Single-cell analysis reveals dynamic changes of neural cells in developing human spinal cord

Abstract

During central nervous system development, neurogenesis and gliogenesis occur in an orderly manner to create precise neural circuitry. However, no systematic dataset of neural lineage development that covers both neurogenesis and gliogenesis for the human spinal cord is available. We here perform single-cell RNA sequencing of human spinal cord cells during embryonic and fetal stages that cover neuron generation as well as astrocytes and oligodendrocyte differentiation. We also map the timeline of sensory neurogenesis and gliogenesis in the spinal cord. We further identify a group of EGFR-expressing transitional glial cells with radial morphology at the onset of gliogenesis, which progressively acquires differentiated glial cell characteristics. These EGFR-expressing transitional glial cells exhibited a unique position-specific feature during spinal cord development. Cell crosstalk analysis using CellPhoneDB indicated that EGFR glial cells can persistently interact with other neural cells during development through Delta-Notch and EGFR signaling. Together, our results reveal stage-specific profiles and dynamics of neural cells during human spinal cord development.

Keywords: EGFR+ glial cell; gliogenesis; human fetal spinal cord; neurogenesis; single-cell RNA sequencing.

Figure 1. Integrated snRNA‐seq data reveal transcriptomic features and changes in the proportions of neural cells in early and mid‐gestation human spinal cord

1. A schematic diagram of the single‐cell sequencing process.

2. Visualization of major classes of spinal cord cells by integrated UMAP plots of cell clusters. Cell types are labeled and circled with dotted lines (top). Violin plots show the most distinct and commonly used marker genes for each cell cluster (bottom). Cell‐type annotations are listed on the right and shown in varying colors.

3. Three‐dimensional visualization of neural subtype developmental trajectories for individual samples from GW7, GW10, GW14, GW18, and GW23. Stage‐dependent differentiation trends changed with development. The red arrow indicates the proliferative neural progenitor cell cluster (MKI67, PAX6, NES, and SOX2). Neural cell types are named and circled with dotted lines. Gray represents non‐neural cell types (e.g., vascular and meningeal).

4. Immunofluorescence staining of the spinal cord at GW7, GW8, GW10, GW11, and GW23 for RBFOX3 (red), OLIG2 (green), and GFAP (white). The appearance of astrocytes and oligodendrocytes was observed from GW8 onward. Scale bars: 100 μm.

Figure 2. Integrated trajectory and expression feature analysis of neuronal cell developmental trajectory in GW7, GW8, GW10, and GW11 samples

1. UMAP plot of cells integrated from datasets of GW7, GW8, GW10, and GW11 samples (GW7S‐NC, GW8S‐02NC, GW10C‐01NC, GW10T‐01NC, GW10L‐01NC, GW10C‐02NC, GW10T‐02NC, GW10L‐02NC, GW11C‐NC, GW11T‐NC, GW11L‐NC) showing neuronal differentiation tendency. Cell types are labeled (left). Heatmap showing differentially expressed genes (DEGs) in neuronal restricted progenitors (NRP) and immature dorsal neurons (IDN) (right).

2. Visualization of selected top gene ontology (GO) enrichment terms for NRP and IDN clusters based on DEG analysis.

3. Immunofluorescence staining of the spinal cord from GW7, GW8, and GW10 for a dorsal marker (PAX7, green), neuronal precursor marker (ASCL1, red), and nuclei marker (DAPI, blue). Images show the neuronal differentiation process on the dorsal side of the spinal cord. Scale bar: 100 μm.

4. Stacked proportional changes of neural progenitor cells (NPC) and neuronal cell classes from GW7–GW11. Cell types were identified as: proliferative neural progenitors (Pro‐NPC), glial restricted progenitors (GRP), IDN, DI4 interneurons (DI4), DI5 interneurons (DI5), motor neuron (MN), DI1 and DI2 interneurons (DI1/2), DI3 interneurons (DI3), DI6 interneurons (DI6), and V0 and V1 interneurons (V0/1).

5. Three‐dimensional developmental trajectories for snRNA‐seq samples from the GW10 cervical sample (top view in the upper right corner). Cells are classified by differentiation stage according to expression of specific markers (neuronal progenitor: PAX3 and PAX7; DI5 interneuron: POU4F1 and TLX3; V3 interneuron: SIM1; and motor neuron: MNX1). Cells in the neuronal development trajectory are colored by gene expression. Distances from progenitor cells to differentiated neurons represent the differing maturation status between dorsal neurons (DI4, DI5) and ventral neurons (MN, V3). Cells with no detectable expression for a given gene were omitted from the plot.

