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Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning

Abstract

The ability of signalling proteins to traverse tissues containing tightly packed cells is of fundamental importance for cell specification and tissue development; however, how this is achieved at a cellular level remains poorly understood1. For more than a century, the vertebrate limb bud has served as a model for studying cell signalling during embryonic development2. Here we optimize single-cell real-time imaging to delineate the cellular mechanisms for how signalling proteins, such as sonic hedgehog (SHH), that possess membrane-bound covalent lipid modifications traverse long distances within the vertebrate limb bud in vivo. By directly imaging SHH ligand production under native regulatory control in chick (Gallus gallus) embryos, our findings show that SHH is unexpectedly produced in the form of a particle that remains associated with the cell via long cytoplasmic extensions that span several cell diameters. We show that these cellular extensions are a specialized class of actin-based filopodia with novel cytoskeletal features that have not been previously described. Notably, particles containing SHH travel along these extensions with a net anterograde movement within the field of SHH cell signalling. We further show that in SHH-responding cells, specific subsets of SHH co-receptors, including cell adhesion molecule downregulated by oncogenes (CDO) and brother of CDO (BOC), actively distribute and co-localize in specific micro-domains within filopodial extensions, far from the cell body. Stabilized interactions are formed between filopodia containing SHH ligand and those containing co-receptors over a long range. These results suggest that contact-mediated release propagated by specialized filopodia contributes to the delivery of SHH at a distance. Together, these studies identify an important mode of communication between cells that considerably extends our understanding of ligand movement and reception during vertebrate tissue patterning.

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Figure 1: Mesenchymal cells of the developing limb bud possess long and highly dynamic cytoplasmic extensions.
Figure 2: Limb mesenchymal cytoplasmic extensions are a class of specialized actin-based filopodia.
Figure 3: Live-cell imaging of SHH ligand production and transport within the limb bud.
Figure 4: Filopodia on SHH-responding cells display an exquisite distribution and co-localization of SHH co-receptors that interact with SHH-producing filopodia.

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Acknowledgements

We thank D. Mullins for discussion on the actin cytoskeleton, as well as G. Martin and members of the Barna laboratory for discussion and critical reading of the manuscript. We thank K. Cabaltera for technical assistance. This work was supported by Spanish Ministry of Education and Science (E.L.), Program for Breakthrough Biomedical Research, UCSF (M.B.), the March of Dimes Basil O’Connor Scholar Research Award (M.B.), and the National Institute of Arthritis and Musculoskeletal and Skin Disease, part of NIH, under award number NIH R21AR062262 (M.B.).

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Authors and Affiliations

Authors

Contributions

M.B. conceived and supervised the project; T.A.S., E.L. and M.B. designed experiments; T.A.S. and E.L. performed experiments. All authors analysed the data, critically discussed the results, and contributed towards the writing and preparation of the manuscript.

Corresponding author

Correspondence to Maria Barna.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-12, Supplementary Methods and Supplementary References. (PDF 20857 kb)

Live in vivo imaging of mosaic labeling of mesenchymal cells from a stage HH21 chick limb bud

pmEGFP and pmKate2 fluorescent proteins label the plasma membrane. There are numerous highly dynamic processes across the imaging field. Individual green and red fluorescence channels are shown to illustrate the the cytoplasmic extensions. Four acquired frames per minute. Scale = 10μm. Time in hr:min:sec. Refer to Fig. 1c-g. (MOV 10238 kb)

Cropped detail from Video S1, demonstrating the cytoplasmic extension growth

Four acquired frames per minute. Scale = 5μm. Time in hr:min:sec. Refer to Fig. 1H. (MOV 3087 kb)

Live in vivo imaging of a mosaic pmEGFP labeled population of a stage HH21 chick limb mesenchymal cells demonstrating interaction among adjacent cells

Two cytoplasmic extensions grow, contact and subsequently retract. Four acquired frames per minute. Scale = 5 μm. Time in hr:min:sec. Refer to Fig. 1i. (MOV 2275 kb)

Cytoplasmic extensions of labeled limb mesenchymal cells interact and make stabilized contacts

Live in vivo imaging of mosaic labeling of mesenchymal cells of a stage HH21 chick limb bud where pmEGFP and pmKate2 label the plasma membrane of two different cells that establish an interaction. Two acquired frames per minute. Scale = 3 μm. Time in hr:min:sec. Refer to Fig. 1j. and Supplementary Video 3. (MOV 1265 kb)

Cytoplasmic extensions of limb mesenchymal cells that make stabilized contacts for over 30 minutes

Live in vivo imaging of mesenchymal cells of the limb bud. The pmEGFP mosaic labeling shows a long-lasting interaction between two cells. Four acquired frames per minute. Scale = 5 μm. Time in hr:min:sec. (MOV 8656 kb)

Cofilin-EGFP labeling reveals a rapid accumulation to the tips of filopodia. Cofilin-EGFP decorates the pmKate2 labeled filopodium in distinct patches with negative territories

During retraction of a filopodium live imaging shows movement of a domain of Cofilin-EGFP back to the cell soma independently from the pmKate2 membrane label. Two acquired frames per minute. Scale = 3μm. Time in hr:min:sec. Refer to Fig. 2d. (MOV 1175 kb)

A panning Z-series through confocal live imaging of ShhCreERT2/+; mT/mG/+ E10.5 mouse limb bud

From dorsal to ventral, demonstrating several mesenchymal cells with multiple long filopodia extending from the cell body. Scale = 10 μm. Refer to Supplementary Fig 1a. (MOV 2226 kb)

Shh is produced as a particle in the ZPA that moves along filopodia

Time lapse acquisition of stage HH21 chick limb ZPA cells, ShhN-GFP showing net anterograde particle movement toward the filopodia tip distal to the cell body. Two acquired frames per minute. Scale = 3μm. Time in hr:min:sec. Refer to Fig. 2c. (MOV 1565 kb)

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Sanders, T., Llagostera, E. & Barna, M. Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature 497, 628–632 (2013). https://doi.org/10.1038/nature12157

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