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Single-cell transcriptomics of adult macaque hippocampus reveals neural precursor cell populations

Nature Neuroscience volume 25, pages 805–817 (2022)Cite this article

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Abstract

The extent to which neurogenesis occurs in adult primates remains controversial. In this study, using an optimized single-cell RNA sequencing pipeline, we profiled 207,785 cells from the adult macaque hippocampus and identified 34 cell populations comprising all major hippocampal cell types. Analysis of their gene expression, specification trajectories and gene regulatory networks revealed the presence of all key neurogenic precursor cell populations, including a heterogeneous pool of radial glia-like cells (RGLs), intermediate progenitor cells (IPCs) and neuroblasts. We identified HMGB2 as a novel IPC marker. Comparison with mouse single-cell transcriptomic data revealed differences in neurogenic processes between species. We confirmed that neurogenesis is recapitulated in ex vivo neurosphere cultures from adult primates, further supporting the existence of neural precursor cells (NPCs) that are able to proliferate and differentiate. Our large-scale dataset provides a comprehensive adult neurogenesis atlas for primates.

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Fig. 1: Dissecting the adult macaque hippocampal cells and molecular signatures.
Fig. 2: Validation of macaque hippocampus cell populations using annotated datasets.
Fig. 3: RGLs and astrocytes are distinct populations.
Fig. 4: Dynamic neurogenesis processes in the adult macaque hippocampal DG.
Fig. 5: HMGB2 correlates with the transition between RGL and IPCs.
Fig. 6: The maturation processes of the GCs in the adult macaque hippocampal DG.
Fig. 7: Morphology and gene expression of cultured hippocampal neurospheres.

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Data availability

The datasets generated in this paper are publicly available at ArrayExpress under accession codes E-MTAB-10225 and E-MTAB-10236. Source data are provided with this paper.

Code availability

The code for generating figures can be found at https://github.com/haozhaozhe/FM_hippo.

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Acknowledgements

This research is supported by research grants from the National Key R&D Program of China (2018YFA0108300), the Natural Science Foundation of China (81870682 and 81961128021), the Guangdong Provincial Key R&D Programs (2018B030335001 and 2018B030337001) and the Science and Technology Program of Guangzhou (202007030011, 202007030010 and 202007030001) to S.L.; the Natural Science Foundation of China (81721003) and the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01S138), CAMS Innovation Fund for Medical Sciences (2019-I2M-5-005) to Y.L.; the Natural Science Foundation of China (81970794), the Science and Technology Program of Guangzhou (201904020036) and the Technology Innovation 2030-Major Project on Brain Science and Brain-Like Computing of the Ministry of Science and Technology of China (2021ZD0202603) to M.X.; the Wellcome BioResource for a ‘Single Cell Gene Expression Atlasʼ (WT 108437/Z/15/Z) and the Open Targets grant (OTAR2067) to Z.M.; and the China Postdoctoral Science Foundation (2019M663256) to Z.-Z.H. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Zhao-Zhe Hao, Jia-Ru Wei, Dongchang Xiao.

Authors and Affiliations

  1. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China

    Zhao-Zhe Hao, Jia-Ru Wei, Dongchang Xiao, Ruifeng Liu, Nana Xu, Lei Tang, Mengyao Huang, Yuhui Shen, Wanjing Huang, Xialin Liu, Mengqing Xiang, Yizhi Liu & Sheng Liu

  2. Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Changsheng Xing

  3. Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China

    Yizhi Liu

  4. Research Unit of Ocular Development and Regeneration, Chinese Academy of Medical Sciences, Beijing, China

    Yizhi Liu

  5. Translational Research Institute of Brain and Brain-Like Intelligence and Department of Anesthesiology, Shanghai Fourth People’s Hospital Affiliated to Tongji University School of Medicine, Shanghai, China

    Zhichao Miao

  6. European Bioinformatics Institute, European Molecular Biology Laboratory, Wellcome Genome Campus, Cambridge, UK

    Zhichao Miao

  7. Guangzhou Laboratory, Guangzhou International Bio Island, Guangzhou, China

    Zhichao Miao

  8. Guangdong Province Key Laboratory of Brain Function and Disease, Guangzhou, China

    Sheng Liu

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  1. Zhao-Zhe Hao
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Contributions

S.L., Z.M., Y.L. and M.X. conceived and supervised the project. Z.-Z.H., Z.M., L.T., M.H., Y.S. and X.L. analyzed the data. J.-R.W., Z.-Z.H., R.L. and W.H. collected the tissues and performed the scRNA-seq experiment. D.X. performed the neurosphere culture experiment. J.-R.W., N.X., C.X. and Z.-Z.H. performed the immunofluorescence staining. S.L., Z.M., Y.L., M.X. and Z.-Z.H. wrote the manuscript, with input from all authors.

