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Comprehensive single-cell transcriptome lineages of a proto-vertebrate

Abstract

Ascidian embryos highlight the importance of cell lineages in animal development. As simple proto-vertebrates, they also provide insights into the evolutionary origins of cell types such as cranial placodes and neural crest cells. Here we have determined single-cell transcriptomes for more than 90,000 cells that span the entirety of development—from the onset of gastrulation to swimming tadpoles—in Ciona intestinalis. Owing to the small numbers of cells in ascidian embryos, this represents an average of over 12-fold coverage for every cell at every stage of development. We used single-cell transcriptome trajectories to construct virtual cell-lineage maps and provisional gene networks for 41 neural subtypes that comprise the larval nervous system. We summarize several applications of these datasets, including annotating the synaptome of swimming tadpoles and tracing the evolutionary origin of cell types such as the vertebrate telencephalon.

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Fig. 1: Overview of single-cell RNA-sequencing assays and cell-type specification at the onset of gastrulation.
Fig. 2: Transcriptome trajectories for defined individual neurons.
Fig. 3: Integration of transcriptome maps and synaptome neuronal circuits.
Fig. 4: Model for the evolution of the telencephalon.

Data availability

Raw sequencing data and the gene-expression matrix are available in the Gene Expression Omnibus (GEO) under accession number GSE131155. Our data can be explored at https://portals.broadinstitute.org/single_cell/study/SCP454/comprehensive-single-cell-transcriptome-lineages-of-a-proto-vertebrate. All other data are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank J. B. Wiggins, J. M. Miller and the Genomics Core Facility for technical support of 10x Chromium platform; IT and Bioinformatics staff at the Lewis-Sigler Institute for Integrative Genomics (LSI) for development of the sequence alignment pipeline; E. G. Gatzogiannis (director of the LSI Imaging Core Facility) for building the two-photon microscope and help with imaging; the Shvartsman laboratory in LSI for Imaris access; A. Sánchez Alvarado at SIMR for providing laboratory support and resources; and members of the Levine Laboratory for helpful discussions, and especially N. Treen for suggesting mNeonGreen as fluorescent reporter. This study was supported by a grant from the NIH (NS076542) to M.L. W.W. (SIMR) was funded by the Stowers Institute.

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Authors

Contributions

K.C. and M.L. conceived the project. K.C., M.L., C.C., L.A.L. and W.W. (SIMR) designed the experiments. L.A.L., P.H.Y., Y.A.C. and K.C. performed Ciona experiments, W.W. (SIMR) performed killifish experiments, C.C. performed computational data analysis. L.R.P., J.C.M. and W.W. (LSI) set up the single-cell RNA-sequencing pipeline. M.L. supervised the project. All authors contributed to interpretation of the results, and C.C., L.A.L., K.C. and M.L. wrote the manuscript.

Corresponding authors

Correspondence to Michael Levine or Kai Chen.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Data quality and biological replicates from mid-tailbud stage.

a, b, Distribution plot of reads numbers, UMIs, gene numbers, correlation coefficient (Spearman) and saturation level per cell from mid-tailbud (a, midTII_biorep1; b, midTII_biorep2). c, The first two principal components were plotted for cells regressed by UMIs (midTII_biorep1, n = 4,929 cells; midTII_biorep2, n = 4,062 cells). d, The first two principal components were plotted for cells regressed by both UMIs and batches. e, The first two canonical correlation vectors were plotted after alignment by canonical correlation analysis. fh, Merged (f) and split (g, h) t-SNE clustering for the biological replicates. i, t-SNE plot of canonical-correlation-analysis-aligned samples of biological replicates (n = 8,991 cells). The numbers indicate different clusters. j, The percentage of cells between replicates within the same cluster (clusters shown in i). k, Box plot of the percentage of cells in each cluster (n = 40 clusters) between replicates. The lower, middle and upper hinges correspond to the first and third quartiles (the 25th and 75th percentiles), and the middle hinge corresponds to the median.

