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Single-cell and spatial atlases of spinal cord injury in the Tabulae Paralytica

Nature volume 631, pages 150–163 (2024)Cite this article

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Abstract

Here, we introduce the Tabulae Paralytica—a compilation of four atlases of spinal cord injury (SCI) comprising a single-nucleus transcriptome atlas of half a million cells, a multiome atlas pairing transcriptomic and epigenomic measurements within the same nuclei, and two spatial transcriptomic atlases of the injured spinal cord spanning four spatial and temporal dimensions. We integrated these atlases into a common framework to dissect the molecular logic that governs the responses to injury within the spinal cord1. The Tabulae Paralytica uncovered new biological principles that dictate the consequences of SCI, including conserved and divergent neuronal responses to injury; the priming of specific neuronal subpopulations to upregulate circuit-reorganizing programs after injury; an inverse relationship between neuronal stress responses and the activation of circuit reorganization programs; the necessity of re-establishing a tripartite neuroprotective barrier between immune-privileged and extra-neural environments after SCI and a failure to form this barrier in old mice. We leveraged the Tabulae Paralytica to develop a rejuvenative gene therapy that re-established this tripartite barrier, and restored the natural recovery of walking after paralysis in old mice. The Tabulae Paralytica provides a window into the pathobiology of SCI, while establishing a framework for integrating multimodal, genome-scale measurements in four dimensions to study biology and medicine.

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Fig. 1: Overview of the Tabulae Paralytica and the snRNA-seq atlas.
Fig. 2: Cell types and subtypes of the uninjured and injured mouse spinal cord.
Fig. 3: Biological principles governing the response to SCI.
Fig. 4: Failure to re-establish a tripartite neuroprotective barrier in old mice.
Fig. 5: A multiomic atlas of SCI.
Fig. 6: A spatial transcriptomic atlas of SCI.
Fig. 7: A 4D spatiotemporal atlas of SCI.
Fig. 8: A rejuvenative gene therapy re-establishes the tripartite barrier to restore walking.

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Data availability

Sequencing data have been deposited to the Gene Expression Omnibus (GSE234774, snRNA-seq and spatial transcriptomics, and GSE230765, multiome). Source data are provided with this paper.

Code availability

Augur, Libra and Magellan are available from GitHub (https://github.com/neurorestore/Augur, https://github.com/neurorestore/Libra and https://github.com/neurorestore/Magellan).

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Acknowledgements

This work was supported by the Swiss National Science Foundation (grant nos. 310030_192558 to G.C., PZ00P3_185728 to M.A.A. and PZ00P3_208988 to J.W.S.); the ALARME Foundation (to M.A.A. and G.C.); the Dr Miriam and Sheldon G. Adelson Medical Foundation (to M.V.S.); Wings for Life (to M.V.S. and M.A.S.); the Wyss Center for Bio and Neuroengineering (to M.A.A. and G.C.); and a Human Frontiers in Science Program long-term fellowship (grant no. LT001278/2017-L to C.K.). We are grateful to J. Ravier and F. Merlos for the illustrations; the Advanced Lightsheet Imaging Center (ALICe) at the Wyss Center for Bio and Neuroengineering, Geneva and E. Bradbury for providing ChABC vectors. This work was supported in part using the resources and services of the Gene Expression Core Facility and the Bertarelli Platform for Gene Therapy at the School of Life Sciences of the EPFL. We acknowledge the foundational work by the many investigators whose work we could not cite due to space limits.

Author information

Author notes

  1. These authors contributed equally: Michael A. Skinnider, Matthieu Gautier

  2. These authors jointly supervised this work: Mark A. Anderson, Jordan W. Squair, Grégoire Courtine

Authors and Affiliations

  1. NeuroX Institute, School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland

    Michael A. Skinnider, Matthieu Gautier, Alan Yue Yang Teo, Claudia Kathe, Thomas H. Hutson, Achilleas Laskaratos, Alexandra de Coucy, Nicola Regazzi, Viviana Aureli, Nicholas D. James, Bernard Schneider, Quentin Barraud, Jocelyne Bloch, Mark A. Anderson, Jordan W. Squair & Grégoire Courtine

