An image montage showing three different kinds of signalling in zebrafish embryos and a breast cancer organoid.

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To understand how TGF-β superfamily ligands function in vivo we are using early zebrafish embryos as a model system. We want to determine how ligand activity is regulated, how the ligands function in a dose-dependent manner and how they contribute to tissue specification.

For a number of years now we have been focusing on spatial and temporal control of Nodal signalling. To visualise Nodal signalling in vivo, we generated a transgenic zebrafish line in which an EGFP reporter is controlled by 3 copies of the Activin-responsive enhancer (ARE), which binds a complex of activated Smad2-Smad4 with the transcription factor FoxH1. We have also used immunostaining of phosphorylated Smad2 to track Nodal signalling activity.

Our work has shown that a Nodal gradient is formed by 50% epiboly that is highest at the margin and weaker towards the anterior. Unexpectedly, it is restricted to the cells that express Nodal signalling and does not extend beyond the Nodal expression domain as was previously thought.

The predominant model to explain the generation of domains of Nodal signalling is the reaction-diffusion model, which is based on the assumption that a highly diffusible inhibitor (in this case Lefty1/2) and a less diffusible activator (Nodal ligands) can create a network as a result of short-range activation and long-range inhibition.

Our findings do not support this model. Instead, we have proposed an alternative model, whereby a temporal delay in the translation of the ligand antagonists Lefty1/2 allows Nodal signalling to become established in four to five cell tiers at the margin, at which point Lefty protein levels reach a sufficiently high threshold to prevent further spread of Nodal signalling. The delay results from translational inhibition of Lefty1/2 by a microRNA, miR-430. Thus, our work clearly shows that temporal and spatial dynamic patterns of ligand/antagonist expression, not ligand/antagonist diffusion, drive morphogen activity (van Boxtel et al., 2015 35, 175-185).

We have gone on to investigate how this Nodal gradient is involved in the specificiation of mesoderm versus endoderm, and have shown that this requires interplay between the Nodal and FGF signalling pathways. We have shown that Nodal induces long-range FGF signaling, whilst simultaneously inducing the cell-autonomous FGF signaling inhibitor Dusp4 within the first two cell tiers from the margin. The consequent attenuation of FGF signaling in these cells allows specification of endoderm progenitors, whilst the cells further from the margin, which receive Nodal and/or FGF signaling, are specified as mesoderm. This elegant model demonstrates the necessity of feedforward and feedback interactions between multiple signalling pathways for providing cells with temporal and positional information (van Boxtel et al., 2018 44, 179-191).