Lineage-resolved atlas of the developing human cortex

Keefe, Matthew G.; Steyert, Marilyn R.; Nowakowski, Tomasz J.

doi:10.1038/s41586-025-09033-8

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Published: 05 November 2025

Lineage-resolved atlas of the developing human cortex

Matthew G. Keefe ORCID: orcid.org/0000-0002-2477-2975^1,2,3,4,5,6^na1,
Marilyn R. Steyert^1,2,3,4,5,6^na1 &
Tomasz J. Nowakowski ORCID: orcid.org/0000-0003-2345-4964^1,2,3,4,5,6

Nature volume 647, pages 194–202 (2025) Cite this article

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Abstract

The human neocortex is composed of diverse cell types¹ that are generated during development according to spatially and temporally organized programmes initiated by neural stem cells^2,3,4,5. Despite the growing number of studies that have captured snapshots of gene expression of single cells along the axis of differentiation and maturation, the underlying map of lineage relationships that link individual progenitor cells to specific subtypes of neurons and glia remains unknown, especially in humans. Here we applied prospective lineage tracing to map the manifold of human neural stem and progenitor cell differentiation across the developmental window encompassing neurogenesis and gliogenesis in human primary tissue. By profiling the clonal output of 6,402 progenitor cells, we created a lineage-resolved map of human cortical development. Here we show that cortical progenitors switch from glutamatergic to GABAergic (involving γ-aminobutyric acid) neurogenesis around midgestation, which coincides with an onset of oligodendrocyte generation. Additionally, we find that truncated radial glia maintain a glutamatergic neurogenic potential for a protracted period during human cortical development. Unexpectedly, we find that late-born glutamatergic neurons derived from truncated radial glia exhibit molecular features of deep cortical layer neurons and may contribute to the expansion of the subplate region during midgestation.

Main

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 cells^4,5,6,7,8. 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 neurons^12,13,14. 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 microscopy^15,16,17. 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 protracted^18,19 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 neurons^21,22,23, 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 tracing^21,25 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.**

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 GW20^27,28. 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 glia^20,27. 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.**

Unexpectedly, our data also revealed a marked shift from glutamatergic neurogenesis to GABAergic neurogenesis around midgestation, with 62% of multi-cell clones containing GABAergic cells (IN_IPCs or inhibitory neurons (INs)) after GW20 (Fig. 2b). To determine what types of GABAergic neurons are generated locally within the germinal zone of the cerebral cortex, we performed iterative clustering that identified five types of GABAergic cells (Fig. 2c and Extended Data Fig. 5a–c). Caudal ganglionic eminence (CGE)-derived GABAergic neurons (NR2F2⁺ and ADARB2⁺) and medial ganglionic eminence (MGE)-derived GABAergic neurons (LHX6⁺ and MAF⁺) were both enriched for ERBB4, a marker of tangential migration, consistent with their presumed ventral origin³³ (Fig. 2d,g). Only a small fraction (0.7%) of these cells were present in multi-cellular clones (Fig. 2e), and no cells in this cluster shared lineage barcodes with glutamatergic neurons or cortical radial glia (Fig. 2f and Extended Data Fig. 2d,e), suggesting that they represent a rare tangentially migrating GABAergic neuroblast derived from CGE or MGE^34,35,36. We also identified OB interneurons (PBX3⁺ and TSHZ1⁺) (Fig. 2g and Extended Data Fig. 5b,d,g,h), a subset of which were found in multi-cell clones, consistent with their dorsal origin^12,13,14 (Fig. 2e and Extended Data Fig. 5e,f). We identified a progenitor population that was enriched for MKI67 (IN_IPC; Fig. 2g), consistent with prior studies^21,37. We validated that these IN_IPCs were present even in uncultured tissue throughout the VZ and OSVZ using immunohistochemistry, especially in post-midgestation samples (Fig. 2h and Extended Data Fig. 6). Finally, we identified a cluster (IN_local) of GABAergic neurons that co-express a subset of CGE (NR2F1), MGE (SOX6) and lateral ganglionic eminence (LGE, MEIS2) markers (Fig. 2g,i and Extended Data Fig. 5c). IN_local cells could be further distinguished from other populations by their enriched expression of transcription factors PAX6 and ST18 (Fig. 2g).

Almost all GABAergic cells that were found in multi-cellular clones (98%) belonged to IN_IPC and IN_local clusters, and only cells in these clusters shared barcodes with glutamatergic lineage cells, including cortical (PAX6⁺ and EMX1⁺ and/or EMX2⁺) radial glia or neurons (Fig. 2e,f and Extended Data Figs. 4a,e and 5e,f). In addition, GABAergic-lineage cells frequently shared lineage relationships with OPCs (Extended Data Fig. 4a,b,e), consistent with a recently described dorsally derived multipotent progenitor that can produce both oligodendrocytes and GABAergic-lineage cells³⁸.

To determine the distribution of presumed locally born GABAergic neurons, we stained uncultured tissue for SCGN, DLX2 and PAX6 proteins. SCGN⁺DLX2⁺ cells were found throughout the telencephalic wall, especially in the cortical plate and marginal zone (Fig. 2i and Extended Data Fig. 7), aligning with the known distribution of CGE-derived cortical interneurons in humans and primates^28,34. Consistent with the lineage tracing data (Fig. 2a–f), the abundance of SCGN⁺DLX2⁺PAX6⁺ triple-positive cells increases significantly around midgestation (Fig. 2i,j and Extended Data Figs. 7 and 8b,c). PAX6⁺ interneurons comprised approximately 15% of SCGN⁺DLX2⁺ cells in post-midgestation samples, indicating that at this point in development, most of the cortical SCGN⁺ population is likely to be derived from CGE^28,34 (Fig. 2j). Notably, SCGN⁺PAX6⁺ cells can also be detected in the cortical plate at GW30 and GW39, suggesting that these cells persist in the cerebral cortex throughout birth (Extended Data Fig. 8d,e).