  2. Defitech Center for Interventional Neurotherapies (.NeuroRestore), CHUV/UNIL/EPFL, Lausanne, Switzerland

    Michael A. Skinnider, Matthieu Gautier, Alan Yue Yang Teo, Claudia Kathe, Thomas H. Hutson, Achilleas Laskaratos, Alexandra de Coucy, Nicola Regazzi, Viviana Aureli, Nicholas D. James, Quentin Barraud, Jocelyne Bloch, Mark A. Anderson, Jordan W. Squair & Grégoire Courtine

  3. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Michael A. Skinnider

  4. Ludwig Institute for Cancer Research, Princeton University, Princeton, NJ, USA

    Michael A. Skinnider

  5. Wyss Center for Bio and Neuroengineering, Geneva, Switzerland

    Thomas H. Hutson & Mark A. Anderson

  6. Department of Neurosurgery, Lausanne University Hospital (CHUV) and University of Lausanne (UNIL), Lausanne, Switzerland

    Viviana Aureli, Jocelyne Bloch, Jordan W. Squair & Grégoire Courtine

  7. Department of Clinical Neuroscience, Lausanne University Hospital (CHUV) and University of Lausanne (UNIL), Lausanne, Switzerland

    Viviana Aureli, Jocelyne Bloch, Mark A. Anderson & Grégoire Courtine

  8. Bertarelli Platform for Gene Therapy, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland

    Bernard Schneider

  9. Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA

    Michael V. Sofroniew

Authors
  1. Michael A. Skinnider
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  2. Matthieu Gautier
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  3. Alan Yue Yang Teo
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  4. Claudia Kathe
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  5. Thomas H. Hutson
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  6. Achilleas Laskaratos
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  7. Alexandra de Coucy
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  8. Nicola Regazzi
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  9. Viviana Aureli
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  10. Nicholas D. James
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  11. Bernard Schneider
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  12. Michael V. Sofroniew
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  13. Quentin Barraud
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  14. Jocelyne Bloch
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  15. Mark A. Anderson
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  16. Jordan W. Squair
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  17. Grégoire Courtine
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Contributions

J.W.S., J.B., M.V.S., M.A.A., M.A.S. and G.C. conceived and designed experiments. J.W.S., M.A.A., C.K., M.G., T.H.H., A.L., A.d.C., N.R., V.A. and N.D.J. conducted experiments. J.W.S., M.A.S., M.G., Q.B. and A.Y.Y.T. analysed the data. B.S. contributed essential resources. J.W.S., M.A.S., M.A.A. and G.C. wrote the manuscript. All authors contributed to the editing of the manuscript.

Corresponding authors

Correspondence to Mark A. Anderson, Jordan W. Squair or Grégoire Courtine.

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The authors declare no competing interests.

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Nature thanks Binhai Zheng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Clustering tree of 180 cell types and subtypes in the snRNA-seq atlas.

a, Clustering tree of the mouse spinal cord, revealing the hierarchical relationships between spinal cord cell types across levels 1 to 3 in the taxonomy, with cell types at level 3 highlighted. Text at the top of the tree shows the clades of the clustering tree corresponding to the major cell types of the mouse spinal cord (i.e., level 1 in the taxonomy). b, As in a, but showing level 4 in the taxonomy. c, As in a, but showing level 5 in the taxonomy.

Extended Data Fig. 2 Neuronal susceptibility and resilience to SCI.

