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Fig. 2. Knockdown of Coco causes germ layer defects at blastula stage. (A-M) Xenopus embryos were injected with either CocoMO or ControlMO and analysed at stage 9.5. Whole-mount in situ hybridisation was performed to identify endoderm (Sox17β), mesoderm (Xbra) and presumptive dorsal tissue (Chordin). Injection of CocoMO causes both a shift in Sox17β expression (A-C, arrowheads; using the blastocoel floor as a reference this shift is clearly seen in sections shown in A′,B′) and an upregulation of expression (D; compare RT-PCR of uninjected embryo and CocoMO-injected embryo). CocoMO-injected embryos were additionally injected with β-Gal in either a dorsal or ventral blastomere at the four-cell stage. Compared with an uninjected control (E, arrows) the shift of Sox17β expression is on the same side as dorsally injected β-Gal (F) but on the opposite side as ventrally injected β-Gal (G). Loss of Coco also caused a reduction of both Xbra (H-J′, arrows) and Chordin (K-M, arrowheads) expression, effects that are not seen following ControlMO injections. The shift of Sox17β expression following CocoMO injection (N,O, arrows) is rescued with an injection of Coco mRNA (P), demonstrating specificity. A-C,E-J,K-P are whole-mount lateral views and A′-B′;H′-J′ are sagittal sections. |
