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.

  • Article
  • Published:

Endfoot regeneration restricts radial glial state and prevents translocation into the outer subventricular zone in early mammalian brain development

Nature Cell Biology volume 22, pages 26–37 (2020)Cite this article

Subjects

Abstract

Neural stem cells, called radial glia, maintain epithelial structure during the early neocortical development. The prevailing view claims that when radial glia first proliferate, their symmetric divisions require strict spindle orientation; its perturbation causes precocious neurogenesis and apoptosis. Here, we show that despite this conventional view, radial glia at the proliferative stage undergo normal symmetric divisions by regenerating an apical endfoot even if it is lost by oblique divisions. We found that the Notch–R-Ras–integrin β1 pathway promotes the regeneration of endfeet, whose leading edge bears ectopic adherens junctions and the Par-polarity complex. However, this regeneration ability gradually declines during the subsequent neurogenic stage and hence oblique divisions induce basal translocation of radial glia to form the outer subventricular zone, a hallmark of the development of the convoluted brain. Our study reveals that endfoot regeneration is a temporally changing cryptic property, which controls the radial glial state and its shift is essential for mammalian brain size expansion.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: LGN knockout causes spindle misorientation and loss of the apical endfoot.
Fig. 2: Spindle misorientation does not impair RG at the proliferative stage.
Fig. 3: RG missing the apical endfoot extend a new process with ectopic adherens junctions.
Fig. 4: Radial glia regenerate the apical endfoot at the early neurogenic stage.
Fig. 5: Regeneration of the apical endfoot depends on the Notch, R-Ras and integrin β1 pathways.
Fig. 6: FRET analysis of R-Ras activity.
Fig. 7: Activation of Notch signalling inhibits oRG generation.
Fig. 8: Apical-endfoot regeneration restricts the timing of oRG generation.

Similar content being viewed by others

Data availability

Source data are available online for Figs. 1–8, Extended Data Figs. 1,2,5–9 and Supplementary Fig. 1. Additional time-lapse movies that support the key findings of this study have been deposited in Figshare (https://figshare.com/projects/Endfoot_Regeneration/71684). All other data supporting the findings of this study are available from the corresponding author on reasonable request.

References

  1. Noctor, S. C., Flint, A. C., Weissman, T. A., Dammerman, R. S. & Kriegstein, A. R. Neurons derived from radial glial cells establish radial units in neocortex. Nature 409, 714–720 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Miyata, T., Kawaguchi, A., Okano, H. & Ogawa, M. Asymmetric inheritance of radial glial fibers by cortical neurons. Neuron 31, 727–741 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. 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 

  4. 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. Cortex 12, 37–53 (2002).

    Article  PubMed  Google Scholar 

  5. Morin, X. & Bellaïche, Y. Mitotic spindle orientation in asymmetric and symmetric cell divisions during animal development. Dev. Cell 21, 102–119 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Chenn, A. & McConnell, S. K. Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis. Cell 82, 631–641 (1995).

    Article  CAS  PubMed  Google Scholar 

  7. Yingling, J. et al. Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division. Cell 132, 474–486 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Postiglione, M. P. et al. Mouse Inscuteable induces apical-basal spindle orientation to facilitate intermediate progenitor generation in the developing neocortex. Neuron 72, 269–284 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Siller, K. H. & Doe, C. Q. Spindle orientation during asymmetric cell division. Nat. Cell Biol. 11, 365–374 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Feng, Y. & Walsh, C. A. Mitotic spindle regulation by Nde1 controls cerebral cortical size. Neuron 44, 279–293 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Xie, Y., Jüschke, C., Esk, C., Hirotsune, S. & Knoblich, J. A. The phosphatase PP4c controls spindle orientation to maintain proliferative symmetric divisions in the developing neocortex. Neuron 79, 254–265 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kosodo, Y. et al. Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells. EMBO J. 23, 2314–2324 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sanada, K. & Tsai, L.-H. G protein βγ subunits and AGS3 control spindle orientation and asymmetric cell fate of cerebral cortical progenitors. Cell 122, 119–131 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. 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 

  15. Morin, X., Jaouen, F. & Durbec, P. Control of planar divisions by the G-protein regulator LGN maintains progenitors in the chick neuroepithelium. Nat. Neurosci. 10, 1440–1448 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Konno, D. et al. Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis. Nat. Cell Biol. 10, 93–101 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. 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 

  18. Hansen, D. V., Lui, J. H., Parker, P. R. L. & Kriegstein, A. R. Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature 464, 554–561 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. 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 

  20. 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. Cortex 21, 1674–1694 (2010).

