Letter

Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size

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Abstract

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability)1, studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans2,3,4, but have little effect on the brains of mice5,6,7,8,9; this probably reflects evolutionarily divergent functions of ASPM10,11. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity12,13,14 than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25–40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients3,4, suggesting that loss of ‘cortical units’ has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates12,13,14,15,16. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.

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Acknowledgements

We thank the late R. W. Guillery, who first introduced ferrets as a model for developmental neuroscience; J. K. Joung for advice on genome editing; J. Bond for the ASPM antibody; L. Vasung, P. Herman, J. Neil and C. D. Kroenke for advice on ferret brain MRI; A. Lee, the G. M. Church laboratory (S. Biwas), the S. McCarroll laboratory (S. Burger), the P. Kharchenko laboratory (J. Fan), and the R. Satija laboratory (A. Butler) for advice on scRNA-seq; S. Wasiuk, E. Feiner, A. S. Kamumbu and M. Lee for technical assistance; Marshall BioResources for animal husbandry; and E. Pollack and the veterinary staff at Boston Children’s Hospital and Yale School of Medicine for surgical support. Animal silhouettes in Fig. 1 were designed by Freepik from https://www.flaticon.com/. This work was supported by P30NS052519 (F.H. and Yale’s QNMR Core Center), 2R01MH067528 (F.H.), 1R24MH114805 (X.P.), R21HD083956 (K.I.), R01EB017337 (P.E.G.), R24HL123482 (J.F.E.), 5R01NS032457 (C.A.W.), 5R21NS091865 (B.-I.B.) and the Allen Discovery Center program through The Paul G. Allen Frontiers Group. C.A.W. is an Investigator of the Howard Hughes Medical Institute.

Reviewer information

Nature thanks S. Juliano, F. Tissir and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Xingshen Sun, Andrew Kodani, Rebeca Borges-Monroy.

Affiliations

  1. Division of Genetics and Genomics, Manton Center for Orphan Disease Research, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    • Matthew B. Johnson
    • , Andrew Kodani
    • , Rebeca Borges-Monroy
    • , Kelly M. Girskis
    • , Steven C. Ryu
    • , Peter P. Wang
    • , Dilenny M. Gonzalez
    • , Richard S. Smith
    • , Christopher A. Walsh
    •  & Byoung-Il Bae
  2. Howard Hughes Medical Institute, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    • Matthew B. Johnson
    • , Andrew Kodani
    • , Rebeca Borges-Monroy
    • , Kelly M. Girskis
    • , Steven C. Ryu
    • , Peter P. Wang
    • , Dilenny M. Gonzalez
    • , Richard S. Smith
    • , Christopher A. Walsh
    •  & Byoung-Il Bae
  3. Department of Anatomy and Cell Biology, Center for Gene Therapy, University of Iowa, Iowa City, IA, USA

    • Xingshen Sun
    • , Ziying Yan
    • , Bo Liang
    •  & John F. Engelhardt
  4. Center for Gene Therapy, University of Iowa, Iowa City, IA, USA

    • Xingshen Sun
    • , Ziying Yan
    • , Bo Liang
    •  & John F. Engelhardt
  5. National Ferret Resource and Research Center, University of Iowa, Iowa City, IA, USA

    • Xingshen Sun
    • , Ziying Yan
    • , Bo Liang
    •  & John F. Engelhardt
  6. Department of Neurosurgery, School of Medicine, Yale University, New Haven, CT, USA

    • Komal Patel
    • , Manavi Chatterjee
    •  & Byoung-Il Bae
  7. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

    • Yu Mi Woo
    •  & Hojoong Kwak
  8. Magnetic Resonance Research Center (MRRC), Yale University, New Haven, CT, USA

    • Daniel Coman
    •  & Fahmeed Hyder
  9. Quantitative Neuroscience with Magnetic Resonance (QNMR) Core Center, Yale University, New Haven, CT, USA

    • Daniel Coman
    • , Xenophon Papademetris
    •  & Fahmeed Hyder
  10. Department of Radiology & Biomedical Imaging, Yale University, New Haven, CT, USA

    • Daniel Coman
    • , Xenophon Papademetris
    • , Lawrence H. Staib
    •  & Fahmeed Hyder
  11. Department of Biomedical Engineering, Yale University, New Haven, CT, USA

    • Xenophon Papademetris
    • , Lawrence H. Staib
    •  & Fahmeed Hyder
  12. Department of Electrical Engineering, Yale University, New Haven, CT, USA

    • Lawrence H. Staib
  13. Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Charlestown, MA, USA

    • Joseph B. Mandeville
  14. Division of Newborn Medicine, Fetal Neonatal Neuroimaging and Developmental Science Center, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

    • P. Ellen Grant
    •  & Kiho Im

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Contributions

B.-I.B., C.A.W. and J.F.E. conceived the project. B.-I.B. generated genome-editing reagents. X.S., Z.Y. and B.L. injected ferret zygotes under J.F.E.’s supervision. M.B.J., K.M.G., P.P.W. and D.M.G. performed immunohistochemistry experiments. R.S.S., M.C., K.I., J.B.M., P.E.G., D.C., X.P., L.H.S. and F.H. performed MRI analyses. M.B.J., R.B.-M., Y.M.W. and H.K. performed scRNA-seq. M.B.J. and R.B.-M. performed single-molecule fluorescence in situ hybridization. A.K. and S.C.R. characterized molecular defects with K.P. and B.-I.B. With input from all authors, M.B.J., B.-I.B. and C.A.W interpreted the data and wrote the paper.

Competing interests

X.P. is a consultant for Electrical Geodesics Inc. The other authors declare no competing interests.

Corresponding authors

Correspondence to Christopher A. Walsh or Byoung-Il Bae.

