Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development
Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. We have proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor. Here we investigate the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. We characterise Xenopus Cic and show that it is widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. A similar mechanism of Cic protein degradation downstream of the Torso receptor tyrosine kinase receptor negatively regulates Cic activity in Drosophila. In keeping with our hypothesis, we show that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. We investigate two of these genes, fos and rasl11b, which, respectively, encode a leucine zipper transcription factor and a ras family GTPase, as putative targets of the proposed Fgf/Erk/Cic axis. We identify Cic consensus binding sites in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. We show that expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, we show that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. Our data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding. Overall design: We undertook triplicate RNA-seq analysis on control water injected, Fgf4 overexpressing and cic knockdown Xenopus tropicalis embryos at neurula stage 14