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Fig. 3. XPIASy functions as an inhibitor of XSmad2. (A) The effect of XPIASy on
embryogenesis. Embryos were injected into the dorsal midline at the four-cell
stage with XPIASy mRNA at a concentration of 0.5 ng (a), 1 ng (b), 5
ng (c) or 10 ng (d), or with dominant-negative XSmad2 mRNA at 5 ng
(f) or 8 ng (g), and developed until stage 27. (e) Uninjected control. (B) The
ventralized phenotype induced by XPIASy is largely rescued by co-injecting
with XSmad2, but not with ß-catenin. Embryos were injected into the DMZ
at the four-cell stage with 1 ng XPIASy (a), 1 ng XPIASy and
0.1 ng XSmad2 (b) or ß-catenin at 0.1 ng (c) or 0.5 ng (d). (e)
Uninjected embryo. (C) XPIASy inhibits animal cap elongation, mediated by the
Smad2/activin pathway. Both blastomeres of two-cell stage embryos were
injected with 0.5 ng of XSmad2 (a), XSmad2 and 0.2 pg of
activin (b), 0.5 ng of XPIASy (c), XPIASy and
activin (d), XSmad2 and XPIASy (e), XSmad2,
XPIASy and activin (f), nothing (g), or activin (h). At
stage 8, animal caps were dissected and cultured until stage 23. (D) XPIASy
inhibits mesoderm marker transcription activated by the Smad2 pathway but only
slightly inhibits transcription induced by the Wnt pathway. Semi-quantitative
RT-PCR was performed to analyze the effect of XPIASy on transcription of the
targets of activin/XSmad2 and Wnt pathways. The mRNAs (0.5 ng) of
XSmad2 (a), activin (b) or ß-catenin (c)
and/or XPIASy were injected into both blastomeres at the two-cell
stage. RT-PCR analysis was performed using animal caps as described in the
Materials and methods. XPIASy largely reduces the transcription of XSmad2 or
activin target genes (Chordin, Mix.2, Xbra1 and Xnr1) but
not Smad1/5 target genes (BMP4, Xvent1 and Msx1), (a) while
it only slightly reduces the level of ß-catenin target genes
(Siamois, Xnr3 and Chordin (c). Un, uninjected caps; WE,
whole embryos. (E) XPIASy inhibits the transcriptional activity of XSmad2 in
luciferase assays using 3TP-luc (a-c) and ARE-luc (d). Reporter plasmid (50
pg) and 0.5 ng of mRNAs of XSmad2 or XPIASy, or both, were
injected into both blastomeres of two-cell stage embryos. Luciferase assay was
performed using whole embryos (a,c,d) or animal caps (b) in the presence (c)
or absence (a,b,d) of activin (0.2 pg) as described in the Materials and
methods. (F) XSmad2 and ß-catenin regulate expression
of XPIASy in positive and negative ways, respectively. The indicated
amount of XSmad2 or ß-catenin mRNA was injected in the
animal side of two-cell stage Xenopus embryos. XPIASy level
in animal caps was analyzed as described in the Materials and methods. This
PCR-cycle number for XPIASy is higher than that in D.
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