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TSC tunes progenitor balance and upper-layer neuron generation in neocortex

Nature volume 650, pages 417–427 (2026) Cite this article

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Abstract

The appropriate generation of upper-layer neurons is necessary to create the circuits that underlie complex brain functions. Radial progenitors divide asymmetrically to generate neurogenic intermediate progenitors (IPs; also known as intermediate precursors), and the symmetric proliferation of IPs rapidly expands the cortical neuronal population. The dynamic maintenance of balanced diversity of cortical progenitors and the resultant generation, placement and connectivity of appropriate numbers of different classes of neurons serve to guide the formation of a properly wired cerebral cortex1,2,3,4,5,6,7,8,9,10,11,12. However, the molecular logic that instructs progenitor balance remains unclear. Here we show that members of the tuberous sclerosis complex (TSC)—proteins that are major regulators of cellular metabolism—function to sculpt radial progenitor–intermediate progenitor balance, radial unit organization and the resultant generation of upper-layer neurons. Developmental deletion of TSC proteins alters the radial progenitor and IP balance and changes radial unit composition, leading to increased upper-layer neuron generation and aberrant cortical connectivity. Human-specific modulation of TSC protein expression through human-gained enhancers affects progenitor balance and generation of upper-layer neurons. Evolutionary downregulation of TSC protein expression may therefore provide an effective route to radial unit sculpting and the expanded generation of upper-layer neurons necessary for higher-order brain functions in humans.

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Fig. 1: Effect of Tsc1/2 deletion on RG/IP balance.
The alternative text for this image may have been generated using AI.
Fig. 2: Tsc1/2 deletion promotes upper-layer neuronal generation.
The alternative text for this image may have been generated using AI.
Fig. 3: Deletion of Tsc1/2 in IPs leads to expansion of upper-layer neurons.
The alternative text for this image may have been generated using AI.
Fig. 4: Effect of Tsc1/2 deletion on radial unit composition and upper-layer neuron generation.
The alternative text for this image may have been generated using AI.
Fig. 5: TSC1/2 mediate the RG/IP balance and lead to the expansion of upper-layer neurons.
The alternative text for this image may have been generated using AI.
Fig. 6: Identification and analysis of the effect of HGEs on TSC2 expression and function.
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Data availability

Raw and processed scRNA-seq data, cell metadata and cluster annotations have been deposited into the GEO under accession GSE281619. The SFARI Gene database (https://gene.sfari.org/) was used identify human genes associated with ASD. Source data are provided with this paper.

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Acknowledgements

We thank J. Guo, J. Ratnasothy and J. Brenman for comments on the study and L. Okay for technical assistance. This research was supported by NIH grants MH132710 (E.S.A.) and NS116859 (E.S.A.), Research Council of Finland grants 336234 (PROFI6 UHBRAIN), 340179, 371089, 365282 (PROFI8 SWAN) and 351966 (T.N.), an ERA-NET NEURON MEPIcephaly grant (T.N.), the JSPS J-PEAKS (Fujita Mind-BRIDGe) (T.N.), a Sigrid Jusélius Foundation grant (T.N.), the HiLIFE Fellow program at the University of Helsinki (T.N.), a Japan Spina Bifida and Hydrocephalus Research Foundation grant (T.N.), a Daiichi Sankyo Foundation of Life Science grant (T.N.), and a Brain Science Foundation grant (T.N.).

Author information

Authors and Affiliations

  1. UNC Neuroscience Center, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Cristine R. Casingal, Naoki Nakagawa, Keiko Yabuno-Nakagawa, Cailyn Meyer, Siling Liu, Su-Ji Cho, Dan Liang, Nana Matoba, Ahana Mallick, Rubal Singla, Jieun Park, Chu-Wei Huang, Hailey Wilson, Janice Lee, Jason L. Stein & E. S. Anton

  2. Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Cristine R. Casingal, Naoki Nakagawa, Keiko Yabuno-Nakagawa, Cailyn Meyer, Siling Liu, Su-Ji Cho, Ahana Mallick, Chu-Wei Huang, Hailey Wilson, Janice Lee & E. S. Anton

  3. Laboratory of Developmental Neurobiology, Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Naoki Nakagawa

  4. Neuroscience Center, HiLIFE–Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland

    Vasiliki Gkini & Takashi Namba

  5. Department of Psychiatry, Yale School of Medicine, New Haven, CT, USA

    Mario Skarica

  6. Department of Genetics, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Dan Liang, Nana Matoba, Rubal Singla, Jieun Park & Jason L. Stein

