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, 34 (6), 2169-90

RAS/ERK Signaling Controls Proneural Genetic Programs in Cortical Development and Gliomagenesis

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RAS/ERK Signaling Controls Proneural Genetic Programs in Cortical Development and Gliomagenesis

Saiqun Li et al. J Neurosci.

Abstract

Neural cell fate specification is well understood in the embryonic cerebral cortex, where the proneural genes Neurog2 and Ascl1 are key cell fate determinants. What is less well understood is how cellular diversity is generated in brain tumors. Gliomas and glioneuronal tumors, which are often localized in the cerebrum, are both characterized by a neoplastic glial component, but glioneuronal tumors also have an intermixed neuronal component. A core abnormality in both tumor groups is overactive RAS/ERK signaling, a pro-proliferative signal whose contributions to cell differentiation in oncogenesis are largely unexplored. We found that RAS/ERK activation levels differ in two distinct human tumors associated with constitutively active BRAF. Pilocytic astrocytomas, which contain abnormal glial cells, have higher ERK activation levels than gangliogliomas, which contain abnormal neuronal and glial cells. Using in vivo gain of function and loss of function in the mouse embryonic neocortex, we found that RAS/ERK signals control a proneural genetic switch, inhibiting Neurog2 expression while inducing Ascl1, a competing lineage determinant. Furthermore, we found that RAS/ERK levels control Ascl1's fate specification properties in murine cortical progenitors--at higher RAS/ERK levels, Ascl1(+) progenitors are biased toward proliferative glial programs, initiating astrocytomas, while at moderate RAS/ERK levels, Ascl1 promotes GABAergic neuronal and less glial differentiation, generating glioneuronal tumors. Mechanistically, Ascl1 is phosphorylated by ERK, and ERK phosphoacceptor sites are necessary for Ascl1's GABAergic neuronal and gliogenic potential. RAS/ERK signaling thus acts as a rheostat to influence neural cell fate selection in both normal cortical development and gliomagenesis, controlling Neurog2-Ascl1 expression and Ascl1 function.

Keywords: Neurog2 and Ascl1; RAS/ERK signaling; bHLH transcription factors; glioma and glioneuronal tumors; neurogenesis versus gliogenesis; proneural genetic switch.

