Fig 5.

Knockdown of Xema protein induces mesoderm formation. (A) Xema morpholinos block the translation of Xema RNA in vitro. Morpholino oligos (500 ng each) and Xema RNA (1 ng) were simultaneously added to a rabbit reticulocyte lysate mix (Promega), in the presence of [³⁵S]Methionine. (B) Effect of Xema morpholinos on exogenous Xema protein levels. Western blot analysis of whole cell lysates extracted from embryos injected with 1ng Xema-Myc RNA. 10 ng MO-1, 10 ng MM-1, and/or 20 ng MO-2 were injected, as listed. (C) Mesoderm and endoderm are induced by injection of Xema MO-1 (10 ng) but not by Xema MM-1 (10 ng). RT-PCR analysis of animal cap explants from embryos injected with Xema morpholinos in the animal pole at early cleavage stages, dissected at blastula stages and harvested during midgastrula stages. (D) Xema MO-1 (10 ng) and MO-2 (20 ng) potently synergize to induce mesendoderm. (E) Co-expression of Xema MO-1 and MO-2 disrupts normal development. The lower left panels show embryos co-injected with 5 ng of MO-1 and 10 ng of MO-2 in the animal pole of early cleavage stage embryos. The top left embryo is uninjected. Second embryo from top is a dorsal view; all other views are lateral; anterior is to right. Note anterior pigmented ectopic lateral structures. The lower right panels show embryos injected with 5 ng of MO-1, 10 ng MO-2, and 166 pg of β-galactosidase RNA in the animal pole of early cleavage embryos. The top embryo is injected with 166 pg of β-galactosidase RNA only. Note the presence of β-galactosidase staining (blue), primarily in the ectopic structures. All views are lateral; anterior is to right. (F) Rescue of Xema morpholino-mediated mesendoderm formation by injection of 1 ng Myc-Xema RNA. 10 ng MO-1 and 20 ng MO-2 were injected, as listed.