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MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundary

Abstract

The midbrain-hindbrain boundary (MHB) is a long-lasting organizing center in the vertebrate neural tube that is both necessary and sufficient for the ordered development of midbrain and anterior hindbrain (midbrain-hindbrain domain, MH). The MHB also coincides with a pool of progenitor cells that contributes neurons to the entire MH. Here we show that the organizing activity and progenitor state of the MHB are co-regulated by a single microRNA, miR-9, during late embryonic development in zebrafish. Endogenous miR-9 expression, initiated at late stages, selectively spares the MHB. Gain- and loss-of-function studies, in silico predictions and sensor assays in vivo demonstrate that miR-9 targets several components of the Fgf signaling pathway, thereby delimiting the organizing activity of the MHB. In addition, miR-9 promotes progression of neurogenesis in the MH, defining the MHB progenitor pool. Together, these findings highlight a previously unknown mechanism by which a single microRNA fine-tunes late MHB coherence via its co-regulation of patterning activities and neurogenesis.

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Figure 1: Gain of miR-9 function causes MHB loss.
Figure 2: Sensor assay to reveal direct interaction of miR-9 with predicted binding sites.
Figure 3: miR-9 overexpression downregulates Fgf signaling.
Figure 4: miR-9 overexpression causes premature neurogenesis across the MHB.
Figure 5: Endogenous miR-9 expression in the MH area avoids the MHB and postmitotic domains.
Figure 6: Morpholino knockdown of miR-9 affects Fgf signaling in a manner opposite to miR-9 gain of function.
Figure 7: Morpholino knockdown of miR-9 affects neurogenesis in a manner opposite to miR-9 gain of function.

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References

  1. Wurst, W. & Bally-Cuif, L. Neural plate patterning: upstream and downstream of the isthmic organizer. Nat. Rev. Neurosci. 2, 99–108 (2001).

    Article  CAS  Google Scholar 

  2. Scholpp, S., Groth, C., Lohs, C., Lardelli, M. & Brand, M. Zebrafish fgfr1 is a member of the fgf8 synexpression group and is required for fgf8 signalling at the midbrain-hindbrain boundary. Dev. Genes Evol. 214, 285–295 (2004).

    Article  CAS  Google Scholar 

  3. Stigloher, C., Chapouton, P., Adolf, B. & Bally-Cuif, L. Identification of neural progenitor pools by E(Spl) factors in the embryonic and adult brain. Brain Res. Bull. 75, 266–273 (2008).

    Article  CAS  Google Scholar 

  4. Bally-Cuif, L., Goridis, C. & Santoni, M.J. The mouse NCAM gene displays a biphasic expression pattern during neural tube development. Development 117, 543–552 (1993).

    CAS  PubMed  Google Scholar 

  5. Vaage, S. The segmentation of the primitive neural tube in chick embryos (Gallus domesticus). A morphological, histochemical and autoradiographical investigation. Ergeb. Anat. Entwicklungsgesch. 41, 3–87 (1969).

    CAS  PubMed  Google Scholar 

  6. Geling, A. et al. bHLH transcription factor Her5 links patterning to regional inhibition of neurogenesis at the midbrain-hindbrain boundary. Development 130, 1591–1604 (2003).

    Article  CAS  Google Scholar 

  7. Geling, A., Plessy, C., Rastegar, S., Strähle, U. & Bally-Cuif, L. Her5 acts as a prepattern factor that blocks neurogenin1 and coe2 expression upstream of Notch to inhibit neurogenesis at the midbrain-hindbrain boundary. Development 131, 1993–2006 (2004).

    Article  CAS  Google Scholar 

  8. Ninkovic, J. et al. Inhibition of neurogenesis at the zebrafish midbrain-hindbrain boundary by the combined and dose-dependent activity of a new hairy/E(spl) gene pair. Development 132, 75–88 (2005).

    Article  CAS  Google Scholar 

  9. Hans, S. et al. her3, a zebrafish member of the hairy-E(spl) family, is repressed by Notch signalling. Development 131, 2957–2969 (2004).

    Article  CAS  Google Scholar 

  10. Hirata, H., Tomita, K., Bessho, Y. & Kageyama, R. Hes1 and Hes3 regulate maintenance of the isthmic organizer and development of the mid/hindbrain. EMBO J. 20, 4454–4466 (2001).