Figures

Figure 1.
Figure 1.
RAS/ERK activation correlates with histological and molecular features of human low-grade gliomas. A–L, Representative sections of adult human neocortex through control (Cx) nondiseased regions (A, D, G, J) or through a ganglioglioma (GG) (B, E, H, K) or pilocytic astrocytoma (PA) (C, F, I, L). Sections were processed for immunolabeling with the neuronal marker NeuN (A–C), GFAP (D–F), Sox9 (G–I), or pERK (J–L). The percentage of cells expressing NeuN (M), Sox9 (N), and pERK (O) was quantitated. *p < 0.05, **p < 0.01, ***p < 0.005. AP-visualized (red) and DAB-visualized (brown) double immunolabeling of GG tissues with pERK/NeuN (P), pERK/GFAP (Q), pERK/Olig2 (R), and pERK/Sox2 (S). Scale bars: 50 μm.
Figure 2.
Figure 2.
RAS signaling is required to regulate a Neurog2-Ascl1 genetic switch in cortical progenitors. A, Dissected E13.5 brain showing coronal plane of section (dotted purple line). B, Schematic illustration of a frontal section through the telencephalon, with the boxed area indicating the targeted area of the neocortex used in all electroporations. C, Cortical progenitors are bipotent, normally selecting a glutamatergic neuronal fate under the proneural activity of Neurog2, but retaining the potential to differentiate into GABAergic neurons or OPCs in response to Ascl1. D, Schematic illustration of signaling pathways activated downstream of RTK/RAS signaling. E–G, Dynamic expression of pERK (E–E″), Etv1 (F–F″), and Etv5 (G–G″) in E13.5, E14.5, and E15.5 cortex. Red arrowheads mark lateral-to-medial expansion of expression. H, I, E15.5 cortical sections double-stained for pERK (red) and Neurog2 (green; H–H″), or Ascl1 (green; I–I″). Arrows mark the cells in which Neurog2 and pErk expression are mutually exclusive. Arrowheads mark the cells in which pErk and Ascl1 are coexpressed. J–P, Schematic illustration of lineage tracing experiments in which an mCherry expression vector was electroporated with or without RasV12 into E12.5 Neurog2GFPKI/+ or Ascl1GFPKI/+ cortical progenitors (J). GFP/mCherry expression in E12.5→E14.5 electroporated Neurog2GFPKI/+ (K, K′, L, L′) and Ascl1GFPKI/+ (M, M′, N, N′) brains. Quantification of GFP and mCherry coexpression in VZ or SVZ/IZ cells of Neurog2GFPKI/+ (O) and Ascl1GFPKI/+ (P) brains. *p < 0.05, **p < 0.01, ***p < 0.005. Scale bars: E–G, E′–G′, E″–G″, 250 μm; H″, I″, 67.5 μm; K–N, K′–N′, 125 μm. A, anterior; ctx, neocortex; D, dorsal; di, diencephalon; dTel, dorsal telencephalon; GF, growth factor; lge, lateral ganglionic eminence; mes, mesencephalon; mge, medial ganglionic eminence; P, posterior; tel, telencephalon; V, ventral; vTel, ventral telencephalon.
Figure 3.
Figure 3.
RAS functions through the ERK branch to regulate the Neurog2-Ascl1 genetic switch in cortical progenitors. E12.5→E15.5 electroporations of a pCIG2 control vector (A, A′, B–B‴), RasV12 (C, C′, D–D‴), bRafV600E (E, E′, F–F‴), MekCA (G, G′, H–H‴), RasN17 (I, I′, J–J″), or MekDN (K, K′, L–L″) vectors (expressing GFP). Transfected brains were analyzed for coexpression of GFP with pErk (A, A′, C, C′, E, E′, G, G′, I, I′, K, K′), or for transcripts for GFP (B, D, F, H, J, L), Etv5 (B′, D′, F′, H′), Neurog2 (B″, D″, F″, H″, J′, J″, L′, L″), and Ascl1 (B‴, D‴, F‴, H‴). Dashed lines outline the transfected region in the neocortex. Red arrowheads mark ectopic pERK (C′, E′, G′), Etv5 (D′, F′, H′), Neurog2 (H″, J′, J″, L′, L″), and Ascl1 expression (D‴, F‴, H‴), whereas yellow arrowheads mark transfected areas in which Neurog2 (D″, F″, H″) or pERK (I, I′, K, K′) expression was extinguished. M, Schematic illustration of repression of Neurog2 expression and induction of Ascl1 expression by RAS/ERK signaling. Scale bars: 250 μm. CP, cortical plate; ctx, neocortex; str, striatum.
Figure 4.
Figure 4.
RAS/ERK signaling inhibits cortical neurogenesis. A–L, E12.5→E15.5 electroporations of a pCIG2 control vector (A, A′, E, E′, I, I′), RasV12 (B, B′, F, F′, J, J′), bRafV600E (C, C′, G, G′, K, K′), and MekCA (D, D′, H, H′, L, L′) vectors (expressing GFP). Transfected brains were analyzed for coexpression of GFP (green) with pan-neuronal marker NeuN (red; A–D, A′–D′) and the cortical neuronal marker Tbr1 (red; E–H, E′–H′), or for transcripts for GFP (I–L) or the subcortical neuronal marker Dlx1 (I′–L′). Dashed lines outline the transfected region in the neocortex. Yellow arrowheads mark transfected areas with reduced expression of NeuN (B′, C′, D′), and Tbr1 (F′, G′, H′), and red arrowheads mark ectopic expression of Dlx1 (L′). Scale bars: 250 μm. M, N, Quantification of GFP+ cells coexpressing NeuN (M) or Tbr1 (N). p values of pCIG2-RasV12, pEF-RasV12, bRafV600E, MekCA, AktCA, RalAV23, and RalBQ72E are relative to pCIG2 control, whereas p values of pCIG2RasV12+pCIG2MekDN, pCIG2RasV12+pCIG2AktDN, pCIG2Rasv12+pCIG2RalADN, and pCIG2RasV12+pCIG2RalBDN are relative to pCIG2RasV12. *p < 0.05, **p < 0.01, ***p < 0.005. ctx, neocortex; str, striatum.
