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Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution

Abstract

Quantitative information on growing organs is required to better understand morphogenesis in both plants and animals. However, detailed analyses of growth patterns at cellular resolution have remained elusive. We developed an approach, multiangle image acquisition, three-dimensional reconstruction and cell segmentation–automated lineage tracking (MARS-ALT), in which we imaged whole organs from multiple angles, computationally merged and segmented these images to provide accurate cell identification in three dimensions and automatically tracked cell lineages through multiple rounds of cell division during development. Using these methods, we quantitatively analyzed Arabidopsis thaliana flower development at cell resolution, which revealed differential growth patterns of key regions during early stages of floral morphogenesis. Lastly, using rice roots, we demonstrated that this approach is both generic and scalable.

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Figure 1: MARS.
Figure 2: ALT.
Figure 3: Validation of MARS-ALT results.
Figure 4: Application of MARS-ALT to flower development.

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References

  1. Coen, E., Rolland-Lagan, A.G., Matthews, M., Bangham, J.A. & Prusinkiewicz, P. The genetics of geometry. Proc. Natl. Acad. Sci. USA 101, 4728–4735 (2004).

    Article  CAS  Google Scholar 

  2. Smith, R. et al. A plausible model of phyllotaxis. Proc. Natl. Acad. Sci. USA 103, 1301–1306 (2006).

    Article  CAS  Google Scholar 

  3. Jonsson, H., Heisler, M.G., Shapiro, B.E., Meyerowitz, E.M. & Mjolsness, E. An auxin-driven polarized transport model for phyllotaxis. Proc. Natl. Acad. Sci. USA 103, 1633–1638 (2006).

    Article  Google Scholar 

  4. Jonsson, H. et al. Modeling the organization of the WUSCHEL expression domain in the shoot apical meristem. Bioinformatics 21 (Suppl. 1), i232–i240 (2005).

    Article  Google Scholar 

  5. Barbier de Reuille, P. et al. Computer simulations reveal properties of the cell–cell signaling network at the shoot apex in Arabidopsis. Proc. Natl. Acad. Sci. USA 103, 1627–1632 (2006).

    Article  CAS  Google Scholar 

  6. Hamant, O. et al. Developmental patterning by mechanical signals in Arabidopsis. Science 322, 1650–1655 (2008).

    Article  CAS  Google Scholar 

  7. Bao, Z. et al. Automated cell lineage tracing in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 103, 2707–2712 (2006).

    Article  CAS  Google Scholar 

  8. Verveer, P.J. et al. High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nat. Methods 4, 311–313 (2007).

    Article  CAS  Google Scholar 

  9. Jaqaman, K. et al. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695–702 (2008).

    Article  CAS  Google Scholar 

  10. Murray, J.I. et al. Automated analysis of embryonic gene expression with cellular resolution in C. elegans. Nat. Methods 5, 703–709 (2008).

    Article  CAS  Google Scholar 

  11. Reddy, G.V., Heisler, M.G., Ehrhardt, D.W. & Meyerowitz, E.M. Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana. Development 131, 4225–4237 (2004).

    Article  CAS  Google Scholar 

  12. Verdeil, J., Alemanno, L., Niemenak, N. & Tranbarger, T. Pluripotent versus totipotent plant stem cells: dependence versus autonomy? Trends Plant Sci. 12, 245–252 (2007).

    Article  CAS  Google Scholar 

  13. Boot, M.J. et al. In vitro whole-organ imaging: 4D quantification of growing mouse limb buds. Nat. Methods 5, 609–612 (2008).

    Article  CAS  Google Scholar 

  14. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    Article  CAS  Google Scholar 

  15. Long, F., Peng, H., Liu, X., Kim, S.K. & Myers, E. A 3D digital atlas of C. elegans and its application to single-cell analyses. Nat. Methods 6, 667–672 (2009).

    Article  CAS  Google Scholar 

  16. Moreno, N., Bougourd, S., Haseloff, J. & Feijo, J. in Handbook of Biological Confocal Microscopy, 3rd edn. (ed., J.B. Pawley) (Springer Science+Business Media, New York, 2006).

