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.

Asymmetric cell divisions promote Notch-dependent epidermal differentiation

Abstract

Stem and progenitor cells use asymmetric cell divisions to balance proliferation and differentiation. Evidence from invertebrates shows that this process is regulated by proteins asymmetrically distributed at the cell cortex during mitosis: Par3–Par6–aPKC, which confer polarity, and Gαi–LGN/AGS3–NuMA–dynein/dynactin, which govern spindle positioning. Here we focus on developing mouse skin, where progenitor cells execute a switch from symmetric to predominantly asymmetric divisions concomitant with stratification. Using in vivo skin-specific lentiviral RNA interference, we investigate spindle orientation regulation and provide direct evidence that LGN (also called Gpsm2), NuMA and dynactin (Dctn1) are involved. In compromising asymmetric cell divisions, we uncover profound defects in stratification, differentiation and barrier formation, and implicate Notch signalling as an important effector. Our study demonstrates the efficacy of applying RNA interference in vivo to mammalian systems, and the ease of uncovering complex genetic interactions, here to gain insights into how changes in spindle orientation are coupled to establishing proper tissue architecture during skin development.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Spindle orientation defects after LGN, Numa1 and Dctn1 depletion.
Figure 2: Impaired stratification in vitro and in vivo when asymmetric cell divisions are impaired.
Figure 3: Differentiation defects after LGN, Numa1 and Dctn1 depletion.
Figure 4: Loss of LGN or Numa1 impairs suprabasal Notch activation.
Figure 5: Genetic interaction between asymmetric cell division and Notch pathways.

References

  1. Neumuller, R. A. & Knoblich, J. A. Dividing cellular asymmetry: asymmetric cell division and its implications for stem cells and cancer. Genes Dev. 23, 2675–2699 (2009)

    PubMed  PubMed Central  Article  Google Scholar 

  2. Knoblich, J. A. Mechanisms of asymmetric stem cell division. Cell 132, 583–597 (2008)

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  4. Yu, F., Morin, X., Cai, Y., Yang, X. & Chia, W. Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization. Cell 100, 399–409 (2000)

    CAS  PubMed  Article  Google Scholar 

  5. Schober, M., Schaefer, M. & Knoblich, J. A. Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts. Nature 402, 548–551 (1999)

    ADS  CAS  PubMed  Article  Google Scholar 

  6. Wodarz, A., Ramrath, A., Kuchinke, U. & Knust, E. Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts. Nature 402, 544–547 (1999)

    ADS  CAS  PubMed  Article  Google Scholar 

  7. Lechler, T. & Fuchs, E. Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature 437, 275–280 (2005)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Smart, I. H. Variation in the plane of cell cleavage during the process of stratification in the mouse epidermis. Br. J. Dermatol. 82, 276–282 (1970)

    CAS  PubMed  Article  Google Scholar 

  9. Poulson, N. D. & Lechler, T. Robust control of mitotic spindle orientation in the developing epidermis. J. Cell Biol. 191, 915–922 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Fuchs, E. Scratching the surface of skin development. Nature 445, 834–842 (2007)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Bowman, S. K., Neumuller, R. A., Novatchkova, M., Du, Q. & Knoblich, J. A. The Drosophila NuMA Homolog Mud regulates spindle orientation in asymmetric cell division. Dev. Cell 10, 731–742 (2006)

    CAS  PubMed  Article  Google Scholar 

  12. Izumi, Y., Ohta, N., Hisata, K., Raabe, T. & Matsuzaki, F. Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization. Nature Cell Biol. 8, 586–593 (2006)

    CAS  PubMed  Article  Google Scholar 

  13. Siller, K. H., Cabernard, C. & Doe, C. Q. The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts. Nature Cell Biol. 8, 594–600 (2006)

    CAS  PubMed  Article  Google Scholar 

  14. Blumer, J. B., Kuriyama, R., Gettys, T. W. & Lanier, S. M. The G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and Giα3 influence cortical positioning of the mitotic spindle poles at metaphase in symmetrically dividing mammalian cells. Eur. J. Cell Biol. 85, 1233–1240 (2006)

