NOTCH, ERK, and SHH signaling respectively control the fate determination of cortical glia and olfactory bulb interneurons

Abstract

During cortical development, radial glial cells (neural stem cells) initially are neurogenic, generating intermediate progenitor cells that exclusively produce glutamatergic pyramidal neurons. Next, radial glial cells generate tripotential intermediate progenitor cells (Tri-IPCs) that give rise to cortical astrocytes and oligodendrocytes, and olfactory bulb interneurons. The molecular mechanisms underlying the transition from cortical neurogenesis to gliogenesis, and the subsequent fate determination of cortical astrocytes, oligodendrocytes, and olfactory bulb interneurons, remain unclear. Here, we report that extracellular signal-regulated kinase (ERK) signaling plays a fundamental role in promoting cortical gliogenesis and the generation of Tri-IPCs. Additionally, sonic hedgehog-smoothened-glioma-associated oncogene homolog (SHH-SMO-GLI) activator signaling has an auxiliary function to ERK during these processes. We further demonstrate that, from Tri-IPCs, NOTCH signaling is crucial for the fate determination of astrocytes, while ERK signaling plays a prominent role in oligodendrocyte fate specification, and SHH signaling is required for the fate determination of olfactory bulb interneurons. We provide evidence suggesting that this mechanism is conserved in both mice and humans. Finally, we propose a unifying principle of mammalian cortical gliogenesis.

Keywords: EGFR; ERK signaling; NOTCH signaling; SHH signaling; cortical gliogenesis.

Fig. 1.

A failure of cortical neurogenesis to gliogenesis switch in the absence of ERK signaling. (A) Strategy for labeling (lateral ventricle injection of FlashTag-CellTrace Yellow at E18.5) and enriching cortical progenitor cells from 1 littermate control brain and 1 Emx1-Cre; Map2k1/2-dcko brain at P2 and scRNA-Seq analysis. (B) Annotation of cortical cell clusters. (C) Fewer fRGCs were present in the cortices of the Emx1-Cre; Map2k1/2-dcko mice, and these cells showed significantly reduced expression of downstream target genes of ERK signaling. Note that genes of BMP7 signaling, SHH signaling, and NOTCH signaling pathways, and genes associated with gliogenesis showed significantly reduced expression in cortical fRGCs. (D) Cells in the Tri-IPC cluster in the control mice expressed marker genes associated with PyN-IPCs, OBIN-IPCs, OPCs, and APCs. In contrast, cells in the Tri-IPC cluster of Emx1-Cre; Map2k1/2-dcko mice showed increased expression of marker genes associate with PyN-IPCs, and greatly reduced expression of marker genes associated with OBIN-IPCs, OPCs, and APCs.

Fig. 2.

NOTCH signaling is required for fRGC maintenance and astrocyte lineage specification. (A) PAX6 and EOMES (TBR2) immunostainings showed that fRGCs and PyN-IPCs (arrowheads) were absent in the cortical VZ/SVZ in the hGFAP-Cre; Rbpj^F/F mice at P0. (B) ALDH1L1 immunostaining showed the absence of fRGCs and APCs in the cortex (arrowheads) in mutant mice. (C) SOX9 immunostaining revealed fRGCs and APCs in the control cortices, whereas these cells were absent in the cortices of hGFAP-Cre; Rbpj^F/F mice. SOX10 immunostaining showed increased OPCs in mutants (arrows). (D) Quantifications of SOX9⁺ fRGCs, SOX9⁺SOX10⁻ APCs, and SOX10⁺ OPCs in cortices of control and hGFAP-Cre; Rbpj^F/F mice. n = 3 mice/genotype; cells in 650 μm-width of cortex were quantified. ***P < 0.001; Student’s t test. (E) Summary of the cortical gliogenesis phenotype in P0 control and hGFAP-Cre; Rbpj^F/F mice. (F) The model summarizing the consequences of blocking NOTCH signaling on cortical gliogenesis. Without NOTCH signaling, cortical gliogenic fRGCs produce Tri-IPCs, but these cells fail to generate APCs; instead, they give rise to more OPCs, and some of OBIN-IPCs.

Fig. 3.

