Movatterモバイル変換

[0]ホーム
URL:

Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Advertisement

Nature Neuroscience
  • Article
  • Published:

Hedgehog signaling promotes basal progenitor expansion and the growth and folding of the neocortex

Nature Neurosciencevolume 19, pages888–896 (2016)Cite this article

Subjects

ACorrigendum to this article was published on 01 August 2016

ACorrigendum to this article was published on 01 August 2016

This article has beenupdated

Abstract

The unique mental abilities of humans are rooted in the immensely expanded and folded neocortex, which reflects the expansion of neural progenitors, especially basal progenitors including basal radial glia (bRGs) and intermediate progenitor cells (IPCs). We found that constitutively active Sonic hedgehog (Shh) signaling expanded bRGs and IPCs and induced folding in the otherwise smooth mouse neocortex, whereas the loss of Shh signaling decreased the number of bRGs and IPCs and the size of the neocortex. SHH signaling was strongly active in the human fetal neocortex but Shh signaling was not strongly active in the mouse embryonic neocortex, and blocking SHH signaling in human cerebral organoids decreased the number of bRGs. Mechanistically, Shh signaling increased the initial generation and self-renewal of bRGs and IPC proliferation in mice and the initial generation of bRGs in human cerebral organoids. Thus, robust SHH signaling in the human fetal neocortex may contribute to bRG and IPC expansion and neocortical growth and folding.

This is a preview of subscription content,access via your institution

Access options

Access through your institution

Subscription info for Japanese customers

We have a dedicated website for our Japanese customers. Please go tonatureasia.com to subscribe to this journal.

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF

Prices may be subject to local taxes which are calculated during checkout

Figure 1:SmoM2 induces neocortical expansion and folding.
Figure 2:SmoM2 expands IPCs and bRGs.
Figure 3:SmoM2 expands bRGs by increasing their self-renewal and changing the aRG division angle toward bRG production.
Figure 4: Retrovirus expressing SMOM2 promotes bRG production at the clonal level.
Figure 5:Smo is required to expand IPCs, bRGs and upper-layer neurons.
Figure 6: Strong SHH signaling in human aRGs.
Figure 7: SHH signaling promotes bRG production in human cerebral organoids.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Change history

  • 28 June 2016

    In the version of this article initially published, the units on thex axis in Figure 3c were given as mm; the correct units are μm. At the end of the legend to Figure 7, the error bars were described as s.d.; they are actually s.e.m. inb and s.d. inc. In the third sentence of the Online Methods section on human cerebral organoids, 10% knockout serum replacement, 1% GlutaMAX and 1% MEM-NEAA should have been 20%, 1× and 1×, respectively. In the sixth sentence, 1% N2 supplement, 1% GlutaMAX and 1% MEM-NEAA should each have been 1×. In the eighth sentence, 6-mm dishes should have been 6-cm dishes, 0.5% N2 supplement and 0.5% MEM-NEAA should each have been 0.5×, and 1% B27 without vitamin A, 1% GlutaMAX and 1% penicillin/streptomycin should each have been 1×. In Supplementary Figure 10b, the graph lacked error bars. The errors have been corrected in the HTML and PDF versions of the article.

References

  1. Lui, J.H., Hansen, D.V. & Kriegstein, A.R. Development and evolution of the human neocortex.Cell146, 18–36 (2011).

    Article CAS PubMed PubMed Central  Google Scholar 

  2. Florio, M. & Huttner, W.B. Neural progenitors, neurogenesis and the evolution of the neocortex.Development141, 2182–2194 (2014).

    Article CAS PubMed  Google Scholar 

  3. Borrell, V. & Götz, M. Role of radial glial cells in cerebral cortex folding.Curr. Opin. Neurobiol.27, 39–46 (2014).

    Article CAS PubMed  Google Scholar 

  4. Sun, T. & Hevner, R.F. Growth and folding of the mammalian cerebral cortex: from molecules to malformations.Nat. Rev. Neurosci.15, 217–232 (2014).