    CAS  PubMed  Article  Google Scholar 

  15. Woodard, G. E. et al. Ric-8A and Giα recruit LGN, NuMA, and dynein to the cell cortex to help orient the mitotic spindle. Mol. Cell. Biol. 30, 3519–3530 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Du, Q. & Macara, I. G. Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins. Cell 119, 503–516 (2004)

    CAS  PubMed  Article  Google Scholar 

  17. Johnston, C. A., Hirono, K., Prehoda, K. E. & Doe, C. Q. Identification of an Aurora-A/PinsLINKER/Dlg spindle orientation pathway using induced cell polarity in S2 cells. Cell 138, 1150–1163 (2009)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Segalen, M. et al. The Fz-Dsh planar cell polarity pathway induces oriented cell division via Mud/NuMA in Drosophila and zebrafish. Dev. Cell 19, 740–752 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Beronja, S., ivshits, G., Williams, S. E. & Fuchs, E. Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nature Med. 16, 821–827 (2010); published online 6 June 2010.

    CAS  PubMed  Article  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  22. Zheng, Z. et al. LGN regulates mitotic spindle orientation during epithelial morphogenesis. J. Cell Biol. 189, 275–288 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006)

    CAS  PubMed  Article  Google Scholar 

  24. Adams, R. J. Metaphase spindles rotate in the neuroepithelium of rat cerebral cortex. J. Neurosci. 16, 7610–7618 (1996)

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. Geldmacher-Voss, B., Reugels, A. M., Pauls, S. & Campos-Ortega, J. A. A 90-degree rotation of the mitotic spindle changes the orientation of mitoses of zebrafish neuroepithelial cells. Development 130, 3767–3780 (2003)

    CAS  PubMed  Article  Google Scholar 

  26. Kaltschmidt, J. A., Davidson, C. M., Brown, N. H. & Brand, A. H. Rotation and asymmetry of the mitotic spindle direct asymmetric cell division in the developing central nervous system. Nature Cell Biol. 2, 7–12 (2000)

    CAS  PubMed  Article  Google Scholar 

  27. Du, Q., Stukenberg, P. T. & Macara, I. G. A mammalian Partner of inscuteable binds NuMA and regulates mitotic spindle organization. Nature Cell Biol. 3, 1069–1075 (2001)

    CAS  PubMed  Article  Google Scholar 

  28. Hardman, M. J., Sisi, P., Banbury, D. N. & Byrne, C. Patterned acquisition of skin barrier function during development. Development 125, 1541–1552 (1998)

    CAS  PubMed  Google Scholar 

  29. 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)

    ADS  CAS  PubMed  Article  Google Scholar 

  30. 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)

    CAS  Article  PubMed  Google Scholar 

  31. Watt, F. M., Estrach, S. & Ambler, C. A. Epidermal Notch signalling: differentiation, cancer and adhesion. Curr. Opin. Cell Biol. 20, 171–179 (2008)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Blanpain, C., Lowry, W. E., Pasolli, H. A. & Fuchs, E. Canonical notch signaling functions as a commitment switch in the epidermal lineage. Genes Dev. 20, 3022–3035 (2006)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Moriyama, M. et al. Multiple roles of Notch signaling in the regulation of epidermal development. Dev. Cell 14, 594–604 (2008)

    CAS  PubMed  Article  Google Scholar 

  34. Demehri, S. et al. Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity. PLoS Biol. 6, e123 (2008)

    PubMed  PubMed Central  Article  Google Scholar 

  35. Pan, Y. et al. γ-secretase functions through Notch signaling to maintain skin appendages but is not required for their patterning or initial morphogenesis. Dev. Cell 7, 731–743 (2004)

    CAS  PubMed  Article  Google Scholar 

  36. Rangarajan, A. et al. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation. EMBO J. 20, 3427–3436 (2001)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Coumailleau, F., Furthauer, M., Knoblich, J. A. & Gonzalez-Gaitan, M. Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division. Nature 458, 1051–1055 (2009)

    ADS  CAS  PubMed  Article  Google Scholar 

  38. Emery, G. et al. Asymmetric Rab11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system. Cell 122, 763–773 (2005)