Enhancing ERK signaling promotes OPC fate specification independent of SHH-SMO signaling. (A and B) The pCAG-MEK1DD-GFP plasmids were electroporated into cortical VZ of control and hGFAP-Cre; Smo^F/F embryos at E14, and brains were analyzed at E17. GFP staining showed electroporated cells. (C and D) pERK staining showed increased ERK activity in the electroporated hemisphere than the contralateral hemisphere. (E–J) EGFR, OLIG2, and SOX10 expression in control mice was significantly increased in the electroporated hemisphere compared with the contralateral hemispheres. In addition, EGFR, OLIG2, and SOX10 expression was severely reduced in the cortices of the hGFAP-Cre; Smo^F/F mice, while their expression was notably increased following electroporation of pCAG-MEK1DD-GFP. Arrows point to electroporation sites and increased expression of markers; n = 3 mice/genotype.

Fig. 4.

Enhancing SHH-GLI^A signaling promotes OPC and OBIN fate specification independent of ERK signaling. (A and B) The pCAG-Gli2^A-GFP plasmids were electroporated into cortical VZ of control and Emx1-Cre; Map2k1/2-dcko embryos at E16, brains were analyzed at P1. GFP staining showed electroporated cells. (C–N) OLIG2, EGFR, SOX10, SOX9, and GSX2 expression was increased in the VZ/SVZ of the electroporated hemisphere in control mice. Note that OLIG2, EGFR, SOX10, SOX9, and GSX2 expression was significantly reduced in the cortical VZ/SVZ of the Emx1-Cre; Map2k1/2-dcko mice, confirming scRNA-Seq data, while electroporating pCAG-Gli2^A-GFP increased their expression (arrows). The dotted lines mark outlines of lateral ventricles and borders of electroporation sites; n = 3 mice/genotype.

Fig. 5.

A proposed model for cellular and molecular mechanisms underlying mammalian (mice and humans) cortical neurogenesis to gliogenesis transition and fate determination of cortical astrocytes, oligodendrocytes, and OBINs. (A) During mouse cortical neurogenic stage, cortical fRGCs produce Neurog2-expressing PyN-IPCs. NEUROG2 represses Ascl1 and Olig2/1 expression in PyN-IPCs and ensures PyN lineage specification. As development proceeds, ERK and SHH signaling activities further increase in cortical fRGCs. Decreased neurogenic signaling leads to reduced expression of Neurog2. pERK and GLI^A together activate Egfr, Ascl1, and Olig2/1 expression. Egfr expression further enhances ERK signaling. Thus, cortical fRGCs generate EGFR⁺ASCL1⁺OLIG2/1⁺ Tri-IPCs, which in turn give rise to APCs, OPCs, and OBIN-IPCs. (B) The similar gliogenesis processes in the human cortex indicate that cellular and molecular mechanisms of mammalian cortical gliogenesis are evolutionarily conserved. (C) Diversification of cortical astrocyte, oligodendrocyte, and OBIN lineages from tRGCs and Tri-IPCs in the human fetal cortex; this process takes place over a much longer period than in the mouse cortex. Top: The early tRGCs (i.e., from GW18 to GW20) generate ASCL1⁺EGFR⁺OLIG2/1⁺ Tri-IPCs. These Tri-IPCs, in general, inherit a relatively high level of HES1, HES5, and HEY1 from tRGCs, which repress ASCL1 and OLIG2/1 expression. Thus, these cells have a high propensity to generate APCs and a lower propensity to generate OPCs. Middle: The tRGCs (i.e., from GW21 to GW23) that are exposed to SHH and even higher ERK signaling generate Tri-IPCs that express higher level of EGFR, OLIG2/1, and ASCL1. These cells have a high tendency to generate OPCs and are less likely to generate APCs and OBIN-IPCs. Bottom: The tRGCs (i.e., from GW24 to first year of life) that are exposed to ERK and persistent high-level SHH signaling generate Tri-IPCs that have a high tendency to express GSX2 and DLX1/2 and give rise to OBIN lineage. SHH signaling represses ERK signaling. GSX2 and DLX1/2 repress OLIG1/2 expression, resulting in a lower probability to generate OPCs (Discussion).