  7. Department of Data Science, Dana-Farber Cancer Institute, Boston, MA, USA

    Jeremy M. Simon

  8. Department of Biostatistics, T. H. Chan School of Public Health, Harvard University, Boston, MA, USA

    Jeremy M. Simon

  9. Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA

    H. Troy Ghashghaei

  10. Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, USA

    Garret D. Stuber

  11. Department of Pharmacology, University of Washington, Seattle, WA, USA

    Garret D. Stuber

  12. Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

    Oskari Heikinheimo

  13. Department of Developmental Biology, Fujita Health University School of Medicine; Division of Developmental Biology, International Center for Brain Science (ICBS), Fujita Health University, Toyoake, Japan

    Takashi Namba

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  1. Cristine R. Casingal
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Contributions

C.R.C., N.N., K.Y.-N., T.N., J.L.S. and E.S.A. designed the experiments and supervised the project. C.R.C., N.N., K.Y.-N., C.M., S.L., S.-J.C., C.-W.H., H.W., J.L., G.D.S., H.T.G. and E.S.A. conducted the experiments with mouse genetic models and analysed the data. C.R.C., S.L., M.S., D.L., N.M. and J.M.S. conducted the scRNA-seq studies and bioinformatic analyses. C.R.C., C.M., A.M., R.S., J.P., V.G., O.H. and T.N. performed the organoid and human cell studies. C.R.C., N.N., K.Y.-N., T.N., J.L.S. and E.S.A. wrote the manuscript.

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Correspondence to E. S. Anton.

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Extended data figures and tables

Extended Data Fig. 1 Altered proliferative niche and astrogliosis in TSC1/2 cKO.

(A,B) Increased density of PH3+ cells in the ventricular zone of TSC1/2 cKO. (C) Quantification of changes in PH3+ cells (n = 6 mice/group). (D-E) An atypical collection of Pax6+, Tbr2+ double-positive cells in TSC1/2 cKO VZ. (F) Quantification of changes in Pax6+, Tbr2+ cells (n = 3 mice/group). (G-H) No changes in caspase-3+ apoptotic cells are evident in the TSC1/2 cKO cortex. (I) Quantification of caspase-3+ cells (n = 6 mice/group). (J,K) Increased density of GFAP+ astrocytes in TSC1/2 cKO cortex. (L) Quantification of changes in GFAP+ cells (n = 5 mice/group). Data shown are mean ± SEM. Unpaired two-sided t-test; **p < 0.01; ***p < 0.001 [p = 0.0026 (C), 0.0087 (F), 0.804 (I), 0.0001 (L)]. VZ, ventricular zone. Scale bar: A-B (50 µm), D-E (10 µm); G-H (50 μm); J-K (20 μm).

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Extended Data Fig. 2 Changes in brain weight and cortical size in TSC1/2 cKO.

(A,B) Increased brain size and weight (C) in TSC1/2 cKO (n = 5 mice/group). (D-F) Increased width (yellow bar) of cerebral cortical wall in TSC1/2 cKO (n = 8 mice/group). (G) Change in cortical size is also reflected in increased cortical circumference [somatosensory cortex level] in TSC1/2 cKO (n = 7 mice/group). Data shown are mean ± SEM. Unpaired two-sided t-test; *p < 0.05, **p < 0.01, and ***p < 0.001 [p = 0.0147 (C), 0.0036 (F), 0.0009 (G)]. Scale bar: A-B (1 mm); D-E (500 μm).

Source data

Extended Data Fig. 3 Effect of TSC1/2 deletion on pS6 expression.

(A,B) Increased pS6 expression in TSC1/2 cKO cortex. (C) Quantification of changes in pS6 expression in TSC1/2 cKO cortex (n = 6 mice/group). (D) Immunoblot analysis and quantification (E) of increased pS6 expression in TSC1/2 cKO cortex (n = 4 mice/group). (F,G) Increased pS6 expression in TSC1/2 cKO RGCs (arrowhead). (H) Quantification of changes in pS6 expression in TSC1/2 cKO RGC soma (n = 25 soma). RGCs were co-labeled with RC2 antibodies. CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Data shown are mean ± SEM. Unpaired two-sided t-test; ***p < 0.001 [p = <0.0001 (C), 0.0001 (E), <0.0001 (H)]. Scale bar: A-B (100 μm); F-G (50 µm). Sample RGC soma are outlined (dotted lines; F-G). A.U., arbitrary unit of pS6 intensity normalized to background. For immunoblot source data, see Supplementary Fig. 1.

