a spatially and temporally regulated piggybac transposition system was developed to express fluorescent fusion proteins in a heritable fashion within the developing chick embryonic limb bud . the regulated expression of introduced transgenes was stable throughout embryogenesis and did not perturb normal limb development . cellular morphology and characterization of specialized filopodia was assessed through the use of membrane - associated fluorescent proteins and selected markers of the actin and tubulin cytoskeleton . molecular components of the shh signaling pathway were visualized as fusion proteins under endogenous regulatory control . high - resolution live imaging of the embryonic limb bud was performed on custom designed spinning disk confocal systems allowing for the visualization of multiple fluorescently coupled proteins in real time , at high spatial resolution .
the ability of signaling 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 over a century , the vertebrate limb bud has served as a paradigm to study cell signaling during embryonic development2 . here 
 we optimize single cell real - time imaging to delineate the cellular mechanisms for how signaling proteins , such as sonic hedgehog ( shh ) , that possess membrane - bound covalent lipid modifications transverse long distances within the limb bud in vivo . by directly imaging shh ligand production under native regulatory control , 
 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 . 
 strikingly , particles containing shh traffic along these extensions with a net anterograde movement within the field of shh cell signaling . 
 we further show that in shh responding cells specific subsets of shh co - receptors , including cdo and 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 significantly extends our understanding of ligand movement and reception during vertebrate tissue patterning .