore Smets  1 , Yves Jossin  1
Affiliations

MKL/SRF and Bcl6 mutual transcriptional repression safeguards the fate and positioning of neocortical progenitor cells mediated by RhoA

Alexia Cossard et al. Sci Adv. .

Abstract

During embryogenesis, multiple intricate and intertwined cellular signaling pathways coordinate cell behavior. Their slightest alterations can have dramatic consequences for the cells and the organs they form. The transcriptional repressor Bcl6 was recently found as important for brain development. However, its regulation and integration with other signals is unknown. Using in vivo functional approaches combined with molecular mechanistic analysis, we identified a reciprocal regulatory loop between B cell lymphoma 6 (Bcl6) and the RhoA-regulated transcriptional complex megakaryoblastic leukemia/serum response factor (MKL/SRF). We show that Bcl6 physically interacts with MKL/SRF, resulting in a down-regulation of the transcriptional activity of both Bcl6 and MKL/SRF. This molecular cross-talk is essential for the control of proliferation, neurogenesis, and spatial positioning of neural progenitors. Overall, our data highlight a regulatory mechanism that controls neuronal production and neocortical development and reveal an MKL/SRF and Bcl6 interaction that may have broader implications in other physiological functions and in diseases.

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Figures

Fig. 1.
Fig. 1.. RhoA reduces neurogenesis and maintains the neural progenitor pool size.
Embryonic brains were in utero electroporated at E15.5 and processed 23 hours later for immunohistological labeling. (A to C, G, and I) Quantification of double staining for (A) GFP+Sox2+, (B) GFP+Tbr2+, (C) GFP+SatB2+, (G) GFP+Ki67− (cell cycle exit), and (I) GFP+p-H3+ (mitotic index) cells. (D) Quantification of the triple-staining GFP+Sox2+ Tbr2− (RGCs), GFP+Sox2+Tbr2+ (committed BPs), GFP+Sox2−Tbr2+ (BPs), and GFP+Sox2−Tbr2− (neurons) cells. (E, F, and H) Coronal sections of E16.5 cerebral cortices electroporated at E15.5 for the expression of NLS-GFP alone (control), dominant-negative form of RhoA (RhoADN), or wild-type RhoA (RhoAWT), along with NLS-GFP and immunostained for (E) Sox2 and Tbr2, (F) Ki67, or (H) p-H3. (A) Control n = 19 of 13 IUE (n = 19//13); RhoAWT n = 8//6; RhoADN n = 9//9; (B) Control n = 21//14; RhoAWT n = 8//6; RhoADN n = 10//8; (C) Control n = 4//3; RhoAWT n = 4//3; RhoADN n = 4//4; (D) Control n = 9//5; RhoAWT n = 7//6; RhoADN n = 8//7; (G) Control n = 12//9; RhoAWT n = 9//7; RhoADN n = 11//6; (I) Control n = 7//4; RhoAWT n = 7//6; RhoADN n = 9//6. Error bars, SEM. ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar, 50 μm.
Fig. 2.
Fig. 2.. RhoA regulates the position of RGCs and BPs.
(A) Coronal sections of E16.5 mice cerebral cortices electroporated with either control, dominant-negative (RhoADN), or wild-type RhoA (RhoAWT) expression vectors at E15.5, coelectroporated with NLS-GFP, and stained for 4′,6-diamidino-2-phenylindole (DAPI). (B to F) Graphics of cerebral walls from electroporated brains, corresponding to the VZ to the upper part of the IZ, and subdivided into 20 bins. The graphics indicate the proportion of cells in each bin of (B) GFP+ cells or (C) GFP+Sox2+ Tbr2− (RGCs), (D) GFP+Sox2+Tbr2+ (Committed BPs), (E) GFP+Sox2−Tbr2+ (BPs), and (F) GFP+Sox2−Tbr2− (neurons) for control, RhoADN, or RhoAWT expression vectors electroporated at E15.5 and observed at E16.5. (B) Control n = 21 of 15 IUE (n = 21//15); RhoAWT n = 14//12; RhoADN n = 20//14; (C to F) Control n = 9//5; RhoAWT n = 7//6; RhoADN n = 6//6. Error bars, SEM. ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar, 50 μm. (G and H) Time-lapse analysis of RGCs in the VZ. Frames are every 20 min. Brains were electroporated at E15.5 and processed for videomicroscopy 14 hours later. (G) Representative pictures of the nuclear movement (red arrowheads) followed by control and RhoA-inhibited GFP+ RGCs in the VZ and with a visible apical process attached to the apical surface. (H) Representative nuclear tracks of the paths followed by control and RhoA-inhibited GFP+ RGCs in the VZ with a visible attached apical process. Distance from the ventricular surface of cell centroids in successive frames (circles) is linked by lines.
