Current Genomics - Volume 4, Issue 8, 2003

During the last century amphibians have served as excellent models for developmental studies, in particular concerning early embryonic development. Since the 1950s (when they were first used in human pregnancy tests), the South-African claw-toed frog Xenopus laevis has been the most popular amphibian model organism. X. laevis is readily maintained in the laboratory, is commercially available, and can be induced to ovulate and mate at any time of the year (most frog species are seasonal breeders). Thousands of the relatively large and robust Xenopus eggs can be produced following a simple injection of the mammalian hormone chorionic gonadotropin. Xenopus embryos efficiently translate injected (synthetic) mRNAs, are transparent, develop externally in a simple salt solution, and are characterized by identifiable blastomeres, a reliable fate map, and ease of microinjection, micromanipulation, surgical intervention (grafting), labelling and culturing in vitro. Furthermore, it has recently become possible to generate hundreds of stable, non-mosaic transgenic Xenopus embryos in a cost-effective and efficient way in a single day [1]. This allows the experimenter to combine the aforementioned traditional advantages of X. laevis with the ability to express a gene at any time and in any place, not only for developmental studies but also to examine cell biological and biochemical processes. X. laevis has therefore, now been added to the list of (transgenic) model systems used for functional analyses (C. elegans, Drosophila, zebrafish and the mouse). Xenopus tropicalis is a close, diploid relative of the pseudotetraploid X. laevis, and has a short generation time making it attractive for creating permanent transgenic lines and performing molecular-genetic studies. The prospect of being able to perform genetic screens and make mutations in known genes will add an important dimension to the Xenopus system. Most anatomical and functional features as well as regulatory pathways are highly conserved between Xenopus and mammals, including humans. Therefore, the majority of information revealed from studies on this lower vertebrate will apply to mammalian systems. For these reasons, interest in Xenopus as a genomics system has increased, as is apparent from recent community-wide initiatives for the generation of large-scale Xenopus expressed sequence tags (ESTs) and genomic sequencing, expression profiling and genetic resources. These research efforts are often coordinated by the Xenopus Initiative of the US National Institutes of Health (NIH) that originates from the Human Genome Project via the NIH Non-Mammalian Models Initiative [2]. A search of Xenopus in the PubMed database revealed citations for over 28,000 articles (for comparison: mouse, 680,000 and zebrafish, 3,600). Nevertheless, in recent years Xenopus has not experienced the great increase in broad interest as seen for other vertebrate model organisms, such as the mouse and zebrafish. This is somewhat surprising since these models have a number of disadvantages when compared to Xenopus. For instance, mice produce fewer eggs, have no external embryonic development, it is not possible to generate genetic mouse chimeras (via tissue transplantation), and mouse transgenesis is cumbersome and costly. Zebrafish have a duplicated genome that is not only more divergent from mammals but the duplicated zebrafish genes have also diverged more from each other (a disadvantage for knock-down approaches), since the duplication occurred >400 million years ago [3]. In contrast, X. tropicalis is a true diploid and the genome duplication in X. laevis occurred ∼30 million years ago [4]. Furthermore, both mouse and zebrafish transgenesis are less efficient than in Xenopus and often mosaic, necessitating the selection of transgenic lines before analysis. Given these facts, it seemed appropriate to pay more attention to Xenopus as a vertebrate model organism and thus, to bring together the latest developments in the Xenopus genomics field. This Current Genomics Hot Topic issue deals with various aspects of X. laevis / tropicalis as a genomics system, including genomic and cDNA library construction and sequencing, bioinformatics, microarray studies, and (insertional) mutagenesis and misexpression approaches. A limiting step for the use of X. tropicalis has been the availability of high-quality cDNA libraries. Bruce Blumberg and colleagues (University of California in Irvine, CA, USA) have recently generated normalized, full-length enriched cDNA libraries from X. tropicalis that will allow functional studies and the identification of transcription units from the genomic sequence (article by Peng et al .). Paul Richardson and colleagues (US Department of Energy Joint Genome Institute, Walnut Creek, CA, USA) lead the X. tropicalis sequencing project, and aim to produce a high-quality sequence (8x coverage) and annotation in 2005. Their efforts are described in the second article of this issue (Richardson and Chapman). The X. laevis and X. tropicalis EST sequencing projects are currently underway with 