Quantifying and perturbing the movement of extracellular proteins in zebrafish embryos

Abstract

Cell-cell communication mediated by secreted signaling molecules is crucial to coordinate early embryonic development. In the classical morphogen model secreted signaling molecules control embryogenesis as follows: After secretion from a source they disperse in the tissue and instruct target cells at a distance to adopt different cell fates that are defined by signaling levels. Thus, the signal’s distribution controls the cellular patterning of the tissue. However, the mechanisms underlying signal distribution in vivo and the requirement for signals acting at a distance remain controversial. Nodals are extracellular signaling molecules of the Transforming growth factor-β (TGF-β) superfamily and are required for mesoderm and endoderm formation in vertebrates. In the zebrafish Danio rerio, Nodals were proposed to function as classical morphogens that disperse from localized Nodal-secreting cells to act on distant cells. However, recent findings suggest that Nodals signal only to neighboring cells and that their signaling is propagated to distant cells by a combination of auto-induction and cell-to-cell signal relay, thus challenging the classical morphogen model. To directly test the two models of Nodal signaling I performed in vivo experiments to observe the endogenous Nodal signaling range. My results suggest that zebrafish Nodals can signal directly – i.e. without relay – to cells at a distance from their source. However, the importance of Nodal dispersal for its function during embryonic patterning remains to be determined. The morphogen model predicts that an altered signal dispersal leads to an altered signaling range and aberrant tissue patterning. To examine whether extracellular signal movement is required for the signal’s biological function, tools that restrict extracellular signal mobility are needed. Recently developed synthetic signal binders can be used to reversibly tether extracellular signals to the cell membrane and perturb signal spreading. I investigated how the transient membrane-tethering can be harnessed to experimentally reduce the effective diffusivity of extracellular proteins and thus regulate their mobility in a tuned manner. This approach allowed me to hinder the diffusion of the long-range Nodal inhibitor Lefty1 and investigate its long-range function in live zebrafish embryos. In zebrafish, Nodals have lower effective diffusion coefficients and a shorter range than their antagonists, the long-range Leftys. To explain the contrasting mobilities of these two TGF-β-related factors that are similar in molecular weight, binding partners in the extracellular matrix were proposed to act as diffusion regulators. My aim was to reveal these hypothetical diffusion regulators. I established a co-immunoprecipitation approach for zebrafish Nodals and Leftys and identified putative interaction partners by mass spectrometry. Surprisingly, known Nodal interaction partners were not identified as diffusion regulators with this approach, raising the possibility that other factors regulate the Nodal/Lefty system. In my work I investigated extracellular signal movement and focused on its modulation by diffusion regulators. My findings highlight that synthetic membrane tethers can be used as experimental diffusion regulators and that they serve as valuable tools to challenge models of long-range morphogen function.