Fig. 3.
Fig. 3.. The MKL/SRF complex regulates neurogenesis downstream of RhoA.
(A) Luciferase assays were performed on lysates from E16.5 cortices electroporated 1 day earlier with control, RhoADN, RhoAWT, or shMKL1/2 expression vectors together with a pGL4-promoter vector containing an SRF-RE that drives transcription of the firefly luciferase reporter gene and a pRL Renilla luciferase internal control. Normalized values are reported as the mean fold expression of luciferase activity ± SD from three independent IUE. Relative Luciferase activity in arbitrary units with control set to 1. (B to E) Brains were in utero electroporated at E15.5 with the indicated plasmids along with NLS-GFP and stained at E16.5 for the indicated markers. (B and D) Quantification of the GFP+Sox2+Tbr2− (RGCs), GFP+Sox2+Tbr2+ (committed BPs), GFP+Sox2−Tbr2+ (BPs), and GFP+Sox2−Tbr2− (neurons) cells. (B) Epistasis experiments are statistically tested against RhoADN or RhoAWT. Controls are shown for comparison. Control n = 9 of 5 IUE (n = 9//5); RhoAWT n = 7//6; RhoADN n = 8//7; RhoADN + MKL1(low) n = 4//3; RhoADN + MKL2 n = 4//3; RhoADN + SRF-TAD (low) n = 4//3; RhoAWT + shMKL1/2 n = 5//3; RhoAWT + SRF-EnRD n = 8//6. (C) Coronal sections of E16.5 cerebral cortices. (D) Control n = 9//5; shMKL1/2 n = 10//5; MKL1 (low) n = 4//3; MKL2 n = 5//4; SRF-TAD (low) n = 8//3; SRF-EnRD n = 6//3. (E) Quantification of GFP+Ki67− cells (exit from cell cycle). Control n = 12//9; RhoAWT n = 9//7; RhoADN n = 11//6; shMKL1/2 n = 9//4; MKL1 (low) n = 4//3; MKL2 n = 6//5; RhoAWT + shMKL1/2 n = 3//3; RhoADN + MKL1 n = 4//3; RhoADN + MKL2 n = 4//3. Error bars, SEM. ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar, 50 μm.
Fig. 4.
Fig. 4.. The MKL/SRF complex regulates the position of RGCs and BPs downstream of RhoA.
E16.5 Coronal sections of mice cerebral cortices electroporated at E15.5 with the indicated plasmids along with NLS-GFP expression vector and stained with DAPI. The associated graphics show the radial distribution of GFP+ cells. (A, A', and A'') MKL1 and SRF-TAD but not MKL2 rescued the positioning defect induced by RhoADN; RhoADN n = 20 of 14 IUE (n = 20//14); RhoADN + MKL1 n = 5//3; RhoADN + MKL2 n = 4//3; RhoADN + SRF-TAD n = 6//3. (B, B', B'', and B''') shMKL1/2, shMKL1, and SRF-EnRD but not shMKL2 rescued the positioning defect induced by RhoAWT; RhoAWT n = 14//12; RhoAWT + shMKL1/2 n = 8//3; RhoAWT + shMKL1 n = 15//5; RhoAWT + shMKL2 n = 5//3; RhoAWT + SRF-EnRD n = 12//6; (C, C', C'', and C''') Inhibition or GoF of MKL1 and SRF but not MKL2 induced similar positional phenotypes as inhibition or GoF of RhoA. Control n = 21//15; shMKL1/2 n = 10//5; MKL1 + MKL2 n = 5//3; shMKL1 n = 9//3; MKL1 n = 12//6; shMKL2 n = 4//3; MKL2 n = 6//6; SRF-EnRD n = 10//6; SRF-TAD n = 9//4. Error bars, SEM. ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar, 50 μm.
Fig. 5.
Fig. 5.. Bcl6 and MKL/SRF mutually repress their transcriptional activity in vitro and in vivo through the formation of a tripartite complex that depends on Bcl6 and SRF direct physical interaction, which prevents each other’s homodimerization.
