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Fig. 2. Maternal Dkk1 inhibits canonical Wnt signaling. (A) Embryos
derived from sibling control (uninj.) and Dkk1-depleted oocytes (5, 7.5 and 10
ng oligo injected) at the early tailbud stage. This phenotype was seen in
seven experiments in a total of 85% of cases (124/165). (B) The
phenotype of Dkk1-depleted (Dkk-) embryos was partially rescued by
the reintroduction of 20 pg human Dkk1 mRNA (Dkk-+mRNA)
before fertilization. Here, Dkk1-depleted embryos (14/17) had the elongated
phenotype shown compared with 4/14 for Dkk1-+mRNA and 0/24
uninjected; 20 pg human Dkk1 mRNA alone (Dkk mRNA) caused enlargement
of head structures (16/18). The experiment was repeated with a similar result.
(C) The relative expression levels of Xnr3 and Xnr5
in control (uninj.), in Dkk1 depleted (Dkk-), in 20 pg human
Dkk1 mRNA (Dkk mRNA) and in Dkk1 depleted+20 pg human Dkk1
mRNA injected (Dkk-+mRNA) embryos assayed by real-time RT-PCR at
the late blastula stage; siblings of those shown in B. (D) TOPflash
reporter activation after injection into two dorsal cells of four-cell stage
control embryos compared with sibling Dkk1-depleted embryos frozen at the
eight-cell, mid-(stage 8), late blastula (stages 9, 9.5) and early gastrula
stages (stages 10, 10.5). (E) Western blot of total β-catenin
protein in control and Dkk1-depleted sibling early blastulae (stage 7), using
-tubulin as a loading control. Quantitation is shown on the right.
(F,G) In situ hybridization of sibling control and Dkk1-depleted
early gastrulae (Xnr5, F; chordin, G). (H) Western
blot of phospho-Smad2 and -Smad1 proteins in control and Dkk1-depleted sibling
at late blastulae (stage 9.5) and early gastrulae (stage 10.5). Before
freezing, embryos were hemisected into batches of four dorsal (dor) and four
ventral (ven) halves.
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