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Drosophila patterning is established by differential association of mRNAs with P bodies

Abstract

The primary embryonic axes in flies, frogs and fish are formed through translational regulation of localized transcripts before fertilization1. In Drosophila melanogaster, the axes are established through the transport and translational regulation of gurken (grk) and bicoid (bcd) messenger RNA in the oocyte and embryo1. Both transcripts are translationally silent while being localized within the oocyte along microtubules by cytoplasmic dynein1,2,3,4. Once localized, grk is translated at the dorsoanterior of the oocyte to send a TGF- α signal to the overlying somatic cells5. In contrast, bcd is translationally repressed in the oocyte until its activation in early embryos when it forms an anteroposterior morphogenetic gradient6. How this differential translational regulation is achieved is not fully understood. Here, we address this question using ultrastructural analysis, super-resolution microscopy and live-cell imaging. We show that grk and bcd ribonucleoprotein (RNP) complexes associate with electron-dense bodies that lack ribosomes and contain translational repressors. These properties are characteristic of processing bodies (P bodies), which are considered to be regions of cytoplasm where decisions are made on the translation and degradation of mRNA. Endogenous grk mRNA forms dynamic RNP particles that become docked and translated at the periphery of P bodies, where we show that the translational activator Oo18 RNA-binding protein (Orb, a homologue of CEPB) and the anchoring factor Squid (Sqd) are also enriched. In contrast, an excess of grk mRNA becomes localized inside the P bodies, where endogenous bcd mRNA is localized and translationally repressed. Interestingly, bcd mRNA dissociates from P bodies in embryos following egg activation, when it is known to become translationally active. We propose a general principle of translational regulation during axis specification involving remodelling of transport RNPs and dynamic partitioning of different transcripts between the translationally active edge of P bodies and their silent core.

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Figure 1: Differential association of bcd and grk with P bodies.
Figure 2: Dynamics of grk, bcd and Me31B particles in live oocytes.
Figure 3: Oocyte P bodies exhibit zones differentially enriched in RNA-associated proteins.
Figure 4: RNA translation fate is regulated through association with P bodies (marked with a dashed black line).
Figure 5: RNA association with P bodies in the early embryo.

References

  1. St Johnston, D. Moving messages: the intracellular localization of mRNAs. Nat. Rev. Mol. Cell Biol. 6, 363–375 (2005).

    CAS  Article  Google Scholar 

  2. Weil, T. T., Forrest, K. M. & Gavis, E. R. Localization of bicoid mRNA in late oocytes is maintained by continual active transport. Dev. Cell 11, 251–262 (2006).

    CAS  Article  Google Scholar 

  3. Cha, B. J., Koppetsch, B. S. & Theurkauf, W. E. In vivo analysis of Drosophila bicoid mRNA localization reveals a novel microtubule-dependent axis specification pathway. Cell 106, 35–46 (2001).

    CAS  Article  Google Scholar 

  4. Clark, A., Meignin, C. & Davis, I. A Dynein-dependent shortcut rapidly delivers axis determination transcripts into the Drosophila oocyte. Development 134, 1955–1965 (2007).

    CAS  Article  Google Scholar 

  5. Neuman-Silberberg, F. S. & Schüpbach, T. The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGFa-like protein. Cell 75, 165–174 (1993).

    CAS  Article  Google Scholar 

  6. St Johnston, D., Driever, W., Berleth, T., Richstein, S. & Nüsslein-Volhard, C. Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte. Development 107 (Suppl.), 13–19 (1989).

    CAS  PubMed  Google Scholar 

  7. Delanoue, R., Herpers, B., Soetaert, J., Davis, I. & Rabouille, C. Drosophila Squid/hnRNP helps Dynein switch from a gurken mRNA transport motor to an ultrastructural static anchor in sponge bodies. Dev. Cell 13, 523–538 (2007).

    CAS  Article  Google Scholar 

  8. Wilsch-Brauninger, M., Schwarz, H. & Nusslein-Volhard, C. A sponge-like structure involved in the association and transport of maternal products during Drosophila oogenesis. J. Cell Biol. 139, 817–829 (1997).

    CAS  Article  Google Scholar 

  9. Snee, M. J. & Macdonald, P. M. Dynamic organization and plasticity of sponge bodies. Dev. Dyn. 238, 918–930 (2009).

    CAS  Article  Google Scholar 

  10. Beckham, C. et al. The DEAD-box RNA helicase Ded1p affects and accumulates in Saccharomyces cerevisiae P-bodies. Mol. Biol. Cell 19, 984–993 (2008).

    CAS  Article  Google Scholar 

  11. Minshall, N., Kress, M., Weil, D. & Standart, N. Role of p54 RNA helicase activity and its C-terminal domain in translational repression, P-body localization and assembly. Mol. Biol. Cell 20, 2464–2472 (2009).

    Article  Google Scholar 

  12. Herpers, B. & Rabouille, C. mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum–Golgi units involved in gurken transport in Drosophila oocytes. Mol. Biol. Cell 15, 5306–5317 (2004).

