Figure 4. nodal, lefty, bmp2/4, chordin, goosecoid, nk2.2, and fgfr1 are direct targets of Nodal while tbx2/3, nk2.2, and smad6 are direct targets of BMP2/4 signaling.

(A,B) Embryos at the late blastula stage were treated for two hours with recombinant Nodal or BMP4 protein in the presence or absence of puromycin. At the end of the treatment the embryos were fixed and the expression of the indicated genes was analyzed by in situ hybridization. In control experiments, DMSO or the translational inhibitor were added alone. (A) A short treatment with recombinant Nodal protein induced strong ectopic expression of early expressed genes such as nodal, lefty, bmp2/4, chordin, goosecoid, nk2.2 and fgfr1. This ectopic expression was still observed in the presence of protein synthesis inhibitor indicating that these genes are early targets of Nodal signaling. In contrast, this 2h treatment with Nodal did not induce ectopic expression of genes expressed later such as foxA and only induced a partial expansion of brachyury expression, an effect that disappeared in the presence of the protein synthesis inhibitor. These genes are therefore likely secondary targets of Nodal signaling. Note that in control embryos the width of the brachyury expression territory encompasses 8–9 cells (red bar) while in Nodal treated embryos the width of this stomodeal field increases to about 12–15 cells. In embryos treated with Nodal and the translation inhibitor, the width of the stomodeal field is similar to that in control embryos. (B) Similarly, BMP4 treatment induced massive ectopic expression of the early expressed genes tbx2/3, nk2.2 and smad6 even in the presence of a translational inhibitor but failed to induce ectopic expression of dorsal markers genes expressed later. tbx2/3, nk2.2 and smad6 are therefore likely direct targets of BMP2/4 signaling while the other dorsally expressed genes are likely indirect targets whose expression requires protein synthesis downstream of activation of the BMP receptors. Identical results were obtained by using emetine as translational inhibitor. lv, lateral view, vv, vegetal pole view fv, frontal view.