Extended Data Fig. 2 t-SNE projections of ten stages from single-cell RNA-sequencing data.

a, t-SNE plot of the entire dataset (n = 90,579 cells). Cells are coloured and labelled by clusters. Differentially expressed genes in each cluster can be found in Supplementary Table 2. b, t-SNE plot of all of the cells, coloured according to tissue type. cl, t-SNE projections of cells, coloured by tissue types at different stages of development (iniG, n = 2,863 cells; midG, n = 3,384 cells; earN, n = 7,154 cells; latN, n = 8,449 cells; iniTI, n = 5,668 cells; earTI, n = 7,109 cells; midTII, n = 8,991 cells; latTI, n = 18,535 cells; latTII, n = 12,635 cells; and larva, n = 15,791 cells). The colour code is the same as in b. m, Violin plots illustrating expression levels of representative marker genes per cell per tissue type (endoderm, n = 14,162 cells; epidermis, n = 26,936 cells; germ cells, n = 396 cells; mesenchyme, n = 19,143 cells; muscle and heart, n = 3,691 cells; nervous system, n = 22,198 cells; and notochord, n = 4,053 cells). Colour code is the same as in b.

Extended Data Fig. 3 Specification of cell types at the onset of gastrulation.

The heat map shows the scaled expression of differentially expressed genes that encode transcription factors (red) and cell-signalling components (green). Many marker genes were newly identified for each tissue.

Extended Data Fig. 4 Reconstructed transcriptional trajectories of muscle, mesenchyme and endoderm.

a, t-SNE projection and expression patterns of representative marker genes of tail muscle, non-canonical muscle and heart (n = 3,691 cells). b, Reconstructed transcriptome trajectories and expression patterns of representative marker genes in muscle. c, t-SNE projection and expression patterns of representative marker genes shown on reconstructed transcriptome trajectories of mesenchyme (n = 19,143 cells). d, Cascade of representative transcription factors and signalling pathway genes along pseudotime in Tll1+ and Hlx+ mesenchyme. Mid-tailbud embryos that express Twist-like-2 (cyan), a mesenchymal marker, and Tll1 (red) reporter gene (top), and an Hlx (cyan) and Tll1 (red) reporter gene (bottom, n = 3 electroporation experiments). e, t-SNE projection and expression patterns of representative marker genes shown on seven reconstructed transcriptome trajectories of endoderm (n = 14,162 cells). Scale bars, 50 μm.

Extended Data Fig. 5 Transcriptome profiles of Ciona notochord cells during development.

a, t-SNE plot of notochord cells. Cells are coloured by developmental stage. The dashed line shows the separation between the primary (n = 3,123 cells) and secondary lineages (n = 627 cells). b, c, The single-cell transcriptome trajectory (top) and pseudotemporal gene-expression profiles (bottom) of the primary notochord and the secondary notochord. Cells were ordered along the trajectory across pseudotime. Only significantly expressed genes (likelihood ratio test) with q < 1 × 10−100 (primary notochord) and q < 1 × 10−20 (secondary notochord) are shown. Selected transcription factors and signalling molecules are labelled in orange. d, Heat map of differentially expressed genes between the primary and secondary notochord. Genes are clustered by Euclidean distance. e, Expression of a Casq1/2 fog>GFP reporter gene in a late-tailbud-stage embryo (left, one optical plane; right, maximum intensity projection). n = 3 electroporation experiments. GFP (green) was present in the muscle and in the secondary notochord (arrow), but no expression was observed in the primary notochord (arrowhead). f, Expression of KH.C9.405>mChCAAX reporter gene in late tailbud II stage embryo. mChCAAX (red) was present in the secondary notochord but not the primary notochord. n = 3 electroporation experiments. Scale bars, 20 μm.

Extended Data Fig. 6 Neural cells.

ac, Expression patterns of representative marker genes for the a- (a), b- (b) and A-lineages (c) are shown in reconstructed transcriptome trajectories of neural cells that span ten developmental stages. d, t-SNE plot of neural cells recovered from the larval stage (n = 1,704 cells). Identified cell types are labelled. e, Heat map of the top-five differentially expressed genes (not including those encoding transcription factors) for each type of neural cell in the larval stage.