In one sample with known regional annotation, we performed lineage tracing in the prefrontal cortex (PFC) and visual cortex (V1). IN_local and IN_IPC cells were present in 36% of multi-cellular clones collected from PFC, versus 15% from V1 (Extended Data Fig. 9a,b), which was consistent with immunostaining validation performed in uncultured tissue (Extended Data Fig. 9c–f). Differences in the abundance of IN_local and IN_IPC cells across areas could reflect the neurogenetic gradient that is known to exist in the developing brain^39,40,41. Together, our study identifies late second trimester as a point of onset of locally generated GABAergic neurons, and identifies molecular features that distinguish these cells from their CGE-derived and MGE-derived counterparts, as well as from presumed OB interneurons (Fig. 2k).

Beyond describing the origins of different subtypes of cortical neurons, we also sought to characterize the progenitors themselves more thoroughly, particularly tRG, a subtype of apical radial glia that emerges around GW17 in humans². In contrast to oRG, whose differentiation trajectories have been studied extensively^{15,27,42,43,44}, the developmental fate of cells derived from tRG remains poorly understood^29,45,46 (Fig. 3a). We identified tRG and oRG in our samples transcriptomically (Extended Data Fig. 3), and utilized clonal barcoding information from STICR to perform careful analysis of the clones that contained each radial glia (RG) subtype (Fig. 3b–e). Both oRG and tRG were capable of producing all cell types including excitatory lineage cells, inhibitory lineage cells and macroglia (Fig. 3f and Extended Data Fig. 4f). Radial glia were most commonly recovered alongside other clonally related radial glia, confirming that both RG subtypes can self-renew (Fig. 3c,e). Indeed, among clones that contain only two recovered cells, we observed global fate restriction, in that recovered cells were more likely to belong to the same general category of cell type than would be expected by chance (Extended Data Fig. 10). Surprisingly, our lineage tracing analysis identified extensive clonal coupling between tRG and EOMES⁺ IPCs (EX_IPCs) or glutamatergic neurons (ENs) (Fig. 3d,e).

**Fig. 3: Ventricular zone progenitors contribute to glutamatergic neurogenesis.**

Because tRG and their clonal coupling to EX_IPCs was identified both pre- and post-midgestation, we explored whether tRG contribute to a persistent late phase of glutamatergic neurogenesis in humans. Immunostaining of primary uncultured tissue sections confirmed an abundance of dividing cells and EOMES⁺ IPCs in the germinal zones of primary tissue sections late in second trimester, notably enriched in the VZ and ISVZ (VZ/ISVZ) region (Fig. 3f and Extended Data Fig. 11). This is consistent with our finding that most IPCs in our dataset originated from VZ infections rather than OSVZ infections when looking across all ages (Fig. 1h). Because IPCs are known to generate excitatory neurons, we also explored whether NEUROD2⁺ cells were present in the germinal zones post-midgestation. Indeed, even in late second trimester, a substantial number of multi-cellular clones contained glutamatergic IPCs or neurons, marked by expression of SLC17A7, NEUROD2 and other canonical markers (Fig. 3b,c and Extended Data Fig. 4a). Immunostaining of uncultured tissue further confirmed the presence of NEUROD2⁺ cells in germinal zones including the VZ/ISVZ region (Fig. 3d, Extended Data Fig. 12). Together, these data reveal that tRG that reside in the ventricular zone remain neurogenic in the late second trimester. Next, we explored the identity of these late-born glutamatergic neurons. Unexpectedly, we found that the presumed late-born glutamatergic neurons that were still in the germinal zones expressed high levels of TBR1, but not SATB2 (Fig. 4a and Extended Data Fig. 13). During mouse development, TBR1⁺ neurons are generated during early development and contribute to deep layers of the cerebral cortex⁴⁷, whereas SATB2⁺ cells contribute to upper layers and are born later according to the inside-out model of cortical development⁴⁸. To better understand how late-born glutamatergic neurons compare to deep layer neurons generated during first trimester in humans, we performed scRNA-seq from microdissected VZ/ISVZ tissue specimens and compared key marker gene expression to data derived from first-trimester specimens⁴⁹, focusing on glutamatergic neurons (Extended Data Fig. 14a). Both late-born and early-born neurons expressed high levels of TBR1 and NEUROD2, as well as more specific subplate neuron markers such as TLE4, NR4A2 (also known as NURR1), CTGF (also known as CCN2) and NFIB (Extended Data Fig. 14b–p). We validated expression of subplate markers CPLX3 and/or NR4A2 and/or CTGF in some VZ/ISVZ-located TBR1⁺ cells using in situ hybridization after midgestation (Fig. 4b–f and Extended Data Fig. 15). Of note, we observed strong expression of CTGF in tRG (Extended Data Fig. 15), consistent with prior reports⁵⁰.

