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Fig. 5. Molecular markers analysis after microinjection of McerP-BMP4, -Xnr1 and -Xwnt8 in frog embryos. (A) RT-PCR analysis at stages 12, 13 and 15 show that the cement gland and anterior neural markers Xag, XBF1, Xemx1 and Nkx2.1 are downregulated in embryos coinjected with McerP-BMP4, McerP-Xnr1 and McerP-Xwnt8 (20 pg each), when compared with uninjected controls, while the levels of more posterior neural markers, like En2 and Krox20, are not changed. ODC was used as a loading control. RNA extracts used for the RT-PCRs were made from pools of 5 randomly picked embryos. (B-K) In situ hybridization analysis for different molecular markers at stages 22/24. The injection of McerP-BMP4, McerP-Xnr1 and McerP-Xwnt8 (20 pg each) leads to the suppression of the anterior domains of expression of XBF1 (B,C), Xotx2 (D,E) and Xnot2 (F,G). Expression of the hindbrain marker, Krox20 (H,I), was not significantly changed in the injected embryos as well as in the controls. (J,K) Xshh expression in injected embryos does not extend as anteriorly as it does with the uninjected sibling embryos. |