Corresponding authors

Correspondence to Mengqing Xiang, Yizhi Liu, Zhichao Miao or Sheng Liu.

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The authors declare no competing interests.

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Nature Neuroscience thanks Gerd Kempermann, Orly Lazarov and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Quality control of the dataset.

a) Distribution of the UMI counts detected in each cell in the whole dataset. b) Distribution of the unique genes detected in each cell in the whole dataset. c) UMAP colored by the 10x version and the animal ID. d) Violin plots of the UMI counts for each sample. The 10x version used for each sample is indicated by the colors. e) Violin plots of the unique genes detected for each sample. The 10x version used for each sample is indicated by the colors. f) The distribution of sample sizes for each animal. g) Proportion of different cell types present in individual animals. Cell types were colored using the same color scheme in Fig. 1d, and labeled at the bottom of panel.

Source data

Extended Data Fig. 2 The enriched gene regulatory networks (GRNs, termed regulons) in the hippocampal cell population.

a) tSNE plot derived from the regulon score, showing the neurogenic-related populations, colored by the cell types or the Gene Regulatory Network enrichment score derived from SCENIC AUcell. b) The dot plot shows the Gene Regulatory Network enrichment score derived from SCENIC AUcell. The color shows the normalized mean enrichment score, while the size of the dots shows the fraction of cells in each group that express the genes. The x-axis shows the name of the enriched regulons (Gene Regulatory Network), while the y-axis shows the cell populations in the dataset. c-f) TF regulatory networks predicted by SCENIC for the RGLs, IPCs and NBs.

Extended Data Fig. 3 The age-related shift in population distribution and gene expression.

a) Sample distribution grouped by age. *, p < 0.05; using two-tailed Mann-Whitney U test for each category between the young adults (age 4–6 year-old, n = 17 biologically independent samples) and the middle-aged adults (age 13–15-year-old, n = 8 biologically independent samples). Error bars, standard error of mean. The p-value for the comparison between young and middle-aged astrocytes is 0.039; for NB, 0.0039. All other comparisons are not significantly different. b) 1,435 DEGs between the Y4–6 and Y13–15 neuroblasts. These genes are visualized using the volcano plot. Genes that are significantly enriched (blue dots, determined by a two-part generalized linear model implemented by MAST) and exhibit > 0.5 log2 fold-change are marked in red. Selective genes are marked with text. c) A strip-chart showing the logarithmic-fold change (Log2 FC) of all genes between young adult (Y4–6) and middle-aged adult (Y13–15) for neurogenic-related cell populations. The dashed line at the top and bottom of the plot indicate the 2-fold change threshold. The dashed line in the middle indicates no differential expression. Genes that are upregulated in young adults have positive FCs. Genes that are differentially expressed (Wilcoxon rank-sum test with FDR correction for multi-comparison) and >30% max FC are colored using the same color scheme in Fig. 1. d) Pair-wise comparison of the neurogenic-related gene (n = 2,081 genes) enrichment between young and middle-aged adults for selective cell populations. Solid line, equal expression; dashed lines: 2-fold enrichment. Gray dots, the mean expression of each neurogenic-related gene of all cells of each population. Blue dots, genes that show >0.5 log2 fold-change enrichment in young adults. Red dots, genes that show >0.5 log2 fold-change enrichment in middle-aged adults. All correlation with a rank-sum based Spearman’s correlation coefficient (r)>0.95 with p < 0.0001. e) Heatmaps showing the selective genes that are upregulated in young adults (upper panel) or middle-aged adults (lower panel) exclusively for selective cell populations. Nomalized FC, normalized fold-change.

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Extended Data Fig.4 The sex-related shift in population distribution and gene expression.