**Fig. 4: Deep layer neurogenesis persists at the ventricular zone throughout the second trimester.**

Finally, because some late-born glutamatergic neurons shared transcriptomic identity with classical subplate and deep layer neurons, we tracked the migration of late-born neurons in culture. To validate subplate localization of these neurons, we performed BrdU labelling of microdissected VZ/ISVZ tissue specimens (Fig. 4g). After 10 days of organotypic slice culture, approximately 5% of BrdU-labelled cells expressed TBR1, whereas only 1.2% expressed SATB2 (Fig. 4h,i). A subset of BrdU-labelled TBR1⁺SATB2⁻ cells were located in the cortical subplate across multiple late-midgestation samples (Fig. 4h,i and Extended Data Fig. 16), suggesting that VZ-derived late-born glutamatergic neurons may contribute new neurons to the cortical subplate (Fig. 4j). Together, our results indicate that late-born glutamatergic neurons adopt similar molecular identities to early-born subplate neurons, supporting a long-standing hypothesis of continued expansion of the subplate throughout the second trimester^51,52.

Discussion

Developmental hierarchy serves as a unifying framework for understanding brain development. Radial glia of the cerebral cortex sequentially generate deep layer neurons and then upper layer neurons before generating astrocytes and oligodendrocytes, a process that has been uncovered through rigorous studies of the mammalian developing brain^{3,53,54,55,56,57,58}. Our findings support prior observations that human radial glia transition from neurogenesis to gliogenesis in a progressive manner^29,59,60, and reveal that dense bulbous protoplasmic astrocytes emerge early, followed by the emergence of dense smooth fibrous astrocytes and OPCs. We further show that in the developing human cortex, the onset of gliogenesis coincides with continued generation of glutamatergic and, increasingly, GABAergic neurons. These findings are consistent with previous reports of GABAergic neuron generation from human cortical progenitors^21,61,62, and provide new insights into the temporal dynamics of their emergence. In mice, locally born GABAergic neurons migrate to the OB^12,13,14 and can be distinguished on the basis of high expression levels of PBX3 and TSHZ1. In addition to these putative OB cells, we identify locally born GABAergic cells that appear to broadly resemble CGE-derived interneurons but exhibit higher levels of PAX6 expression and lack the tangential migration marker ERBB4. Locally born GABAergic cells also highly express SCGN, which is known to be more abundant in human than in mouse^63,64. Although more work is needed to determine the terminal fates of these cells in the human brain, we expect that some GABAergic cells may remain in the dorsal telencephalon (Extended Data Fig. 7), consistent with the unequal scaling hypothesis^65,66.

It is important to acknowledge that the tissue culture methods required to conduct such lineage tracing experiments remove many aspects of the full context of an in vivo experiment such as sensory inputs, gradient patterning factors and mechanical forces, all of which are known to influence cell-type proportions^67,68. It is therefore important that findings from our prospective lineage study have been independently validated using orthogonal methods^22,23. Where possible, we utilized immunohistochemistry to identify the locations and prevalences of cell types that we described transcriptomically. Acknowledging this limitation, it remains to be established whether GABAergic neurogenesis occurs in vivo in the human brain, as opposed to being a consequence of culture⁶¹. However, emerging analyses of somatic mutations in postmortem tissue^22,23 provide further evidence in support of the phenomenon.

Our study provides new evidence in support of protracted glutamatergic neurogenesis extending into late second trimester. Our data implicate tRG as a possible neural stem cell of origin of intermediate progenitor cells and late-born glutamatergic neurons. This is an unexpected finding, given that tRG have so far been shown to give rise to astrocytes and ependymal cells^45,69, and were thought to become largely quiescent by midgestation⁵³. Future studies are needed to determine what molecular programmes maintain the capacity of tRG to generate glutamatergic neurons late into second trimester, and the ultimate fate and function of late-born glutamatergic neurons, which were found to express markers of deep layer and subplate neurons. In rodents, subplate neurons are among the first neurons to be generated in the cerebral cortex, and support the establishment of thalamocortical circuits during development^70,71. Generation of subplate neurons in primates and humans has been proposed to take place throughout corticogenesis^54,72,73,74. Our data point towards a continued production of glutamatergic neurons that express subplate neuron markers TBR1, CTGF and NURR1. These markers are expressed in the ancestral subplate cells found in sauropsids and mammals⁷². A recent study of human entorhinal cortex suggested that TBR1-positive glutamatergic intermediate neuronal progenitors may persist through neonatal stages⁷⁵, although additional analyses are needed to determine whether subplate neuron generation continues into infancy in humans. Together, these results suggest that the late second trimester is a critical timepoint for developmental transitions in cortical neurogenesis, with many processes distinguishing humans from mice.

Methods

Tissue source

De-identified tissue samples were collected with previous patient consent in strict observance of the legal and institutional ethical regulations. Protocols were approved by the Human Gamete, Embryo, and Stem Cell Research Committee (institutional review board) at the University of California San Francisco. GW30 and GW39 tissue samples were collected from de-identified tissue samples with previous patient consent to institutional ethical regulations of the University of California San Francisco Committee on Human Research.

STICR lentiviral library preparation

STICR plasmid libraries containing three bits of diverse barcodes were prepared as described²¹. In this study, we used two pools: one with index E (Addgene #180483) and one with index 3 (Addgene #186335). In brief, STICR lentivirus was produced using a second-generation lentivirus packaging system, pMD2.G (Addgene #12259), psPAX2 (Addgene #12260), and we included pcDNA3.1 puro Nodamura B2 plasmid (Addgene #17228) along with the other plasmids to enhance titre. Plasmids were transfected into Lenti-X HEK293T cells (Takara Bio, 632180) using Lipofectamine 3000 (Thermo, L3000075). Lenti-X 293T cells were grown and transfected in in DMEM (Fisher, MT10017CV) supplemented with 10% FBS (Hyclone, SH30071.03) and 1% penicillin/streptomycin (Fisher, 15070063). Twenty-four hours after transfection, medium was replaced with DMEM supplemented with 2% FBS, sodium pyruvate (0.11 mg ml⁻¹ final concentration, Sigma P2256–25G) and sodium butyrate (0.005 M final molarity, Sigma B5887–1G). Seventy-two hours after transfection, medium was collected, passed through a 0.45-μm filter (Corning, 431220) and then ultracentrifuged at 22,000g for 2 h. The pellet was resuspended in 50 μl sterile PBS (Thermo, 14190250) overnight at 4 °C, then aliquoted and stored at −80 °C.

