Measuring morphogen transport over multiple spatial scales in live zebrafish embryos

Morphogenesis is controlled by signalling morphogens that form gradients across the embryo. The gradient formation requires that the low molecular weight morphogens slow down their diffusion by at least one order of magnitude. However, the precise slow-down of diffusion across the relevant micrometre scales has not been directly observed. Here, we develop and employ Single-Plane Illumination Microscope based spatial Fluorescence Cross-Correlation Spectroscopy (SPIM-sFCCS) to directly measure the diffusion coefficient of the morphogen Squint in early zebrafish embryos as a function of topography and length scale. We show that Squints diffusion coefficient changes on length scales that are commensurate with the diameter of the intercellular spaces in the embryo and that the slow-down is regulated by receptor binding. The slowdown can be reduced by either the knockdown of Activin receptor 2b, a receptor for Squint, or the overexpression of Lefty2, an inhibitor of Squint. Based on our results and supporting simulations, we propose an interstitial space-dependent transient receptor binding and diffusing mechanism to explain this slowdown, which is crucial for gradient formation and embryonic patterning.