Figure 5.
Figure 5.
RAS/ERK signaling promotes proliferation and a glial cell fate in cortical progenitors. E12.5→E15.5 electroporations of a pCIG2 control vector (A, A′, F, F′, K, K′, P, P′), RasV12 (B, B′, G, G′, L, L′, Q, Q′), bRafV600E (C, C′, H, H′, M, M′, R, R′), MekCA (D, D′, I, I′, N, N′, S, S′), and Etv5 (E, E′, J, J′, O, O′, T, T′) vectors (expressing). Transfected brains were analyzed for coexpression of GFP with Sox9 (A–E, A′–E′), BrdU (30 min pulse; F–J, F′–J′), Olig2 (K–O, K′–O′), or GFAP (P–T, P′–T′). Dashed lines outline the transfected region in the neocortex. Red arrowheads mark transfected cells ectopically expressing Sox9 (B′–E′), BrdU (G′–J′), Olig2 (L′–N′), or GFAP (Q′–S′). Scale bars: 250 μm. U, V, Quantification of GFP+ cells coexpressing Sox9 (U) or Olig2 (V). p values of pCIG2RasV12, pEFRasV12, pCIG2bRafV600E, pCIG2MekCA, pCIG2AktCA, pCIG2RalAV23, and pCIG2RalBQ72E are relative to pCIG2 control, whereas p values of pCIG2RasV12+pCIG2MekDN, pCIG2RasV12+pCIG2AktDN, pCIG2Rasv12+pCIG2RalADN, and pCIG2RasV12+pCIG2RalBDN are relative to pCIG2RasV12. *p < 0.05, **p < 0.01, ***p < 0.005. ctx, neocortex; str, striatum.
Figure 6.
Figure 6.
Activation of RAS signaling in cortical progenitors results in tumorigenesis. A–E, E18.5 control (C) and E12.5→E18.5 brains electroporated with pCIG2RasV12 (A, B, D, E, E′) were processed for H&E staining (B–E, E′), or GFP transcripts (A). F–H, E12.5→E16.5 electroporations of a pCIG2 control vector (F–F‴) and pCIG2RasV12 (G–G‴, H–H‴). Transfected brains were analyzed for coexpression of GFP (green), proliferation marker Ki67 (red), and pan-neuronal marker Tuj1 (blue). White arrowheads mark RasV12 transfected cells expressing Ki67 instead of Tuj1 (H–H‴). I–P, E12.5→E15.5 brains electroporated with a pCIG2 control vector (I, I′, K, K′, M, M′, O, O′) or pCIG2RasV12 (J, J′, L, L′, N, N′, P, P′) were processed for immunostaining of pan-neural progenitor markers Sox2 (I, I′, J, J′) and Nestin (K, K′, L, L′), cortical-specific progenitor markers Pax6 (M, M′, N, N′), and Tbr2 (O, O′, P, P′). Red arrowheads mark ectopic expression of Sox2 (J′) and Nestin (L′), while yellow arrowheads mark transfected areas with reduced expression of Pax6 (N′) and Tbr2 (P′). Q–V, E12.5→E15.5 electroporations of pCIG2RasV12 with (T–V, V′) or without Neurog2 (Q–S, S′). Transfected brains were analyzed for the transcripts of Spry2 (Q, T), Pdgfa (R, U), or coexpression of GFP and Tuj1 (S, S′,V, V′). Red arrowheads mark ectopic expression of Spry2 (Q, T) or Pdgfa (R). W–Z, E12.5→E15.5 electroporations of pCIG2 (W, W′), AktA (X, X′), RalAV23 (Y, Y′), and RalBQ72L (Z, Z′) followed by analysis of BrdU incorporation. Red arrowheads mark ectopic BrdU incorporation. Dashed lines outline the transfected region in the neocortex. Scale bars: I–V, I′–V′, F–F‴, G–G‴ 250 μm; H–H‴, 67.5 μm. CP, cortical plate; ctx, neocortex; str, striatum.
Figure 7.
Figure 7.
RAS/ERK levels influence Ascl1's fate specification properties and target gene selection. A–C, Western blot analysis of lysates from HEK293 cells transfected with pEFRasV12 or pCIG2RasV12 and analyzed for pERK, ERK, and β-actin expression (A). Expression levels were quantified by densitometry and normalized to β-actin and total ERK (B, C). D–E, E12.5→E14.5 brains electroporated with pEFRasV12 and GFP were processed for the expression of GFP (D), pERK (D, D′), and Ascl1 (E). F–K, E12.5→E14.5 electroporations of Neurog2GFPKI/+ cortices with pEFRasV12 and mCherry, analyzed for expression of mCherry (F, J, K, red), Neurog2 (G), Dlx1 (H, H′), Tuj1 (I, K, blue), and GFP (J, K). Dashed red lines outline the transfected region in the neocortex. Red arrowheads mark ectopic Dlx1 (H, H′) or Tuj1 (I). Yellow arrowheads mark transfected areas with reduced Neurog2 (G) and GFP (J) expression. Scale bars: D–H, 250 μm; I–K, H′, 125 μm. L–W, E18.5 control (L, O, R, U) and E12.5→E18.5 brains electroporated with pEFRasV12 (M, P, S, V) or pCIG2RasV12 (N, Q, T, W) were processed for H&E staining (L–N), or immunohistochemistry for Olig2 (O–Q), GFAP (R–T), or TUJ1/pERK (U–W). Scale bars: 500 μm. X–Z, Transcriptional reporter assays in P19 cells using Dll1 (X), Dlx1/2 I12b intergenic enhancer (Y), and Sox9 (Z) reporters. *p < 0.05, **p < 0.01, ***p < 0.005. AA, Schematic representation of the effects of RAS/ERK activity levels on Ascl1 target gene selection. ctx, neocortex; str, striatum.
