Conserved Genetic Signatures Parcellate Cardinal Spinal Neuron Classes Into Local and Projection Subsets Peter J

RESEARCH

NEURODEVELOPMENT bined sample (Fig. 1A and fig. S1). At P0, spinal neurons have been generated and the Conserved genetic signatures parcellate cardinal basic functional features of adult spinal circuitry have been established, but expression spinal neuron classes into local and persists for many developmental markers of neuronal subtype diversity (2, 9, 10, 18). Using projection subsets marker analyses, 6743 cells passed quality control and were identified as neurons (Fig. 1A). Peter J. Osseward II1,2†, Neal D. Amin1‡, Jeffrey D. Moore3, Benjamin A. Temple1,2, Glutamatergic and GABAergic neurons largely Bianca K. Barriga1,4, Lukas C. Bachmann1, Fernando Beltran Jr.1, Miriam Gullo1, Robert C. Clark1, segregated in the uniform manifold approxi- Shawn P. Driscoll1, Samuel L. Pfaff1*, Marito Hayashi1†§ mationandprojection(UMAP)space,anda graph-based comparison using the highly vari- Motor and sensory functions of the spinal cord are mediated by populations of cardinal neurons arising from able genes conservatively identified 45 distinct separate progenitor lineages. However, each cardinal class is composed of multiple neuronal types with clusters(Fig.1,BandC).Asexpected,manyof distinct molecular, anatomical, and physiological features, and there is not a unifying logic that systematically these clusters were enriched for genes that have accounts for this diversity. We reasoned that the expansion of new neuronal types occurred in a stepwise been previously identified as marker genes for manner analogous to animal speciation, and we explored this by defining transcriptomic relationships using cardinal classes (Fig. 1D and table S1) (1, 2). a top-down approach. We uncovered orderly genetic tiers that sequentially divide groups of neurons by To explore the conserved relationships their motor-sensory, local-long range, and excitatory-inhibitory features. The genetic signatures defining that exist among lumbar spinal neurons, we neuronal projections were tied to neuronal birth date and conserved across cardinal classes. Thus, the developed a divisive clustering pipeline using intersection of cardinal class with projection markers provides a unifying taxonomic solution for the k-means algorithm to split spinal neurons Downloaded from systematically identifying distinct functional subsets. into pairwise groups iteratively on the basis of their overall transcriptional composition (Fig. 1, E and F). We focused on transcription factors rom a functional perspective, spinal neu- One perspective of neuronal diversity is that becausetheyregulateneuronalidentityand rons can be divided along several axes, it arose through evolution in order to expand function (2, 14, 15). We anticipated that this

including motor-sensory, excitatory- neural functions (14–16). In this view, primi- approach would first reveal the glutamatergic http://science.sciencemag.org/ F inhibitory, and locally connected neurons tive neuron types served as the precursors and GABAergic neurotransmitter division in for intracord processing versus projection for more specialized subtypes, leading to the the cell population because it is such a promi- neurons for communication with the brain prediction that individualneuronalattributes nent phenotypic difference, and the two cell (1–6). Neuron heterogeneity is often charac- mayhaveemergedinastepwisefashionwith types were isolated from separate litters. How- terized on the basis of landmarks such as ordered hierarchical relationships (14, 16, 17). ever, the neuronal populations split along an their neurotransmitter type, connectivity, cyto- This analogy to animal speciation prompted us axis unrelated to neurotransmitter identity (Fig. architecture, morphology, physiology, develop- to investigate whether molecular and cellular 1, B and G) (11, 19). Differential gene expression mental origin, and genetic profile (7). In the correlates for this type of stepwise diversifica- of the first division indicated that transcription spinal cord, separate cardinal neuron classes tion could be detected among large heteroge- factor Ebf1 is highly expressed in a cell group

