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.

  • Letter
  • Published:

Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway

A Corrigendum to this article was published on 03 October 2011

This article has been updated

Abstract

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing1,2,3. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: miR-449 microRNAs are the most upregulated microRNAs during multiciliogenesis.
Figure 2: miR-449 and CDC20B are localized in multiciliated cells of human airway epithelium.
Figure 3: miR-449 knockdown inhibits multiciliogenesis.
Figure 4: miR-449 microRNAs repress the Notch pathway in HAECs.
Figure 5: miR-449 microRNAs repress the Notch pathway in Xenopus embryonic epidermis.

Similar content being viewed by others

Change history

  • 14 September 2011

    In the version of this Letter initially published online and in print, an article by Lizé et al. (Cell Cycle 9, 4579–4583; 2010), which reports that miR 449 microRNAs accumulate during mucociliary differentiation of human airway epithelia, was inadvertently omitted from the references list. On pages 1–2, the following text has replaced the previous text: “miR 449a, miR 449b and miR 449c (collectively named miR 449), constitute by far the most strongly induced microRNAs during epithelium differentiation in both species. Although representing less than 0.01% of all microRNA sequences in proliferating HAECs, miR 449 accounted for more than 8% of the microRNA reads in differentiated HAECs (Fig. 1a and Supplementary Fig. S1c,d; see also ref. 13).” The omitted reference has now been added to the reference list: 13. Lizé, M., Herr, C., Klimke, A., Bals, R. & Dobbelstein, M. MicroRNA 449a levels increase by several orders of magnitude during mucociliary differentiation of airway epithelia. Cell Cycle 9, 4579–4583 (2010). References 13–40 have been changed to 14–41, respectively.

References

  1. Fliegauf, M., Benzing, T. & Omran, H. When cilia go bad: cilia defects and ciliopathies. Nat. Rev. Mol. Cell. Biol. 8, 880–893 (2007).

    Article  CAS  Google Scholar 

  2. Shah, A. S. et al. Motile cilia of human airway epithelia are chemosensory. Science 325, 1131–1134 (2009).

    Article  CAS  Google Scholar 

  3. Wanner, A., Salathe, M. & O’Riordan, T. G. Mucociliary clearance in the airways. Am. J. Respir. Crit. Care Med. 154, 1868–1902 (1996).

    Article  CAS  Google Scholar 

  4. Hayes, J. M. et al. Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development. Dev. Biol. 312, 115–130 (2007).

    Article  CAS  Google Scholar 

  5. Satir, P., Mitchell, D. R. & Jekely, G. How did the cilium evolve? Curr. Top Dev. Biol. 85, 63–82 (2008).

    Article  CAS  Google Scholar 

  6. Stubbs, J. L., Oishi, I., Izpisua Belmonte, J. C. & Kintner, C. The forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish embryos. Nat. Genet. 40, 1454–1460 (2008).

    Article  CAS  Google Scholar 

  7. Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004).

    Article  CAS  Google Scholar 

  8. Eulalio, A., Huntzinger, E. & Izaurralde, E. Getting to the root of miRNA-mediated gene silencing. Cell 132, 9–14 (2008).

    Article  CAS  Google Scholar 

  9. Ortholan, C. et al. MicroRNAs and lung cancer: new oncogenes and tumor suppressors, new prognostic factors and potential therapeutic targets. Curr. Med. Chem. 16, 1047–1061 (2009).

    Article  CAS  Google Scholar 

  10. LeSimple, P. et al. Trefoil factor family 3 peptide promotes human airway epithelial ciliated cell differentiation. Am. J. Respir. Cell. Mol. Biol. 36, 296–303 (2007).

    Article  CAS  Google Scholar 

  11. Marcet, B. et al. Extracellular nucleotides regulate CCL20 release from human primary airway epithelial cells, monocytes and monocyte-derived dendritic cells. J. Cell. Physiol. 211, 716–727 (2007).