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Extended Data Fig. 4 Increased width of the corpus callosum following deletion of TSC1/2 in intermediate progenitors.

(A,D) Consistent with the increase in upper layer neurons (tdTomato+) in TSC1/2Tbr2 cKO, the width of the corpus callosum (arrow), containing the axonal projections of these neurons, is increased. (E) Quantification of increased width of tdTom labeled corpus callosum (CC). Data shown are mean ± SEM (n = 10 mice/group). Unpaired two-sided t-test; ***p < 0.0001. Scale bar: A-D (100 μm).

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Extended Data Fig. 5 Altered cortical size, laminar organization, neuronal morphology, neuronal projections, and connectivity in MADM labeled Tsc1/2 Emx-cKO cortex.

(A,B) Expansion of cortical size in MADM labeled Tsc1/2 Emx-cKO brains. (C,D) TSC1/2 deletion leads to disrupted laminar organization and neuronal misplacement (circle; D) in MADM labeled Tsc1/2 Emx-cKO brains. (E,F) Changes in the morphology and increased branching of MADM labeled upper layer neurons (compare areas indicated by asterisk [E, F]). (G) Quantification of increased neurite branching in Tsc1/2 Emx-cKO cortex (n = 12 mice/group). (H,I) Consistent with the expanded upper layer neurons, the thickness of corpus callosum (CC) containing axons of upper layer neurons traversing to the contralateral cortex is increased in Tsc1/2 Emx-cKO. Bar (H, I) indicates callosal width. (J) Quantification of increased callosal width in Tsc1/2 Emx-cKO. Data shown are mean ± SEM (n = 5 mice/group). Unpaired two-sided t-test; **p < 0.01 and ***p < 0.001 [p = 0.0002 (G), 0.0022 (J)]. Scale bar: A-B (200 μm); C-D (50 μm); E-F (20 µm); H-I (100 µm).

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Extended Data Fig. 6 Effect of inactivation of TSC1, 2, and TBC1D7.

Embryonic cortices were electroporated with control and TBC1D7 shRNAs at E14.5 and analyzed at E18.5. (A-H) TBC1D7 knockdown (GFP+) further disrupts cortical malformation in TSC1/2 cKO. Compared to control (A-B), expanded upper layers and upper layer neuronal misplacement are evident in TSC1/2 cKO (C-D; arrow [A vs. C]). Inactivation of TBC1D7 in TSC1/2 cKO background (E-H) further disrupts cortical plate organization (compare areas indicated by arrows; C vs. E, G) and leads to ectopic collection of neurons near the ventricular zone (compare areas indicated by asterisks; C vs. E, G). (B, D, F, H) Quantification of GFP+ cell position in the developing cortical wall. Data shown are mean ± SEM (n [mice]: control = 5, TSC1/2cKO = 6, TSC1/2cKO+Tbc1d7shRNA1 = 9, and TSC1/2cKO+Tbc1d7shRNA2 = 7). Two-way ANOVA; Control [B] vs. TSC1/2cKO [D], TSC1/2cKO [D] vs. TSC1/2cKO+Tbc1d7 shRNA1 [F] or TSC1/2cKO+Tbc1d7 shRNA2 [H], *P < 0.0001. (I-M) Co-labeling of GFP+ neurons in the cortical plate with upper layer neuronal marker Satb2, indicates increased GFP+, Satb2+ upper layer neurons in TSC1, 2, and TBC1D7 deficient cortices (K, L, M; yellow arrowheads) as compared to control (I, M; white arrowheads) or TSC1/2 cKO cortices (J, M; purple arrowheads). Data shown are mean ± SEM (n [mice]: Control = 5, TSC1/2cKO = 5, TSC1/2cKO+Tbc1d7shRNA1 = 7, and TSC1/2cKO+Tbc1d7shRNA2 = 7). Unpaired two-sided t-test; **p < 0.01 and ***p < 0.001 ([M: Control vs. TSC1/2cKO, p = 0.0003; TSC1/2cKO vs. TSC1/2cKO+Tbc1d7 shRNA1, p = 0.0017, or TSC1/2cKO+Tbc1d7 shRNA2, p = 0.0024]) (N-R) Co-labeling of GFP+ neurons with deep layer neuronal marker Tbr1, indicates no changes in GFP+, Tbr1+ neurons. Data shown are mean ± SEM (n [mice]: Control = 5, TSC1/2cKO = 5, TSC1/2cKO+Tbc1d7shRNA1 = 5, and TSC1/2cKO+Tbc1d7shRNA2 = 5). Unpaired two-sided t-test; **p < 0.01 and ***p < 0.001 ([R: Control vs. TSC1/2cKO, p = 0.9385; TSC1/2cKO vs. TSC1/2cKO+Tbc1d7 shRNA1, p = 0.7682, or TSC1/2cKO+Tbc1d7 shRNA2, p = 0.7790]). CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: A, C, E, G (50μm); I-L (20μm); N-Q (40 µm).

