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Spatiotemporal clonal architecture of the newborn mouse forebrain

Nature作者：Guohua Yuan2026年9月30日正文已收录本站

Data availability

scRNA-seq transcriptomic data and STICR barcode data are available at the Gene Expression Omnibus (GEO) under accession number GSE341480. An interactive browser of single-cell data and MERFISH data can be found at the University of California, Santa Cruz (UCSC) cell browser (https://p4-brain-lineage.cells.ucsc.edu). The MERFISH dataset is available at Brain Image Library (BIL; https://api.brainimagelibrary.org/web/view?bildid=ace-let-lax).

Code availability

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Acknowledgements

We thank all members of the Nowakowski laboratory for their insight and advice while conducting these experiments and preparing the manuscript; the UCSF Helen Diller Family Comprehensive Cancer Center Flow and Cell Sorting Core Facility (RRID:SCR_026372) for their assistance with FACS; and R. Delgado for his pioneering work in the initial design of STICR. This work was also supported by the Allen Institute. T.J.N. is a New York Stem Cell Foundation Robertson Neuroscience Investigator.

Funding

This project was supported by the National Institute of Mental Health (NIMH) and National Institute of Neurological Disorders and Stroke (NINDS), of the National Institutes of Health (NIH), with grant numbers U01MH130962, R01NS123263 and R01MH128364, the California Institute for Regenerative Medicine (CIRM) DISC0-14429, as well as by gifts from the Esther A. and Joseph Klingenstein Fund, the Shurl and Kay Curci Foundation, the Sontag Foundation and the William K. Bowes Jr Foundation.

Author information

Authors and Affiliations

  1. Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA

    Guohua Yuan, Marilyn R. Steyert, Matthew G. Keefe, Yasmin Fukushima, Adam Kazerounian, Arturo Alvarez-Buylla & Tomasz J. Nowakowski

  2. Allen Institute for Brain Science, Seattle, WA, USA

    Michael Kunst, Rémi Mathieu, Cindy T. J. van Velthoven, Delissa McMillen, Jack Waters & Hongkui Zeng

  3. The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA

    Arturo Alvarez-Buylla & Tomasz J. Nowakowski

  4. Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA

    Tomasz J. Nowakowski

  5. Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, San Francisco, CA, USA

    Tomasz J. Nowakowski

  6. Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA

    Tomasz J. Nowakowski

  7. Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, San Francisco, CA, USA

    Tomasz J. Nowakowski

Authors

  1. Guohua Yuan
  2. Michael Kunst
  3. Marilyn R. Steyert
  4. Matthew G. Keefe
  5. Rémi Mathieu
  6. Cindy T. J. van Velthoven
  7. Delissa McMillen
  8. Jack Waters
  9. Yasmin Fukushima
  10. Adam Kazerounian
  11. Arturo Alvarez-Buylla
  12. Hongkui Zeng
  13. Tomasz J. Nowakowski

Contributions

Conceptualization: G.Y., M.G.K., M.K., J.W., C.T.J.v.V., H.Z. and T.J.N. Investigation and experimental design: G.Y., M.K., M.R.S., M.G.K. and C.T.J.v.V. Methodology: G.Y., M.K., M.R.S., M.G.K., C.T.J.v.V., D.M., J.W., Y.F., A.K. and R.M. Data analysis: G.Y., M.K., M.G.K., R.M. and C.T.J.v.V. Visualization: G.Y. and M.R.S. Funding acquisition: T.J.N. and H.Z. Project administration: T.J.N., H.Z. and A.A.-B. Supervision: T.J.N., H.Z. and A.A.-B. Writing—original draft: G.Y. and T.J.N. Writing—review and editing: G.Y., T.J.N., H.Z., A.A.-B., M.K., M.R.S., M.G.K. and C.T.J.v.V.