a) Sample distribution grouped by sex. *, p < 0.05; using two-tailed Mann-Whitney U test for each category between the females (n = 7 biologically independent samples) and the males (n = 18 biologically independent samples). Error bars, standard error of mean. The p-value for the comparison between female and male IPC is 0.0015; for pre-OPC, 0.049; for PVM, 0.008. All other comparisons are not significantly different. b) Strip-chart showing the logarithmic-fold change (Log2FC) for all genes between females and males for neurogenic-related cell populations. The dashed line at the top and bottom of the plot indicate the 2-fold change threshold. The dashed line in the middle indicates not differentially expressed. Genes that are upregulated in females have potived FCs. Genes that are significantly expressed (Wilcoxon rank-sum test with FDR correction for multi-comparison) and >30% max FC are colored using the same color scheme in Fig. 1. c) Pair-wise comparison of the neurogenic-related gene (n = 2,081 genes) enrichment between females and males for selected cell populations. Solid line, equal expression; dashed lines: 2-fold enrichment. Gray dots, mean expression of each gene of all cells of each population. Blue dots, genes that show >0.5-fold enrichment in females. Red dots, genes that show >0.5-fold enrichment in males. All correlation with a rank-sum based Spearman’s correlation coefficient (r)>0.95 with p < 0.0001. d) Heatmaps showing the selective genes that are upregulated in female adults (upper panel) or male adults (lower panel) exclusively for selective cell populations. Normalized FC, normalized fold-change.

Extended Data Fig. 5 Immunostaining of canonical neural markers validated the existence of neural progenitor cells in SGZ.

a) NESTIN + cells in SGZ with apical processes (white arrowheads) touch the capillaries (yellow arrows), Scale bar, 10 μm. n = 4 brains. b) NESTIN + MKI67 + cells in SGZ with apical processes crossing the GCL into the ML, the processes touch capillaries of tubular morphology (yellow arrows). Scale bar, 10 μm. n = 4 brains. c) HOPX protein (gray), progenitor marker SOX2 (green), and neural stem cell marker GFAP (red) are colocalized in RGLs of macaques. Scale bar, 10 μm. n = 4 brains. Dashed lines indicate the outline of the cell. d) Immunolabeling for ASCL1, SOX2 and GFAP in the subgranular zone (SGZ) of the dentate gyrus (DG) of macaque. White arrowheads indicate ASCL1 + SOX2 + GFAP- IPC; yellow arrowheads indicate ASCL1 + SOX2 + GFAP + RGL. Scale bars, 25 μm (low magnification), 10 μm (high magnification). n = 4 brains.

Extended Data Fig. 6 Macaque adult hippocampal transcriptomic cell types are aligned with well-established mouse datasets.

a) UMAP visualization of macaque hippocampal transcriptomic cell types aligned with the Hochgerner_2 dataset. UMAP visualization of macaque cells (n = 15,878) and mouse cells (n = 3,132), stained with the species. b) UMAP visualization of macaque cell annotations using the same UMAP coordinates in panel a, stained according to the color scheme as in Fig. 1d. Astro, Astro_1–4; Astro_im, Astro_im1, Astro_im2; GC, GC_1–3; Microglia, Microglia_1 and 2; OPC, OPC_1 and 2; Pyr, Pyr_1 and 2; RGL, RGL_1, RGL_2. c) UMAP visualization of mouse cell annotations using the same UMAP coordinates in panel a, stained according to the color scheme as in Fig. 1d. d) Cell-type homologous heatmap between macaque (in rows) and mouse (in columns) for the Hochgerner_2 dataset. Gray shades correspond to the minimum proportion of co-clustering between mouse and macaque cells. Rows show macaque populations and columns show mouse populations. Colorbar indicates the alignment score. e) Macaque hippocampal transcriptomic cell types aligned with the Joglekar dataset, visualization of macaque cells (blue, n = 15,878) and mouse cells (orange, n = 5,305) f) UMAP visualization of macaque cells, stained according to the color scheme in Fig. 1d using the same coordinates as in panel d. g) UMAP visualization of mouse cells, stained according to the color scheme in Fig. 1d using the same coordinates as in panel d. h) Cell-type homologous heatmap between macaque (in rows) and mouse (in columns) for the Joglekar dataset. Gray shades correspond to the minimum proportion of co-clustering between mouse and macaque cells. Rows show macaque populations and columns show mouse populations. Colorbar indicates the alignment score. i) Macaque hippocampal transcriptomic cell types aligned with the Shin dataset, visualization of macaque cells (blue, n = 800) and mouse cells (orange, n = 132). j) UMAP visualization of macaque cells, stained according to the color scheme in Fig. 1d using the same coordinates as in panel i. k) UMAP visualization of mouse cells, stained according to the color scheme in Fig. 1d using the same coordinates as in panel i. Gray dots are macaque cells to facilitate the visualization of the coordinates.