    Article CAS PubMed PubMed Central  Google Scholar 

  5. Dehay, C., Kennedy, H. & Kosik, K.S. The outer subventricular zone and primate-specific cortical complexification.Neuron85, 683–694 (2015).

    Article CAS PubMed  Google Scholar 

  6. Smart, I.H.M., 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. Cortex12, 37–53 (2002).

    Article PubMed  Google Scholar 

  7. Zecevic, N., Chen, Y. & Filipovic, R. Contributions of cortical subventricular zone to the development of the human cerebral cortex.J. Comp. Neurol.491, 109–122 (2005).

    Article PubMed PubMed Central  Google Scholar 

  8. Lukaszewicz, A. et al. G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex.Neuron47, 353–364 (2005).

    Article CAS PubMed PubMed Central  Google Scholar 

  9. Hansen, D.V., Lui, J.H., Parker, P.R.L. & Kriegstein, A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature464, 554–561 (2010).

    Article CAS PubMed  Google Scholar 

  10. 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).

    Article CAS PubMed  Google Scholar 

  11. Reillo, I., de Juan Romero, C., García-Cabezas, M.Á. & Borrell, V. A role for intermediate radial glia in the tangential expansion of the mammalian cerebral cortex.Cereb. Cortex21, 1674–1694 (2011).

    Article PubMed  Google Scholar 

  12. Wang, X., Tsai, J.-W., LaMonica, B. & Kriegstein, A.R. A new subtype of progenitor cell in the mouse embryonic neocortex.Nat. Neurosci.14, 555–561 (2011).

    Article CAS PubMed PubMed Central  Google Scholar 

  13. Shitamukai, A., Konno, D. & Matsuzaki, F. Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors.J. Neurosci.31, 3683–3695 (2011).

    Article CAS PubMed PubMed Central  Google Scholar 

  14. García-Moreno, F., Vasistha, N.A., Trevia, N., Bourne, J.A. & Molnár, Z. Compartmentalization of cerebral cortical germinal zones in a lissencephalic primate and gyrencephalic rodent.Cereb. Cortex22, 482–492 (2012).

    Article PubMed  Google Scholar 

  15. 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 One7, e30178 (2012).

    Article PubMed PubMed Central CAS  Google Scholar 

  16. Stashinko, E.E. et al. A retrospective survey of perinatal risk factors of 104 living children with holoprosencephaly.Am. J. Med. Genet.128A, 114–119 (2004).

    Article PubMed  Google Scholar 

  17. Heussler, H.S., Suri, M., Young, I.D. & Muenke, M. Extreme variability of expression of a Sonic Hedgehog mutation: attention difficulties and holoprosencephaly.Arch. Dis. Child.86, 293–296 (2002).

    Article CAS PubMed PubMed Central  Google Scholar 

  18. Derwińska, K. et al. PTCH1 duplication in a family with microcephaly and mild developmental delay.Eur. J. Hum. Genet.17, 267–271 (2009).

    Article PubMed CAS  Google Scholar 

  19. Komada, M. et al. Hedgehog signaling is involved in development of the neocortex.Development135, 2717–2727 (2008).

    Article CAS PubMed  Google Scholar 

  20. Englund, C. et al. Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex.J. Neurosci.25, 247–251 (2005).

    Article CAS PubMed PubMed Central  Google Scholar 

  21. Nonaka-Kinoshita, M. et al. Regulation of cerebral cortex size and folding by expansion of basal progenitors.EMBO J.32, 1817–1828 (2013).

    Article CAS PubMed PubMed Central  Google Scholar 

  22. Betizeau, M. et al. Precursor diversity and complexity of lineage relationships in the outer subventricular zone of the primate.Neuron80, 442–457 (2013).

    Article CAS PubMed  Google Scholar 

  23. Gertz, C.C., Lui, J.H., LaMonica, B.E., Wang, X. & Kriegstein, A.R. Diverse behaviors of outer radial glia in developing ferret and human cortex.J. Neurosci.34, 2559–2570 (2014).

    Article CAS PubMed PubMed Central  Google Scholar 

  24. Stahl, R. et al. Trnp1 regulates expansion and folding of the mammalian cerebral cortex by control of radial glial fate.Cell153, 535–549 (2013).

