The human neocortex contains hundreds of types of neurons and glia1 that emerge early in development from what appears to be a more limited set of progenitor types2. Birthdating studies conducted in the 1970s have provided a blueprint for our understanding of cortical neurogenesis by revealing the sequential, inside-out production of cortical layers and subsequent generation of glia3, but were unable to resolve the exact developmental origins of these cells. Clonal lineage tracing studies in model organisms, especially in mice, have increased our understanding of development, revealing that radial glia in the ventricular zone (VZ) of the developing brain act as neural stem cells4,5,6,7,8. Radial glia differentiate according to an intrinsic neurodevelopmental hierarchy9 that involves not only deep and upper cortical layer neurogenesis, but also generation of astrocytes10, oligodendrocytes11 and olfactory bulb (OB) GABAergic neurons12,13,14. Extending these studies to primates and humans has been constrained by the low throughput of experimental approaches for mapping the clonal output of individual progenitor cells using observational methods such as time-lapse microscopy15,16,17. This limits our understanding of the degree of conservation of neurodevelopmental processes from mice to humans, which is important for three main reasons.
First, it has long been known that neurogenesis in the human cortex is protracted18,19 to support the expansion of the cerebral cortex. However, the cellular mechanisms that underlie this extended neurogenic window and the temporal dynamics associated with the transition to gliogenesis are poorly understood20. Second, cortical progenitor cells of humans and primates appear to disproportionately generate large numbers of GABAergic neurons21,22,23, but the cellular and temporal origins of these cells during development remain unknown. Finally, the developing human cortex contains truncated radial glia (tRG)—a distinct subtype of radial glia called that emerge during the second trimester2—but their contributions to corticogenesis are poorly characterized. Addressing these questions would provide important insights into the possible mechanisms of human cortical expansion.
Here we have applied massively parallel lineage tracing21 to profile the differentiation patterns of 6,402 neural stem and progenitor cells across periods of late second trimester neurogenesis and gliogenesis, capturing progenitors that reside in the major stem cell niches. Our lineage-resolved atlas of the developing human brain uncovers three novel insights into progenitor cell dynamics of the developing human cerebral cortex. First, we show that GABAergic neurons generated from cortical progenitors emerge after midgestation and their generation from cortical progenitor cells constitutes a previously unappreciated developmental switch from glutamatergic to GABAergic neurogenesis. Second, we uncover that tRG are capable of producing all major cortical cell types, and are particularly important for glutamatergic neurogenesis via generation of intermediate progenitor cells. Third, we show that in the late phases of cortical neurogenesis, VZ and inner subventricular zone (ISVZ) progenitors generate glutamatergic neurons. A subset of these neurons show transcriptomic similarity to deep cortical layer neurons, suggesting a possible late phase of cortical neurogenesis that might reactivate deep cortical layer programmes. Together, our work provides insight on the developmental dynamics of neural stem cell differentiation in humans and uncovers previously unappreciated relationships between neurogenic and gliogenic trajectories of neural stem niches.
To create a lineage-resolved atlas of the developing human neocortex, we acquired 9 primary tissue specimens from 8 individuals before midgestation (n = 5, up to gestation week 20 (GW20)) and after midgestation (n = 4, after GW20) (Extended Data Table 1). These timepoints include middle and late stages of cortical neurogenesis and early gliogenesis, and harbour enriched expression of genes implicated in neurodevelopmental disorders and autism spectrum disorder24. To investigate how differentiation patterns of neural stem and progenitor cells change at these critical stages of development, we utilized STICR (single-cell RNA-sequencing-compatible tracer for identifying clonal relationships), a recently established tool for massively parallel clonal cell lineage tracing21,25 in which a molecularly barcoded lentiviral library with error-correctable barcodes enables tracing of clonal cell lineage of up to 250,000 individual cells per experiment with barcode collision probability of less than 0.5% (Fig. 1a). Across all nine tissue samples, we applied independent and molecularly indexed viral libraries of STICR to the germinal zones using VZ and outer subventricular zone (OSVZ)-specific labelling methods (Fig. 1a). We isolated GFP-positive cells after 10–12 days of ex vivo organotypic slice culture and processed the cells for single-cell RNA sequencing (scRNA-seq) to recover transcriptomic identities and lineage barcodes (Extended Data Figs. 1 and 2). Across all samples, we recovered 97,540 single cells that passed stringent quality control criteria, including 63,725 cells from specimens before midgestation and 33,815 cells after midgestation (Fig. 1c,d and Extended Data Fig. 1). We recovered STICR barcodes from 60% of cells passing quality control criteria (Extended Data Fig. 2c,d).