    Article  PubMed  Google Scholar 

  21. 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 

  22. Martínez-Martínez, M. Á. et al. A restricted period for formation of outer subventricular zone defined by Cdh1 and Trnp1 levels. Nat. Commun. 7, 11812 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Meng, W., Mushika, Y., Ichii, T. & Takeichi, M. Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts. Cell 135, 948–959 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Shioi, G. et al. Apical constriction in distal visceral endoderm cells initiates global, collective cell rearrangement in embryonic visceral endoderm to form anterior visceral endoderm. Dev. Biol. 429, 20–30 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. Alexandre, P., Reugels, A. M., Barker, D., Blanc, E. & Clarke, J. D. W. Neurons derive from the more apical daughter in asymmetric divisions in the zebrafish neural tube. Nat. Neurosci. 13, 673–679 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Das, R. M. & Storey, K. G. Mitotic spindle orientation can direct cell fate and bias Notch activity in chick neural tube. EMBO Rep. 13, 448–454 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kraut, R., Chia, W., Jan, L. Y., Jan, Y. N. & Knoblich, J. A. Role of inscuteable in orienting asymmetric cell divisions in Drosophila. Nature 383, 50–55 (1996).

    Article  CAS  PubMed  Google Scholar 

  28. Wilsch-Brauninger, M., Peters, J., Paridaen, J. T. M. L. & Huttner, W. B. Basolateral rather than apical primary cilia on neuroepithelial cells committed to delamination. Development 139, 95–105 (2011).

    Article  PubMed  CAS  Google Scholar 

  29. Paridaen, J. T. M. L., Wilsch-Bräuninger, M. & Huttner, W. B. Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division. Cell 155, 333–344 (2013).

    Article  CAS  PubMed  Google Scholar 

  30. 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 

  31. Nakamura, Y. et al. The bHLH gene Hes1 as a repressor of the neuronal commitment of CNS stem cells. J. Neurosci. 20, 283–293 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gaiano, N., Nye, J. S. & Fishell, G. Radial glial identity is promoted by Notch1 signaling in the murine forebrain. Neuron 26, 395–404 (2000).

    Article  CAS  PubMed  Google Scholar 

  33. Hitoshi, S. et al. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev. 16, 846–858 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mizutani, K.-I. & Saito, T. Progenitors resume generating neurons after temporary inhibition of neurogenesis by notch activation in the mammalian cerebral cortex. Development 132, 1295–1304 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Ohtsuka, T. et al. Visualization of embryonic neural stem cells using Hes promoters in transgenic mice. Mol. Cell. Neurosci. 31, 109–122 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Ohtsuka, T. et al. Gene expression profiling of neural stem cells and identification of regulators of neural differentiation during cortical development. Stem Cells 29, 1817–1828 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Konno, D. et al. The mammalian DM domain transcription factor Dmrta2 is required for early embryonic development of the cerebral cortex. PLoS ONE 7, e46577 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Takahashi, S. et al. Rab11 regulates exocytosis of recycling vesicles at the plasma membrane. J. Cell Sci. 125, 4049–4057 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Zhang, Z., Vuori, K., Wang, H., Reed, J. C. & Ruoslahti, E. Integrin activation by R-ras. Cell 85, 61–69 (1996).

    Article  CAS  PubMed  Google Scholar 

  40. Conklin, M. W., Ada-Nguema, A., Parsons, M., Riching, K. M. & Keely, P. J. R-Ras regulates β1-integrin trafficking via effects on membrane ruffling and endocytosis. BMC Cell Biol. 11, 14 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kawaguchi, A. et al. Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis. Development 135, 3113–3124 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Hodkinson, P. S. et al. Mammalian NOTCH-1 activates β1 integrins via the small GTPase R-Ras. J. Biol. Chem. 282, 28991–29001 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Ohata, S. et al. Dual roles of Notch in regulation of apically restricted mitosis and apicobasal polarity of neuroepithelial cells. Neuron 69, 215–230 (2011).

