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A human-specific enhancer fine-tunes radial glia potency and corticogenesis

Nature volume 643, pages 1321–1332 (2025)Cite this article

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Abstract

Humans have evolved an extraordinarily expanded and complex cerebral cortex associated with developmental and gene regulatory modifications1,2,3. Human accelerated regions (HARs) are highly conserved DNA sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to species-specific cortical development remains largely unknown4,5. HARE5 is a HAR transcriptional enhancer of the WNT signalling receptor Frizzled8 that is active during brain development6. Here, using genome-edited mouse (Mus musculus, Mm) and primate models, we demonstrated that human (Homo sapiens, Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacities of neural progenitor cells. Hs-HARE5 knock-in mice have significantly enlarged neocortices, containing more excitatory neurons. By measuring neural dynamics in vivo, we showed that these anatomical features result in increased functional independence between cortical regions. We assessed underlying developmental mechanisms using fixed and live imaging, lineage analysis and single-cell RNA sequencing. We discovered that Hs-HARE5 modifies radial glial cell behaviour, with increased self-renewal at early developmental stages, followed by expanded neurogenic potential. Using genome-edited human and chimpanzee (Pan troglodytes, Pt) neural progenitor cells and cortical organoids, we showed that four human-specific variants of Hs-HARE5 drive increased enhancer activity that promotes progenitor proliferation. Finally, we showed that Hs-HARE5 increased progenitor proliferation by amplifying canonical WNT signalling. These findings illustrate how small changes in regulatory DNA can directly affect critical signalling pathways to modulate brain development. Our study uncovered new functions of HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.

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Fig. 1: Hs-HARE5 promotes cortical expansion and neocortical connectivity in mice.
Fig. 2: Hs-HARE5 promotes neural progenitor expansion during cortical development.
Fig. 3: Hs-HARE5 causes RGCs to increase production of IPs and neurons.
Fig. 4: Human-specific HARE5 variants directly affect NPC proliferation in humans and chimpanzees by altering enhancer activity.
Fig. 5: Hs-HARE5 promotes progenitor proliferation through canonical WNT signalling.

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Data availability

Bulk RNA-seq datasets generated in this study are available under Gene Expression Omnibus accession number GSE287748, whereas scRNA-seq datasets are available under Gene Expression Omnibus accession number GSE288849. Source data are provided with this paper.

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Acknowledgements

We thank Silver Laboratory past and present members, and J. Huang and C. Lowe for helpful discussions, C. Sheehan for technical assistance and A. Pollen for technical advice on iPS cells. We are grateful for the non-human primate biological materials provided by the Emory National Primate Research Center, formerly known as the Yerkes National Primate Research Center. The chimpanzee iPS cell line was a gift from Y. Gilad (funded by the Emory National Primate Research Center grant no. ORIP/OD P51OD011132). We thank the Duke University Mouse Transgenic Facility for mouse generation and maintenance, Duke University Light Microscopy Core Facility for the use of shared equipment, Duke University Sequencing and Genomic Technologies core for scRNA-seq library sequencing, and Duke Regeneromics core for bioinformatics. The following funding supported this study: NIH R01NS083897, R01NS120667, R37NS110388 and R01MH132089-02 (for D.L.S.), The Ruth K. Broad fellowship (for J.L.), NIH R00NS109323 and Pilot Project Award from the NIH COBRE in Neurodevelopment and Its Disorders P20GM148302 (for E.S.) and T32HG002760 (for H.M.D.).

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Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA

    Jing Liu, Federica Mosti, Davoneshia Lollis, Carla F. Escobar-Tomlienovich, Camila M. Musso, Nicole D. Moss & Debra L. Silver

  2. Department of Neurobiology, Duke University Medical Center, Durham, NC, USA

    Federica Mosti & Debra L. Silver

  3. Department of Neuroscience, Medical University of South Carolina, Charleston, SC, USA

    Hanzhi T. Zhao & Ewoud R. E. Schmidt

  4. Department of Biology, Duke University, Durham, NC, USA

    Jesus E. Sotelo-Fonseca, Abdull J. Massri & Gregory A. Wray

  5. Department of Cell Biology, Duke University Medical Center, Durham, NC, USA

    Yiwei Mao & Debra L. Silver

  6. Department of Neuroscience, University of Wisconsin–Madison, Madison, WI, USA

    Hannah M. Doll & Andre M. M. Sousa

  7. Waisman Center, University of Wisconsin–Madison, Madison, WI, USA

    Hannah M. Doll & Andre M. M. Sousa

  8. Duke Institute for Brain Sciences and Duke Regeneration Center, Duke University Medical Center, Durham, NC, USA

    Debra L. Silver

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  1. Jing Liu
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Contributions