arise from molecularly distinct progenitor neous populations of spinal neurons. Thus, we defined as group-E neurons, whereas cells arrayed along the dorsoventral axis of rather than focusing on fine-grained molecular transcription factor Hoxc10 is enriched in a on April 30, 2021 the neural tube (2, 8). Neurons within each differences that may or may not correspond different group we termed group-H neurons cardinal class share properties such as the to functional cellular features, we used a top- (Fig. 1G). Histological analysis of Ebf1+ and same neurotransmitter identity and have been down approach to identify the transcriptomic Hoxc10+ neurons identified a spatial divi- targeted in functional studies (1, 2). However, signatures linked to the emergence of differ- sion of these groups (Fig. 2, A and B, and fig. it is increasingly apparent that each cardinal ent neuronal types. At each branch point in S2). Ebf1+ neurons were located in laminae I class is itself composed of heterogeneous popu- our established hierarchy, we defined the to III of the superficial dorsal horn, corresponding lations of neuron types, impeding a detailed molecular, developmental, cytoarchitectural, to the site where many exteroceptive sensory understanding how spinal circuits function neurotransmitter, and connectivity properties inputs terminate. By contrast, Hoxc10+ neurons (9–13). We sought to determine whether there of the neurons. Our analysis uncovered molec- were predominantly located in laminae IV to was a coherent logic for spinal neuron diver- ular markers for local and projection neurons X, areas involved in proprioception and motor sification in mice that could be used to sys- regardless of cardinal class or neurotransmitter. control (3, 4). Although group-E and group-H tematically describe the heterogeneity within By combining markers for cardinal classes neurons broadly differ in motor-sensory func- all the cardinal classes. with those for conserved genetic signatures tions associated with their positions, both types for projection status, we established a simple arecomposedofamixtureofglutamatergic combinatorial matrix that systematically iden- and GABAergic neurons (Fig. 1, B and G, and 1Gene Expression Laboratory, Salk Institute for Biological tified discrete subsets of spinal neurons. fig. S3) (3, 4). Studies, La Jolla, CA 92037, USA. 2Neurosciences Graduate Program, University of California, San Diego, 9500 Gilman Drive, Group-E and -H neurons divide the spinal Group-N and -Z neurons are arrayed along the 3 La Jolla, CA 92037, USA. Howard Hughes Medical Institute, motor-sensory circuitry mediolateral axis of the spinal cord Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge, MA, USA. 4Biological To enrich for neurons while minimizing tech- Using the hierarchical-divisive strategy outlined Sciences Graduate Program, University of California, San Diego, nical and biological variability, we genetically above (Fig. 1F), we further split the group-E 9500 Gilman Drive, La Jolla, CA 92037, USA. †These authors contributed equally to this work. labeled glutamatergic and g-aminobutyric neurons into two subgroups and found that ‡Present address: Department of Psychiatry and Behavioral acid (GABA)–ergic neurons in postnatal day this division identifies glutamatergic-related Sciences, Stanford University, Stanford, CA 94305, USA. §Present 0 (P0) mice, sorted lumbar spinal neurons and GABAergic-related genes and their cor- address: Howard Hughes Medical Institute, Department of Cell 2 4 Biology, Harvard Medical School, Boston, MA 02115, USA. separately, pooled them, and performed single- responding neurons (Fig. 1, B and H) ( , ). *Corresponding author. Email: [email protected] cell RNA-sequencing (scRNA-seq) on the com- Division of the group-H population produced

Osseward et al., Science 372, 385–393 (2021) 23 April 2021 1of8 RESEARCH | RESEARCH ARTICLE

A postnatal day 0 B Glutamatergic GABAergic 24 C 12 D Glutamatergic Vglut2:Cre; Vgat:Cre; 21 tdTomato tdTomato 40 dI1 18 02 dI3 17 31 28 01 29 16 dI5/LB 010203 09 12 16 19 21 24 28 3229 40 32 V2a 35 39 MN 15 31 18 27 09 03 38 35 15 19 V3 17 37 25 23 pool 38 GABAergic 22 06 scRNA-seq 11 04 03 dI4 0405 10 200733 34 36 41 100% 43 13 44 dILA 08 302614 42 41 34 10 05 dI6 22 50% 36 20 07 33 V1 11 13 23 37

0% V2b 11 23 UMAP2 08 14 30 26 45 neuron UMAP1

non-neuron E F Group-E Group-H functions? spinal neurons enriched enriched k-means 2 Group-E 300 Tshz2 cluster 1 Ebf1 Ebf2 Fig. 1G Cck Hoxc10 ... Npy UMAP 2 Nefl cluster 4 Frzb Nap1l5 200 UMAP 1 k-means 2 k-means 2 Zfhx3 Elavl2 Fig. 1H Fig. 1I Hoxd10

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Group-Z Group-N 200 Zic2 200 Sst Hoxb9 Lhx1 Prox1 Gad1 Nefl Sncg Tlx3 Pax2 100 100 Neurod2 −log10(p−value) Slc6a1 −log10(p−value) Zfhx4 Slc17a6 Otp Esrrg Foxp2 0 0 −2 −1 0 1 2 Group-E (GABA) −2 0 2 log2 Fold Change log2 Fold Change Group-N (Glut) Group-N (GABA) Group-Z (GABA) Group-Z (Glut)

