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Fig. 1. Crescent inhibits CEMs without affecting mesodermal or neural specification in whole embryos. (A) Lineage tracer analysis of CEMs after overexpression of Crescent or Frzb1. (B) Analysis with FLAG-tagged proteins, Crescent-FLAG (Cres-F) and Frzb1-FLAG (Frzb1-F). (a) Comparison of expression levels of Cres-F and Frzb1-F in injected embryos by Western blot analysis. Homogenates from embryos injected with cres-F or frzb1-F mRNA (100 pg/embryo) were electrophoresed (0.5 embryo equivalent per lane), FLAG-tagged proteins were detected with an anti-FLAG antibody. α and δ non-specific bands; β, Cres-F band overlapping a non-specific band; γ, Frzb1-F band. (a′) Intensities of bands were quantified with an image analyzer. The net volume of Cres-F band (β–α) is equivalent to that of Frzb1-F band (γ). Vertical axis, band intensity in arbitrary units. (b–d) FLAG-tagged proteins, Cres-F and Frzb1-F show activities similar to the wild type (see A). (e–m) Embryos at neurula stages 16–17 were stained for β-gal (red), and subjected to whole-mount in situ hybridization for XmyoDa (e–g), XPA26 (h–j), or sox2 (k–m). Dorsal view; anterior is upwards. (C) and (D) Expression of early marker genes in injected embryos. Four-cell-stage embryos were injected with mRNA, as indicated, with nβ-gal mRNA on the left (C) or both sides (D) of the dorsal equatorial region. Embryos at the gastrula (stage 10.5 or 11) or mid neurula stage (stage 14), as indicated, were stained for β-gal and subjected to whole-mount in situ hybridization for goosecoid (gsc) and Xotx2 (C), or for Xbra (D). An arrowhead indicates the spot-like expression of Xbra. (E) Crescent inhibits axis-inducing activity of Xwnt8. Four-cell-stage embryos were injected into the ventral equatorial region with mRNA as indicated. Embryos at the tailbud stage (stage 40) are shown. Numbers in each panel indicate the frequency of the represented phenotype. Amounts of injected mRNAs (pg/embryo): β-globin, 100 (A,Ba,D,E) or 25 (Be,h,k,C); crescent, 100; frzb1, 100; cres-F, 100 (Bb), 25 (Bf,i,l,C); frzb1-F, 100 (Bc), 25 (Bg,j,m); Xwnt8, 2; dkk1, 50. |
