Article Figures & Data
Figures
- Figure 1.
Retrograde labeling and FANS purification of supraspinal nuclei. A, AAV2-retro-H2B-mScarlet was injected to lumbar spinal cord, followed 2 weeks later by microdissection of fluorescently labeled brain regions in sagittal section. Middle panels, Schematics represent the anatomic ROIs. Right, Tissue slices with retrograde fluorescent label. Red lines indicate regions of microdissection. B, Representative image of gates used for isolation of mScarlet-labeled nuclei. C, Visual inspection of nuclei after FANS shows mScarlet label in nearly all nuclei. LHA, Lateral hypothalamic area; PVH, paraventricular hypothalamus; MB, midbrain; DP, dorsal pons; Ret, medullary reticular formation. Scale bars: A, 1 mm; C, 50 µm.
- Figure 2.
snRNA-seq analysis indicates that samples are mostly glutamatergic neurons. A, UMAP clustering of 7748 supraspinal nuclei identifies 18 distinct groups. B, Separation of the three merged samples confirms that each contributes to all 18 clusters, supporting replicate consistency. C, Dotplot represents expression of neuronal markers in all clusters and very low expression of non-neuronal markers, as expected from the retrograde labeling strategy. D, E, Violin plots (D) and feature plots (E) represent expression of glutamatergic markers in most clusters, and expression of GABAergic, serotonergic, or noradrenergic markers in only small clusters.
- Figure 3.
Corticospinal neurons identified by established layer V markers. A-C, Feature plots of Bcl11b (A), Crym (B), and Fezf2 (C) show high enrichment in Clusters 0 and 2 (red circles). D-K, ISH detection from the Allen Brain Atlas of transcripts with enrichment in layer V of cortex (red arrows). Corresponding violin plots indicate high expression of transcripts in Clusters 0 and 2 (dotted red rectangles), consistent with corticospinal identity.
- Figure 4.
ISH data from the Allen Brain Atlas enables preliminary identification of transcriptional clusters. A, E, I, M, Q, U, Violin plots (left) and feature plots (right) of transcripts with high enrichment in single clusters. B, F, J, N, R, V, ISH data from the Allen Brain Atlas. Red boxes represent ROIs. C, G, K, O, S, W, Corresponding anatomic registration to regions that are known to supply supraspinal input. D, H, L, P, T, X, Higher-magnification views with detection of putative marker genes. Arrows point to areas of specific gene expression: LHA, Lateral hypothalamic area; PVH, paraventricular hypothalamus; RN, red nucleus; LC, locus coeruleus; PCG, pontine central gray. Scale bars: V, 1 mm; W, X, 200 µm.
- Figure 5.
Identification of hindbrain populations. A, Dotplot of Hox transcript levels represents selective expression in a subset of HOX gene clusters, indicating hindbrain populations of caudal rhombomeric origin. B-I, Feature plots with putative hindbrain populations outlined and specific transcripts enriched in subregions indicated by red arrows.
- Figure 6.
Anatomical assignment and marker genes for populations of supraspinal neurons. A, UMAP clustering of supraspinal nuclei with anatomic designations for each. B, Dotplot of gene expression showing examples of transcripts with enriched expression in each cluster relative to other supraspinal populations. HB, Hindbrain; PVH, paraventricular hypothalamus; RN, red nucleus; LC, locus coeruleus; DP, dorsal pons.
- Figure 7.
Visualization of candidate marker genes in subcortical supraspinal populations. Adult mice received lumbar injection AAV2-retro-H2B-mScarlet followed 2 weeks later by tissue sectioning and ISH detection of candidate marker genes. In all panels, red represents retrograde H2B-mSc; green represents transcript detection. A, D, G, J, M, Brain sections with regions of predicted expression indicated in blue and a predicted nonexpressing region in orange. B, E, H, K, N, Higher-magnification view of target populations with insets. C, F, I, L, O, Higher magnification of nonexpressing supraspinal populations. P, Percent of supraspinal cells in which transcript was detected, indicating selective expression of Plagl1 in the lateral hypothalamic area, Ttc6 Emx2 in the red nucleus, and Prdm6 in the dorsal region of the pons. LHA, Lateral hypothalamic area; HB, hindbrain; SN, solitary nucleus; DP, dorsal pons. Scale bars: M, 2 mm; N, 50 µm; N′, O, 10 µm. N = 3 animals; >60 cells per region. Error bars are SEM.
- Figure 8.
Visualization of candidate marker genes in hindbrain supraspinal populations. Adult mice received lumbar injection AAV2-retro-H2B-mScarlet followed 2 weeks later by tissue sectioning and ISH detection of candidate marker genes. A, D, G, J, Overview of brain sections with medullary ROIs (arrows). B, E, H, K, Higher-magnification views of target regions. C, F, I, L, High-resolution images of supraspinal cell nuclei (red) with transcript detection (green) in some neurons (arrows) but absent in nearby supraspinal cells (empty arrowheads). Scale bars: A, D, G, J, 1 mm; B, E, H, K, 50 µm; C, F, I, L, 20 µm.
- Figure 9.
Differential expression of TFs, growth factor, axon guidance, and growth inhibitory receptors in supraspinal populations. A, Dotplot represents the expression levels of selected TFs in each of 14 distinct types of supraspinal neurons. B, Dotplot represents the level of expression of selected receptors in supraspinal populations. Relevant ligands are listed to the left and include neurotrophins, axon guidance cues, and growth-inhibitory cues. C, Dotplot represents expression of voltage-gated ion channels. Extended Data Fig. 9-1 provides average expression of all transcripts in all populations.
Extended Data
Figure 9-1
Average expression values of all detected transcripts in all clusters. The associated Excel file provides the average transcript abundance of all detected features for each of the 14 identified cell types. A column indicates whether each transcript is identified as a TF (Lambert et al., 2018) or voltage-gated ion channels. Download Figure 9-1, XLSX file.
