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Figure 2. Role of BMP in ectodermal cell fate commitment.a, Expression of Nodal, Vg1, Zic, EvxA, SoxB1a, K1, Neurogenin and Hu/Elav in G1, G4 and N2 stage control embryos and in dorsomorphin-treated N2 stage embryos. The white arrow head indicates the early ectodermal Neurogenin expression domain in control embryos. b, Schematic partial representation of the results presented in (a). c, Expression of K1 and Neurogenin at the N2 stage in control embryos and embryos treated with both dorsomorphin and zBMP4. All in situ hybridization images are side views with anterior towards the left. Scale bar, 50 µm.
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Figure 3. Role of Nodal/Activin and FGF signalling pathways in ectoderm specification. Expression at G4 and N2 stages of Neurogenin, Hu/Elav, K1 and Otx in control embryos, in embryos treated with recombinant Activin protein, and in embryos treated with both Activin and SU5402. All in situ hybridization images are side views with anterior towards the left. Enlargements of the anterior region (white frame) of N2 stage embryos are presented below the pictures of whole embryos to highlight Otx expression in the ectoderm. Scale bar, 50 µm.
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Figure 4. Nodal/Activin is the main signal triggering neural induction.a, Expression of K1 and Neurogenin in control, dorsomorphin-treated, Activin-treated, FGF1/2-treated or Activin+FGF1/2-treated GE and BE. b, Expression of Neurogenin in FGF1/2+SB505124-treated and Activin+SU5402-treated GE and BE. c, Neurogenin expression induction by the graft of the dorsal blastoporal lip on GE is lost after SB505124 treatment in most cases, but maintained after SU5402 treatment. Number of explants showing the presented expression pattern is indicated on each panel. Scale bar, 50 µm.
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Figure 5. Nodal induces neural tissue in Xenopus.
a, Expression of gsc, Xbra and Xnot-2 at early neurula stage 13 in control embryos and in embryos injected with Nodal recombinant protein at stage 10 or 11. Dorsal views for gsc and Xnot-2, ventral views for Xbra.
b, Expression of sox2 and foxD5 (left: front view; right: dorsal view) and N-tubulin (ectopic neurons indicated by a white asterisk) in control and Nodal-injected embryos.
c, Embryos were injected with GR-t-Smad2 mRNA, animal caps were explanted at early gastrula stage, induced or not with dexamethasone at stage 11 and processed for sox2 in situ hybridization.
d, Expression of sox2 and k81 in control embryos and in embryos injected with zBMP4, Nodal or both recombinant proteins. Scale bar, 250 µm.
e, Confocal images of pSmad1 immunostaining and nuclear DAPI staining in control embryos and in embryos injected with zBMP4, Nodal or both recombinant proteins. Scale bar, 50 µm.
f, in situ hybridization of sox2 and foxD5 in animal caps treated with cycloheximide, Activin, or both. Scale bars, 200 µm. The number of embryos showing the phenotype displayed over the total number of embryos analyzed is indicated on each panel.
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Figure 6. Activin/Nodal signaling is required within the ectoderm for neural induction in Xenopus.
a, Expression of chd, sox2 and foxD5 at stage 10 in control embryos and in embryos injected with dnAlk4 mRNA in dorsal animal cells. Vegetal view for chd and dorsal view for sox2 and foxD5.
b, Expression of chd at stage 13 and N-tubulin at stage 15 and 25 in control embryos and in embryos injected with dnAlk4 mRNA in dorsal animal cells. Dorsal view for chd and N-tubulin at stage 15 and stage 25 (left) and lateral view for N-tubulin at stage 25 (right).
c, Expression of sox2 at stage 10 in control embryos and in embryos injected with dnAlk4 mRNA in dorsal animal cells. Noggin recombinant protein was injected in the blastocoele at stage 8 to induce neural tissue. Embryos are shown in dorsal view. In all cases, embryos were injected with fixable fluorescein lysine dextran and revealed by immunostaining (orange). The number of embryos showing the phenotype displayed over the total number of embryos analyzed is indicated on each panel. Scale bars, 250µm.
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Supplementary Figure 1. K1 expression pattern. In situ hybridization of K1 at blastula (a), G1 (b), G4 (c), G6 (d-e) and N1 (f) stages. e, cross section of (d) at the level of the arrow. Whole mount in situ hybridization pictures are all side views with anterior to the left and dorsal to the top. Scale bars, 50 µm. Expression of K1 starts at the G4 stage and is restricted to
the future epidermis.
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Supplementary Figure 2. SoxB1a expression pattern. In situ hybridization at different developmental stages. (a), (b), (d), (f) and (h) are side views. (c) is a blastoporal view of the embryo shown in (b). (e) and (g) are dorsal views of the embryos shown in (d) and (f), respectively. Scale bar, 50µm.
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Supplementary Figure 3. Interaction between Nodal/Activin and BMP pathways.
a, Immunostaining against pSmad1/5/8 and nuclear DAPI staining in control, dorsomorphintreated and Activin-treated N2 stage amphioxus embryos.
c, Expression of Neurogenin at
the G4 and N2 stages in control embryos and after zBMP4 and Activin + zBMP4 treatments,
and at the N4 stage in control embryos and embryos treated with both Activin and
dorsomorphin. In situ hybridization images are side views with anterior towards the left. Scale
bar, 50 µm.
