A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex

Abstract

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr⁺ and Olig2⁺ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

Keywords: Olig2; enhancer; gliogenesis; lineage switch; neurogenesis.

Fig. 1.

Fewer Ascl1⁺Egfr⁺ and Egfr⁺Olig2⁺ IMPs were present in the cortical VZ/SVZ of E17.5 Smo cko mice. (A–L) Immunostaining for Ascl1, Egfr, and Olig2 in Smo^fl/+ (A–C and G–I) and Smo cko cortices (D–F and J–L). Images were taken at the rostral-middle position along the rostral-caudal axis. (M and N) Quantification of the Ascl1⁺Egfr⁺ and Egfr⁺Olig2⁺ cells. Numbers represent means + SEM (n = 3 mice per genotype). *P < 0.05; unpaired Student’s t test. (Scale bars: 100 μm in L, applies to A–L.)

Fig. 2.

Olig2 deletion leads to fewer cells of oligodendrocyte, astrocyte, and olfactory bulb interneuron lineages at E18.5. (A–T), Immunostaining for Olig2 (A and F), Sox10 (B and G), Ascl1 (C and H), Egfr (D and I), phosphorylated MAPK (E and J), Sox9 (K and P), Olig1 (L and Q), Id1 (M and R), Aldh1l1 (N and S), and Sp8 (O and T) in wild-type (A–E and K–O) and Olig2^−/− (F–J and P–T) cortices. Images were taken at the rostral-middle position along the rostral-caudal axis. (U) Quantification of Ascl1⁺, Egfr⁺, phosphorylated MAPK⁺, Sox9⁺, Olig1⁺, Id1⁺, Aldh1l1⁺, and Sp8⁺ cells. Numbers represent means + SEM (n = 3 mice per genotype). *P < 0.05; unpaired Student’s t test. (Scale bars: 100 μm in J and T, applies to A−J and K−T, respectively.

Fig. 3.

Pax6 and Gli3 repress olfactory bulb interneuron lineage. (A–P), Immunostaining of Olig2 (A, E, I, and M), Gsx2 (B, F, J, and N), Sp8 (C, G, K, and O), and Tbr2 (D, H, L, and P) in the cortices of P0 wild-type (A–D), hGFAPcre; Pax6^fl/fl (E–H), hGFAPcre; Gli3^fl/+; Pax6^fl/+ (I–L), and hGFAPcre; Gli3^fl/fl; Pax6^fl/fl (M–P) mice. Images were taken at the rostral-middle position along the rostral-caudal axis. (Q) Quantification of Olig2⁺, Gsx2⁺, Sp8⁺, and Tbr2⁺ cells in 300-μm wide VZ/SVZ regions. Dotted lines demarcate the VZ and SVZ. Numbers represent means + SEM (n = 3 mice per genotype). ns, not significant; *P < 0.05; unpaired Student’s t test. (Scale bar: 100 μm in P, applies to A–P.)

Fig. 4.

Gli3 and Pax6 binding sites in the Olig1/2 loci have enhancer activity and their interactions with the Olig1/2 promoters are conserved between the mouse and human. (A) ChIP-seq analysis showed Gli3 binds to three sites in the Olig1/2 loci (GBS1/e14414, GBS2, and GB3/e14416), and Pax6 binds to multiple sites including e14412, PBS, e14414, e14415, and e14416. The e14412, e14414, e14415, e14416, and e14417 are enhancers predicted by the ENCODE project, and are shown in gray. The red blocks represent the regions that interacted with Olig2 or Olig1 promoters in the cortical cells, as revealed in the 4C experiments. Note that GBS1 is part of e14414, and GBS3 overlaps with e14416. (B) Single-cell ATAC-seq revealed that the 3 GBS sequences are differentially accessible in RGCs, MIPCs, astrocyte, oligodendrocyte, and olfactory bulb interneuron lineages. (C) Enhancer reporter assays showed that the Gli3 and Pax6 binding sites have enhancer activity in E11.5 or E16.5 mouse embryos. Note that hs1528 (e14412) and hs1188 (e14414) are human sequences, and mm817 is the mouse sequence for e14414. All the enhancer data have been loaded in the Vista Enhancer browser: mm2287 (PBS), mm2288 (e14415), and mm2289 (e14416). Abbreviations: FB, forebrain; NT, neural tube; MB, midbrain; HB, hindbrain; AB, abdomen; LB, limb. (D) H3K4me3 PLAC-seq experiments revealed that the Gli3 and Pax6 binding sites interact with OLIG1 and OLIG2 promoters in human fetal cortical glial lineage cells. The OLIG1 and OLIG2 promoters are shown in pink bars, the green bars represent the homologous sequences of the Gli3 and Pax6 binding peaks in human cells, and the wavy lines represent the interactions detected in H3K4me3 PLAC-seq. ATAC peaks in OC, oRG, and vRG cells are also shown. OC, glial lineage cells; oRG, outer radial glial cells; vRG, ventricular radial glial cells.

Fig. 5.

Enhancers e14414 and GBS2 regulate Olig2 and Olig1 expression in cortical VZ/SVZ. P0 images were shown. (A–X) Immunostaining for Pax6, Olig2, Olig1 in Olig2^−/+ (A–D), Olig2^−/Δe14414 (E–H), Olig2^−/Δe14415 (I–L), Olig2^−/ΔGBS2 (M–P), Olig2^−/Δe14416 (Q–T), and Olig2^−/− (U–X) cortices. Pax6 expression delineates the VZ and SVZ. Images were taken at the rostral-middle position along the rostral-caudal axis. (Y and Z) Quantification Olig2⁺ and Olig1⁺ cells in 350-μm wide cortical VZ/SVZ regions. Numbers represent means + SEM (n = 3 mice per genotype). *P < 0.05; unpaired Student’s t test. (Scale bar: 20 μm in X, applies to A–X.)

Fig. 6.

Deletion of e14414 or GBS2 leads to defective lineage switch in the cortex. P0 images were shown. (A–H) Immunostaining for Olig2 (A-1 to A-5), Olig1 (B-1 to B-5), Egfr (C-1 to C-5), Sox9 (D-1 to D-5), Aldh1l1 (E-1 to E-5), Id1 (F-1 to F-5), Sox10 (G-1 to G-5), and Sp8 (H-1 to H-5) in the cortices of Olig2^+/− (A-1 to H-1), Olig2^−/Δe14414 (A-2 to H-2), Olig2^−/Δe14415 (A-3 to H-3), Olig2^−/ΔGBS2 (A-4 to H-4), and Olig2^−/Δe14416 (A-5 to H-5) mice. Images were taken at the rostral-middle position along the rostral-caudal axis. (I–P) Quantification Olig2⁺, Olig1⁺, Egfr⁺, Sox9⁺, Aldh1l1⁺, Id1⁺, Sox10⁺, and Sp8⁺ cells. Numbers represent means + SEM (n = 3 mice per genotype). *P < 0.05; unpaired Student’s t test. (Scale bar: 100 μm in H-5, applies to A-1 to H-5.)