294,924 ESTs available for laevis and 170,927 ESTs for tropicalis, as of July 25, 2003. Initially, Xenopus EST sequencing was relatively slow but the recent efforts have placed Xenopus high in the species ranking of available EST sequences. The availability of the ESTs is crucial for genome-wide expression screens and the construction of Xenopus UniGene sets (i.e. forming clusters of unique gene sequences) for efficient microarray analysis and allowing ‘digital (in silico) differential display’. Having the sequence of the Xenopus genome will enable identification of conserved DNA regulatory elements, which can then be directly assayed in an experimental system. The genome sequencing effort will also provide a vital resource in mapping and cloning mutant genes in X. tropicalis. In addition, the Xenopus genome sequence will add important insights into vertebrate evolution, since amphibians occupy a key phylogenetic position between mammals and fish. In both their ontogeny and phylogeny, most amphibians are quasiterrestrial with a double life in aquatic and land environments. For example, the amphibian limb skeleton clearly resembles that of mammals, but is very different from the fin of fish. A further interesting aspect of the availability of the amphibian genome may be the possibility of developing high-throughput tools to be used in toxicological and environmental research (Xenopus toxicogenomics), since frogs are considered to be good indicators of environmental damage caused by pollution. The main website resource with a compendium of useful Xenopus data is Peter Vize's Xenbase (University of Calgary, Alberta, Canada) and includes sections on genetics and genomics, as described in the article by Bowes and Vize. A large-scale gene-expression analysis was performed by Ken Cho and colleagues (University of California in Irvine, CA, USA) using Xenopus microarrays containing 42,000 sequenced cDNAs prepared from embryos at the neurula stage of development, to address several key aspects of Xenopus embryogenesis (Peiffer et al.). The article by Takuya Nakayama and Rob Grainger (University of Virginia, Charlottesville, VA, USA) presents the main features of X. tropicalis, in particular its genetic possibilities. X. tropicalis is a small, fast-growing frog with the only diploid genome among the 14 Xenopus species and one of the smallest genomes (∼1,700 million base pairs or half the size of that of X. laevis) among amphibians (>4,500 species), simplifying genetic studies. This amphibian may thus, make fundamental contributions to vertebrate comparative and functional genomics. In general, mutations in model organisms are either being identified in wild-caught animals, or introduced by chemical mutagenesis and gene-trapping strategies. Michelle Hamlet and Paul Mead (St. Jude Children's Research Hospital, Memphis, TN, USA) adapted the transgenesis technique in order to trap genes and describe transposons to increase the rate of gene trapping (article by Hamlet and Mead). Many early-developmental studies involve microinjection of mRNAs, antibodies, or (morpholino) antisense oligonucleotides but these (macro)molecules are degraded with time (transient expression). The generation of stable and non-mosaic transgenic Xenopus embryos, tadpoles and frogs has allowed cell- and developmental stage-specific transgene expression, and the transgene is reliably transferred from the parent (F0) to subsequent generations. Often the green fluorescent protein GFP is used as the reporter to study transgene expression in the living embryo. In the last article (Dirks et al.), Ron Dirks and colleagues (University of Nijmegen, The Netherlands) describe efforts to accomplish in X. laevis transgene-driven RNA interference and the use of stable, cell-specific transgene expression to study the role of proteins of unknown function (Functional Genomics). Finally, I would like to take the opportunity to thank Current Genomics Editor-in-Chief Christian Neri for his invitation to act as a Guest Editor, Sadia Masoom for editorial assistance and all colleagues for their contributions to this Hot Topic issue on Xenopus Genomics. As will be apparent from the information presented in the various articles of this issue, it is conceivable that Xenopus will develop into an attractive alternative of other model organisms, including zebrafish and mouse, to study the functioning of the vertebrate genome. References [1] Kroll, K.L. and Amaya, E. Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 1996, 122: 3173-3183. [2] Klein, S.L., Strausberg, R.L., Wagner, L., Pontius, J., Clifton, S.W. and Richardson, P. Genetic and genomic tools for Xenopus research: The NIH Xenopus initiative. Dev. Dyn. 2002, 225: 384-391. [3] Amores, A., Force, A., Yan, Y.L., Joly, L., Amemiya, C., Fritz, A., Ho, R.K., Langeland, J., Prince, V., Wang, Y.L., Westerfield, M., Ekker, M. and Postlethwait, J.H. Zebrafish hox clusters and vertebrate genome evolution. Science 1998, 282: 1711-1714. [4] Bisbee, C.A., Baker, M.A., Wilson, A.C., Haji-Azimi, I. and Fischberg, M. Albumin phylogeny for clawed frogs (Xenopus). Science 1977, 195: 785-787.

