Nodal/Activin Pathway is a Conserved Neural Induction Signal in Chordates

Abstract

Neural induction is the process through which pluripotent cells are committed to a neural fate. This first step of Central Nervous System formation is triggered by the "Spemann organizer" in amphibians and by homologous embryonic regions in other vertebrates. Studies in classical vertebrate models have produced contrasting views about the molecular nature of neural inducers and no unifying scheme could be drawn. Moreover, how this process evolved in the chordate lineage remains an unresolved issue. In this work, by using graft and micromanipulation experiments, we definitively establish that the dorsal blastopore lip of the cephalochordate amphioxus is homologous to the vertebrate organizer and is able to trigger the formation of neural tissues in a host embryo. In addition, we demonstrate that Nodal/Activin is the main signal eliciting neural induction in amphioxus, and that it also functions as a bona fide neural inducer in the classical vertebrate model Xenopus. Altogether, our results allow us to propose that Nodal/Activin was a major player of neural induction in the ancestor of chordates. This study further reveals the diversity of neural inducers deployed during chordate evolution and advocates against a universally conserved molecular explanation for this process.

Figure 1. The dorsal blastoporal lip of amphioxus is homologous to the vertebrate organizer.

a, Graft experiments were undertaken by dissecting and inserting the blastopore lip of a G2 gastrula stage embryo in the ventral archenteron of a host embryo at the same developmental stage. Some embryos showed partial double axis as indicated using the neural marker Netrin. Other embryos formed a complete double axis as indicated by expression of the notochordal marker Brachyury and by immunostaining of axons for acetylated α-tubulin. The arrowhead indicates the position of the transverse section and the asterisks the ectopic expression of Netrin. b, Ectodermal explants were obtained through micro-dissection of the animal pole of G1 gastrula (gastrula explants, GE). Half of the ectodermal explants showed an inner cell mass expressing the mesoderm marker Brachyury. The external cell layer expressed the epidermal marker K1, whereas no expression of the neural marker Neurogenin was detected. c, Graft on GE of a fluorescently labelled (Texas Red) dorsal blastoporal lip induced the expression of Neurogenin exclusively in host cells. Green signal corresponds to auto-fluorescence. d, Negative control grafts of the mesendoderm part of the ventral blastopore lip onto GE did not induce Neurogenin expression. Anterior is to the left. Scale bar, 50 µm and 10 µm for section. Numbers correspond to embryos presenting similar labelling.

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.

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.

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.

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.

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.