A harmonized atlas of mouse spinal cord cell types and their spatial organization

May combine two claims.

The authors validated a combinatorial marker code for each neuronal cell type and mapped their spatial distributions in the adult spinal cord.

“We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord.”

May combine two claims.

The authors generated an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework.

“Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework.”

May combine two claims.

The three studies that FACS sorted neurons from the spinal cord predominantly gave rise to cells in the neuronal sub-clusters, as well as non-neural cells that most likely represented doublets.

“The three studies that FACS sorted neurons from the spinal cord (Hayashi, Haring, and Baek datasets) predominantly gave rise to cells in the neuronal sub-clusters, as well as non-neural cells that most likely represented doublets.”

May combine two claims.

The authors present SeqSeek, a web-based resource for querying the data by gene or cell type and for accessing an automated classification algorithm of any spinal cord cell or nucleus from raw sequencing data.

“We now present SeqSeek, a web-based resource for querying this data by gene or cell type and for accessing automated classification algorithm of any spinal cord cell or nucleus from raw sequencing data.”

May combine two claims.

Co-integration with embryonic cell types allowed inference of putative lineage relationships for each postnatal cell type and uncovered complex convergent contributions from multiple lineages to many cell types.

“Co-integration with embryonic cell types allowed us to infer putative lineage relationships for each postnatal cell type and uncovered complex convergent contributions from multiple lineages to many cell types.”

May combine two claims.

The motoneuron family includes alpha motoneurons (MNa) with relatively higher levels of Poln and Spp134, gamma motoneurons (MNg) with relatively higher levels of Esrrg and Htr1f, and preganglionic cells (PGC) expressing Gfra3, Nos1, and Fbn2.

“The motoneuron (MN) family includes alpha motoneurons (MNa) which had relatively higher levels of Poln and Spp134, gamma motoneurons (MNg) which had relatively higher levels of Esrrg and Htr1f35, and the related preganglionic cells (PGC) which expressed Gfra3, Nos1, and Fbn236,37.”

• cites [35] Friese, A. et al. Gamma and alpha motor neurons distinguished by expression of transcription factor Err3. Proc. Natl Acad. Sci. USA106, 13588–13593 (2009). sourceNot checked yet

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• cites [36] Schaller, S. et al. Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons. Proc. Natl Acad. Sci. USA 114, E2486–E2493 (2017). sourceNot checked yet

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• cites [37] Springell, D. A., Powers-Martin, K., Phillips, J. K., Pilowsky, P. M. & Goodchild, A. K. Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity identifies a novel subpopulation of sympathetic preganglionic neurons. Neuroscience 133, 583–590 (2005). sourceNot checked yet

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May combine two claims.

Dorsal clusters showed very high robustness with the bootstrapped co-clustering test, whereas mid and ventral clusters showed low to moderate robustness.

“Dorsal clusters showed very high robustness with this measure, whereas mid and ventral clusters showed low to moderate robustness.”

May combine two claims.

Integration-adjusted gene expression values markedly improved the ability to identify relationships between the clusters of the original studies and improved the top correlation score for each original cluster.

“Moreover, the integration-adjusted gene expression values markedly improved the ability to identify relationships between the clusters of the original studies and also improved the top correlation score for each original cluster (Supplementary Fig. 1c, d).”

May combine two claims.

Putative dorsal clusters were well separated from each other by long dendrogram branches, while putative mid and ventral clusters were much closer to each other in the reduced gene expression space.

“This analysis also revealed that putative dorsal clusters were well separated from each other by long dendrogram branches, while putative mid and ventral clusters were much closer to each other in this reduced gene expression space (Fig. 3a).”

May combine two claims.

The work revealed striking differences between dorsal and ventral neuronal cell types, both in their cell-type relationships and molecular trends.

“This work revealed striking differences between dorsal and ventral neuronal cell types, both in their cell-type relationships and molecular trends.”

May combine two claims.

The authors tested a range of automated classification algorithms and identified a two-tiered model based on label transfer and neural networks as the best method for classifying spinal cord cell types.

“Finally, we tested a range of automated classification algorithms and identified a two-tiered model based on label transfer and neural networks as the best method for classifying spinal cord cell types.”

May combine two claims.

High throughput studies detected fewer genes per cell/nucleus than single well technical approaches, and studies using cells detected more genes per cell/nucleus but had higher levels of immediate early gene and stress gene expression than studies using nuclei.

“A comparison across studies revealed that these high throughput studies detected fewer genes per cell/nucleus than studies that used single well technical approaches (the Hayashi, Haring, and Baek datasets), and studies that used cells (the Hayashi, Haring, Zeisel, and Baek datasets) detected more genes per cell/nucleus but had higher levels of immediate early gene and stress gene expression than”

May combine two claims.

The merged dataset contains more cells and nuclei than were analyzed in the original studies and a uniform set of genes.

“As a result, this merged dataset contains more cells and nuclei than were analyzed in the original studies and a uniform set of genes.”

The quote only partly states this claim.

The Sathyamurthy dataset was the only source of Schwann and peripheral glia cells.

“The only study to dissect the spinal cord including the dorsal and ventral spinal roots (the Sathyamurthy dataset) was the only source of Schwann and peripheral glia cells that would be located in these roots.”

May combine two claims.

The initial motoneuron family comprised only nuclei from the Sathyamurthy and Rosenberg datasets and contained only 565 MNs, and no refined sub-populations of motoneurons could be detected.

“This family was only comprised of nuclei from the Sathyamurthy and Rosenberg datasets but we could not detected refined sub-populations of motoneurons. However, this included only 565 MNs.”

May combine two claims.

Among the three studies that examined all cell types, the early postnatal Rosenberg study showed enrichment of immature cells of oligodendrocyte lineage relative to the adult Sathyamurthy study, while the adolescent Zeisel study showed an intermediate distribution.

“Moreover, among the three studies that examined all cell types, the early postnatal Rosenberg study showed enrichment of immature cells of oligodendrocyte lineage relative to the adult Sathyamurthy study, while the adolescent Zeisel study showed an intermediate distribution.”

May combine two claims.

The authors observed 20 dorsal excitatory clusters, 14 dorsal inhibitory clusters, 10 deep dorsal/mid excitatory clusters, 7 deep dorsal/mid inhibitory clusters, 8 ventral excitatory clusters, 6 ventral inhibitory clusters, 3 cholinergic motoneuron clusters, and 1 cluster of cerebrospinal fluid contacting neurons.

“We observed 20 dorsal excitatory clusters, 14 dorsal inhibitory clusters, 10 deep dorsal/mid excitatory clusters, 7 deep dorsal/mid inhibitory clusters, 8 ventral excitatory clusters, 6 ventral inhibitory clusters, 3 cholinergic motoneuron clusters, and 1 cluster of the cerebrospinal fluid contacting neurons (CSF-cN).”

May combine two claims.

Using Seurat integration, cells and nuclei from all six studies were spatially interposed in a UMAP visualization of principal component space and separated into groupings that each expressed a panel of well-established cell type markers.

“With this approach, the cells and nuclei from all six studies were spatially interposed in a UMAP visualization of principal component space (Fig. 1c) and separated into groupings that each expressed a panel of well-established cell type markers such as Snap25 (neurons), Mbp (oligodendrocytes), Aqp4 (astrocytes), and Ctss (microglia) (Fig. 1d, e).”

• cites [22] Nguyen, Q. H., Pervolarakis, N., Nee, K. & Kessenbrock, K. Experimental considerations for single-cell RNA sequencing approaches. Front Cell Dev. Biol. 6, 108 (2018). sourceNot checked yet

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• cites [23] Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902.e21 (2019). sourceNot checked yet

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May combine two claims.

