Investigating the Cellular and Molecular Mechanisms of Wound Healing in Xenopus Oocytes and Embryos

Abstract

The African clawed frog Xenopus has remarkable capacities to heal wounds rapidly and to regenerate complex tissues. Because of its experimental tractability, studies using Xenopus oocytes, embryos, and larvae have contributed extensively to our understanding of the molecular and cellular mechanisms underpinning wound healing and tissue regeneration. In this protocol, we describe wound-healing assays following mechanical or laser injuries of oocytes and multicellular epithelia in Xenopus laevis embryos. We also explain how to perform assays aimed at investigating the cellular and molecular events during wound healing, including gene knockdown and overexpression experiments. In the latter assays, we explore the use of biochemical pull-down assays to investigate the activity of Rho GTPases, as well as the injection of mRNAs encoding fluorescent proteins or probes, followed by quantitative confocal image analyses to assay the dynamics of cytoskeletal components and their regulators. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. ReagentsAgarose Antibodies, anti-Cdc42 (Cell Signaling Technology 2462) (for GST pull-down assays only) Antibodies, anti-Rac1/2/3 (Cell Signaling Technology 2465) (for GST pull-down assays only) Antibodies, anti-RhoA (Santa Cruz Biotechnology SC-179) (for GST pull-down assays only) Bovine serum albumin (BSA) Constructs (optional; see Steps 1, 7.ii, 7.iv, 11) Embryos can be injected with moesin-gfp to observe actin dynamics ), gfp-α-tubulin to observe microtubule dynamics (Woolner and Papalopulu 2012), GEM-GECO or C2-mrfp to observe calcium dynamics (Clark et al. 2009; Soto et al. 2013), or gfp/mcherry-caax to label the plasma membrane and thus outline the cell 3 Correspondence: enrique.amaya@manchester.ac.uk From the Xenopus collection, edited by Hazel L. Sive.