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Fig. 3. XGrhl1 expression is dependent on the BMP4 signaling pathway.
Microinjection of a dominant-negative BMP receptor mutant (tBR)
results in a decrease in XGrhl1 and XK81A1 expression with a
concomitant increase in transcripts encoding the NCAM neural marker.
ODC was used as a control for RNA recovery. Uninjected, uninjected
cap; WE, whole embryo. (A) Enforced expression of the BMP4 antagonist
noggin represses both XGrhl1 and XK81A1 expression.
(B) RT-PCR analysis of animal pole explants at stage 21 injected at the
one-cell stage with noggin mRNA. (C) Factors antagonizing BMP4
signaling block XGrhl1 expression in vivo. In situ hybridization
analysis (blue) for XGrhl1 in embryos injected with neuralizing
[noggin (600 pg), Ngeminin (1 ng)] or epidermal-inducing
factors [XVent-2 (400 pg), BMP4 (1 ng)]. Embryos were
co-injected with ß-galactosidase mRNA (50 pg; stained red) for lineage
tracing. Vent-2 and geminin, anteroventral view;
noggin, lateral view; BMP4, ventral view. (D) Co-injection
of xMad1 rescues XGrhl1 expression in
tBR-expressing explants. RT-PCR analysis of animal pole explants
injected at the one-cell stage with either tBR alone (2 ng), or
tBR with increasing concentrations of xMAD1 encoding RNA.
Induction of XLMO2 indicates functional XMad1 transcripts
(Mead et al., 2001). (E) Ectopic expression of XGrhl1 does not alter
epidermal specification or neuralization in vivo. In situ hybridization for
XK81A1 expression (blue) of stage 14 embryos injected at the one- to
four-cell stage in the animal pole with XGrhl1-encoding transcripts
(4 ng). ß-Galactosidase (red stain) was used as a lineage tracer. The
upper panels illustrate representative embryos injected in blastomeres with an
epidermal fate (lateral orientation); lower panels are representative of
blastomeres with a neural fate (anterior orientation). (F) Co-injection of
XGrhl1 transcripts does not rescue tBR-induced
neuralization. RT-PCR analysis of animal cap explants injected at the one-cell
stage with either tBR (2 ng) alone or tBR with
XGrhl1 encoding RNA (4 ng).
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