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Extended Data Fig. 7 Macaque adult hippocampal transcriptomic cell types are aligned with well-established human datasets.

a) UMAP visualization of macaque hippocampal transcriptomic cell types aligned with the Li dataset. UMAP visualization of macaque cells (n = 2,400) and human embryonic/fetal cells (n = 323), stained with the species. b) UMAP visualization of macaque cell annotations using the same UMAP coordinates in panel a, stained according to the color scheme as in Fig. 1d. Astro, Astro_1–4; GC, GC_1–3; MG, Microglia_1 and 2; OPC, OPC_1 and 2; Pyr, Pyr_1 and 2; RGL, RGL_1 and 2. c) UMAP visualization of human cell annotations using the same UMAP coordinates in panel a, stained according to the color scheme as in Fig. 1d. d) Macaque hippocampal transcriptomic cell types aligned with the Zhong dataset, visualization of macaque cells (blue, n = 15,878) and human cells (orange, n = 6,383) e) UMAP visualization of macaque cells, stained according to the color scheme in Fig. 1d using the same coordinates as in panel d. f) UMAP visualization of human cells, stained according to the color scheme in Fig. 1d using the same coordinates as in panel d. g) Cell-type homologous heatmap between macaque (in rows) and human (in columns) for the Zhong et al. dataset. The color of each cell represents the alignment score between human and macaque cells. The larger value (darker in the heatmap) indicates a better alignment. Rows show macaque populations and columns show human populations. Colorbar indicates the alignment score.

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Extended Data Fig. 8 The lineage of the neurogenic-related populations.

a) The PCA visualization of the neurogenic-related population stained by cell populations, following the same color scheme as in Fig. 1. b) The PCA visualization of the neurogenic populations stained by canonical neurogenic marker genes. Colorbar indicates log-normalized gene-expression. c) the PCA visualization with the RNA velocities visualized as arrows. d) The heatmap shows the expression of the pseudotime related genes as Viridis (light yellow as a high expression), while the colorbar on top shows the cells in the neurogenic stages using the same color scheme in a. Selective genes are labeled to the left of the heatmap. Colorbar indicates gene expression level. e) The neurogenic lineage inferred by Slingshot. f) Pseudotime analysis derived from Slingshot suggests trajectories from RGL, via IPC to NB. Colorbar indicates the derived pseudotime.

Extended Data Fig. 9 Comparison of the gene enrichment between macaques and mice.

a) Neurogenic markers that are conserved between mice and macaques. b) Species-specific marker genes that are only enriched in macaques or in mice. c) Comparison of the gene enrichment between macaques and mice for the RGLs, IPCs, neuroblasts, and GCs, together with astrocytes and mature oligodendrocytes. Solid line, equal expression; dashed lines: 2-fold enrichment. Blue dots, high-diversity genes that are >2-fold enrichment in mice. Top five genes labeled by text. Gray dots, mean expression of each neurogenic-related gene (n = 2,081 genes) of all cells of each population. Red dots, high-diversity genes that are >2-fold enrichment in macaques with the top five genes labeled by text. The “r” value, the Spearman’s correlation coefficient for each comparison. All comparisons are significant with p < 0.001. d) Neurogenic-related cell composition for macaques (n = 39,555) and mice (n = 3,711) of the Hochgerner datasets.

Extended Data Fig. 10 Process of neurosphere formation and gene expression of cultured hippocampal neurospheres.

a-d) In vitro recording process of a cultured neurosphere clone from the adult macaque hippocampi. n = 4 clones. Scale bars, 40 μm. D, day. e) UMAP visualization of the cultured sphere cells (n = 6,988 cells), stained by the expression of selective marker genes. VIM, RGL and astrocyte marker; NNAT, novel neuroblast marker; SOX4, neuroblast marker; TUJ1, neuronal marker. Colorbar indicates log-normalized gene expression level. f-h) The cultured neurosphere cells from macaque hippocampus were immunoreactive for RGL and astrocyte marker VIMENTIN in f, neuroblast marker NNAT and SOX4 in g and h. n = 4 clones. Scale bars, 40 μm. i) Immunostaining analysis of EdU long-term labeling of the cultured neurospheres from the adult macaque hippocampus showed a small number of cells in the neurosphere were labeled by fluorescence for both TUJ1 and EdU in i (indicated by arrows). Scale bars, 40 μm. n = 4 clones for each staining. j) UMAP visualization of transcriptomic cell types projected from the Hochgerner_1 dataset. Red dots represent the distribution of the predicted cell type. IPC, intermediate progenitor cell; NB, neuroblast; GC, granule cell; Astro, astrocyte.

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Hao, ZZ., Wei, JR., Xiao, D. et al. Single-cell transcriptomics of adult macaque hippocampus reveals neural precursor cell populations. Nat Neurosci 25, 805–817 (2022). https://doi.org/10.1038/s41593-022-01073-x

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