Figure EV1. Alignment analysis of cell‐type conservation and divergent expression between human and mouse

1. UMAP visualization of human (n = 50,533 nuclei) and mouse (n = 26,039 cells) neuronal and neural progenitor cell (NPC) clusters after alignment.

2. Radar chart showing similar distribution of neuronal subtype proportions among the two species during the corresponding developmental period.

3. Human and mouse cell‐type homologies for NPCs predicted from shared cluster membership. Gray shade corresponds to the minimum proportion of co‐clustering between species. Columns show human clusters and rows show mouse clusters. Homologous clusters are labeled based on human and mouse cluster membership. Inferred cell‐type homologies highlighted in red boxes. Orange boxes indicate subclasses of PD4/5‐related neuronally restricted progenitors in human and mouse data.

4. Violin plots showing GFAP expression in human and mouse integrated data. Blue boxes indicate astrocyte subclasses in human and mouse.

5. Comparison of expression levels of orthologous genes between species for NPC‐Ependymal and EXC‐DI4 types.

6. Gene families exhibiting the most divergent expression patterns included neurotransmitter receptors, extracellular matrix elements, ion channels, and fatty acid transporters.

7. Expression of ionotropic glutamate receptors differed between homologous cell types. Scores are listed on the right.

Figure EV2. Alignment analysis of cell‐type conservation and divergent expression between human and mouse

1. A, B

   Integrated UMAP plots of NPCs and neuronal cell types from developing human (GW7–GW11) and mouse (E9.5–E13.5) spinal cord data. Red line show cell subtypes base on marker gene expression.

2. C, D

   Human and mouse cell‐type homologies for inhibitory and excitatory neuron types, predicted based on shared cluster membership. Inferred cell‐type homologies highlighted in red boxes. Gray color corresponds to the minimum proportion of co‐clustering between species. Columns show human clusters, and rows show mouse clusters. Homologous clusters are labeled on the basis of human and mouse cluster membership.

3. E

   Taxonomy of 22 neuronal and seven NPC‐homologous cell types and cell classes. Color icons represent different neural types. Arrows indicate one‐to‐one matches. Asterisks indicate subclasses lacking homologous types.

4. F

   Similar functional gene families (n = 384 gene sets) discriminate inhibitory neuron types in human and mouse. Error bars correspond to the standard deviation of mean MetaNeighbour AUROC scores across ten subsamples of cells.

5. G

   Violin plots showing expression of PIEZO2 in human and mouse integrated data (left). Red box indicates human Exc‐DI5 PIEZO2 subclass.

6. H

   Time split UMAP plots of mouse data show the gradual appearance of an astrocyte progenitor cluster.

Figure 3. Neural cell‐type integration analysis and characterization

1. UMAP plot of snRNA‐seq data from 64,381 combined neural cells from GW7 to GW23. Cell cluster phenotypes are labeled and the red arrow indicates the EGFR‐expressing glial cluster (left). Selected UMAP plots from different development stages (GW7, GW11, GW18, and GW23) represent the neural development trend (right). Gray represents non‐specific cell types.

2. The stacked proportions of neural cell classes showing changes in stage‐dependent differentiation trends with developmental stage.

3. Violin plot showing the expression of selected genes. EGFR⁺ Cluster 14 is circled with a red dotted line.

4. Visualization of selected top gene ontology (GO) enrichment terms of astrocyte clusters based on DEG analysis.

Figure 4. Characterization of GW7 progenitor cell populations and EGFR‐expressing transitional glial cells

1. UMAP plot of Cluster 0 and Cluster 14 populations from the GW7 sample.

2. Heatmap of differentially expressed genes (DEGs) in Clusters 0 and 14 at GW7. On the right of the heatmap are the top‐twelve DEGs. Selected top gene ontology (GO) terms related to the corresponding DEGs (right).

3. Single‐cell resolution trajectory of EGFR⁺ transitional glial cells (Cluster 14) was constructed from sample (GW11T‐NC, GW14T‐NC, GW18T‐NC, GW20T‐NC, GW21T‐NC) during the transitional period using Monocle. Linear arrangement of the transition trajectory with developmental stages represents the transition process of a regulatory cell type. The arrow indicates the developmental trend.

4. Violin plots produced by VlnPlot function of Seurat package representing changes in expression of EGFR, DLL1, MIAT, and FAM189A2 in cluster 14 with development based on snRNA counts data.

5. Overview of selected statistically significant interactions between neural cell types according to cell–cell communication analysis from statistical test of CellPhoneDB v.2.0. Size indicates P value, and color indicates the means of the average expression level of the receptor–ligand pairs. The EGFR⁺ glial cell type exhibited the most prominent interactions with Delta‐Notch and EGFR signaling within the network.

