Morphogen gradients provide positional cues for cell fate specification and tissue patterning during embryonic development. One important aspect of morphogen function, the mechanism by which long-range signalling occurs, is still poorly understood. In Xenopus, members of the TGF-β family such as the nodal-related proteins and activin act as morphogens to induce mesoderm and endoderm. In an effort to understand the mechanisms and dynamics of morphogen gradient formation, we have used fluorescently labelled activin to study ligand distribution and Smad2/Smad4 bimolecular fluorescence complementation (BiFC) to analyse, in a quantitative manner, the cellular response to induction. Our results indicate that labelled activin travels exclusively through the extracellular space and that its range is influenced by numbers of type II activin receptors on responding cells. Inhibition of endocytosis, by means of a dominant-negative form of Rab5, blocks internalisation of labelled activin, but does not affect the ability of cells to respond to activin and does not significantly influence signalling range. Together, our data indicate that long-range signalling in the early Xenopus embryo, in contrast to some other developmental systems, occurs through extracellular movement of ligand. Signalling range is not regulated by endocytosis, but is influenced by numbers of cognate receptors on the surfaces of responding cells.
- Nodal-related proteins
- Bimolecular fluorescence complementation (BiFC)
We thank all present and former members of our laboratory, especially Elizabeth Callery and Kevin Dingwell, for their helpful comments. We are also grateful to Mike Gilchrist (Gurdon Institute) for discussion and to Marcos Gonzalez-Gaitan and Roger Tsien for cDNA constructs. This work is supported by the EU sixth framework project `EndoTrack' (FP6 Grant LSHG-CT-2006-019050), by a Wellcome Trust Programme Grant awarded to J.C.S., and by the Foundation for Science and Technology (FCT), Portugal. A.I.H. is a student of the Gulbenkian PhD Program in Biomedicine, Portugal. Deposited in PMC for release after 6 months.
- Accepted June 12, 2009.