Corresponding author

Correspondence to Tomasz J. Nowakowski.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Collection of virus-labeled cells with lineage barcodes and clone size of broad cell types.

a, Representative image showing GFP-positive cells labeled by the STICR virus. The injection was performed at E11, and the tissue was collected at P4 (n = 2 samples). Scale bar = 500 μm. b, Representative FACS plot illustrating the gating strategy used to sort GFP-positive cells. The final sorted GFP+ population was used for all scRNA-seq analyses in this study. c, Heatmap showing the percentage of identical clones shared between pairs of brains. For each pairwise comparison, the percentage was calculated relative to the sample with the lower total clone count. Each row and column represents an independent brain. This analysis assesses the extent of barcode overlap across independent samples. d, Bar plot showing the average clone size for each injection date. Clones from each injection date were subsampled to the same number. Error bars indicate the SEM, and each dot represents an individual clone. Only multicellular clones are shown. The number of points in each group from left to right, are: 4106, 3348, 1898, 2468, and 1165. e, Bar plot showing the average clone size for each cell type. Error bars indicate the SEM, and each dot represents an individual clone. Only multicellular clones are shown. The number of points in each group from left to right, are: 562, 535, 24962, 500, 9004, 328, 24508, 12697, 13942, 799, 504, and 2158.

Extended Data Fig. 2 Simulations evaluating the effects of capture rate and the number of recovered multicellular clones.

a, Box plots showing cell loss across experimental steps. Boxes show the median and interquartile range; whiskers extend to the lowest and highest observations within 1.5× the interquartile range of the lower and upper quartiles, respectively. Dots represent individual samples (n = 93). Dots outside of displaying range were not shown. b, Schematic of the sampling simulation framework using ground-truth clones. The simulation models how clone features inferred from repeated partial sampling under different capture rates recover the ground-truth lineage structure. c, Probability of recovering all cell types across different numbers of recovered multicellular clones. Panels represent different capture rates or ground-truth clone sizes. This analysis estimates how many observed multicellular clones are required to recover the constituent cell types of a lineage. Each point represents the proportion of 500 independent simulations in which all cell types were detected. d, Correlation between cell-type proportions in simulated and ground-truth clones across different numbers of recovered multicellular clones. Panels represent different capture rates and ground-truth clone sizes. Boxes show the median and interquartile range; whiskers extend to the lowest and highest observations within 1.5× the interquartile range of the lower and upper quartiles, respectively. Each point represents one independent simulation and shows the Pearson correlation between the observed and true cell-type proportions. Up to 500 simulations were included per condition; simulations with undefined correlations were excluded. Error bars indicate the standard error of the mean (SEM). e, Heatmap from simulations based on a ground-truth configuration in which all cells belong to a single clone, showing the number of shared clones between two cell types. Each row of panels represents an independent sampling simulation, and each column of panels represents the number of recovered multicellular clones. f, Heatmap from simulations based on a ground-truth configuration in which cells belong to two separate clones, showing the number of shared clones between two cell types. Each row of panels represents an independent sampling simulation, and each column of panels represents the number of recovered multicellular clones. g, False fate-bias rate, calculated from the ground-truth clones in panel a, across different capture ratios, clone sizes, and numbers of multicellular clones. False fate bias is defined as the probability that >25% of cell-type pairs exhibit either zero shared clones or a shared-clone fraction below 10% of the smaller of the two clone counts.

Extended Data Fig. 3 Spatial location of broad cell types in MERFISH data.

a, MERFISH spatial maps of the cell types in coronal sections from rostral to caudal. Cells are colored by broad cell type categories, same as Fig. 1c. Sections are ordered from rostral to caudal, with AP order = 0 denoting the most rostral section. Sections out of the forebrain were not shown. b, Cell-type proportions across MERFISH sections along the rostral–caudal axis. Sections are ordered from rostral to caudal, with AP = 0 denoting the most rostral section. Cell-type proportions were normalized to the total number of cells in each section.

