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Patterning of the Xenopus gastrula marginal zone in the axis running equatorially from the Spemann organizer-the so--called "dorsal/ventral axis"--has been extensively studied. It is now evident that patterning in the animal/vegetal axis also needs to be taken into consideration. We have shown that an animal/vegetal pattern is apparent in the marginal zone by midgastrulation in the polarized expression domains of Xenopus brachyury (Xbra) and Xenopus nodal-related factor 2 (Xnr2). In this report, we have followed cells expressing Xbra in the presumptive trunk and tail at the gastrula stage, and find that they fate to presumptive somite, but not to ventrolateral mesoderm of the tailbudembryo. From this, we speculate that the boundary between the Xbra- and Xnr2-expressing cells at gastrula corresponds to a future tissue boundary. In further experiments, we show that the level of mitogen-activated protein kinase (MAPK) activation is polarized along the animal/vegetal axis, with the Xnr2-expressing cells in the vegetal marginal zone having no detectable activated MAPK. We show that inhibition of MAPK activation in Xenopus animal caps results in the conversion of Xnr2 from a dorsal mesoderm inducer to a ventralmesoderm inducer, supporting a role for Xnr2 in induction of ventralmesoderm.
FIG. 2. Xbra is essential for establishing and/or maintaining
animal/vegetal mesoderm polarity. (A) MAPK activation in
stage-11 embryos that had been injected in both the AB4 and CD4
blastomeres with 250 pg of Xbra-EnR RNA (total 1 ng). X-Gal
staining for the product of coinjected LacZ RNA (blue punctate
staining) marks the AB4 and CD4 descendants. MAPK activation
was detected with an antibody (diffuse purple staining). We conclude
that MAPK remains activated despite Xbra function being
blocked. (B) Close up of the marginal zone of the embryo shown in
(A). (Câ G) In situ hybridization for Xnr2 expression at stage 10.5.
The Spemann organizer is to the top for all embryos. (C) Control
(noninjected) embryo. (D) Embryo injected with 250 pg of Xbra-EnR
RNA into both the AB4 and CD4 blastomeres. Xnr2 expression was
expanded into the animal marginal zone of the embryo in comparison
to the control embryo (arrow). (E) Embryo injected with 1 ng of
Xbra RNA into both the CD4 blastomeres (total 2 ng of Xbra) at the
16-cell stage. Xnr2 expression was decreased in the descendents of
the injected blastomeres. LacZ staining (blue) indicates cells that
inherited the injected RNA. (F) Same treatment for the embryo as
in (E), except in this example Xnr2 expression is completely
eliminated in the injected cells. (G) Embryos were injected with
200 pg of XFD RNA into the AB3 and CD3 blastomeres (total 400
pg) on one side, and with 200 pg of XFD plus 1 ng of Xbra RNAs
into the other AB3 and CD3 (total 400 pg of XFD and 2 ng of Xbra)
blastomeres. Xnr2 expression is expanded into the animal marginal
zone on the side in which XFD RNA alone had been injected (left
side of the embryos in this picture, arrows), while the expression is
decreased or eliminated on the other side in which Xbra RNA had
been injected together with XFD RNA (right side of the embryos in
this picture, arrowheads). (H) Stage-11 embryo injected with 1 ng of
Xbra RNA into both the CD4 blastomeres (total 2 ng) at the 16-cell
stage. Immunostaining with an anti-activated MAPK antibody is
shown. Note that the gap between the domain of activated MAPK
(purple) and the blastopore lip still exists (arrow) (compare to A).
FIG. 4. Xnr2 expression overlaps a region of low MAPK activation. (A) Immunostaining of a stage-11 embryo with an anti-activated MAPK
antibody. Staining (blue) was observed in a ring throughout the marginal zone with a distinct gap between the vegetal limit of the staining
and the blastopore lip. Because involution of cells has progressed further in the Spemann organizer region at this stage, no gap was seen.
Shown is a vegetal view. The Spemann organizer is to the top. (B) Xnr2 expression at stage 11 by in situ hybridization was detected in a
narrow band just above the blastopore lip. Again, expression in the Spemann organizer was not seen because gastrulation had proceeded
further in this region. Shown is a vegetal view with the Spemann organizer at the top. (C) A higher magnification view of the marginal zone
from (A) showing staining with anti-activated MAPK antibody. (D) A higher magnification view of the marginal zone from (B) stained for
Xnr2 expression by in situ hybridization.
