FoxO6 affects Plxna4-mediated neuronal migration during mouse cortical development

Abstract

The forkhead transcription factor FoxO6 is prominently expressed during development of the murine neocortex. However, its function in cortical development is as yet unknown. We now demonstrate that cortical development is altered in FoxO6^+/- and FoxO6^-/- mice, showing migrating neurons halted in the intermediate zone. Using a FoxO6-directed siRNA approach, we substantiate the requirement of FoxO6 for a correct radial migration in the developing neocortex. Subsequent genome-wide transcriptome analysis reveals altered expression of genes involved in cell adhesion, axon guidance, and gliogenesis upon silencing of FoxO6 We then show that FoxO6 binds to DAF-16-binding elements in the Plexin A4 (Plxna4) promoter region and affects Plxna4 expression. Finally, ectopic Plxna4 expression restores radial migration in FoxO6^+/- and siRNA-mediated knockdown models. In conclusion, the presented data provide insights into the molecular mechanisms whereby transcriptional programs drive cortical development.

Keywords: FoxO6; Plxna4; cortex; development; radial migration.

Fig. 1.

FoxO6 is present in the developing cortex, and ablation does not lead to abnormalities in layering as shown by Ctip2 and Satb2 distribution. (A) During cortical development, FoxO6 is broadly expressed, showing expression in the proliferating (sub)ventricular zone and the postmitotic cortical plate at E16.5 and E18. Next to the cortical expression of FoxO6, expression is primarily found in the nasal epithelium and the ganglionic eminences at E16.5 and E18. (B) Immunohistochemistry for Ctip2 reveals no clear differences in the amount of subcortical projection neurons in FoxO6^−/− cortices. Cortices were counterstained with DAPI. (C) Immunohistochemistry for Satb2 reveals no clear differences in the amount of callosal-projecting pyramidal neurons in FoxO6^−/− cortices. Satb2-expressing area was divided into two bins covering the upper layers and deep Satb2-expressing layers. Cortices were counterstained with DAPI. (D) Quantification of the number of DAPI-positive cells for the Ctip2 region in FoxO6^+/+ and FoxO6^−/− cortices. (E) Quantification of the number of DAPI-positive cells for a fixed region covering the upper and deep Satb2-expressing layers in FoxO6^+/+ and FoxO6^−/− cortices. (F) Quantification of the number of Ctip2-positive cells for a fixed region covering layers V and VI in FoxO6^+/+ and FoxO6^−/− cortices. Data were normalized against the number of DAPI cells in that region. (G) Quantification of the number of Satb2-positive cells for a fixed region covering the upper and deep Satb2-expressing layers in FoxO6^+/+ and FoxO6^−/− cortices. Data were normalized against the number of DAPI cells in that region. All quantifications were performed on n = 4 per condition. The error bars show the SEM. Two-tailed Student’s t test. c, caudate putamen; dn, dorsal neocortex; ge, ganglionic eminences; h, hippocampus; hy, hypothalamus; ne, nose epithelium; th, thalamus.

Fig. S1.

Numbers of apical and basal progenitor cells are unchanged in FoxO6^−/− cortices. (A) Immunostaining for Sox2 reveals no clear difference in the amount of multipotent apical progenitor cells in FoxO6^−/− cortices. Cortices were counterstained with DAPI. (B) Immunostaining for Tbr2 reveals that the pool of basal progenitors is not clearly affected in FoxO6^−/− cortices. Cortices were counterstained with DAPI. (C) Quantification of the number of Sox2-positive cells for a fixed region covering the VZ and SVZ in FoxO6^+/+ and FoxO6^−/− cortices. Data were normalized against the number of DAPI cells in that region. (D) Quantification of the number of Tbr2-positive cells for a fixed region covering the VZ and SVZ in FoxO6^+/+ and FoxO6^−/− cortices. Data were normalized against the number of DAPI cells in that region. (E) Quantification of the average width of the subventricular zone/ventricular zone, intermediate zone, and cortical plate. Previously performed DAPI counterstaining was used to identify and measure the width of the indicated layers in E18 FoxO6^+/+ and FoxO6^−/− brains. The error bars show the SD. Two-tailed Student’s t test; no significant differences were observed.

Fig. 2.