VZ and OSVZ infections

Cortical tissue samples from midgestation (GW16-24) were initially maintained in ice cold artificial cerebrospinal fluid (ACSF: 125 mm NaCl, 2.5 mm KCl, 1 mm MgCl₂, 2 mm CaCl₂, 1.25 mm NaH₂PO₄, 25 mm NaHCO₃, 25 mm d-(+)-glucose) bubbled with 95% O₂ and 5% CO₂. Samples were selected to have intact VZ and dorsal cortical gross anatomy. Selected samples were embedded in 3% low-melt agarose (Sigma, BP165-25) in ACSF, then sectioned to 300 μm thickness using a vibratome (Leica, VT1200S). Next, the VZ was manually dissected off of the slices with a scalpel under a dissecting scope (Leica, MZ10F). VZs were then incubated at 37 °C for 1 h with STICR virus (index E) diluted 1:100 in slice culture medium (60% Basal Medium Eagle (Gibco) 32% Hanks buffered saline solution (Lonza), 5% heat-inactivated FBS (Hyclone), 1% glucose (Sigma), 1% N2 (Thermo), 1% Glutamax (Gibco) and 1% Penicillin-Streptomycin (Gibco)). The remainder of the microdissected slices, including OSVZ to CP were plated onto Millicell (Millipore) inserts in a 6-well plate with 1.2 ml slice culture medium, then VZs were added back to each slice in their initial orientation. OSVZ infections were performed the next day by microinjection of 1:100 STICR virus with index 3 using needles pulled from capillary tubes (Sutter P-80, Drummond 5-000-1001-X10). Volumes were in the 3–5 μl range, and we aimed to infect the whole OSZV on each slice. Slices were maintained for 10–12 days at 37 °C with 5% CO₂ and 8% O₂, with medium half-changes every other day. BrdU labelling of the VZ was performed with microinjection using pulled needles. Slices were labelled with 3–5 μl of 10uM BrdU (Thermo Fisher, B23151) at the time of plating. Medium was changed the following day and BrdU was not refreshed.

Dissociation and FACS

In preparation for fluorescence-activated cell sorting (FACS) and single-cell sequencing, slices were individually dissociated in 500 μl of a solution of papain (Worthington, LK003178) and apoptosis inhibitor cocktail CEPT (50 nM chroman 1, 5 µM emricasan, 0.1% polyamine supplement and 0.7 µM trans-ISRIB), as described⁷⁶, diluted with HBSS (Gibco) at 37 °C. After an initial 30 min incubation, samples were gently triturated and 1:100 DNase (Sigma) was added. Following another 15 min incubation at 37 °C, papain was quenched using 500 μl of a solution containing HBSS with 1% BSA (Miltenyi) and DNase was added and gentle trituration was continued until samples had no visible chunks. Samples were spun down at 300g for 5 min and supernatant was removed. Cell pellets were resuspended in 1 ml HBSS plus 0.1% BSA and passed through the 35-μm filter into a FACS tube (Falcon, 352235). 1 ml of additional HBSS plus 0.1% BSA was used to rinse the filter and collect all single cells into the bottom of the FACS tube.

A standard gating strategy was used to restrict to single cells and collect only GFP⁺ (STICR-labelled) cells on the FACS Aria Fusion with a 100-μm nozzle. We used NERL saline pH 7.4 (Thermofisher 8505) FACS buffer, and cells were sorted into HBSS plus 0.1% BSA. Samples were kept on ice before and after FACS, and the chamber and collection tubes were maintained at 4 °C during sorting. All GFP⁺ cells from each slice were collected, and immediately analysed by scRNA-seq. Following FACS, cells were spun in a swinging bucket centrifuge at 300g for 5 min and resuspended in a small volume of HBSS before proceeding with single-cell sequencing.

scRNA-seq

Next, we used 10x Chromium Next GEM Single Cell 3′ kits (PN-1000121) to sequence the GFP⁺ population following protocols according to v3.1 chemistry. In brief, we input between 10,000 and 20,000 cells per lane on a Chromium Chip G and followed the manufacturer protocol for transcriptomic library preparation. After cDNA generation and quantification, STICR barcodes were amplified using custom primers and PCR conditions as described²¹. Transcriptomic libraries were sequenced on Illumina Novaseq X or NextSeq 2000, and barcode libraries were sequenced on the NextSeq 2000. We used paired-end sequencing on Illumina platforms to sequence both cDNA and barcode libraries, targeting 25,000 reads per cell for cDNA libraries and 5,000 reads per cell for barcode libraries.

We included three samples that were not barcoded with STICR. For these samples, we microdissected the VZ as described above and dissociated to single cells in papain plus CEPT in HBSS. Cells were immediately sequenced with either 10x, as described above, or with PIPseq T10 3′ Single Cell RNA Kit v5.0. For the sample processed with PIPseq, we followed the manufacturer protocol for library preparation, and libraries were sequenced on NextSeq 2000.

