The spinal cord is a fascinating structure that is responsible for coordinating movement in vertebrates. Spinal motor neurons control muscle activity by transmitting signals from the spinal cord to diverse peripheral targets. In this study, we profiled 43,890 single-nucleus transcriptomes from the adult mouse spinal cord using fluorescence-activated nuclei sorting to enrich for motor neuron nuclei. We identified 16 sympathetic motor neuron clusters, which are distinguishable by spatial localization and expression of neuromodulatory signaling genes. We found surprising skeletal motor neuron heterogeneity in the adult spinal cord, including transcriptional differences that correlate with electrophysiologically and spatially distinct motor pools. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neuron (γ*). Collectively, these data provide a single-cell transcriptional atlas (http://spinalcordatlas.org) for investigating the organizing molecular logic of adult motor neuron diversity, as well as the cellular and molecular basis of motor neuron function in health and disease.
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All custom code used to analyze data and generate figures is available in the form of a Jupyter Notebook at https://github.com/neurojacob/blum_et_al_2021.
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This work was supported by National Institutes of Health (NIH) grants R35NS097263 (to A.D.G.) and R01NS083998 (to J.A.K.), the Robert Packard Center for ALS Research at Johns Hopkins (to A.D.G.), the Blavatnik Family Foundation (to J.A.B.) and the Brain Rejuvenation Project of the Wu Tsai Neurosciences Institute. Sorting was performed on an instrument in the Shared FACS Facility obtained using NIH S10 Shared Instrument Grant S10RR025518-01. Portions of Figs. 1d and 2b were generated using objects from BioRender.com.
A.D.G. has served as a consultant for Aquinnah Pharmaceuticals, Prevail Therapeutics and Third Rock Ventures and is a scientific founder of Maze Therapeutics. W.J.G. has affiliations with 10x Genomics (consultant), Guardant Health (consultant) and Protillion Biosciences (co-founder and consultant). J.A.B. has served as a consultant for Maze Therapeutics.
Peer review information Nature Neuroscience thanks Alain Chédotal and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Single-nucleus transcriptional analysis of the adult mouse spinal cord reveals canonical cell types.
a, Canonical cell class labels, visualized on UMAP. b, Average log-normalized marker gene expression across canonical cell classes. c-d, Representative in situ hybridization against Chat/Nos1 in transverse sacral (c) and thoracic (d) spinal cord hemi-sections. n = 3 biologically independent animals. e-f, Average log-normalized expression of Zeb2 (e) and Fbn2 (f) across all cholinergic clusters (labeled), overlaid on UMAP. Dotted line surrounds clusters corresponding to visceral motor neurons. g, Representative in situ hybridization against Chat/Fbn2 in transverse thoracic spinal cord hemi-section. n = 3 biologically independent animals. Scale bars=200 µm (c-d) and 100 µm (g). LAC = lateral autonomic column (c,d), VH = ventral horn (c,d).
Extended Data Fig. 2 Novel markers of skeletal motor neurons confirmed by Allen Spinal Cord Atlas in situ hybridizations.
a-d, Transverse schematic illustrating expected positions of skeletal motor neurons in ventral horn (VH, green) in lumbar spinal cord. Second row—corresponding in situ hybridization against Tns1 (a), Bcl6 (b), Syn1 (c), and Actb (d). Third row—expression mask shows relative enrichment of Tns1 and Bcl6 in small and large cell bodies in the VH.
