Loss of Foxg1 causes premature gliogenesis.

(A–D) Cre electroporation at E15.5 in control (A, Foxg1lox/+; R26FRT-GFP) and Foxg1 LOF (B, Foxg1lox/lox; R26FRT-GFP) embryos, followed by analysis at P14. (C, D) 67.3% of GFP+ cells colocalised with NeuN in control brains and 1% in Foxg1 LOF brains. 16% of GFP+ cells colocalised with ALDH1L1 in control brains and 56% in Foxg1 LOF brains. n= 2151 (Control), 2761 (Foxg1 LOF) cells from N=3 brains (biologically independent replicates). (E–H) Cre electroporation at E14.5 in control (E, Foxg1lox/+; R26FRT-GFP) and Foxg1 LOF (F, Foxg1lox/lox; R26FRT-GFP) embryos, followed by analysis at P14. (G, H) 77.7% of GFP+ cells colocalised with NeuN in control brains and 0% in Foxg1 LOF brains. 20.1% of GFP+ cells colocalised with ALDH1L1 in control brains and 55.7% in Foxg1 LOF brains. n= 3160 (Control), 2978 (Foxg1 LOF) cells from N=3 brains (biologically independent replicates). In each row (A, B, E, F), the boxes in the leftmost low magnification panels indicate approximate regions shown in either the NEUN or ALDH1L1 high magnification panels. Filled arrowheads depict colocalisation, and open arrowheads depict non-colocalisation of marker and electroporated cells. Statistical test: two-tailed unpaired t-test. *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001). All scale bars are 50 μm.

Foxg1 LOF leads to premature glial fate acquisition in progenitors but no proliferation defects.

(A) Schematic depicting the two alternative mechanisms that could result in enhanced gliogenesis upon loss of Foxg1: a change in cell type produced by the progenitor, i.e. “fate switch” or increase in proliferation of astrocytes accompanied by neuronal cell death. (B, B’, C, C’) Images of the ventricular and sub-ventricular zone (the dashed line indicates the ventricle boundary). Cre electroporation at E15.5 in control (Foxg1lox/+; R26FRT-GFP) and Foxg1 LOF (Foxg1lox/lox; R26FRT-GFP) embryos, followed by analysis at E18.5. Proliferation marker KI67 (B, B’) colocalises with similar numbers of GFP+ cells in control and Foxg1 LOF brains. Cell death marker Cleaved Caspase 3 (C, C’) does not reveal differences in colocalisation with GFP+ control and Foxg1 LOF cells. In contrast, glial progenitor markers NFIA (D, D’) display increased colocalisation with GFP+ cells in Foxg1 LOF (51.7%) compared with controls (12.5%). Neurogenic intermediate progenitor marker TBR2 (E, E’) displays decreased colocalisation with GFP+ cells in Foxg1 LOF brains (0%) compared with controls (8%). n= 3590 (control), 2100 (mutant) cells from N=3 brains (biologically independent replicates). (G) Schematic depicting the genotype and corresponding fluorescent labels resulting from the MADM recombination events. (E–F) Cre electroporation at E14.5/E15.5 in Foxg1-MADM brains (M12GT/TG, Foxg1) analysed at P14. Green (Foxg1—/—) and red (Foxg1+/+) cells were scored based on neuronal (open arrowheads, E) or glial (arrowheads, E) morphology. (F) represents the number of neurons or glia as a percentage of the total population of neurons+glia of each genotype: control (red; +/+) or Foxg1 mutant (green;–/–) neurons. n= 354 cells from N=5 brains (biologically independent replicates). Statistical test: two-tailed unpaired t-test. *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001). All scale bars: 50μm.

FOXG1 binds and regulates the expression of Fgfr3.

(A) RNA-seq analysis of FACS-purified Control and Foxg1 LOF progenitors harvested two days after labelling at E15.5. Gliogenic factors such as Nfia, Id3, and Olig3 are upregulated, and neuronal markers such as Pou3f1 and Robo4 are downregulated. (B–D) Multimodal analysis comparing FOXG1 occupancy (ChIP-seq) and bivalent epigenetic marks (H3K4Me3 and H3K27Me3) and astrocyte-enriched genes from (48) reveals a list of 19 genes common to each dataset (B). Four of these are upregulated upon loss of Foxg1, including the known gliogenic gene Fgfr3 (D). FOXG1 occupies a -26kb enhancer region of Fgfr3 (C). (E) KEGG analysis of the upregulated genes from (A) identifies the MAPK signalling pathway downstream of FGF signalling. (F) Loss of Foxg1 from E15.5 progenitors (hGFAPCreERT2, tamoxifen at E15.5) causes upregulation of FGFR3 receptor by E17.5 as seen in cells near the VZ of the somatosensory cortex. Boxes (F) indicate the regions in high magnification shown in the adjacent panels (G). Dashed circles outline the ROIs identified in the DAPI channel used for intensity quantification in (H). (G; n=50 (Control and Foxg1 LOF) ROIs from N=3 brains) and phosphorylated-ERK1/2 (H; n= 67 (Control) and 89 (Foxg1 LOF) cells from N=3 brains (biologically independent replicates). Statistical test: Mann-Whitney test *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001). All scale bars: 50μm.

Foxg1 suppresses FGF-induced gliogenesis.