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Extended Data Fig. 7 Changes in the average expression of the ASD genes in RG, IP, and UL cells of control and TSC1/2 cKO cortices.

(A) Log2-fold changes in high confidence ASD gene expression levels in cKO cells compared to controls are illustrated (P < 0.05, two-tailed hierarchical permutation test). High confidence ASD gene list is from SFARI ASD gene database (https://gene.sfari.org/). SFARI genes with no significant expression changes in any of the 3 cell classes are not included. RGC, radial glial cells; IP, intermediate precursors; UL, upper layer neurons. Circle size indicates the value of log fold change. Red-blue gradient indicates the direction of change.

Extended Data Fig. 8 Expression profile and epigenomic regulation of TSC genes.

(A) Sequence conservation analysis shows that the TSC complex genes are highly conserved between mice and humans. (B) Expression profile of the TS complex genes in human and mouse neural cell types44. Data shown are mean ± SEM (n = 10 [human], 22 [mouse] brain cell clusters). Unpaired two-sided t-test; **p < 0.01 and ***p < 0.001 [p = 0.0083 (TSC1), <0.0001 (TSC2)]. (C) Histone modifications around the transcription start site of TS complex genes. H3k27me3 peaks (orange box) are present in TS complex genes only in the human embryonic brain. In contrast, H3k27ac peaks (teal box) are present in Tsc1, Tsc2, and Tbc1d7 of the mouse embryonic and adult brain. They are also present in TSC1, TSC2, and TBC1D7 of the human embryonic and adult brain tissue. RG, radial progenitors; IP, intermediate progenitors.

Source data

Extended Data Fig. 9 Identification of candidate gene regulatory elements in TBC1D7 and TSC1.

(A) ATAC-seq peaks showing a candidate HGE (teal box; TBC1D7-P1) in the TBC1D7 promoter56. The black lines indicate the significantly correlated peak pairs across a population of human neural progenitors and their correlation values (r). (B) HGEs were not detected near TSC1 in ATAC-seq or Hi-C data from neurons, progenitors, or germinal zone (GZ). (C) qPCR analysis of relative TBC1D7 expression in hNPCs after lentiviral transduction with dCas9-KRAB-gRNAs targeted to the candidate TBC1D7 human-gained enhancer. No changes in TBC1D7 expression were detected. Data shown are mean ± SEM (n = 9 unique donors). Unpaired two-sided t-test p > 0.05 (0.6363 [Cntrl sg vs sg1]; 0.6559 [Cntrl sg vs sg2]; 0.0702 [Cntrl sg vs sg3]; 0.3921 [Cntrl sg vs sg4]; 0.3130 [Cntrl sg vs sg5]; 0.4876 [Cntrl sg vs sg6]).

Source data

Supplementary information

Supplementary Fig. 1 (download TIF )

Raw images of pS6 and β-actin immunoblots. (A-B) Scans of pS6 (A) and β-actin (B) immunoblots used in Extended Data Fig. 3D. Outlined area indicates cropped area for pS6 and β-actin. Genotypes and molecular weight markers are indicated (related to Extended Data Fig. 3D).

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Supplementary Tables (download DOCX )

This file contains Supplementary Tables 1 and 2. Supplementary Table 1: gRNAs targeting the HGEs of TSC2 and TBC1D7 (related to Fig. 6 and Extended Data Fig. 9). Supplementary Table 2: Viral titres of HGE gRNAs (related to Fig. 6 and Extended Data Fig. 9).

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Casingal, C.R., Nakagawa, N., Yabuno-Nakagawa, K. et al. TSC tunes progenitor balance and upper-layer neuron generation in neocortex. Nature 650, 417–427 (2026). https://doi.org/10.1038/s41586-025-09810-5

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