The transcriptomic landscape of spinal V1 interneurons reveals a role for En1 in specific elements of motor output

SUMMARY Neural circuits in the spinal cord are composed of diverse sets of interneurons that play crucial roles in shaping motor output. Despite progress in revealing the cellular architecture of the spinal cord, the extent of cell type heterogeneity within interneuron populations remains unclear. Here, we present a single-nucleus transcriptomic atlas of spinal V1 interneurons across postnatal development. We find that the core molecular taxonomy distinguishing neonatal V1 interneurons perdures into adulthood, suggesting conservation of function across development. Moreover, we identify a key role for En1, a transcription factor that marks the V1 population, in specifying one unique subset of V1Pou6f2 interneurons. Loss of En1 selectively disrupts the frequency of rhythmic locomotor output but does not disrupt flexion/extension limb movement. Beyond serving as a molecular resource for this neuronal population, our study highlights how deep neuronal profiling provides an entry point for functional studies of specialized cell types in motor output.

(I-N) Positive identification of V1 interneurons using label transfer.(I) E9.5-E13.5 whole spinal cord scRNA-seq data 21 was used as a reference data set.(J) E13.5 En1::Cre INTACT snRNA-seq query data (black) enriched for V1 interneurons by flow cytometry.(K) Data set from (J) projected onto reference data (I).(L) Expression of the transcription factor En1 overlaid on the projected data set.(M-N) V1 label transfer prediction scores and final assignments for the E13.5 snRNAseq data set, used for subsequent positive and negative selection of our postnatal data.

Figure S2 .
Figure S2.Quality Control (QC) statistics on V1 interneuron nuclei, related to Figure 1.(A) Table showing the total number of nuclei identified as V1 interneurons grouped by age and anatomical location.Colors represent low (blue) to high (red) proportions across all categories.(B) Overall QC statistics on V1 interneurons showing total number of unique molecular identifier (UMI)-corrected counts per nuclei, number of genes detected per nuclei, and the percentage of mitochondrial reads per nuclei.(C-E) Similar to (B) except the same statistics were parsed by the cluster identification shown in Figure 1C.(F)Table displaying the number and percentage of nuclei in each cluster.

Figure S3 .
Figure S3.Analysis of transcription factor expression and V1 Sp8 clade structure, related to Figures 1 and 3. (A) Dot plot showing scaled average expression of the 19 transcription factors (TFs) described previously in Bikoff et al., 29 all of which were validated via immunohistochemistry (IHC).Note that several genes were detected at low levels but still showed biased expression.(B) V1 Sp8 cluster

Figure S4 .
Figure S4.Taxonomic relationship and similarity of V1 clusters, related to Figure 3. (A) Dendrogram showing phylogenetic analysis based on the top 3000 most variable genes.(B) Number of differentially expressed genes (DEGs) per cluster based on non-parametric Wilcoxon rank sum test for differential gene expression.(C) Heatmap of Pearson correlation coefficients based on the log-normalized average expression of all genes per cluster identified cluster #13 as unique.(D) Principal component analysis (PCA) plot of aggregated counts per cluster showing that cluster #13 drives most of the variance along PC1.

Figure S5 .
Figure S5.Additional gene expression analysis in En1 Het and En1 KO V1 interneurons, related to Figure 5. (A) The total number of V1 interneurons is similar in En1 Het and En1 KO mice (p = 0.815, unpaired two-tailed t-test).(B) Dot plot showing similar gene expression of 19 transcription factors in V1 nuclei from En1 Het and En1 KO mice.Note that the remaining En1 KO V1 nuclei in cluster #10 retained a normal Pou6f2 + and Nr5a2 + identity.(C) Pseudo-bulk analysis of differentially expressed genes revealed that Nr5a2 and Pou6f2 are significantly downregulated in En1 KO V1 nuclei, compared with that in En1 Het nuclei.(D) Spatial distributions of the overall V1 interneuron population (top), V1 Pou6f2 interneurons (middle), and V1 Sp8 interneurons (bottom) in P0 lumbar

Figure S6 .
Figure S6.Validation of V1 ablation and En1 conditional knockout experiments, and additional behavioral data, related to Figure 6.(A) Schematic of the V1 ablation paradigm.Diptheria toxin (DT) was administered at P0, followed by either physiological analysis via fictive locomotion 4 days post-DT administration, or limb kinematic analysis during tail suspension 7 days post-DT administration.All mice in the study were heterozygous for the Tau::ds-DTR and Ai65D alleles.Littermates lacking Cre, Flpo, or both were pooled and used as controls.(B-C) Immunohistochemical analysis and quantification in P7 lumbar spinal cords of control (+PBS) or V1-ablated (+DT) quadruple-heterozygous mice showed near complete ablation of V1 interneurons at P7 (n = 6 mice, ****p < 0.0001, unpaired two-tailed