Abstract
Precise generation of excitatory neurons and inhibitory interneurons is crucial for proper formation and function of neural circuits in the mammalian brain. Because of the size and complexity of the human brain, it is a challenge to reveal the rich diversity of interneurons. To decipher origin and diversity of interneurons in the human fetal subpallium, here we show molecular features of diverse subtypes of interneuron progenitors and precursors by conducting single-cell RNA sequencing and in situ sequencing. Interneuron precursors in the medial and lateral ganglionic eminence simultaneously procure temporal and spatial identity through expressing a combination of specific sets of RNA transcripts. Acquisition of various interneuron subtypes in adult human brains occurs even at fetal stages. Our study uncovers complex molecular signatures of interneuron progenitors and precursors in the human fetal subpallium and highlights the logic and programs in the origin and lineage specification of various interneurons.
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Data availability
The raw single-cell sequencing data are available in the Gene Expression Omnibus at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE165388, with the accession code GSE165388. All images of ISS are available upon request at https://github.com/yuanbell/Single-cell-sequencing-of-subpallium.
Code availability
All codes generated during this study are available upon request at https://github.com/yuanbell/Single-cell-sequencing-of-subpallium.
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Acknowledgements
This study was supported by the Scientific Research Funds of Huaqiao University (Z16Y0017 to T.S.), the Fundamental Research Funds for the Central Universities (ZQN-715 to Y.S.), the Innovation Awards of Quanzhou Talents (2018C057R to Y.C.), the Natural Science Foundation of Fujian Province of China (2018J01585 to S.H.), the National Key Research and Development Program of China (2017YFA0106800 to R.K.) and the National Natural Science Foundation of China (31771141 to T.S., 11802096 to Y.S. and 31770927 to R.K.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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Y.Y. and T.S. conceived and designed the study. Y.Y. performed the single-cell sequencing experiments. Y.Y., Z.Z., R.C. and S.H. analyzed sequencing data. Y.Y., D.X. and R.K. performed the ISS experiment. Y.S., W.J.C., W.H.C. and W.L. prepared brain tissues. Y.Y. and T.S. wrote the paper. T.S. supervised the entire study.
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Extended data
Extended Data Fig. 1 Single-cell transcriptomic maps of the human fetal subpallium.
a, Cell clustering of human fetal subpallial samples at gestational weeks (GW) 9 to 12 integrated using harmony and depicted using t-SNE. b, The distribution of each subclusters in the subpallium at four developing stages. Colors indicate cell clusters as shown in a. c, Clustering of all cells from four developing stages after batch correction visualized using t-SNE. d, Heatmap illustrating differentially expressed genes (DEGs) in five major clusters and 17 subclusters. e, Assessment of 71 genes from 2,922 DEGs using Random Forest Classifier from scikit-learn of the remaining 16 subclusters after removing excitatory lineages. ‘True label’ indicates the manual annotation based on the 71 genes.
Extended Data Fig. 2 Expression patterns of canonical genes from each cell types.
a-f, Representative genes expressed in cell clusters from human fetal subpallium visualized using t-SNE. Each dot represents one cell. Major clusters include excitatory lineages (a), neural progenitor cells (NPCs, b) and interneuron precursors (INPs, c) derived from the medial ganglionic eminence (MGE_INPs, d), caudal ganglionic eminence (CGE_INPs, e) and lateral ganglionic eminence (LGE_INPs, f).
Extended Data Fig. 3 Heterogeneity of interneuron progenitors in the human fetal subpallium.
a-c, Expression of DLX2 and GAD2 at gestational weeks (GW) 9 (a), GW11 (b), GW12 (c), visualized by the t-SNE plot. d, Violin plots of expression patterns of progenitor markers expressed at the ventricular zone (VZ) and subventricular zone (SVZ) in the subpallium at GW12. e, Violin plots of expression patterns of ganglionic eminence regional markers in the progenitors from GW9 to GW12.
Extended Data Fig. 4 Expression patterns of subpallial genes in the ganglionic eminence of the human fetal brain as detected by in situ sequencing (ISS).
a and b, Expression patterns of 6 subpallial genes in the medial ganglionic eminence (MGE) (a), lateral ganglionic eminence (LGE) (a) and caudal ganglionic eminence (CGE) (b) at gestational week 12 (GW12). c-e, Boxed areas in a and b are shown in high power views. Scale bars: 1 mm in a and b; 100 µm in c-e.
