Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord

Abstract

In vertebrates, motor control relies on cholinergic neurons in the spinal cord that have been extensively studied over the past hundred years, yet the full heterogeneity of these neurons and their different functional roles in the adult remain to be defined. Here, we develop a targeted single nuclear RNA sequencing approach and use it to identify an array of cholinergic interneurons, visceral and skeletal motor neurons. Our data expose markers for distinguishing these classes of cholinergic neurons and their rich diversity. Specifically, visceral motor neurons, which provide autonomic control, can be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons is also reflected in our analysis with alpha, gamma, and a third subtype, possibly corresponding to the elusive beta motor neurons, clearly distinguished. In combination, our data provide a comprehensive transcriptomic description of this important population of neurons that control many aspects of physiology and movement and encompass the cellular substrates for debilitating degenerative disorders.

Fig. 1. Strategy for single nucleus RNA sequencing of spinal cord cholinergic neurons.

Spinal cords were extruded from Chat-IRES-Cre::CAG-Sun1-sfGFP mice and separated into 3 regions (Cervical, Thoracic, and Lumbar/Sacral) that were processed separately (1). Image from a whole cleared spinal cord (SC) immunolabeled for GFP (Supplementary Movie 1). Tissue was lysed (2) and nuclei were isolated by density gradient centrifugation (3). Nuclear suspensions (4) were sorted using fluorescent-activated cell sorting (FACS) (5) to select singlet GFP-positive DRAQ5-positive nuclei (6) that were processed for single nucleus RNA sequencing using the 10X Genomics Chromium platform (7). Single nucleus cDNA libraries were sequenced together on an Illumina HiSeq (7), then analyzed (8). Figure created with BioRender.com. Scale bars, (1) 200 µm, (5) 50 µm.

Fig. 2. Classification of cholinergic neurons reveals the extensive diversity of motor neurons and interneurons.

a UMAP of all 16,042 cholinergic nuclei showing 21 distinct clusters. b Expression of marker genes for cholinergic interneurons (Pax2), visceral MNs (Zeb2), and skeletal MNs (Tns1) in the 21 cholinergic clusters. c Dot plot showing the top 15 marker genes for each main cholinergic subtype. d Feature plots depicting expression of the top novel marker gene for each cholinergic subtype. e RNAScope for novel marker genes for a subset of interneurons (Slc6a1, red) and visceral MNs (Fbn2, cyan) co-expressing with Chat (yellow). Interneurons in the intermediate zone (box 3) are double positive for Slc6a1 and Chat (arrowhead) or only express Chat (arrow); visceral MNs in the lateral horn (box 2) are double positive for Fbn2 and Chat, and skeletal MNs in the ventral horn (box 1) are only Chat+. f ISH for novel marker gene Tns1 (cyan) highlights skeletal MNs, showing restricted localization to the ventral horn and co-expression with Chat in cells with the distinctive cell body shape of skeletal MNs. Co-labeling with Prph (red) and Chat (yellow) highlights skeletal MNs in the ventral horn (box 1) that are triple positive, visceral MNs in the lateral horn (box 2) that are double positive for Chat and Prph, and interneurons in the intermediate zone (box 3) that are only positive for Chat (arrow) or occasionally double-positive for Chat and Prph (arrowhead). e, f: thoracic spinal cord; all images are merged for the 3 colors except for Fbn2 and Tns1, shown alone, as marked. Full single-color images and merges are shown in Supplementary Fig. 6. Low magnification image scale bars, 200 µm. High magnification scale bars, 50 µm.

Fig. 3. Novel markers for distinguishing alpha, gamma, and Type 3 motor neurons.

a Expression of known alpha and gamma markers by skeletal clusters. b Dot plot for the top marker genes for each skeletal cluster, showing more shared expression between clusters S1 and S2. c Expression of novel and specific marker genes for each skeletal MN subtype. d ISH showing expression of novel alpha MN markers Stk32a (red) and Sv2b (yellow) co-expressed with known alpha marker Rbfox3 (magenta) in large diameter Chat+ (cyan) ventral horn neurons. Arrowheads highlight examples of large diameter, triple-positive alpha MNs; arrow highlights the example of a small diameter Chat+ MN that is negative for all 3 alpha markers (i.e., gamma or Type 3). e ISH image overlayed on endogenous Chat-Cre::Ai14 labeling showing expression of S1 marker Gpr149 (yellow), S2 marker Stk32a (cyan), and S3 marker Nrp2 (red). High magnification images highlight a large MN expressing Stk32a and surrounded by C-boutons (examples indicated by arrows), characteristic of alpha MNs, and a small MN expressing Nrp2 and lacking C-boutons, characteristic of gamma MNs. ISH from the ventral horn of lumbar spinal cord sections. Low magnification scale bars, 100 µm. High magnification scale bars, d 50 µm, e 20 µm.