J.L. and D.L.S. conceived of and designed the study. D.L.S. supervised the experiments. J.L., D.L., Y.M., C.F.E.-T. performed the experiments and analyses of the mouse model. J.E.S.-F., A.J.M., G.A.W. and J.L. performed scRNA-seq and analysis. J.L. performed RNA-seq and analysis with help from N.D.M., H.M.D. and A.M.M.S. performed FZD8 expression analysis across primates. H.T.Z. and E.R.E.S. performed adult neural connectivity experiments. F.M. and J.L. performed HARE5 mutation cloning and luciferase experiments. F.M., C.M.M. and J.L. performed CRISPR editing on human and chimpanzee ES cells/iPS cells, F.M. and J.L. performed cortical organoid experiments with the help of C.F.E.-T. for data analysis. J.L. performed human and chimpanzee NPC differentiation experiments with the help of C.F.E.-T. for data analysis. J.L. and D.L.S. wrote the paper. All authors reviewed, edited and approved the paper.

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Correspondence to Debra L. Silver.

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Extended data figures and tables

Extended Data Fig. 1 FZD8 is the main target of HARE5 in neural cells.

a, HiC data showing FZD8 is the highest frequency target of HARE5 in human neural cells. b, Experimental paradigm for CRISPRi of HARE5 by lentivirus in SH-SY5Y cell line. c, ChIP-qPCR showing the levels of H3K27me3 at HARE5, FZD8 promoter, GJD4 promoter and CCNY promoter, after 3 days transduction with control and Hs-HARE5 gRNAs (n = 3) individual transductions. *P = 0.0102. d, RT-qPCR of FZD8, GJD4 and CCNY after 3 days transduction with control and HARE5 gRNAs (n = 3) individual transductions. **P = 0.0079. All values represent the mean ± s.d. Statistics on independent experiments. Student’s unpaired, two-tailed t-test (c, d).

Source data

Extended Data Fig. 2 FZD8 specifically expressed in radial glia.

a, Published bulk RNA-sequencing analyses confirm FZD8 expression is comparable in cerebellar regions across human and macaque. Processed bulk RNA-seq data for human and macaque brains was obtained from a previous study33. Graph generated for human (n = 311) and macaque (n = 257) cortical areas across 34 and 26 brains respectively. b, Fzd8 expression in mouse reveals a pattern of elevated expression prenatally compared to postnatally. Processed bulk RNA-seq data for mouse was obtained from published dataset35. Each data point represents a measurement in RPKM taken from the frontal cortex of a different wild-type mouse per stage (n = 5 each stage). This data was integrated into Fig. 1d with TranslatingTime. c-e, Published single-cell RNA-sequencing analyses show Fzd8 expression is restricted to RGCs in mouse cortex from E12-E1537 (c) and in human38 (d) and macaque34 (e) fetal cortices at peak neurogenesis stages.

Extended Data Fig. 3 Hs-HARE5 impacts cortical size and neuronal distribution in mice.

a, qPCR of Ccny mRNA in E14.5 control (n = 3), HARE5Hs/Hs (n = 4), and HARE5cKO (n = 3) cortices from 3 litters. b, Quantification of cortical area in 3-month control (n = 6) and HARE5Hs/Hs (n = 4) brains from 3 litters, with each animal normalized to their own body weight. *P = 0.0298. c, Representative whole mount images of control, HARE5Hs/Mm, HARE5Hs/Hs, HARE5cHET and HARE5cKO brains at P0. Dotted line from control is superimposed on all brains. d, Quantification of cortical area at P0 of control (n = 35), HARE5Hs/Mm (n = 18), HARE5Hs/Hs (n = 8), HARE5cHET (n = 16) and HARE5cKO (n = 4) brains across 9 litters. ****P < 0.0001 (cHet); **P = 0.0061 (cKO). e, Quantification of cortical area at P0 of control (n = 35), and HARE5Hs/Hs (n = 8) brains across 9 litters. f, Quantification of neuronal number at P21 of layer II/III (BRN2) in control (n = 6) and HARE5Hs/Hs (n = 9) cortices; layer IV (ROR-β) in control (n = 4) and HARE5Hs/Hs (n = 5) cortices; layer V (CTIP2) in control (n = 4) and HARE5Hs/Hs (n = 5) cortices; layer VI (TBR1) in control (n = 5) and HARE5Hs/Hs (n = 7) cortices from 8 litters. g-j, Lamination analysis of P21 control and HARE5Hs/Hs brains stained for BRN2 (magenta) (g); ROR-β (red) (h); CTIP2 (cyan)(i); TBR1 (green) (j). The bracket on j indicates region quantified for TBR1. Scale bars: 1 mm (c); 50 µm (g-j). All values represent the mean ± s.d. Each litter normalized to control littermates (a,d,e). Statistics on animals (a,b,d,e,f); colors (a,b,d,e), littermates. One-way ANOVA with Dunnett’s multiple comparisons test (a,d), student’s unpaired, two-tailed t-test (b,e,f).