Extended Data Fig. 7 The identification of rare neuronal subtypes in the larval stage.

a, Distribution of cells that express Galr2 in the t-SNE plot. Cells within the dashed circle show Galr2 expression in bipolar tail neurons (n = 26 cells). Reporter assay with a bipolar-tail-neuron minimal enhancer for Galr2 shows the specific activity of Galr2 in bipolar tail neurons (n = 3 electroporation experiments). b, Distribution of cells that express Dmbx in the t-SNE plot. Cells within the dashed circle show Dmbx expression in decussating neurons (n = 4 cells). The 5′ regulatory sequences of Dmbx are active in decussating neurons (red, n = 3 electroporation experiments). c, Distribution of cells that express NP in the t-SNE plot. Cells within the dashed circle show NP expression in VP+ posterior sensory vesicle (n = 11 cells). Reporter assay for NP (green) shows the specific expression of NP in neurons in the posterior sensory vesicle (n = 3 electroporation experiments). d, Distribution of cells that express Prop in the t-SNE plot. Cells within the dashed circle show Prop expression in Eminens neurons (n = 17 cells). Expression of the Prop reporter gene is specific to Eminens neurons (green) (n = 3 electroporation experiments). e, t-SNE plot of the larval nervous system showing cells that express Ptf1a (top) and VP (bottom). The dotted circle corresponds to coronet cells (top, n = 72 cells) and VP+ posterior sensory vesicle cluster (bottom, n = 11 cells). f, Expression of the reporter Ptf1a>mChCAAX (red) for coronet cells and NP>GFPCAAX (green) for VP+ posterior sensory vesicle shows that these cell populations do not contact each other, but are in close vicinity (top, n = 3 electroporation experiments; the GFP channel is shown in c). Expression of the reporter Prop>mChCAAX (red) for Eminens neurons and NP>GFPCAAX (green) for VP+ posterior sensory vesicle. NP+ cells are also in proximity to Eminens neurons (bottom, n = 2 electroporation experiments). Scale bars, 10 μm.

Extended Data Fig. 8 Expression of marker genes for Eminens neurons.

ah, Expression levels of eight marker genes in the larval nervous system, shown in t-SNE plots (left, n = 1,704 cells), and their corresponding reporter assays (mChCAAX for vGat and H2B-mCherry for the other genes, red) with a Prop>GFPCAAX reporter (green, right). n = 2 electroporation experiments for Gad, S39aa 2.2 kb, Znt3 and Asic1b; n = 3 electroporation experiments for vGat, Calm, Fgf13 and Galr2. The dashed circle in the t-SNE plots identifies Eminens neurons. Scale bars, 20 μm.

Extended Data Fig. 9 Manipulation of Eminens gene regulatory network.

a, Pseudotemporal expression profiles of regulatory genes and signalling components in Eminens neurons. b, Diagram of the Prop regulatory sequences with their length indicated on the left. A representative embryo is shown for the different fusion genes (GFPCAAX, green). The minimal Prop enhancer has weak expression in Eminens neurons (arrow). When the binding site for FoxH-a was mutated (260 bp FoxH-a mut), these regulatory sequences show even less activity. c, Bar plot of the percentage of the embryos that express GFP shown in b. Numbers on the right of the column correspond to the percentage of GFP+ embryos. χ2 test with four degrees of freedom was performed (P < 2.2 × 10−16), followed by two-sided Fisher’s exact test with Bonferroni adjustment for multiple comparisons. P values: 900 bp versus 700 bp, P = 1.05 × 10−7; 900 bp versus 300 bp, P = 3.47 × 10−13; 900 bp versus 260 bp, P = 2.36 × 10−4; 900 bp versus 260-bp FoxH-a mut, P = 1.81 × 10−19; 700 bp versus 300 bp, P = 0.011; 700 bp versus 260 bp, P = 0.36; 700 bp versus 260-bp FoxH-a mut, P = 0.088; 300 bp versus 260 bp, P = 5.59 × 10−6; 300 bp versus 260-bp FoxH-a mut, P = 0.69; 260 bp versus 260-bp FoxH-a mut, P = 1.27 × 10−7. Numbers of embryos: 900 bp, n = 207; 700 bp, n = 300; 300 bp, n = 160, all pooled over 2 electroporation experiments; 260 bp, n = 440, 260-bp FoxH-a mut, n = 750, all pooled over 3 electroporation experiments. d, Overexpression of Prop using Dmrt1 regulatory sequences causes supernumerary Prop+ cells (bottom panel) compared to control embryos expressing LacZ (top). The 2-kb Prop reporter gene shows specific expression in Eminens neurons (H2B–YFP, green). The images show representative embryos for both conditions. e, Quantification of Prop+ cells from the experiments in d. Dmrt1>LacZ, n = 269 embryos; Dmrt1>Prop, n = 210 embryos, pooled over 3 electroporation experiments. The orange dots indicate the mean and the bars indicate the s.d. Dmrt1>LacZ, 1.5 ± 1.4 cells; Dmrt1>Prop, 4.2 ± 4.8 cells. Mann–Whitney U-test, P = 3.65 × 10−9. Scale bars, 20 μm.

