Exosome secretion is required for sonic hedgehog dispersal and signal gradient formation in the embryonic limb mesenchyme

SUMMARY Carrier-assisted diffusion and cytoneme transport have been postulated to disperse Hedgehog across diverse embryonic tissues, yet their relative contributions to patterning mesenchymal tissues remains poorly understood. Here, we combine novel signaling assays with quantitative microscopy to establish exosome secretion as a predominant and adaptable mechanism for Sonic Hedgehog (Shh) dispersal. Introducing a novel synchronous release system to visualize Shh trafficking in embryonic tissues, we demonstrate that Shh traffics through the exosome biogenesis pathway in the limb bud mesenchyme. Shh-bound exosomes diffuse through extracellular space, and can also bind and travel along cytonemes, providing a potential mechanism for directed and/or long-range transport. Using a synthetic patterning assay, we show that exosome secretion is essential to establish short-range Shh gradients in vitro. We propose that exosome-based Shh secretion, combined with different modes of extracellular transport, provides a tunable mechanism to sculpt Shh gradients on different length and time scales, across different embryonic tissues. HIGHLIGHTS A novel synchronous release system reveals trafficking dynamics in embryonic cells Sonic hedgehog is packaged and secreted on exosomes for extracellular dispersal Exosome secretion is required to establish short-range Hedgehog gradients Diffusion and cytoneme transport provide tunable exosome dispersal strategies Competing Interest Statement The authors have declared no competing interest.