Extended Data Fig. 4 Lineage analysis of CR cells.

a, Clonal coupling correlation among broad cell types. Data from all brain regions and injection dates were combined. Correlations were calculated using the Pearson coefficient based on the normalized number of shared clones between each pair of cell types. b, Venn diagram showing shared clones of CR cells between the cortex and hippocampus. c, Heatmap showing the number of shared clones between CR cells across different injection timepoints. Clone counts are normalized by the total number of clones detected at each injection age. d, MERFISH spatial maps of representative CR cell clusters (top) and astrocyte and ependymal cell clusters (bottom). Cells are colored by cluster identity. e, Heatmap showing shared-clone counts between hippocampal CR cell clusters and astrocyte or ependymal cell clusters. Color indicates shared-clone counts. Right and bottom color bars indicate the number of multicellular clones containing each cell type. Cell-type pairs with at least two shared clones are shown. Data is pooled across all injection time points. f, Heatmap showing shared-clone counts between cortical CR cell clusters and astrocyte or ependymal cell clusters. Color indicates shared-clone counts. Right and bottom color bars indicate the number of multicellular clones containing each cell type. Cell-type pairs with at least two shared clones are shown. Data is pooled across all injection time points.

Extended Data Fig. 5 Lineage analysis of microglia subtypes.

a, Expression of marker genes across microglia subtypes. Each dot represents a single cell. b, UMAP visualization of microglia in clones from five brain regions. Cells are colored by subtypes of microglia. c, UMAP visualization of microglia in clones from five brain regions. Cells are colored by brain regions. d, Stacked bar plot showing the distribution of different microglial subtypes across regions. e, Bar plot showing the average clone size for microglia subtypes. Error bars indicate the SEM, and each dot represents an individual clone. The number of points in each group from left to right, are: 73, 104, 272, and 143. f, Venn diagram showing shared clones of microglia between different subtypes. g, UpSet plot showing shared clone counts between microglia from different brain regions. h, Heatmap showing the number of shared clones between microglia across different injection timepoints. Diagonal values indicate clones with at least two cells of that type. Clone counts are normalized by the total number of clones detected at each injection age. i, Heatmap showing the number of shared clones between endothelial cells, epithelial cells, and vasculature cells across different injection timepoints. Diagonal values indicate clones with at least two cells of that type. Clone counts are normalized by the total number of clones detected at each injection age.

Extended Data Fig. 6 MERFISH spatial maps showing the subregional distribution of representative clusters.

This figure provides anatomical context for cluster annotations and supports interpretation of region- and subregion-resolved clonal analyses in the main figures.

Extended Data Fig. 7 Clonal lineage fates of glutamatergic neurons in different brain regions.

a, Relationship between clonal coupling and transcriptomic similarity for glutamatergic neuron subtypes across regions. Color indicates whether each pair of subtypes comes from the same brain region or from different regions. This analysis evaluates whether transcriptionally similar populations are also clonally coupled. b, Heatmap showing the temporal generation of glutamatergic neurons in major cortical layers. Colors indicate the ratio of cells in different categories on each date, normalized across each row using z-scores. c, Heatmap showing clonal coupling between clusters of cortical layer glutamatergic neurons, with colors representing the number of shared clones. Diagonal values indicate clones with at least two cells of that type. d, BrdU labelling showing the temporal difference of deep layer generation in cingulate cortex and other cortical regions. The data was downloaded from Neuron Birthdate Database36 (https://www.neurobirth.org/). e, Venn diagram showing shared clones of hippocampal glutamatergic neurons between DG, CA1, and CA3. f, Heatmap showing the dynamics of postnatally born glutamatergic neuron cluster proportions relative to all glutamatergic neurons. Colors represent row-wise z-score normalized ratios. g, MERFISH spatial maps of OB glutamatergic neurons. Cells are colored by the temporal stages that they are primarily labelled. Embryo indicates being labelled only in embryo stages. Embryo-Postnatal indicates being labelled both in embryo and postnatal stages. Postnatal indicates being labelled primarily in postnatal stages.

Extended Data Fig. 8 Cluster-resolution clonal map of glutamatergic neuron lineage progression.

Heatmap of clonal coupling between clusters of glutamatergic neurons within each region. Clone counts are normalized by the total number of glutamatergic neuron clones in each region at each injection age. Diagonal values indicate clones with at least two cells of that type. This cluster-resolution map expands the glutamatergic-neuron analysis by showing within-region lineage relationships across developmental labeling windows. The thalamic sample included both thalamic and epithalamic tissue. The dissections may have included a small amount of tissue from adjacent regions.