Extended Data Fig. 10 Pseudotemporal gene-expression cascade of the peripheral nervous system.

Representative transcription factors and signalling pathway genes along pseudotime in the reconstructed developmental trajectories of the peripheral nervous system are shown.

Extended Data Fig. 11 Pseudotemporal gene-expression cascade of the central nervous system of a-lineage.

Representative transcription factors and signalling pathway genes along pseudotime in reconstructed developmental trajectories of the central nervous system of a-lineage are shown.

Extended Data Fig. 12 Model for the evolution of the telencephalon.

a, Gene-expression cascade of regulatory genes and signalling components of palp sensory cells (also known as axial columnar cells). Genes implicated in the development of the vertebrate telencephalon are labelled in red. b, Gene-expression cascade of regulatory genes and signalling components in the anterior-most regions of the sensory vesicle (Six3/6+ pro-anterior sensory vesicle). Genes implicated in vertebrate telencephalon development are labelled in blue. c, d, The putative regulatory interactions among transcription factors from the cascade of palp sensory cells (c) and Six3/6+ pro-anterior sensory vesicle (d) along their developmental trajectories. e, The FoxG reporter gene with Ciona enhancer sequence exhibits restricted expression in a subset of cells in the olfactory bulb of the killifish telencephalon (arrowheads) and in the eye lens (left, GFP channel; right, merged image of bright-field and GFP channel images). n = 3 independent transgenic lines (Methods). D, diencephalon; M, midbrain; T, telencephalon. Scale bar, 400 μm.

Supplementary information

Supplementary Information

This file contains a guide to Supplementary Tables 1-7.

Reporting Summary

Supplementary Table 1

scRNA-Seq data summary – see Supplementary Information for full description.

Supplementary Table 2

Differentially expressed genes (DEGs) in different tissues and developmental stages – see Supplementary Information for full description.

Supplementary Table 3

The estimated binding score of Tbx6 binding motif on 2kbp DNA sequence upstream of TSS of muscle related genes expressed in the secondary notochord – see Supplementary Information for full description.

Supplementary Table 4

Numbers of recovered neural cells from different stages – see Supplementary Information for full description – see Supplementary Information for full description.

Supplementary Table 5

Neuropeptide, neurotransmitter receptor, transporter and neurotransmitters identified in neural cells in larva stage – see Supplementary Information for full description.

Supplementary Table 6

Gene name used in the manuscript and matched KH number – see Supplementary Information for full description.

Supplementary Table 7

Primer sequences used in molecular cloning – see Supplementary Information for full description.

Supplementary Video 1

Eminens neuron migration: Time-lapse video using two-photon microscopy of Eminens neuron migration (from late tailbud I to late tailbud III, n = 2 embryos). The embryo expressed H2B-mApple under the regulatory sequences of Dmrt1 (red) and PH-nG driven by Prop regulatory sequences (green). The Eminens cell migrates from the anterior part of the sensory vesicle toward its posterior side before axon starts to elongate. The number in the bottom left corner indicates the time in hours:minutes:seconds since fertilization. Scale bar 15um.

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Cao, C., Lemaire, L.A., Wang, W. et al. Comprehensive single-cell transcriptome lineages of a proto-vertebrate. Nature 571, 349–354 (2019). https://doi.org/10.1038/s41586-019-1385-y

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