Cell Tissue Res DOI 10.1007/s00441-005-0031-1 REGULAR ARTICLE Dana M. García . Hermann Bauer . Thomas Dietz . Thomas Schubert . Jürgen Markl . Michael Schaffeld Identification of keratins and analysis of their expression in carp and goldfish: comparison with the zebrafish and trout keratin catalog Received: 21 March 2005 / Accepted: 23 May 2005 # Springer-Verlag Abstract With more than 50 genes in human, keratins zebrafish, and the cyprinid fishes being more similar to each make up a large gene family, but the evolutionary pres- other than to the salmonid trout. Because of the detected sure leading to their diversity remains largely unclear. Nev- similarity of keratin expression among the cyprinid fishes, ertheless, this diversity offers a means to examine the we propose that, for certain experiments, they are inter- evolutionary relationships among organisms that express changeable. Although the zebrafish distinguishes itself as keratins. Here, we report the analysis of keratins expressed being a developmental and genetic/genomic model organ- in two cyprinid fishes, goldfish and carp, by two-dimen- ism, we have found that the goldfish, in particular, is a more sional polyacrylamide gel electrophoresis, complementary suitable model for both biochemical and histological stud- keratin blot binding assay, and immunoblotting. We further ies of the cytoskeleton, especially since goldfish cytoskel- explore the expression of keratins by immunofluorescence etal preparations seem to be more resistant to degradation microscopy. Comparison is made with the keratin expres- than those from carp or zebrafish. sion and catalogs of zebrafish and rainbow trout. The keratins among these fishes exhibit a similar range of mo- Keywords Keratin . Intermediate filaments . Goldfish lecular weights and isoelectric points, with a similar overall (Carassius auratus) . Carp (Cyprinus carpio) . Zebrafish pattern on two-dimensional gels. In addition, immunofluo- (Danio rerio)(Teleostei) rescence microscopy studies of goldfish and carp tissues have revealed the expression of keratins in both epithelial and mesenchymally derived tissues, as reported previously Introduction for zebrafish and trout. We conclude that keratin expression is qualitatively similar among these fishes, with goldfish Intermediate filament (IF) proteins belong to a large and carp patterns being more similar to each other than to multigene family with diverse members that exhibit tis- sue- and developmental-phase-specific expression patterns (Moll et al. 1982; Hoffmann and Franz 1984; Hoffmann et al. 1985; Winkles et al. 1985; Franz and Franke 1986; This work was supported by grants to J.M. from the Stiftung Miyatani et al. 1986; Fouquet et al. 1991; Markl and Rheinland-Pfalz für Innovation (836-386261/138) and the Deutsche Forschungsgemeinschaft (Ma 843/5-1) and a grant to D.G. Franke 1988; Fuchs and Weber 1994; Parry and Steinert from the National Science Foundation (INT-0078261). 1999; Watanabe et al. 2001, 2002; Herrmann et al. 2003). According to their amino acid sequence, six IF protein D. M. García types are generally distinguished: type I and II (keratins), Department of Biology, Texas State University-San Marcos, San Marcos, TX, 78666, USA type III (vimentin, desmin, glial fibrillary acidic protein, e-mail: [email protected] peripherin, and plasticin), type IV (neurofilament pro- teins, α-internexin, gefiltin, and xefiltin), type V (nuclear H. Bauer lamins), and the heterogeneous type VI (including nestin, Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, synemin, paranemin, syncoilin, and tanabin). In mammals, 14195 Berlin, Germany keratins are largely restricted to epithelial cells and tissues, whereas vimentin is primarily expressed in mesenchymal T. Dietz . T. Schubert . J. Markl . M. Schaffeld (*) cells (Franke et al. 1978; Moll et al. 1982). Other IF pro- Institute of Zoology, Johannes Gutenberg University, teins also show tissue specificity. Assigning functions to Johannes-von-Müller-Weg 6, 55099 Mainz, Germany IFs beyond that of structural support has been difficult, in e-mail: [email protected] part because of the size and diversity of the keratin gene e-mail: [email protected] family. Over 50 keratin genes have been identified in humans, representing almost 80% of the IF genes dis- amined vertebrate groups, including lamprey, shark, bony covered thus far (Hesse et al. 2001, 2004; Rogers et al. fish, and terrestrial vertebrates (e.g., Schaffeld et al. 1998, 2004, 2005). 