Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

作者：Fellgett Simon W; Ram**ottom Simon A; Maguire Richard J; Cross Stephen; O'Toole Peter; Pownall Mary E^*

来源：Jove-Journal of Visualized Experiments, 2015, (106): e53162.

DOI：10.3791/53162

• Developmental Biology
• Issue 106
• Hedgehog
• wingless
• GFP
• RFP
• developmental biology
• patterning
• secreted ligands
• cell signalling

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.

• 出版日期2015-12