title: Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients.
abstract: This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.
authors.affiliation: Department of Biomedical Science, The Bateson Centre, University of Sheffield.
authors.forename: Simon W
authors.initials: SW
authors.lastname: Fellgett
authors.affiliation: Institute of Genetic Medicine, Newcastle University.
authors.forename: Simon A
authors.initials: SA
authors.lastname: Ramsbottom
authors.affiliation: Department of Cardiovascular Science, The Bateson Centre, University of Sheffield.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Maguire
authors.affiliation: School of Biochemistry, University of Bristol.
authors.forename: Stephen
authors.initials: S
authors.lastname: Cross
authors.affiliation: Biology Department, University of York.
authors.forename: Peter
authors.initials: P
authors.lastname: O'Toole
authors.affiliation: Biology Department, University of York; betsy.pownall@york.ac.uk.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Pownall
mesh_terms: D000818:Animals; D004475:Ectoderm / Q000378:metabolism; D005260:Female; D053823:Hedgehog Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051153:Wnt Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism*
chemical_list: D053823:Hedgehog Proteins; D008024:Ligands; D012333:RNA, Messenger; C558166:Shh protein, Xenopus; D051153:Wnt Proteins; D029867:Xenopus Proteins