    Article CAS PubMed  Google Scholar 

  25. Lui, J.H. et al. Radial glia require PDGFD-PDGFRβ signalling in human but not mouse neocortex.Nature515, 264–268 (2014).

    Article CAS PubMed PubMed Central  Google Scholar 

  26. Florio, M. et al. Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion.Science347, 1465–1470 (2015).

    Article CAS PubMed  Google Scholar 

  27. LaMonica, B.E., Lui, J.H., Hansen, D.V. & Kriegstein, A.R. Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex.Nat. Commun.4, 1665 (2013).

    Article PubMed CAS  Google Scholar 

  28. Gal, J.S. et al. Molecular and morphological heterogeneity of neural precursors in the mouse neocortical proliferative zones.J. Neurosci.26, 1045–1056 (2006).

    Article CAS PubMed PubMed Central  Google Scholar 

  29. Pilz, G.A. et al. Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type.Nat. Commun.4, 2125 (2013).

    Article PubMed CAS  Google Scholar 

  30. Wang, H., Ge, G., Uchida, Y., Luu, B. & Ahn, S. Gli3 is required for maintenance and fate specification of cortical progenitors.J. Neurosci.31, 6440–6448 (2011).

    Article CAS PubMed PubMed Central  Google Scholar 

  31. Dave, R.K. et al. Sonic hedgehog and notch signaling can cooperate to regulate neurogenic divisions of neocortical progenitors.PLoS One6, e14680 (2011).

    Article CAS PubMed PubMed Central  Google Scholar 

  32. Yabut, O.R., Fernández, G., Huynh, T., Yoon, K. & Pleasure, S.J. Suppressor of fused is critical for maintenance of neuronal progenitor identity during corticogenesis.Cell Rep.12, 2021–2034 (2015).

    Article CAS PubMed PubMed Central  Google Scholar 

  33. Shikata, Y. et al. Ptch1-mediated dosage-dependent action of Shh signaling regulates neural progenitor development at late gestational stages.Dev. Biol.349, 147–159 (2011).

    Article CAS PubMed  Google Scholar 

  34. Yu, W., Wang, Y., McDonnell, K., Stephen, D. & Bai, C.B. Patterning of ventral telencephalon requires positive function of Gli transcription factors.Dev. Biol.334, 264–275 (2009).

    Article CAS PubMed  Google Scholar 

  35. Johnson, M.B. et al. Single-cell analysis reveals transcriptional heterogeneity of neural progenitors in human cortex.Nat. Neurosci.18, 637–646 (2015).

    Article CAS PubMed PubMed Central  Google Scholar 

  36. de Juan Romero, C., Bruder, C., Tomasello, U., Sanz-Anquela, J.M. & Borrell, V. Discrete domains of gene expression in germinal layers distinguish the development of gyrencephaly.EMBO J.34, 1859–1874 (2015).

    Article CAS PubMed PubMed Central  Google Scholar 

  37. Huang, X. et al. Transventricular delivery of Sonic hedgehog is essential to cerebellar ventricular zone development.Proc. Natl. Acad. Sci. USA107, 8422–8427 (2010).

    Article CAS PubMed PubMed Central  Google Scholar 

  38. Lancaster, M.A. et al. Cerebral organoids model human brain development and microcephaly.Nature501, 373–379 (2013).

    Article CAS PubMed  Google Scholar 

  39. Corbit, K.C. et al. Vertebrate Smoothened functions at the primary cilium.Nature437, 1018–1021 (2005).

    Article CAS PubMed  Google Scholar 

  40. Pollen, A.A. et al. Molecular identity of human outer radial glia during cortical development.Cell163, 55–67 (2015).

    Article CAS PubMed PubMed Central  Google Scholar 

  41. Wong, F.K. et al. Sustained Pax6 expression generates primate-like basal radial glia in developing mouse neocortex.PLoS Biol.13, e1002217 (2015).

    Article PubMed PubMed Central CAS  Google Scholar 

  42. Noctor, S.C., Martínez-Cerdeño, V., Ivic, L. & Kriegstein, A.R. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.Nat. Neurosci.7, 136–144 (2004).

