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The hindered diffusion model postulates that the movement of a signaling molecule through an embryo is affected by tissue geometry and binding-mediated hindrance, but these effects have not been directly demonstrated in vivo. Here, we visualize extracellular movement and binding of individual molecules of the activator-inhibitor signaling pair Nodal and Lefty in live developing zebrafish embryos using reflected light-sheet microscopy. We observe that diffusion coefficients of molecules are high in extracellular cavities, whereas mobility is reduced and bound fractions are high within cell-cell interfaces. Counterintuitively, molecules nevertheless accumulate in cavities, which we attribute to the geometry of the extracellular space by agent-based simulations. We further find that Nodal has a larger bound fraction than Lefty and shows a binding time of tens of seconds. Together, our measurements and simulations provide direct support for the hindered diffusion model and yield insights into the nanometer-to-micrometer-scale mechanisms that lead to macroscopic signal dispersal.

The influential hindered diffusion model postulates that the global movement of a signaling molecule through an embryo is affected by local tissue geometry and binding-mediated hindrance, but these effects have not been directly demonstratedin vivofor any signaling molecule. Nodal and Lefty are a prime example of an activator-inhibitor signaling pair whose different global diffusivities are thought to arise from differential hindrance. Here, we used single-molecule tracking of Nodal and Lefty to directly probe the tenets of the hindered diffusion model on the nanoscale. We visualized individual fluorescently-tagged Nodal and Lefty molecules in developing zebrafish embryos using reflected light-sheet microscopy. Single-particle tracking revealed molecules in three states: molecules diffusing in extracellular cavities, molecules diffusing within cell-cell interfaces, and molecules bound to cell membranes. While the diffusion coefficients of molecules were high in extracellular cavities, mobility was reduced and bound fractions were higher within cell-cell interfaces; counterintuitively, molecules nevertheless accumulated in cavities. Using agent-based simulations, we identified the geometry of the extracellular space as a key factor influencing the accumulation of molecules in cavities. For Nodal, the fraction of molecules in the bound state was larger than for Lefty, and individual Nodal molecules had binding times of tens of seconds. Together, our single-molecule measurements and simulations provide direct support for the hindered diffusion model in a developing embryo and yield unprecedented insights into the nanometer to micrometer scale transport mechanisms that together lead to macroscopic signal dispersal and gradient formation.