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Fig. 5. Knockdown of Xema protein induces mesoderm formation. (A) Xema morpholinos
block the translation of Xema RNA in vitro. Morpholino
oligonucleotides (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) The effect of Xema morpholinos on
exogenous Xema protein levels. Western blot analysis of whole-cell lysates
extracted from embryos injected with 1 ng 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. (Left column) Lower 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 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. (Right column)
Lower panels show embryos injected with 5 ng of MO-1, 10 ng MO-2 and 166 pg of
lacZ RNA in the animal pole of early cleavage embryos. The top embryo
is injected with 166 pg of lacZ RNA only. Note the presence of
ß-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.
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