Fig. 1: Temporally resolved STICR lineage tracing reveals differences in progenitor output across midgestation. a, Experimental design for lineage tracing from dorsal cortical tissue samples across midgestation. Created in BioRender; Nowakowski, T. (2024) https://BioRender.com/hmdw1ry. b, Uniform manifold approximation and projection (UMAP) embedding and clustering of STICR-labelled cells following scRNA-seq. c, UMAP by sample, demarcated by gestational week at tissue acquisition. d, UMAP by pre- and post-midgestation (grouped). e, Bar chart showing the proportion of cells that belong to each cluster for each individual sample. f, Bar chart showing the proportion of cells from pre- or post-midgestation samples that contribute to each cluster. The dotted line represents the proportion of all cells obtained from pre-midgestation samples, regardless of cluster identity. g, UMAP comprising cells identified transcriptomically as radial glia and astrocytes, which were further subclustered to identify more specific subtypes (oRG and tRG). h, Bar chart showing the proportion of subclustered glial cells from g that were derived from the VZ or OSVZ. The dotted line represents the proportion of all cells obtained from VZ samples, regardless of cluster identity. i, UMAP of subclustered glial cells from g coloured by pre- and post-midgestation. j, Volcano plot showing differentially expressed genes between pre-midgestation radial glia (left) and post-midgestation radial glia (right). P values adjusted for multiple comparisons with Bonferroni correction. Selected genes are highlighted. METTL7B is also known as TMT1B. Full size image
To assign cell identities, we performed unbiased clustering and then analysed gene expression profiles of individual clusters and projected marker gene expression for specific cell types across all cells (Extended Data Fig. 1c,d and Supplementary Table 1). We thus annotated radial glia (RGs, HES1-positive (HES1+)), intermediate progenitor cells (EX_IPCs, EOMES+), glutamatergic neurons (excitatory neurons (ENs), SLC17A7+), GABAergic neurons (inhibitory neurons (INs), GAD2+), astrocytes (SPARCL1+), oligodendrocyte precursor cells (OPCs, OLIG2+) and oligodendrocytes (MBP+) (Fig. 1b, Extended Data Fig. 3a and Supplementary Table 2). Cells from all samples were found across all clusters (Fig. 1e and Extended Data Fig. 1e,f). As expected26, we found greater proportions of intermediate precursor cells (IPCs) and glutamatergic neurons in specimens before midgestation than after midgestation (85% versus 15% for EX_IPC and 75% versus 25% for EN) (Fig. 1f and Extended Data Fig. 1f). In parallel, we observed a higher proportion of macroglia (astrocytes, oligodendrocytes and OPCs) after midgestation (Fig. 1f and Extended Data Fig. 1f), consistent with the onset of gliogenesis around GW2027,28. We also observed a higher proportion of GABAergic cells in post-midgestation samples, increasing from 6.7% to 33% of all cells (Fig. 1f and Extended Data Fig. 1f), as expected on the basis of the progressive migration of interneurons from the ganglionic eminences over time28.
To better understand the progenitor cells that were present in these samples, we performed further analysis of the progenitor and glial cells, which are closely related transcriptomically. Progenitors and macroglia were iteratively subclustered from the full dataset on the basis of marker gene expression, focusing on putative astrocytes (SPARCL1+ or CD44+) and radial glia (HES1+, VIM+, FOXG1+ and EMX2+) (Fig. 1g and Extended Data Fig. 3a,b). Analysis of these subclusters revealed a population of putative outer radial glia (oRG) (INPP1+ and PPM1K+), tRG (CRYAB+ and ANXA1+), putative early OPCs (PDGFRA+) and two distinct subpopulations of astrocytes corresponding to grey matter ‘dense bulbous’ astrocytes (S100A11+) and white matter ‘dense smooth’ astrocytes (ANGPTL4+ and TIMP3+)29 (Extended Data Fig. 3c–g). During initial STICR labelling, germinal zones were labelled with separate indices to track the spatial origins of daughter cells (Fig. 1a). We observed that the majority of tRG were derived from the VZ, whereas oRG were derived from both the VZ and the OSVZ (Fig. 1h). Consistent with prior reports, the grey matter dense bulbous astrocytes were more commonly derived from the VZ, whereas white matter dense smooth astrocytes were derived from the OSVZ29 (Fig. 1h).