Data processing

10x transcriptomic libraries were processed using CellRanger v7.0.1 (RRID:SCR_017344) and were aligned to human transcriptome GRCh38-2020-A. Cellranger outputs were processed using Seurat v4.3.0.9002 (RRID:SCR_016341). Datasets were first processed to remove cells with fewer than 800 or greater than 30,000 reads, fewer than 500 or greater than 30,000 genes, or more than 10% of reads corresponding to mitochondrial genes. Libraries were integrated using Harmony v0.1.1⁷⁷, and Seurat was used to identify clusters and perform UMAP dimensional reduction. Clusters driven by high read counts or mitochondrial reads were removed. Differential expression analysis was conducted using Seurat FindAllMarkers function, and gene expression in clusters was manually analysed for known markers of given cell types. Iterative clustering to identify subtypes of cells was performed by subsetting the full Seurat object to only cells that were present in desired clusters, then re-running Harmony integration, cluster identification, and dimensional reduction. Cell identities determined through analysis of iterative clusters were re-assigned to cells in the full Seurat object.

STICR barcode analysis was performed using custom scripts, as partially described²¹. First, BBMap (https://sourceforge.net/projects/bbmap/; RRID:SCR_016965) was used to remove low quality reads and extract reads aligning to STICR barcode sequences. Next, individual STICR barcode fragments (‘bits’) were aligned to reference sequences and renamed according to their bit index. The three STICR bits were then merged with the cell barcode and unique molecular identifiers (UMIs) for each read using Awk. UMI-tools (v0.5.1) was used to collapse duplicate STICR barcode or cell barcode reads by UMI, allowing for 1 bp mismatch in the UMI.

To ensure high fidelity of barcode assignments to cells, several steps were taken with custom downstream processing steps. Final outputs from the first script were converted into a sparse matrix of cell barcodes (CBC) by STICR barcode (barcode), with UMI counts at the intersection of each CBC/barcode combination. SCTransform (v0.4.1) was used to read the raw feature matrix output from CellRanger and classify droplets into four categories: cell-free droplets (<50 total UMIs), droplet II (>50, <100 total UMIs), droplet I (>100, <500 total UMIs) and cells (>500 total UMIs). Droplets were plotted on a kneeplot to validate assignments. Next, scAR⁷⁸ (v0.2.3) was used to assign STICR barcodes to cells. Broadly, scAR identifies barcodes that are highly present in empty droplets and subtracts that presence from legitimate cells, ensuring that barcodes that are present in the library as a result of lysed cells are not causing aberrant assignment of cells to clones. While several cells in each sample were found with multiple STICR barcodes, none were present in multi-cellular barcodes, and these barcodes were filtered out due to possible contamination from ambient signal. The next step taken to ensure high-quality barcodes was filtering based on read counts. An output from the first STICR pipeline that identifies CBC and STICR barcode before collapsing reads on UMIs was used to identify the total number of reads for each CBC/barcode combination, irrespective of UMIs. The distribution of reads was then plotted for each CBC/barcode combination. While the majority of CBC/barcode combinations exceeded thousands of reads and were normally distributed, there was a subset of CBC/barcode combinations that had very few (<10) total reads. In some instances, these cells were still identified as having multiple UMIs. CBC/barcode combinations with fewer than 10 reads were removed from downstream analysis. The final filtering step was to remove barcode calls with fewer than 3 UMIs. Overall, this process yielded high-confidence barcodes in ~60% of all cells (Extended Data Fig. 2d). Barcodes and barcode UMI counts were mapped to cells in the Seurat object by aligning on the cell barcode. Index E was used in all VZ injections and Index 3 for all OSVZ injections. One sample (GW18) did not have any barcodes recovered but was included in the single-cell sequencing object and in analyses that did not require barcodes. Uncultured samples sequenced with 10x were processed with CellRanger as described above. Uncultured samples sequenced with PIPseq were processed with PipSeeker 3.2.0. After initial processing, datasets were processed in Seurat as described above. GW18 and GW21 samples sequenced with 10x were merged using Harmony as described above. After clustering, marker genes for clusters were examined to identify putative excitatory neuron clusters. To further these analyse excitatory neurons, we utilized Seurat’s DoHeatmap function to visualize expression of known deep layer markers based off of literature reports and other unpublished data from the Nowakowski lab. We additionally utilized a previously published atlas of first-trimester cortical development (https://cells-test.gi.ucsc.edu/?ds=early-brain⁴⁹). The dataset was downloaded, then reclustered in Seurat. Excitatory neuron clusters were processed in the same way as the other uncultured samples.

Immunostaining and imaging

Primary slices with BrdU were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 1 h at 4 °C, then washed 3 times with PBS. Primary slices were stained floating in 200 μl of each solution, and washed with 1 ml PBS per wash. Cryosections were stained directly on slides with enough of each solution to cover full tissue, and maintained in a humid chamber for all incubations. For BrdU-containing slices, antigen retrieval steps were performed prior to staining. This includes a 1 h incubation in 2 N HCl, then a 1 h incubation in 1 M boric acid solution followed by 3× wash with PBS. All samples were blocked in a solution of PBS with 10% donkey serum (Jackson Immunoresearch, 017-000-121) plus 0.2% gelatin (Sigma, G6144-100G) plus 0.1% Triton X-100 (Fisher, AC215680010) for 30 min. Primary and secondary antibodies were diluted in blocking solution. Samples were incubated in primary antibody solution overnight at 4 °C, then washed 3 times with PBS at room temperature. Samples were then incubated in secondary antibody solution for 3 h at room temperature, followed by a solution of PBS with 1 μg ml⁻¹ DAPI for 5 min and then washed 3 times with PBS before mounting samples on slides with Fluoromount (Invitrogen, 0100-20). Primary antibodies included in this study are detailed in Extended Data Table 2. Secondary antibodies in this study include: donkey anti mouse 647 (1:500, A31571, Thermo, RRID:AB_162542), donkey anti rabbit 594 (1:500, A11012, Thermo, RRID:AB_2534079) and donkey anti sheep 647 (1:500, A21448, Thermo, RRID:AB_10374882). Images were collected using 10× and 20× air objectives on a Leica SP8 confocal system and processed using Fiji (RRID:SCR_002285).

