The neural crest is an evolutionary novelty that fostered the emergence of vertebrate anatomical innovations such as the cranium and jaws1. During embryonic development, multipotent neural crest cells are specified at the lateral borders of the neural plate before delaminating, migrating and differentiating into various cell types. In invertebrate chordates (cephalochordates and tunicates), neural plate border cells express conserved factors such as Msx, Snail and Pax3/7 and generate melanin-containing pigment cells2,3,4, a derivative of the neural crest in vertebrates. However, invertebrate neural plate border cells have not been shown to generate homologues of other neural crest derivatives. Thus, proposed models of neural crest evolution postulate vertebrate-specific elaborations on an ancestral neural plate border program, through acquisition of migratory capabilities and the potential to generate several cell types5,6,7. Here we show that a particular neuronal cell type in the tadpole larva of the tunicate Ciona intestinalis, the bipolar tail neuron, shares a set of features with neural-crest-derived spinal ganglia neurons in vertebrates. Bipolar tail neuron precursors derive from caudal neural plate border cells, delaminate and migrate along the paraxial mesoderm on either side of the neural tube, eventually differentiating into afferent neurons that form synaptic contacts with both epidermal sensory cells and motor neurons. We propose that the neural plate borders of the chordate ancestor already produced migratory peripheral neurons and pigment cells, and that the neural crest evolved through the acquisition of a multipotent progenitor regulatory state upstream of multiple, pre-existing neural plate border cell differentiation programs.
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The authors would like to thank F. Razy-Krajka for assistance with Kaede photoconversion and comments on the manuscript, T. Tolkin for constructing the Mrf reporter plasmid, Z. Lu for ultramicrotomy, and C. Desplan, A. Di Gregorio and all members of the Christiaen and Meinertzhagen labs for feedback and suggestions. We thank H. Hashimoto, F. Robin and N. Takatori for embryo illustration template files. This work was funded by a National Science Foundation Postdoctoral Fellowship in Biology (under grant NSF-1161835) to A.S., by National Institutes of Health award GM096032 to L.C., and by grant DIS0000065 from NSERC (Ottawa) to I.A.M.
The authors declare no competing financial interests.
Extended data figures and tables
a, Immunolabelling for β-galactosidase (red) and in situ hybridization for Snail mRNA (green) in stage 12 embryo electroporated with Msx>lacZ, revealing Snail expression in the BTN progenitors (b9.36 cells, arrowheads). Dashed area enlarged in a′. b, Double in situ hybridization for Snail (green on merged image) and Msx (red on merged image) in stage 12 embryos counterstained with DAPI (blue on merged image), showing co-expression in neural plate border cells, including BTN progenitors. Scale bars, 25 μm.
a, Photoconversion of Kaede::nls driven by the Msx driver was used to follow the cell divisions of the BTN progenitors from the late gastrula stage to the early tailbud stage. Both b10.71 and b10.72 divide once. b11.141 will give rise to a definitive anterior BTN (see Extended Data Fig. 4). Numbers in each panel represent time in minutes elapsed from the initial photoconversion event. Scale bar, 50 μm. b, Lineage tree showing specification of aBTNs in relation to other cells of the posterior neural plate borders. For simplicity, only one side of the embryo is depicted. c, Lateral view of a 110-cell-stage embryo showing the positions of blastomeres in b. Red lines connect sibling cells. d, Dorsal view of a neurula-stage embryo showing zippering of posterior neural-plate-border-derived capstone cells18 as neural tube closure is initiated. Panels b and d are courtesy of H. Hashimoto and F. Robin (University of Chicago) and N. Takatori (Tokyo Metropolitan University), and partially modelled after ref. 17. Panel c modelled after ref. 49.
