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

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 ϩ 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.

C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation

“…The S861/864 phosphorylation site is a consensus site for phosphorylation of the MAP kinases ERK1/2. Further, during neurogenesis, REST turnover increases, whereas concomitantly, growth factors such as EGF and FGF mediate transcriptional changes via the Ras-ERK signaling pathway (24). Our results, using growth-factor stimulation in cells as a surrogate for signaling pathways, suggest that S861/864 phosphorylation occurs, at least in part, via ERK1 and ERK2.…”

Neurog2 and Ascl1 together regulate a postmitotic derepression circuit to govern laminar fate specification in the murine neocortex

“…Ascl1 also promotes a glutamatergic fate in other contexts, including in E10.5 cortical progenitors [CajalRetzius neurons (23)] and in direct neuronal reprogramming studies in fibroblasts (46). Ascl1 thus is promiscuous in its choice of target genes, promoting the formation of Ctip2 + glutamatergic projection neurons in early cortical progenitors (this study) as well as GABAergic interneurons (25) and proliferative Sox9 + glioblasts (33) and Cux1 + neurons in later progenitors (this study). How Ascl1 selects its downstream transcriptional targets to promote different cell fates is not entirely clear, although RAS/ERK signaling has been implicated in biasing Ascl1-expressing progenitors toward glioblast lineages (33).…”

“…We first focused on Neurog2, generating a conditional gain-of-function transgenic line to assess its laminar fate-specification properties. We did not generate Ascl1 transgenics, because Ascl1 promotes the ectopic expression of both neuronal and glial markers, complicating analyses in vivo (32,33). A conditional Neurog2 gain-of-function allele was generated using a transgene carrying a floxed "stop" cassette and the Neurog2 coding sequence (flSTOP::Neurog2) (Fig.…”

“…Highlighting the instructive properties of the proneural genes, Neurog2 promotes the ectopic expression of glutamatergic neuronal markers, including Tbr1, in ectopic sites in the ventral telencephalon (42), whereas Ascl1 induces the expression of GABAergic markers in dorsal domains (25,33). To test whether Neurog2 and Ascl1 were sufficient to induce a deep-layer neuronal identity outside the normal temporal window, we performed E15.5→P4 cortical electroporations, targeting progenitors that give rise to upper layer neurons.…”

“…Brains were washed three times in 1× PBS, cryoprotected in 20% sucrose/1× PBS overnight at 4°C, blocked in optimum cutting temperature (OCT) compound, and cryosectioned at 10 μm. RNA in situ hybridization (33,48) and immunohistochemistry (33) were performed as described previously.…”