Fig. 3. Partial depletion of maternal XTcf3 and FoxH1 together causes a complete loss of Xnr3 expression. (A) Oocytes were injected with 6 ng of XTcf3 oligo, 2.5 ng of FoxH1 oligo or both, cultured for 58 hours and then fertilized by the host transfer method. Embryos derived from these oocytes were frozen at two-hourly intervals through the late blastula and gastrula stages and analysed by real-time RT-PCR for the expression of Xnr3 and goosecoid mRNAs. Double-depleted embryos have complete loss of expression of Xnr3 mRNA throughout the gastrula stages. In comparison, goosecoid mRNA was reduced but not eliminated in double-depleted embryos. (B) Sibling embryos to those analyzed in (A) were allowed to develop to the tailbud stage. Phenotypically, partially XTcf3-depleted embryos have an anteriorized phenotype (TCF3 depleted), Partial FoxH1-depleted embryos have reduced or absent anterior structures and double-depleted embryos have more severe axial defects, than those caused by the depletion of XTcf3 or FoxH1 alone. (C) Nodal signaling was inhibited in control and FoxH1-depleted oocytes by the injection of 500 pg of CerS mRNA into oocytes. Embryos were frozen at the late blastula and early gastrula stages and assayed for Xnr3 and Xnr1 mRNA expression. Xnr3 continues to be expressed in control embryos in which nodal signaling is blocked by CerS. In contrast, Xnr1 expression is dependent on nodal signaling. (D) Western blot analysis with anti-phospho-Smad2 (pSmad2) antibody of wild-type embryos and embryos injected as oocytes with 500 pg CerS mRNA and analysed at two stages during gastrulation. CerS blocks nodal signaling and completely prevents Smad2 phosphorylation (as well as Smad2^* phosphorylation; a second phosphorylated form). The blot was reprobed for actin as a loading control.