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Fig. 1. scRNA-seq identifies relationships among groups of spinal neurons. populations can be used to define branch points. The functional differences at the (A) Lumbar segments from P0 Vglut2:Cre; Ai14 and Vgat:Cre; Ai14 neonates (two branch points can be used to identify increasingly more specific neuronal females and one male from each litter) were microdissected. tdTomato+ cells were attributes linked to function. (F) Schematic of analysis used to investigate the sorted and pooled as one sample before scRNA-seq. Using marker analyses and higher-order relationships across spinal neuron populations conducted with quality control measures, 6743 cells were identified as neurons (94.4% of total iterative k-means 2 divisive clustering. Data for each color-coded division are cells detected). These neurons had 1012 median genes per cell and 1606 median indicated for (G) to (K). (G to K) (Left) UMAP color coded according to identified unique molecular identifiers (UMIs) per cell. (B) UMAP visualization of neurons division. (Right) Volcano plot depicting differentially expressed genes across each identifies that glutamatergic neurons and GABAergic neurons largely segregate k-mean division. [(G) and (I)] Further analysis of highlighted genes are available in UMAP space. (C) Forty-five identified clusters are color coded in UMAP space. in Fig. 2 and fig. S2. [(H), (J), and (K)] Glutamatergic- and GABAergic-related (D) Colored dots with numbers correspond to clusters from Fig. 1C. As expected, genes are highlighted. (G) Group-E: 3360 cells, Jaccard similarity [across clusters corresponded to known cardinal neuron classes, defined by their marker bootstraps (supplementary materials, materials and methods)] = 0.960; group-H: expression. V1 and V2b markers were found in the same clusters (12). Several 3383 cells, Jaccard similarity = 0.959. (H) Group-E (Glut): 1851 cells, Jaccard clusters are not shown because they were not enriched for obvious cardinal class similarity = 0.960; group-E (GABA): 1509 cells, Jaccard similarity = 0.922. (I) marker genes (table S1). (E) Schematic outlining methods to identify neuronal Group-Z: 1707 cells, Jaccard similarity = 0.867; group-N: 1676 cells, Jaccard subtypes. (Top) Dimensionality reduction tools can be used to define the similarity = 0.809. (J) Group-N (Glut): 1040 cells, Jaccard similarity = 0.791; group- maximum number of genetically distinct clusters. Functions must be independently N (GABA): 636 cells, Jaccard similarity = 0.613. (K) Group-Z (Glut): 768 cells, defined for each cluster. (Bottom) Hierarchical comparisons of neuronal Jaccard similarity = 0.769; group-Z (GABA): 939 cells, Jaccard similarity = 0.760.

Osseward et al., Science 372, 385–393 (2021) 23 April 2021 2of8 RESEARCH | RESEARCH ARTICLE a different result. Rather than splitting group- (group-Z) (Fig. 1I and table S2). Histological group-Z neurons occasionally intermingle H neurons into glutamatergic and GABAergic analysis found that these two sets of genes (Fig.2,CtoH,andfig.S5),butfewcells cell populations, we found a subgroup of neu- spatially divided the spinal cord into large wedge coexpressed markers for both group-N and rons enriched for Nfib, NeuroD2, and Prox1 patterns: group-N cells located dorsomedially group-Z neurons (Fig. 2, E and G, and fig. (designated group-N) and a separate sub- and group-Z cells positioned ventrolaterally S5). Although group-N and group-Z neurons group enriched for Zfhx3, Zfhx4, and FoxP2 (Fig. 2, C and D, and fig. S4). Group-N and are defined by gene sets, we found that NeuroD2 and Zfhx3 were representative markers of ACGroup-E BDGroup-H Group-N Group-Z group-N and -Z neurons, respectively (Figs. 1I and 2, C and D; and figs. S2 to S5). Our Ebf1 Hoxc10 NeuroD2 Zfhx3 sequencing was conducted on cells from the lumbar spinal cord, but we found that NeuroD2 and Zfhx3 labeled neurons in the same mediolateral pattern along the entire rostrocaudal axis of the cord (figs. S6 and S7). Further k- means splitting of group-N and -Z neurons resolved the glutamatergic and GABAergic subsets in each population (Fig. 1, J and K, 500 500 500 500 and fig. S3) (2, 4). Our findings unmask a Hoxc10 Zfhx3 stepwise hierarchy of genetic divisions among 250 250 250 250 spinal neurons with layers that supersede neu- Ebf1 rotransmitter physiology (Fig. 2H). Downloaded from µm 0 µm 0 µm 0 µm 0

NeuroD2 Group-N and -Z neurons have distinct -250 -250 -250 -250 birth dates Although the group-E–group-H division ap- 0 300 600 0 300 600 0 300 600 0 300 600 µm µm µm µm peared to correspond to neurons involved in

exteroception and motor control, respectively, http://science.sciencemag.org/ G E colocalization Zfhx3 / NeuroD2 we hypothesized that the group-N and -Z sub- 100 FoxP2 sets derived from the group-H population might 80 Group-Z Zfhx3 segregate along alternative features. To inves- Zfhx4 60 tigate a possible developmental distinction NeuroD2 40 between group-N and group-Z neurons, we