Cells or nuclei from each study were segregated from each other almost completely, indicating that the study of origin is a major source of variability in the dataset.

“Unfortunately, the cells or nuclei from each study were segregated from each other almost completely, indicating that the study of origin is a major source of variability in the dataset (Fig. 1b).”

May combine two claims.

The authors validated the differentially expressed patterns of several genes using in situ hybridization and at the protein level for CAMK2α and WFA-lectin, confirming predicted gene expression signatures that would differentially regulate plasticity in the dorsal and ventral horns.

“We validated the differentially expressed patterns of several of these genes using in situ hybridization and also at the protein level for CAMK2α and WFA-lectin to reveal perineuronal nets, thereby confirming predicted gene expression signatures that would differentially regulate plasticity in the dorsal and ventral horns of the spinal cord (Fig. 3c–f).”

May combine two claims.

When either all genes or the top 500 highly variable genes were analyzed, there were weak overall correlations and very few alignments between clusters from different studies.

“When either all genes or the top 500 highly variable genes were analyzed, there were weak overall correlations and very few alignments between clusters from different studies (Supplementary Fig. 1a, b, d).”

May combine two claims.

Excit-17 included almost exclusively nuclei from the Rosenberg dataset and showed enriched expression of Dcx.

“Excit-17 included almost exclusively nuclei from the Rosenberg dataset and showed enriched expression of the immature neuron marker Dcx.”

May combine two claims.

Excit-5 expressed Pvalb and Cck, Excit-6 expressed Rorb and Cpne4, and Excit-7 had only nuclei from the Rosenberg dataset and expressed Dcx, suggesting an immature phenotype.

“Excit-5 also expressed Pvalb and Cck, Excit-6 expressed Rorb and Cpne4, and Excit-7 was distinguished by having only nuclei from the Rosenberg dataset and expressed the immature neuron marker Dcx, suggesting an immature phenotype.”

May combine two claims.

The Reln family clusters expressed enriched levels of Car12 (particularly Excit-9 and Excit-10), Trhr (Excit-8), Npr1 (Excit-9 and Excit-10), Nmur2 (Excit-10), and Grp (Excit-9).

“These clusters expressed enriched levels of Car12 (in particular in Excit-9 and Excit-10), the neuropeptide receptors Trhr (Excit-8), Npr1 (Excit-9 and Excit-10), and Nmur2 (Excit-10), and the neuropeptide Grp (Excit-9).”

May combine two claims.

Inhib-6 and Inhib-7 expressed Cdh3 and were distinguished by co-expression of Kcnip2 and Pvalb in Inhib-7.

“Inhib-6 and Inhib-7 expressed Cdh3 and were distinguished by co-expression of Kcnip2 and Pvalb in Inhib-7.”

Classified as probably not a substantial claim.

The Prkcg family was defined by Prkcg together with Cck and Trh (Excit-3) and Nts (Excit-4).

“Prkcg is a classic marker gene in the spinal cord and defined this family together with the neuropeptides Cck and Trh (Excit-3) and Nts (Excit-4).”

May combine two claims.

Inhib-8 contained only low levels of Cdh3 in this analysis, but Cdh3 expression was confirmed by in situ hybridization, and Inhib-8 was included in the Cdh3 family based on proximity in principal component space; Inhib-8 also expressed Klhl14.

“While Inhib-8 contained only low levels of Cdh3 in this analysis, Cdh3 expression was confirmed by in situ hybridization and this cluster was included in this family based on proximity in principal component space as reflected in the UMAP and dendrogram analysis. Inhib-8 also expressed Klhl14.”

May combine two claims.

The Cpne4 dorsal excitatory family comprised Excit-1 and Excit-2; Excit-1 was a rare subset expressing Dach2, and Excit-2 was more prevalent and co-expressed Prkcg and Cbln2.

“The Cpne4 dorsal, excitatory family was comprised of Excit-1 and Excit-2. Excit-1 was a rare subset, both in the harmonized clusters and in the in situ counts, that also expressed Dach2. Excit-2 was more prevalent and co-expressed Prkcg as well as Cbln2.”

May combine two claims.

The Megf11 cluster (Excit-20) displayed features of dorsal and mid excitatory neurons, being located in lamina 4/5 and grouped with mid neurons in principal component space.

“The Megf11 cluster (Excit-20) displayed features of dorsal excitatory neurons and mid excitatory neurons, being located in lamina 4/5 and being grouped with mid neurons in principal component space in the UMAP and dendrogram analysis.”

May combine two claims.

There were two discrepancies between sequencing and in situ hybridization data in the Prkcg family: Cck was present at high levels in Excit-3 in sequencing but not detected in most Prkcg-expressing cells of adult spinal cord, and Prkcg was not enriched in Excit-4 in sequencing but was readily detected with Nts and Calb1 in adult tissue.

“Of note, there were two discrepancies in this family between the sequencing data and the in situ hybridization data: Cck was present at high levels in the Excit-3 in the sequencing data but we did not detect Cck in most Prkcg-expressing cells of the adult spinal cord and Prkcg was not enriched in Excit-4 in the sequencing data but was readily detected with Nts and Calb1 in this cluster in adult t”

May combine two claims.

Inhib-1 expressed Sorcs3, Inhib-3 expressed Nppc and Nrgn, Inhib-4 expressed Rxfp2, Inhib-5 did not express these other genes, and Inhib-2 expressed Sorcs3 and Adamts5.

“In addition to Rorb, Inhib-1 expressed Sorcs3, Inhib-3 expressed Nppc as well as Nrgn, Inhib-4 expressed Rxfp2, and Inhib-5 did not express these other genes. Inhib-2 expressed Sorcs3 and Adamts5.”

May combine two claims.

Both Excit-3 and Excit-4 expressed Calb1, although Calb1 was not specific to these clusters.

“Both subsets also expressed Calb1, although it was not specific to these clusters.”

May combine two claims.

The CSF-cN cluster was very distinct from other neuronal populations, inhibitory, and expressed Sox2, Gata2, and Gata3, suggesting an immature phenotype.

“This cluster was very distinct from other neuronal populations, inhibitory, and also expressed the early neuron marker Sox2 and the V2b lineage markers Gata2 and Gata3, suggesting an immature phenotype.”

May combine two claims.

The Rreb1 dorsal excitatory family comprised Excit-12 and Excit-13, which expressed Satb1 and either Zim1 (Excit-12) or Nmur2 and Crh (Excit-13).

“The Rreb1 dorsal, excitatory family was comprised of Excit-12 and Excit-13. These clusters also expressed Satb1 and either Zim1 (Excit-12) or Nmur2 and Crh (Excit-13).”

May combine two claims.

PGCs were clustered into 23 subtypes that varied by spinal cord level, and MNa were clustered into 14 subtypes that also varied by spinal level.

“PGCs were clustered into 23 subtypes that varied by spinal cord level and MNa were clustered into 14 subtypes that also varied by spinal level.”

May combine two claims.

Within the Sox5 family, Excit-14 and Excit-15 were slightly separated and similar to Rreb1 family clusters, expressing Col5a2 (Excit-14) or Col5a2 and Enpp1 (Excit-15).

“Within this family, Excit-14 and Excit-15 were slightly separated and also similar to the Rreb1 family clusters and expressed Col5a2 (Excit-14) or Col5a2 and Enpp1 (Excit-15).”

May combine two claims.

Excit-16, Excit-18, and Excit-19 expressed Tac1 (Excit-16), Nmu-hi/Tac2-lo (Excit-18), and Tac2hi/Nmu-lo (Excit-19).

