Technical Reference #1690

1690.

Rab5-Mediated Endocytosis of Activin Is Not Required For Gene Expression or Long-Range Signalling in Xenopus Anja I. Hagemann; Xin Xu; Oliver Nentwich; Marko Hyvonen and James C. Smith, University of Cambridge, Development, 136(1690), (2009)
Link To Paper

Abstract:
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 longrange 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.

Keywords:
Xenopus; TGF- ; Smads; Activin; Nodal-related proteins; Bimolecular fluorescence complementation (BiFC); Morphogen

Materials & Methods:
Xenopus embryos and manipulation Xenopus embryos were obtained injected dissected and staged as described (Nieuwkoop and Faber 1975; Tada et al. 1997). For animal cap assays animal pole regions were transferred to agarose-coated dishes and cultured in 0.75 normal amphibian medium (NAM) (Slack 1984) containing 0.2% bovine serum albumin (BSA). To investigate long-range signalling animal pole regions were transferred to fibronectin-coated (10 dilution of 0.1% fibronectin from bovine plasma Sigma) glassbottomed dishes (MatTek) in 0.75 NAM essentially as described (Williams et al. 2004). For ‘bead’ experiments Affi-Gel beads (Affi-Gel Blue Gel Bio-Rad 100-200 mesh) were incubated overnight with 1 mM Alexa488-activin protein at 4°C. For dissociated cell experiments animal caps were dissociated in Ca2+- and Mg2+-free medium (75 mM Tris pH 7.5 880 mM NaCl 10 mM KCl 24 mM NaHCO3) for 30-45 minutes at room temperature and were then transferred to 0.75 NAM containing 0.2% BSA before being treated with activin. Dissociated cells were seeded onto a fibronectin-coated or 0.75 μg/ml E-cadherin-coated (recombinant human E-cadherin R&D Systems) (Ogata et al. 2007) glass bottom dish (MatTek) in 0.75 NAM containing 0.2% BSA in the presence or absence of 1 μg/ml Alexa488-activin or 5 μg/ml transferrin.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
embryos