Methods of Calpain Inhibition to Determine the Role of Calpains in Embryo Development in Amphibians
Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that the role of calpains in cell migration and other processes has been extensively studied in vitro, the same does not apply to cell migration and morphogenetic events during embryogenesis, in vivo. Herein, we describe the use of three different methods to selectively block calpain activity in vivo in order to investigate the impact on Xenopus gastrulation and neurulation, namely, a calpain inhibitor, a dominant negative, and a morpholino antisense oligonucleotide (MO). We also provide methods to determine the effectiveness of the calpain inhibition and effect on cell fate specification and morphogenetic movements, during embryogenesis in vivo.
Key wordsXenopus Calpain Protein downregulation CI3 inhibitor Whole-mount in situ hybridization Immunofluorescence Casein zymography Mesoderm migration Convergent extension
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