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Patterning events that occur before the mid-blastula transition (MBT) and that organize the spatial pattern of gene expression in the animal hemisphere have been analyzed in Xenopus embryos. We present evidence that genes that play a role in dorsoventral specification display different modes of activation. Using early blastomere explants (16-128-cell stage) cultured until gastrula stages, we demonstrate by RT-PCR analysis that the expression of goosecoid (gsc), wnt-8 and brachyury (bra) is dependent on mesoderm induction. In contrast, nodal-related 3 (nr3) and siamois (sia) are expressed in a manner that is independent of mesoderm induction, however their spatially correct activation does require cortical rotation. The pattern of sia and nr3 expression reveals that the animal half of the 16-cell embryo is already distinctly polarized along the dorsoventral axis as a result of rearrangement of the egg structure during cortical rotation. Similar to the antagonistic activity between the ventral and the dorsal mesoderm, the ventral animal blastomeres can attenuate the expression of nr3 and sia in dorsal animal blastomeres. Our data suggest that no Nieuwkoop center activity at the blastula stage is required for the activation of nr3 and sia in vivo.
Fig. 1. The expression pattern of dorsal and ventral marker genes in
explants of animal blastomeres from 16-cell embryos. (A) Schematic illustration
of the experimental procedure; (B) analysis of the expression of
marker genes by RT-PCR. Dorsal and ventral explants were harvested and
lysed immediately after isolation (lanes 2 and 3), cultured until stage 8.5
(lanes 4 and 5) or stage 10.5 (lanes 6 and 7). RNA isolated from whole
embryos at stage 10 (lane 1).
Fig. 2. Descendants of 16-cell stage animal blastomeres can be identified and isolated at the 128-cell stage. (A) In early embryos the descendants of the
animal blastomeres show darker pigmentation than the vegetal cells at the 128-cell stage. (B) Injection of fluorescent dextran (FDA) as a lineage tracer into
the dorsal vegetal blastomeres at the 16-cell stage demonstrates that at the128-cell stage the pigment border seen in (A) separates the descendants of animal
and vegetal blastomeres. (C) Descendants of the 16-cell stage dorsal animal blastomeres explanted at the 128-cell stage which were dissected using the
pigment border as a morphological marker do not contain fluorescent cells from the vegetal region. (D) The vegetal part of this 128-cell embryo contains the
FDA lineage tracer. The dotted line indicates the pigment border that separates animal and vegetal blastomeres.
Fig. 3. The expression of gsc, bra and wnt-8 is induced in animal blastomeres
explanted from 128-cell embryos. (A) Schematic illustration of
the experimental procedure; (B) dorsal and ventral blastomeres of 128-cell
embryos corresponding to the progeny of the dorsal or ventral animal
blastomeres of 16-cell embryos were isolated and cultured until stage
8.5 (lanes 1 and 2) or stage10.5 (lanes 3 and 4). gsc expression is induced
in dorsal-and wnt-8 in ventral explants. bra is expressed in dorsal and
ventral explants.
Fig. 4. Early mesodermal marker genes are activated differentially in
animal tier explants cultured in combination with dorsal or ventral vegetal
blastomeres. (A) Schematic diagram of the experimental procedure
describing the explantation and recombination of blastomeres from 16-
cell embryos. (B) RT-PCR analysis of explants: animal layer (AL), dorsal
vegetal blastomeres (DVB), vegetal ventral blastomeres (VVB) (lanes 1, 2
and 4) and combinations of AL and DVB (lane 3), AL and VVB (lane 5)
were cultured until stage 10.5. For the RT-PCR reaction, RNA corresponding
to 0.02 AL explant equivalents, 0.1 DVB or VVB equivalents and 0.04
AL/DVB or AL/VVB equivalents were used. The DVB can induce the
expression of gsc, bra and wnt-8 in AL/DVB combinations but VVB is not
sufficient to induce gsc and bra expression in the AL/VVB combinations
(lanes 3 and 5).
Fig. 5. Tilting rescues axis formation in UV-treated embryos by the redistribution of dorsal and mesodermal determinants. (A) Embryos were ventralized by
UV-treatment (DAI 0.5) or were tilted 90immediately after UV-treatment and reared to stage 38 (B). The tilting fully restores the body axis (DAI 5); (C)
antibody whole mount staining for b-catenin in the tilted UV-embryos at stage 8.5. The arrow head indicates the position of the nuclear b-catenin patch which
is encircled by the dotted line. This region corresponds to the vegetal pole of the UV-treated embryo. Dotted line in (B) indicates the position of the dorsal lip
in the tilted embryos.
Fig. 6. Tilting of UV-treated embryos restores the expression of sia and nr-
3 in 16-cell stage animal tier explants. (A) Schematic illustration of the
explanting and tilting procedure. (B) The expression profile of marker
genes analyzed by RT-PCR at blastula and gastrula stages. Animal tier
explants from untreated (lanes 2 and 3), UV-treated (lanes 4 and 5) and
UV-treated-tilted embryos (lanes 6 and 7) were analyzed at stages 8.5 and
10.5. The expression of sia and nr-3 which is abolished by UV-treatment
can be restored in the embryos by tilting.
