Tissue and Cell 42 (2010) 275–281 Contents lists available at ScienceDirect Tissue and Cell journal homepage: www.elsevier.com/locate/tice Calpains expression during Xenopus laevis development E.N. Moudilou, N. Mouterfi, J.-M. Exbrayat, C. Brun ∗ Université de Lyon, UMRS 449, Laboratoire de Biologie Générale, Université Catholique de Lyon, Reproduction et développement comparés, EPHE, 25 rue du Plat, F-69288 Lyon Cedex 02, France article info Article history: Received 15 January 2010 Received in revised form 28 June 2010 Accepted 1 July 2010 Keywords: Xenopus laevis Development Immunochemistry Calpains Organogenesis abstract Calpains are cytoplasmic proteases activated by calcium, implicated in cell differentiation and apoptosis. The best characterized enzymes are calpains 1–3. The aim of this work was to localize calpains 1–3 during the development of Xenopus laevis in order to clarify the function of these three proteases. For the first time, we detected the localization of the three proteases at the protein level between one- cell stage and adult age. Their expression was weak at early stages, then increased at tadpole stage and decreased through metamorphosis and adult life. The calpain’s expression was maximal during the period characterized by the appearance of organs and modelling process. These observations suggest that calpains play a crucial role during development. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Calpains are a superfamily of 14 different cysteine proteases, widely expressed from Bacteria to Mammals (Sorimachi et al., 1997). Their name originated from their calcium requirement for activation and their papain-like activity (Croall and Ersfeld, 2007; Goll et al., 2003). The calpain family is implicated in numerous bio- logical processes, in particular cell proliferation, programmed cell death, cell differentiation (Honda et al., 2008; Liang et al., 2006; Yajima and Kawashima, 2002) and cell migration (Toyota et al., 2003; Franco and Huttenlocher, 2005; Suzuki et al., 2004). In Mam- mals, calpain 1 (-calpain), encoded by Capn1 gene, and calpain 2 (m-calpain), encoded by Capn2 gene, are activated in vitro by micromolar and millimolar Ca 2+ concentrations, respectively. Both calpains consist of a specific catalytic large subunit (80 kDa) and an invariant regulatory small subunit (30 kDa), encoded by Capn4 gene (Sorimachi and Suzuki, 2001; Franco and Huttenlocher, 2005). These enzymes are negatively regulated by a specific endogenous inhibitor, the calpastatin (Perrin and Huttenlocher, 2002). Unlike the ubiquitous calpains 1 and 2 (Reed et al., 2003), the human calpain 3 is mainly expressed in skeletal muscles (Sorimachi et al., 1989), and its mutation causes type 2A limb-girdle muscular dystrophy (Ono et al., 1998). Several reports showed a crucial role of calpains in developmen- tal process in a variety of species. Different homologs of human ∗ Corresponding author. Tel.: +33 04 72 32 50 72; fax: +33 04 72 32 50 66. E-mail address: cbrun@univ-catholyon.fr (C. Brun). calpains have been described in Drosophila melanogaster (inverte- brate) and in zebra-fish Danio rerio (vertebrate) in the first days of development (Emori and Saigo, 1995; Dutt et al., 2006; Lepage and Bruce, 2008). The Capn4 gene disruption results in a simulta- neous abolition of calpains 1 and 2 and leads to embryonic lethality (Zimmerman et al., 2000). Furthermore, it has been shown that the disruption of mouse Capn1 has no particular effect on the embryogenesis, whereas that of Capn2 leads to embryonic lethality between morula and blastocyste stages (Dutt et al., 2006). In Xenopus laevis, calpain large subunit genes have been sequenced and are known as Capn1 mu/I and mu/m (Klein et al., 2002) for the calpain 1, CL-2 and Capn2.2 for the calpain 2 (Cao et al., 2001; Klein et al., 2002). The monomeric calpain 3 is encoded by the Capn3 gene (Klein et al., 2002). In order to evaluate the functional role of the different calpains during the development in X. laevis, a detailed analysis of the pro- teases’ distribution during embryonic development was performed by use of immunohistochemistry method on transversal sections of embryos. 2. Materials and methods 2.1. Animals Sexually mature X. laevis was obtained from CNRS (UPRES A6026, Université de Rennes I, France). Embryos were obtained by artificial fertilization according to the French legislation concerning animal welfare. Oocytes were stripped from females injected 12 h earlier with human chorionic gonadotropin (Organon SA, Puteaux, France) and fertilized with the minced testis of males. Animals 0040-8166/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2010.07.001