    Article  CAS  PubMed  Google Scholar 

  45. Takaya, A. et al. R-Ras regulates exocytosis by Rgl2/Rlf-mediated activation of RalA on endosomes. Mol. Biol. Cell 18, 1850–1860 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Tsunekawa, Y. et al. Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain. Development 143, 3216–3222 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Tsai, J.-W., Chen, Y., Kriegstein, A. R. & Vallee, R. B. LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages. J. Cell Biol. 170, 935–945 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Toyo-Oka, K. et al. Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation. J. Cell Biol. 180, 1133–1147 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Sahara, S. & O’Leary, D. D. M. Fgf10 regulates transition period of cortical stem cell differentiation to radial glia controlling generation of neurons and basal progenitors. Neuron 63, 48–62 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Marsden, M. & DeSimone, D. W. Regulation of cell polarity, radial intercalation and epiboly in Xenopus: novel roles for integrin and fibronectin. Development 128, 3635–3647 (2001).

    CAS  PubMed  Google Scholar 

  51. Stubbs, J. L., Davidson, L., Keller, R. & Kintner, C. Radial intercalation of ciliated cells during Xenopus skin development. Development 133, 2507–2515 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Walck-Shannon, E. & Hardin, J. Cell intercalation from top to bottom. Nat. Rev. Mol. Cell Biol. 15, 34–48 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Sedzinski, J., Hannezo, E., Tu, F., Biro, M. & Wallingford, J. B. Emergence of an apical epithelial cell surface in vivo. Dev. Cell 36, 24–35 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Werner, M. E. et al. Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1. J. Cell Biol. 206, 367–376 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Tavano, S. et al. Insm1 induces neural progenitor delamination in developing neocortex via downregulation of the adherens junction belt-specific protein Plekha7. Neuron 97, 1299–1314 (2018).

  56. Takahashi, T., Nowakowski, R. S. & Caviness, V. S. The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall. J. Neurosci. 15, 6046–6057 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Lakso, M. et al. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl Acad. Sci. USA 93, 5860–5865 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Yusa, K., Zhou, L., Li, M. A., Bradley, A. & Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. Proc. Natl Acad. Sci. USA 108, 1531–1536 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Laviv, T. et al. Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins. Nat. Methods 13, 989–992 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Fiordalisi, J. J., Johnson, R. L., Ulkü, A. S., Der, C. J. & Cox, A. D. Mammalian expression vectors for Ras family proteins: generation and use of expression constructs to analyze Ras family function. Methods Enzymol. 332, 3–36 (2001).

    Article  CAS  PubMed  Google Scholar 

  61. Jüschke, C., Xie, Y., Postiglione, M. P. & Knoblich, J. A. Analysis and modeling of mitotic spindle orientations in three dimensions. Proc. Natl Acad. Sci. USA 111, 1014–1019 (2014).

    Article  PubMed  CAS  Google Scholar 

  62. Kamentsky, L. et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics 27, 1179–1180 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  64. Pau, G., Fuchs, F., Sklyar, O., Boutros, M. & Huber, W. EBImage—an R package for image processing with applications to cellular phenotypes. Bioinformatics 26, 979–981 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Boulanger, J. et al. Patch-based non-local functional for denoising fluorescence microscopy image sequences. IEEE Trans. Med. Imaging 29, 442–454 (2010).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Lin and M. Matsuda for plasmids; T. Miyata, A. Kawaguchi, Y. Tsunekawa and Q. Wu for their technical advice; and M. Takeichi, S. Yonemura and S. Hayashi for critical discussion of the data. Some of the imaging experiments were performed at the RIKEN Kobe Light Microscopy Facility. TEM analysis was supported by the Laboratory for Ultrastructural Research at RIKEN BDR. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (KAKENHI) grant nos 18H04003, 17H05779 and 24113006 to F.M.; grant nos 16K21627 and 18K14828 to I.F. and grant no. 16K18381 to A.S. In addition, I.F. was a recipient of the RIKEN Special Postdoctoral Researcher Program.

