Zebrafish and Xenopus are both undoubtedly well suited to whole-organism based small-molecule screens because large numbers of embryos can be arrayed in multiwell plates, along with compounds from a chemical library. The first large-scale chemical library screen using a vertebrate model was reported by Peterson et al. (2000) who screened 1,100 compounds against zebrafish embryos arrayed in 96-well plates to identify molecules that modulate embryogenesis. The use of Xenopus as an alternative vertebrate model for chemical screens has been proposed in the past (Brändli,2004; Tomlinson et al.,2005), but until recently zebrafish has remained the only vertebrate model used for large-scale chemical screens (Zon and Peterson,2005). This has been in part due to the availability of chemically induced zebrafish mutants that model human disease (Shin and Fishman,2002; Rubinstein,2003). The versatility of Xenopus must, therefore, be compared to the well-established properties of the zebrafish system. The recent report of genetic screens performed in Xenopus tropicalis (Goda et al.,2006) indicate that Xenopus mutants that phenocopy human disorders may be recovered in the future. The X. tropicalis genome is now nearly complete (JGI X. tropicalis genome assembly 4.1; http://genome.jgi-psf.org/Xentr4/), public databases harbor the nucleotide sequences of almost 2 million expressed sequence tag (EST) from X. tropicalis and X. laevis (dbEST; http://www.ncbi.nlm.nih.gov/dbEST/), and DNA microarrays are available for both Xenopus species. Thus, Xenopus is rapidly becoming a model organism armed with an impressive collection of genomic and transcriptomic tools.
The closer a model organism is in evolutionary terms to the target organism, i.e., humans, the more reliably results from studies in the model organism translate to humans. Figure 1 shows a phylogenetic tree highlighting the evolutionary distances between various animal model organisms used in biomedical research. Xenopus has a common evolutionary history with mammals that is an estimated 90–100 million years longer than between zebrafish and mammals. Since both are tetrapods, Xenopus and mammals share extensive synteny at the level of the genomes and have many similarities in organ development, anatomy, and physiology (Christensen et al.,2008; Raciti et al.,2008). A high degree of sequence conservation and structure between the proteins of model species and humans is desirable, since this will increase the likelihood that bioactive compounds recovered in chemical screens in model organisms will retain activity and specificity for preclinical studies in mice and eventually for clinical studies in humans. As stated, Xenopus as a tetrapod is evolutionarily closer to humans than zebrafish. There are also significant differences at the genomic level. Genome and EST sequence data support the notion that the X. tropicalis genome is diploid in nature (Hellsten et al.,2007). In contrast, the teleost lineage of fish underwent a genome duplication event after diverging from tetrapods about 450 million years ago (Postlethwait et al.,1998; Postlethwait,2007). Zebrafish, therefore, possess two copies of many mammalian genes. Such redundancy can confound extrapolation of the effect of single gene knockdown or mutation from fish to mammals and complicate the generation of zebrafish models of congenital human disease. In addition, comparative genomics has identified entire gene families, such as the KRAB domain containing C2H2 zinc finger (KRAB-ZF) transcription factors (Emerson and Thomas,2009) that first arose in tetrapod vertebrates and are absent in the zebrafish genomes.
There are drawbacks that apply to both animal models. Most advantages of zebrafish and Xenopus as a model for chemical screens are limited to embryonic and larval stages and do not apply to adult organisms, since they are typically too large to be employed in whole-organism-based screens. Despite these limitations, zebrafish larvae and Xenopus tadpoles contain most organs and tissues affected by common human diseases, including a cardiovascular system, a digestive tract, excretory organs, sensory organs, a hematopoietic system, and a central nervous system. Being tetrapods, Xenopus tadpoles will develop lungs and limbs, which are absent from zebrafish. Importantly, the organs of Xenopus tadpoles are morphologically and functionally more similar to their human counterpart than those found in zebrafish, as we will illustrate with three examples: the heart, immune system, and the kidneys. The zebrafish heart contains two chambers, a single atrium and a single ventricle, with endocardial cushions forming an atrioventricular valve (Stainier,2001). In contrast to amphibians and mammals, the zebrafish heart does not progress to septation. The heart of Xenopus tadpoles is an evolutionary intermediate between the two-chambered heart of fish and the four-chambered heart of mammals (Mohun et al.,2000; Warkman and Krieg,2007). It consists of a single ventricle, and left and right atrial chambers separated by a septum. In general, the events of Xenopus heart development closely resemble the equivalent processes of heart development in mammals including a leftward bend of the outflow tract, the presence of a spiral valve in the outflow tract and an atrioventricular valve to separate the atria and ventricle, asymmetric division of the atria early in development (with the right side being larger), and the presence of trabeculae within the thickened wall of the ventricular myocardium (Mohun et al.,2000; Warkman and Krieg,2007). Differences are also evident with the immune system. While the antibody-based, adaptive immune system is present in all jawed vertebrates (gnathostomes) (Klein and Nikolaidis,2005), zebrafish and Xenopus differ considerably with regard to the presence of major lymphoid tissues, immunoglobulin gene organization, and the ability to perform immunoglobulin class switching (Du Pasquier et al.,1989; Traver et al.,2003). Bone marrow, gut-associated lymphoid nodules, and primitive lymph nodes are absent from zebrafish but present in Xenopus. There are also significant differences in the gene organization, gene usage, and gene number of immunoglobulin genes, whereby Xenopus shares many features with mammals (Du Pasquier,2001). Zebrafish express a new immunoglobulin heavy chain subtype, immunoglobulin Z, which does not have a counterpart in mammals (Danilova et al.,2005). Furthermore, isotype class switching is a process that has its earliest evolutionary roots in amphibians and does not occur in fishes (Traver et al.,2003). In summary, Xenopus display an efficient immune system that is very similar to mammals and includes rearranging T-cell receptors (TCR) and immunoglobulin genes, as well as major histocompatibilitiy complex (MHC) class I- and class II-restricted T cell recognition. With regard to the excretory system, the pronephric kidneys of zebrafish and Xenopus kidneys differ in their global structure from the adult mammalian kidneys, but fulfill the same essential physiological functions in solute reabsorption, water and ion homeostasis, pH regulation, and waste product excretion. This is underscored at the molecular level by a recent large-scale gene expression study that revealed a remarkable conservation between the nephron organization of Xenopus tadpole kidneys and adult mammalian kidney (Raciti et al.,2008). Overall, these traits favor the use of amphibians for large-scale in vivo drug screens and suggest that they may unveil mechanisms and pathways relevant to human disease and therapy. There is no doubt that mammals, such as mice and rats, clearly reflect human physiology better. However, there are no other vertebrates of the tetrapod superclass that offer the same advantages of Xenopus. Their free-living embryos are amenable to large-scale, high-throughput chemical screens. Therefore, Xenopus provides the optimal trade-off between experimental use, cost-efficient performance, and biological relevance to humans.