a, Boxplot highlighting the proportion of neurons within individual libraries from the severity experiment, as compared to other major cell types. b, Boxplot showing the log2-odds ratio comparing the proportions of neurons from each level 4 subtype between the uninjured spinal cord, for all comparisons involving the injured spinal cord at 7 days post-injury. Cerebrospinal fluid-contacting neurons are the lone subpopulation to exhibit statistically significant resilience following SCI. **, p < 0.01; ***, p < 0.001. c, Scatterplot highlighting an individual comparison from b, showing the proportions of neurons from each level 4 subtype in the uninjured spinal cord, x-axis, and 7 days after a complete injury, y-axis. Color shows the log2-odds ratio. Cerebrospinal fluid-contacting neurons are highlighted. d, Proportion, y-axis, and absolute number, point size, of cerebrospinal fluid-contacting neurons recovered from each experimental condition. Dotted line shows the proportion of cerebrospinal fluid-contacting neurons in the uninjured spinal cord. e, Representative histological photomicrographs show injured spinal cords across injury severities after staining for NeuN and PKD1L2, a marker of cerebrospinal fluid-contacting neurons. f, Quantification of histological data demonstrating increasing proportions of cerebrospinal fluid-contacting neurons across injury severities. g, Volcano plot showing differentially expressed genes in cerebrospinal fluid-contacting neurons following spinal cord injury, as compared to other neuron subtypes.

Extended Data Fig. 3 Conserved and divergent neuronal responses to SCI.

a, Heatmap showing fold changes for all genes differentially expressed after SCI in at least one level 4 neuron subtype at 1 day, top, and 1 month, bottom, after injury. Patterns of differential expression are broadly conserved at 1 day, but more subtype-specific at 1 month. b, Heatmap showing fold changes for selected genes with broadly conserved patterns of differential expression across level 4 neuron subtypes at 1 day post-injury. c, Heatmap showing coefficients estimated by linear mixed models within each neuron subtype for up- or downregulation of the early-conserved neuronal module over the injury timecourse. d, Heatmap showing coefficients estimated by linear mixed models within each neuron subtype for selected GO term modules with broadly conserved patterns of up- or downregulation across level 4 neuron subtypes at 1 day post-injury. e, Dot plot showing median expression of the circuit reorganization module in each level 4 neuron subtype across timepoints. Point color and size both show median expression of the circuit reorganization module. f, Boxplots showing expression of the axon development, left, and dendrite development, right modules in Vsx2-expressing neurons across timepoints. g, Boxplot showing expression of the circuit reorganization module in each level 4 neuron subtype within the uninjured spinal cord. Vsx2-expressing neurons display the highest expression of the circuit reorganization module in the uninjured spinal cord. h, Dendrogram showing expression of the growth factor Gdnf across levels 1 to 4 of the neuron taxonomy. Point color shows mean expression in each neuron subtype, while point size reflects the proportion of neurons of that subtype with detectable expression. i, Scatterplots comparing basal expression of the circuit reorganization module in the uninjured spinal cord, x-axis, with the SCI-induced upregulation of this module at each timepoint after injury, y-axis, for each level 4 neuron subtype. Inset text shows the Pearson correlation. Basal and induced expression of the circuit reorganization module is maximally correlated at 1 month post-injury, coinciding with the temporal window of opportunity for natural recovery after SCI. j, Timeline of Vsx2ON neuron diphtheria toxin ablation experiments. Two weeks before complete crush SCI, animals received an injection of AAVs expressing DTR. At eight weeks, animals received daily injections of diphtheria toxin for 7 days. Kinematics were then recorded and tissue was collected for evaluation. k, Histological verification of Vsx2ON neuron ablation in the lower thoracic region. Images show loss of Vsx2ON neurons in the thoracic spinal cord, above and below the level of the crush SCI. Bar graph shows the number of Vsx2ON neurons found in each animal (n = 4 mice per group, independent samples two-tailed t-test, t = 11.7, p = 2.4 × 10–5). l, Locomotor performance in the Vsx2ON ablation experiment, as quantified in Supplementary Fig. 3 (n > 10 gait cycles per mouse, n = 4 mice per group).