a, Schematic diagram representing Neurog locus and 5′ cis-regulatory sequences including b-line and b-line minimal cis-regulatory modules. Peaks represent nucleotide sequence conservation with Ciona savignyi genome. b, Late gastrula embryo (stage 13) electroporated with full-length Neurog (blue) and Nodal b-line (red) reporter constructs. Reporter co-expression is seen in b9.36 descendants on either side of the neural plate. Neurog expression also marks tail-tip lineages of uncertain provenance, previously reported to be descended from b8.21 (ref. 10). Scale bar, 25 μm. c, Neurog b-line reporter. d, Neurog b-line minimal reporter. Scale bars in c, d, 50 μm.
a, Lateral view of in situ hybridization (ISH) for Neurog (green) in embryo electroporated with Neurog b-line>H2B::mCherry (red) shows that Neurog expression is selectively maintained in only a subset of initially Neurog-expressing neural plate border cells. a′, In the b9.36 lineage, the anterior-most cell (b11.141, solid arrowhead) is always the sole one to express Neurog at this stage, and will go on to become the anterior BTN. Dashed arrowhead indicates b11.142, the sister cell of b11.141, which has downregulated Neurog relative to its sibling. b, b′, The identities of the cells in the tail tip (presumed b8.21-derived) lineages are unclear, but Neurog is similarly restricted (arrowheads) to a single cell on either side of the midline, which we interpret as the definitive posterior BTNs. c, Control embryo treated with DMSO vehicle, showing wild-type pattern of Neurog expression only in b11.141. d, Neurog is expanded to b11.142 upon treatment with the MEK inhibitor U0126 at 7 h.p.f. This condition also results in specification of supernumerary BTNs, presumably due to expanded Neurog expression (see text for details). Thus, downregulation of Neurog in b11.142 also requires MEK/ERK signalling. e, Diagram of the aBTN lineage, descended from the b8.18 blastomere. Scale bars in a, b, 25 μm. Scale bars in c, d, 10 μm.
Extended Data Figure 5 Perturbation of Notch signalling does not alter Neurogenin expression or bipolar tail neuron specification and differentiation.
a, Top, lateral view of a stage 23 embryo electroporated with Msx>H2B::mCherry (magenta nuclei), Neurog b-line>unc-76::eGFP (green) and Msx>nls::lacZ, serving as the wild-type control condition. Bottom, embryo electroporated with same reporters as upper panel, plus Msx>Su(H)-DBM, which encodes a DNA-binding mutant form of the Notch co-activator Rbpj. No discernable difference in Neurog activation or BTN specification was observed between control and Su(H)-DBM conditions (1 of 32 versus 2 of 42 embryos showing ectopic Neurog+ BTNs, respectively). b, Late overexpression of Su(H)-DBM using the Neurog b-line driver similarly did not alter BTN specification/differentiation, as monitored by Asic>unc-76::eGFP reporter expression (0 of 50 control versus 0 of 50 Su(H)-DBM embryos showed ectopic Asic+ BTNs). Scale bars, 50 μm.
a, Embryo at 11.5 h.p.f. (18 °C) with BTNs displaced from clonally related epidermal cells (epid.) labelled by UNC-76::VenusYFP (red), Galnt7ΔC::CFP (green), and H2B::mCherry (blue) driven by Neurog b-line cis-regulatory module. Targeted localization of CFP by the Galnt7 N-terminal signal sequence reveals polarized subcellular distribution of Golgi apparatus on posterior side of BTN nuclei as migration and proximal process extend in an anterior direction. This is distinct from the apical (dorsal) location of the Golgi apparatus in epidermal cells. b, Embryo at 12.5 h.p.f. (18 °C) showing 180° inversion of Golgi apparatus localization to the anterior side of the nucleus, immediately preceding distal process extension. Scale bars in a, b, 50 μm. c, Still frames from a confocal image stack time lapse movie (Supplementary Video 4) showing inversion of Golgi complex (Galnt7ΔC::VenusYFP, green) relative to nuclei (H2B::mCherry, red) in migrating BTNs. Time lapse imaging initiated at 11.5 h.p.f. (18 °C). Time in minutes elapsed from start shown at bottom right of each panel. Anterior BTN (aBTN) indicated by magenta arrowhead, posterior BTN (pBTN) indicated by white arrowhead. Scale bar, 25 μm. d, Diagram showing correlation of average length of proximal (left) and distal (right) processes and angle of Golgi apparatus location relative to cell nucleus along the anterior–posterior axis in BTNs at different time points. Locations of Golgi apparatus represented by rose plots of bins of 20° spanning anterior (0°) and posterior (180°) endpoints around dorsal edge of BTN nucleus. Bin diameters indicate number of cells. Embryos analysed belong to the same pool as embryos in a and b. See Supplementary Table 1 for source data.
