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Fig. 4
Pou5f3 and Nanog promote ventral fate.
(a) nanog MO-injected larvae show severely affected (Class (C) IV), less-severely affected (C III), mildly affected (C II) and least affected (C I) phenotypes. (b) Relative percentages of C I, C II, C III and C IV larvae according to dose of nanog MO injected (1.6 ng of nanog MO injection, n = 167; 0.8 ng of nanog MO injection, n = 186; 0.4 ng of nanog MO injection, n = 143). Co-injection of nanog MO (0.4 ng) with nanog* mRNA (0.1 ng) leads to over 80% wt-like larvae as opposed to 20% wt-like larvae in its absence (n = 126). (c-e) Embryos are at 50%-epiboly except where indicated. Embryos are in top views except lateral views for bmp2b-, oct4- and sox17-stained embryos. Dorsal is to the right-hand side. Markers were analysed following injection of 0.8 ng of nanog MO at the 1-cell stage. (c) chd expression in the dorsal margin is expanded ventrally in 30%-epiboly nanog morphants relative to wt embryos (86%, n = 40), and is uniformly expressed in the blastoderm of nanog morphants at 50%-epiboly relative to wt embryos (94%, n = 66). gsc expression in the prospective shield is expanded ventrally within the germ ring in nanog morphants relative to wt embryos at the early gastrula stage (71%, n = 47). bmp2b expression in the ventral ectoderm and organizer is markedly reduced in nanog morphants relative to wt embryos at mid-gastrulation (96%, n = 45). Expression of vox, a BMP target, is greatly diminished in nanog morphants relative to wt embryos (86%, n = 68). Expression of vent in the ventral margin is nearly absent in nanog morphants relative to wt embryos (95%, n = 56). At the early-gastrula stage, pou5f3 expression in the blastoderm is reduced in nanog morphants compared to wt embryos (97%, n = 44). (d) Effect of MZspg, nanog MO and MZspg/nanog MO on the expression of chd and vox. chd expression in the organizer of wt embryos (100%, n = 92) is ventrally expanded in MZspg embryos (98%, n = 75) and nanog morphants (90%, n = 97). In nanog MO-injected MZspg embryos, chd expression is further expanded in the entire blastoderm (92%, n = 62). vox expression in the ventral margin of wt embryos (100%, n = 96) is markedly reduced in MZspg embryos (97%, n = 60) and nanog morphants (88%, n = 84). In nanog MO-injected MZspg embryos, vox expression is completely lost (94%, n = 58). (e) Effect of oct4 mRNA in nanog MO, and nanog mRNA in MZspg mutant on chd expression. chd expression is ventrally expanded in MZspg embryos (98%, n = 75) relative to wt embryos (100%, n = 92). nanog mRNA cannot cause chd expansion when injected into wt embryos (93%, n = 65) and cannot rescue chd ventral expansion when injected into MZspg embryos (94%, n = 52). chd expression is ventrally expanded in nanog morphants (90%, n = 97) relative to wt embryos (100%, n = 92). Pou5f3 mRNA cannot cause chd expansion when injected into wt embryos (92%, n = 67) and cannot rescue chd ventral expansion caused by nanog depletion when co-injected with nanog MO (88%, n = 64). Data are from three to five independent experiments (n = 40-150). See also Figure 4-figure supplement 1.