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Figure 5. Depletion of XTcf-3 and XCIRP results in a lateral expansion of sox2. (A) In situ hybridization revealed that both, XTcf-3 and XCIRP are required for proper neural development. The expression of sox2 was laterally expanded (arrow) at the injected side (asterisk). Two picomoles Tcf3Mo, eight picomoles Tcf4Mo or two picomoles CIRP-Mo or two picomoles LefMo1 + two picomoles LefMo2 were coinjected with 4 pg dextrane (to trace the injected side) and 1000 pg XTcf-3 mRNA or 200 pg XCIRP DNA into one blastomere of 2-cell stage embryos. (B) Quantification of the lateral expansion. 71,2% of the Tcf3Mo, 51,5% Tcf3Mo+XTcf-3, 36% Tcf3Mo+XTcf-3+XCIRP, 53% of the CIRP-Mo and 30% of the CIRP-Mo+CIRP and 0% of the Tcf4Mo and LefMo, injected embryos showed a lateral expansion of sox2. n: number of analyzed embryos. (C) The alignment of the XCIRP antisense morpholino oligonucleotide (CIRP-Mo) with XCIRP and XCIRP2 indicates that it is supposed to block both isoforms. The Western Blot demonstrates that the amount of endogenous XCIRP protein is reduced following CIRP-Mo injection. The reduction of XCIRP protein by blocking XTcf-3 translation (Tcf3Mo) can be restored by co-injection of XTcf-3 mRNA (Tcf3Mo + XTcf-3). Two picomoles CIRP-Mo, two picomoles Tcf3Mo and two picomoles Tcf3Mo + 1000 pg XTcf-3 mRNA were injected into both blastomeres of 2-cell stage embryos. RIPA lysates corresponding two 1/3 embryo where separated on a 15% SDS page and either stained with coomassie (CBB) as loading control or transferred to nitrocellulose and stained with anti-CIRP polyclonal antiserum (α-CIRP).(D) The anti-CIRP polyclonal antiserum (α-CIRP), originally directed against XCIRP2 [18] recognizes both, endogenous XCIRP/XCIRP2 (blue arrow) and overexpressed XCIRP (black arrow). Staining with the anti-myc antibody shows the overexpressed myc-tagged XCIRP (black arrow) and some faster migrating proteins, which correspond most likely to degradation products. |
