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. 2013 May 30;497(7451):628-32.
doi: 10.1038/nature12157. Epub 2013 Apr 28.

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

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

Timothy A Sanders et al. Nature. .

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 understood. For more than a century, the vertebrate limb bud has served as a model for studying cell signalling during embryonic development. 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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Figures

Figure 1
Figure 1
Mesenchymal cells of the developing limb bud possess long and highly dynamic cytoplasmic extensions. (a) Left: HH14 chick embryo indicating the site of DNA injection. The red dashed line indicates the cross-sectional plane, Right: Microinjected DNA in the coelom is shown in green, electrodes position is indicated (LPM)(NC: Notochord; NT: Neural Tube). (b) Diagram of piggyBac transposon system resulting in integration of transposon inverted terminal repeat (ITR) flanked expression cassettes by piggyBac transposase. Cre recombinase flanked by loxP sites (black triangles) results in mosaic labeling from a loxP-containing reporter construct, containing membrane palmitoylated mKate2 or mEGFP expressed via the tetracycline responsive element (TRE). Ubiquitous promoter (CAGP), doxycycline (DOX) inducible transactivator protein (3G). (c) Confocal z-series acquired in vivo from a HH21 limb bud reveals an intricate network of cellular extensions (Supplementary Movie 1). Scale = 10µm. (d) Single x-y plane, from c, highlighting the network of long cytoplasmic extensions among mesenchymal cells. (e) A representative long extension, from c, 75 µm marked by line. Scale = 10µm. (f) Example of an interaction between two cytoplasmic extensions. Interaction starts at tips (f1, asterisk) and then extends until both extensions overlap (f2, asterisks) (see also Supplementary Movie 4). Scale = 3µm. Time in min:sec. (g) Frequency distribution of extending (black) and retracting (grey) velocities, n = 8. (h) Extension dynamics. Gray bars represent net length change in µm. Red line represents the mean velocity, nm/sec. X-axis ticks = 1 minute intervals.
Figure 2
Figure 2
Limb mesenchymal cytoplasmic extensions are a class of specialized actin-based filopodia. (a1–3) UCHD-EGFP demonstrating that membrane labeled pmKate2 filopodia extensions contain actin filaments. Scale = 3µm. (b1–3) Myosin X-EGFP is localized to each pmKate2 labeled filopodium and is concentrated at the distal tip. (c1–3) LifeAct-Kate2 marks only the proximal aspect of pmEGFP labeled filopodia and does not label the entire extension, shown by bracket. (d1–3) Cofilin-EGFP is present in interrupted domains along the filopodia, negative regions shown with brackets. Scale = 5µm.
Figure 3
Figure 3
Live cell imaging of Shh ligand production and trafficking within the limb bud. (a) Schematic of piggyBac-mediated integration of transposon flanked expression cassettes (ITR). The G. gallus Shh minimal promoter (ShhP) and ZRS element direct spatial expression in the limb ZPA of doxycycline (DOX) inducible transactivator protein (3G) which in turn allows for the temporal control of ShhN-EGFP or ShhP-EGFP and pmKate2. (b1) A representative Shh producing cell harboring multiple long filopodia with ShhP-EGFP present in discrete particles as well as in a more diffuse form localized along these extensions. (b2) ShhN-EGFP is produced as a particle visualized within the cell soma (arrows) as well as along the filopodia. (c1–4) A representative timelapse showing anterograde ShhN-EGFP particle movement (indicated by arrow) that accumulates at the tip of the certain filopodia but not others (arrows). (Supplementary Movie 8). Time in min:sec, interval 4 frames/sec. (d) Graph demonstrating the movement of ShhN-EGFP particles relative to the filopodium, normalized distance to filopodia base = 0 and filopodia tip = 1. (e) Graph demonstrating the net vectors of particle (n=38) displacement represented as a percentage of the total filopodia length that particles transverse, anterograde (green), no displacement (blue, < 5%) and retrograde displacement (red). The relative thickness of each vector reflects the percentage of particles within each category.. There is a statistically significant, net anterograde movement of Shh particles away from the cell soma, p < 0.002. (f) Shh-harboring filopodia (red line) are statistically more stabilized than filopodia without Shh (black line), p < 0.001, n=200 timepoints. Scale = 3µm.
Figure 4
Figure 4
Filopodia on Shh responding cells display an exquisite distribution and co-localization of Shh co-receptors that interact with Shh producing filopodia. (a1) Live imaging of Cdo-GFP and (b1) Boc-GFP expression in defined microdomains along the filopodial membrane, within subsets of filopodia but not others (arrows). (a2–b2) Higher magnification of Fig. a1 and b1 showing multiple positive microdomains of co-receptor localization (brackets) interspersed along the filopodia membrane. (c1–4) Cdo-GFP and Boc-Kate2 are colocalized along microdomains of filopodia (arrow) labeled with membrane-associated near-infrared fluorescent protein (pmiRFP). (d) Boc-harboring filopodia (red line) are statistically more stabilized than filopodia without Boc (black line), p < 0.001, n=160 timepoints. (e) Expression system to specifically label Shh producing cells and Shh responding cells in the same limb bud. (f) Representative 3D image of a filopodia from a Shh producing cell (pmKate2 - red) that interacts with domains of Boc-GFP (green) along the filopodia membrane (pmiRFP, fuscia) of Shh responding cell. Arrows show interaction along Boc microdomains (brackets). (g) Shh producing cell, indicated by pmKate2 and marked by bracket, with a long filopodium containing ShhN-EGFP particles (arrows) that contacts a Smoothened positive cell, Smo+, (outlined by a dashed line). Smoothened-BFP localization to the cilium is a marker of Shh pathway activation. Scale = 3µm.

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