Statistics and reproducibility

Contributions to lineage tracing data by sample are detailed in Extended Data Table 1. All successful biological replicates were included in the lineage tracing data. Images shown in main figures were representative of multiple images taken across replicates, and additional biological replicates for given staining panels are shown in extended data, when available. In general, we believe these results to be replicable across multiple samples of the same gestational age, stained and processed in either the same or a different batch. Statistical methods were not used to predetermine sample sizes. Statistics in Fig. 2 were performed on a total of 24 images taken from 8 different samples, aged GW17, GW19, GW20, GW21, GW23, GW23, GW24 and GW24. Pearson’s correlation was performed using the actual age of each sample. Student’s t-test was performed with a two-sided alternative hypothesis after binning samples into two groups (≤GW20 and >GW20). Statistics in Fig. 4i were performed on a total of 9 images from 3 samples aged GW20.5, GW21 and GW24. A paired Student’s t-test was performed with a two-sided alternative hypothesis, since cell counts with different co-stains were from the same tissue area (same images).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The STICR plasmid is available on Addgene with three separate index sequences (Addgene #180483, #186334 and #186335). The single-cell data generated for this study are publicly available at https://cortex-midgestation.cells.ucsc.edu/ and on dbGap with accession code phs002624.v4.p1. In the study, we compare our dataset to a publicly available dataset from Eze et al.⁴⁹ (Extended Data Fig. 14), which can be found at https://cells.ucsc.edu/?ds=early-brain.

Code availability

Code generated for this project is available at https://github.com/mattgkeefe/sticr_paper_code. Software used for data analysis was publicly available and is described in detail in the Methods.

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Acknowledgements

The authors thank all members of the Nowakowski lab for their insight and advice while conducting these experiments and preparing the manuscript, in particular D. Shin and C. Kim; the UCSF Helen Diller Family Comprehensive Cancer Center Flow and Cell Sorting Core Facility (RRID:SCR_026372) for their assistance with FACS; M. Paredes for providing additional tissue samples; R. Delgado for his pioneering work in the initial design of STICR; and D. Allen for her innovative application of STICR to distinct germinal zones. This project was supported by the National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH) R01NS123263, UM1MH130991, the California Institute for Regenerative Medicine (CIRM) DISC0-14429, as well as gifts from Esther A. and Joseph Klingenstein Fund, the Shurl and Kay Curci Foundation, the Sontag Foundation, and William K. Bowes Jr Foundation. T.J.N. is a New York Stem Cell Foundation Robertson Neuroscience Investigator.

Author information

These authors contributed equally: Matthew G. Keefe, Marilyn R. Steyert

Authors and Affiliations

Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, USA
Matthew G. Keefe, Marilyn R. Steyert & Tomasz J. Nowakowski
Department of Anatomy, University of California San Francisco, San Francisco, CA, USA
Matthew G. Keefe, Marilyn R. Steyert & Tomasz J. Nowakowski
Department of Psychiatry and Behavioral Sciences, University of California San Francisco, San Francisco, CA, USA
Matthew G. Keefe, Marilyn R. Steyert & Tomasz J. Nowakowski
Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA, USA
Matthew G. Keefe, Marilyn R. Steyert & Tomasz J. Nowakowski
The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, USA
Matthew G. Keefe, Marilyn R. Steyert & Tomasz J. Nowakowski
Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, CA, USA
Matthew G. Keefe, Marilyn R. Steyert & Tomasz J. Nowakowski

Authors

Matthew G. Keefe
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Marilyn R. Steyert
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Tomasz J. Nowakowski
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Contributions

Conceptualization: M.G.K., M.R.S. and T.J.N. Methodology: M.G.K. and M.R.S. Investigation: M.G.K. and M.R.S. Visualization: M.G.K. and M.R.S. Funding acquisition: T.J.N. Project administration: T.J.N. Supervision: T.J.N. Writing, original draft: M.G.K. M.R.S. and T.J.N. Writing, review and editing: M.G.K., M.R.S. and T.J.N.

Corresponding author

Correspondence to Tomasz J. Nowakowski.

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Nature thanks Wolfgang Enard, Zoltan Molnar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Single cell quality control and clustering.

(a) Violin plots of number of genes per cell (nFeature_RNA), number of transcripts per cell (nCount_RNA), and percent of reads aligned to mitochondrial reads per cell (percent.mt) by sample. (b) Same as (a), but plotting by cell type. (c) Heatmap showing gene expression in a random subset of 1,000 cells from each manually defined cell type. Genes represent the top 10 most uniquely expressed genes per cell type compared to all other cell types. (d) Feature plots showing markers of progenitors and glia (HES1, MKI67, SPARCL1, OLIG2, MBP), excitatory lineage (EOMES, SLC17A7), and inhibitory lineage (GAD2, LHX6). (e) Percentage of cell types found across all samples. (f) Percentage of cell types found across all pre-midgestation or post-midgestation samples.

Extended Data Fig. 2 STICR barcode recovery.

(a) Matrix of barcode collisions across samples. The first number represents the total number of barcodes found in both samples, the second number represents the percentage of shared barcodes from the total pool of barcodes from both samples. Empty boxes had no barcodes found across both samples. (b) Sankey diagram showing recovery of clones across all samples, divided by sample age and then by clone size (multi-cell clones have barcodes shared between 2 or more cells). (c) Sankey diagram showing recovery of barcodes from all cells. (d) Cells with barcodes recovered and (e) cells in multi-cellular clones projected in UMAP space. (f-h) Clone size histogram showing number of clones with given number of cells in all samples, pre-midgestation samples, or post-midgestation samples.