Group-N Prox1 20 performed scRNA-seq of embryonic day Nfib 12.5 (E12.5) spinal cord cells composed of the Ebf1 Group-E N.M. embryonically defined cardinal cell classes x1 xP2 Nfib Ebf1 (Fig.3,AandB;fig.S8;andtableS3).TheE-H Fo Zfhx3Zfhx4 Pro NeuroD2

division was not evident at E12.5; however, the group-N and group-Z neuronal populations on April 30, 2021 F H spatial distance weredetected(Fig.3,CandD,andfigs.S8and S9). We birth-dated the neurons marked by 1.0 lumbar neurons FoxP2 NeuroD2 and Zfhx3 at P0 using EdU pulses 0.8 + Group-Z Zfhx3 embryonically. The peak of Zfhx3 neuron birth Group-E Group-H + Zfhx4 0.6 was at E10.5, whereas the peak of NeuroD2 NeuroD2 0.4 neuron generation was later at E13.5 (Fig. 3E

Group-N Prox1 0.2 Group-N Group-Z and fig. S10). To examine whether group-N and Glut

Nfib GABA 0 group-Z identity is dependent on neuronal Group-E Ebf1 Group-E connectivity, we genetically ablated propri- Glut Glut x1 GABA GABA Group-N oceptive sensory neurons or motor neurons. o Nfib Ebf1 FoxP2Zfhx3Zfhx4 Pr NeuroD2 Group-Z Despite the absence of sensory and motor neurons, Zfhx3 and NeuroD2 expression pat- Fig. 2. Branch points in the hierarchical comparisons define spinal neuron groups with discrete loca- terns were unaltered (fig. S11). Furthermore, tions. (A to D) (Top) Immunostaining against representative markers for group-E, -H, -N, and -Z neurons we found that Zfhx3 and NeuroD2 expression in lumbar segments at P0. All images are 20-mm cryosections. Scale bars, 100 mm. (Bottom) Spatial analysis was maintained in adult spinal neurons (P70), of representative markers displayed on right half of lumbar spinal cord. Contours indicate density of approximating the labeling pattern observed representative markers at the 10th to 90th percentiles. y axis = 0 represents the top end of the central canal, at P0 (Fig. 3F). Taken together, our findings and x axis = 0 represents the midline. (E) Matrix of pairwise mean colocalization rates between group-Z, -N, indicate that the group-N and -Z populations and -E markers. “N.M.” (blue boxes with X) indicates not measured because of antibody incompatibility have distinct timings of neurogenesis and (figs. S4 and S5). NeuN+ Nfib+ neurons were quantified to exclude Nfib+ glial cells. (F) Matrix of pairwise are present embryonically through adulthood weighted Jaccard distance between group-Z, -N, and -E markers. 1.0, no overlap; 0, complete overlap. (Fig. 3G). Hierarchical clustering dendrogram reveals closer spatial associations among marker genes within each group than between separate groups. (G) Immunostaining of Zfhx3 and NeuroD2 in lumbar spinal cord at Group-N and -Z neurons divide each cardinal P0 reveals low incidences of colocalization. Cryosection, 20 mm. Scale bar, 100 mm. This image is a composite neuron class into subsets of Fig. 2, C and D. (H) (Left) Transcriptomic hierarchy of lumbar spinal neurons. (Right) Schematic of spatial We set out to understand how group-E, group- enrichments of group-Z, -N, and -E neurons. N, and group-Z neurons identified here spatially

Osseward et al., Science 372, 385–393 (2021) 23 April 2021 3of8 RESEARCH | RESEARCH ARTICLE and molecularly intersect with the previously and Zfhx3 (group-Z) (Fig. 4A). Group-E neu- neurons derived from the group-H population characterized cardinal classes. Cre lines label- ronswerefoundtoberestrictedtothedI4/ (Fig.4,BtoF,andfigs.S12andS13).Analysis ing the cardinal classes dI1, dI3, dI4/dILA, dILA and dI5/dILB classes (figs. S12 and S13), of E12.5 scRNA-seq data confirmed that the dI5/dILB, V1, V2a, and V3 were immuno- whereas all tested cardinal classes were com- dI2, dI6, and V2b classes also were a mixture stained for Ebf1 (group-E), NeuroD2 (group-N), posedofamixtureofbothgroup-Nandgroup-Z of both group-N and group-Z neurons (fig.