    CAS  Article  Google Scholar 

  13. Nakamura, A., Amikura, R., Hanyu, K. & Kobayashi, S. Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development 128, 3233–3242 (2001).

    CAS  PubMed  Google Scholar 

  14. Dobbie, I. et al. in Live Cell Imaging: A Laboratory Manual Second edn (eds Goldman, Robert D., Swedlow, Jason R. & Spector, David L.) 203–214 (Cold Spring Harbor Laboratory Press, 2010).

    Google Scholar 

  15. Jaramillo, A. M., Weil, T. T., Goodhouse, J., Gavis, E. R. & Schüpbach, T. The dynamics of fluorescently labeled endogenous gurken mRNA in Drosophila. J. Cell Sci. 121, 887–894 (2008).

    CAS  Article  Google Scholar 

  16. Forrest, K. M. & Gavis, E. R. Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr. Biol. 13, 1159–1168 (2003).

    CAS  Article  Google Scholar 

  17. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

    CAS  Article  Google Scholar 

  18. Teixeira, D., Sheth, U., Valencia-Sanchez, M. A., Brengues, M. & Parker, R. Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA 11, 371–382 (2005).

    CAS  Article  Google Scholar 

  19. Chang, J. S., Tan, L., Wolf, M. R. & Schedl, P. Functioning of the Drosophila orb gene in gurken mRNA localization and translation. Development 128, 3169–3177 (2001).

    CAS  PubMed  Google Scholar 

  20. Neuman-Silberberg, F. S. & Schüpbach, T. Dorsoventral axis formation in Drosophila depends on the correct dosage of the gene gurken. Development 120, 2457–2463 (1994).

    CAS  PubMed  Google Scholar 

  21. Wilkie, G. S. & Davis, I. Drosophila wingless and pair-rule transcripts localize apically by dynein-mediated transport of RNA particles. Cell 105, 209–219 (2001).

    CAS  Article  Google Scholar 

  22. MacDougall, N., Clark, A., MacDougall, E. & Davis, I. Drosophila gurken (TGFα) mRNA localizes as particles that move within the oocyte in two dynein-dependent steps. Dev. Cell 4, 307–319 (2003).

    CAS  Article  Google Scholar 

  23. Bokel, C., Dass, S., Wilsch-Brauninger, M. & Roth, S. Drosophila Cornichon acts as cargo receptor for ER export of the TGFα-like growth factor Gurken. Development 133, 459–470 (2006).

    Article  Google Scholar 

  24. Weil, T. T., Parton, R., Davis, I. & Gavis, E. R. Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition. Curr. Biol. 18, 1055–1061 (2008).

    CAS  Article  Google Scholar 

  25. Horner, V. L. et al. The Drosophila calcipressin sarah is required for several aspects of egg activation. Curr. Biol. 16, 1441–1446 (2006).

    CAS  Article  Google Scholar 

  26. Moser, J. J. & Fritzler, M. J. Cytoplasmic ribonucleoprotein (RNP) bodies and their relationship to GW/P bodies. Int. J Biochem. Cell Biol. 42, 828–843 (2010).

    CAS  Article  Google Scholar 

  27. Buchan, J. R. & Parker, R. Eukaryotic stress granules: the ins and outs of translation. Mol. Cell 36, 932–941 (2009).

    CAS  Article  Google Scholar 

  28. King, M. L., Messitt, T. J. & Mowry, K. L. Putting RNAs in the right place at the right time: RNA localization in the frog oocyte. Biol. Cell 97, 19–33 (2005).

    CAS  Article  Google Scholar 

  29. Kloc, M., Bilinski, S. & Etkin, L. D. The Balbiani body and germ cell determinants: 150 years later. Curr. Top Dev. Biol. 59, 1–36 (2004).

    CAS  Article  Google Scholar 

  30. Updike, D. & Strome, S. P granule assembly and function in Caenorhabditis elegans germ cells. J. Androl. 31, 53–60 (2010).

    CAS  Article  Google Scholar 

  31. Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009).

    CAS  Article  Google Scholar 

  32. Eulalio, A., Behm-Ansmant, I., Schweizer, D. & Izaurralde, E. P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol. Cell Biol. 27, 3970–3981 (2007).

    CAS  Article  Google Scholar 

  33. Parker, R. & Sheth, U. P bodies and the control of mRNA translation and degradation. Mol. Cell 25, 635–646 (2007).

    CAS  Article  Google Scholar 

  34. Balagopal, V. & Parker, R. Polysomes, P bodies and stress granules: states and fates of eukaryotic mRNAs. Curr. Opin. Cell Biol. 21, 403–408 (2009).

    CAS  Article  Google Scholar 

  35. Stoecklin, G., Mayo, T. & Anderson, P. ARE-mRNA degradation requires the 5′- 3′ decay pathway. EMBO Rep. 7, 72–77 (2006).

    CAS  Article  Google Scholar 

  36. Buszczak, M. et al. The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics 175, 1505–1531 (2007).

    CAS  Article  Google Scholar 

  37. Theurkauf, W. E. & Hazelrigg, T. I. In vivo analyses of cytoplasmic transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization pathway. Development 125, 3655–3666 (1998).