Author information

Author notes

  1. Daijiro Konno

    Present address: Division of Pathophysiology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

  2. These authors contributed equally: Ikumi Fujita, Atsunori Shitamukai, Fumiya Kusumoto.

Authors and Affiliations

  1. Laboratory for Cell Asymmetry, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Ikumi Fujita, Atsunori Shitamukai, Fumiya Kusumoto, Shun Mase, Taeko Suetsugu, Ayaka Omori, Daijiro Konno & Fumio Matsuzaki

  2. Laboratory of Molecular Cell Biology and Development, Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

    Fumiya Kusumoto, Shun Mase & Fumio Matsuzaki

  3. Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Tokyo, Japan

    Kagayaki Kato

  4. Laboratory for Animal Resource Development, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Takaya Abe

  5. Laboratory of Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan

    Takaya Abe & Go Shioi

Authors
  1. Ikumi Fujita
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Atsunori Shitamukai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fumiya Kusumoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shun Mase
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Taeko Suetsugu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ayaka Omori
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kagayaki Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Takaya Abe
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Go Shioi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Daijiro Konno
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Fumio Matsuzaki
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

I.F., A.S. and F.M. designed the project. I.F., F.K. and A.S. engaged in and performed all of the experiments. I.F. and F.K. observed the regeneration of RG. A.S. observed the effects of R-Ras and integrin β1. I.F. and T.S. performed the electron-microscopy analyses. T.S., I.F. and A.S. performed the ferret experiments. D.K., T.A. and G.S. generated the LGN-null mutant. D.K., F.K., T.S., I.F. and A.O. analysed the LGN-mutant phenotype in the fixed sections. S.M. and A.S. performed the Notch/NICD experiments. K.K. created the program for calculating spindle orientation. I.F and F.M. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Fumio Matsuzaki.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Generation of the LGN-null mutant mouse line and analysis of mitosis.

a, Schematic representation of the strategies for the generation of LGN-null mutant mice. The generation of the LGN-∆C allele was previously described16. See Methods for details. The directions of genotyping primers are indicated by arrows. b, A typical result of genotyping PCR for the LGN-null allele using Primers 1–3. Four independent littermates showed similar results, of which unprocessed gel images are indicated in Source data. c, Offspring from LGN-null heterozygous parents. The ratio of genotypes showed no inconsistency with the Mendelian ratio. Two-tailed χ2 test, P = 0.79. d, Cumulative plots of spindle orientations in the WT, LGN-heterozygous and the LGN-homozygous mutants at E11.5, E13.5, and E15.5. The data set is the same as Fig. 1b and n is indicated in Fig. 1b. The uniformly random orientation model is shown with dotted lines. Transparent bands, 95% confidence intervals by Wilson score interval. e, Time-lapse imaging of EGFP-Plekha7 mice at E12.5. Embryos were electroporated with an expression vector for CyRFP at E11.5. Arrows, a mitotic RG and its daughter cells. Arrowheads, the apical endfeet of the cells. A mitotic RG was distinguished from interphase cells by transient expansion of the apical membrane due to mitotic swelling and weakening of the EGFP-Plekha7 signal (10 and 20 min); the weakened signal of EGFP-Plekha7 at mitosis reflects the release of Plekha7 from the adherens junction into the cytoplasm at mitosis in the RG. The cell body split in two at 20 min, followed by apical adhesion belt splitting up to 50 min. Two biologically independent samples showed similar results. Scale bar, 5 µm.

Source data

Extended Data Fig. 2 Analysis of the LGN-mutant phenotypes.

