![]() P-R are in situ hybridizations showing rasl11b expression in early (stage 10+) and mid-gastrula (stage 11.5) embryos. Gastrula stage 10.5 embryos showing fos mRNA expression, H-K, or dpErk staining, L-O, in control embryos and embryos treated from stage 8 with 0.2% DMSO drug vehicle, 200μM SU5402 Fgfr inhibitor or 25 μM PD0325901 Mek inhibitor. D and G show immunohistochemical staining for dpErk in early (stage 10+) and mid-gastrula (stage11.5) embryos. B, C, E and F, are in situ hybridizations showing fos mRNA expression in early (stage 10+) and mid-gastrula (stage 10.5 and stage 11.5) Xenopus tropicalis embryos. Orange arrow indicates the level at which the clustering tree was cut. Red shading indicates increased and blue shading decreased expression relative to the average expression level (white). Grey points indicate genes below the significance thresholds.Ĭandidate targets of a Fgf/Erk/Cic pathway during gastrulation.Ī, Cluster analysis of the temporal expression patterns (4 to 23 hours post-fertilisation at 24☌) of genes up-regulated by both Fgf4 overexpression and Cic knockdown. Significantly up-regulated genes are indicated in red and down-regulated genes in blue. D and E, Volcano plots of gene expression in control water injected embryos versus Fgf4 overexpressing embryos and control versus Cic knockdown embryos. B and C, Venn diagrams of genes in Xenopus tropicalis embryos at early neurula stage 14 significantly up-regulated (p≤0.01, effect size ≥1.75) or down-regulated (p≤0.01, effect size ≤0.571) in Fgf4 overexpressing (Cska-Fgf4 injected) or Cic knockdown (Cic-TALEN injected) embryos, p-values of overlaps are indicated. Red and blue dots, respectively, indicate congruent up and down regulation. Gene expression changes resulting from Fgf4 overexpression and Cic knockdown at early neurula stage 14.Ī, Scatterplot of changes in gene expression (log 2 effect size) at early neurula stage 14 following injection of 1 ng each of forward and reverse Cic-TALENs versus log 2 effect size in genes significantly changing in embryos injected with of 5 pg Cska-Fgf4 plasmid (p≤ 0.01, effect size ≥1.75 or ≤0.571). Red bars indicated increased expression relative to control embryos. ![]() Log 2 effect size and p values as determined by Sleuth analysis of RNA-seq data. D, changes in marker gene expression in embryos injected with the Cic-TALEN pair at neurula stage 14. n values are combined from three separate experiments. C, Bar chart showing classification of embryo phenotype in control uninjected, forward TALEN only and forward+reverse injected TALEN embryos. The most severe class with blastopore defects exhibit varying degrees of blastopore non-closure with resulting failure of axial elongation and reduced anterior development. Axial defects is an intermediate effect class that generally show loss of anterior structures and is accompanied by more general effects on axial elongation. Anterior defects is mildest effect class characterised by loss of anterior structures, including cement gland and eye structures, while the rest of the axis relatively unaffected. B, Stage 41 larval stage Xenopus tropicalis embryos showing phenotypes used to classify the effects of Cic-TALEN injection. The linker region between the binding sites is targeted by the reformed bipartite endonuclease of the TALEN pair and subject to error prone non-homologous end joining repair. Binding sites of the forward and reverse TALENs are indicated. Phenotypic effects of TALEN mediated Cic knockdown.Ī, HMG box sequence in exon 6 of Xenopus cic gene targeted for the Cic-TALEN pair. Embryos were collected 30 minutes after wounding. F, Western blot showing that Cska-Fgf4 injection or puncture wounding reduces the levels of myc-Cic protein relative to control myc-Cic expressing gastrula stage 10.5 embryos. D, localised injection of 10 pg Cska-Fgf plasmid or E, needle puncture wounding of the presumptive ectoderm results in ectopic Erk activation in cells of the animal hemisphere. ![]() C, no dpErk is detected in the animal pole region of control embryos. Staining is restricted to a known region of Fgf signalling. B, vegetal view of immunohistochemistry for activated diphospho Erk (dpErk) showing the staining in the nascent mesoderm around the blastopore in control embryos at gastrula stage 10.5. Activation of Erk signalling reduces Cic protein in Xenopus embryonic cells.Ī, Immunohistochemistry showing myc-tagged murine CIC-S expressed from injected synthetic mRNA (500 pg) localises to the nuclei of cells in the animal pole region of a Xenopus laevis embryo at gastrula stage 10.5. ![]()
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