    CAS  PubMed  Google Scholar 

  38. Norvell, A., Debec, A., Finch, D., Gibson, L. & Thoma, B. Squid is required for efficient posterior localization of oskar mRNA during Drosophila oogenesis. Dev. Genes Evol. 215, 340–349 (2005).

    CAS  Article  Google Scholar 

  39. Herpers, B., Xanthakis, D. & Rabouille, C. ISH–IEM: a sensitive method to detect endogenous mRNAs at the ultrastructural level. Nat. Protoc. 5, 678–687 (2010).

    CAS  Article  Google Scholar 

  40. Slot, J. W. & Geuze, H. J. Cryosectioning and immunolabeling. Nat. Protoc. 2, 2480–2491 (2007).

    CAS  Article  Google Scholar 

  41. Rabouille, C. Quantitative aspects of immunogold labeling in embedded and nonembedded sections. Methods Mol Biol. 117, 125–144 (1999).

    CAS  PubMed  Google Scholar 

  42. Filardo, P. & Ephrussi, A. Bruno regulates gurken during Drosophila oogenesis. Mech. Dev. 120, 289–297 (2003).

    CAS  Article  Google Scholar 

  43. Hammond, L. E., Rudner, D. Z., Kanaar, R. & Rio, D. C. Mutations in the hrp48 gene, which encodes a Drosophila heterogeneous nuclear ribonucleoprotein particle protein, cause lethality and developmental defects and affect P-element third-intron splicing in vivo. Mol. Cell Biol. 17, 7260–7267 (1997).

    CAS  Article  Google Scholar 

  44. Rehwinkel, J., Behm-Ansmant, I., Gatfield, D. & Izaurralde, E. A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing. RNA 11, 1640–1647 (2005).

    CAS  Article  Google Scholar 

  45. Court, F. A., Hendriks, W. T., MacGillavry, H. D., Alvarez, J. & van Minnen, J. Schwann cell to axon transfer of ribosomes: toward a novel understanding of the role of glia in the nervous system. J. Neurosci. 28, 11024–11029 (2008).

    CAS  Article  Google Scholar 

  46. Parton, R. M., Valles, A. M., Dobbie, I. M. & Davis, I. Live cell imaging in Drosophila melanogaster. Cold Spring Harb. Protoc. 2010, 387–418 (2010).

    Google Scholar 

  47. Weil, T. T., Parton, R. M. & Davis, I. Preparing individual Drosophila egg chambers for live imaging. J. Vis. Exp.http://dx.doi.org/10.3791/3679 (2012).

  48. Gross, D. J. & Webb, W. W. Spectroscopic Membrane Probe, Vol. II. 19–48 (CRC Press Inc., 1988).

    Google Scholar 

  49. Saxton, M. J. & Jacobson, K. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).

    CAS  Article  Google Scholar 

  50. Tadakuma, H., Ishihama, Y., Shibuya, T., Tani, T. & Funatsu, T. Imaging of single mRNA molecules moving within a living cell nucleus. Biochem. Biophys. Res. Commun. 344, 772–779 (2006).

    CAS  Article  Google Scholar 

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Acknowledgements

We are grateful to: E. R. Gavis (Princeton University, USA), D. Ish-Horowicz (CRUK, UK), T. Schüpbach (Princeton University, USA), A. Jaramillo (Princeton University, USA), J. van Minnen (University of Calgary, Canada), L. Cooley (Yale University, USA), R. Singer (Albert Einstein College of Medicine, USA) and A. Nakamura (RIKEN, Japan) for fly stocks, antibodies and reagents; all members of the Cell Microscopy Center in Utrecht, Netherlands for assistance with electron microscopy; J. W. Sedat (UCSF, USA) and D. Agard (UCSF, USA) for advise on super-resolution imaging; E. R. Gavis for experimental advice; A. Ephrussi (EMBL, Germany) for discussions of the data. This work was supported by: Marie Curie International Incoming Fellowship (ROXA0) to T.T.W.; Wellcome Trust Senior Research Fellowship (081858) to I.D. and supporting R.M.P. and T.T.W.; Cancer Research UK funding to R.H.; studentship from Wellcome Trust to J.M.H.; Darwin Trust to J.S.

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T.T.W., R.M.P., C.R. and I.D. provided the intellectual basis of the work and designed the experiments. T.T.W., R.M.P., B.H., D.X. and T.V. performed experiments resulting in figures. J.S., R.H. and J.M.H. preformed experiments that contributed intellectually but did not result in figures. R.H. generated reagents. I.M.D. and R.M.P. provided technical expertise for equipment, advanced microscopy and data analysis. T.T.W., R.M.P., C.R. and I.D. wrote and edited the manuscript.

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Correspondence to Catherine Rabouille or Ilan Davis.

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Weil, T., Parton, R., Herpers, B. et al. Drosophila patterning is established by differential association of mRNAs with P bodies. Nat Cell Biol 14, 1305–1313 (2012). https://doi.org/10.1038/ncb2627

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