a, Dot plots and box plots of the spindle orientations in WT and LGN-homozygous mutant dorsal cortices at E10.5 (n = 47 and 51 cells, respectively), E11.5 (n = 58 and 62), and E14.5 (n = 95 and 87). Two-tailed Kolmogorov–Smirnov test, P = 5.9 × 10−6 (E10.5), 4.3 × 10–14 (E12.5) and 4.7 × 10–9 (E14.5). b, Cumulative plots of a, as shown in Extended Data Fig. 1d. c, “Non-apical” mitotic RG (Pax6+, Tbr2– and PH3+ cells) residing at a distance of > 30 µm from the ventricular surface but remains in the VZ (arrows) in the LGN mutant dorsal cortex at E14.5. The dotted line indicates the VZ margin. Scale bar, 100 µm. Similar results were observed in two biologically independent experiments. d, e, Percentages of apical and non-apical mitotic RG (Pax6+, Tbr2– and PH3+ cells) in WT (n = 6 embryos) and LGN-heterozygous (n = 13 embryos) mice at E14.5, as shown in Fig. 2d. Two-tailed Mann–Whitney U-test, p = 0.53. f, WT and LGN-homozygous mutant cortices at E10.5 and E12.5 stained with TUNEL, an anti-Pax6 antibody and DAPI. Scale bar, 100 µm. No induction of apoptotic cells was observed in the LGN mutant at either stage. Similar results were observed in two biologically independent experiments. g, h, mInsc overexpression modulates spindle orientations in RG. WT dorsal cortices were electroporated with the control or mInsc overexpression plasmid along with the EGFP marker plasmid at E11.5 and analysed at E12.5. EGFP+ cells at anaphase were analysed as described in Fig. 1b. g, Dot and box plots of the spindle orientation in the control (n = 63 cells from 6 embryos) and mInsc overexpression (n = 59 cells from 7 embryos). Box plots are defined as described in Fig. 1b. Two-tailed Kolmogorov–Smirnov test, P < 2.2 × 10–16. ***P < 0.001. h, Cumulative plot of (g). The uniformly random orientation model is shown with a dotted line. Transparent bands, 95% confidence intervals by Wilson score interval.

Source data

Extended Data Fig. 3 mInsc overexpression causes spindle misorientation, and a resultant off-apical cell extends a new apical process at the early neurogenic stage.

a, b, Time-lapse imaging of control (a) and mInsc-overexpressing (b) RG at E12.5. WT embryos were electroporated with the indicated plasmids at E11.5. The dorsal cortices were sliced at E12.5 and observed from a transverse view. White dotted lines indicate the ventricular surface. Each colour of arrowheads and asterisks indicates an identical apical process and a cell body of each daughter cell. a, In the control, most RG divisions split the apical endfoot into both daughters. 20 out of 20 cells showed similar results in three experiments. b, mInsc overexpression frequently caused bypassing divisions, which generated a basal daughter cell that lost the apical endfoot (yellow asterisk). In most cases of such bypassing divisions, the migrating basal daughter newly extended a process toward the apical endfoot (arrows). 10 out of 11 cells showed similar results in one experiment. c–f, WT dorsal cortices electroporated at E11.5 with plasmids for mInsc overexpression and sparse labelling with mem-EGFP were analysed at E12.5 by immunostaining for Sox2, Plekha7 and EGFP. In a pair of sister RG (Sox2+ and Tbr2–, e, f), one sister cell shows an apical extension with Plekha7 accumulation (d, arrows). g–m, WT dorsal cortices electroporated by the same condition as in (c–f). Cell shapes visualized by mem-EGFP were analysed in every focal plane and overlaid with colours separately (pseudocolor in g, j, k). g, k, Projection images of 20-µm thick sections. h–j, l, m, Single focal images. g–j, Immunostaining for EGFP, ZO-1 and Pax6, showing a pair of Pax6+ cells (h, i), in which one sister cell (h) extended an apical process detaching from the ventricular surface and contacted the other sister cell at its apical tip, where ZO-1 accumulated (j). k–m, Immunostaining for EGFP, ZO-1 and Tbr2, showing a pair of Tbr2– and Tbr2+ cells that were assumed to be asymmetric neurogenic division products (l, m). The Tbr2– daughter cell exhibited ZO-1 accumulation at the tip of the apical extension, suggesting that the process with ZO-1 accumulation was derived from RG, but not from IP. Arrows, ZO-1 accumulation at the tips of apically extending processes. Asterisks, background staining of vascular endothelial cells. c–m, Similar results were observed in at least two biologically independent samples. Scale bars, 10 µm (a–i, k–m) and 2 µm (j).