FoxO6^−/− mice display hampered radial migration during cortical development, which is exemplified by the altered presence of Cux1-positive callosal projection neurons. (A) Tracing newborn neurons by in utero electroporation (IUE) at E14.5 with a GFP expression vector revealed an altered distribution of cortical cells in FoxO6^−/− cortices compared with FoxO6^+/+ cortices at E18. Magnifications are shown for the cortical plate, intermediate zone, and ventricular/subventricular zone. (B) Quantification of triplicate experiments as shown in A. Ratios between the ventricular zone, intermediate zone, and cortical plate are shown. (C) Immunostaining for Cux1 in FoxO6^−/− cortices compared with FoxO6^+/+ cortices. (D) Quantification of the number of Cux1-positive cells for a fixed region covering layers II–IV in FoxO6^+/+ and FoxO6^−/− cortices. Data were normalized against the number of DAPI cells in that region. (E) Cortices in utero electroporated with siFoxO6-1 at E14.5 and cotransfected with a GFP expression vector display altered neuronal distribution at E18 compared with siScrambled (control) electroporated cortices. Magnifications are shown for the cortical plate, intermediate zone, and ventricular/subventricular zone. (F) Quantification of triplicate experiments as shown in E. Ratios between the ventricular zone, intermediate zone, and cortical plate are shown. The error bars show the SD. Two-tailed Student’s t test; *P < 0.05 (Bonferroni-corrected significance level in B and F).

Fig. S2.

siFoxO6-1 and siFoxO6-2 down-regulate FoxO6-GFP protein expression. (A and B) HEK293 cells were transfected with FoxO6-GFP and either siScrambled, siFoxO6-1, or siFoxO6-2. Western blot revealing down-regulation of FoxO6-GFP upon treatment with siFoxO6-1 and siFoxO6-2. β-Actin was used as a loading control. GFP was used to show that the siRNAs target FoxO6 and not GFP. (C) Densitometric analysis of the Western blot analysis; n = 3 vs. n = 3. The error bars show the SD. Two-tailed Student’s t test; *P < 0.05.

Fig. S3.

siRNA-mediated down-regulation of FoxO6 does not induce additional migration defects in FoxO6^−/− cortices. (A) E18 FoxO6^−/− cortices in utero electroporated with siScrambled at E14.5 and cotransfected with a GFP expression vector display altered neuronal distribution at E18. (B) E18 cortices in utero electroporated with siFoxO6 at E14.5 and cotransfected with a GFP expression vector did not display additional migration deficits.

Fig. S4.

Knockdown of FoxO6 via siFoxO6-2 results in defective radial migration, confirming data of siFoxO6-1. (A and B) GFP protein expression at E18 in cortices in utero electroporated with siScrambled or siFoxO6-2, cotransfected with a GFP expression vector. siFoxO6-2 displays an altered distribution of GFP-positive cells at E18 compared with siScrambled (control) electroporated cortices. (C) Quantification of duplicate experiments as shown in A and B. Ratios of GFP⁺ cells between the ventricular zone, intermediate zone, and cortical plate are shown. Note the reduction in cortical plate-positioned neurons upon treatment with siFoxO6-2, resembling the phenotype observed with siFoxO6-1. The error bars show the SD.

Fig. 3.

Genome-wide transcriptional profiling of FoxO6 knockdown neurons. (A) A graphical representation of the methodology used. FoxO6 is down-regulated by in utero electroporation at E14.5 using two different siRNAs targeting FoxO6 expression. Brains are dissected and dissociated at E16.5. Cortices are coelectroporated with GFP to FACsort the transfected cells followed by RNA isolation and microarray analysis. Three electroporated cortices were pooled for each N per condition to minimize the effects due to electroporation differences and between-litter differences. (B) Regulated genes by FoxO6 (FDR cutoff 0.1), identified as a result of the two different siRNAs, display a strong correlation indicating that in both datasets similar genes are represented, which are changed in a similar direction; siFoxO6-2, R = 0.85, P < 0.01; siFoxO6-1, R = 0.89, P < 0.01. (C) GO term analysis reveals that similar processes are regulated by FoxO6 knockdown through the use of siFoxO6-2 and siFoxO6-1. (D, Left) Graphical representation of the microarray analysis setup and resulting amount of regulated genes (243, siFoxO6-2; 140, siFoxO6-1) and the exact overlap (24 genes) in the P < 0.05 FDR-restricted datasets. (D, Right) Heatmap of the 24 genes that are present in such an overlap, including FoxO6. ctx, cortex; FACS, fluorescence-activated cell sorting; FSC, forward scatter; GFP, green fluorescent protein; lv, lateral ventricle.

Fig. S5.

Validation of a selection of genes as identified in the FoxO6 knockdown genome-wide transcriptome analysis. In an independent set of experiments, cortices were in utero electroporated with siFoxO6-1 and cotransfected with a GFP expression vector, followed by FACsorting and RNA isolation. Genes significantly regulated according to the transcriptome data were investigated by qPCR. Significant down-regulation of FoxO6, Plxna4, Nfia, and Pik3ca was confirmed. Significant up-regulation of Xlr3c was also confirmed. Lmx1a was used as an unchanged control. The error bars show the SD. Two-tailed Student’s t test; *P < 0.05.

Fig. 4.