Extended Data Fig. 9 Molecular identities of postnatally born glutamatergic neurons.

a, Violin plots showing expression of canonical excitatory and inhibitory neuronal marker genes in postnatally born excitatory and inhibitory neurons isolated from dissected cortical regions. b, Diagram highlighting regions enriched for postnatally generated glutamatergic neurons in the validation dataset. Created in BioRender; Nowakowski, T. https://biorender.com/b6902xe (2026). c–e, Immunostaining validation of postnatally born glutamatergic neurons in representative coronal sections. BrdU was administered intraperitoneally at P0, and whole-mount analysis was performed at P4 (n = 3 samples). Double-positive cells are indicated by arrows. Scale bar = 100 μm. f, Immunostaining validation of postnatally born glutamatergic neurons in representative sagittal sections. BrdU was administered intraperitoneally at P0, and whole-mount analysis was performed at P4 (n = 3 samples). Double-positive cells are indicated by arrows. Scale bar = 1 mm.

Extended Data Fig. 10 Regional dispersion of GABAergic neurons across regions.

a, Violin plots showing the expression of canonical CGE, MGE, and LGE marker genes across inhibitory neuron clusters. b, Relationship between clonal coupling and transcriptomic similarity for subtypes of GABAergic neurons across regions. Color indicates whether each pair of subtypes comes from the same brain region or from different regions. c, MERFISH spatial maps showing the distribution of different GABAergic neuron subtypes across distinct striatal subregions. d, Heatmap displaying GABAergic neurons clones dispersed across multiple regions. Each row represents a single clone, and colors indicate the proportion of cells from each region within that clone. e, Left: UpSet plot showing shared clone counts between GABAergic neurons from different regions. Right: dotplot showing the subtype composition of GABAergic neurons within each clone category defined in the top panel. Intersection counts of more than 200 were shown. f, Boxplot showing the average cosine distance of cells within each clone in the PCA space of transcriptomic data. Boxes show the median and interquartile range; whiskers extend to the lowest and highest observations within 1.5× the interquartile range of the lower and upper quartiles, respectively. Each dot indicates one single clone. The number of points in each group from left to right, are: 4103, 90, 3725, 549, 5269, and 4885. This analysis tests whether cells within dispersed clones are transcriptionally more diverse than cells within regionally restricted clones. g, Barplot showing the temporal dynamics of the proportions of different clone categories across injection timepoints. Proportions are calculated as the number of clones in each category relative to the total number of GABAergic neurons clones at each timepoint.

Extended Data Fig. 11 Cluster-resolution clonal map of GABAergic neuron lineage progression.

Heatmap of clonal coupling between clusters of GABAergic neurons within each region. Clone counts are normalized by the total number of GABAergic neuron clones in each region at each injection age. Diagonal values indicate clones with at least two cells of that type. This cluster-resolution map expands the GABAergic-neuron analysis by showing within-region lineage relationships across developmental labeling windows. The thalamic sample included both thalamic and epithalamic tissue. The dissections may have included a small amount of tissue from adjacent regions. Striatal GABAergic neurons were divided into two major clonal groups. The first group was enriched at E14 and comprised LGE-derived neurons in the striatal core. The second arose between E12 and E16, persisted until P0, and was transcriptomically similar to migrating OB neurons. OB GABAergic neurons formed two major clonal groups: an early group, including two LGE-derived clusters labeled between E11 and E16 and enriched at E11–E12, which disappeared by P0; and a second group enriched at E16 and P0, likely originating from the V-SVZ or locally.

