In early vertebrate development, signalling cues from the extracellular environment direct pluripotent cells along distinct differentiation pathways. Understanding how pluripotent cells respond to signals is a fundamental challenge in biology with obvious bearing on the genesis of developmental defects, or on the in vitro derivation of tissues from stem cells for therapeutic purposes. Among the major current goals of stem cell biology is to define the mechanisms that direct pluripotent cells to form precursors of smooth muscle and other mesodermal tissues. To address this question we study cells of the chick neural crest (NC). NC stem cells within the cephalic region of the neural fold generate all head structures and express factors required during formation and expansion of the higher vertebrate forebrain1. However, the signalling mechanisms that underpin this developmental program are incompletely resolved.
During normal chick development NC stem cells develop within a region of the embryo that expresses fibroblast growth factor (FGF). Bone morphogenetic protein (BMP) signalling inhibits FGF expression, whereas BMP antagonists increase FGF production. Loss of BMP antagonists reduces FGF signalling and causes forebrain hypoplasia2. FGF signalling activates the MAP kinase signalling pathway to ERK. The goal of this project is to determine the role of ERK signaling in NC stem cells and its contribution to forebrain development.
We recently showed that ERK is recruited to transcription complexes at target gene promoters3 and have identified novel ERK substrates phosphorylated in these promoter complexes. These phospho-proteins may thus enable us to define target gene sets for ERK signalling in different cell backgrounds. One molecule of particular interest is MED14, a subunit of the Mediator complex that modulates signalling inputs to direct transcription. We identified an ERK-specific target site in MED14 and generated antibodies to the phosphorylated form of this molecule. Chromatin immunoprecipitation (ChIP) experiments have confirmed MED14 phosphorylation at target promoters in response to ERK signalling4. In this project we will identify ERK responsive genes within the chick genome that regulate PGC development.
The phospho-specific MED14 antibody will be used for ChIP-seq experiments on NC stem cells in the presence or absence of the inhibitor UO126. These experiments will identify the genes associated with MED14 phosphorylation. In follow up studies in chick embryos we will determine the role of these genes in the specification of NC stem cell derivatives. We will also produce NC cell lines expressing a phospho-mimetic variant of MED14 and test how this affects their differentiation in response to FGF and BMP signals.
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for information on specific International or EU scholarships.
1 Sauka-Spengler, T. Bronner-Fraser, M. (2008) A gene regulatory network orchestrates neural crest formation. Nat Rev Mol Cell Biol 9:557-568
2 Creuzet, S.E. (2009) Neural crest contribution to forebrain development. Semin Cell Dev Biol. 20:751-9
3 Zhang, H.M., Li, L., Papadopoulou, N., Hodgson, G., Evans, E., Galbraith, M., Dear, M., Vougier, S., Saxton, J., Shaw, P.E. (2008) Mitogen-induced recruitment of ERK and MSK to SRE promoter complexes by ternary complex factor Elk-1. Nucleic Acids Res. 36, 2594-607.
4 Galbraith, M.D., Saxton, J., Li, L., Shelton, S.J., Zhang, H., Espinosa, J.M., Shaw, P.E. (2013). ERK phosphorylation of MED14 in promoter complexes during mitogen-induced gene activation by Elk-1. Nucleic Acids Res. 41, 10241-53.