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

Simon W. Fellgett; Simon A. Ramsbottom; Richard J. Maguire; Stephen Cross; Peter O'Toole; Mary E. Pownall

doi:10.3791/53162

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Method Article

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

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DOI:

10.3791/53162

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December 14th, 2015

Simon W. Fellgett¹ , Simon A. Ramsbottom² , Richard J. Maguire³ , Stephen Cross⁴ , Peter O'Toole⁵ , Mary E. Pownall⁵

¹Department of Biomedical Science, The Bateson Centre, University of Sheffield, ²Institute of Genetic Medicine, Newcastle University, ³Department of Cardiovascular Science, The Bateson Centre, University of Sheffield, ⁴School of Biochemistry, University of Bristol, ⁵Biology Department, University of York

Summary

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating 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.

Introduction

During early embryonic development, cells are progressively committed to follow specific lineages of differentiation: this means a group of totipotent (or pluripotent) cells become gradually restricted to establishing populations of progenitor cells determined to give rise to one cell type. Cell-cell signalling is central to the regulation of lineage specification during embryonic development. Manipulation of these signals will be required to direct stem cells toward particular fates to support novel medical treatments.

A relatively small number of signalling pathways are reiterated during development, including pathways responding to the T....