“Excit-16, Excit-18, and Excit-19 expressed the neuropeptides Tac1 (Excit-16), Nmu-hi/Tac2-lo (Excit-18), and Tac2hi/Nmu-lo (Excit-19).”

May combine two claims.

Each cluster in the Pdyn family expressed Pdyn; Inhib-10 also expressed Gal and Mlxipl, and Inhib-11 also expressed Gal only; the family clusters also expressed Rorb and Nrgn.

“Each of these clusters expressed Pdyn, while Inhib-10 also expressed Gal and Mlxipl and Inhib-11 also expressed Gal only. Of note, the clusters in this family also expressed Rorb and Nrgn.”

May combine two claims.

Dual contributions of dI5.5 and dI2.1 to Excit-20 and of V1 and V2b to Inhib-27 were observed.

“Dual contributions of dI5.5 and dI2.1 to Excit-20 and of V1 and V2b to Inhib-27 were also observed.”

• AssumptionRests on an assumption: Developmental lineage is a powerful and influential framework for categorizing spinal neurons.

May combine two claims.

The model showed very high confidence scores for correct predictions, but performance varied with cell type prevalence.

“The model showed very high confidence scores for correct predictions; however, performance varied with cell type prevalence suggesting a target for improving the model in the future (Supplementary Fig. 11).”

• AssumptionRests on an assumption: There is no existing gold standard for spinal cord cell identities.
• AssumptionRests on an assumption: The harmonized analysis cell type labels are adequate as training labels for machine learning classifiers.

May combine two claims.

Excit-35 also expressed Vamp1, Pou3f1, Shox2, and Pou6f2, and Excit-36 expressed Esrrg.

“Excit-35 also expressed Vamp1, Pou3f1, Shox2, and Pou6f2 and Excit-36 expressed Esrrg.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Nearly all cells and nuclei in the remaining ME family were from the Rosenberg and Sathyamurthy datasets, and Excit-26 was predominantly from the Rosenberg dataset.

“Of note, nearly all of the cells and nuclei in this family were from the Rosenberg and Sathyamurthy datasets and Excit-26 in particular was predominantly from the Rosenberg dataset.”

May combine two claims.

A neural network model with one hidden layer (256 nodes) and an SGD optimizer showed the best performance (overall accuracy 80%) for the second tier and was selected for further work.

“A neural network model with one hidden layer (256 nodes) and an SGD optimizer showed the best performance (overall accuracy of 80%) and was selected for further work.”

• AssumptionRests on an assumption: There is no existing gold standard for spinal cord cell identities.
• AssumptionRests on an assumption: The harmonized analysis cell type labels are adequate as training labels for machine learning classifiers.

May combine two claims.

Pou6f2 was expressed in the deep dorsal horn and dorsal part of the ventral horn and was enriched in mid-excitatory (Excit-21, Excit-28, Excit-30), ventral excitatory (Excit-34 and Excit-35), and ventral inhibitory (Inhib-24) clusters located within this domain.

“For example, Pou6f2 was expressed in the deep dorsal horn and in the dorsal part of the ventral horn and was enriched in mid-excitatory (Excit-21, Excit-28, and Excit 30), ventral excitatory (Excit-34 and Excit-35), and ventral inhibitory (Inhib-24) clusters that are located within this domain.”

May combine two claims.

The Chat dorsal inhibitory cluster Inhib-14 was a deep dorsal (lamina 4), inhibitory and cholinergic population that also expressed Nos1.

“The Chat dorsal inhibitor cluster Inhib-14 was a deep dorsal (lamina 4), inhibitory and cholinergic population and also expressed Nos180–82.”

• cites [80] Laing, I., Todd, A. J., Heizmann, C. W. & Schmidt, H. H. Subpopulations of GABAergic neurons in laminae I-III of rat spinal dorsal horn defined by coexistence with classical transmitters, peptides, nitric oxide synthase or parvalbumin. Neuroscience 61, 123–132 (1994). sourceNot checked yet

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• cites [81] Kluchová, D. et al. Partial colocalization of NADPH-diaphorase and acetylcholinesterase positivity in spinal cord neurons. Physiol. Res. 49, 151–155 (2000).Not checked yet

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• cites [82] Mesnage, B. et al. Morphological and functional characterization of cholinergic interneurons in the dorsal horn of the mouse spinal cord. J. Comp. Neurol. 519, 3139–3158 (2011). sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Nearly all cells and nuclei in the ME/Lmx1b family were from the Rosenberg and Sathyamurthy datasets.

“Of note, nearly all of the cells and nuclei in this family were from the Rosenberg and Sathyamurthy datasets.”

May combine two claims.

Nfib was expressed in the medial deep dorsal horn (mid) spinal cord and was enriched in both excitatory (Excit-23, Excit-25, Excit-30) and inhibitory (Inhib-15 and Inhib-21) clusters.

“Similarly, Nfib was expressed in the medial deep dorsal horn (mid) spinal cord and was enriched in both excitatory (Excit-23, Excit-25, and Excit-30) and inhibitory (Inhib-15 and Inhib-21) clusters.”

May combine two claims.

Inhib-12 and Inhib-13 expressed Npy and were distinguished by low levels of Vgf (Inhib-12) or expression of Qrfpr (Inhib-13).

“These clusters expressed Npy and were distinguished by low levels of Vgf (Inhib-12) or by expression of Qrfpr (Inhib-13).”

May combine two claims.

Other markers subdivided the ME/Lmx1b clusters: Nms (Excit-21), Bcl11a (Excit-22 through Excit-25), Satb1 and Cdh23 (Excit-23, Excit-24, and Excit-25), Cep112 (Excit-23 and Excit-24), and Prox1 (Excit-25).

“Other markers sub-divided the clusters in a combinatorial manner, including Nms (Excit-21), Bcl11a (Excit-22 through Excit-25), Satb1 and Cdh23 (Excit-23, Excit-24, and Excit-25), Cep112 (Excit-23 and Excit-24), and Prox1 (Excit-25).”

May combine two claims.

All three strategies (label transfer, SVM, neural network) performed well on coarse cell type classification, with label transfer showing the best performance (overall accuracy 89%), followed by neural network (83%) and SVM (80%).

“All three strategies performed well, with label transfer showing the best performance (overall accuracy of 89%), followed by the neural network (83%), and then the SVM (80%) (Fig. 6a and Supplementary Table 6).”

• AssumptionRests on an assumption: There is no existing gold standard for spinal cord cell identities.
• AssumptionRests on an assumption: The harmonized analysis cell type labels are adequate as training labels for machine learning classifiers.

May combine two claims. The quote only partly states this claim.

Co-integration of the merged dataset of six postnatal studies with neurons from a single-cell sequencing atlas of e9.5 to e13.5 mouse spinal cord and analysis of seven studies together revealed long trajectories of embryonic cells leading towards particular cluster families, allowing inference of developmental relationships.

“We found that long trajectories of embryonic cells led towards particular cluster families, which allowed us to infer developmental relationships (Fig. 5b).”

• AssumptionRests on an assumption: Developmental lineage is a powerful and influential framework for categorizing spinal neurons.

May combine two claims.

The molecular identities of the cardinal classes were confirmed by classic marker analysis, and these marker genes often extended into the postnatal cell type domains.

“The molecular identities of the cardinal classes were confirmed by classic marker analysis (Fig. 5c) and these marker genes often extended into the postnatal cell type domains as well.”

• AssumptionRests on an assumption: Developmental lineage is a powerful and influential framework for categorizing spinal neurons.

May combine two claims.