Extended Data Fig. 12 Subregion-resolved analysis in OB.

a, MERFISH spatial maps showing the distribution of different GABAergic neuron subtypes across distinct OB subregions. b, Heatmap showing clonal coupling between OB GABAergic neuron clusters. Color indicates shared-clone counts. Diagonal values indicate the number of clones containing each cell type. Cell-type pairs with at least two shared clones are shown. OB GABAergic neurons collected within the OB from the P0 labelling showed very limited clone sharing with other regions, including the striatum and cortex (Fig. 1d). This suggests that many OB GABAergic neurons captured at P4 (showing here) were born embryonically or locally generated, whereas postnatally generated neurons migrating toward the OB through the RMS had largely not yet reached the OB at P4. c, Proportion of cell clusters across temporal windows within each OB subregion. Proportions were calculated relative to all OB GABAergic neurons and min–max normalized for visualization. This panel summarizes temporal differences in subtype abundance across OB subregions. d and e, UpSet plots showing shared-clone counts among OB GABAergic neuron clusters. Counts were calculated from pooled data across all injections. f, Heatmap showing clonal coupling between GABAergic neuron clusters in the OB and striatum. Color indicates shared-clone counts. Right and bottom color bars indicate the number of multicellular clones containing each cell type. Cell-type pairs with at least two shared clones are shown. This analysis identifies the clonal coupling of OB and striatal subtypes across temporal windows.

Extended Data Fig. 13 Regional disperse and clonal fate biases of glial cells.

a, MERFISH spatial maps showing glial subtypes in thalamic regions in representative coronal sections. Cells are colored by subtype. b, Relationship between clonal coupling and transcriptomic similarity for glial subtypes across regions. Color indicates whether each pair of subtypes comes from the same brain region or from different regions. c, Heatmap showing clonal coupling between clusters of mitotic cells across regions. Colors represent the number of shared clones. Diagonal values indicate clones with at least two cells of that type. d, Heatmap showing clonal coupling between clusters of astrocytes across regions. Colors represent the number of shared clones. Diagonal values indicate clones with at least two cells of that type. e, Heatmap showing clonal coupling between clusters of ependymal cells across regions. Colors represent the number of shared clones. Diagonal values indicate clones with at least two cells of that type. f, MERFISH spatial maps showing the distribution of OPC clusters 16 and 5 across brain regions. Cells are colored by subtype. g Clonal enrichment of different sub cell types with the astrocyte lineage and OPC lineage (including OPCs and oligodendrocytes) across regions. The clonal enrichment was calculated from the hypergeometric test.

Extended Data Fig. 14 Cluster-resolution clonal map of glia lineage progression.

Heatmap of clonal coupling between clusters of glial cells within each region. Clone counts are normalized by the total number of glial cells clones in each region at each injection age. Diagonal values indicate clones with at least two cells of that type. This cluster-resolution map expands the glial analysis by showing within-region lineage relationships across developmental labeling windows. The thalamic sample included both thalamic and epithalamic tissue. The dissections may have included a small amount of tissue from adjacent regions. At P0, the postnatal group was further divided into two subgroups. Subtype annotation of B cells (adult neural stem cells) reveals cluster 21 (Crym+) as ventral subpallial, cluster 2 (Gsx2+, Dio2+) as dorsal subpallial, and clusters 26, 25, 27, and 1 (Emx1+, Hopx+, Thap2c+) as pallial.

Extended Data Fig. 15 Clonal relationships between OPC and astrocytes across temporal labeling in thalamus and cortex.

a, MERFISH spatial maps showing the distribution of OPC and astrocytes clusters across distinct thalamus and epithalamus regions. b, Heatmap showing clonal coupling between OPC and astrocyte clusters in thalamic and epithalamic regions. Color indicates shared-clone counts. Diagonal values indicate the number of clones containing each cell type. Cell-type pairs with at least two shared clones are shown. c, MERFISH spatial maps showing the distribution of OPC and astrocytes clusters across distinct cortical regions. d, Heatmap showing clonal coupling between OPC and astrocyte clusters in the cortex. Color indicates shared-clone counts. Diagonal values indicate the number of clones containing each cell type. Cell-type pairs with at least two shared clones are shown. e, MERFISH spatial maps showing the distribution of cell clusters of hippocampus OPC, Astrocytes, and neurons across distinct hippocampus subregions.

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Yuan, G., Kunst, M., Steyert, M.R. et al. Spatiotemporal clonal architecture of the newborn mouse forebrain. Nature (2026). https://doi.org/10.1038/s41586-026-11064-8

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