Source data

Extended Data Fig. 4 Correlation analysis of resting state activity of each individual mouse.

a, Spatial maps show the pixelwise correlation value threshold of independence across the imaging field of view for each mouse. b-d, A similar analysis as Fig. 1 with (b) maps showing the temporal correlation between each pixel and the average time course of the functional cluster to which it belongs; and (c) correlation maps were spatially binned into a grid of 10×10 pixels and compared between control(n = 3) and HARE5Hs/Hs and HARE5Hs/Mm (n = 4) brains from 2 litters. P-value map shows statistically significant spatial grids as calculated using two-tailed t-test (All non-black pixels are statistically significant, p < 0.05). (d) Functional clusters were grouped into known anatomical regions based on the Allen Atlas Common Coordinate Framework. Secondary motor cortex (M2) was split rostral-caudally and evaluated separately. Graphs show average Pearson’s correlation (r) within regions rM2; cM2; S1bf; RSP, between control (n = 3) and HARE5Hs/Hs and HARE5Hs/Mm (n = 4) from 2 litters. **P = 0.0078 (S1bf); **P = 0.0058 (cM2); **P = 0.0097 (RSP). e, Spatial maps show pixelwise correlation of each pixel to its own functional cluster (within region correlation) for each individual mouse. All values represent the mean ± s.d. Statistics on animals (d). Student’s unpaired, two-tailed t-test (d).

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Extended Data Fig. 5 Global synapse and glia number are unaffected by Hs-HARE5.

a, Representative images of adult brain sections stained for SYN1 (green) and DRAQ5 (red), Mask used to exclude cells from synapse quantifications (black). b-c, Quantification of SYN1+ puncta in layer II/III (b) and layer V (c) of S1 in adult control (n = 3), HARE5Hs/Mm and HARE5Hs/Hs (n = 5) brains from 3 litters. Values are calculated as fraction of non-DRAQ5 labeled pixels. d-g, Glia staining on P21 control and HARE5Hs/Hs brains. Representative images of OLIG2 (grey) (d) and SOX9 (grey)(e). f-g, Quantification of global cell number of oligodendrocytes (f) and astrocytes (g) in P21 control (n = 4) and HARE5Hs/Hs (n = 3) brains from 3 litters. Scale bars: 50 µm (a),100 µm (d, e). All values represent the mean ± s.d., Statistics on animals; colors (f,g), littermates. Student’s unpaired, two-tailed t-test (b, c, f, g). Images of sections were captured in entirety using tiling, which creates appearance of some artificial lines.

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Extended Data Fig. 6 Hs-HARE5 impact on RGC and IP proliferation.

