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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Embryonic mouse medial neocortex as a model system for studying the radial glial scaffold in fetal human neocortex
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Faheem, M. et al. Molecular genetics of human primary microcephaly: an overview. BMC Med. Genomics 8, S4 (2015).
Bond, J. et al. ASPM is a major determinant of cerebral cortical size. Nat. Genet. 32, 316–320 (2002).
Passemard, S. et al. Abnormal spindle-like microcephaly-associated (ASPM) mutations strongly disrupt neocortical structure but spare the hippocampus and long-term memory. Cortex 74, 158–176 (2016).
Desir, J., Cassart, M., David, P., Van Bogaert, P. & Abramowicz, M. Primary microcephaly with ASPM mutation shows simplified cortical gyration with antero-posterior gradient pre- and post-natally. Am. J. Med. Genet. A 146A, 1439–1443 (2008).
Pulvers, J. N. et al. Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline. Proc. Natl Acad. Sci. USA 107, 16595–16600 (2010).
Fujimori, A. et al. Disruption of Aspm causes microcephaly with abnormal neuronal differentiation. Brain Dev. 36, 661–669 (2014).
Capecchi, M. R. & Pozner, A. ASPM regulates symmetric stem cell division by tuning cyclin E ubiquitination. Nat. Commun. 6, 8763 (2015).
Williams, S. E. et al. Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice. Development 142, 3921–3932 (2015).
Jayaraman, D. et al. Microcephaly proteins Wdr62 and Aspm define a mother centriole complex regulating centriole biogenesis, apical complex, and cell fate. Neuron 92, 813–828 (2016).
Montgomery, S. H. & Mundy, N. I. Microcephaly genes evolved adaptively throughout the evolution of eutherian mammals. BMC Evol. Biol. 14, 120 (2014).
Bae, B. I., Jayaraman, D. & Walsh, C. A. Genetic changes shaping the human brain. Dev. Cell 32, 423–434 (2015).
Fietz, S. A. et al. OSVZ progenitors of human and ferret neocortex are epithelial-like and expand by integrin signaling. Nat. Neurosci. 13, 690–699 (2010).
Reillo, I., de Juan Romero, C., García-Cabezas, M. A. & Borrell, V. A role for intermediate radial glia in the tangential expansion of the mammalian cerebral cortex. Cereb. Cortex 21, 1674–1694 (2011).
Johnson, M. B. et al. Single-cell analysis reveals transcriptional heterogeneity of neural progenitors in human cortex. Nat. Neurosci. 18, 637–646 (2015).
Smart, I. H., Dehay, C., Giroud, P., Berland, M. & Kennedy, H. Unique morphological features of the proliferative zones and postmitotic compartments of the neural epithelium giving rise to striate and extrastriate cortex in the monkey. Cereb. Cortex 12, 37–53 (2002).
Hansen, D. V., Lui, J. H., Parker, P. R. & Kriegstein, A. R. Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature 464, 554–561 (2010).
Bond, J. et al. Protein-truncating mutations in ASPM cause variable reduction in brain size. Am. J. Hum. Genet. 73, 1170–1177 (2003).
Hutchinson, E. B. et al. Population based MRI and DTI templates of the adult ferret brain and tools for voxelwise analysis. Neuroimage 152, 575–589 (2017).
Martínez-Cerdeño, V. et al. Comparative analysis of the subventricular zone in rat, ferret and macaque: evidence for an outer subventricular zone in rodents. PLoS ONE 7, e30178 (2012).
Martínez-Martínez, M. A. et al. A restricted period for formation of outer subventricular zone defined by Cdh1 and Trnp1 levels. Nat. Commun. 7, 11812 (2016).
Pollen, A. A. et al. Molecular identity of human outer radial glia during cortical development. Cell 163, 55–67 (2015).
Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015).
Kodani, A. et al. Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication. eLife 4, e07519 (2015).
Paridaen, J. T., Wilsch-Bräuninger, M. & Huttner, W. B. Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division. Cell 155, 333–344 (2013).
Wang, X. et al. Asymmetric centrosome inheritance maintains neural progenitors in the neocortex. Nature 461, 947–955 (2009).
Singh, S. & Solecki, D. J. Polarity transitions during neurogenesis and germinal zone exit in the developing central nervous system. Front. Cell. Neurosci. 9, 62 (2015).
Dauber, A. et al. Novel microcephalic primordial dwarfism disorder associated with variants in the centrosomal protein ninein. J. Clin. Endocrinol. Metab. 97, E2140–E2151 (2012).
Zhang, X. et al. Cell-type-specific alternative splicing governs cell fate in the developing cerebral cortex. Cell 166, 1147–1162 (2016).
Lehtinen, M. K. et al. The cerebrospinal fluid provides a proliferative niche for neural progenitor cells. Neuron 69, 893–905 (2011).
Mashiko, D. et al. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci. Rep. 3, 3355 (2013).
Li, Z., Sun, X., Chen, J., Leno, G. H. & Engelhardt, J. F. Factors affecting the efficiency of embryo transfer in the domestic ferret (Mustela putorius furo). Theriogenology 66, 183–190 (2006).
Chahboune, H. et al. Neurodevelopment of C57B/L6 mouse brain assessed by in vivo diffusion tensor imaging. NMR Biomed. 20, 375–382 (2007).
Petersen, K. F. et al. The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome. Proc. Natl Acad. Sci. USA 104, 12587–12594 (2007).
Rueckert, D. et al. Nonrigid registration using free-form deformations: application to breast MR images. IEEE Trans. Med. Imaging 18, 712–721 (1999).
Papademetris, X., Jackowski, A. P., Schultz, R. T., Staib, L. H. & Duncan, J. S. Integrated intensity and point-feature nonrigid registration. In International Conference on Medical Image Computing and Computer-Assisted Intervention Vol. 3216 (eds Barillot, C., et al.) 763–770 (Springer, Berlin, 2001).
Sawada, K. & Watanabe, M. Development of cerebral sulci and gyri in ferrets (Mustela putorius). Congenit. Anom. (Kyoto) 52, 168–175 (2012).
Joshi, A. et al. Unified framework for development, deployment and robust testing of neuroimaging algorithms. Neuroinformatics 9, 69–84 (2011).
Rahman, A. S., Parvinjah, S., Hanna, M. A., Helguera, P. R. & Busciglio, J. Cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures. J. Vis. Exp 45, e2384 (2010).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc 7, 562–578 (2012).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol. 33, 495–502 (2015).
Villani, A. C. et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science 356, eaah4573 (2017).
Higgins, J. et al. Human ASPM participates in spindle organisation, spindle orientation and cytokinesis. BMC Cell Biol 11, 85 (2010).
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.
Nature thanks S. Juliano, F. Tissir and the other anonymous reviewer(s) for their contribution to the peer review of this work.
X.P. is a consultant for Electrical Geodesics Inc. The other authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Figures and Tables
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).
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.
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.
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 (PAX6−Ki-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.
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).
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.
a, t-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.
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.
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Johnson, M.B., Sun, X., Kodani, A. et al. Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size. Nature 556, 370–375 (2018). https://doi.org/10.1038/s41586-018-0035-0
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