Fig. 3. Analysis of gene expression in Mixer-depleted gastrulae (A) and in mRNA rescued gastrulae (B). (A) Embryos were injected at the two-cell stage with 20 ng or 40 ng of MO-1 and cultured until the early or mid-gastrula stage. The resulting Mixer-depleted embryos were collected at indicated stages and analyzed by real time RT-PCR for the expression of various mesodermal markers Eomesodermin, Fgf8, Xnot1, Gata2, Fgf3, eFGF, antipodean, mesendodermal markers Xnr1, Xnr5, Antivin, Bix1, Bix4, endodermal markers Xsox17, Gata5, Endodermin, Cerberus, Xlim-1 and Ectodermal markers Xlim-5 and epidermal keratin. Expression levels are normalized to ODC. (B) The rescue experiment was carried out by injecting MO-1 into the vegetal region of both cells of the two-cell embryos. Embryos were cultured until the eight-cell stage and injected into the four vegetal blastomeres with either 50, 125 or 500 pg of Mixer MO-R mRNA. Embryos were cultured until the mid-gastrula stage (stage 10.5), frozen and assayed by real-time RT-PCR. Expression levels were normalized to ODC.