ABCD Cluster identification E12.5 spinal neurons Group-N/Z neuron classifier (P0) E12.5 k-means 2 embryos E12.5 neurons Cardinal class assignment scRNA-seq Population 1 Population 2 Group-Z Group-N Indeterminant subset neurons dILA Glutamatergic dI4/dILA GABAergic Indeterminant V2b d-GABA dI6 pdI4/pdILA V1 V2a pdI6

progenitor . dI2 V3 E12.5 scRNA-seq pdI5/pdILB

dI5/dILB dI1 dI3 MN Downloaded from Tac1+ dI5

UMAP2 Phox2a+ dI5 UMAP1

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Fig. 3. The group-N and -Z division is linked to neuronal birth date. (A)Cells group-Z markers, and 84% of population 2 [(C), red cells] neurons expressed along the entire spinal cord were isolated from two E12.5 embryos from two litters group-N markers. Thus, the k-means 2 division identifies group-N and group-Z and were subjected to scRNA-seq as separate samples. These datasets were neurons at an early embryonic stage. (E) EdU was injected into multiple pregnant merged post hoc. We bioinformatically identified 2945 neurons, which were females (E10.5 to E14.5), and lumbar segments were collected from their pups selected for further analysis (fig. S8). These neurons had 3566 median genes per at P0. Representative markers Zfhx3 and NeuroD2 were used to birth date group-Z cell and 12,189 median UMIs per cell. Glutamatergic and GABAergic neurons are and group-N neurons, respectively. The peak birth date for group-Z neurons was labeled on the UMAP. (B) Cardinal classes segregate in UMAP space. Twenty- E10.5, whereas the peak for group-N neurons was E13.5. Each dot represents seven clusters were identified (fig. S8). On the basis of the top 20 differentially the colocalization rate for a single animal. Welch one-way analysis of variance expressed genes for each cluster, cardinal classes were identified. Clusters from (ANOVA) comparing every time point to every other time point, with Dunnett T3 the same cardinal class were merged (table S3). (C) Higher-order groups of spinal correction for multiple comparisons. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. neurons were identified with k-means 2 divisive clustering (Fig. 1F). Embryonic Cryosections, 20 mm. Scale bars, 100 mm. (F) Immunostaining of Zfhx3 and NeuroD2 neurons are color coded according to the first k-means 2 division. Population 1: in lumbar spinal cord at P70. The mediolateral location and marker expression 1971 cells, Jaccard similarity = 0.978; population 2: 974 cells, Jaccard similarity = patterns in adult P70 animals were similar to P0. Cryosection, 25 mm. Scale bar, 0.955. Further divisions are provided in fig. S9. (D) The first k-means 2 division 100 mm. (G) Summary. Group-Z neurons are born before group-N neurons. of embryonic neurons largely corresponded to group-N and group-Z identity Molecular markers for both groups are expressed embryonically, postnatally, and into established from P0: 79% of population 1 [(C), blue cells] neurons expressed adulthood. Markers for group-Z and group-N neurons are not coexpressed.

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Fig. 4. Each cardinal class is a ACB mixture of both group-N and Cardinal Class Analysis dI1 (Atoh1:Cre) -Z neurons. (A) Summary of mouse Transcription Neurotransmitters dI1 / Zfhx3 / NeuroD2 all dI1 (tdTomato) Zfhx3 / NeuroD2 lines used to label cardinal classes. Class Factor & Projections Each cardinal class has a characteristic transcription dI1 Atoh1 Glut Mixed ML * 500 factor expression, neurotransmitter, dI3 Isl1 Glut Ipsilateral and axon laterality profile. dI4 / dILA Ptf1a GABA Mixed (B) Atoh1:Cre was crossed to a 250 tdTomato reporter to indelibly label dI5 / dILB Lmx1b Glut Mixed dI1 neurons. Immunostaining for V1 En1 GABA Ipsilateral μm 0 NeuroD2 (group-N) and Zfhx3 V2a Vsx2 (Chx10) Glut Ipsilateral (group-Z) reveals discrete dI1 pop- -250 V3 Sim1 Glut Mixed ulations marked by the two genes. 03006000300600 Asterisk in the dorsal horn μm μm indicates ectopic sensory fibers from the DRG (29). Cryosection, 20 mm. D dI3 (Isl1) dI4/dILA (Ptf1a) dI5/dILB (Lmx1b) V1 (En1) V2a (Chx10) V3 (Sim1) Scale bar, 100 mm. (C) (Left) Contours indicate density of dI1 neurons at the 10th to ML

90th percentiles. Mediolateral 500 Downloaded from density displayed above a two-dimensional contour plot. 250 (Right) Contours indicate densities of group-N and group-Z dI1 μm 0 neuronsat50thto90thpercentiles. -250 http://science.sciencemag.org/

Group-N dI1 neurons were all cardinal class (tdTomato) generally located medially, whereas 0 300 600 0 300 600 0 300 600 030060003006000300600 group-Z dI1 neurons were located μm μm μm μm μm μm laterally. (D) Contours and mediolateral densities of indicated cardinal classes. Contours indicate E density of cardinal class neurons at the 10th to 90th percentiles.

