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Fig. 2. Assays on Klf4 function with animal caps. (A-C) Uninjected control whole embryos (Uninj. WE), uninjected control animal caps (uninj. caps), and caps injected with Klf4 mRNA (Klf4 caps), were assays for the expression of Xbra (A), Sox17α (B) and Sox2 (C), and their respective quantification. (D) Mixer expression in uninjected control whole embryos (Uninj. WE), Klf4 mRNA-injected whole embryos (Klf4. WE), uninjected control animal caps (uninj. caps) and injected caps (Klf4 caps), and respective quantification. In A-D, embryos were placed vegetally to view normal expression of marker genes; except those in D, embryos were also orientated to animal view (An) to show the staining for Mixer in animal pole. Graphs represent the numbers of WE or caps with (positive) or without (negative) gene expression in three experiments. (E) qPCR detection of gene expression to analyse tissue differentiation in animal caps injected with Klf4 RNA. Error bars represent s.d. in triplicate. A Student's t-test was conducted to compare the changes in gene expression between uninjected (Uninj. caps) and Klf4-injected (Klf4) caps. Asterisks indicate P<0.01. NS: not significant. In the experiments above, 400 pg of Klf4 mRNA was injected close to the animal pole of all blastomeres of four-cell embryos, and animal caps were removed at stage 8.5. For whole-mount in situ hybridization assays, caps were cultured until sibling control embryos reached stage 10.5. For qPCR assays, caps were cultured until sibling control embryos reached stage 15. |