    Article  CAS  Google Scholar 

  12. Coraux, C., Roux, J., Jolly, T. & Birembaut, P. Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration. Proc. Am. Thorac. Soc. 5, 689–694 (2008).

    Article  Google Scholar 

  13. Lizé, M., Herr, C., Klimke, A., Bals, R. & Dobbelstein, M. MicroRNA-449a levels increase by several orders of magnitude during mucociliary differentiation of airway epithelia. Cell Cycle 9, 4579–4583 (2010).

    Article  Google Scholar 

  14. Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).

    Article  CAS  Google Scholar 

  15. Cardinaud, B. et al. miR-34b/miR-34c: a regulator of TCL1 expression in 11q- chronic lymphocytic leukaemia? Leukemia 23, 2174–2177 (2009).

    Article  CAS  Google Scholar 

  16. Flicek, P. et al. Ensembl 2008. Nucleic Acids. Res. 36, D707 (2008).

    Article  CAS  Google Scholar 

  17. Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599 (2007).

    Article  CAS  Google Scholar 

  18. Dawe, H. R., Farr, H. & Gull, K. Centriole/basal body morphogenesis and migration during ciliogenesis in animal cells. J. Cell. Sci. 120, 7–15 (2007).

    Article  CAS  Google Scholar 

  19. Pearson, C. G., Culver, B. P. & Winey, M. Centrioles want to move out and make cilia. Dev. Cell. 13, 319–321 (2007).

    Article  CAS  Google Scholar 

  20. Vladar, E. K. & Stearns, T. Molecular characterization of centriole assembly in ciliated epithelial cells. J. Cell. Biol. 178, 31–42 (2007).

    Article  CAS  Google Scholar 

  21. Hajj, R. et al. Basal cells of the human adult airway surface epithelium retain transit-amplifying cell properties. Stem. Cells. 25, 139–148 (2007).

    Article  CAS  Google Scholar 

  22. Le Brigand, K., Robbe-Sermesant, K., Mari, B. & Barbry, P. MiRonTop: mining microRNAs targets across large scale gene expression studies. Bioinformatics 26, 3131–3132 (2010).

    Article  CAS  Google Scholar 

  23. Zhen, G. et al. IL-13 and epidermal growth factor receptor have critical but distinct roles in epithelial cell mucin production. Am. J. Respir. Cell. Mol. Biol. 36, 244–53 (2007).

    Article  CAS  Google Scholar 

  24. Feng, M. & Yu, Q. miR-449 regulates CDK-Rb-E2F1 through an auto-regulatory feedback circuit. Cell Cycle 9, 213–214 (2010).

    Article  CAS  Google Scholar 

  25. Lize, M., Pilarski, S. & Dobbelstein, M. E2F1-inducible microRNA 449a/b suppresses cell proliferation and promotes apoptosis. Cell. Death. Differ. 17, 452–458 (2009).

    Article  Google Scholar 

  26. Yang, X. et al. miR-449a and miR-449b are direct transcriptional targets of E2F1 and negatively regulate pRb-E2F1 activity through a feedback loop by targeting CDK6 and CDC25A. Genes Dev. 23, 2388–2393 (2009).

    Article  CAS  Google Scholar 

  27. Choi, W. Y., Giraldez, A. J. & Schier, A. F. Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430. Science 318, 271–274 (2007).

    Article  CAS  Google Scholar 

  28. Tsao, P. N. et al. Notch signalling controls the balance of ciliated and secretory cell fates in developing airways. Development 136, 2297–2307 (2009).

    Article  CAS  Google Scholar 

  29. Guseh, J. S. et al. Notch signalling promotes airway mucous metaplasia and inhibits alveolar development. Development 136, 1751–1759 (2009).

    Article  CAS  Google Scholar 

  30. Deblandre, G. A., Wettstein, D. A., Koyano-Nakagawa, N. & Kintner, C. A two-step mechanism generates the spacing pattern of the ciliated cells in the skin of Xenopus embryos. Development 126, 4715–4728 (1999).