Extended Data Fig. 3 Visceral motor neuron populations express selective repertoires of neuropeptides and are spatially distinct.
a, Estimated relative density of visceral motor neurons along the rostral-caudal axis of the spinal cord based on Allen Spinal Cord Atlas10. Density functions are combined density estimates of marker genes for each cluster (see Methods). Clusters were grouped according to shape of density function, with clusters 3,7, and 10 clearly enriched in the sacral spinal cord. b, Validation of visceral spatial modeling from (a) via high-resolution in situ hybridization for Chat and visceral cluster markers (Piezo2, Cdh8, Creb5, Cpne4, Fbn2). Plots show number of motor neurons counted in the autonomic column, added across three counted slides in each region. Individual data points for total visceral motor neurons shown with filled circles, while marker gene-positive cell numbers shown with filled triangles. n = 2 (Piezo2, Cdh8, Creb5, Cpne4) or 5 (Fbn2) biologically independent replicates. Error bars are SEM. c, Average log-normalized expression of Rxfp1 and Nts across all visceral motor neuron clusters (labeled), overlaid on UMAP. d, Representative in situ hybridization against Chat/Fbn2/Rxfp1 in transverse sacral spinal cord shows coexpression in the autonomic column but not in the ventral horn (VH). n = 3 biologically independent animals. Scale bar=100 µm. e, Average log-normalized expression of Adra2a across all visceral clusters (labeled) shows that sporadic expression exists across populations, overlaid on UMAP. f, Representative in situ hybridization against Chat/Piezo2/Cdh8 in cholinergic cells around the central canal (CC). Scale bar=50 µm. n = 2 biologically independent animals. g, Average log-normalized expression of Gldn across all cells in spinal cord shows clear enrichment in partition cell cluster (arrowhead), overlaid on UMAP. h, Average log-normalized expression of Nrxn3 across cholinergic interneurons shows that Nrxn3 expression is limited to half of partition cells (arrowhead), overlaid on UMAP.
Extended Data Fig. 4 Vipr2 and Npas1 are novel, robust, and specific markers of α and γ motor neurons in the spinal cord.
a, Average expression of Vipr2 and Npas1 across all spinal cord cell populations (labeled), overlaid on UMAP. Arrow points to α and γ motor neuron clusters, respectively. b, Representative in situ hybridization against Chat/Rbfox3/Vipr2 in transverse spinal cord shows coexpression in the ventral horn (VH). n = 4 biologically independent animals. Scale bar=100 µm. c, Representative in situ hybridization against Chat/Htr1d/Vipr2 in transverse spinal cord shows mutual exclusion. Scale bar=50 µm (inset) and 200 µm (overview). n = 4 biologically independent animals. d, Representative in situ hybridization against Chat/Npas1/Rbfox3 in transverse spinal cord shows mutual exclusion of Rbfox3 and Npas1 in Chat + cells. n = 5 biologically independent animals. Scale bar=20 µm (inset) and 200 µm (overview). e, Representative in situ hybridization against Chat/Npas1/Vipr2 in transverse spinal cord shows mutual exclusion of novel markers Vipr2 and Npas1 in Chat + cells. n = 4 biologically independent animals. Scale bar=20 µm (inset) and 200 µm (overview).
a, UMAP with 3 subclustered γ motor neurons populations. b, Novel marker gene expression across γ motor neuron subpopulations. Dot size is proportional to the percent of each cluster expressing the marker gene, while blue color intensity is correlated with expression level. c, Average log-normalized expression of genes enriched in γ* motor neurons over γ overlaid on UMAP. α, γ, and γ* populations are labeled. d, Average log-normalized expression of genes enriched in γ motor neurons over γ* overlaid on UMAP. α, γ, and γ* populations are labeled. e-h, Average expression of novel γ markers Stxbp6 (e) and Plch1 (f), as well as novel γ* markers Pard3b (g) and Creb5 (h) by cluster. i, Representative in situ hybridization against Htr1d/Creb5/Stxbp6 in transverse spinal cord shows mutual exclusion of novel markers Creb5 and Stxbp6 in Htr1d + cells. n = 4 biologically independent animals. Scale bar=20 µm (inset) and 200 µm (overview). j, Representative in situ hybridization against Htr1d/Pard3b/Stxbp6 in transverse spinal cord shows mutual exclusion of novel markers Pard3b and Stxbp6 in Htr1d + cells. Arrowheads label canonical γ motor neurons and *labels γ*. n = 5 biologically independent animals. Scale bar=20 µm (inset) and 100 µm (overview). Differentially expressed genes determined by Wilcoxon rank sum test implementation in Seurat and adjusted for multiple comparisons (Bonferroni method) (p_adj<0.01, log2-fold change >0.5).