Inhib-15 expressed Prox1, Gabra1, and Nfib; Inhib-16 expressed Gpc3 and Sema5b; Inhib-17 expressed Satb2; Inhib-18 expressed Sema5b; Inhib-19 expressed Ccbe1 and Pou6f2; Inhib-20 expressed higher levels of Tfap2b and Zfhx3; Inhib-21 expressed Nfib and had only Gad2 and not Slc6a5 and was mainly from the Rosenberg dataset.

“Inhib-15 expressed Prox1, Gabra1, and Nfib, Inhib-16 expressed Gpc3 and Sema5b, Inhib-17 expressed Satb2, Inhib-18 expressed Sema5b, Inhib-19 expressed Ccbe1 and Pou6f2, Inhib-20 expressed higher levels of Tfap2b as well as Zfhx3, and Inhib-21 expressed Nfib and was distinguished by having only Gad2 and not Slc6a5 and was mainly derived from the Rosenberg dataset.”

May combine two claims.

Excit-26 expressed Nfib, Excit-27 expressed Adamts2, Excit-28 expressed Chat and Pitx2 and likely corresponds to V0c neurons, and Excit-29 expressed Pmfbp1.

“Excit-26 expressed Nfib, Excit-27 expressed Adamts2, Excit-28 expressed Chat and Pitx2 and thus likely corresponds to V0c neurons, and Excit-29 expressed Pmfbp1.”

May combine two claims.

The Clarke's column marker gene Gdnf was expressed at very low levels in the merged dataset but was present in several Excit-30 cells.

“The Clarke’s column marker gene, Gdnf, was expressed at very low levels in the merged dataset, but was present in several Excit-30 cells.”

May combine two claims.

The analysis supported a division of deep dorsal horn excitatory neurons into two overall groups (ME/Lmx1b family more closely related to dI5, other ME clusters more closely related to dI1-dI3) and a division of ventral excitatory neurons into two groups (Excit-31 through Excit-34 closer to dI1-3 and Excit-35 through Excit-37 closer to V0, V2a, and V3 populations).

“This analysis also supported a division of deep dorsal horn excitatory neurons into two overall groups (with the ME/Lmx1b family being more closely related to dI5, while the other ME clusters are more closely related to dI1-dI3) and a division of ventral excitatory neurons into two groups (with Excit-31 through Excit-34 being closer to dI1-3 and Excit-35 through Excit-37 being closer to V0, V2a, a”

• AssumptionRests on an assumption: Developmental lineage is a powerful and influential framework for categorizing spinal neurons.

May combine two claims.

Model performance varied with the co-clustering frequency of each cluster and with the ability to identify cell types in situ; these measures can be used to set a reasonable expectation for neural network performance.

“We found that the model performance varied with the co-clustering frequency of each cluster and with the ability to identify cell types in situ and we propose that these measures can be used to set a reasonable expectation for neural network performance.”

• AssumptionRests on an assumption: There is no existing gold standard for spinal cord cell identities.
• AssumptionRests on an assumption: The harmonized analysis cell type labels are adequate as training labels for machine learning classifiers.
• AssumptionRests on an assumption: Perfect performance would require perfect and invariable biological data: discrete cell types that express completely distinct patterns of gene expression and experimental data without natural heterogeneity, doublets, low-quality cells, or other sources of indeterminate data.

May combine two claims.

Excit-37 expressed low levels of the V3 marker gene Sim1 as well as Rnf220.

“Excit-37 expressed low levels of the V3 marker gene Sim1 as well as Rnf220.”

May combine two claims.

These ventral excitatory clusters could be distinguished by Gm26673, Syt2, and Prlr (Excit-31), Mdga1 and Prlr (Excit-32), and Bnc2 and Pou6f2 (Excit-34).

“These clusters could be distinguished by Gm26673, Syt2, and Prlr (Excit-31), Mdga1 and Prlr (Excit-32), and Bnc2 and Pou6f2 (Excit-34).”

May combine two claims.

For classification of 69 neuronal sub-types, a linear model (no regularization, SGD optimizer) showed the best performance, with an overall test accuracy of 85%.

“We found that a linear model (with no regularization and with an SGD optimizer) showed the best performance, with an overall test accuracy of 85% (Fig. 6b and Supplementary Table 6).”

• AssumptionRests on an assumption: There is no existing gold standard for spinal cord cell identities.
• AssumptionRests on an assumption: The harmonized analysis cell type labels are adequate as training labels for machine learning classifiers.

May combine two claims.

Ventral clusters are much more similar to each other, with close or overlapping distributions in principal component space and overlapping gene expression patterns.

“In contrast, ventral clusters are much more similar to each other, with close or overlapping distributions in principal component space and overlapping gene expression patterns.”

May combine two claims.

Dorsal clusters are distinct from each other with clearly separated individual cell types that can be grouped loosely into families.

“Dorsal clusters are distinct from each other with clearly separated individual cell types that can be grouped loosely into families.”

May combine two claims.

Three very different methods - single-cell data clustering, multiplexed in situ hybridization, and an artificial intelligence neural network - all showed a relatively weak ability to classify ventral cell types into discrete types and a relatively strong but still imperfect ability to classify dorsal cell types.

“For example, three very different methods – single-cell data clustering, multiplexed in situ hybridization, and an artificial intelligence neural network – all showed a relatively weak ability to classify ventral cell types into discrete types and a relatively strong but still imperfect ability to classify dorsal cell types.”

May combine two claims.

Differential gene expression and gene ontology analysis revealed that learning-related genes are enriched in the dorsal horn while structural stability-related genes are enriched in the ventral horn.

“Differential gene expression and gene ontology analysis revealed that learning-related genes are enriched in the dorsal horn while structural stability-related genes are enriched in the ventral horn.”

May combine two claims.

The neural network model was at least as accurate as other methods such as Seurat-based clustering and high-content in situ hybridization and was orders of magnitude faster.

“The neural network model was at least as accurate as other methods such as Seurat-based clustering and high-content in situ hybridization and was orders of magnitude faster.”

• AssumptionRests on an assumption: There is no existing gold standard for spinal cord cell identities.
• AssumptionRests on an assumption: The harmonized analysis cell type labels are adequate as training labels for machine learning classifiers.

May combine two claims.

Several cell types were not expected to be present in the independent dataset (Schwann cells, peripheral glia, meninges 2, PGC, Excitatory-7, Excitatory-17) based on dissection method, lumbar region, and adult age; as expected, these cell types were not predicted by the two-tiered model.

“Of note, several cell types were not expected to be present in the independent dataset, including Schwann cells, peripheral glia, and meninges 2 (based on the surgical dissection method used that did not include spinal roots or outer layers of meninges) and including PGC, Excitatory-7, and Excitatory-17 (based on the lumbar region and adult age that was used). As expected, these cell types were”

• AssumptionRests on an assumption: There is no existing gold standard for spinal cord cell identities.
• AssumptionRests on an assumption: The harmonized analysis cell type labels are adequate as training labels for machine learning classifiers.

May combine two claims.

Several mid/ventral cell types were not detected in the independent dataset while two ventral clusters (Excitatory-31 and Inhibitory-27) were over-represented.

“In particular, several mid/ventral cell types were not detected in the independent dataset while two ventral clusters (Excitatory-31 and Inhibitory-27) were over-represented (Fig. 6 and Supplementary Fig. 13).”

• AssumptionRests on an assumption: There is no existing gold standard for spinal cord cell identities.
• AssumptionRests on an assumption: The harmonized analysis cell type labels are adequate as training labels for machine learning classifiers.

May combine two claims.

