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. 2015 Oct 1;6:8434. doi: 10.1038/ncomms9434

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(A) Phospho-Smad1/5/8 immunostaining at the mesenchyme blastula stage of control embryos (a) or nodal morphants (b). Strong nuclear staining is observed in a D/V gradient in one half of the control embryo, both in the ectoderm and in skeletogenic mesenchyme cells (a). Most of the signal disappears in the nodal morphants (b). (B) Phospho-Smad1/5/8 immunostaining of nodal morphants rescued by single injection of alk4/5/7QD mRNA at the 4-cell stage (c–g) and nodal morphants rescued by double injection of alk4/5/7QD mRNA (h-l). (c,h). The nodal morpholino was injected together with RLDX and the alk4/5/7QD mRNA with FLDX. Both tracers can be seen in the fully rescued embryo in (c). Single injection of alk4/5/7QD mRNA rescues psmad1/5/8 staining in nodal morphants on the opposite side of the injection clone (c–g) while double injection of alk4/5/7 mRNA results in nuclear pSmad1/5/8 staining in two groups of cells located in lateral regions (h–l). Note that, unlike rescued embryos displaying high pSmad 1/5/8 signalling opposite to the ventral side (b,c), the highest intensity of pSmad1/5/8 staining is found in the midline separating the two organizers. Embryos in c, d, h and i are confocal stacks acquired with the exact same parameters. Embryos in e–g and j–l are maximum projections of confocal stacks acquired with the exact same parameters. Embryos in g and l are the same as in f and I but using the Fiji ‘Thermal' lookup table. (C) Accumulation of pSmad1/5/8 in the lateral regions of the doubly injected embryos requires Chordin function. Chordin morphants doubly injected with alk4/5/7QD do not show the strong bilateral pSmad staining but show instead a uniform and low staining. (D) Scheme summarizing the pattern of pSmad1/5/8 of wild type embryos (a) or of embryos doubly injected with Alk4/5/7QD (b). Both local repression of BMP signalling ventrally by Chordin and active shuttling of BMP2/4 from the ventral side to the dorsal side shape the steep gradient of pSmad1/5/8. Note that this configuration is partially similar to that of a Drosophila embryo in which Chordin produced in two ventrolateral territories shuttles Dpp to the dorsal midline. Scale bar, 10 μm.