Dorsoventral densities are provided ML 500 in fig. S14. (E) Distributions of group- N and group-Z neurons within each

250 on April 30, 2021 cardinal class. Contours indicate density of interneuron μm 0 subpopulations at the 50th to 90th Zfhx3 / NeuroD2 percentiles. Each cardinal class -250 was split into medial group-N and lateral group-Z neurons. (F) Quantifi- 0 300 600 0 300 600 0 300 600 030060003006000300600 cation of group-Z and group-N neu- μm μm μm μm μm μm rons within each cardinal class. Each dot indicates one animal. FGcolocalization H The ratio of group-N to -Z neurons varies across cardinal classes. Zfhx3 (Group-Z) NeuroD2 (Group-N) lumbar neurons N-Z Division (G) Summary of the cardinal class (ML positioning) types within the hierarchical dI1 Group-N Group-Z comparisons of neurons. (H) Stylized Group-E Group-H dI3 overview of cell spatial relationships dI5 defined by coexpression of cardinal dI4 Group-N Group-Z dI4

and N-Z markers. Cardinal classes Glut have stereotyped dorsoventral GABA dI1 dI5 settling positions. Neurons compos- dI3 Glut ing each cardinal class are split into Glut V1 GABA medial group-N and lateral group-Z GABA V3 Cardinal Class cells. V2a (DV positioning) V2a dI5-E dI4-E dI1-N V1-N dI1-Z V1-Z dI3-N dI4-N dI3-Z dI4-Z V1 V3 dI5-N dI5-Z V2a-N V2a-Z 0 0 0 20 4 6 V3-N V3-Z Group-N/Z subtype / cardinal class (%)

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A B C CTB-555 Group-Z enriched GO terms 1 DEF G SYNAPTIC SIGNALING Ina CTB injection sites AXON DEVELOPMENT Nefh 0 D. forebrain (thalamus) CELL CELL ADHESION Nefl E. cerebellum (vermis) SYNAPSE ORGANIZATION Nefm −1 Group-ZGroup-NGroup-E F. medulla NEURON PROJECTION GUIDANCE G. cervical spinal cord 0246810 enrichment score D E spinothalamic neurons spinocerebellar neurons

spinothalamic neurons spinothalamic neurons 100 *** spinocerebellar neurons spinocerebellar neurons 80 **** Zfhx3 NeuroD2 ** Zfhx3 NeuroD2 ****

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60 60

40 40

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0 0 Zfhx3Ebf1 NeuroD2 Prox1 Zfhx3Ebf1 NeuroD2 Prox1 EZ N EZ N

Fig. 5. Projection and long-range neurons express group-Z markers. Each dot on the graph indicates the colocalization rate of one animal. Cryosections, on April 30, 2021 (A) Neuronal morphology-related GO terms are enriched in group-Z compared 20 mm. Scale bars, 100 mm. Welch one-way ANOVA comparing Zfhx3 to every with group-N neurons (table S4). (B) Heatmap of neurofilament genes expressed in other marker, with Dunnett T3 correction for multiple comparisons. *P ≤ 0.05, spinal neurons showing enrichment in group-Z neurons (log2 scale). (C)Schematic **P ≤ 0.01, ***P ≤ 0.001. (D) Spinothalamic neurons within cervical segments of retrograde tracing analysis. CTB was injected into the forebrain (thalamus), (region of greatest abundance) expressed group-Z markers. Spinothalamic cerebellum (vermis), medulla, and cervical spinal cord, respectively, to retrogradely neurons within lumbar segments are shown in fig. S17. (E) Spinocerebellar neurons label long-range projection neurons within the spinal cord. Injections were done in within lumbar segments expressed group-Z markers. Additional markers are neonates, and tissue was collected 4 to 5 days later. (D to G) Immunostaining provided in fig. S18. (F) Spinomedullary neurons within lumbar segments expressed against group-Z (Zfhx3), -E (Ebf1), and -N (NeuroD2 and Prox1) markers combined group-Z markers. Additional markers are provided in fig. S19. (G) Cervicolumbar with indicated CTB labeling. White arrowheads indicate group-Z long-range neurons. neurons expressed group-Z markers. Additional markers are provided in fig. S20.