    Article CAS PubMed  Google Scholar 

  43. Haubensak, W., Attardo, A., Denk, W. & Huttner, W.B. Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis.Proc. Natl. Acad. Sci. USA101, 3196–3201 (2004).

    Article CAS PubMed PubMed Central  Google Scholar 

  44. Miyata, T. et al. Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells.Development131, 3133–3145 (2004).

    Article CAS PubMed  Google Scholar 

  45. Lewitus, E., Kelava, I., Kalinka, A.T., Tomancak, P. & Huttner, W.B. An adaptive threshold in mammalian neocortical evolution.PLoS Biol.12, e1002000 (2014).

    Article PubMed PubMed Central CAS  Google Scholar 

  46. Tong, C.K. et al. Primary cilia are required in a unique subpopulation of neural progenitors.Proc. Natl. Acad. Sci. USA111, 12438–12443 (2014).

    Article CAS PubMed PubMed Central  Google Scholar 

  47. Han, Y.-G. & Alvarez-Buylla, A. Role of primary cilia in brain development and cancer.Curr. Opin. Neurobiol.20, 58–67 (2010).

    Article CAS PubMed PubMed Central  Google Scholar 

  48. Han, Y.-G. et al. Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells.Nat. Neurosci.11, 277–284 (2008).

    Article CAS PubMed  Google Scholar 

  49. Spassky, N. et al. Primary cilia are required for cerebellar development and Shh-dependent expansion of progenitor pool.Dev. Biol.317, 246–259 (2008).

    Article CAS PubMed PubMed Central  Google Scholar 

  50. Hu, W.F., Chahrour, M.H. & Walsh, C.A. The diverse genetic landscape of neurodevelopmental disorders.Annu. Rev. Genomics Hum. Genet.15, 195–213 (2014).

    Article PubMed CAS  Google Scholar 

  51. Zhu, G. et al. Pten deletion causes mTorc1-dependent ectopic neuroblast differentiation without causing uniform migration defects.Development139, 3422–3431 (2012).

    Article CAS PubMed PubMed Central  Google Scholar 

  52. Chow, L.M.L., Zhang, J. & Baker, S.J. Inducible Cre recombinase activity in mouse mature astrocytes and adult neural precursor cells.Transgenic Res.17, 919–928 (2008).

    Article CAS PubMed PubMed Central  Google Scholar 

  53. Marszalek, J.R. et al. Genetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian photoreceptors.Cell102, 175–187 (2000).

    Article CAS PubMed  Google Scholar 

  54. McKinsey, G.L. et al. Dlx1&2-dependent expression of Zfhx1b (Sip1, Zeb2) regulates the fate switch between cortical and striatal interneurons.Neuron77, 83–98 (2013).

    Article CAS PubMed PubMed Central  Google Scholar 

  55. Anders, S., Pyl, P.T. & Huber, W. HTSeq–a Python framework to work with high-throughput sequencing data.Bioinformatics31, 166–169 (2015).

    Article CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Baker at St. Jude Children's Research Hospital for theGFAP::CreER andNestin::CreER mice; L.S. Goldstein at the University of California San Diego for theKif3aloxP/loxP mice; M.E. Hatley at St. Jude Children's Research hospital for the pBABE-GFP (originally a gift from William Hahn) and pBABE-SmoM2 vectors; J.L. Rubenstein and S. Pleasure at the University of California, San Francisco, for the Dlx2 antibody and the protocol for Ascl1 immunostaining, respectively; and D. Finkelstein and J. Peng at St. Jude Children's Research Hospital for help with the RNA-seq analyses and human embryonic stem cell culture, respectively. Human tissue was obtained from the NIH NeuroBioBank Brain at the University of Maryland, Baltimore, MD. We thank the staff of the Cell and Tissue Imaging Center, the Small Animal Imaging Center, the Hartwell Center for Bioinformatics and Biotechnology, and the Veterinary Pathology Core at St. Jude Children's Research Hospital for technical assistance. We thank S. Baker, X. Cao, M. Dyer, and K.A. Laycock for comments on the manuscript. Y.-G.H. is supported by NIH/NCI Cancer Center Core Support grant CA021765 (SJCRH), the Sontag Foundation Distinguished Scientist Award, Whitehall Foundation research grant, and ALSAC.

