Fig. 4. Mixer depletion causes ectopic expression of VegT target genes. (A) Embryos injected with Mixer MO-1 were cultured until the blastula stage, and dissected into vegetal and equatorial explants. These explants were cultured until the mid-gastrula stage, frozen and analyzed for expression of mesodermal and endodermal markers by real-time RTPCR. Expression levels were normalized to ODC. we, wild-type embryo; eq, equatorial explant; bs, vegetal explant. Expression levels were normalized to ODC. (B) Half-mount in situ for Cerberus, eomesodermin, Bix3, Fgf8 and Xbra in Mixer MO-1-injected embryos. Arrows indicate dorsal. Embryos were injected with 25 ng of Mixer MO-1 into both vegetal cells of a two-cell embryo. These embryos were cultured until stage 10 and 11, fixed in MEMFA, bisected, re-fixed in MEMFA and assayed for gene expression by whole-mount in situ hybridization for the indicated gene. (C) Whole-mount in situ for eomesodermin in Mixer MO-1-injected embryos. Embryos were injected with 6 ng of Mixer MO-1 into one vegetal cell of an eight-cell embryo. These embryos were cultured until stage 10.5, fixed in MEMFA and analyzed for eomesodermin expression by whole-mount in situ hybridization. The arrow indicates the expanded region of eomesodermin mRNA. The pinkish color is due to ß-gal staining from co-injection of ß-galactosidase mRNA as a lineage tracer.