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Supplementary Figure 3. Interaction between Nodal/Activin and BMP pathways.
b, Heat map of log2 fold changes
for selected genes that are up- (red) or down-regulated (green) in whole embryos at the G4
and N2 stages after Activin and dorsomorphin treatments. For both treatments the expression
of genes orthologous to classical Nodal and BMP targets in vertebrates was up and downregulated,
respectively, confirming that BMP and Nodal are globally opposing signals.
Concerning ectoderm cell fate commitment, the expression of epidermal genes was downregulated
after both treatments whereas neural genes were overexpressed only in Activintreated
embryos. In dorsomorphin-treated embryos the expression of pan-ectoderm genes,
such as SoxB1a, POU2 or Dll, was up-regulated. Expression of dorsal mesendoderm genes
was up-regulated mainly in dorsomorphin-treated embryos.
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Supplementary Figure 4. Fate of animal and vegetal blastomeres at the 8-cell stage.
Schematic representation of the four labeled animal cells (micromeres) or vegetal cells
(macromeres) at the 8-cell stage. Cells were labeled by contact with DiI (Invitrogen). Green
auto-fluorescence of the labeled embryo at the G2 stage is shown. Red fluorescence is
observed in the ectoderm in embryos in which animal cells were labeled (a) whereas red fluorescence is observed in the mesendoderm when vegetal cells were labeled (b). Scale bar,
50 µm.
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Supplementary Figure 5. Brachyury expression in explants. Brachyury expression in FGF1/2 (a, d), Activin (b, e) and Activin-FGF1/2 (c, f) treated GE and BE, respectively. In GE or BE that show inner cell mass, these cells are expressing Brachyury, in wild-type as in treated explants. Scale bar, 50 µm.
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Supplementary Figure 6. Timing of Nodal/Activin signal requirement for neural
induction. a, Expression of Neurogenin at the N2 stage in embryos after continuous treatment with Activin or SB505124 from 3 hpf, 4 hpf, 5 hpf or 7 hpf at 19°C. b, Expression of Neurogenin in G4 and N2 stage embryos after treatment with Activin or SB505124 between 3 hpf and 4 hpf or between 3 hpf and 6 hpf at 19°C. c, Expression of Neurogenin in G4 and N2 control embryos. d, Drawing of amphioxus embryos at the stages at which treatments were performed, adapted from Conklin. When two pictures are presented, the left one is side view with anterior to the left and dorsal to the top whereas the right picture is dorsal view with anterior to the left. Blue arrows correspond to the period of treatment. Our results show that inhibiting the Nodal/Activin pathway via the application of SB505124 at any time between cleavage stage and G1 stage impeded the formation of Neurogenin positive neural tissue (a, cd). Moreover, one-hour treatment between cleavage stage and morula stage was also sufficient to preclude neural tissue formation (b, c-d). Conversely, treatment with Activin between cleavage stage and the beginning of gastrulation (G0-G1 stage) induced neural commitment in the whole ectoderm at G4, although neural fate was lost in the anterior region at N2 (b, c-d). In contrast, activating the Nodal/Activin pathway after the morula stage was not sufficient to neuralize all ectodermal cells (a, c-d). Altogether these results suggest that Nodal/Activin signal is required very early for neural induction to occur, and that the maintenance of the neural fate in the anterior region is also dependent upon this signal.
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Supplementary Figure 7. Nodal/Activin induces neural tissue in Xenopus animal cap
assays. a, RT-qPCR from animal caps treated from stage 11 to 15 with Nodal. Bars represent SEM of 5 independent experiments, represents undetectable (background) gene expression levels. Statistical analysis has been performed using Paired t-test, * p<0.05; ** p<0.005. b, RT-qPCR from animal caps injected with GR-t-Smad2, explanted at early gastrula stage and induced or not with dexamethasone at stage 10 or 11. Uninjected animal caps were treated with ethanol and served as reference. Bars represent SEM of 4 independent experiments, except for chd and otx2 for which they represent 2 independent experiments. Statistical analysis has been performed using Two-way ANOVA followed by Bonferroni post-test; *
p<0.05; ** p<0.005; ***p<0.001. c, RT-qPCR analyses of sox2 and foxD5 expression in
animal caps treated with cycloheximide, Activin, or both. Error bars indicate SEM.
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Supplementary Figure 8. Activin/Nodal signaling is required for proper neural tissue formation in Xenopus. a, Expression of foxD5 at stage 13, N-tubulin at stage 15 and sox2 at stage 13 (left) and stage 25 (right) in control embryos and in embryos treated with 200µM SB505124 from midgastrula stage 11. Note the decreased intensity of neural marker gene expression in SB505124-treated embryos. Dorsal view for foxD5, N-tubulin and sox2 at stage 13 and stage 25 (right) and lateral view for sox2 at stage 25 (left). b, Expression of chd, myoD and sox2 at early neurula stage 14 in control embryos and in embryos treated with 800µM SB431542 from stage 11. Note the reduced neural plate in SB431542-treated embryos despite normal axial and paraxial mesoderm formation. Dorsal view for chd, myoD and sox2 (left), and anterior view for sox2 (right). Scale bar, 200µm.
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sox2 (SRY box 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
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sox2 (SRY box 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, dorsal view, anterior left.
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