Source data

Extended Data Fig. 4 Neurons remain differentiated after SCI.

a, Volcano plots showing differential expression for all level 4 neuron subtypes simultaneously across timepoints. Enlarged points represent the key marker genes for each neuron subtype shown in Supplementary Fig. 11a. Marker genes shown in grey show no evidence of differential expression after SCI in their respective neuron subtype. b, Differential expression of key marker genes for level 4 neuron subtypes across timepoints and injury severities (log2-fold change, y-axis, and false discovery rate, point color). Timepoints or severities for which key marker genes show evidence of statistically significant differential expression, compared to uninjured neurons, are plotted with light grey backgrounds. Genes without statistical evidence of differential expression (i.e., FDR greater than 5%) are shown as white points. n.d., genes that were not detectably expressed and could not be subjected to DE analysis. c, Volcano plot showing differential expression for averaged gene expression modules comprising the top 5, top 10, or top 50 marker genes identified for each level 4 neuron subtype by unbiased comparisons with all other neurons, simultaneously across all timepoints. The vast majority of marker gene modules show no evidence of downregulation after SCI in their respective neuron subtype. d, As in c, but showing differential expression for averaged gene expression modules comprising the top 50 marker genes for each level 4 neuron subtype, shown separately for each condition in the timecourse experiment.

Extended Data Fig. 5 Failure of tripartite barrier formation in old mice.

a, Sankey diagram showing the proportions of immune cell subtypes in young and old mice at seven days post-injury. b, Volcano plot showing the statistical significance of changes in immune cell subtype proportions between young and old mice. c, Composite tiled scans and confocal insets of albumin and GFAP in horizontal sections from representative old and young mice at two weeks after SCI. d, Line graph demonstrates albumin intensity at specific distances rostral and caudal to lesion centers. Bottom right, bar graph indicates the area under the curve (independent samples two-tailed t-test, n = 5 per group, t = –3.47, p = 0.022). e, Dendrogram showing cell type prioritizations assigned by Augur across the cellular taxonomy of the spinal cord in comparisons of young and old mice at seven days post-injury. The eight level 5 cell types with the highest AUCs are annotated. f, Sankey diagram showing the proportions of vascular cell subtypes in young and old mice at seven days post-injury. g, Sankey diagram showing the proportions of astroependymal cells expressing Id3 in young and old mice at seven days post-injury (p = 1.9 × 10–3, χ2 test). h, Average expression of the BBB identity module, left, and the peripheral vascular identity module, right, in capillary endothelial cells from young and old mice at seven days post-injury. i, Heatmap showing log-fold changes for all genes differentially expressed in at least one level 4 cell type in comparisons of injured versus uninjured mice, top, and old versus young mice, bottom. j, Heterogeneity of differential expression in comparisons of injured versus uninjured mice, top, and old versus young mice, bottom. Each point shows a gene differentially expressed in at least one level 4 cell type. The x-axis shows the average log2-fold change across all cell types; the y-axis shows the standard deviation of the log2-fold change across cell types (“response heterogeneity”); point size reflects the total number of cell types in which the gene is differentially expressed; and point color reflects the proportion of cell types in which the sign of the log2-fold change was the same as the modal sign (“direction consistency”).

Extended Data Fig. 6 Shared and distinct programs of gene expression across lesion compartments.

a, Left, total number of differentially expressed genes detected within each lesion compartment at 7 days and 2 months after SCI. Right, legend showing the position of each lesion compartment, as in Fig. 6c. b-c, Volcano plots showing differentially expressed genes for all lesion compartments at 7 days, b, and 2 months, c. The top three genes per lesion compartment, as ranked by the product of the log2-fold change and the –log10 p-value, are annotated. d-e, Heatmap showing log2-fold changes for all genes differentially expressed in at least one lesion compartment at 7 days, d, and 2 months, e. f-g, Heatmap showing log2-fold changes for the top five genes differentially expressed in each lesion compartment at 7 days, f, and 2 months, g. h-i, Visualization of selected differentially expressed genes specific to individual lesion compartments at 7 days, h, and 2 months, i, on the two- dimensional coordinate system of the injured spinal cord.