Author information

Authors and Affiliations

  1. Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Lei Wang, Shirui Hou & Young-Goo Han

  2. Division of Brain Tumor Research, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Lei Wang, Shirui Hou & Young-Goo Han

Authors
  1. Lei Wang

    You can also search for this author inPubMed Google Scholar

  2. Shirui Hou

    You can also search for this author inPubMed Google Scholar

  3. Young-Goo Han

    You can also search for this author inPubMed Google Scholar

Contributions

L.W. and Y.-G.H. designed and performed the experiments and wrote the manuscript. S.H. performed thein utero retroviral injections. Y.-G.H. conceived and supervised the study.

Corresponding author

Correspondence toYoung-Goo Han.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Boundaries used to quantify cells in E16.5 brains and SmoM2 expression patterns induced by multiple Cre lines.

a. Tangential boundaries were determined based on the trajectories of radial processes from the VZ to the pial surface. The processes of RGs were visualized by RC2 staining. Note that radial processes originating from the medial roof of the lateral ventricle curve to reach the medial surface of the hemisphere instead of extending straight to the dorsal surface of the brain. We used that boundary point (arrow) as our landmark to define the medial (M) and dorsal (D) parts of the cortex. The arrowhead indicates the dorsal medial corner of the lateral ventricle. A small part of the medial roof of the lateral ventricle was omitted in the RC2 tilting (*).b. Separate channels for images shownFig. 2a. The thin dotted lines indicate the boundary between the medial (M) and dorsal (D) cortex. The thick dotted lines indicate a boundary between the SVZ and VZ.c. Immunofluorescence showing SmoM2 expression induced by different Cre lines. Anti-GFP antibody was used to detect SmoM2-YFP fusion protein expressed bySmoM2 mutants. TheGFAP::Cre;SmoM2loxP/+ cortex displays a high-medial to low-lateral gradient, whereas the cortices ofNestin::CreER;SmoM2loxP/+ (injected with tamoxifen at E12.5) orNestin::Cre; SmoM2loxP/+ brains showed no clear gradient of SmoM2 expression. All the micrographs have been repeated for more than 3 times.

Supplementary Figure 2 Diverse morphology of bRGs (oRGs) inSmoM2 mutants.

a. E16.5SmoM2 cortex labeled for RC2 (green), Glast (blue), and Sox2 (red). InsetA shows an example of a Sox2+ cell attached to the pial surface by a single basal process that resembles the classic morphology of bRGs. InsetB shows a Sox2+ cell that has just divided and bears a basal process with a growth cone–like structure at the end (arrowhead). InsetC shows a Sox2+ cell with bipolar processes positioned tangentially. Scale bar = 20 μm.b. Diverse morphology of bRGs inGFAP::CreER; SmoM2loxP/+; tdTomatoloxP/+ cortex at E16.5 after tamoxifen injection at E13.5, as shown by labeling for tdTomato reporter (red), Pax6+ (green), and Tbr2− (blue): bRGs bearing apical (A), bipolar (B), basal (C), or multipolar (D) processes. The multipolar cells may correspond to transient bRGs observed in monkeys, which alternate between stages showing unipolar or bipolar radial processes and stages without a radial process22; however, we cannot rule out the possibility that these cells may be mis-differentiated bRGs or IPCs. The arrows point to the processes. Note that the bRGs are Pax6+ (green) Tbr2− (blue). The pie chart quantifies Pax6+ Tbr2− tdTomato+ cells in each morphologic category. All the micrographs have been repeated for more than 3 times.