This work led to the discovery of gene expression signatures for plasticity in the dorsal horn and for structural stability in the ventral horn.

“This work also led to the surprising discovery of gene expression signatures for plasticity in the dorsal horn and for structural stability in the ventral horn.”

May combine two claims.

The deep dorsal horn excitatory neurons can be divided into two families, with Excit-21 through Excit-25 in one family that is likely derived from dI5 precursors and Excit-26 through Excit-29 in a looser family that includes dI1/2 and V0 precursors.

“For example, the deep dorsal horn excitatory neurons can be divided into two families, with Excit-21 through Excit-25 in one family that is likely derived from dI5 precursors and Excit-26 through Excit-29 in a looser family that includes dI1/2 and V0 precursors.”

May combine two claims.

Within the ventral horn, structural stability-related genes included components of perineuronal nets such as Tnr (Tenascin-R), as well as adhesion molecules such as Sdk1 and intracellular signaling components such as Ptpn5 (STEP).

“Within the ventral horn, this included components of perineuronal nets (which are thought to restrict plasticity and had been previously detected in the ventral spinal cord110,111) such as Tnr (Tenascin-R), as well as adhesion molecules such as Sdk1 and intracellular signaling components such as Ptpn5 (STEP) which de-phosphorylates CAMK2α, NMDA receptors, and ERK kinases33.”

• cites [110] Sorg, B. A. et al. Casting a wide net: role of perineuronal nets in neural plasticity. J. Neurosci. 36, 11459–11468 (2016). sourceNot checked yet

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• cites [111] Galtrey, C. M., Kwok, J. C. F., Carulli, D., Rhodes, K. E. & Fawcett, J. W. Distribution and synthesis of extracellular matrix proteoglycans, hyaluronan, link proteins and tenascin-R in the rat spinal cord. Eur. J. Neurosci. 27, 1373–1390 (2008). sourceNot checked yet

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• cites [33] Won, S. & Roche, K. W. Regulation of glutamate receptors by striatal-enriched tyrosine phosphatase 61 (STEP61). J. Physiol. 599, 443–451 (2021). sourceNot checked yet

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May combine two claims.

The harmonized analysis afforded a much deeper characterization of cell type relationships and robustness, the validation of these molecular distinctions in tissue, and through machine learning.

“Building on these preliminary findings, the scope of the harmonized analysis here afforded a much deeper characterization of cell type relationships and robustness, the validation of these molecular distinctions in tissue, and through machine learning.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Adding regularization lowered the gap between training and validation accuracy.

“Adding regularization helped to lower the gap between the training and validation accuracy”

Probably not a substantial claim: may not pass S3 (load-bearing).

Lowering the batch size from 128 to 32 improved validation accuracy by around 5%.

“Whereas the training was faster, validation accuracy improved by around 5% when we lowered the batch size to 32.”

Probably not a substantial claim: may not pass S3 (load-bearing).

No additional improvement was seen at a batch size of 16.

“No additional improvement was seen at a batch size of 16, so the batch size was set to 32 for the rest of the study.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All enriched gene annotation clusters from GO analysis were associated with basic cell metabolism terms such as the ribosome, metabolic pathways, and proton transport.

“GO analysis revealed that all of the enriched gene annotation clusters were associated with basic cell metabolism terms such as the ribosome, metabolic pathways, and proton transport (Supplementary Table 6).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The support vector machine pipeline achieved an overall accuracy of 80% on the validation data.

“This pipeline achieved an overall accuracy of 80% on the validation data.”

May combine two claims.

It is not clear whether the cell types from the original studies are comparable in their current forms, resulting in a fragmented set of incomplete and conflicting atlases for the spinal cord.

“Indeed, it is not clear whether the cell types from the original studies are comparable in their current forms, resulting in a fragmented set of incomplete and conflicting atlases for the spinal cord.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The general feature of lower robustness in mid and ventral clusters is consistent with previous observations and likely reflects similar patterns of gene expression amongst mid and ventral clusters.

“This general feature was consistent with previous observations and likely reflects similar patterns of gene expression amongst mid and ventral clusters1,4.”

• cites [1] Sathyamurthy, A. et al. Massively parallel single nucleus transcriptional profiling defines spinal cord neurons and their activity during behavior. Cell Rep. 22, 2216–2225 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [4] Rosenberg, A. B. et al. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science 360, 176–182 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The authors began with raw sequencing reads from each study and performed their own data processing with uniform methods and filters.

“To best compare the data from these studies, we began with the raw sequencing reads from each study and performed our own data processing with uniform methods and filters.”

May combine two claims.

Integration of publicly available datasets can be used to harmonize spinal cord sequencing data, preserve important biological differences between studies, and uncover a robust set of shared cell types.

“Together, these analyses demonstrated that integration of publicly available datasets can be used to harmonize spinal cord sequencing data, preserve important biological differences between studies, and uncover a robust set of shared cell types.”

May combine two claims.

The authors discovered general differences in the relationships between clusters in the dorsal versus the ventral horn and molecular trends that could confer differential plasticity control in these two regions.

“Thus, we discovered general differences in the relationships between clusters in the dorsal versus the ventral horn and molecular trends that could confer differential plasticity control in these two regions.”

May combine two claims.

The Npy dorsal inhibitory family comprised Inhib-12 and Inhib-13; studies suggest this family's markers identify neurons involved in mechanical itch and pain.

“The Npy dorsal, the inhibitory family was comprised of Inhib-12 and Inhib-13. Studies suggest this family’s markers identify neurons involved in mechanical itch and pain73,78,79.”

• cites [73] Boyle, K. A. et al. A quantitative study of neurochemically defined populations of inhibitory interneurons in the superficial dorsal horn of the mouse spinal cord. Neuroscience 363, 120–133 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [78] Bourane, S. et al. Gate control of mechanical itch by a subpopulation of spinal cord interneurons. Science 350, 550–554 (2015). sourceNot checked yet

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• cites [79] Tashima, R. et al. A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior. Proc. Natl. Acad. Sci. USA 118, e2021220118 (2021). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The Reln dorsal excitatory family comprised Excit-8, Excit-9, Excit-10, and Excit-11 and expressed markers indicative of a role in chemical itch sensation.

“The Reln dorsal, excitatory family was comprised of Excit-8, Excit-9, Excit-10, and Excit-11 and expressed markers indicative of a role in chemical itch sensation58–62.”

• cites [58] Bell, A. M. et al. Expression of green fluorescent protein defines a specific population of lamina II excitatory interneurons in the GRP::eGFP mouse. Sci. Rep. 10, 13176–14 (2020). sourceNot checked yet

  Not checked in the free run.

• cites [59] Christensen, A. J. et al. In vivo interrogation of spinal mechanosensory circuits. Cell Rep. 17, 1699–1710 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [60] Fatima, M. et al. Spinal somatostatin-positive interneurons transmit chemical itch. Pain 160, 1166–1174 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [61] Huang, J. et al. Circuit dissection of the role of somatostatin in itch and pain. Nat. Neurosci. 21, 707–716 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [62] Mishra, S. K. & Hoon, M. A. The cells and circuitry for itch responses in mice. Science 340, 968–971 (2013). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The Pdyn dorsal inhibitory family comprised Inhib-9, Inhib-10, and Inhib-11 and expressed markers suggestive of a role in chemical itch.

“The Pdyn dorsal, inhibitory family was comprised of Inhib-9, Inhib-10, and Inhib-11 and expressed markers suggestive of a role in chemical itch52,61,73–77.”