a, Quantification of proportion of RGCs (PAX6+ or SOX2+) and IPs (TBR2+) that are EGFP+ in E14.5 Hs-HARE5::EGFP (n = 4) cortices from 3 litters. **P = 0.0024. b, Quantification of RGC number within a 100 µm wide radial column in E12.5 control (n = 4), HARE5Hs/Hs (n = 5) and HARE5cKO (n = 3) cortices from 3 litters. c, Quantification of mitotic RGCs in E12.5 control (n = 7) and HARE5cKO (n = 3) cortices from 3 litters. Same controls used in Fig. 2e. d, Cartoon shows overview of BrdU pulse chase from E11.5 to E12.5. e, Representative sections of E12.5 control, HARE5cKO cortices stained for BrdU (green), KI67 (red) and DAPI (blue). f, Quantification of cell cycle exit in E12.5 control (n = 6) and HARE5cKO (n = 3) cortices from 3 litters. ***P = 0.0002. Same controls used in Fig. 2h. g, Representative sections of E14.5 control and HARE5Hs/Mm cortices stained for PH3 (green) and DAPI (blue). h, Quantification of basally located mitotic progenitors in E14.5 control (n = 7) and HARE5Hs/Mm (n = 5) cortices from 2 litters. **P = 0.0081. i, Representative sections of E14.5 control and HARE5Hs/Hs cortices stained with TBR2 (green), PH3 (red) and DAPI (blue). j, Quantification of mitotic IPs in E14.5 control (n = 6) and HARE5Hs/Hs (n = 5) cortices from 4 litters. Each litter is normalized with control littermates. *P = 0.0244. k-m, Quantification of the proportion of all cells that are RGCs (k), IPs (l) and newborn IPs (m) in E14.5 control (n = 10), HARE5Hs/Mm (n = 7) and HARE5Hs/Hs (n = 4) cortices from 5 litters. (l) *P = 0.0379 (HARE5Hs/Mm); **P = 0.0018 (HARE5Hs/Hs); (m) ****P < 0.0001 (HARE5Hs/Mm and HARE5Hs/Hs). Scale bars: 50 µm (e, g, i), 10 µm (i-inset). All values represent the mean ± s.d., Statistics on animals; colors, littermates. P values were calculated based on the One-way ANOVA with Dunnett’s multiple comparisons test (b, k, l, m), student’s unpaired, two-tailed t-test (a, c, f, h, j). Colors indicated littermates.

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Extended Data Fig. 7 scRNA-seq indicates cell composition of Hs-HARE5 cortices.

a, Experimental paradigm for scRNA sequencing of E14.5 control (n = 2) and HARE5Hs/Hs (n = 2) cortices from 2 litters. b-e, UMAP of RGCs (b) and IPs (d) from all 4 samples. Quantification of subcluster proportions of RGCs (c) and IPs (e). *P = 0.02 (RGC-9); *P = 0.04 (RGC-11). *P = 0.01 (IP-0,1,9); *P = 0.02 (IP-6). Cell clusters were defined by gene lists from Supplementary Table 1. f, Gene Ontology analysis of molecular function showing statistically Differentially Expressed Genes (DEGs) in HARE5Hs/Hs RGCs. The x-axis indicates the fold change in expression. DEGs are listed in Supplementary Table 1. P values were calculated based on a two-tailed Fisher test (c, e).

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Extended Data Fig. 8 Hs-HARE5 promotes progenitor cell cycle exit at mid-stages of cortical development.

a, Cartoon shows an overview of BrdU pulse chase from E13.5 to E14.5. b, Representative sections of E14.5 control and HARE5Hs/Hs brains stained for BrdU (green), KI67 (red) and DAPI (blue). c, Quantification of cell cycle exit in E14.5 control (n = 4) and HARE5Hs/Hs (n = 4) cortices from 2 litters. d, Cartoon shows an overview of BrdU pulse chase from E14.5 to E15.5. Below is an overview of cell types labeled and generated from this pulse-chase experiment. e, Representative sections of E15.5 control and HARE5Hs/Hs brains stained for BrdU (green), KI67 (red), TBR2 (grey) and DAPI (blue). f, Quantification of cell cycle exit of TBR2+ cells in E15.5 control (n = 4) and HARE5Hs/Hs (n = 5) cortices from 3 litters. BrdU+TBR2 + KI67- cells reflect a subset of TBR2+ cells which have exited the cell cycle. g, Representative sections of E15.5 control and HARE5Hs/Hs brains stained for BrdU (green), KI67 (red), SOX2 (grey) and DAPI (blue). High magnification view of progenitors, right. h, Quantification of cell cycle exit of SOX2+ cells in E15.5 control (n = 4) and HARE5Hs/Hs (n = 5) cortices from 3 litters. **P = 0.0086. BrdU+SOX2 + KI67- cells reflect a subset of SOX2+ cells which have exited the cell cycle. For data in (f,h), this is an underestimate of all exiting progenitors due to the labels used and timing of BrdU pulse relative to the cell cycle of progenitors. Scale bars: 50 µm (b, e, g), 20 µm (g-inset). All values represent the mean ± s.d. Statistics on animals; colors, littermates. P values were calculated based on the student’s unpaired, two-tailed t-test (c, f, h).