    Article  CAS  Google Scholar 

  11. Hirate, Y. & Okamoto, H. Canopy1, a novel regulator of FGF signaling around the midbrain-hindbrain boundary in zebrafish. Curr. Biol. 16, 421–427 (2006).

    Article  CAS  Google Scholar 

  12. Lun, K. & Brand, M. A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary. Development 125, 3049–3062 (1998).

    CAS  PubMed  Google Scholar 

  13. Reifers, F. et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125, 2381–2395 (1998).

    CAS  PubMed  Google Scholar 

  14. Fjose, A. & Drivenes, O. RNAi and microRNAs: from animal models to disease therapy. Birth Defects Res. C. Embryo Today 78, 150–171 (2006).

    Article  CAS  Google Scholar 

  15. Kawakami, Y. et al. MKP3 mediates the cellular response to FGF8 signaling in the vertebrate limb. Nat. Cell Biol. 5, 513–519 (2003).

    Article  CAS  Google Scholar 

  16. Munchberg, S.R., Ober, E.A. & Steinbeisser, H. Expression of the Ets transcription factors erm and pea3 in early zebrafish development. Mech. Dev. 88, 233–236 (1999).

    Article  CAS  Google Scholar 

  17. Yan, X. et al. Improving the prediction of human microRNA target genes by using ensemble algorithm. FEBS Lett. 581, 1587–1593 (2007).

    Article  CAS  Google Scholar 

  18. Tallafuss, A. & Bally-Cuif, L. Tracing of her5 progeny in zebrafish transgenics reveals the dynamics of midbrain-hindbrain neurogenesis and maintenance. Development 130, 4307–4323 (2003).

    Article  CAS  Google Scholar 

  19. 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 

  20. Valencia-Sanchez, M.A., Liu, J., Hannon, G.J. & Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515–524 (2006).

    Article  CAS  Google Scholar 

  21. Bae, Y.K., Shimizu, T. & Hibi, M. Patterning of proneuronal and inter-proneuronal domains by hairy- and enhancer of split–related genes in zebrafish neuroectoderm. Development 132, 1375–1385 (2005).

    Article  CAS  Google Scholar 

  22. Wienholds, E. et al. MicroRNA expression in zebrafish embryonic development. Science 309, 310–311 (2005).

    Article  CAS  Google Scholar 

  23. Darnell, D.K. et al. MicroRNA expression during chick embryo development. Dev. Dyn. 235, 3156–3165 (2006).

    Article  CAS  Google Scholar 

  24. Kapsimali, M. et al. MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system. Genome Biol. 8, R173 (2007).

    Article  Google Scholar 

  25. Kloosterman, W.P., Wienholds, E., de Bruijn, E., Kauppinen, S. & Plasterk, R.H. In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat. Methods 3, 27–29 (2006).

    Article  CAS  Google Scholar 

  26. Mueller, T. & Wullimann, M.F. Anatomy of neurogenesis in the early zebrafish brain. Brain Res. Dev. Brain Res. 140, 137–155 (2003).

    Article  CAS  Google Scholar 

  27. Stark, A., Brennecke, J., Bushati, N., Russell, R.B. & Cohen, S.M. Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3′ UTR evolution. Cell 123, 1133–1146 (2005).

    Article  CAS  Google Scholar 

  28. Leaman, D. et al. Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development. Cell 121, 1097–1108 (2005).

    Article  CAS  Google Scholar 

  29. Li, Y., Wang, F., Lee, J.A. & Gao, F.B. MicroRNA-9a ensures the precise specification of sensory organ precursors in Drosophila. Genes Dev. 20, 2793–2805 (2006).

    Article  CAS  Google Scholar 

  30. 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  Google Scholar 

  31. Wu, J. & Xie, X. Comparative sequence analysis reveals an intricate network among REST, CREB and miRNA in mediating neuronal gene expression. Genome Biol. 7, R85 (2006).

    Article  Google Scholar 

  32. Conaco, C., Otto, S., Han, J.J. & Mandel, G. Reciprocal actions of REST and a microRNA promote neuronal identity. Proc. Natl. Acad. Sci. USA 103, 2422–2427 (2006).

    Article  CAS  Google Scholar 

  33. Brand, M. et al. Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain. Development 123, 179–190 (1996).

    CAS  PubMed  Google Scholar 

  34. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B. & Schilling, T.F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310 (1995).