• cites [52] Duan, B. et al. Identification of spinal circuits transmitting and gating mechanical pain. Cell 159, 1417–1432 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [61] Huang, J. et al. Circuit dissection of the role of somatostatin in itch and pain. Nat. Neurosci. 21, 707–716 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [73] Boyle, K. A. et al. A quantitative study of neurochemically defined populations of inhibitory interneurons in the superficial dorsal horn of the mouse spinal cord. Neuroscience 363, 120–133 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [74] Brewer, C. L., Li, J., O’Conor, K., Serafin, E. K. & Baccei, M. L. Neonatal injury evokes persistent deficits in dynorphin inhibitory circuits within the adult mouse superficial dorsal horn. J. Neurosci. 40, 3882–3895 (2020). sourceNot checked yet

  Not checked in the free run.

• cites [75] Brewer, C. L., Styczynski, L. M., Serafin, E. K. & Baccei, M. L. Postnatal maturation of spinal dynorphin circuits and their role in somatosensation. Pain 161, 1906–1924 (2020). sourceNot checked yet

  Not checked in the free run.

• cites [76] Kardon, A. P. et al. Dynorphin acts as a neuromodulator to inhibit itch in the dorsal horn of the spinal cord. Neuron 82, 573–586 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [77] Serafin, E. K. et al. Transcriptional profile of spinal dynorphin-lineage interneurons in the developing mouse. Pain 160, 2380–2397 (2019). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The Prkcg dorsal excitatory family comprised Excit-3 and Excit-4 and likely corresponded to neurons involved in light static touch and allodynic pain in pathological situations.

“The Prkcg dorsal, excitatory family was comprised of Excit-3 and Excit-4 and likely corresponded to neurons involved in light static touch and allodynic pain in pathological situations46,48–56.”

• cites [46] Gatto, G. et al. A functional topographic map for spinal sensorimotor reflexes. Neuron 109, 91–104.e5 (2021). sourceNot checked yet

  Not checked in the free run.

• cites [48] Abraira, V. E. et al. The cellular and synaptic architecture of the mechanosensory dorsal horn. Cell 168, 295–310.e19 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [49] Acton, D. et al. Spinal neuropeptide Y1 receptor-expressing neurons form an essential excitatory pathway for mechanical itch. Cell Rep. 28, 625–639.e6 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [50] Bourane, S. et al. Identification of a spinal circuit for light touch and fine motor control. Cell 160, 503–515 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [51] Cheng, L. et al. Identification of spinal circuits involved in touch-evoked dynamic mechanical pain. Nat. Neurosci. 20, 804–814 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [52] Duan, B. et al. Identification of spinal circuits transmitting and gating mechanical pain. Cell 159, 1417–1432 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [53] Gutierrez-Mecinas, M., Furuta, T., Watanabe, M. & Todd, A. J. A quantitative study of neurochemically defined excitatory interneuron populations in laminae I-III of the mouse spinal cord. Mol. Pain. 12, 174480691662906 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [54] Liu, Y. et al. Touch and tactile neuropathic pain sensitivity are set by corticospinal projections. Nature 561, 547–550 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [55] Malmberg, A. B., Chen, C., Tonegawa, S. & Basbaum, A. I. Preserved acute pain and reduced neuropathic pain in mice lacking PKCgamma. Science 278, 279–283 (1997). sourceNot checked yet

  Not checked in the free run.

• cites [56] Peirs, C. et al. Dorsal horn circuits for persistent mechanical pain. Neuron 87, 797–812 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The Rorb and Adamts5 dorsal inhibitory family comprised Inhib-1, Inhib-2, Inhib-3, Inhib-4, and Inhib-5, with markers of neurons involved in dampening of dynamic touch.

“The Rorb and Adamts5 dorsal, the inhibitory family was comprised of Inhib-1, Inhib-2, Inhib-3, Inhib-4, and Inhib-5, with markers of neurons involved in the dampening of dynamic touch48,68,69.”

• cites [48] Abraira, V. E. et al. The cellular and synaptic architecture of the mechanosensory dorsal horn. Cell 168, 295–310.e19 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [68] Cui, L. et al. Identification of early RET+ deep dorsal spinal cord interneurons in gating pain. Neuron 91, 1137–1153 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [69] Gutierrez-Mecinas, M., Polgár, E., Bell, A. M., Herau, M. & Todd, A. J. Substance P-expressing excitatory interneurons in the mouse superficial dorsal horn provide a propriospinal input to the lateral spinal nucleus. Brain Struct. Funct. 223, 2377–2392 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The Cdh3 dorsal inhibitory family comprised Inhib-6, Inhib-7, and Inhib-8 and are likely involved in dampening of dynamic touch and mechanical allodynia.

“The Cdh3 dorsal, inhibitory family was comprised of Inhib-6, Inhib-7, and Inhib-8 and are likely to be involved in the dampening of dynamic touch and therefore in mechanical allodynia70–72.”

• cites [70] Boyle, K. A. et al. Defining a spinal microcircuit that gates myelinated afferent input: implications for tactile allodynia. Cell Rep. 28, 526–540.e6 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [71] Hughes, D. I. et al. Morphological, neurochemical and electrophysiological features of parvalbumin-expressing cells: a likely source of axo-axonic inputs in the mouse spinal dorsal horn. J. Physiol. 590, 3927–3951 (2012). sourceNot checked yet

  Not checked in the free run.

• cites [72] Petitjean, H. et al. Dorsal horn parvalbumin neurons are gate-keepers of touch-evoked pain after nerve injury. Cell Rep. 13, 1246–1257 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The Maf dorsal excitatory family comprised Excit-5, Excit-6, and Excit-7, which expressed markers of neurons involved in light touch.

“The Maf dorsal, the excitatory family was comprised of Excit-5, Excit-6, and Excit-7 which expressed markers of neurons involved in light touch46,48,50,56,57.”

• cites [46] Gatto, G. et al. A functional topographic map for spinal sensorimotor reflexes. Neuron 109, 91–104.e5 (2021). sourceNot checked yet

  Not checked in the free run.

• cites [48] Abraira, V. E. et al. The cellular and synaptic architecture of the mechanosensory dorsal horn. Cell 168, 295–310.e19 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [50] Bourane, S. et al. Identification of a spinal circuit for light touch and fine motor control. Cell 160, 503–515 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [56] Peirs, C. et al. Dorsal horn circuits for persistent mechanical pain. Neuron 87, 797–812 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [57] Peirs, C. et al. Mechanical allodynia circuitry in the dorsal horn is defined by the nature of the injury. Neuron 109, 73–90.e7 (2021). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The Sox5 dorsal excitatory family comprised Excit-14, Excit-15, Excit-16, Excit-17, Excit-18, and Excit-19 and expressed markers suggestive of a role in coping pain and mechanical nociception.

“The Sox5 dorsal, the excitatory family was comprised of Excit-14, Excit-15, Excit-16, Excit-17, Excit-18, and Excit-19, and expressed markers suggestive of a role in coping pain and mechanical nociception46,52,56,63–67.”

• cites [46] Gatto, G. et al. A functional topographic map for spinal sensorimotor reflexes. Neuron 109, 91–104.e5 (2021). sourceNot checked yet

  Not checked in the free run.

• cites [52] Duan, B. et al. Identification of spinal circuits transmitting and gating mechanical pain. Cell 159, 1417–1432 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [56] Peirs, C. et al. Dorsal horn circuits for persistent mechanical pain. Neuron 87, 797–812 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [63] Huang, T. et al. Identifying the pathways required for coping behaviours associated with sustained pain. Nature 565, 86–90 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [64] Moriyama, M. et al. Presynaptic modulation by neuromedin U of sensory synaptic transmission in rat spinal dorsal horn neurones. J. Physiol. 559, 707–713 (2004). sourceNot checked yet

  Not checked in the free run.