(A, B, J, and K) TriFC assay. Epifluorescence microscopic images of HEK293T cells 8 hours after transfection for the coexpression of the GFP (S1 to S9) fragment and the indicated proteins fused with either S10 or S11 GFP fragments and untagged proteins. Cells were stained with DAPI. The graphics indicate the relative GFP complementation with the condition “SRF + MKL1” set at 1 ± SD from three independent experiments. (C to E) Coimmunoprecipitation assays were performed using tagged Bcl6, SRF, and MKL1 expressed in HEK293T. Twenty-four hours after transfection, cell lysates were analyzed directly or after pull-down with an HA antibody. Samples were immunoblotted with HA, Myc, or glutathione S-transferase (GST) antibodies. n ≥ 3. (F to I) Luciferase assays performed with either control, RhoAWT, RhoADN, MKL1, MKL2, shBcl6, or Bcl6 expression vectors together with a pRL Renilla luciferase internal control and (F and G) a pGL4-promoter vector containing SRF-RE (results are expressed as relative fold-change of luciferase activity) or (H and I) a pTA vector containing a Bcl6(BS) in front of a cytomegalovirus (CMV) promoter that drives the transcription of the firefly luciferase reporter gene (normalized values are reported as the mean fold repression of luciferase activity). The luciferase assays were performed (F and H) on HEK293T lysates 24 hours after transfection or (G and I) on lysates from E16.5 cortices electroporated 1 day earlier. Relative luciferase activity in arbitrary units ± SD from three independent experiments. Error bars, SEM. ***P < 0.001, **P < 0.01, *P < 0.05, NS, not significant. Scale bar, 50 μm.
Fig. 6.
Fig. 6.. Identification of Bcl6 and SRF domains involved in their interaction.
(A and D) Schematic representation of the different domain constructions. (B, C, and E) TriFC assay. The graphics indicate the comparison of fluorescence from GFP complementation with the conditions “SRF + Bcl6” in (B), “SRF + MKL1” in (C), and “Bcl6 + SRF” in (E) set at 1. n = 8 independent experiments. Error bars, SEM.
Fig. 7.
Fig. 7.. Bcl6 activity counteracts the effect of RhoA on neurogenesis and cell position in vivo.
(A, C, G, I, M, and O) Coronal sections of E16.5 cerebral cortices in utero electroporated at E15.5 with the indicated plasmids along with NLS-GFP. (B, D, H, J, N, and P) The graphics indicate the proportion of cells in each bin of GFP+ cells for the indicated expression vectors electroporated at E15.5 and observed at E16.5. (B) RhoADN n = 20 of 14 IUE (n = 20//14); RhoADN + shBcl6 n = 10//8; (D) RhoAWT n = 14//12; RhoAWT + Bcl6 n = 7//5; (H) shMKL1/2 n = 10//5; shMKL1/2 + shBcl6 n = 11//4; (J) MKL1 n = 12//7; MKL1 + Bcl6 n = 4//3; (N) SRF-EnRD n = 10//6; SRF-EnRD + shBcl6 n = 5//3; (P) SRF-TAD n = 9//4; SRF-TAD + Bcl6 n = 7//3. (E, F, K, L, Q, and R) Quantification of the GFP+Sox2+Tbr2−, GFP+Sox2+Tbr2+, GFP+Sox2−Tbr2+, GFP+Sox2−Tbr2−, and GFP+Ki67− cells. (E) Control n = 9//5; RhoAWT n = 7//6; RhoADN n = 8//7; RhoADN + shBcl6 n = 10//4; RhoAWT + Bcl6 n = 9//5; (F) Control n = 12//9; RhoAWT n = 9//7; RhoADN n = 11//6; RhoADN + shBcl6 n = 12//6; RhoAWT + Bcl6 n = 9//5; (K) Control n = 9//5; shMKL1/2 n = 7//4; MKL1 (low) n = 4//3; MKL2 n = 5//4; shMKL1/2 + shBcl6 n = 8//3; MKL1 (low) + Bcl6 n = 10//4; MKL2 + Bcl6 n = 3//3; (L) Control n = 12//9; shMKL1/2 n = 6//3; MKL1 (low) n = 4//3; MKL2 n = 6//5; shMKL1/2 + shBcl6 n = 15//5; MKL1 (low) + Bcl6 n = 10//4; MKL2 + Bcl6 n = 3//3; (Q) Control n = 9//5; SRF-TAD n = 8//3; SRF-EnRD n = 6//3; SRF-EnRD + shBcl6 n = 7//3; SRF-TAD+Bcl6 n = 5//3; (R) Control n = 12//9; SRF-TAD n = 6//3; SRF-EnRD n = 7//4; SRF-EnRD + shBcl6 n = 6//3; SRF-TAD+Bcl6 n = 6//3. Error bars, SEM. ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar, 50 μm.
Fig. 8.
Fig. 8.. Bcl6 induces neurogenesis in vivo.
