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Evolutionary origin of the chordate nervous system revealed by amphioxus developmental trajectories

Nature Ecology & Evolution volume 8, pages 1693–1710 (2024)Cite this article

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Abstract

The common ancestor of all vertebrates had a highly sophisticated nervous system, but questions remain about the evolution of vertebrate neural cell types. The amphioxus, a chordate that diverged before the origin of vertebrates, can inform vertebrate evolution. Here we develop and analyse a single-cell RNA-sequencing dataset from seven amphioxus embryo stages to understand chordate cell type evolution and to study vertebrate neural cell type origins. We identified many new amphioxus cell types, including homologues to the vertebrate hypothalamus and neurohypophysis, rooting the evolutionary origin of these structures. On the basis of ancestor–descendant reconstruction of cell trajectories of the amphioxus and other species, we inferred expression dynamics of transcription factor genes throughout embryogenesis and identified three ancient developmental routes forming chordate neurons. We characterized cell specification at the mechanistic level and generated mutant lines to examine the function of five key transcription factors involved in neural specification. Our results show three developmental origins for the vertebrate nervous system: an anterior FoxQ2-dependent mechanism that is deeply conserved in invertebrates, a less-conserved route leading to more posterior neurons in the vertebrate spinal cord and a mechanism for specifying neuromesoderm progenitors that is restricted to chordates. The evolution of neuromesoderm progenitors may have led to a dramatic shift in posterior neural and mesodermal cell fate decisions and the body elongation process in a stem chordate.

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Fig. 1: Deep scRNA-seq of amphioxus embryos.
Fig. 2: Single-cell resolution of neurula stage amphioxus neural cell types.
Fig. 3: Reconstruction of amphioxus neural trajectories.
Fig. 4: CNS neuron development across species.
Fig. 5: HyPTh and DiMes neurons in T1 stage Lhx3/4, Msxlx and FoxQ2a mutants.
Fig. 6: scRNA-seq and in situ characterization of Brachyury KO embryos.

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Data availability

Plasmids, amphioxus lines and polyclonal antibodies generated in this study are available with a completed materials transfer agreement. To facilitate further exploration of this dataset, we have uploaded the data to make it publicly available (https://lifeomics.shinyapps.io/single_cell_atla_app/). Users can visualize integration results for different samples on reduced dimensions and compare gene expression based on cell types annotated in this study. The data generated in this study can be downloaded in raw form from the Genome Sequence Archive hosted by the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences, and are publicly accessible at https://ngdc.cncb.ac.cn/gsa under accession number CRA010773. Processed data are available at the Science Data Bank94 at https://www.scidb.cn/en/list under DOI number https://doi.org/10.57760/sciencedb.08801.

Code availability

Publicly available datasets and common, freely available data analysis software packages used in this project are listed in Methods. Python and R codes are freely available at https://github.com/daiyc-zoo/amphioxus_singlecell.

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Acknowledgements

We are grateful to P. Holland, Y.-J. Luo, T. Lewin and K. Jindrich for advice and feedback on the preliminary dataset, and we thank J. Huang and M. Zhu for advice and recommendations for single-cell platforms and protocols. We thank F. Guo, Q. Zhang and Z. Zhang for feedback on data analysis methods, and we thank D. Chen for helping set up the interactive website for amphioxus single-cell data display. This research was supported by grants from the National Science Foundation of China under grant numbers 32100335 to Y.D.; 32070458, 32270439 and 32061160471 to G. Li; and 82050002 to X.Z. This study was also supported by the Office of China Postdoc Council 2021 Postdoctoral International Exchange Program, Talent-Introduction Program awarded to Y.D. under postdoc number 273565 and the Institute of Zoology Chinese Academy of Sciences Bingzhi Postdoctoral Fellowship Program awarded to Y.D.

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Authors and Affiliations

  1. State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China

    Yichen Dai, Yanhong Zhong, Rongrong Pan, Liang Yuan, Yongheng Fu, Yuwei Chen, Huimin Liu, Chenggang Shi & Guang Li

  2. CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Beijing, China

    Yichen Dai, Juan Du, Meng Li, Xiao Wang, Gaoming Liu, Pingfen Zhu & Xuming Zhou

  3. School of Life Sciences, Xinjiang Normal University, Urumqi, China

    Liang Yuan

  4. Department of Biology, University of Oxford, Oxford, UK

    Sebastian Shimeld

Authors
  1. Yichen Dai
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  2. Yanhong Zhong
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  3. Rongrong Pan
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  4. Liang Yuan
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  5. Yongheng Fu
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  7. Juan Du
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  9. Xiao Wang
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  10. Huimin Liu
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  11. Chenggang Shi
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  14. Sebastian Shimeld
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  15. Xuming Zhou
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  16. Guang Li
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Contributions

Conceptualization: Y.D. and G. Li. Sample collection: Y.D., Y.Z., H.L., R.P., H.L. and C.S. Experimental investigation: Y.D., Y.Z., H.L., R.P., Y.C. and J.D. Lhx3/4 and Msxlx gRNA design and relevant in situ analysis: Y.F. and R.P. Maintenance of Brachyury knockout animals: Y.Z. and L.Y. Maintenance of FoxQ2a knockout animals: R.P. Computational analysis: Y.D. Visualization: Y.D. Supervision: G. Li, X.Z., M.L., P.Z., G. Liu and X.W. Data interpretation: Y.D., G. Li, S.S. and X.Z. Writing—original draft: Y.D. Writing—review and editing: Y.D., G. Li, X.Z. and S.S.