Extended Data Fig. 4 Visualization of regenerating apical processes in LGN mutants with EGFP–Plekha7.

a, b, Two-photon imaging of dissected dorsal cortices from (a) LGN-heterozygous and (b) LGN-homozygous EGFP-Plekha7 mice at E10.5. Projection images of en face views and transverse views of the square regions with white dotted lines are shown. The yellow dotted lines indicate the basal edges of the tissues. Arrows, non-apical EGFP-Plekha7 foci. c, Immunostaining of LGN-homozygous EGFP-Plekha7 mice at E11.5 for EGFP, Plekha7 and β-catenin. Arrows, colocalization of EGFP and Plekha7 with β-catenin. Scale bar, 10 µm. d, Time-lapse imaging of EGFP-Plekha7 in the LGN-homozygous mutant at E11.5. Max projection images from en face and transverse views are shown. Arrows, a non-apical EGFP-Plekha7 focus; arrowhead, a newly emerged apical endfoot. e, Schematic image of the three-dimensional analysis of EGFP-Plekha7 foci. Images were taken from the apical side (en face view). The x- and y-axes are parallel to the apical surface, and the z-axis is parallel to the apico-basal axis of the tissue. Kymographs were generated by projecting signals in a defined volume, as indicated by the yellow box. See Methods for details. f, Kymographs and en face view of EGFP-Plekha7 during delamination in LGN-heterozygous or homozygous mice at E13.5. Delaminating apical endfeet were coloured by green. Only a faint EGFP-Plekha7 signal was observed during delamination (arrowheads), which occurred most likely due to cell differentiation. Each data was obtained from at least two biologically independent samples with similar results. Scale bars, 50 µm (a, b), 10 µm (c–e), and 5 µm (f).

Extended Data Fig. 5 Quantification of adhesion plaques in the LGN mutant and WT.

a, Schematic illustration of the analysis of EGFP-Plekha7 foci in the LGN mutant. The dynamics of each individual focus were analysed by using three-dimensional positions every 10 minutes. b, c, Trajectories and angles of EGFP-Plekha7 foci in the LGN mutant at E11.5. b, Trajectories were analysed along the z-axis as depicted in (a). c, Angles were analysed for every 10 minutes. 90 and –90 degrees represent movements toward the apical and basal sides of a tissue, respectively. n = 128 displacements in 10 min of 8 plaques from 2 embryos. d, e, Trajectories and angles of EGFP-Plekha7 foci in the LGN mutant at E13.5. Note that E13.5 data were obtained every 5 minutes but the analysis in (e) was performed every 10 minutes. n = 146 displacements in 10 min of 9 plaques from 2 embryos. Two-tailed Kolmogorov–Smirnov test comparing (c) and (e), P = 9.7×10–13. f, Extension of the apical process at E14.5 under mInsc overexpression. WT dorsal cortices were electroporated with the indicated plasmids at E13.5 and analysed from transverse view at E14.5. Left, a kymograph of EGFP-ZO-1. Right, images of a bypassing division (1 h 40 min) and its progenies (green and blue asterisks) at the indicated time points. The arrowheads indicate adhesion plaques. The arrow indicates weak signal of a delaminating endfoot (as shown in Extended Data Fig. 4f). The descending rate of the apical process at E14.5 was approximately 0.2-fold of that in E11.5 (compare with Fig. 4b). Two independent experiments showed similar results. g, h, Inheritance (g) and origin (h) of the apical endfeet in the WT background at E11.5, E13.5, and E15.5. Data from 4 (E11.5), 4 (E13.5), and 5 (E15.5) embryos. g, n = 620 (E11.5), 465 (E13.5), and 360 (E15.5) cells. h, n = 1,323 (E11.5), 857 (E13.5), and 960 (E15.5) cells. h, Two-tailed Fisher's exact test between E11.5 and E13.5 or E15.5, P = 5.5×10–3 or 4.9×10–4, respectively. *P < 0.05, **P < 0.01. Error bars, 95% confidence intervals.

Source data

Extended Data Fig. 6 Regeneration of the apical endfoot in the LGE.