Plxna4 is expressed in the developing cortex and restores radial migration defects in FoxO6^+/− animals. (A) In situ hybridization for Plxna4 at E16.5 and E18. At E16.5, from rostral to caudal, Plxna4 is most prominently expressed in the superficial layers of the cortical plate and intermediate zone with an apparent gradient from lateral to medial regions. At E18, expression is not restricted to the lateral regions of the developing cortex but is now prominently expressed in the entire outer layer of the cortical plate (20). (B) Pseudooverlay of adjacent sections analyzed for FoxO6 and Plxna4 expression indicate regional overlap. (C) GFP-traced neurons in FoxO6^−/− cortices of E18 embryos in utero electroporated with a GFP expression vector and either empty vector control or Plxna4 expression vector. Cortices were counterstained with DAPI. (D) The distribution of the transfected cells was quantified for the indicated layers for at least three brains per condition using multiple sections per brain. (E) GFP-traced neurons in FoxO6^+/− cortices of E18 embryos in utero electroporated with a GFP expression vector and either empty vector control or Plxna4 expression vector. Cortices were counterstained with DAPI. (F) The distribution of the transfected cells was quantified for the indicated layers for at least three brains per condition using multiple sections per brain. The error bars show the SD. Two-tailed Student’s t test; *P < 0.05.

Fig. S6.

Plxna4 expression is significantly reduced specifically in FoxO6^+/− FACsorted cortical cells. FoxO6^+/+, FoxO6^+/−, and FoxO6^−/− cortices were in utero electroporated with GFP at E14.5, and labeled cells were FACsorted at E16.5 and followed by RNA isolation. Plxna4 expression was measured by qPCR. Only in FoxO6^+/− animals was a significant down-regulation of Plxna4 observed. The error bars show the SD. Two-tailed Student’s t test; *P < 0.05.

Fig. 5.

Plxna4 restores radial migration in FoxO6 knockdown-induced radial migration defects. (A) Schematic overview of cortical migration phenotypes in FoxO6^+/+ cortices transfected with the indicated siRNAs showing hampered radial migration in cortices transfected with siFoxO6-1. Schematic representation of the hypothesis that Plxna4 rescues altered radial migration in cells down-regulated for FoxO6 (Right). Arrowheads point to the hypothesized positions of the neurons in the different treatment situations. (B) GFP-traced neurons in cortices of E18 embryos in utero electroporated with siFoxO6-1, a GFP expression vector, and either empty vector control (20) or Plxna4 expression vector. Cortices were counterstained with DAPI. (C) The distribution of the transfected cells was quantified for the indicated layers for at least three brains per condition using multiple sections per brain. The error bars show the SD. Two-tailed Student’s t test; *P < 0.05 and **P < 0.001. (D) Meta-analysis of the distribution of GFP-labeled cells in cortices treated with siScrambled, siFoxO6-1, and either empty vector control or Plxna4 expression vector. The analysis represents the CP minus IZ value (%). (E) Similar representation as in D for FoxO6^+/+, FoxO6^+/−, and FoxO6^−/−, electroporated with either EV or Plxna4. Introducing Plxna4 restores in part the percentage of GFP-labeled cells in the cortical plate in FoxO6^+/− cortices. The error bars show the SD. Wt, wild type.

Fig. S7.

Validation of Plxna4 protein overexpression after IUE with a Plxna4 expression plasmid. (A) Cortices were electroporated with both GFP and Plxna4 expression plasmids. Immunostaining for GFP and Plxna4 reveals that both genes are expressed in transduced cortical cells. (B) Magnifications showing GFP and Plxna4 colocalization.

Fig. S8.

Wild-type cortices overexpressing Plxna4 are unaffected in cortical migration. (A) Cortical migration is unaffected at E16.5 in embryos in utero electroporated at E14.5 with a vector expressing Plxna4 compared with empty vector control. (B) Cortical migration is unaffected at E18 in embryos in utero electroporated at E14.5 with a vector expressing Plxna4 compared with empty vector control. (C) Quantification of duplicate experiments as shown in B. Ratios of GFP⁺ cells between the (sub)ventricular zone, intermediate zone, and cortical plate are shown. The error bars show the SD.

Fig. 6.

FoxO6 binds and is able to regulate the Plxna4 promoter via an identified FoxO6 DNA-binding element. (A) Schematic representation of four putative FoxO DBEs in a 10-kb region surrounding the mouse Plxna4 transcription start site. (B) In vivo FoxO6 ChIP-qPCR in E16.5 cortices showing enrichment of DBE2 and DBE4 (n = 2). (C) FoxO6 ChIP-qPCR showing highest enrichment (n = 3) at DBE4 of the Plxna4 promoter. (D) Dual-luciferase assays in 3T3 cells with the different DBEs showing increased activity for DBE4 (n = 3, P < 0.05), which is diminished by the addition of serum (n = 3). Values are normalized to Renilla. DBE, DAF-16–binding element; S+, serum added. The error bars show the SD. Two-tailed Student’s t test; *P < 0.05 and **P < 0.001.