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Correspondence to Sebastian Shimeld, Xuming Zhou or Guang Li.

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Nature Ecology & Evolution thanks Jacob Musser, Ulrich Technau and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Cell filtering and cell cluster identification.

(A) Doublet cells were identified according to described methods. T1 stage data from five replicate samples show that predicted doublet cells are likely to cluster together on the global embedding. (B) Cells with more than 20% reads mapping to the same gene were filtered prior to clustering. T1 stage data show that cells with a high percentage of reads mapped to one gene also have a low total gene count in each cell, suggesting that these cells are likely low-quality. (C) Cells with less than 750 genes identified tend to cluster together on the global embedding. These cells were also removed prior to downstream analysis. (D) All five repeat samples for the T1 stage are shown here, with the percentage of cells from each replicate in each T1 stage cell type depicted in different colors. (E) Developmental timeline indicating stages in this dataset and the Ma et al. dataset16 in different colors. The reference time scale uses timepoints for sample collection recorded for this study. Exact time used for sample collection was not reported in the Ma et al. dataset. However, all terminology used to describe specific developmental stages conform to staging standards in a previous paper81, and we deduced time points for sample collection in the Ma et al. dataset by experience in our lab. Stages under which the embryo is undergoing gastrulation are highlighted in pink. (F) Difference between median gene number detected per cell and cell number per stage between our dataset and the Ma et al. dataset16. Each dot represents one biological sample. The developmental stage that each sample corresponds to is shown on the horizontal axis. B: blastula; G0: early gastrula; G3: early/mid-gastrula (cap-shaped); G4: mid-gastrula (cup-shaped); G5: mid-/late gastrula (vase-shaped); G6: late gastrula (bottle-shaped); N0: early neurula (unsegmented); N1: early neurula (three pairs of somites); N2: early/mid-neurula (four to five somite pairs); N3: early/mid-neurula (six to seven pairs of somites); N4: mid-neurula (eight to nine somite pairs); T1: late neurula (appearance of first pigment spot); L0: early larvae (mouth open).

Extended Data Fig. 2 Cross-species comparison of neural cell types.

(A) Developmental timeline for four chordate species, mouse, zebrafish, tunicate, and amphioxus, showing all scRNA-seq samples used for SAMap analysis. Developmental stage for each sample with corresponding time after fertilization are shown in parallel, with whole-embryo datasets shown in red and vertebrate anterior tissue and hypothalamus tissue-only datasets shown in blue. Gastrula stages for each species are highlighted in pink. (B) Global UMAP of all neural cells from each of the four chordate species projected into the same 2D space in a pairwise manner. Species identity is indicated by color. (C) Homology of pigment cells, motor neurons, and glia-like neurons amongst four chordate species. Cell-cell homologies with mapping scores higher than the three thresholds are displayed, and names of cell clusters of interest are shown. The width of links between cell clusters correspond to mapping score value, with wider links representing higher mapping scores in the same circular layout. (D) Expression of orthologous genes in tunicate and amphioxus scRNA-seq datasets. The orthologous genes displayed are enriched in both tunicate Arx+ pro-aSV neurons and amphioxus HyPTh neurohypophysis-like neurons (T1: 42, 82, 92). (E) Expression of orthologous genes enriched in tunicate coronet cells and amphioxus HyPTh dopamine neurons (T1: 83).

Extended Data Fig. 3 Reconstruction of amphioxus embryogenesis cellular trajectories using transcriptome similarity and scVelo.

(A) Inferred relationships between cell clusters across amphioxus development predicted using transcriptome similarity. Colored rectangles represent “nodes” that correspond to one of the cell cluster annotations defined in Supplementary Table 2. All nodes are linked to at least one line (“edge”) with edge weight above 0.2. Developmental stages were treated as distinct time points, and the stage that each node belongs to is shown at the bottom of the figure, with nodes from the same stage aligned vertically. Node color corresponds to germ layer ancestry in Supplementary Table 2. All edges with weights above 0.2 are shown in color scale. Nodes containing an asterisk (“*”) are neuromesoderm cell clusters while those containing a hash (“#”) are prechordal plate-like cell clusters that co-express endoderm and axial mesoderm marker genes. We assigned an artificial “fertilized egg” cell cluster linked to B stage cell clusters in dotted lines to ensure graph connectivity. (B - D) scVelo results for ectoderm, mesoderm, and endoderm-origin cell types across four developmental stages (N0, N2, N4, and T1), respectively. Key cell cluster identities are shown in the left panel, while the corresponding stages are shown in the right panel. HyPTh, Hypothalamo-Prethalamic primordium; DiMes, Di-Mesencephalic primordium; RhSp, Rhombencephalo-Spinal primordium; CNS, central nervous system; PNS, peripheral nervous system; PGC, primordial germ cell.