    CAS  Google Scholar 

  31. Marnellos, G., Deblandre, G. A., Mjolsness, E. & Kintner, C. Delta-Notch lateral inhibitory patterning in the emergence of ciliated cells in Xenopus: experimental observations and a gene network model. Pac. Symp. Biocomput. 329–340 (2000).

  32. Essner, J. J et al. Conserved function for embryonic nodal cilia. Nature 418, 37–38 (2002).

    Article  CAS  Google Scholar 

  33. Marchal, L., Luxardi, G., Thome, V. & Kodjabachian, L. BMP inhibition initiates neural induction via FGF signalling and Zic genes. Proc. Natl Acad. Sci. USA 106, 17437–17442 (2009).

    Article  CAS  Google Scholar 

  34. Mitchell, B. et al. A positive feedback mechanism governs the polarity and motion of motile cilia. Nature 447, 97–101 (2007).

    Article  CAS  Google Scholar 

  35. Irizarry, R. A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).

    Article  Google Scholar 

  36. Le Brigand, K. & Barbry, P. Mediante: a web-based microarray data manager. Bioinformatics 23, 1304–1306 (2007).

    Article  CAS  Google Scholar 

  37. Kloosterman, W. P. et al. In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat. Methods 3, 27–29 (2006).

    Article  CAS  Google Scholar 

  38. Pohl, B. S. & Knochel, W. Isolation and developmental expression of Xenopus FoxJ1 and FoxK1. Dev. Genes. Evol. 214, 200–205 (2004).

    Article  CAS  Google Scholar 

  39. Morichika, K. et al. Perturbation of Notch/Suppressor of Hairless pathway disturbs migration of primordial germ cells in Xenopus embryo. Dev. Growth. Differ. 52, 235–244 (2010).

    Article  CAS  Google Scholar 

  40. Lopez, S. L. et al. Notch activates sonic hedgehog and both are involved in the specification of dorsal midline cell-fates in Xenopus. Development 130, 2225–2238 (2003).

    Article  CAS  Google Scholar 

  41. Pottier, N. et al. Identification of keratinocyte growth factor as a target of microRNA-155 in lung fibroblasts: implication in epithelial-mesenchymal interactions. PLoS One 4, e6718 (2009).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by CNRS, INSERM, Région Champagne-Ardenne, Région PACA, CG06 and by grants from ANR, Vaincre la Mucoviscidose, ARC and INCa.

We thank V. Magnone, G. Rios, S. Fourré, K. LeBrigand and J. Maurizio, from the IBISA Functional Genomics Platform, S Antipolis, for help with transcriptome analyses and bioinformatics, F. Brau and J. Cazareth, for cellular imaging, V. Thomé for in situ hybridization experiments, F. Aguila for artwork and B. Mari for discussions.

This work is the object of a CNRS patent N°09/03723.

Author information

Authors and Affiliations

Authors

Contributions

B.M. led the project. P. Barbry, the Principal Investigator (IPMC), initiated and managed the entire project. L.K. is the Principal Investigator (IBDML) of the Xenopus section. B.M., L.K. and P. Barbry planned experiments, analysed and interpreted data and wrote the paper. B.M. and B. Chevalier carried out cell culture, cellular and molecular biology and cellular imaging in human, C.C., B.N-R. and T.J. carried out in situ hybridization experiments, cell culture and cell sorting on human tissues, G.L., M.C. and L.K. carried out Xenopus experiments, K.R-S. contributed to bioinformatics analyses, L-E.Z. and R.W. carried out HTS experiments, R.W. helped with manuscript correction, B. Cardinaud helped with molecular cloning, C.M. carried out Affymetrix transcriptome experiments and L.G-C. helped with PCR experiments. P. Birembaut provided critical discussion.

Corresponding author

Correspondence to Pascal Barbry.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2064 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Marcet, B., Chevalier, B., Luxardi, G. et al. Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway. Nat Cell Biol 13, 693–699 (2011). https://doi.org/10.1038/ncb2241

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2241

This article is cited by

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