S13) (13). Moreover, the group-N neurons from Local versus long-range projection neuron a variety of inputs (for example, dorsal root each cardinal class were always medial to the types underlie the N-Z division ganglia sensory, descending serotonergic, dI1, group-Z cells (Fig. 4, C to E and H, and fig. S14) To further understand the cellular features of dI4-6, V1, and V3) (fig. S16, B and C) (24). Gene (9, 10, 20). Although each cardinal class con- group-N and group-Z neurons, we explored Ontology (GO) term analysis revealed that tained both group-N and group-Z neurons, the the connectivity patterns of spinal neurons. group-Z neurons were enriched for genes ratio of these types varied within each class We first tested whether the group-N and involved in axon and synapse development, (Fig. 4, F and H, and figs. S13 and S15) (10, 20). group-Z neurons represented premotor inter- including high neurofilament (Nef-l, -m, and -h) Group-N and group-Z neurons do not obey neurons, using monosynaptic rabies virus expression (Fig. 5, A and B). Consequently, we cardinal class divisions; rather, all cardinal tracing. We found that both the group-N and speculated that group-Z neurons may contain classes are a mix of group-N and group-Z cells group-Z populations contained ipsi- and con- one or more populations of long-range projec- with discrete mediolateral positions. Thus, the tralateral premotor neurons (fig. S16A) (21–23). tion neurons (25). Subsets of neurons from group-N–group-Z division is an orthogonal Next, using immunostaining and mouse genet- multiple cardinal classes have been shown to molecular and spatial axis that intersects with ics to visualize presynaptic terminals, we found share projection targets, further raising the cardinal class identity (Fig. 4H). that both group-N and group-Z neurons receive possibility that group-Z neurons represent

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A B Progenitor Domain DV location N-Z Division N-Z Division Cardinal Wnt Class Bmp Group-N Group-Z DV NeuroD2, Nfib, Prox1 Zfhx3, Zfhx4, FoxP2, Otp Birthdate Shh ipsi / contra Local Projection

Lmx1b dI5 Thalamus Ptf1a dI4

ML Atoh1 dI1 Cerebellum Isl1 dI3

Sim1 V3 Brainstem (LRN) Chx10 Cardinal Class V2a ML location e.phys. property En1 V1 Cervicolumbar

Glut / GABA projection range Downloaded from

Fig. 6. A unified taxonomic index of spinal neurons. (A) Summary of and green lines). (B) Summary of neuronal subsets defined by coexpression relationships of neuronal attributes. Neuronal subtypes can be viewed as a of cardinal class markers and N-Z labels. This matrix provides a simple composite of distinct properties that fall into a range of categories (circles). labeling index to define neuronal subsets. The postsynaptic targets of group-Z Neuronal subtypes arise by joining different combinations of these attributes. neurons within each cardinal population are predicted from projection

Cardinal class and N-Z markers simplify the task of defining neuronal neuron–tracing studies (26, 29, 31, 37, 38). Local connections and collaterals http://science.sciencemag.org/ subtypes because both correspond to multiple neuronal features (magenta are not shown (26, 27). long-range projections across neuronal classes with axon projection in the thoracic region. N and group-Z is not absolute, and there is (7, 9, 26, 27). We labeled spinothalamic, spino- Namely, a fraction of spinocerebellar projection sparse intermingling of NeuroD2+ and Zfhx3+ cerebellar, spinomedullary, and lumbocervical Clarke’s column neurons was found to express neurons. To further test the relationship be- projection neurons with retrograde tracer chol- the group-N marker NeuroD2 (fig. S18) (28). tween group-N and group-Z markers and their era toxin subunit b (CTB) in combination with The reason for this discrepancy is unclear; how- axonal projection patterns, we examined the group-E, -N, and -Z marker immunostaining ever, these cells form a distinct medial nucleus marker profile of projection neurons in this