Radial glia were observed throughout the second trimester, but exhibited substantial variations in gene expression across time (Fig. 1i,j, Extended Data Fig. 3d and Supplementary Table 3). Pre-midgestation radial glia were enriched for genes associated with excitatory neurogenesis, including PAX6, FEZF2, NEUROG2, NEUROD2 and NEUROD6, and with genes that are characteristic of intermediate progenitor cells including EOMES and PPP1R17 (Fig. 1k). These early cells were also enriched in genes associated with the Wnt pathway, consistent with prior reports30 (Supplementary Table 3). By contrast, post-midgestation radial glia were enriched for genes associated with astrocytes (S100B, SPARCL1, GJA1 and AQP4) and oligodendrocyte precursor cells (OLIG2) (Fig. 1j). These later radial glia also showed an increase in expression of HES1 and HES5, which have been shown to repress excitatory neurogenesis31. Together, these findings show that both germinal niche and developmental age are correlated with different patterns in lineage outputs from neural stem and progenitor cells.
To determine how progenitor output changes across midgestation, we focused our analysis on multi-cellular clones (those with at least two cells that share the same lineage barcode) before and after midgestation (Fig. 2a,b). Our analysis identified 4,209 multi-cell clones from specimens obtained up to GW20, and 2,193 from specimens obtained after GW20 (Extended Data Fig. 2a,b). Before midgestation, the vast majority (77.6%) of clones contained glutamatergic cells (EX_IPCs or ENs) (Fig. 2a and Extended Data Fig. 4a). By contrast, only 9.9% of clones contained glutamatergic lineage cells after midgestation, whereas 40% of clones comprised OPCs, oligodendrocytes or astrocytes (Fig. 2b and Extended Data Fig. 4a). Although we captured relatively few multi-cellular clones that contain astrocytes (1% of all pre-midgestation clones and 3.6% of all post-midgestation clones), 26% of astrocyte-containing clones also included OPCs, consistent with the recently discovered dual-potency glial progenitor in the human neocortex32 (Extended Data Fig. 4c). In mice, OPCs are known to derive from both the ventral ganglionic eminences and from cortical radial glia20,27. In our dataset, 58% and 46% of OPC-containing clones shared barcodes with radial glia before and after midgestation, respectively, suggesting a strong contribution of dorsally derived OPCs in humans throughout midgestation (Extended Data Fig. 4b). Together, these findings suggest that in contrast to mice, the onset of gliogenesis in human cortical development occurs gradually and coincides with ongoing neurogenesis.
Fig. 2: Dorsally born GABAergic neurons emerge after midgestation. a,b, Left, chord diagrams for pre-midgestation (a) and post-midgestation (b) samples in which the thickness of connecting lines represents the frequency of clonal relationships between the two linked cell types. Right, upset plots representing the cell-type abundance (bottom left), types of clones (bottom right) and the abundance of each clone type (top right) for multi-cellular clones in pre- and post-midgestation samples. c, UMAP embedding and subclustering of GABAergic neurons. d, GABAergic neuron UMAP coloured by pre- and post-midgestation (grouped). e,f, GABAergic neurons in multi-cellular clones (e) and GABAergic neurons in multi-cellular clones (f) where one or more of the other cells in the same clone are in the excitatory lineage (EX_IPC or EN), projected in UMAP space. g, Dot plot of expression of cell-type markers and percentage of cells expressing the marker within each subcluster. h, Immunohistochemical staining of DLX2, MKI67 and PAX6 at GW17 and GW24. Example cells with triple-positive co-localization are marked by circles. Scale bars, 20 μm. i, Immunohistochemical staining of DLX2, SCGN and PAX6 in the cortical plate (CP) of GW17 and GW24 samples. Example cells with triple-positive co-localization are marked by circles. Scale bars, 20 μm. j, Left, ratio of DLX2+SCGN+PAX6+ cells to DLX2+ cells quantified from immunohistochemical stains such as those in i. Dots represent mean cell counts from the cortical plate (excluding the marginal zone) from each 20× image that was quantified (n = 3 images per sample; n = 8 samples; samples from GW17, GW19, GW20, GW21, GW23, GW23, GW24 and GW24). The black line represents linear regression, with 95% confidence interval in pink. Pearson’s correlation coefficient (R) is shown, P = 0.0029. Right, ratio of DLX2+SCGN+PAX6+ cells to DLX2+SCGN+ cells in pre- and post-midgestation samples. Dots represent mean counts from each biological replicate (n = 3 images per sample) and coloured bars represent means per binned age group (n = 3 samples for pre-midgestation, mean = 0.021, n = 5 samples for post-midgestation, mean = 0.14). Error bars show s.e.m. for each age group. Two-sided Student’s t-test, P = 0.046. k, Model of how dorsally born GABAergic neurons emerge and migrate in the cortex. Dotted lines represent a lineage relationship. Created in BioRender; Nowakowski, T. (2024) https://BioRender.com/4fgwqb2. Full size image
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