a–f, Immunostaining of LGE (a, b, e, f) and MGE (c, d) in WT (e), LGN-heterozygous (a, c) or LGN-homozygous (b, d, f) mutants for PH3, Sox2 and Ascl1, at E11.5 (a–d) and E14.5 (e, f). Arrows indicates Sox2+ Ascl1– PH3+ cells outside the VZ. Scale bars, 100 µm. g, Kymographs of EGFP-Plekha7 in the E11.5 LGE of LGN-heterozygous and LGN-homozygous mutants. Arrowheads indicate adhesion plaques moving away from the apical surface. Scale Bar, 10 µm. h, i, Inheritance (h) and origin (i) of the apical endfeet in the LGE of LGN-heterozygous and LGN-homozygous mutants at E11.5 and E14.5. Analyses were performed over 13.5 h (E11.5) or 24 h (E14.5). Data of LGN-heterozygous and LGN-homozygous mutants were obtained respectively from 2 and 3 (E11.5), or 1 and 3 (E14.5) embryos. Sample sizes of LGN-heterozygous and LGN-homozygous are respectively, 263 and 409 cells (h, E11.5); 124 and 192 cells (h, E14.5); 528 and 731 cells (i, E11.5); 412 and 1,215 cells (i, E14.5). Two-tailed Fisher's exact test, h, P < 2.2×10–16 (E11.5) and P = 2.8×10–6 (E14.5); i, p = 1×10–6 (E11.5) and 1 (E14.5), respectively. ***P < 0.001; n.s., P ≥ 0.05.

Source data

Extended Data Fig. 7 Regeneration of the apical endfoot in the thalamus.

a, Immunostaining of the developing thalamus in LGN-heterozygous or LGN-homozygous mutants for PH3, Sox2 and Ascl1 at E11.5. Scale bar, 100 µm. Two independent experiments showed the similar results. b, c, Inheritance (b) and origin (c) of apical endfeet in the thalamus of LGN-heterozygous and LGN-homozygous mutants at E11.5. Analyses were performed over 13.5 h. b, n = 226 (LGN-heterozygous) and 161 (LGN-homozygous) cells from 2 and 3 embryos, respectively. c, n = 252 (LGN-heterozygous) and 417 (LGN-homozygous) cells from 2 and 3 embryos, respectively. Two-tailed Fisher's exact test, b, P < 1×10–15, c, P = 9.4×10–7. ***P < 0.001.

Source data

Extended Data Fig. 8 Effects of NICD overexpression, integrin β1 knockdown and R-Ras knockout on apical-endfoot regeneration.

a, FACS analysis of dorsal cortical cells using pHes1-d2EGFP mice. EGFP+ fraction was defined according to the range outside the WT cell distribution (left). The EGFP+ fraction was separated into EGFP-high and EGFP-low populations based on the peak of the E11.5 distribution. Percentages of EGFP high and low populations are indicated. n = 92,009 (WT, E11.5), 92,155 (pHes1-d2EGFP, E11.5), 94,721 (E13.5), 90,876 (E14.5) and 89,649 (E15.5) cells from 7, 6, 7, 8, and 6 embryos, respectively. ****P <0.0001. Fisher's exact test comparing with E11.5, P < 2.2×10–16. b, Time-lapse imaging of a cell expressing mInsc and NICD at E14.5 from en face and transverse views. Arrows, the tip of an extending process, which finally formed an adhesion belt of EGFP-ZO-1 at the apical surface of the tissue (at 6 h 00 min). Two independent experiments showed similar results. c, Ratio of Pax6– and Pax6+ cells inside or outside the VZ among all EGFP+ cells in the control, mInsc overexpression, NICD expression, and expression of both. The same data set as in Fig. 7a, b. Data are mean ± s.d. of embryos. d–d’’, Immunostaining for Rab11 (magenta) and ZO-1 (green) in an LGN-homozygous mutant mouse at E11.5. Rab11 accumulated adjacent to ZO-1 ectopic foci (d’) and the apical adhesion belt (d’’). Two independent experiments showed similar results. Scale bars, 5 µm. e, f, Origin (g) and inheritance (h) of the apical endfeet in Itgb1 knockdown under mInsc overexpression at E14.5. Data from 5 embryos each. e, n = 452 (control) and 628 (Itgb1 knockdown) cells. f, n = 249 (control) and 347 (Itgb1 knockdown) cells. Two-tailed Fisher's exact test, P = 0.18. n.s., P ≥ 0.05.

