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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References
Zhu, A. J. & Scott, M. P. Incredible journey: how do developmental signals travel through tissue? Genes Dev. 18, 2985–2997 (2004)
Niswander, L. Pattern formation: old models out on a limb. Nature Rev. Genet. 4, 133–143 (2003)
Hsiung, F., Ramirez-Weber, F.-A., Iwaki, D. D. & Kornberg, T. B. Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic. Nature 437, 560–563 (2005)
RamÃrez-Weber, F. A. & Kornberg, T. B. Cytonemes: cellular processes that project to the principal signaling center in Drosophila imaginal discs. Cell 97, 599–607 (1999)
Roy, S., Hsiung, F. & Kornberg, T. B. Specificity of Drosophila cytonemes for distinct signaling pathways. Science 332, 354–358 (2011)
Riddle, R. D., Johnson, R. L., Laufer, E. & Tabin, C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401–1416 (1993)
Yang, Y. et al. Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb. Development 124, 4393–4404 (1997)
Yusa, K., Rad, R., Takeda, J. & Bradley, A. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nature Methods 6, 363–369 (2009)
Kerber, M. L. & Cheney, R. E. Myosin-X: a MyTH-FERM myosin at the tips of filopodia. J. Cell Sci. 124, 3733–3741 (2011)
Mogilner, A. & Rubinstein, B. The physics of filopodial protrusion. Biophys. J. 89, 782–795 (2005)
Munsie, L. N., Caron, N., Desmond, C. R. & Truant, R. Lifeact cannot visualize some forms of stress-induced twisted f-actin. Nature Methods 6, 317 (2009)
Breitsprecher, D. et al. Cofilin cooperates with fascin to disassemble filopodial actin filaments. J. Cell Sci. 124, 3305–3318 (2011)
Niswander, L., Jeffrey, S., Martin, G. R. & Tickle, C. A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371, 609–612 (1994)
Maas, S. A., Suzuki, T. & Fallon, J. F. Identification of spontaneous mutations within the long-range limb-specific Sonic hedgehog enhancer (ZRS) that alter Sonic hedgehog expression in the chicken limb mutants oligozeugodactyly and silkie breed. Dev. Dyn. 240, 1212–1222 (2011)
Ingham, P. W. Hedgehog signaling: a tale of two lipids. Science 294, 1879–1881 (2001)
Li, Y., Zhang, H., Litingtung, Y. & Chiang, C. Cholesterol modification restricts the spread of SHH gradient in the limb bud. Proc. Natl Acad. Sci. USA 103, 6548–6553 (2006)
Berg, J. S. & Cheney, R. E. Myosin-X is an unconventional myosin that undergoes intrafilopodial motility. Nature Cell Biol. 4, 246–250 (2002)
Lewis, P. M. et al. Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. Cell 105, 599–612 (2001)
Cabantous, S., Terwilliger, T. C. & Waldo, G. S. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotechnol. 23, 102–107 (2005)
Kaddoum, L., Magdeleine, E., Waldo, G. S., Joly, E. & Cabantous, S. One-step split GFP staining for sensitive protein detection and localization in mammalian cells. Biotechniques 49, 727–736 (2010)
Marigo, V., Davey, R. A., Zuo, Y., Cunningham, J. M. & Tabin, C. J. Biochemical evidence that patched is the Hedgehog receptor. Nature 384, 176–179 (1996)
Allen, B. L. et al. Overlapping roles and collective requirement for the coreceptors GAS1, CDO, and BOC in SHH pathway function. Dev. Cell 20, 775–787 (2011)
Kavran, J. M., Ward, M. D., Oladosu, O. O., Mulepati, S. & Leahy, D. J. All mammalian Hedgehog proteins interact with cell adhesion molecule, down-regulated by oncogenes (CDO) and brother of CDO (BOC) in a conserved manner. J. Biol. Chem. 285, 24584–24590 (2010)
Tenzen, T. et al. The cell surface membrane proteins Cdo and Boc are components and targets of the Hedgehog signaling pathway and feedback network in mice. Dev. Cell 10, 647–656 (2006)
Yao, S., Lum, L. & Beachy, P. The Ihog cell-surface proteins bind Hedgehog and mediate pathway activation. Cell 125, 343–357 (2006)
Miller, J., Fraser, S. E. & McClay, D. Dynamics of thin filopodia during sea urchin gastrulation. Development 121, 2501–2511 (1995)
Boehm, B. et al. The role of spatially controlled cell proliferation in limb bud morphogenesis. PLoS Biol. 8, e1000420 (2010)
Kelley, R. O. & Fallon, J. F. Identification and distribution of gap junctions in the mesoderm of the developing chick limb bud. J. Embryol. Exp. Morphol. 46, 99–110 (1978)