Supplementary Figure 3 Increase of RGs at the expense of IPCs and neurons in the VZ and increase of RGs dividing non-apically.

a. Sections rostral and caudal to the images shown inFig. 3d labeled for TuJ1 (white or red), Pax6 (blue), and Tbr2 (green). Both rostral and caudal sections showed patterns of cell composition in the VZ similar to that in the medial section shown inFig. 3d. Arrows point to examples of bRGs. Scale bar = 50 μm.b. Non-apically dividing RGs at E15.5 indicated by the M-phase marker phospho-histone 3 (PH3, grey or red), Sox2 (green), and Tbr2 (blue). The arrows indicate examples of non-apically dividing RGs (PH3+ Sox2+ Tbr2−). Scale bar = 20 μm.c. Higher magnification of cellsA,B, andC in panel (b).d. Quantification of dividing IPCs (PH3+ Tbr2+) and RGs (PH3+ Sox2+ Tbr2−). Mann Whitney test, for IPC (PH3+ Tbr2+),P = 0.0004, Sum of ranks = 45, 126, Mann-Whitney U = 0.0000, for AP RGs (PH3+ Sox2+ Tbr2−),P = 0.2878, Sum of ranks = 98, 73, Mann-Whitney U = 28.00, for nonAP RGs (PH3+ Sox2+ Tbr2−),P = 0.0016, Sum of ranks = 50, 121, Mann-Whitney U = 5.000. 9 sections from 4 pairs of control and mutant mice were analyzed. AP, apical; ns,P > 0.05; ***P < 0.001. Error bars = standard error of the mean.

Supplementary Figure 4 SmoM2 induces folding outside the cingulate cortex.

a. Nissl staining of brain sections ofNestin::Cre;SmoM2loxP/+ mice at P7. Only a fewNestin::Cre;SmoM2loxP/+ mutant embryos survived to birth. The boxed regions showing folds (A–D) are enlarged below. The arrows in (B) and (D) point to folds in the lateral cortex. Scale bar = 1 mm. These were only observed in two rare survivors asNestin::Cre;SmoM2loxP/+ mice die prenatally.b. Cortex corresponding to the boxed area in panel (B) labeled for layer-specific markers, Satb2 (white or red, layers II–IV), Ctip2 (blue, layer V), and Tbr1 (green, layer VI). The cortices of the rare survivingNestin::Cre;SmoM2loxP/+ mice maintained normal layering.c. Nissl staining of coronal sections of control (SmoM2loxP/+) andNestin::CreER; SmoM2loxP/+ brains at P3. A relatively low dose of tamoxifen (1.5 mg/40 g of body weight, IP injection at E12.5) was used to avoid embryonic lethality. The arrows point to folds in the lateral cortices that are enlarged in the images on the right. Scale bar = 0.5 mm. Cortical folding was observed in approximately 30% of theNestin::CreER; SmoM2loxP/+ brains examined.

Supplementary Figure 5 Expression of Ascl1 and Dlx2 in the cortices ofGFAP::Cre; SmoM2loxP/+ mice.

a. qPCR quantification of Ascl1 and Dlx2 mRNA in microdissected medial E14.5 cortices of control andSmoM2 mutants.b. E16.5 brains labeled for Ascl1 (green), Pax6 (red), and Tbr2 (blue).c. E16.5 cortices labeled for Dlx2 (green) and Tbr2 (purple) or Sox2 (purple). All the micrographs have been repeated for more than 3 times. GE: ganglionic eminence

Supplementary Figure 6 Primary cilia and Smo are required for neocortical growth.

a. The upper pair of images show the whole brains of aSmoM2 mutant (GFAP::Cre;SmoM2loxP/+) and aSmoM2 mutant lacking cilia (GFAP::Cre;SmoM2loxP/+; Kif3aloxP/loxP) at P2. The lower row pair shows the cingulate cortex stained with hematoxylin. Note the absence of folding in theSmoM2 mutants without cilia.b. Whole brains of a mutant lackingSmo (GFAP::Cre;SmoloxP/loxP) and a control mouse at P21. Images represent results from more than 3 pairs of mice. Scale bar = 2 mm.