    Article  CAS  Google Scholar 

  35. Enright, A.J. et al. MicroRNA targets in Drosophila. Genome Biol. 5, R1 (2003).

    Article  Google Scholar 

  36. Rusinov, V., Baev, V., Minkov, I.N. & Tabler, M. MicroInspector: a web tool for detection of miRNA binding sites in an RNA sequence. Nucleic Acids Res. 33, W696–700 (2005).

    Article  CAS  Google Scholar 

  37. Miranda, K.C. et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell 126, 1203–1217 (2006).

    Article  CAS  Google Scholar 

  38. Rehmsmeier, M., Steffen, P., Hochsmann, M. & Giegerich, R. Fast and effective prediction of microRNA/target duplexes. RNA 10, 1507–1517 (2004).

    Article  CAS  Google Scholar 

  39. Pfaffl, M.W., Horgan, G.W. & Dempfle, L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30, e36 (2002).

    Article  Google Scholar 

  40. Müller, M., von Weizsäcker, E. & Campos-Ortega, J.A. Transcription of a zebrafish gene of the hairy-Enhancer of split family delineates the midbrain anlage in the neural plate. Dev. Genes Evol. 206, 153–160 (1996).

    Article  Google Scholar 

  41. Tonou-Fujimori, N. et al. Expression of the FGF receptor 2 gene (fgfr2) during embryogenesis in the zebrafish Danio rerio. Mech. Dev. 119 Suppl 1: S173–S178 (2002).

    Article  Google Scholar 

  42. Krauss, S. et al. Zebrafish pax(zf-a): a paired box-containing gene expressed in the neural tube. EMBO J. 10, 3609–3619 (1991).

    Article  CAS  Google Scholar 

  43. Molven, A., Njolstad, P.R. & Fjose, A. Genomic structure and restricted neural expression of the zebrafish wnt-1 (int-1) gene. EMBO J. 10, 799–807 (1991).

    Article  CAS  Google Scholar 

  44. Ekker, M., Wegner, J., Akimenko, M.A. & Westerfield, M. Coordinate embryonic expression of three zebrafish engrailed genes. Development 116, 1001–1010 (1992).

    CAS  PubMed  Google Scholar 

  45. Hammerschmidt, M., Bitgood, M.J. & McMahon, A.P. Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. Genes Dev. 10, 647–658 (1996).

    Article  CAS  Google Scholar 

  46. Cheesman, S.E., Layden, M.J., Von Ohlen, T., Doe, C.Q. & Eisen, J.S. Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation. Development 131, 5221–5232 (2004).

    Article  CAS  Google Scholar 

  47. Strahle, U., Blader, P., Henrique, D. & Ingham, P.W. Axial, a zebrafish gene expressed along the developing body axis, shows altered expression in cyclops mutant embryos. Genes Dev. 7, 1436–1446 (1993).

    Article  CAS  Google Scholar 

  48. Oxtoby, E. & Jowett, T. Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucleic Acids Res. 21, 1087–1095 (1993).

    Article  CAS  Google Scholar 

  49. Giraldez, A.J. et al. MicroRNAs regulate brain morphogenesis in zebrafish. Science 308, 833–838 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to members of the L.B.-C. laboratory for discussions and to M. Götz, W. Norton and M. Wassef for their insightful ideas and critical reading of the manuscript. This work was funded by a junior group grant from the Volkswagen Association, the EU 6th framework integrated project ZF-Models (contract No. LSHC-CT-2003-503466), the Life Science Association (No. GSF 2005/01), a special research grant from the Institut du Cerveau et de la Moelle épinière, the Excellence Center for Protein Science, Munich, and the Helmholtz 'Impuls und Vernetzungsfond'.

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C.L. and C.S. jointly conducted the experiments. A.W. and R.K. conducted parallel analyses in chicken to support the findings described here. A.F. provided technical assistance and L.B.-C. supervised the project. C.L. and L.B.-C. wrote the manuscript.

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Correspondence to Christoph Leucht or Laure Bally-Cuif.

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Leucht, C., Stigloher, C., Wizenmann, A. et al. MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundary. Nat Neurosci 11, 641–648 (2008). https://doi.org/10.1038/nn.2115

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