Li, X. et al. piggyBac internal sequences are necessary for efficient transformation of target genomes. Insect Mol. Biol. 14, 17–30 (2005)
Yusa, K., Zhou, L., Li, M. A., Bradley, A. & Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. Proc. Natl Acad. Sci. USA 108, 1531–1536 (2011)
Matsuda, T. & Cepko, C. L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl Acad. Sci. USA 101, 16–22 (2004)
Harvey, C. D., Yasuda, R., Zhong, H. & Svoboda, K. The spread of Ras activity triggered by activation of a single dendritic spine. Science 321, 136–140 (2008)
Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnol. 24, 79–88 (2005)
Filonov, G. S. et al. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nature Biotechnol. 29, 757–761 (2011)
Hesselson, D., Anderson, R. M., Beinat, M. & Stainier, D. Y. R. Distinct populations of quiescent and proliferative pancreatic beta-cells identified by HOTcre mediated labeling. Proc. Natl Acad. Sci. USA 106, 14896–14901 (2009)
Riedl, J. et al. Lifeact mice for studying F-actin dynamics. Nature Methods 7, 168–169 (2010)
Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nature Methods 5, 605–607 (2008)
Bohil, A. B., Robertson, B. W. & Cheney, R. E. Myosin-X is a molecular motor that functions in filopodia formation. Proc. Natl Acad. Sci. USA 103, 12411–12416 (2006)
Hao, J.-J. et al. Phospholipase C-mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane. J. Cell Biol. 184, 451–462 (2009)
Callejo, A., Quijada, L. & Guerrero, I. Detecting tagged Hedgehog with intracellular and extracellular immunocytochemistry for functional analysis. Methods Mol. Biol. 397, 91–103 (2007)
Vincent, S., Thomas, A., Brasher, B. & Benson, J. D. Targeting of proteins to membranes through hedgehog auto-processing. Nature Biotechnol. 21, 936–940 (2003)
Vyas, N. et al. Nanoscale organization of hedgehog is essential for long-range signaling. Cell 133, 1214–1227 (2008)
Chamberlain, C. E., Jeong, J., Guo, C., Allen, B. L. & McMahon, A. P. Notochord-derived SHH concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning. Development 135, 1097–1106 (2008)
Okada, A. et al. Boc is a receptor for sonic hedgehog in the guidance of commissural axons. Nature 444, 369–373 (2006)
Pinaud, F. & Dahan, M. Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins. Proc. Natl Acad. Sci. USA 108, E201–E210 (2011)
Barna, M. & Niswander, L. Visualization of cartilage formation: insight into cellular properties of skeletal progenitors and chondrodysplasia syndromes. Dev. Cell 12, 931–941 (2007)
Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009)
Visel, A., Minovitsky, S., Dubchak, I. & Pennacchio, L. A. VISTA Enhancer Browser—a database of tissue-specific human enhancers. Nucleic Acids Res. 35, D88–D92 (2007)
Cao, D. et al. The expression of Gli3, regulated by HOXD13, may play a role in idiopathic congenital talipes equinovarus. BMC Musculoskelet. Disord. 10, 142 (2009)
Hamburger, V. A series of normal stages in the development of the chick embryo. J. Morphol. 195, 231–272 (1951)
Krull, C. E. A primer on using in ovo electroporation to analyze gene function. Dev. Dyn. 229, 433–439 (2004)
Auerbach, R., Kubai, L., Knighton, D. & Folkman, J. A simple procedure for the long-term cultivation of chicken embryos. Dev. Biol. 41, 391–394 (1974)
Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007)
Harfe, B. D. et al. Evidence for an expansion-based temporal SHH gradient in specifying vertebrate digit identities. Cell 118, 517–528 (2004)
Ahn, S. & Joyner, A. L. Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning. Cell 118, 505–516 (2004)
Chen, L. et al. Cdc42 deficiency causes Sonic hedgehog-independent holoprosencephaly. Proc. Natl Acad. Sci. USA 103, 16520–16525 (2006)
Nobes, C. D. & Hall, A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81, 53–62 (1995)
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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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.
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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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DOI: https://doi.org/10.1038/nature12157
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