Source data

Extended Data Fig. 9 FRET analysis of the R-Ras activity.

a, b, The R-Ras activity in the dorsal cortex and LGE. Each WT brain region was electroporated with R-Ras FRET probe at E13.5 and analysed at E14.5. Data are mean ± s.d. of n = 98 (dorsal cortex) and 71 (LGE) cells from one embryo each. Two-tailed Mann–Whitney U-test, P < 1×10–15. c, d, The R-Ras activity in the dorsal cortex under NICD expression. WT brains were electroporated with R-Ras FRET probe and the control or NICD expression plasmids at E13.5 and analysed at E14.5. Data are mean ± s.d. of n = 115 (control) and 166 (NICD expression) cells from two embryos each. Two-tailed Mann–Whitney U-test, P = 8.1×10–6. e–h, Effects of Fgf10 in the endfoot regeneration. e, WT brains were electroporated with the control or Fgf10 overexpression plasmids at E11.5 and analysed by immunostaining for BLBP at E12.5. Similar results were observed in two biologically independent samples for each condition. f, g, Inheritance (f) and origin (g) of the apical endfeet in Fgf10 overexpression under mInsc overexpression at E12.5. Data from 6 embryos each. f, n = 170 (control) and 206 (Fgf10 overexpression). g, n = 213 (control) and 277 (Fgf10 overexpression). Two-tailed Fisher's exact test, P = 1. h, FRET analysis of the R-Ras activity under Fgf10 overexpression at E12.5. Data are mean ± s.d of n = 133 (control) and 138 (Fgf10 overexpression) cells from 2 embryos each. Two-tailed Mann–Whitney U-test, P = 0.75. *P < 0.05; ***P < 0.001; n.s., P ≥ 0.05. Scale bars, 10 µm (a, c) and 100 µm (e).

Source data

Supplementary information

Supplementary Information

Supplementary Figure 1

Reporting Summary

Supplementary Video 1

Apical-endfoot dynamics in the LGN-heterozygous dorsal cortex at E11.5. The E11.5 LGN-heterozygous dorsal cortex was observed at 10-min intervals using EGFP-Plekha7. A z-projection movie of a 30 × 30 µm square region was shown. Two independent experiments showed similar results .

Supplementary Video 2

Apical-endfoot dynamics in the LGN-homozygous dorsal cortex at E11.5. The E11.5 LGN-homozygous dorsal cortex was observed at 10-min intervals using EGFP-Plekha7. A z-projection movie of a 30 × 30 µm square region was shown. Two independent experiments showed similar results .

Supplementary Video 3

Apical-endfoot dynamics in the WT background at E11.5. The E11.5 WT dorsal cortex was observed at 5-min intervals using EGFP-Plekha7. A z-projection and transverse projection movies of a 50.7 × 50.7 µm square region were shown at 5-min intervals. The arrow indicates an adhesion plaque that finally generated a new endfoot (2:50) in the right images. Drifting was adjusted as described in Methods. Four independent experiments showed similar results .

Source data

Source Data Fig. 1

Statistical Source Data of Fig. 1

Source Data Fig. 2

Statistical Source Data of Fig. 2

Source Data Fig. 3

Statistical Source Data of Fig. 3

Source Data Fig. 4

Statistical Source Data of Fig. 4

Source Data Fig. 5

Statistical Source Data of Fig. 5

Source Data Fig. 6

Statistical Source Data of Fig. 6

Source Data Fig. 7

Statistical Source Data of Fig. 7

Source Data Fig. 8

Statistical Source Data of Fig. 8

Unprocessed Gels Extended Data Fig. 1

Unprocessed gels of Extended Data Fig. 1

Source Data Extended Data Fig. 2

Statistical Source Data of Extended Data Fig. 2

Source Data Extended Data Fig. 5

Statistical Source Data of Extended Data Fig. 5

Source Data Extended Data Fig. 6

Statistical Source Data of Extended Data Fig. 6

Source Data Extended Data Fig. 7

Statistical Source Data of Extended Data Fig. 7

Source Data Extended Data Fig. 8

Statistical Source Data of Extended Data Fig. 8

Source Data Extended Data Fig. 9

Statistical Source Data of Extended Data Fig. 9

Source Data Supplementary Fig. 1

Statistical Source Data of Supplementary Fig. 1

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fujita, I., Shitamukai, A., Kusumoto, F. et al. Endfoot regeneration restricts radial glial state and prevents translocation into the outer subventricular zone in early mammalian brain development. Nat Cell Biol 22, 26–37 (2020). https://doi.org/10.1038/s41556-019-0436-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-019-0436-9

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature 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