Extended Data Fig. 7 Cell type deconvolution of the 2D spatial atlas.

a, Major cell types assigned to each spatial barcode, visualized for each experimental condition on the two-dimensional coordinate system of the injured spinal cord. b, Sankey diagram showing the cellular composition of each lesion compartment at 7 days, for major (level 1) cell types. c, Sankey diagram showing the cellular composition of each lesion compartment at 2 months, for level 1 cell types. d, Sankey diagram showing the cellular composition of each lesion compartment at 2 months, for level 2 cell types. e, Sankey diagram showing the evolution of the cellular composition of the entire injured spinal cord between 7 days and 2 months, for level 1 cell types. f, Sankey diagram showing the evolution of the cellular composition of the entire injured spinal cord between 7 days and 2 months, for level 2 cell types. g, Visualization of the deconvolution weights assigned by RCTD for selected level 2 cell types at each timepoint, on the two-dimensional coordinate system of the injured spinal cord.

Extended Data Fig. 8 Molecular basis of spatial prioritization at the gene level.

a-c, Volcano plots showing correlations between the AUCs assigned by Magellan at each spatial barcode and transcriptome-wide gene expression across the same spatial barcodes (a, 7 days versus uninjured; b, 2 months versus uninjured; c, 7 days versus 2 months). Inset pie charts show the proportions of all tested genes that are significantly correlated with the spatial prioritizations. d, Heatmap showing Pearson correlations between spatial prioritizations and gene expression for each pairwise comparison of experimental conditions, for the top 40 most recurrently correlated genes across all comparisons. e, Heatmap showing Pearson correlations between spatial prioritizations and gene expression for each pairwise comparison of experimental conditions, for the top 40 most variably correlated genes across all comparisons. f, Visualization of selected genes prioritized by their correlation to spatial prioritizations on the two-dimensional coordinate system of the injured spinal cord.

Extended Data Fig. 9 Inhibitory and facilitating molecules in the 3D spatial atlas.

Expression of selected inhibitory and facilitating molecules across the 3D spatial transcriptomic atlas at 7 days. a, Cspg4; b, Cspg5; c, Ncan; d, Acan; e, Lama1.

Extended Data Fig. 10 A rejuvenative gene therapy reestablishes the tripartite barrier to restore walking.

a, Left, experimental design of a gene therapy intervention to promote the formation of the tripartite barrier, reproduced from Fig. 8a. Right, a second chronophotography series showing walking in old mice without (top) and with (bottom) a gene therapy intervention to promote the formation of the tripartite barrier. b, Composite tiled scans of GFAP and CD45 in horizontal sections from representative old and treated mice. c, Horizontal sections from representative old and treated mice identifying a restoration of Sox9ONId3ON cells in the astrocyte border region in treated mice. d, Composite tiled scans and confocal insets of albumin and GFAP in horizontal sections from representative old and treated mice after SCI. e, Line graph demonstrates albumin intensity at specific distances rostral and caudal to lesion centers. Bottom right, bar graph indicates the area under the curve (independent samples two-tailed t-test, n = 5 per group, t = 4.07, p = 0.0099). f, Locomotor performance in the gene therapy experiment, as quantified in Supplementary Fig. 3 (n > 10 gait cycles per mouse, (n = 5 mice per group; Tukey’s honestly significant difference test). *, p < 0.05; **, p < 0.01; ***, p < 0.001. g, Left, schematic overview of the classification pipeline using high-resolution kinematics data from young and old mice. Right, experimental conditions assigned to individual steps in old mice that received gene therapy by a machine-learning model trained on kinematics data from untreated animals.

Source data

Supplementary information

Supplementary information

These files contain Supplementary Notes 1–6, Figs. 1–42 and References.

Reporting Summary

Supplementary Table 1

Raw kinematics data for all experiments presented in the paper.

Supplementary Table 2

List of genes used to calculate gene module scores reported in the paper.

Supplementary Video 1

The Tabulae Paralytica: multimodal single-cell and spatial atlases of SCI.

Supplementary Video 2

Ablating Vsx2ON neurons prevents the natural recovery of walking after SCI.

Supplementary Video 3

A rejuvenative gene therapy re-establishes the tripartite barrier to restore walking in old mice.

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Skinnider, M.A., Gautier, M., Teo, A.Y.Y. et al. Single-cell and spatial atlases of spinal cord injury in the Tabulae Paralytica. Nature 631, 150–163 (2024). https://doi.org/10.1038/s41586-024-07504-y

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