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Fig. 1. LRP6 is necessary for dorsal axis formation. (A) Two
wild-type whole oocytes, and four animal and vegetal halves, or six animal,
equatorial and vegetal thirds, were frozen and assayed by real-time RT-PCR for
the expression of LRP6 and Wnt11 mRNAs. LRP6 mRNA
was expressed throughout the oocytes. (B) Groups of two control and
LRP6 antisense oligo-injected oocytes (2.5, 5 and 10 ng oligo) were incubated
for 24 hours, and assayed for the expression of LRP6 and the related
LRP5 mRNA. LRP6, but not LRP5, mRNA levels were
reduced by the antisense oligo. (C) The phenotype of tailbud-stage
embryos derived from oocytes injected with 500 pg mouse LRP6 mRNA
(dorsalized) or 5 ng LRP6 antisense oligo (LRP6-; ventralized).
(D) The LRP6-depleted ventralized phenotype at tailbud stage caused by
the injection of LRP6 antisense oligo (middle row; 3 ng antisense oligo
injected) was rescued by the injection of 100 pg mouse LRP6 mRNA
(bottom row). LRP6 mRNA was injected 48 hours after oligo injection
and 24 hours before oocyte maturation. (E) The expression of Wnt target
genes (siamois, Xnr3, chordin and goosecoid) assayed by
real-time RT-PCR at the early gastrula stage in sibling embryos to those in D.
(F) The expression of endoderm marker Xsox17
and
ventral mesodermal marker Xwnt8 was delayed in LRP6-depleted embryos
at the late blastula stage, but reached wild-type levels of expression by the
early gastrula stage in LRP6-depleted embryos. By comparison, Xnr3
expression remained off in LRP6-depleted embryos. (G) Transverse
sections of tailbud-stage embryos derived from a control, LRP6-depleted and
LRP6- + LRP6-mRNA-injected oocytes. LRP6 depletion
resulted in a lack of dorsal structures, which was rescued by
LRP6-mRNA injection. (H) TOPflash reporter activation in
control and LRP6-depleted late blastulae after injection into two dorsal cells
at the 4-cell stage. Error bars indicate the standard deviation from the mean
(s.e.m.).
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