Extended Data Fig. 3 Subclustering of radial glia and astrocytes.

(a) Progenitors and glia of all types (HES1+ radial glia, EOMES + IPCs, OLIG2 + OPCs, NPY+ GABAergic IPCs, MKI67+ dividing cells) were identified from the main UMAP for subclustering. Identified progenitor cells are highlighted in red in UMAP space. (b) Identification of non-cycling radial glia (HES1/VIM+, DLX2/EOMES/OLIG2/MKI67−) from all progenitors. Identified radial glia highlighted are highlighted in red in UMAP space. (c) Subclustering of radial glia identified 10 clusters. (d) Progenitor and glia UMAP colored by sample age. (e) Progenitor and glia UMAP colored by determined identities. (f) Feature plot of known marker genes for radial glia subtypes (VIM, HES1, INPP1, ANXA1, CRYAB), OPCs (PDGFRA), astrocytes (GJA1, SPARCL1), and dividing cells (MKI67). (g) Dotplot showing expression of known glial marker genes including markers for dorsal identity, ventral identity, pan-radial glia, truncated radial glia, outer radial glia, astrocytes, as well as markers for cycling cells, OPCs, and IPCs.

Extended Data Fig. 4 Clonal composition by sample age for various cell types.

(a) Upset plots representing the cell type abundance (bottom left), types of clones (bottom right), and the abundance of each clone type (top right) for all multi-cellular clones. Cell types are grouped as follows: RG: radial glia; Glia: astrocytes and oligodendrocytes; OPCs: OPCs; EX: EX_IPCs and ENs; IN: IN_IPCs, IN_local, IN_MGE, IN_CGE, IN_OB. (b-d) Upset plots for all multi-cellular clones containing (b) OPCs, (c) astrocytes, (d) radial glia, (e) IN_IPCs, and (f) oRGs across all ages only.

Extended Data Fig. 5 Subclustering of GABAergic lineage cells.

(a) Identification of non-cycling GABAergic lineage cells (GAD2+, OLIG2−, MKI67−) from the main UMAP. Cells taken for subclustering highlighted in red. (b) Subclustering of GABAergic cells identified 10 clusters. (c) GABAergic cell UMAP colored by sample age. (d) Feature plots showing markers of GABAergic neurons (GAD2) and expression of subtype marker genes including: CGE-derived (NR2F2, ADARB2), olfactory bulb destined (PBX3), MGE-derived (LHX6, SOX6), and locally-derived (NPY, PAX6, SCGN). (e) UMAP of all cells; highlighted cells are in a shared clone that contains at least one GABAergic lineage cell and one dorsal lineage cell (RG, EX_IPC, or EN). (f) Number of cells in dorsal shared clones from each GABAergic cell subcluster. (g) Heatmap of top 10 most uniquely expressed genes for each subcluster. (h) Dotplot of gene expression from putative locally-born GABAergic neuron subclusters. Genes from Delgado et al²¹ correspond to DLX+ IPCs, IN.3 (CGE-like), or IN.2 (OB-like).

Extended Data Fig. 6 Inhibitory intermediate progenitors present in germinal zones in late second trimester.

(a) Immunostaining for DLX2, MKI67, and PAX6 shown in a GW17 and GW24 sample. Insets marked by white squares correspond to immunostaining shown in Fig. 2 (b) GW21 sample VZ and OSVZ region stained for inhibitory intermediate progenitor markers DLX2, KI67, and PAX6. Triple-positive cells are demarcated with white circles, while DLX2 + KI67 + PAX6- cells are denoted with salmon circles. Two example insets (yellow square and purple square) are shown on the right.

Extended Data Fig. 7 Dorsally-born GABAergic neuron signature across the developing cortical wall in late second trimester.

Immunostaining for DLX2, SCGN, and PAX6 shown in a GW17 (a) and GW24 (b) sample, tiling from the ventricular zone (bottom) to the cortical plate (top). Dotted white lines demarcate the bottom and top of tissue, as well as the SP/CP boundary, all identified with DAPI. Insets marked by white squares in the upper layers of the developing cortex correspond to immunostaining shown in Fig. 2i.

Extended Data Fig. 8 Dorsally-born GABAergic neuron signature increases during midgestation and persists into third trimester.

(a) Additional GW22 sample demonstrates morphology of SCGN + PAX6+ cells in the CP and MZ. (b-c) Additional examples of pre- (b) and post- (c) midgestation samples co-stained with SCGN and PAX6. Full cortical thickness is shown, from VZ (bottom) to CP (top). (d-e) Third trimester samples stained with SCGN and PAX6. Cortical plate and marginal zone are shown with a dotted line at the border. Yellow arrows indicate representative SCGN + PAX6+ cells.

Extended Data Fig. 9 Generation of dorsally-born GABAergic neurons across cortical areas.

(a, b) Upset plots showing the composition of multi-cellular clones from the prefrontal cortex (PFC) (a) and visual cortex (V1) (b) of a GW20 sample. (c, d) Immunostaining for IN_IPC markers at the germinal zones of the PFC (c) or V1 (d) of a GW20 sample. White circles denote DLX2/MKI67/PAX6 triple positive cells. (e, f) Immunostaining for IN_local markers at the cortical plate of the PFC (e) or V1 (f) of the same GW20 sample shown in (c, d). White circles denote DLX2/SCGN/PAX6 triple positive cells.