2002a,b). The tissue specificity of expression of the various IF However, for goldfish and carp, only cytoskeletal pro- proteins has been hypothesized to indicate their diversity of teins from the optic nerve and oocytes, respectively, have function. This supposition has been challenged by Owaribe been biochemically characterized in detail. In this study, we et al. (1988) who have observed that the retinal pigment present a further evaluation of the carp and the goldfish, by epithelium of some species expresses keratins, whereas in examining many different organs representing tissues de- others, vimentin is expressed. Since the retinal pigment rived from the ecto-, endo-, and mesoderm. These fish epithelium is presumed to perform similar functions (e.g., species have been chosen because they are closely related metabolic support of the retina, phagocytosis of photore- to the genetic and developmental model organism, the ceptor discs) in these various species, the idea that IF zebrafish, but are, in several respects, easier to work with diversity reflects the functional diversity of cells and tis- because of their larger size. We find the types of keratins sues seems less likely. This idea has further been called into expressed to be qualitatively similar when examined by question following Markl and Franke’s(1988) discovery two dimensional polyacrylamide gel electrophoresis (2D- that, in trout, mesenchymally derived cells express keratins PAGE), complementary keratin blot-binding (CKBB) as- as a rule and vimentin as an exception to the rule. This say, and immunoblotting. Furthermore, tissues examined finding has since been extended to zebrafish (Cerdá et al. by immunofluorescence microscopy exhibit similar label- 1998; Conrad et al. 1998) and carp (Groff et al. 1997a,b). ing patterns to those previously observed in zebrafish, with Although the hope of assigning functions to IFs based on few exceptions, making goldfish in particular an appealing their tissue-specific expression has dimmed, the possibility organism to represent cyprinid fishes in future studies of that IFs function with respect not only to cell structure, but keratins. also to, for example, cell signaling, is gaining support (Paramio and Jorcano 2002; Kirfel et al. 2003). Never- theless, IF expression patterns may yet contain information Materials and methods regarding the evolution of IFs and the evolutionary relationships among the organisms in whose tissues they Animals and preparation of tissues are expressed. In addition, evolutionary comparisons might elucidate structure-function relationships, e.g., by illustrat- Mature carp (Cyprinus carpio) were obtained from a local ing a role for IFs in adapting to osmotic stress (see fish dealer and killed by a blow to the head. Mature Schaffeld and Markl 2004; Schaffeld et al. 2004, 2005)or goldfish (Carassius auratus) were a gift from Carlos Mora- enabling nerve, fin, and limb regeneration (e.g., Ferretti et Ferrer (University of Mainz) and were killed by spinal al. 1989, 1993; Tsonis et al. 1992; Corcoran and Ferretti section. Both fish types were immediately dissected on 1997, 1999; Markl and Schechter 1998; Martorana et al. ice. Organs to be used in immunofluorescence microscopy 2001). Furthermore, the reduction in the number of keratin were immediately snap-frozen in isopentane that had been genes by loss/decrease of function in some fish lines should pre-cooled in liquid nitrogen to approximately −130°C. be considered as a possibility in evolutionary comparisons Tissues to be used in biochemical preparations were fro- (see Winter et al. 2001 for an example in hominids). With zen directly in liquid nitrogen. All tissues were stored at this in mind, the expression of IFs, particularly keratins and −80°C until use. vimentin, has been evaluated in a number of fishes representing various branches of the evolutionary tree. The fishes most extensively studied include the shark Analytical procedures Scyliorhinus stellaris (Schaffeld et al. 1998, 2001, 2004), the zebrafish Danio rerio (Cerdá et al. 1998; Conrad et al. Procedures for isolating the insoluble cytoskeletal proteins 1998; Schaffeld et al. 2003), the rainbow trout Oncorhyn- and the following two-dimensional polyacrylamide gel cus mykiss (Markl and Franke 1988; Markl et al. 1989; electrophoresis (2D-PAGE), by using isoelectric focusing Herrmann et al. 1996; Schaffeld et al. 2002a,b), the com- (IEF) for the first dimension and sodium dodecylsulfate mon goldfish Carassius auratus (Giordano et al. 1989, (SDS) for the second dimension, were carried out as pre- 1990; Cohen et al. 1991; Druger et al. 1992, 1994; Fuchs et viously described (Schaffeld and Markl 2004). For the al. 1994), the carp Cyprinus carpio (Groff et al. 1997a,b; identification of keratins, polypeptides separated by 2D- Mencarelli and Cotelli 1997), and the lungfish Protopterus PAGE were electrically blot-transferred to nitrocellulose aethiopicus (Alibardi 2001, 2002; Alibardi and Joss 2003; sheets and exposed either to various keratin antibodies