Extended Data Fig. 5 Expression patterns of genes in the subpallium of the human fetal brain as detected by in situ sequencing (ISS).
a-d, Expression patterns of genes expressed in the ventricular zone (VZ) and subventricular zone (SVZ) at gestational week 12 (GW12) are shown in green and purple pseudo-colors, respectively, in the medial ganglionic eminence (MGE), lateral ganglionic eminence (LGE) and caudal ganglionic eminence (CGE). Scale bars: 1 mm.
Extended Data Fig. 6 Expression patterns of genes expressed in the ventricular zone (VZ) in the subpallium of the human fetal brain as detected by in situ sequencing (ISS).
a, Merged expression of CLU, LIX1, PTN and SPARC in the medial ganglionic eminence (MGE), lateral ganglionic eminence (LGE) and caudal ganglionic eminence (CGE) at gestational week 12 (GW12). b-e, Expression of CLU (b), LIX1 (c), PTN (d) and SPARC (e) in the MGE, LGE and CGE. Scale bars: 100 µm.
Extended Data Fig. 7 Specification of medial ganglionic eminence (MGE)-derived interneuron precursors.
a, Clustering of MGE-derived interneuron precursors from GW9 to GW12 visualized by t-SNE embedding. Cluster 5, 1, 2, 4 and 7 were similar to ANGPT2+/CRABP1+, ZEB2+/MAF+, POU3F2+/CNTNAP2+, NR2F1+/MEIS2+ and LHX8+/NKX2-1+, respectively, in the Fig. 4a, cluster 3, 6, 8 were raised from the GW12. b, Heatmap of differentially expressed genes from subclusters in a. c, Diffusion map of the most variable genes and reconstruction of the cell lineages. Dots represent cells, and black lines represent predicted cell lineage-1 and -2. As examples, the cluster 6 was predicted to the lineage 1 and the cluster 3 to the lineage 2. d. Expression patterns of genes highly expressed in the dorsal (MEIS2 and NR2F1) and ventral (LHX8 and ZIC4) MGE in human fetal brains at gestational week 12 (GW12) as detected using in situ sequencing (ISS). e, Box plots representing relative numbers of positive dots of MGE marker genes in boxed areas in the dorsal and ventral MGE in d. Box: 25–75th percentiles, whiskers: 10–90th percentiles, horizontal line in box: median. Scale bars: 1 mm.
Extended Data Fig. 8 Expression patterns of medial ganglionic eminence (MGE) specific genes of the human fetal brain as detected by in situ sequencing (ISS).
a, Coronal sections labeled with DAPI to illustrate cell nucleoli of a human fetal brain at gestational week 12 (GW12). Boxed areas highlight the MGE. The dorsal (D), ventral (V), medial (M) and lateral (L) orientations of the sections are labeled. b, High expression of CNTNAP2 and MAF at the root of lineage-1 and -2 based on the diffusion map in lineage analyses. c-e, Expression patterns of 10 MGE-precursor marker genes in coronal sections of human fetal brains. The dorsal and ventral MGEs are labeled as dMGE (c) and vMGE (d). Scale bars: 1 mm.
Extended Data Fig. 9 Lateral ganglionic eminence (LGE)-derived interneuron precursors.
a and b, Expression patterns of 8 LGE specific genes in coronal sections of the human fetal brain at gestational week 12 (GW12) as detected by in situ sequencing (ISS). The ventral (green box) and dorsal (blue box) LGE are shown as vLGE and dLGE. The boxed areas also are shown in high power views. Scale bars: 1 mm and 100 µm.
Extended Data Fig. 10 Caudal ganglionic eminence (CGE)-derived interneuron precursors.
a, The transcription network regulated by CGE-specific transcription factors in interneuron precursors. b, Gene ontology (GO) enrichment analysis of target genes for CGE-specific transcription factors. c, A coronal section labeled with DAPI to illustrate cell nucleoli of one human fetal brain at gestational week 12 (GW12). d, Expression patterns of CALB2, NPAS3, ST18 and SP9 in the CGE in coronal sections of the human fetal brain at GW12, as detected by in situ sequencing (ISS). Scale bar: 1 mm.
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Yu, Y., Zeng, Z., Xie, D. et al. Interneuron origin and molecular diversity in the human fetal brain. Nat Neurosci 24, 1745–1756 (2021). https://doi.org/10.1038/s41593-021-00940-3
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DOI: https://doi.org/10.1038/s41593-021-00940-3
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