E15.5 brains were electroporated with the indicated plasmids along with NLS-GFP and processed at E16.5. (A and E) Coronal sections stained for the indicated markers. (B to D) Quantification of the GFP+Sox2+Tbr2−, GFP+Sox2+Tbr2+, GFP+Sox2−Tbr2+, GFP+Sox2−Tbr2−, GFP+Ki67−, and GFP+p-H3+ cells. (B) Control n = 9 of 5 IUE (n = 9//5); shBcl6 n = 4//3; Bcl6 n = 5//3; (C) Control n = 12//9; shBcl6 n = 5//5; Bcl6 n = 13//7; (D) Control n = 7//4; shBcl6 n = 3//3; Bcl6 n = 12//6. (F) The graphics indicate the percentage of cells in each bin of GFP+ cells from brains electroporated with the indicated expression vectors. Control n = 21//15; shBcl6 n = 10//7; Bcl6 n = 12//6. Error bars, SEM. ***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant. Scale bar, 50 μm.
Fig. 9.
Fig. 9.. Bcl6 and SRF physical interaction is necessary for the RhoA/MKL/SRF pathway and Bcl6 mutual inhibition in vitro and in vivo.
(A) Schematic representation of Bcl6 protein structure and location of mutations in the Bcl6 mutant (Bcl6*). (B) TriFC assay. The graphics indicate the percentage of GFP+ complemented cells with the condition “Bcl6 + SRF” set at 100%. n = 4. (C to E) Luciferase assays performed with either control, MKL1, Bcl6, or Bcl6 bearing the mutations C121F and P421L (Bcl6*) expression vectors together with a pRL Renilla luciferase internal control and a pGL4-promoter vector containing (C) a pTA vector containing a Bcl6(BS) in front of a CMV promoter that drives the transcription of the firefly luciferase reporter gene (normalized values are reported as the mean fold repression of luciferase activity) or (D and E) as SRF-RE (results are expressed as relative fold-change of luciferase activity in arbitrary units). The luciferase assays were performed (C and D) on HEK293T lysates 24 hours after transfection or (E) on lysates from E16.5 cortices electroporated 1 day earlier. n = 3. (F and L) Coronal sections of E16.5 cerebral cortices in utero electroporated at E15.5 with the indicated plasmids along with NLS-GFP. (G and M) The graphics indicate the proportion of cells in each bin of GFP+ cells for the indicated expression vectors electroporated at E15.5 and observed at E16.5. RhoADN n = 20 of 14 IUE (n = 20//14); RhoADN + Bcl6* n = 6//3; Control n = 21//15; Bcl6* n = 11//4. (H to K and N to Q) Quantification of the GFP+Ki67−; GFP+Sox2+; GFP+Tbr2+; GFP+Sox2+Tbr2−; GFP+Sox2+Tbr2+; GFP+Sox2−Tbr2+; GFP+Sox2−Tbr2−; cells. (H and N) RhoAWT n = 9//7; RhoAWT + Bcl6* n = 4//3; RhoAWT + Bcl6 n = 9//5; Control n = 12//9; Bcl6 n = 13//7; Bcl6* n = 9//3; (I to K and O to Q) RhoAWT n = 7//6; RhoAWT + Bcl6* n = 9//3; Bcl6* n = 11//4; RhoAWT + Bcl6 n = 9//5; Bcl6 n = 5//3; Control n = 9//5. Error bars, SEM; ***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant. Scale bar, 50 μm.
Fig. 10.
Fig. 10.. RhoA, MKL, and SRF increase the length of the G1 phase of the cell cycle.
(A) Representative diagram of the G1 phase length measurement protocol. Electroporation transfects a cohort of cells in late S, G2-M phases (green circles) at time T0. The GFP+ cohort progresses through the G1 phase and observed at different timing (example here, T1 or T2). A 30-min EdU pulse before dissection at T1 or T2 labels S phase cells (red circles). T2 represents the time for electroporated cells to reenter the S phase and observed by the earliest appearance of double staining for GFP and EdU (yellow circles). (B to H) Brains were in utero electroporated at E15.5. (B) Coronal sections of E16.5 cerebral cortices electroporated with either a control, RhoADN, or RhoAWT expression plasmids at E15.5 along with NLS-GFP and stained for EdU. (C to E) Quantification of GFP+EdU+ cells after a single 30-min EdU injection at 8, 10, 12, 18, or 23 hours after surgery. n ≥ 5 for EdU at 23 hours; n ≥ 3 for EdU at 8, 10, 12, and 18 hours after surgery. (F) Illustration of the regulation of the expression of the fluorescent proteins mKO2-hCdt1 (red) and mAG1-hGem (green) of the Fucci plasmid according to the different phases of the cell cycle. (G) Coronal sections of E16.5 cerebral cortices injected with either a control, RhoADN, or RhoAWT expression plasmids at E15.5 along with Fucci plasmid. (H) Quantification of the mAG1+ (S, G2, or M phase), mKO2+ (G1 phase), or mAG1+ mKO2+ (S phase entry) cells. n ≥ 14. Error bars, SEM. ***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant. Scale bar, 50 μm.
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