• cites [65] Petitjean, H. et al. Recruitment of spinoparabrachial neurons by dorsal horn calretinin neurons. Cell Rep. 28, 1429–1438.e4 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [66] Polgár, E. et al. Substance P-expressing neurons in the superficial dorsal horn of the mouse spinal cord: insights into their functions and their roles in synaptic circuits. Neuroscience 450, 113–125 (2020). sourceNot checked yet

  Not checked in the free run.

• cites [67] Smith, K. M. et al. Calretinin positive neurons form an excitatory amplifier network in the spinal cord dorsal horn. Elife 8, 1750 (2019). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

All ME/Lmx1b clusters except Excit-25 expressed Tacr1, and Excit-21 also expressed Lypd1, suggesting these are candidate ascending populations.

“All of the clusters except Excit-25 expressed Tacr1 and Excit-21 also expressed Lypd1, suggesting that these are candidate ascending populations3.”

• cites [3] Häring, M. et al. Neuronal atlas of the dorsal horn defines its architecture and links sensory input to transcriptional cell types. Nat. Neurosci. 21, 869–880 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The joint analysis of spinal cord neurons from embryonic through adult stages began to align these two perspectives on cell types while highlighting complex relationships between genetically defined cellular origins and mature transcriptional signatures.

“Together, the joint analysis of spinal cord neurons from embryonic through adult stages began to align these two perspectives on cell types while also highlighting the complex relationships that exist between genetically defined cellular origins and mature transcriptional signatures.”

• AssumptionRests on an assumption: Developmental lineage is a powerful and influential framework for categorizing spinal neurons.

May combine two claims.

Excit-33 was comprised almost entirely of nuclei from the Rosenberg dataset and may represent an immature cell type.

“Excit-33 was comprised almost entirely of nuclei from the Rosenberg dataset and may represent an immature cell type.”

May combine two claims.

Excit-35, Excit-36, and Excit-37 are likely derived from the V2a lineage, as they expressed Vsx2 (Chx10) and included many cells from the Hayashi dataset sorted based on Chx10 expression, and thus may play a role in skilled reaching.

“Excit-35, Excit-36, and Excit-37 are likely derived from the V2a lineage, as they expressed Vsx2 (Chx10) and included many cells from the Hayashi dataset that sorted cells based on Chx10 genetic expression and thus may play a role in skilled reaching87.”

• cites [87] Azim, E., Jiang, J., Alstermark, B. & Jessell, T. M. Skilled reaching relies on a V2a propriospinal internal copy circuit. Nature 508, 357–363 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Several cluster markers of ventral cell types, such as Sim1, were not observed in adult spinal cord tissue by in situ hybridization; while detected in harmonized sequencing data, they likely represent lingering RNA from developmental samples.

“Of note, several cluster markers of ventral cell types, such as Sim1, were not observed in adult spinal cord tissue by in situ hybridization and while they are detected in the harmonized sequencing data, they likely represent lingering RNA from developmental samples.”

May combine two claims.

These ME/Lmx1b clusters expressed Lmx1b, suggesting a dI5/dILB embryonic origin.

“These clusters expressed Lmx1b, suggesting a dI5/dILB embryonic origin.”

May combine two claims.

Excit-28 and Excit-29 also expressed Onecut2 and Pou6f2, potentially revealing a link with ventral cell types.

“Excit-28 and Excit-29 also express Onecut2 and Pou6f2, potentially revealing a link with ventral cell types.”

May combine two claims.

Ventral clusters had less distinct gene expression patterns and were less robust than dorsal and mid clusters, so their final identities should be considered with caution.

“In general, we found that the ventral clusters had less distinct gene expression patterns and were less robust than dorsal and mid clusters; therefore, the final identities of these clusters should be considered with caution.”

May combine two claims.

The remaining ME family comprised Excit-26, Excit-27, Excit-28, and Excit-29; these clusters do not express Lmx1b and are likely derived from dI1-3 or ventral embryonic lineages.

“The remaining ME family was comprised of mid, excitatory clusters were comprised of Excit-26, Excit-27, Excit-28, and Excit-29. These clusters do not express Lmx1b, in contrast to the other mid excitatory family and are likely derived from dI1-3 or ventral embryonic lineages.”

May combine two claims.

The mid inhibitory cell types were grouped as one family comprising Inhib-15, Inhib-16, Inhib-17, Inhib-18, Inhib-19, Inhib-20, and Inhib-21, which expressed Slc6a5 (except Inhib-21) and Gad2, implicating these neurons in sensorimotor processing.

“The mid inhibitory cell types were grouped as one family comprised of Inhib-15, Inhib-16, Inhib-17, Inhib-18, Inhib-19, Inhib-20, and Inhib-21, which expressed the glycinergic marker Slc6a5 (with the exception of Inhib-21) and also the gaba-ergic marker Gad2, implicating these neurons in sensorimotor processing84–86.”

• cites [84] Hilde, K. L. et al. Satb2 is required for the development of a spinal exteroceptive microcircuit that modulates limb position. Neuron 91, 763–776 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [85] Koch, S. C. et al. RORβ spinal interneurons gate sensory transmission during locomotion to secure a fluid walking gait. Neuron https://doi.org/10.1016/j.neuron.2017.11.011 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [86] Fink, A. J. P. et al. Presynaptic inhibition of spinal sensory feedback ensures smooth movement. Nature 509, 43–48 (2014). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In situ hybridization experiments were performed on mid/lower lumbar spinal cord sections, so markers for Excit-30 were not validated.

“As the in situ hybridization experiments were performed on mid/lower lumbar spinal cord sections, we did not validate markers for this cluster.”

May combine two claims.

Inhib-22 and Inhib-27 also expressed Gad2, Pax2, and Pou6f2; they were distinguished by low levels of Gata3 in Inhib-27, which may represent V2b lineage.

“Inhib-22 and Inhib-27 also expressed the gaba-ergic marker Gad2 as well as Pax2 and Pou6f2. They were distinguished by low levels of Gata3 expression in Inhib-27, which may represent V2b lineage.”

May combine two claims.

Inhib-23 was identified by expression of Gm26673 and Pvalb, which may suggest that this cluster included Ia-inhibitory neurons.

“They were identified by expression of Gm26673 and Pvalb in Inhib-23, which may suggest that this cluster included Ia-inhibitory neurons89.”

• cites [89] Alvarez, F. J. et al. Postnatal phenotype and localization of spinal cord V1 derived interneurons. J. Comp. Neurol. 493, 177–192 (2005). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

These results must be interpreted with caution because each of these datasets were included in the overall training data, which could lead to artificially high performance.

“These results must be interpreted with caution because each of these datasets were included in the overall training data which could lead to artificially high performance.”

• AssumptionRests on an assumption: There is no existing gold standard for spinal cord cell identities.
• AssumptionRests on an assumption: The harmonized analysis cell type labels are adequate as training labels for machine learning classifiers.

May combine two claims.

The mid excitatory (ME)/Lmx1b family comprised Excit-21, Excit-22, Excit-23, Excit-24, and Excit-25 and corresponds to cells suggested to be involved in pain.

“The mid excitatory (ME)/Lmx1b family was comprised of Excit-21, Excit-22, Excit-23, Excit-24, and Excit-25 and corresponds to cells suggested to be involved in pain3,83.”

• cites [3] Häring, M. et al. Neuronal atlas of the dorsal horn defines its architecture and links sensory input to transcriptional cell types. Nat. Neurosci. 21, 869–880 (2018). sourceNot checked yet

  Not checked in the free run.