Extended Data Fig. 4 Cross-species comparison across all six species.

Similarity of gastrula (G0, G4, N0 stage) amphioxus predicted neural precursors to cell clusters in other species. Cell-cell similarities with mapping scores higher than the three thresholds are displayed, and the name of each cell cluster assigned the highest mapping score in each species is shown. The width of links between cell clusters corresponds to mapping score value, with wider links representing higher mapping scores in the same circular layout. A: anterior/apical; P: posterior/body.

Extended Data Fig. 5 Cross-species comparison of TFs enriched in predicted anterior and posterior neuron precursors.

Expression pattern of several TFs of interest across all six species. Expression of each ortholog in each species is min-max normalized, with its highest expression value in the stages and cell clusters shown here assigned a value of “1” and its lowest value assigned “0”. Genes with empty boxes (for example FoxQ2 in mouse) represent cases where the ortholog is missing or not expressed in any of the cells analyzed in that species. Gene IDs corresponding to each ortholog shown in this figure are listed in Supplementary Table 6. Several early-mid gastrula and mid-late gastrula stages for mouse and sea urchin are omitted in this figure, and for the full expression pattern please refer to Supplementary Fig. 23.

Extended Data Fig. 6 Brachyury double knock-out embryos.

(A) SoxB1c and Bra2 protein location in N2 stage wild type embryos. The same embryo is shown in lateral and dorsal viewpoints with focus on the posterior tailbud. Overlap of SoxB1c (green) and Bra2 (red) is present in the tailbud region (arrow). Scale bar = 20 µm. (B) Expression of SoxB1c and Bra2 in N2 and N4 stage scRNA-seq data in global 2D UMAP. Cells are colored by either SoxB1c or Bra2 expression. Cells with normalized SoxB1c expression above 1 and Bra2 expression above 2 are highlighted in red in the same 2D UMAP. (C, C’) Brachyury protein presence in N0 and N2 stage Brachyury control and KO embryos. Location of Brachyury protein is indicated in red, while DAPI staining is in blue. Wild type Bra2 protein expression is detected in control embryos (C) while Bra2 is not detectable in Brachyury double knockout embryos of the same developmental stage (C’). Both lateral and dorsal view are shown for the same embryo. Scale bar = 20 µm. (D, D’) N2 stage Brachyury control and mutant embryos. Somites are distinguishable in Brachyury control embryos (D). Brachyury double mutants (D’) lack clear somite boundaries and have an enlarged area around the anterior somites. Embryos are shown in lateral and dorsal views. Scale bar = 50 µm. (E) Differences in cell abundance between Brachyury control and double knockout embryos. Cell clusters with significantly decreased abundance in knockout embryos are shown in red, and those with significantly increased abundance in knockout embryos are in blue. (F) Number of differentially expressed genes in all mutant embryo cell clusters compared to control. Genes up-regulated in mutant embryos are shown in red while down-regulated genes are in green. (G) Differential expression of genes in all non-anterior neural ectoderm, neuromesoderm, tailbud mesoderm, and notochord mesoderm cell clusters in Brachyury double knockout embryos compared to control. Genes down-regulated in knockout embryos are shown in green on the left of each panel, and up-regulated genes are shown in red on the right side. Differentially expressed genes shown in Fig. 6c are highlighted.

Extended Data Fig. 7 Summary of key findings.

(A) In T1 stage amphioxus embryos, the anterior-most and posterior-most tips of the CNS (highlighted in photo) follow different developmental trajectories that can be traced back to early gastrula (G0 stage). In early gastrula, TF gene FoxQ2a and Bra2 are key for establishing identity of predicted anterior and posterior neural precursors, respectively. Scale bar = 50 µm. (B) In T1 stage amphioxus embryos, the previously described “hypothalamus-like” region67 is composed of three cell groups including hypothalamus-like neurons (T1: 90, red), neurohypophysis-like neurons (T1: 42, 82, 92, blue), and dopamine neurons (T1: 83, yellow). The amphioxus posterior CNS is made up of a group of neuromesoderm cells (T1: 7, 66, 69, green) first reported in this study. Scale bar = 50 µm. (C) Comparisons between cnidarian, ambulacrarian, and chordate animals at single-cell level provide support for the dual evolutionary origin of the vertebrate CNS46. Anterior marker FoxQ2 expression is conserved in invertebrates only. Chordate-specific cell types include neuromesoderm cells and hypothalamus or hypothalamus-like neurons. A: anterior; P: posterior.

Supplementary information

Supplementary information

Supplementary text and table legends, and Supplementary Figs. 1–25.

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Supplementary Data 1

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Dai, Y., Zhong, Y., Pan, R. et al. Evolutionary origin of the chordate nervous system revealed by amphioxus developmental trajectories. Nat Ecol Evol 8, 1693–1710 (2024). https://doi.org/10.1038/s41559-024-02469-7

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