Supplementary Figure 7Gli1 expression in the mouse embryonic forebrain andGLI1 expression in the human fetal forebrain.

a.In situ hybridization forGli1 mRNA (dark brown dots) on mouse brain (E15.5) (Images obtained from the Allen Institute for Brain Science website athttp://developingmouse.brain-map.org/experiment/siv&quest;id=100051605&imageId=101024922&initImage=ish). The boxed areas are enlarged on the right. Note thatGli1 was only detectable in the ventral forebrain, including the ganglionic eminence.b. Levels ofGLI1 mRNA expression (purple dots) in the ganglionic eminence were similar to those in the cortex (Fig. 6b) in the human fetal brain. The boxed area in the upper image is enlarged in the lower image. CP, cortical plate; CTX, cortex; GE, ganglionic eminence; VZ, ventricular zone. Images represent results from 3 independent tissue samples.

Supplementary Figure 8 Relative levels ofGLI1 in the human fetal neocortex are higher than are those ofGli1 in the mouse embryonic cortex.

a. Relative levels ofGLI1 mRNA in the human fetal neocortex over time from 8 pcw to 38 pcw.GLI1 expression was normalized to that ofSOX2,NES, orPAX6. We constructed these graphs by using RNAseq data from the BrainSpan Developmental Transcriptome database (http://www.brainspan.org).b. Comparisons ofGli1 andGLI1 expression in different cortical areas of the mouse and human brain. Mouse mRNA expression levels were obtained from RNAseq analyses of E14.5 medial and lateral cortices. The human fetal brain results were obtained from the BrainSpan database (12–19 pcw).c.GLI1 andGli1 expression in sorted human and mouse RGs. Calculations were based on RNAseq data fromFlorio et al.28.

Supplementary Figure 9SHH mRNA and SHH protein are expressed in the human hypothalamic VZ.

a.In situ hybridization images forSHH mRNA (purple dots) on human fetal brain at 14 pcw.SHH mRNA was detected in the hypothalamic VZ (*). Each boxed area is enlarged in the adjacent image to the right. Images represent results from 2 independent tissue samples.b. Human fetal hypothalamus at 14 pcw stained with anti-SHH antibody (green) and DAPI (purple). CP, cortical plate; VZ, ventricular zone. Scale bar = 20 μm. Pictures represent at least 3 repeats.

Supplementary Figure 10 Blocking SHH signaling decreases SATB2+ neurons in human cerebral organoids.

a. Organoids are labeled for SOX2 (green), TBR2 (blue), and phospho-vimentin (red). SOX2+ RGs formed a VZ-like structure surrounding a lumen. TBR2+ IPCs formed an SVZ-like layer basal to the VZ-like structure. The phospho- vimentin labeled RGs in mitosis (arrowheads). Similar to what is observedin vivo, most RGs divided at the apical surface lining lumen; however, some RGs divided outside the VZ, resembling bRGs. The arrows indicate radial fibers of RGs expressing phospho-vimentin.b. The experimental scheme and organoids labeled for SATB2 (green) and CldU (purple) and quantification of SATB2+ CldU+ cells normalized to the total number of SATB2+ cells. Organoids were treated with SANT1 (400 nM) or DMSO for 10 days from 29 days to 39 days after differentiation. To label a cohort of neurons produced during treatment, we treated organoids with CldU (3 μg/mL) for 48 h from 35 days to 37 days after differentiation. The organoids were fixed at 64 days after differentiation. Scale bar = 50 μm. Two tailed unpaired t test,P = 0.0000, t(20) = 5.126; 10 (DMSO) and 12 (SANT-1) 'cortical' regions of 4 organoids each from 2 independent experiments were analyzed; KS normality test,P > 0.1; F test for variance,P = 0.5469, F(9, 11) = 1.458. Error bars = standard error of the mean.

Supplementary information

Rights and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, L., Hou, S. & Han, YG. Hedgehog signaling promotes basal progenitor expansion and the growth and folding of the neocortex.Nat Neurosci19, 888–896 (2016). https://doi.org/10.1038/nn.4307

Download citation

This article is cited by

Access through your institution
Buy or subscribe

Advertisement

Search

Advanced search

Quick links

Nature Briefing

Sign up for theNature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox.Sign up for Nature Briefing
[8]ページ先頭
©2009-2025 Movatter.jp