Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model

Abstract

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.

Regulation of Nodal signaling propagation by receptor interactions and positive feedback

“…Overexpression of acvr2b-a did not markedly change the effective diffusivity of Squint-GFP ( Figure 6B ), suggesting that the strong interaction between Squint and Acvr2b-a previously shown in vivo ( Wang et al, 2016 ) is not sufficient to modulate Squint diffusivity. In contrast, oep overexpression reduced the effective diffusivity of Squint-GFP from about 2 µm 2 /s to ~1 µm 2 /s, consistent with the increased Nodal signaling range and distribution in the absence of oep ( Lord et al, 2021 ; Figure 5C ; Figure 5—figure supplement 1C ), the decreased Nodal distribution with overexpressed oep ( Lord et al, 2021 ) and the increased bound fraction upon oep overexpression in single-molecule tracking experiments ( Kuhn et al, 2022 ). Strikingly, the effective diffusivity of Squint-GFP in the absence of acvr1b-a/acvr1b-b increased to >3 µm 2 /s, consistent with the broader Squint-GFP distribution in the absence of these Type I receptors ( Figure 5C ; Figure 5—figure supplement 1C ).…”

“…The diffusion of signals through tissues can be hindered by their transient binding (rapid binding and unbinding) to extracellular molecules ( Bläßle et al, 2018 ; Kuhn et al, 2022 ; Mörsdorf and Müller, 2019 ; Müller et al, 2013 ). To test whether receptor interactions can affect Nodal diffusion, we performed Fluorescence Recovery After Photobleaching (FRAP) assays ( Almuedo-Castillo et al, 2018 ; Bläßle et al, 2018 ; Müller et al, 2012 ; Müller et al, 2013 ; Pomreinke et al, 2017 ; Soh and Müller, 2018 ).…”

“…There are currently two major models that can explain the propagation of Nodal signaling in this context. In the hindered diffusion model, Nodal is secreted from source cells and its free diffusion through the embryo is hindered by interactions with immobile diffusion regulators ( Müller et al, 2012 ; Müller et al, 2013 ; Rogers and Müller, 2019 ; Wang et al, 2016 ; Lord et al, 2021 ; Kuhn et al, 2022 ). In the second model, Nodal ligands only act in a juxtacrine fashion, and propagation of Nodal signaling to adjacent cells is mediated by a relay mechanism involving positive feedback of Nodal expression ( Liu et al, 2022 ; Lord et al, 2021 ; Rogers and Müller, 2019 ; van Boxtel et al, 2015 ; van Boxtel et al, 2018 ).…”

“…In this hindered diffusion model, Nodal’s free diffusivity of approximately 40 µm 2 /s ( Müller et al, 2013 ; Wang et al, 2016 ) would be slowed down by an order of magnitude through interactions with immobile diffusion regulators such as receptors ( Wang et al, 2016 ) or heparin sulfate proteoglycans ( Marjoram and Wright, 2011 ). Indeed, our recent single-molecule tracking experiments showed that individual Nodal molecules have binding times of tens of seconds in the extracellular space, leading to hindered diffusion on the nanometer to micrometer scale ( Kuhn et al, 2022 ). Here, we directly assessed the influence of Nodal receptors on the dispersal of Nodal ligands at the tissue scale.…”

“…It has been proposed that this mobility difference is due to interactions between Nodal ligands and membrane-bound diffusion regulators, whereas Lefty proteins move more freely in the extracellular space ( Müller et al, 2012 ; Müller et al, 2013 ). Indeed, Nodal’s signaling range and distribution dramatically increase in the absence of the zebrafish Tdgf1 co-receptor homolog Oep ( Lord et al, 2021 ), and single-molecule imaging has recently shown that the fraction of molecules in the bound state is larger for Nodal than for Lefty ( Kuhn et al, 2022 ). Since Nodals strongly bind to the zebrafish Type II receptor Acvr2b-a in vivo ( Wang et al, 2016 ), the main Nodal receptors themselves might also act as diffusion regulators.…”