ERK signaling expands mammalian cortical radial glial cells and extends the neurogenic period

Abstract

The molecular basis for cortical expansion during evolution remains largely unknown. Here, we report that fibroblast growth factor (FGF)-extracellular signal-regulated kinase (ERK) signaling promotes the self-renewal and expansion of cortical radial glial (RG) cells. Furthermore, FGF-ERK signaling induces bone morphogenic protein 7 (Bmp7) expression in cortical RG cells, which increases the length of the neurogenic period. We demonstrate that ERK signaling and Sonic Hedgehog (SHH) signaling mutually inhibit each other in cortical RG cells. We provide evidence that ERK signaling is elevated in cortical RG cells during development and evolution. We propose that the expansion of the mammalian cortex, notably in human, is driven by the ERK-BMP7-GLI3R signaling pathway in cortical RG cells, which participates in a positive feedback loop through antagonizing SHH signaling. We also propose that the relatively short cortical neurogenic period in mice is partly due to mouse cortical RG cells receiving higher SHH signaling that antagonizes ERK signaling.

Keywords: BMP7; ERK singling; FGF signaling; SHH; cortical evolution.

Fig. 1.

Overexpression of Fgf8 expands the cortical progenitor pool and induces Bmp7 expression in cortical RG cells. (A) mRNA in situ hybridizations on coronal sections through the telencephalon of E14.5 littermate control and hGFAP-Cre; Rosa^Fgf8 mouse embryos (n = 3), showing significant upregulation of Bmp7 expression in the cortex (arrows). Note that Bmp7 expression is only detected in the medial cortex of littermate controls. LGE, lateral ganglionic eminence; LV, lateral ventricle. (B–G) Expression of pERK, SP8, HOPX, EGFR, and OLIG2 was greatly increased (arrows), whereas expression of NR2F1 and Fgfr3 was down-regulated (arrowheads) in the cortex following Fgf8 overexpression. (H and I) The heat map of bulk RNA-seq data showing expression of neurogenesis genes was down-regulated, whereas expression of FGF8 signaling response genes, Etv1, Etv4, Etv5, Mest, Sp8, Spry1, Spry2, and Spry4 was up-regulated in the hGFAP-Cre; Rosa^Fgf8 cortex (n = 5) compared with littermate controls (n = 5) at E14.5.

Fig. 2.

Constitutive activity of ERK signaling induces Bmp7 expression in cortical RG cells. (A–F) Expression of pERK, Bmp7, HOPX, ETV5, EGFR, and OLIG2 was up-regulated in the cortex of hGFAP-Cre; Rosa^MEK1DD mice at E14.5 (arrows). (G and H) NR2F1 and Fgfr3 expression was down-regulated (arrowheads). (I and J) Heat map of selected differentially expressed genes in the E14.5 cortex of hGFAP-Cre; Rosa^MEK1DD mice (n = 4) relatively to controls (n = 5).

Fig. 3.

Deletion of Map2k1 and Map2k2 genes in the developing cortex leads to loss of ERK signaling and premature neural differentiation. (A–C) Expression of pERK, HOPX, and Bmp7 was lost specifically in the cortex of Emx1-Cre; Map2k1/2-dcko mice at E14.5 (arrowheads). (D and E) NR2F1 and Fgfr3 expression was increased in the cortex, especially in the medial cortex (arrows). (F) Immunostaining of EOMES, a marker for PyN IPCs, showing that PyN IPCs located in the VZ surface in Emx1-Cre; Map2k1/2-dcko mice at E14.5 (arrows). (G and H) scRNA-Seq analysis and heat map showing expression of selected ERK signaling response genes in cortical RG cells of Emx1-Cre; Map2k1/2-dcko mice relative to littermate controls at E14.5. Note that the expression of Etv1, Etv5, Hopx, Spry2, and Tnc was completely lost in cortical RG cells in Emx1-Cre; Map2k1/2-dcko mice.

Fig. 4.

The SHH-SMO signaling antagonizes ERK signaling in cortical RG cells. (A–H) Expression of Gli1, Fgf15, NR2F1, Fgfr3, EGFR, and OLIG2 was greatly up-regulated (arrows), whereas expression of pERK and HOPX was severely reduced (arrowheads) in the cortex of hGFAP-Cre; Rosa^SmoM2 mice compared with littermate controls at E14.5. (I and J) scRNA-Seq analysis and heat map showing expression of key signature genes in cortical RG cells of hGFAP-Cre; Rosa^SmoM2 mice relative to controls at E14.5.

Fig. 5.

Stronger ERK signaling activity in human cortical oRG cells than in tRG cells. (A) The diagram showing brain sections spanning the rostral-caudal extent of the human telencephalon at GW18. CGE, caudal ganglionic eminence; MGE, medial ganglionic eminence; RMS, rostral migratory stream. (B) The coronal section through the rostral telencephalon (the outlined area in A) at GW18 double immunostained with pERK and HOPX. ISVZ, inner subventricular zone, IFL, inner fiber layer. (C and D) Higher magnification images showing that pERK and HOPX immunoreactivity is stronger in the cortical OSVZ (C) than cortical VZ (D).

Fig. 6.

The proposed principle of human and mouse cortical development and evolution. (A) During mouse early telencephalon patterning and development, FGFs are expressed in the rostral patterning center, whereas SHH is expressed in the POA and MGE-derived neurons. In general, FGF-ERK signaling exhibits in a rostral^high-caudal^low gradient, whereas SHH-SMO signaling exhibits in a ventral^high-dorsal^low and caudal^high-rostral^low gradient. 4 V, fourth ventricle; POA, preoptic area. (B) We propose that ERK signaling drives the expansion and evolution of the human cortex. At the beginning of cortical neurogenesis in the most recent ancestor to all mammals, it is assumed that there is already a subset of cortical fRG cells that express relatively higher levels of pERK. Elevated ERK signaling in these cortical fRG cells promotes BMP7 expression, which increases GLI3R generation and represses SHH signaling. A decrease in SHH signaling in cortical fRG cells further enhances ERK signaling. Therefore, the ERK-BMP7-GLI3R signaling pathway in cortical fRG cells participates in a positive feedback loop, which expands the cortical fRG cell pool, increases the length of the cortical neurogenic period, and thus drives cortical development, expansion, and evolution. (C) We propose that SHH signaling may drive mouse cortical evolutionary dwarfism. During mouse cortical development and evolution, smaller brains with the smaller lateral ventricle results in relatively higher levels of SHH signaling in the cortex, which antagonizes ERK signaling. Relatively weak ERK signaling fails to induce Bmp7 expression in the dorsal cortical RG cells. Therefore, mouse cortical neurogenesis is mainly protected by GLI3R, but not BMP7, resulting in a shortened period of cortical neurogenesis (for example, from more than 130 d in humans to about 7 d in mice), which is associated with a greatly reduced the number of cortical neurons and cortical size.