Differential spreading behaviors of Nodal signalling molecules in the extracellular space cooperatively shape left-right asymmetry

Left-right (LR) patterning of vertebrate embryos is mediated by diffusive proteins secreted mainly from the left-right organizer (LRO), such as TGF-{beta} superfamily ligands Nodal and Gdf1, as well as their inhibitors Dand5 and Lefty. However, precise modes of action for these proteins remain unclear due to difficulties in visualizing and manipulating their extracellular behaviors. Here we visualize and manipulate the extracellular dynamics of these proteins employing novel transgenic zebrafish lines and extracellular trapping molecules including the morphotrap. We demonstrate that the heterodimer of Nodal and Gdf1, which is the major signal transducer in vertebrate Nodal signalling, spreads from the LRO through the extracellular space in the paraxial mesoderm (PM) to its target tissue, the lateral plate mesoderm (LPM), in a free-diffusion-like manner. By contrast, Dand5 is highly enriched in the right PM, where it is immobilized through interaction with heparan sulfate proteoglycans and asymmetrically suppresses Nodal signalling. Our study uncovers diverse modes of action for LR-related secreted proteins in the extracellular space, providing a new framework for understanding how spatially organized Nodal signalling molecules shape embryonic LR asymmetry.