Abstract
Neural tissue formation is induced by growth factors that activate networks of signal transduction cascades that ultimately lead to the expression of early neural genes, including transcription factors of the SoxB family. Here, we report that fibroblast growth factor (FGF)-induced Erk1/2 (Mapk3 and Mapk1, respectively) mitogen-activated protein kinase (MAPK), but not phosphatidylinositol 3'-OH kinase (PI3K, Pik3r1), signalling is required for neural specification in mouse embryonic stem (ES) cells and in the chick embryo. Further, blocking Erk1/2 inhibits the onset of key SoxB genes in both mouse ES cells (Sox1) and chick embryos (Sox2 and Sox3) and, in both contexts, Erk1/2 signalling is required during only a narrow time window, as neural specification takes place. In the absence of Erk1/2 signalling, differentiation of ES cells stalls following Fgf5 upregulation. Using differentiating ES cells as a model for neural specification, we demonstrate that sustained Erk1/2 activation controls the transition from an Fgf5-positive, primitive ectoderm-like cell state to a neural progenitor cell state without attenuating bone morphogenetic protein (BMP) signalling and we also define the minimum period of Erk1/2 activity required to mediate this key developmental step. Together, these findings identify a conserved, specific and stage-dependent requirement for Erk1/2 signalling downstream of FGF-induced neural specification in higher vertebrates and provide insight into the signalling dynamics governing this process.
Highlights
Neural specification is a fundamental developmental process during which cells embark on the neural differentiation programme
After an initial drop in activation following overnight culture in serum-free N2B27 media, phospho-Erk1/2 and phosphoPKB(S473) (PKB is known as Akt – Mouse Genome Informatics; a key mediator of PI3K signalling) levels increased as differentiation proceeded, while phospho-phospholipase C gamma (PLC␥)1(Y783) (PLC␥1 is known as Plcg1 – Mouse Genome Informatics) levels gradually decreased (Fig. 1B)
Erk1/2 is activated by FGF receptors (FGFRs) and drives neural specification In order to determine which pathways are activated in response to endogenous fibroblast growth factor (FGF) during the period of neural specification, we employed the specific FGF receptor inhibitor PD173074 (Mohammadi et al, 1998). 46C cells were grown under differentiation conditions in the presence of PD173074 and analysed daily by flow cytometry for Sox1-enhanced green fluorescent protein (EGFP), as well as by western blotting
Summary
Neural specification is a fundamental developmental process during which cells embark on the neural differentiation programme. This is indicated by the onset of expression of genes characteristic of neuroepithelium, which include members of the SoxB family of transcription factors (Pevny et al, 1998; Rex et al, 1997; Wood and Episkopou, 1999). There is piecemeal evidence that implicates all three of these pathways in the induction of neural tissue in vertebrate embryos Most of this comes from work in the frog embryo, in which signalling via Ras (upstream of Erk1/2 and PI3K) is required for the induction of posterior neural tissue (Delaune et al, 2005; Ribisi, Jr et al, 2000), whereas PKC activators can turn on neural genes via an unknown mechanism (Otte et al, 1988). Early (preneural) genes that identify potential neural tissue include a different SoxB gene, Sox, whereas, in this vertebrate embryo, the later-expressed Sox gene is a marker of definitive neural tissue (Rex et al, 1997)
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