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Extended Data Fig. 9 Hs-HARE5-dependent transcriptome supports neurogenic transition across cortical development.

a, Experimental paradigm for sample collection for Bulk-RNA sequencing of control (n = 3) and HARE5Hs/Hs (n = 3) cortices for each stage, E12.5 and E14.5, from 5 litters. b, Scatter plot shows the correlation of log2Fold Change values for all transcripts differentially expressed from E12.5 to E14.5 of control and HARE5Hs/Hs cortex. Colored dots represent significant changes in differential expression in indicated genotypes. The gene Pakap was omitted from the plot for clarity as it had log2Fold change <-10 in both genotypes. c, Criteria used to identify statistically significant gene expression in HARE5Hs/Hs cortices from E12.5 to E14.5. d, Proportional Venn diagram of the genes expressed in controls, HARE5Hs/Hs cortices, and their overlap. The diagram was drawn using BioVenn. e,f, Gene Ontology analysis (biological process) showing all statistically significant enriched categories from E12.5 to 14.5 in HARE5Hs/Hs cortices for upregulated transcripts (e) and downregulated genes (f). The x-axis indicates the fold change in expression. g, Gene Ontology analysis showing all statistically significant enriched categories for upregulated (top) and downregulated (bottom) transcripts from E12.5 to 14.5 in control cortices. The x-axis indicates the fold change in expression. DE genes are listed in Supplementary Table 2. Statistics for RNA-seq differential gene expression (b,c) were performed using the DE-seq2 package (see Supplemental Table 2 for Padj values).

Extended Data Fig. 10 ASD variants in the HARE5 locus.

a, Table showing location, some clinical information, and known enhancer impact of ASD variants in Hs-HARE5 (either from this study or indicated source). b, Substitutions and mutations in HARE5 sequence including 4 human-specific substitutions (red underlined) and ASD related mutations (yellow highlight).

Extended Data Fig. 11 HARE5 knock-in hiPSC/hESC lines and cortical NPCs/organoid differentiation.

a, Experimental paradigm for genotyping after CRISPR editing. b, EcoRI enzyme digestion shows the successful mutation between Hs-HARE5 and Pt-HARE5 (gained EcoRI site within the 3rd substitution) in the control and mutant cell lines. Blue arrow, Pt, Red arrow, Hs. c, Sanger sequencing showing the successful knock-in with all 4 mutations in Hs-HARE5Pt/Pt lines and Hs-HARE5Hs/Pt lines. This is representative of genotyping of 200 colonies. d,e, Colonies stained with OCT4 (red) and DAPI (blue). This is representative of multiple stainings. f, Experimental paradigm for human NPC differentiation. g-i, Human NPC identity characterization by RT-qPCR on D12-14 of control (n = 4), HARE5Pt/Pt (n = 7) NPCs from 2 and 3 cell lines, respectively, across 3 differentiations, showing FOXG1 (g) PAX6 (h) and OCT4 (i) *P = 0.0239 (i). RNA levels normalized with D0 controls. j-k, RT-qPCR of FZD8 (j) and CCNY (k) of D12-14 human NPC of control (n = 4), HARE5Pt/Pt (n = 7) NPCs from 2 and 3 cell lines, respectively, across 3 differentiations. Each mutant line normalized with controls. **P = 0.0098. l, Experimental paradigm for chimpanzee NPC differentiation. m-o, Chimpanzee NPC identity characterization by RT-qPCR on D15 of controls and Pt-HARE5Hs/Hs lines of control (n = 9) and Pt-HARE5Hs/Hs (n = 5) NPCs from 2 and 1 cell lines, respectively, across 5 differentiations, showing FOXG1 (m) PAX6 (n) and OCT4 (o). RNA levels normalized with D0 controls. p-q, RT-qPCR of FZD8 (p) and CCNY (q) mRNA of D15 NPCs of control (n = 9) and Pt-HARE5Hs/Hs (n = 5) chimpanzee NPCs from 2 and 1 cell lines, respectively, across 5 individual differentiations. Each mutant line normalized with controls. *P = 0.0491 (p); *P = 0.0306 (q). r, Experimental paradigm for human 3D cortical organoids differentiation. s, D30 cortical organoids in bright field. t, RT-qPCR of FOXG1 mRNA on D30 human cortical organoids of controls (n = 14) and Hs-HARE5Pt/Pt (n = 12) from 2 and 2 cell lines, respectively, across 2 differentiations. Normalized with D0 H9. u, Images showing different neural rosette fields for control and Hs-HARE5Pt/Pt cortical organoids (n = 2 lines each shown), stained for SOX2 (green), PH3 (magenta) and DAPI (blue). This is representative of 16 independent stainings. Scale bars: 100 µm (d,e,s), 50 µm (u). All values represent the mean ± s.d. Statistics on independent NPC experiments (g-q), organoids (t); colors, cell lines; shapes, differentiations. P values were calculated based on the student’s unpaired, two-tailed t-test (g-k, m-q, t).