Extended Data Fig. 10 Global analysis of cellular architectures of clones.

a) Summary of approach to randomizing cell types across clones and iteratively generating these clones. Created in BioRender; Nowakowski, T. (2024) https://BioRender.com/moeg7qu. b) Number of unique cell types found per individual clone plotted against the size of the individual clone. Point size represents the number of clones with a given size and number of unique cell types. c) Number of unique cell types per clone after shuffling cell types found in each clone. d) Number of clones identified with a given number of unique cell types. Clones were randomly generated 1,000 times. Gray points indicate the mean number of clones from the 1,000 iterations and error bars represent the 95% CI. P-values represent the Bonferroni-corrected significance for a two-sided permutation test. e) Composition of clones that contain either an oRG or tRG. Randomized distributions were generated by first restricting to clones that include either an oRG or tRG, then shuffling cell types found in each clone 1,000 times. Graph shows all clone types that were found at least twice in either oRG- or tRG-containing clones in the observed data. f-g) Number of clones for each two-cell clone type for e) ≤GW20 or f) >GW20 samples, comparing mean and 95% CI of a randomized distribution vs. observed counts. Randomized distributions were generated by first restricting to ≤GW20 or >GW20 samples, then shuffling cell types found in each clone 1,000 times. Cell types were broadly reclassified as progenitor (P: RG, EX_IPC, IN_IPC), neuron (N: EX, INs), or glia (G: OPC, Oligodendrocyte, Astrocyte).

Extended Data Fig. 11 Robust neurogenesis at the VZ/ISVZ persists post-midgestation.

Representative images of the VZ/ISVZ region and the OSVZ region from pre-midgestation (GW15, 16, 17, 18) and post-midgestation (GW22, 24, 24.5) samples stained for EOMES, MKI67, and PAX6.

Extended Data Fig. 12 Neurogenesis at the VZ/ISVZ across mid-gestation.

Samples from across mid-gestation stained with NEUROD2, EOMES, and PAX6.

Extended Data Fig. 13 Deep and upper layer neurogenesis throughout second trimester.

(a) Full cortical thickness of pre-midgestation samples co-stained with PAX6, TBR1 and SATB2 from VZ (bottom) to CP (top). GW16, GW18, and GW24 samples correspond to images in Fig. 4. (b) Full cortical thickness of post midgestation samples co-stained with PAX6, TBR1 and SATB2 from VZ (bottom) to CP (top). GW24 sample corresponds to images in Fig. 4. (c) Insets of the VZ and ISVZ showing TBR1 and SATB2 from full thickness images for samples in (b). In all panels, the ventricular surface is labeled with a white dotted line.

Extended Data Fig. 14 Transcriptomic identity of nascent VZ-resident neurons in midgestation resembles that of first trimester SP neurons.

(a) Schematic explaining how data for uncultured excitatory neurons was generated. Data was either obtained from a first trimester study (Eze et al.⁴⁹) or generated in this study by dissecting the VZ and proceeding directly to sequencing, either with 10x or Pip-seq. Created in BioRender; Nowakowski, T. (2024) https://BioRender.com/xpwdppm. (b) Uniform manifold approximation and projection (UMAP) embedding and clustering of first trimester samples from Eze et al.⁴⁹. (c) UMAP by sample age. (d) Feature plots showing markers of radial glia (HES1), dividing cells (MKI67), excitatory IPCs (EOMES), and excitatory neurons (SLC17A7). (e) UMAP with excitatory neuron clusters in red. (f-i) same as (b-e) for midgestation VZ sample processed with 10x. (j-m) same as (b-e) for midgestation VZ sample processed with PIP-seq. (n-p) Heatmaps with manually selected subplate excitatory neuron marker genes in Eze et al.⁴⁹ first trimester dataset (n), midgestation VZ sample processed with 10x (o), and midgestation VZ sample processed with PIP-seq (p).

Extended Data Fig. 15 Some nascent excitatory neurons in the germinal zones express markers of subplate excitatory neurons.

(a) Full column of tissue VZ to CP from RNAscope shown in Fig. 4. Highlighted squares correspond to the insets shown in Fig. 4. (b) Examples of two additional VZ/ISVZ FOVs. White circles with solid lines represent cells that are positive for a given marker, while dashed lines indicate that cells were positive for TBR1 but negative for the given marker.

Extended Data Fig. 16 Generation of TBR1+ve neurons from the VZ in slice culture.

(a-c) In three different samples aged (a) GW21 (also shown in Fig. 4), (b) GW24, or (c) GW20, slices were labeled at the VZ with BrdU and cultured for 10 days. Images of SP region were taken after 10 days in culture. White arrowheads indicate representative TBR1/BrdU double-positive cells.

Extended Data Table 1 Specimens for STICR lineage tracing experiment

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Extended Data Table 2 Antibodies used for immunohistochemistry

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Supplementary information

Supplementary Tables (download XLSX )

Supplementary Tables 1–3. Supplementary Table 1: Marker genes for de novo clusters; Supplementary Table 2: Marker genes for assigned cell types; Supplementary Table 3: Differentially expressed genes between pre- (pct.2) and post- (pct.1) midgestation radial glia.

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Keefe, M.G., Steyert, M.R. & Nowakowski, T.J. Lineage-resolved atlas of the developing human cortex. Nature 647, 194–202 (2025). https://doi.org/10.1038/s41586-025-09033-8

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Received: 31 January 2024
Accepted: 16 April 2025
Published: 05 November 2025
Version of record: 05 November 2025
Issue date: 06 November 2025
DOI: https://doi.org/10.1038/s41586-025-09033-8

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