Fig. 2

The Tg(ARE:eGFP) Zebrafish Line Is a Sensitive and Specific In Vivo Nodal Reporter

(A) Top: schematic representation of the Nodal reporter gene construct. Bottom: WISH for eGFP in Tg(ARE:eGFP) embryos at 50% epiboly (above, lateral view; below, animal view) and 22 somite stages. Dashed line indicates the midline. A, anterior; D, dorsal; lpm, lateral plate mesoderm; P, posterior; V, ventral.

(B) WISH for eGFP in control-treated Tg(ARE:eGFP) embryos or after inhibition of Nodal signaling by SB-505124 from the 32-cell stage (left), injection of foxh1 MO (middle), or in a tdgf1^-/- background (right) at 50% or 30% epiboly. For the tdgf1^-/- experiment, a clutch of 16 embryos from a heterozygous incross was analyzed. Note that 25% of embryos lack eGFP staining.

(C) qPCR for eGFP, ta, and odc1 on dissociated blastula-stage Tg(ARE:eGFP) embryonic cells. Depicted is the mean relative expression compared with untreated cells, normalized to eef1a1l1 levels from one representative experiment ± SD.

(D) Polystyrene beads soaked in recombinant human NODAL were implanted into the animal pole of 1000-cell Tg(ARE:eGFP) embryos. Once the embryos had reached ring stage, they were stained for the indicated genes. Animal views are shown. A red spot indicates the position of the bead.

(E) eGFP expression at 8-cell and 1,000-cell stages.

(F) eGFP expression in sphere, 30% and 50% epiboly Tg(ARE:eGFP) embryos. Arrow indicates expression in dorsal cells.

(G) Western blot for phosphorylated Smad2 (P-Smad2) in blastula stage embryos treated with or without SB-505124. Mcm6 is a loading control.

See also Figures S2–S4.