    CAS  PubMed  Article  Google Scholar 

  39. Mummery-Widmer, J. L. et al. Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi. Nature 458, 987–992 (2009)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Nickoloff, B. J. et al. Jagged-1 mediated activation of notch signaling induces complete maturation of human keratinocytes through NF-κB and PPARγ. Cell Death Differ. 9, 842–855 (2002)

    CAS  PubMed  Article  Google Scholar 

  41. Knoblich, J. A., Jan, L. Y. & Jan, Y. N. Asymmetric segregation of Numb and Prospero during cell division. Nature 377, 624–627 (1995)

    ADS  CAS  PubMed  Article  Google Scholar 

  42. Wang, H., Ouyang, Y., Somers, W. G., Chia, W. & Lu, B. Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon. Nature 449, 96–100 (2007)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Wirtz-Peitz, F., Nishimura, T. & Knoblich, J. A. Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization. Cell 135, 161–173 (2008)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Rhyu, M. S., Jan, L. Y. & Jan, Y. N. Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76, 477–491 (1994)

    CAS  PubMed  Article  Google Scholar 

  45. Clayton, E. et al. A single type of progenitor cell maintains normal epidermis. Nature 446, 185–189 (2007)

    ADS  CAS  PubMed  Article  Google Scholar 

  46. Mizutani, K., Yoon, K., Dang, L., Tokunaga, A. & Gaiano, N. Differential Notch signalling distinguishes neural stem cells from intermediate progenitors. Nature 449, 351–355 (2007)

    ADS  CAS  PubMed  Article  Google Scholar 

  47. Murtaugh, L. C., Stanger, B. Z., Kwan, K. M. & Melton, D. A. Notch signaling controls multiple steps of pancreatic differentiation. Proc. Natl Acad. Sci. USA 100, 14920–14925 (2003)

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  48. Tanigaki, K. et al. Notch-RBP-J signaling is involved in cell fate determination of marginal zone B cells. Nature Immunol. 3, 443–450 (2002)

    CAS  Article  Google Scholar 

  49. Vasioukhin, V., Degenstein, L., Wise, B. & Fuchs, E. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc. Natl Acad. Sci. USA 96, 8551–8556 (1999)

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank N. Stokes, L. Polak and D. Oristian for their assistance in the mouse facility; S. Lanier and T. Gettys for providing AGS3 and Gαi antibodies; N. Gaiano and C. Cepko for constructs; D. Melton and T. Honjo for mice; S. Chai for Numb constructs; E. Ezhkova for sharing microarray data; and J. Knoblich for sharing unpublished results and reagents. We are grateful to M. Schober, D. Devenport, E. Ezratty, C. Luxenburg and members of the Fuchs laboratory for discussions and critical reading of the manuscript. We thank S. Mazel and the RU Flow Cytometry Resource Center for assistance with cell sorting, A. North and the RU Bioimaging Resource Center for assistance with image acquisition and the Comparative Biology Center (AAALAC accredited) for veterinary care of our mice. S.E.W. was supported by an American Cancer Society postdoctoral fellowship and S.B. was a Human Frontier Science Program postdoctoral fellow. E.F. is an investigator in the Howard Hughes Medical Institute. Work in the Fuchs laboratory was supported by a grant from the National Institutes of Health (E.F. R01AR27883).

Author information

Authors and Affiliations

Authors

Contributions

S.E.W., E.F. and S.B. designed experiments. S.E.W. performed the experiments and analysed their raw data. H.A.P. conducted ultrastructural analyses. S.E.W. and S.B. performed lentiviral injections. S.E.W. and E.F. wrote the paper. All authors provided intellectual input, read and approved the manuscript.

Corresponding author

Correspondence to Elaine Fuchs.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

The file contains Supplementary Figures 1-12 with legends. (PDF 12996 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Williams, S., Beronja, S., Pasolli, H. et al. Asymmetric cell divisions promote Notch-dependent epidermal differentiation. Nature 470, 353–358 (2011). https://doi.org/10.1038/nature09793

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09793

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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