Extended Data Figure 7 Proposed evolution of neural crest through the acquisition of multipotency by neural plate border cells.
a, Cartoon diagram depicting a hypothetical path for neural plate border and neural crest evolution, starting with the reconstructed last common olfactorean ancestor, which could have had neural plate borders lined with committed progenitor cells giving rise to several pigmented ocelli and BTN-like peripheral neurons, a condition that may be conserved in extant cephalochordates50. These cells would have been reduced in the highly miniaturized embryos of extant tunicates, while vertebrates are proposed to have co-opted a mesenchymal, multipotency program to bestow these cells with the potential to give rise to pigment cells, peripheral neurons or other derivatives, after a prolonged period of EMT and migration. b, Diagram representing idealized cell lineages in the neural plate borders of tunicate and hypothetical urolfactorean ancestor, in which segregated lineages at the neural plate borders give rise to committed pigment cell or peripheral neuronal progenitors. c, Diagram of simplified neural crest cell lineage deploying a multipotency program downstream of neural plate border specification and upstream of cell differentiation. Thus, neural crest cells could have evolved through redeployment of a multipotency program (intercalation hypothesis)1, or through its maintenance from earlier embryonic stages (heterochrony hypothesis)30.
This table contains Golgi apparatus repositioning and bipolar tail neuron process extension source data. It contains all measurements, when possible, of Golgi apparatus angle relative to nucleus (measure in degrees), and proximal and distal process lengths (measured in μm), for anterior (aBTN) and posterior (pBTN) bipolar tail neuron precursors on a single side (right or left, not indicated) of each embryo imaged. Embryos were grown to three different stages (11.5, 12.5, and 13.5 hours post-fertilization). See main text, methods, and Extended Data Figure 6 for more details. (XLSX 18 kb)
Time-lapse confocal imaging of embryo electroporated with Neurog b-line>unc-76::VenusYFP (green), imaged starting at 9.5 hours post-fertilization (at 18 °C). Confocal Z-stacks were acquired every 3 minutes for roughly 2 hours and 20 minutes. (MOV 336 kb)
Time-lapse confocal imaging of embryo electroporated with and Fog>H. sapiens CD4::eGFP (green), imaged starting at 11 hours, 21 minutes post-fertilization (at 18°C). Confocal Z-stacks were acquired every 2 minutes, for roughly 1 hour, 45 min. The bipolar tail neuron and related epidermis midline cells are labelled by Neurog b-line>unc-76::mCherry (red), while the cell membranes of the entire epidermis is labeled by Fog>H. sapiens CD4::eGFP. (MOV 423 kb)
A 3D confocal projection of embryo imaged in Figure 2c, showing a Neurog reporter-labeled bipolar tail neuron (magenta) migrating along Mrf reporter-labeled paraxial mesoderm-derived muscles (green). The embryo was electroporated only on the right side. Cell outlines are stained by Alexa Fluor 633 phalloidin. (MOV 2126 kb)
Time-lapse confocal imaging of embryo electroporated with Neurog b-line>H2B::mCherry (red) and Neurog b-line>Galnt7ΔC::VenusYFP (green), imaged starting at 11.5 hours post-fertilization (at 18°C). Confocal Z-stacks were acquired every minute for roughly 1 hour. (MOV 182 kb)
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Stolfi, A., Ryan, K., Meinertzhagen, I. et al. Migratory neuronal progenitors arise from the neural plate borders in tunicates. Nature 527, 371–374 (2015). https://doi.org/10.1038/nature15758
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