Extended Data Figures and Tables

  1. Extended Data Fig. 1 Cytoarchitecture and neuronal subtype lamination in the cortex of mature Aspm knockout ferrets.

    a, Nissl stains of coronal sections from the brains of P41 littermates, as shown in Fig. 1l, with additional Aspm+/− and Aspm−/− littermates shown. b, c, Brain sections of P41 littermates immunostained for cortical layer-specific projection neurons including SATB2 (layer II–IV), CTIP2 (layer V), and FoxP2 (layer VI). The experiments were repeated independently three times with similar results. Scale bars, 2 mm (a, top), 200 μm (a, bottom) and 100 μm (b, c).

  2. Extended Data Fig. 2 SOX2 and Ki-67 immunostaining in additional E35 and P0 littermates dorsal cortex.

    Additional results to Fig. 2e, f. Each set of panels is from the brain of a different littermate, showing the high penetrance of the neural progenitor cell basal displacement phenotype. The experiments were repeated independently three times with similar results. Scale bar, 200 μm.

  3. Extended Data Fig. 3 Displaced progenitors in Aspm knockout ferrets have basal fibres.

    Additional results for Fig. 2h, i. Immunostaining of SOX2, Ki-67 and VIM shows that displaced neural progenitors have basal radial fibres. The experiments were repeated independently three times with similar results. Scale bars, 100 μm.

  4. Extended Data Fig. 4 Aspm knockout mice do not demonstrate displaced progenitors in the intermediate zone.

    a, b, Unlike Aspm−/− ferrets, Aspm−/− mice do not have displaced NPCs in the intermediate zone. However, they show a variable increase in the number of intermediate progenitors (PAX6Ki-67+ cells in a and TBR2+ cells in b), which is enhanced by heterozygous, compound mutation in Wdr62, a microcephaly gene causing more severe microcephaly9. The experiments were repeated independently three times with similar results. Scale bars, 100 μm.

  5. Extended Data Fig. 5 Modest increase in apoptosis throughout the germinal zones of the Aspm knockout telencephalon.

    Apoptotic cells (yellow) are indicated by enzymatic fluorescence detection of double-stranded DNA damage with DAPI nuclear counterstaining (blue). The experiments were repeated independently three times with similar results. a, Whole section. b, c, Cortical wall columns. Scale bars, 500 μm (a) and 100 μm (b, c).

  6. Extended Data Fig. 6 Additional immunohistochemical analyses of displaced progenitors in the Aspm knockout cortex.

    a, E35 knockout cortex stained for VRG and ORG markers SOX2 and HOPX reveals extensive co-labelling in both the ventricular zone (VZ) and SVZ, including in displaced OSVZ progenitors. b, In the E35 knockout OSVZ, clusters of supernumerary displaced neural progenitor cells include numerous TBR2+ intermediate progenitors and are surrounded by DCX+ newborn neurons, indicating preserved neurogenesis within the precocious OSVZ niche of the Aspm knockout cortex. The experiments were repeated independently three times with similar results. Scale bars, 50 μm.

  7. Extended Data Fig. 7 scRNA-seq batch, sample and cluster analyses.

    at-SNE plot from Fig. 3a with cells coloured by biological replicate (that is, animal). Most clusters include cells from all samples, except for a cluster expressing blood genes and a cluster expressing choroid plexus epithelial cells that are mostly from animal WT5E. These two cell clusters were not included in downstream analyses. HET, heterozygote; KO, knockout; WT, wild type. Numbers and letters indicate litter and animal identification number, respectively. b, t-SNE plot from Fig. 3a with cells coloured by the batch they were processed in. Clusters are composed of cells from all batches. c, Per-cell gene count and UMI count per sample. Each violin plot is one biological replicate and each dot is one cell. Sample WT5D was not included in the analysis due to the lower gene and UMI count compared to other samples as well as the inconsistent clustering compared to other wild-type samples (data not shown). d, Per-cell gene count and UMI count for identified clusters. Each violin plot is one cell cluster and each dot is one cell. The three clusters in grey (EN4, BL, CPE) were not included in downstream analyses. See Methods for details. This scRNA-seq experiment was performed once with n = 22,211 cells (8,037 cells from two Aspm+/+ and one Aspm+/− ferrets and 14,174 cells from four Aspm−/− ferrets). RG1, cycling radial glial progenitors; RG2, interphase radial glial progenitors; IP, intermediate progenitors; EN1, upper-layer excitatory neurons; EN2, deep-layer excitatory neurons; EN3, Cajal–Retzius cells; IN1, immature inhibitory neurons; IN2, SST+ inhibitory neurons; IN3, ventral/inhibitory progenitors; ENDO1, endothelial cells 1; ENDO2, endothelial cells 2; OPC, oligodendrocyte precursors; MG, microglia; EN4, mixed excitatory neuron identity; BL, blood cells; CPE, choroid plexus epithelial cells.

  8. Extended Data Fig. 8 Loss of Aspm disrupts centriole duplication in FEFs.

    Mitotic Aspm knockout FEFs, identified by staining for pH3 and co-stained for the centriolar marker centrin, display a significant loss of centrioles. The percentage of cells with an abnormal number (less than 4) of centrioles is increased eightfold in Aspm−/− FEFs compared to Aspm+/+ FEFs (n = 100 cells per genotype for three independent experiments; P = 0.003). The experiments were repeated independently three times with similar results. Statistical analysis was performed using a two-tailed t-test; data are mean ± s.e.m.

  9. Extended Data Table 1 Region-specific changes in volume and surface area by loss of Aspm in ferrets
  10. Extended Data Table 2 Cluster identifiers of E35 ferret cerebral cortical cells analysed by scRNA-seq

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