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Extended Data Fig. 12 Hs-HARE5-dependent control of WNT signaling during neurogenesis.

a, Experimental paradigm for testing canonical WNT signaling using TCF/LEF-H2B-EGFP reporter, following inhibition by IWR1 in HEK cells. b, Quantification of WNT activated cells treated with DMSO, 3 µM IWR1 and 30 µM IWR1. (n = 3) independent transfections. Each dot represents the average value of multiple imaging fields from each transfection. *P = 0.0378. c, Experimental paradigm for testing canonical WNT signaling in E10.5 mouse primary NPCs in vitro. d, Representative image of E11.5 control and HARE5Hs/Mm NPCs labeled with WNT reporter (green), mCherry (red), SOX2 (grey) and DAPI (blue). Inset, high magnification view of canonical WNT activated cells. e, Quantification of EGFP+ RGCs in E11.5 control (n = 5) and combined HARE5Hs/Mm and HARE5Hs/Hs (n = 8) cortices from 3 litters. Each litter is normalized with control littermates. **P = 0.0029. f, Experimental paradigm for testing canonical WNT signal activity of E14.5 by IUE in vivo. g, Representative sections of E15.5 control and HARE5Hs/Mm brains electroporated with pCAG-mCherry (red), WNT reporter (green), SOX2 (grey) and DAPI (blue). h, Quantification of EGFP + SOX2+ RGCs in E15.5 control (n = 5) and combined HARE5Hs/Mm and HARE5Hs/Hs (n = 7) cortices from 3 litters. i, Volcano plots showing the differential RNA expression levels of various WNT signaling ligands across E12.5 to E14.5 of control (left) and HARE5Hs/Hs (right). Results derived from RNAseq experiment in Extended Data Fig. 9 with n = 3 embryos each genotype and developmental stage, from 5 litters. j-k, RT-qPCR of mFzd8 (j) and canonical WNT signaling targets (k) after 2 days of transfection in E10.5 mouse NPCs with control and mFzd8 for (n = 3) individual transfections. *P = 0.0152 (j); *P = 0.0484 (Tcf1); *P = 0.025 (Dkk1); *P = 0.0157 (cMyc); *P = 0.0221 (Sox2). i, Experimental paradigm for measuring canonical WNT signaling by WNT reporter using TCF/LEF-H2B-EGFP reporter, following activation by CHIR99021 in human NPCs. m, Quantification of WNT activated cell proportion in D12 NPCs treated with DMSO (n = 3) and 5 µM CHIR99021 (n = 3) from 2 differentiations of 2 control lines. Each dot represents the average value of imaging fields from each cell line. **P = 0.0033. Scale bars: 100 µm (a,d), 50 µm (g-inset), 20 µm (d-inset), 10 µm (g). All values represent the mean ± s.d., Statistics on independent NPC experiments; colors, littermates (e), cell lines (m). P values were calculated based on the one-way ANOVA with Dunnett’s multiple comparisons test (b). Student’s unpaired, two-tailed t-test (e, h, j, k,m). Panel c adapted with permission from ref. 63, The Company of Biologists.

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Supplementary information

Supplementary Fig. 1

Raw agarose gel electrophoresis image. Related to Extended Data Fig. 11b.

Reporting Summary

Supplementary Table 1

Differential gene expression and Gene Ontology analyses for single-cell RNA sequencing data. Related to Fig. 2m–o and Extended Data Fig. 7.

Supplementary Table 2

Differential gene expression and Gene Ontology analyses for bulk RNA sequencing data. Related to Extended Data Fig. 9.

Supplementary Table 3

Primers and oligonucleotides used in this study.

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Liu, J., Mosti, F., Zhao, H.T. et al. A human-specific enhancer fine-tunes radial glia potency and corticogenesis. Nature 643, 1321–1332 (2025). https://doi.org/10.1038/s41586-025-09002-1

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