Fig. 4. Alpha motor neurons sub-cluster into eight transcriptional groups.

a Alpha MNs can be re-clustered into 8 sub-clusters. b UMAPs displaying the alpha MN clusters in each separately barcoded segment of the spinal cord. Clusters 2 and 7 are most prominent in cervical, 3 and 4 are sparse or missing from thoracic. c Dot plot showing the top marker genes for each cluster. No markers were identified for clusters 0 or 1. Genes in bold are shown by ISH. d Quantification of cluster marker expression in alpha MNs retrogradely labeled from various muscles (Axial = lumbar extensors of the spine, TA = tibialis anterior). For each muscle, N = 2 animals were injected with the retrograde tracer FastBlue. Percent of FastBlue+ alpha MNs, based on Stk32a expression, expressing cluster markers from each injected muscle. Cpne4 (axial n = 66 neurons, digit n = 56, phrenic n = 175, soleus n = 91, TA n = 78) and Erbb4 (axial n = 138 neurons, digit n = 77, phrenic n = 177, soleus n = 63, TA n = 119) stand out as markers for digit-innervating and phrenic MNs, respectively. Grm5 was relatively enriched in alpha MNs projecting to the soleus compared with the other muscles (axial n = 138 neurons, digit n = 77, phrenic n = 177, soleus n = 63, TA n = 119), and C1qtnf4/Gulp1 was expressed in double the number of alpha MNs projecting to digit muscle (axial n = 94 neurons, digit n = 67, phrenic n = 142, soleus n = 72, TA n = 100). e ISH examples of images quantified in d. Alpha MNs innervating the digits express the cluster 4 marker Cpne4 (yellow) but lack the cluster 5 marker C1qtnf4 (cyan). f Alpha MNs innervating the diaphragm (phrenic MNs) express the cluster 7 marker Erbb4 (yellow) but lack the cluster 2 marker Grm5 (cyan). *p = 0.0053 by Shapiro-Wilk test comparing by muscle. #p = 0.0031 by Shapiro-Wilk test comparing by the gene. Scale bars, e 50 µm, f 25 µm.

Fig. 5. Spinal cholinergic interneurons separate into 8 transcriptionally distinct subtypes.

a Top markers of the 8 cholinergic interneuron clusters. b ISH showing expression of two top markers for cholinergic interneurons (Fig. 2c), Slc6a1 (cyan) and Mpped2 (yellow) in Chat+ neurons (red) in the intermediate zone. Boxes highlight a neuron expressing Chat and Mpped2, but lacking Slc6a1 (left), and a neuron expressing Chat, Mpped2, and Slc6a1 (right). c ISH showing expression of Pitx2 and Tox near the central canal (outlined) of the lumbar spinal cord. Co-expression of Pitx2 and Tox in Chat+ cells exemplifies cluster I5 (left) whereas Pitx2 and Chat without Tox identify a cluster I6 cell (right). d High-magnification images showing co-expression of Piezo2, Reln, and Chat in an I8 neuron found near the central canal (outlined) of the thoracic spinal cord. e, f Piezo2 and Reln co-expression are diagnostic of a rare population of interneurons, cluster I8. g Pitx2, a known marker for partition cells, is expressed only in clusters I5 and I6. h Tox is expressed only in interneuron cluster I5 and thus differentiates between the two Pitx2-expressing clusters. Low magnification scale bars, 50 µm. High magnification scale bars, 10 µm.

Fig. 6. Visceral motor neurons exhibit extensive diversity in their gene expression profiles and anatomic localization.

a Visceral motor neurons can be re-clustered into 16 distinct sub-clusters. b Dot plot showing the top three marker genes for each cluster. Genes in bold font are shown by ISH below. c UMAPs displaying the visceral motor neuron clusters derived from each separately barcoded segment of the spinal cord. Clusters 0, 1, 3, 4, 10, 11, 12, 14, 15 are sparse or missing from the lumbar/sacral segment; conversely, cluster 5 is unique to the lumbar/sacral segment, where cluster 2 is also most abundant. Cluster 8 is most sparse in thoracic, while clusters 3 and 15 are most abundant in thoracic, and cluster 14 is most abundant in cervical. d Dot plot showing expression of marker genes for uniquely cervical/thoracic (C/T) or lumbar/sacral (L/S) clusters in each segment of the spinal cord, with Gpc3, Dach2, and Sema5a most highly expressed in rostral levels and Sst most highly expressed in caudal levels. e Quantification of each level-specific cluster marker in visceral MNs of each spinal cord level, shown as percent of Fbn2 + MNs expressing each gene (N = 2 animals), highlighting rostral expression of Gpc3 (cervical n = 100 neurons, thoracic n = 64, lumbar n = 41, sacral n = 140), Dach2 (cervical n = 175 neurons, thoracic n = 139, lumbar n = 92, sacral n = 91), and Sema5a (cervical n = 180 neurons, thoracic n = 142, lumbar n = 72, sacral n = 92) and caudal expression of Sst (cervical n = 144 neurons, thoracic n = 77, lumbar n = 75, sacral n = 110), as predicted by the sequencing data. f ISH showing Gpc3 + (yellow) visceral MNs, likely representing cluster 3 neurons, in the cervical spinal cord. g ISH showing Sst+ visceral MNs (yellow), likely representing cluster 5 neurons, in the sacral spinal cord. Scale bars, 50 µm.