• cites [83] Barik, A. et al. A spinoparabrachial circuit defined by Tacr1 expression drives pain. Elife 10, e61135 (2021). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Excit-31, Excit-32, Excit-33, and Excit-34 expressed low levels of Lhx2, Lhx9, and Isl1, potentially suggesting dorsal dI1/dI2/dI3 embryonic lineages.

“Of these, Excit-31, Excit-32, Excit-33, and Excit-34 expressed low levels of Lhx2, Lhx9, and Isl1, potentially suggesting dorsal dI1/dI2/dI3 embryonic lineages for these clusters.”

May combine two claims.

Many cells from the Baek dataset, which sorted cells based on spinocerebellar status, were found in Excit-35, suggesting this population includes ascending projection neurons that target the cerebellum.

“Intriguingly, many cells from the Baek dataset, which sorted cells based on spinocerebellar status were found in Excit-35, suggesting that this population includes ascending projection neurons that target the cerebellum.”

May combine two claims.

Inhib-26 was the most robust ventral cluster and expressed the Renshaw marker genes Chrna2, Chrna7, and Calb1, suggesting it corresponded to Renshaw cells.

“Inhib-26 was the most robust ventral cluster and expressed the Renshaw marker genes Chrna2, Chrna7, and Calb1, suggesting that this cluster corresponded to Renshaw cells89,90.”

• cites [89] Alvarez, F. J. et al. Postnatal phenotype and localization of spinal cord V1 derived interneurons. J. Comp. Neurol. 493, 177–192 (2005). sourceNot checked yet

  Not checked in the free run.

• cites [90] Perry, S. et al. Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK. Eur. J. Neurosci. 41, 889–900 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Excit-30 contained only two defined spinocerebellar cells from the Baek et al. dataset while the majority was from the Hayashi dataset, arguing against Clarke's column identity and suggesting a V2 embryonic lineage.

“However, this cluster only contained two defined spinocerebellar cells from the Baek et al. dataset while the majority of this cluster was from the Hayashi dataset, arguing against Clarke’s column identity and also suggesting a V2 embryonic lineage.”

May combine two claims.

The Excit-30 cluster was marked by Gbx2, Neurod2, and Sp8, and there was partial evidence that it corresponded to Clarke's column (CC).

“The Excit-30 cluster was marked by Gbx2, Neurod2, and Sp8 and there was partial evidence that it corresponded to Clarke’s column (CC).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The difference between in situ hybridization and harmonized sequencing data may reflect differences in developmental patterns.

“In a few instances, this data differed from the harmonized sequencing data (for example in Excit-3 and Excit-4) which may reflect differences in developmental patterns.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Animal experiments were performed in accordance with institutional guidelines and approved (protocol #1384) by the National Institute of Neurological Disorder and Stroke's Institutional Animal Care and Use Committee.

“Animal experiments were performed in accordance with institutional guidelines and approved (protocol #1384) by the National Institute of Neurological Disorder and Stroke’s Institutional Animal Care and Use Committee.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

An even balance of male and female mice that were 9 weeks old and of mixed C57BL/6 J and BALB/cJ background were used for single nucleus sequencing (four mice) and validation studies (two groups of three mice).

“An even balance of male and female mice that were 9 weeks old and of mixed C57BL/6 J and BALB/cJ background were used for single nucleus sequencing (four mice) and validation studies (two groups of three mice).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The adult cell types of Ia inhibitory neurons and CSF-contacting neurons each have dual origins.

“It is already known that the adult cell types of Ia inhibitory neurons and CSF-contacting neurons each have dual origins43,45,121, suggesting that this could be a common occurrence.”

• cites [43] Petracca, Y. L. et al. The late and dual origin of cerebrospinal fluid-contacting neurons in the mouse spinal cord. Development 143, 880–891 (2016).Not checked yet

  Not checked in the free run.

• cites [45] Djenoune, L. et al. The dual developmental origin of spinal cerebrospinal fluid-contacting neurons gives rise to distinct functional subtypes. Sci. Rep. 7, 719–14 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [121] Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

For all three phases, the authors used a dendrogram based on mean gene expression and UMAP location to identify closely related clusters and then probed for differential gene expression to determine whether candidate pairs of clusters should be merged.

“For all three phases, we used the following method to determine whether candidate pairs of clusters should be merged: a dendrogram based on mean gene expression and UMAP location were used to systemically identify closely related clusters and we then probed for differential gene expression (for example, see Supplementary Fig. 4).”

Classified as probably not a substantial claim.

The range of resolutions was examined by the range of average silhouette scores for the clusters, visual inspection of UMAP cluster distribution, and comparison of cluster markers with known markers and with known co-expression patterns in the literature.

“Here, the range of resolutions were examined by (1) the range of average silhouette scores for the clusters, (2) visual inspection of UMAP cluster distribution, and, most importantly (3) comparison of cluster markers with known markers and with known co-expression patterns in the literature. (Supplementary Fig. 4).”

May combine two claims.

This analysis is limited in scope to RNA expression in the postnatal mouse spinal cord and reflects a merged study of multiple time points.

“Most specifically, this analysis is limited in scope to RNA expression in the postnatal mouse spinal cord and reflects a merged study of multiple time points.”

Classified as probably not a substantial claim. May combine two claims.

Annotations from Seurat integration were overlapped on the integrated UMAP projections obtained from other integration methods and the cell type clusters were compared for reproducibility.

“Annotations from Seurat integration were then overlapped on the integrated UMAP projections obtained from other integration methods and the cell type clusters were compared for reproducibility.”

May combine two claims.

Ventral neuron cell types may be organized at a second, nested level of spatial trends that overlay embryonic lineage-defined cell types: a Pou6f2-Esrrg trend along the dorsal-ventral axis and a Nfib-Zfhx3/4 and birthdate trends along the medial-lateral axis.

“Ventral neuron cell types may be organized at a second, nested level of spatial trends that overlay embryonic lineage-defined cell types: a Pou6f2-Esrrg trend along the dorsal-ventral axis and a Nfib-Zfhx3/4 and birthdate trends along the medial-lateral axis, consistent with a recent report108.”

• cites [108] Osseward, P. J. et al. Conserved genetic signatures parcellate cardinal spinal neuron classes into local and projection subsets. Science 372, 385–393 (2021). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

This reflects some technical limitations but also a fundamental complexity and diversity in how gene expression is controlled within individual cells and in cell-type populations.

“We propose that this reflects some technical limitations but also a fundamental complexity and diversity in how gene expression is controlled within individual cells and in cell-type populations.”

Classified as probably not a substantial claim.

Cell type annotations for the non-neuronal cell types were based on the presence of well-established marker genes and on the gene expression patterns in the Allen in situ hybridization database.

“Cell type annotations for the non-neuronal cell types were based on the presence of well-established marker genes (Supplementary Table 1) and on the gene expression patterns in the Allen in situ hybridization database (for meningeal, ependymal, Schwann cell, and peripheral glia clusters).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The main integration was compared to three independent methods: Harmony, Conos, and LIGER.

“This integration was compared to three independent methods (Harmony25, Conos26, and LIGER27).”

• cites [25] Korsunsky, I. et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat. Meth 16, 1289–1296 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [26] Barkas, N. et al. Joint analysis of heterogeneous single-cell RNA-seq dataset collections. Nat. Meth 16, 695–698 (2019). sourceNot checked yet

  Not checked in the free run.

• cites [27] Welch, J. D. et al. Single-cell multi-omic integration compares and contrasts features of brain cell identity. Cell 177, 1873–1887.e17 (2019). sourceNot checked yet

  Not checked in the free run.