Figure EV3. Altered gene expression levels and differentiation trajectory of EGFR⁺ glial cells at different gestational time points

1. A–D

   (A) Violin plots produced by VlnPlot function of Seurat package representing expression levels of HES6, NES, GFAP, and AQP4 in the EGFR⁺ glial cluster across developmental process based on snRNA counts data. The EGFR⁺ glial cluster cell type (DLL1 ⁺ EGFR ⁺ HES6 ⁺) could be found in GW9T (B), GW16T (C), and GW20T (D) scRNA‐seq datasets (left, red circle). Dot plots showing expression of DLL1 and EGFR were predominantly found within the EGFR⁺ glial cell type (middle). Pseudotime analysis suggesting that EGFR⁺ glial cells serve as neuronal progenitors in neuronal differentiation trajectory during neurogenesis stage and jion astrocyte differentiation trajectories in gliogenesis stage (right).

Figure 5. Expression profile of EGFR‐expressing glia during spinal cord development

1. Schematic diagram showing the spatial and temporal dynamics of EGFR‐expressing cells during spinal cord development. The solid green line represents EGFR‐expressing radial glial cells, while the green dotted line represents EGFR‐expressing transitional glial cells. The blue line represents the gray matter boundary.

2. Immunofluorescence staining of spinal cord sections at GW7, GW11, GW17, and GW23 for EGFR (green) and nuclei (DAPI, blue). EGFR expression on the ventral side was observed in GW7 and GW11 spinal cord. EGFR expression on the dorsal side was observed in GW11, GW17, and GW23 spinal cord. Dorsal EGFR expression was mainly concentrated within the dorsal horn area. Scale bar: 100 μm. (Top) Higher‐magnification view of boxes at bottom. White dashed lines indicate dorsal horn borders.

3. UMAP plot of GW11 sample. Dot plot shows the expression of featured genes in the EGFR‐expressing progenitor cluster mapped on the UMAP plot. Expression of PAX3 and OLIG2 displays a complementary pattern, while PDGFRA was expressed at a low level within the EGFR⁺ glial cluster.

4. Representative immunofluorescence staining of a GW11 spinal cord section. Most dorsal EGFR‐expressing cells at GW11 co‐expressed GFAP and SOX9, but not OLIG2. Scale bars: 100 μm.

Figure EV4. Characterization of EGFR‐expressing cells in GW11 human spinal cord

1. A–E

   Co‐staining of EGFR with GFAP (A) SOX9 (B) OLIG2 (C) IBA1 (D) and NOTCH1 (E). Higher‐magnification view of boxes at the bottom. The SOX9 staining image is the same as the immunofluorescence image shown in Fig 6A.

2. F

   Immunofluorescence staining of GW11 spinal cord with glial lineage marker SOX2 (green) and neuronal lineage marker NEUN (red). Higher‐magnification view of boxes at the bottom. Scale bars: 100 μm.

Source data are available online for this figure.

Figure 6. Comparison of EGFR‐expressing cell features between GW11 and GW17 spinal cord

1. A–D

   GW11 EGFR‐expressing cells exhibited radial glia morphology and most co‐expressed SOX9 (A) but not OLIG2 (B). However, EGFR co‐expression with SOX9 or OLIG2 was found in GW17 EGFR‐expressing cells (C, D). White dashed lines marked dorsal horn borders. Higher‐magnification view of boxes on left. Scale bars: 100 μm.

Figure EV5. Characterization of spinal cord EGFR‐expressing cells based on brain cortex data

1. UMAP plot of spinal cord EGFR⁺ dataset mapped alongside human brain cortex EGFR⁺ dataset (Fu et al, 2021). Cycling cells, cells not in G0 of the cell cycle; IPC2, intermediate progenitor cell 2; IN, immature inhibitory neuron; PriOPC, primitive oligodendrocyte progenitor cell; OPC, oligodendrocyte progenitor cell; OAPC, HOPX ⁺ SPARCL1 ⁺ glial progenitor cell; AstroEpen, astrocyte ependymal cell; Endo, endothelial cell.

2. Separate UMAP plots showing the subtype distribution of the reference brain cortex EGFR⁺ dataset (left) and spinal cord EGFR⁺ dataset (right).

3. Heatmap showing differentially expressed genes (DEGs) of spinal cord EGFR⁺ subtypes.

4. Time split UMAP plots of the spinal cord EGFR⁺ dataset shows the subtype distribution EGFR⁺ cells at GW11, GW16, GW20, and GW23.

5. Histogram showing that the composition of spinal cord EGFR⁺ subtypes changes with developmental stage.