cebpa does not induce mesodermal markers in animal caps. (A) One-cell stage embryos were injected with either gfp or cebpa mRNA. Animal caps were excised, and RNA was collected when sibling embryos reached stage 8, 9, 10, and 11. Real-time PCR showed that xbra (i), gsc (ii), and eomes (iii) were highly up-regulated during gastrulation stage in the whole embryo (stage 8-11). However, these mesodermal marker genes were not induced in either the gfp or the cebpa mRNA-injected animal caps (i-iii). In contrast, the myeloid marker spib (iv) was rapidly up-regulated in cebpa-injected animal caps but remained undetectable in gfp-injected animal caps and whole embryos. (B) One-cell stage embryos were injected with either gfp or cebpa mRNA, and treated with dimethyl sulfoxide or a combination of 2 inhibitors (SU5402 and SB505124), which inhibit the FGF and nodal receptors, respectively, from the 2-cell stage onward. RNA was isolated from whole embryos and animal caps at stage 11. Real-time PCR analysis showed that cebpa induced the expression of the myeloid markers spib (iii), lurp (iv), and mmp7 (v), even when the combination of FGF and nodal inhibitors inhibited the expression of the early mesodermal genes xbra (i) and gsc (ii). Expression levels and ΔCt values were normalized relative to rpl8. Error bars represent SEM of 3 independent experiments.