  17. Kwiatkowska, D. Surface growth at the reproductive shoot apex of Arabidopsis thaliana pin-formed 1 and wild type. J. Exp. Bot. 55, 1021–1032 (2004).

    Article  CAS  Google Scholar 

  18. Grandjean, O. et al. In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis. Plant Cell 16, 74–87 (2004).

    Article  CAS  Google Scholar 

  19. Barbier de Reuille, P., Bohn-Courseau, I., Godin, C. & Traas, J. A protocol to analyse cellular dynamics during plant development. Plant J. 44, 1045–1053 (2005).

    Article  CAS  Google Scholar 

  20. Ourselin, S., Roche, A., Prima, S. & Ayache, N. Block matching: a general framework to improve robustness of rigid registration of medical images. in Third International Conference on Medical Robotics, Imaging And Computer Assisted Surgery (eds., DiGioia, A.M. and Delp, S.) 557–566 (Springer, 2000).

  21. Commowick, O. & Malandain, G. From statistical atlases to personalized models. in Proceedings of the SA2PM Workshop (2006).

  22. Soille, P. Morphological Image Analysis: Principles and Applications 89–125 (Springer-Verlag, Berlin, 1999).

  23. Rebouillat, J. et al. Molecular genetics of rice root development. Rice 2, 15–34 (2009).

    Article  Google Scholar 

  24. Steeves, S. & Sussex, I.M. Patterns in Plant Development (Cambridge University Press, 1989).

  25. Preibisch, S., Saalfeld, S., Rohlfing, T. & Tomancak, P. Bead-based mosaicing of single plane illumination microscopy images using geometric local descriptor matching. Proc. SPIE 72592S, 1–10 (2009).

    Google Scholar 

  26. Das, P. et al. Floral stem cell termination involves the direct regulation of AGAMOUS by PERIANTHIA. Development 136, 1605–1611 (2009).

    Article  CAS  Google Scholar 

  27. Edmonds, J. & Karp, R.M. Theoretical improvements in algorithmic efficiency for network flow problems. J. Association Computing Machinery 19, 248–264 (1972).

    Article  Google Scholar 

  28. Tarjan, R. Data structures and Network Algorithms. (Society for Industrial and Applied Mathematics, 1983).

    Book  Google Scholar 

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Acknowledgements

We thank A. Berger for help with establishing plant growth and imaging protocols; A. Lacroix for help with plant growth; and C. Lionnet for help with imaging. This work was funded by grants from the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (to J.-L.V.), the Region Languedoc-Roussillon (to R.F.) and from the Institut National de la Recherche en Informatique et en Automatique (to C.G.). P.D. was funded by a European Union Marie Curie Incoming International Fellowship grant (IIF-022002). The work of J.T. and C.G. was also funded by grants from the Agence Nationale de Recherche (Virtual Carpel, GeneShape and Flower model) and the European Union (Morphex).

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Authors and Affiliations

Authors

Contributions

P.D., J.T., J.-L.V., G.M. and C.G. conceived the experiments; P.D. carried out the Arabidopsis experiments; J.-L.V. carried out the rice experiments; R.F., G.M. and C.G. conceived the software pipeline; R.F. wrote the software; R.F., E.M. and V.M. carried out the software experiments; E.M. and R.F. wrote the MARS-ALT documentation; P.D. and V.M. analyzed the output; P.D., G.M. and C.G. wrote the paper with input from the other authors.

Corresponding author

Correspondence to Christophe Godin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Table 1 and Supplementary Notes 1–7 (PDF 2916 kb)

Supplementary Video 1

Four-dimensional development of a flower from stage 0 to stage 3. The video shows the flower-A time course tracked over 70 h. Cells colored in red are those that are going to divide before the next time point. (MPG 4764 kb)

Supplementary Video 2

Backward tracing of sepal cells from early stage 3 to stage 0 (MPG 4354 kb)

Supplementary Video 3

Cut-away rendering of a rice root meristem (MPG 2542 kb)

Supplementary Software

MARS-ALT pipeline and documentation (ZIP 168685 kb)

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Fernandez, R., Das, P., Mirabet, V. et al. Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution. Nat Methods 7, 547–553 (2010). https://doi.org/10.1038/nmeth.1472

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