Extended Data Fig. 6 Retrograde CTB labeling of motor pools connects transcriptional subpopulations with motor pools.
a, Proportion of CTB-labeled cells from GLUT and IF that are labeled with Cdh8 and Sema3e. The GLUT has a significantly larger proportion of Cdh8 + and Sema3e + cells than the IF. n = 5 biologically independent animals. b, Lower power view of in situ hybridization against Prkcd/Sv2a, and Kcnq5/Chodl (insets=Fig. 4h) in longitudinal sections demonstrates the specificity of CTB injections into the Soleus (SOL) and Tibialis anterior (TA). n = 5 (TA) and 4 (SOL) biologically independent animals. One-way ANOVA with post-hoc Sidak multiple comparison test between same-gene conditions. Adjusted p-values=0.0212 (Cdh8) and 0.0499 (Sema3e). c, Expression of FF and SF gene modules overlaid on UMAP of all α motor neurons. d, Average log-normalized canonical marker expression of Chodl (fast-firing) and Sv2a (slow-firing). Scale bars = 250 µm. e, Proportion of Prkcd + cells positive for Sv2a (left) and proportion of Chodl + cells that are positive for Kcnq5 (right). n = 3 biologically independent animals. f, Representative in situ hybridization showing Kcnq5 is expressed in a subset of Chodl + cells. n = 4 biologically independent animals. Scale bar=50 µm inset and 200 µm overview. *=p value<0.05, **=p value <0.01, ***=p value<0.001, ****=p value<0.0001. Error bars are SEM.
Extended Data Fig. 7 Retrograde CTB labeling of motor pools enables the identification of transcriptionally distinct classes of fast and slow-firing motor neurons in the adult spinal cord.
a, Representative in situ hybridization against Chat/Mmp9/Kcnq5 in transverse spinal cord shows that Kcnq5 is expressed in a subset of Mmp9 + fast-firing motor neurons. n = 4 biologically independent animals. Scale bar=20 µm inset and 200 µm overview. b, Representative in situ hybridization against Chat/Sv2a/Prkcd in transverse spinal cord shows that Prkcd is expressed in almost every Chat + /Sv2a + slow-firing motor neuron. n = 2 biologically independent animals. Scale bar=20 µm inset and 200 µm overview. c, Representative in situ hybridization against Chat/Mmp9/Prkcd in transverse spinal cord shows that Prkcd is excluded from almost every Chat + /Mmp9 + fast-firing motor neuron. n = 4 biologically independent animals. Scale bar=30 µm inset and 200 µm overview. d-e, Proportion of cells expressing fast and slow-firing markers in the CTB-labeled TA (d) and SOL (e) motor pools. There is a significantly higher proportion of cells expressing both known and novel fast-firing markers in TA than SOL (d), and a higher proportion of cells expressing both known and novel slow-firing markers in SOL than TA. Adjusted p-value=0.0456 (Chodl + >Kcnq5 + ). f, Total number of CTB-positive cells labeled across biologically independent animals. One-way ANOVA with post-hoc Tukey multiple comparison test among all conditions. n = 4-5 biologically independent animals (d-f). *=p value<0.05, **=p value <0.01, ***=p value<0.001, ****=p value<0.0001. Error bars are SEM.
a-e, Each spinal cord sequencing replicate, plotted side-by-side and visualized by UMAP. Note that we observe minimal batch-to-batch variability along sex, age, or replicate number axes in terms of cluster identification and overall shape of the dimensionality reduced data. This does not preclude sex or age-related transcriptional changes but demonstrates that they do not fundamentally alter the transcriptomic classes that we focus on in the manuscript.
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Blum, J.A., Klemm, S., Shadrach, J.L. et al. Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons. Nat Neurosci 24, 572–583 (2021). https://doi.org/10.1038/s41593-020-00795-0
Clinical Autonomic Research (2021)