Figure 8.
Figure 8.
RAS/ERK regulates Ascl1 transcriptional activity via direct phosphorylation. A, Distribution of the six SP sites within wild-type Ascl1 (Ascl1-wt). Annotation of the serines mutated to alanines in Ascl1-SA3 and Ascl1-SA6 (mutated sites in red). B, Characterization of a phospho-specific Ascl1 antibody in HEK293 cells transfected with Ascl1 or Ascl1-SA3 along with pCIG2 or RasV12. C, Phospho-Ascl1 antibody recognizes in vitro-transcribed/translated Ascl1 (and not Ascl1-SA3) phosphorylated by recombinant ERK. D–F, Transcriptional reporter assays in P19 cells using Dll1 (D), Dlx1/2 I12b intergenic enhancer (E), and Sox9 (F) reporters, demonstrating that the effects of RAS/ERK activity levels on Ascl1 target gene selection are abrogated when all six SP sites in Ascl1 are mutated. *p < 0.05, **p < 0.01, ***p < 0.005.
Figure 9.
Figure 9.
Ascl1 fate specification properties depend on the six SP phosphoacceptor sites. A–I, E12.5→E15.5 electroporations of pCIG2 (A, A′, D, D′, G, G′), wild-type Ascl1 (B, B′, E, E′, H, H′), and Ascl1-SA6 (C, C′, F, F′, I, I′) vectors (expressing GFP). Transfections were analyzed for ectopic expression of the glioblast marker Sox9 (A–C, A′–C′), proliferation marker BrdU (30 min pulse; D–F, D′–F′), and OPC marker Olig2 (G–I, G′–I′). Quantitation of GFP+ cells coexpressing Sox9 in the SVZ/IZ (S). J–R, E12.5→E15.5 electroporations of pCIG2 (J, J′, M, M′, P, P′), wild-type Ascl1 (K, K′, N, N′, Q, Q′), and Ascl1-SA6 (L, L′, O, O′, R, R′) vectors (expressing). Transfections were analyzed for expression of the pan-neuronal marker NeuN (J, J′, K, K′, L, L′), cortical neuronal marker Tbr1 (M, M′, N, N′, O, O′), or for transcripts for GFP (P–R) or Dlx1 (P′–R′). Quantification of GFP+ cells coexpressing NeuN (T) or Tbr1 (U). Dashed lines outline the transfected region in the neocortex. Yellow arrowheads mark ectopic expression of Sox9 (B′), BrdU (E′), NeuN (L′), or Dlx1 (Q′, R′). Scale bars: 250 μm. *p < 0.05, **p < 0.01, ***p < 0.005. ctx, neocortex; str, striatum.
Figure 10.
Figure 10.
Ascl1 is not required for OPC differentiation in response to RAS/ERK signaling. A–J, E12.5→15.5 electroporations of Ascl1+/− (A, B, D, F, G, I) or Ascl1−/− (C, E, H, J) cortices with mCherry (A, F), or pEFRasV12+mCherry (B, C, G, H), or pCIG2RasV12 (D, E, I, J). Transfections were analyzed for the transcripts of Etv1 (A–E) or Etv5 (F–J). K–O, E12.5→15.5 electroporations of Ascl1+/+ (K–K″), Ascl1GFPKI/+ (L–L″), and Ascl1GFPKI/KI (M–M″) cortices with pEFRasV12+mCherry imaged for the coexpression of mCherry (red), GFP (green), and Olig2 (blue). Quantitation of percentage of double-positive cells (Olig2+mCherry+ cells/mCherry+ cells; N) and triple-positive cells (Olig2+mCherry+GFP+ cells/mCherry+GFP+ cells; O) in Ascl1+/+, Ascl1GFPKI/+, and Ascl1GFPKI/KI cortices. p > 0.05. Dashed lines outline the transfected region in the neocortex. Yellow arrowheads mark the cells with ectopic expression of Etv1 (B–E), Etv5 (G–J), or Olig2 (K′–M′). Scale bars: A–J, 500 μm; K–M, K′–M′, K″–M″, 250 μm. ctx, cortex; str, striatum.
Figure 11.
Figure 11.
Model of RAS/ERK-proneural interactions in cortical development and gliomagenesis. In normal embryonic cortical progenitors, RAS/ERK signaling is low at early developmental stages, and Neurog2 is expressed, promoting robust glutamatergic neuronal differentiation. In the abnormal context of elevated RAS/ERK signals, Neurog2 expression is switched to Ascl1 expression. Moderate levels of RAS/ERK signals drive cortical progenitor cells to interneuronal fate with a few glioblast cells also differentiating, whereas high levels of RAS/ERK signals not only promote Neurog2-Ascl1 genetic switch in cortical progenitor cells, but also convert Ascl1 to a proglioblast molecule via direct phosphorylation, driving aberrant glioblast-like, and not neuronal differentiation, resulting in the formation of astrocytomas.

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