(A–D) In-utero electroporations were performed in wild-type embryos at E15.5, and the brains were analysed at P7. (A) GFP electroporation labels LII/III cells that are NEUN+ (arrowheads) and SOX9 ー (open arrowheads). (B) Overexpression of Fgf8 leads to premature gliogenesis, and the GFP+ cells are NEUN ー (open arrowheads) and SOX9+ (arrowheads). (C) Overexpression of Foxg1 produced NEUN+ (arrowheads) and SOX9ー (open arrowheads) neurons, some of which displayed delayed migration (black asterisk), and others migrated to the cortical plate (white asterisk), as shown in (37). (D) Overexpression of Foxg1 together with FGF8 partially rescued neuronal fate such that GFP+ cells also displayed NEUN (arrowheads) but not SOX9 (open arrowheads). In A–D, the boxes in the leftmost low magnification panels indicate approximate regions shown in the adjacent high magnification panels. (E) Quantifications of GFP+ cells that are also NEUN+ in each condition: 98.6% (GFP); 1.8% (Fgf8); 98.3% (Foxg1); 74.1% (Foxg1+Fgf8). (F) Quantifications of GFP+ cells that are also SOX9+ in each condition: 0% (Egfp); 87.7% (Fgf8+Egfp); 0% (Foxg1-Egfp); 24.2% (Foxg1-Egfp+Fgf8). n= 2123 (Egfp), 1643 (Fgf8+Egfp), 1357 (Foxg1-Egfp), 1924 (Foxg1-Egfp+Fgf8) cells each from N=3 brains (biologically independent replicates). Statistical test: Two-way ANOVA with Tukey’s correction. *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001). All scale bars: 50μm.

Postmitotic neuron Foxg1 LOF leads to premature gliogenesis and upregulation of the MAPK pathway.

(A) SOX9 staining in the control P0 cortex identifies gliogenic progenitors at the ventricular zone (white asterisk) and scattered cells throughout the cortical plate. (B–D) NexCre-driven loss of Foxg1 is specific to postmitotic neurons, as seen by GFP reporter expression (C, white bars) and causes a non-autonomous upregulation of nuclear SOX9 in the ventricular zone progenitors and an increase in the numbers of SOX9+ cells cortical plate (B; quantifications: D, E). B and C are images of the same section showing SOX9 alone (B) and together with the GFP reporter (C). (F) Transcriptomic analysis of cortical plate tissue from control and NexCre/+; Foxg1lox/lox; R26FRT-GFP reveals a significant upregulation of Fgf18 upon loss of Foxg1. (G) Fgf18 expression in CUX2+ upper layer cells peaks at P7, as seen in the RNA seq dataset from (52). (H) Examination at E18.5 reveals increased levels of phosphorylated p42/44-ERK1/2 (pERK1/2) within the ventricular zone of NexCre/+; Foxg1lox/lox brains, indicative of enhanced FGF signalling. This upregulation of pERK1/2 is reversed upon treatment with the FGF Inhibitor NVP-BGJ398 (H; Quantifications: J). (I) In sections from the same brains, levels of SOX9 within the ventricular zone (VZ) are increased upon postmitotic loss of Foxg1, and this is restored to baseline levels upon administration of the inhibitor. (I; quantifications: K). Quantifications of pERK1/2 in each condition: 233 (Control); 248 (NexCre/+; Foxg1lox/lox); 207 (Control+NVP-BGJ398); 223 (NexCre/+; Foxg1lox/lox+NVP-BGJ398) cells from N=2 brains (biological replicates from 2 independent experiments). Quantifications of SOX9 levels in each condition: 233 (Control); 234 (NexCre/+; Foxg1lox/lox); 228 (Control+NVP-BGJ398); 205 (NexCre/+; Foxg1lox/lox+NVP-BGJ398) cells from N=3 brains (biological replicates from 2 independent experiments). Statistical test: Mann-Whitney Test (D, E); Two-way ANOVA with Tukey’s correction (J, K). *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001). All scale bars: 50μm.

Foxg1-Fgf double LOF leads to premature oligogenesis.

(A–C) Cre electroporation at E15.5 in control (A, Foxg1lox/+; R26FRT-GFP) and Foxg1 LOF (B, Foxg1lox/lox; R26FRT-GFP) embryos, followed by analysis at P14. GFP+ cells in control brains do not colocalise with ALDH1L1, OLIG2, and PDGFRA staining (A), whereas most GFP+ cells in Foxg1 LOF brains display these markers (B). Co-electroporation of Cre together with a construct encoding soluble FGFR3c (an FGF-chelator) in Foxg1lox/lox; R26FRT-GFP causes a significant increase in the co-localisation of GFP+ cells with Oligodendrocyte Precursor Cells (OPCs) markers such as OLIG2 and PDGFRA (C). In each row (A–C), the boxes in the leftmost low magnification panels indicate approximate regions from the same section or serial sections shown in the adjacent high magnification panels. A quantitative analysis reveals a drastic reduction of neurogenesis at the expense of gliogenesis (astrocytes+OPCs) upon loss of Foxg1 and an additional increase in the percentage of OPCs with the additional abrogation of FGF signalling (D, D’). n= 4069 (Control), 3970 (Foxg1 LOF), 3332 (Foxg1 LOF + sFGFR3c) from N=3 brains (biologically independent replicates). Statistical test: Two-way ANOVA.with Tukey’s correction *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001). All scale bars: 50μm.

Schematic depicting the regulation of gliogenesis by FOXG1.

In neurogenic progenitors, FGFR3 levels are suppressed by FOXG1. As time progresses, FOXG1 levels decrease within progenitors, and FGFR3 levels increase, making progenitors more sensitive to FGF signalling. Concomitantly, postmitotic neurons secrete factors, including FGF18, which is also under FOXG1 regulation.FGF signalling drives progenitors towards astrogliogenesis. Later, when both FOXG1 and FGF levels are low, the progenitors transition to oligogenesis.