Letter | Published:

Luminal signalling links cell communication to tissue architecture during organogenesis

Nature volume 515, pages 120124 (06 November 2014) | Download Citation

Abstract

Morphogenesis is the process whereby cell collectives are shaped into differentiated tissues and organs1. The self-organizing nature of morphogenesis has been recently demonstrated by studies showing that stem cells in three-dimensional culture can generate complex organoids, such as mini-guts2, optic-cups3 and even mini-brains4. To achieve this, cell collectives must regulate the activity of secreted signalling molecules that control cell differentiation, presumably through the self-assembly of microenvironments or niches. However, mechanisms that allow changes in tissue architecture to feedback directly on the activity of extracellular signals have not been described. Here we investigate how the process of tissue assembly controls signalling activity during organogenesis in vivo, using the migrating zebrafish lateral line primordium5. We show that fibroblast growth factor (FGF) activity within the tissue controls the frequency at which it deposits rosette-like mechanosensory organs. Live imaging reveals that FGF becomes specifically concentrated in microluminal structures that assemble at the centre of these organs and spatially constrain its signalling activity. Genetic inhibition of microlumen assembly and laser micropuncture experiments demonstrate that microlumina increase signalling responses in participating cells, thus allowing FGF to coordinate the migratory behaviour of cell groups at the tissue rear. As the formation of a central lumen is a self-organizing property of many cell types, such as epithelia6 and embryonic stem cells7, luminal signalling provides a potentially general mechanism to locally restrict, coordinate and enhance cell communication within tissues.

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Acknowledgements

We are grateful to J. Ellenberg, S. de Renzis and F. Peri for suggestions and comments on the manuscript, A. Aulehla for advice about timing, and E. Karsenti and the Gilmour laboratory for discussion. We thank M. Brand for advice about FGF tagging, the EMBL Advanced Light Microscopy Facility, in particular Y. Belyaev, for imaging assistance, the European Molecular Biology Laboratory (EMBL) Monoclonal Antibody (MACF) and Protein Expression Facilities for Fgfr1a antibody, K. Miura from the EMBL Centre for Cell and Molecular Imaging for advice with data analysis, E. Dona and T. Gregor for advice with the smFISH protocol, and A. Gruia for fish care. We acknowledge funding from the European Molecular Biology Organization and EMBL Interdisciplinary Postdocs (EIPOD) (to C.R.) and the Deutsche Forschungsgemeinschaft SFB 488 (to D.G.).

Author information

Author notes

    • Celine Revenu

    Present address: Institut Curie, 26 rue d’Ulm, 75248 Paris, France.

Affiliations

  1. European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany

    • Sevi Durdu
    • , Murat Iskar
    • , Celine Revenu
    • , Nicole Schieber
    • , Andreas Kunze
    • , Peer Bork
    • , Yannick Schwab
    •  & Darren Gilmour

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Contributions

D.G. and S.D. designed the study. S.D. performed all experiments, with the exception of CLEM experiments performed with N.S. and Y.S., and antibody-based analysis of the microlumen performed by C.R. S.D. and M.I. developed the data analysis methods with input from P.B. A.K. developed the LexPR inducible gene expression system and C.R. generated the Cxcr4b–RFP line. D.G. and S.D. interpreted the data and wrote the paper with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Darren Gilmour.

Extended data

Supplementary information

Videos

  1. 1.

    Parameters affecting lateral line organ spacing

    Embryo growth, primordium migration velocity and organ deposition timing are the potential parameters to underlie organ spacing. Time-lapse movie of cldnb:lynGFP embryo (bright field lower panel and fluorescence imaging upper panel). Corresponds to Extended Data Fig. 1e. Scale bar = 200 μm. Time interval = 20 min.

  2. 2.

    Tracking organs with peak detection

    Organs were tracked automatically using signal intensity profiles of cell membranes based on a peak detection method. Upper panel is time-lapse movie of cldnb:lynGFP embryo and lower panel is maximum intensity profile along the thickness of the upper panel image over time. Relates to Method "analysis of migration and organ patterning". Scale bar = 100 μm. Time interval = 10 min.

  3. 3.

    Decreasing FGF signaling with FGFR1 inhibitor increases organ deposition time in a dose-dependent way

    Time-lapse movies of FGF inhibitor treated embryos (DMSO control, 0.5 μM and 1 μM SU5402 treatments). Corresponds to Fig 1d. Scale bar 100 μm. Time interval = 10 min.

  4. 4.

    Decreased FGF signaling in FGFR1a mutant increases organ deposition time

    Time-lapse movies of fgfr1at3R705H mutant embryos. Corresponds to Extended Data Fig. 2. Scale bar = 100μm. Time interval = 10 min.

  5. 5.

    Increasing FGF signaling decreases deposition time in a dose-dependent way

    Time-lapse movies of FGF inducer treated embryos. (10 μM RU486 treated no target gene carrier control, 5, 10, 20 μM RU486 treated Fgf3-GFP transactivation treatments) Corresponds to Fig1e. . Scale bar = 100 μm. Time interval = 10 min.

  6. 6.

    Appearance of secGFP and Fgf3-GFP accumulation in apical spheres correlates with organ deposition

    Time-lapse movies of secGFP and Fgf3-GFP expressing embryos with red nuclei counter-label. Panel 1 and 4 corresponds to Figure 2b. Second movie panel shows luminal opening of organ 1, corresponds to Extended Data Fig.10b. Scale bar 100 μm. Time interval = 25 min.

  7. 7.

    3D CLEM with secGFP filing the microlumen structure

    3D stacks of EM images of an organ center, secGFP overlay with EM stacks, luminal space and kinocilia segmentation and overlay of segmented microlumina with secGFP signal are shown. Relates to Method "correlative light-electron microscopy". Scale bar indicated in the movie.

  8. 8.

    Organ patterning response to ectopic FGF3 source clones

    Time-lapse movie showing that ectopic Fgf3-GFP3 expressing clones (bottom, in green) accelerate the deposition of individual organs. (membrane counter-label in red). Corresponds to Extended Data Fig. 7a. Scale bar = 100 μm. Time interval = 30 min.

  9. 9.

    3D segmentation for smFISH data analysis

    Volume masking of lateral line tissue and spot segmentation of nuclei and smFISH signal (membranes in green, nuclei in blue and pea3 transcripts in grey). Relates to Method "single molecule fluorescent in situ hybridization". Scale bar = 10 μm.

  10. 10.

    Lumen micropuncture on Fgf3-GFP pool

    Time-lapse movie shows Fgf3-GFP leakage upon micropuncture (Fgf3-GFP green, lynRFP red). Arrow indicates image acquired right after the laser pulse. Corresponds to Figure 4c. Scale bar = 5 μm. Time interval = 0.5 sec.

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DOI

https://doi.org/10.1038/nature13852

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