(Fig. 5C). Although group-N and group-Z neu- with unusual molecular features, such as the region of the spinal cord. Retrograde fills of rons are approximately equal in number (fig. expression of Vglut1 rather than Vglut2 (29, 30). projection neurons preferentially labeled the on April 30, 2021 S6), the long-range projection neurons in both Previous gene profiling of Clarke’s column Zfhx3+ neurons but not the NeuroD2+ neurons the lumbar and cervical cord were preferentially neurons also revealed that they cluster sepa- (Fig. 5, E to G). Thus, the molecular features enriched for Zfhx3+ neurons (Fig. 5, D to G, and rately from other thoracic spinal neurons (28). that distinguish group-N from group-Z neurons figs. S17 to S20). Because the single marker Zfhx3 The non–Clarke’s column spinocerebellar reveal a core phenotypic difference: Group-N does not label all group-Z neurons, we also per- neurons in the thoracic cord express group-Z (and group-E) neurons are restricted to form- formed the supraspinal labeling and immu- genes that are typical for projection neurons ing local connections within the spinal cord, nostaining with additional group-Z markers, (fig. S18) (28). whereas group-Z neurons can also form long- including Zfhx4, Otp, and Foxp2. We found In principle, the correlation between group- range connections with distant targets (Fig. 6). that multiple group-Z markers identify addi- Z markers and projection neuron labeling might tional long-range projection neurons, providing have arisen as a byproduct of long-range neu- Discussion further evidence that many group-Z cells project rons predominantly being located laterally In this study, we sought to identify a unifying long axons (figs. S18 and S19). By contrast, within the spinal cord where group-Z markers logic for spinal neuron diversification. We used markers for group-N (NeuroD2 and Prox1) are expressed for independent reasons. There- a top-down approach to identify the conserved and group-E (Ebf1) rarely (0 to 7.9%) labeled fore,wesoughttodefinetheaxonprojection genetic relationships among heterogeneous long-range neurons (Fig. 5, D to G, and figs. pattern of group-Z neurons in atypical loca- cell types and used the branch points of S17 to S20). As a positive control, injection of tions within the spinal cord. A sparse popula- this hierarchical system to find marker genes CTB directly into the cord labeled group-N tion of Zfhx3+ neurons were situated in the that were linked to neuronal birth date, cyto- neurons near the injection site (fig. S20). Taken very superficial lamina of the spinal cord (Figs. architecture, neurotransmitter, and projection together, our labeling studies indicate that 2D and 3E). The superficial Zfhx3+ neurons status. The divide between group-E neurons group-Z neurons are composed of long-range were labeled after injection of CTB into the marked by Ebf1 and group-H cells marked by projecting cells, whereas group-N and group-E thalamus (Fig. 5D). These unusual Zfhx3+ pro- Hoxc10 corresponded to a split between sen- neurons appear to be preferentially composed jection neurons were further confirmed to rep- sory and motor circuits, respectively (Fig. 2H). of local interneurons. resent spinothalamic cells by using Nk1r/Tacr1 The H-neuron population was further divided Although most of our studies focused on labeling and were also born early like other into group-N and -Z types, in which NeuroD2 the lumbar spinal cord, we found one counter- group-Z neurons (Fig. 3E and fig. S17) (5, 31). labeled medial, late born, locally projecting example to the group-N–group-Z correlation Near the midline, the boundary between group- interneurons and Zfhx3 marked lateral, early

Osseward et al., Science 372, 385–393 (2021) 23 April 2021 7of8 RESEARCH | RESEARCH ARTICLE

born, projection neurons (Figs. 2H, 3G, and 6, genetic status (Fig. 6B and fig. S21). By identify- 35. L. C. Greig, M. B. Woodworth, M. J. Galazo, H. Padmanabhan, AandB). ing markers associated with diversification for J. D. Macklis, Nat. Rev. Neurosci. 14, 755–769 (2013). – The N-Z division giving rise to local versus the purpose of expanding cellular functions— 36. C. Q. Doe, Annu. Rev. Cell Dev. Biol. 33, 219 240 (2017). — 37. R. B. Roome et al., Cell Rep. 33, 108425 (2020). projection neurons may have occurred early in this case, evolution and development it 38. L. Ruder, A. Takeoka, S. Arber, Neuron 92,1063–1078 in evolution because the genetic signatures for may be possible to create more unified and (2016). these two groups are conserved across broad practical systematics for identifying function- ACKNOWLEDGMENTS populations of spinal neurons. Consistent with ally different cell types in many systems. We thank K. Lettieri, B. O’Leary, H. Forman, A. J. 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Osseward et al., Science 372, 385–393 (2021) 23 April 2021 8of8 Conserved genetic signatures parcellate cardinal spinal neuron classes into local and projection subsets Peter J. Osseward II, Neal D. Amin, Jeffrey D. Moore, Benjamin A. Temple, Bianca K. Barriga, Lukas C. Bachmann, Fernando Beltran Jr., Miriam Gullo, Robert C. Clark, Shawn P. Driscoll, Samuel L. Pfaff and Marito Hayashi

Science 372 (6540), 385-393. DOI: 10.1126/science.abe0690

Neuronal identities Neurons of the mouse spinal cord can be identified by any of several metrics, including what neurotransmitters

they use, what cells they connect to, where they are located, and what neuroprogenitor gave rise to them. Osseward et Downloaded from al. generated a different metric, genetic signatures, and identified classes of local and projection neurons that were otherwise heterogeneous by other classification systems. With this focus on a cell's genetic signature, its neurotransmitter phenotype, which is accessible by a variety of transcriptional routes, can be seen as a parallel to convergent evolution in development. Science, this issue p. 385 http://science.sciencemag.org/

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