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In Xenopus, mRNAs synthesized during oocyte differentiation are inherited by the egg and direct all protein synthesis until the late-blastula stage. This provides an opportunity to study the roles of maternally expressed genes in embryonic development of a vertebrate. Oocytes can be depleted of specific mRNAs by the injection of antisense deoxyoligonucleotides and then fertilized to assay for developmental abnormalities. The ease of experimental manipulation of early Xenopus embryos in culture gives considerable opportunity for the analysis of the abnormalities seen.
FIGURE 1
Method used to generate fertilized eggs depleted of a specific maternally encoded
mRNA. Oocytes are manually dissected free of their follicles and injected with
antisense deoxyoligonucleotides (oligos). After 24 h, they are stimulated to mature
in vitro with progesterone. They are then transplanted into the abdominal cavity of a
female frog that has been stimulated to lay eggs. Together with the host's own eggs,
the experimental eggs pass down the oviducts of the host female and can be
fertilized after release from the cloaca.
Figure 2
The antisense-oligo-oocyte-transfer method described here depends crucially on the ability to distinguish the experimental oocytes
from the host oocytes. To do this, we artificially colour the experimental oocytes (a) using the vital dyes Nile blue sulphate, neutral
red and Bismarck brown. In combination, these yield other colours, giving five useful colours in total. The dyes are non-toxic, so that
eggs will fertilize (b) and embryos will develop to the swimming larva stages (c) carrying the markers. Oocytes and embryos shown in
(a) and (b) are 1.2 mm in diameter; larvae shown in (c) are 10 mm in length.
Figure 3
Illustration of the effects on development of depleting the mRNA coding for 13-catenin in the oocyte. 13-catenin mRNA (band I in each
lane) is depleted after injection of either 0.5, 1 or 2 ng of antisense oligo (a). In the oocyte, the wild-type level of mRNA is seen in
lane 1 and after antisen~e iniectio~q in lanes 2 (2 ng), 3 (1 rig) and 4 (0.5 ng). After fertilization, the mRNA level is still reduced
(compare the wild-type level in lane 5 with the 2-ng-oligo-injected level in lane 6). At the late-blastula stage, zygotic transcription has
stared, and ~-catenin mRNA is appearing again in the embryo [compare wild-type levels in lane 7 with oligo-injected levels in
bands 8 (1 ng) and 9 (0.5 ng)]. By the neurula stage, the mRNA levels are the same in wild-type and oligo-injected embryos (bands
10-12). Band II in each lane is a loading control. Reproduced, with permission, from Ref. 10. (b) Control embryos at the neurula
stage; the neural folds are seen forming on the upper surfaces. (c) Effect of the depletion of the maternal mRNA for 13-catenin. The
central nervous system, shown by the neural folds in (b), is missing. In addition, all other dorsal structures, including notochord and
somites, are absent. Embryos shown in (b) and (c) are each 1.2 mm in diameter.
