gpc4 is expressed in the posterior endoderm and transgenic lines that expresses Gpc4 in the endoderm. (A–C′′) Expression of gpc4 transcript in the posterior region of a Tg(sox17:EGFP) embryo at the 10 somite (s) stage, as detected by WISH. Cryosection was performed on the embryos after gpc4 WISH. (A)gpc4 expression in embryos. Posterior dorsal view, with anterior up. Red line shows the estimated plane for cross sectioning. (B–C′′) Transverse sections of the embryo. (C–C′′) Higher-magnification images of the region shown in red dashed box in B. (C) Overlay of WISH panel (C′) and anti-GFP IF staining panel (C′′). Yellow arrows, endoderm; NT, neural tube; NC, notochord. (D) Schematic depiction of the transgene sox17:GFP-Gpc4. GFP (green box) is inserted after the N-terminal signal peptide of Gpc4, and expression is driven by the endoderm-specific promoter sox17 (gray box). (E–E′′) Expression of transgenic GFP-Gpc4 at 1 somite (s) stage. (E) Bright-field image. (E′) Epifluorescence image of GFP expression. (E′′) Overlay of E and E′. White arrows, Kupffer’s vesicle. (F–F′′) A representative confocal z-stack image at 14 somite (s) stage, showing the expression of GFP-Gpc4 (F) and mem-mCherry (F′) on the plasma membranes of endodermal cells, and overlay of F and F′ (F′′). (G) Levels of gpc4 mRNA in embryos expressing the transgene relative to those that do not express it, as detected by qPCR at 3 somite (s) stage. Data are mean ± SEM. *, P < 0.05; unpaired Student's t test.