A large variety of mammalian and non-mammalian animal models have been used in research designed to uncover fundamental mechanisms underlying development and disease. Genomics tools have become increasingly necessary for the molecular genetic analysis of important biological questions. However, there are few genomics resources available for the emerging vertebrate model Xenopus tropicalis. Here we discuss our approach towards making a collection of full-length cDNAs from X. tropicalis that will serve as a resource for EST sequencing, microarray development and large-scale functional genomics analysis of Xenopus development.

The Human Genome Project has resulted in the elucidation of the genomic sequence of a number of model organisms as well as a reference sequence for the human genome. The utility of these available genomes has been demonstrated by researchers throughout the world, and spurred the desire to obtain additional genomic information from a number of sources. The United States Department of Energy's Joint Genome Institute has undertaken a project to sequence the genome of the amphibian Xenopus (Silurana) tropicalis. The primary goal of the project is to produce a high-quality genome sequence and annotation to meet the needs of the research community. In March of 2002, a number of Xenopus researchers from around the world met at the JGI Production Genomics Facility in Walnut Creek, California to discuss goals and strategies for the project. The project is designed to make use of a whole-genome shotgun approach supplemented with extensive BAC end sequences and shotgun sequence from selected BACs. A high-quality draft genome is desired that will meet minimal criteria for contiguity and long-range linking information. At depths of 6-8X sequence coverage, we expect that a large fraction of features of interest (exons, promoters and regulatory regions) will be covered in large contigs of high sequence quality without gaps. In addition, long-range linking of contigs will be achieved through paired end gap-spanning clones so that contigs are ordered and oriented into large scaffolds with gaps of defined size. These scaffolds typically contain multi megabase-sized regions of the genome. This approach has led to high-quality draft genomes of the pufferfish (Fugu rubripes), Ciona intestinalis and the mouse. Since there will be extensive coverage of large inserts for Xenopus including BAC and Fosmid end sequencing, clones will be readily available for finishing selected regions of the genome.

The many advantages of the Xenopus experimental system for investigating vertebrate development are limited by the small number of database resources that exist to support this work. This deficit also affects the usefulness of Xenopus data to researchers using other model organism systems. A number of database resources are available, and the applicability of these resources to Xenopus researchers is described. The power of relational databases is also explored as well as the methods that are being used by Xenbase to build an object-relational database that allows users to integrate data from multiple sources, such as literature, gene sequence, sequence annotation and gene expression.

Recent advances in DNA microarray technology have opened the door for large-scale gene expression screening, functional analysis and genomic profiling. Microarrays offer a new opportunity for genome-wide studies and are quickly revolutionizing biological analyses. This review will focus on several important issues related to Xenopus DNA microarray technology, including the advantages of using this model system for array studies, the current status of Xenopus microarrays, experimental designs, and the future of this method in the Xenopus field.

Amphibians have been favored organisms by experimental embryologists for more than a century. Their large, resilient embryos are ideal for manipulations, such as tissue transplantation, explantation and recombination, methods that have been used to demonstrate the existence and location of inducing centers in developing embryos and to define important embryological principles. Molecular biological approaches developed primarily in Xenopus, including a highly efficient method for transgenesis, have provided another dimension to our understanding of a multitude of cellular and developmental problems. Although there have been many developmental mutants reported in amphibians, mainly in axolotl and Xenopus laevis, amphibians have not been as widely used for genetic approaches as other vertebrates, like the mouse and zebrafish. This has been in part because of the long generation time of these species and their large genome size, especially in the case of axolotl. The duplicated genome of X. laevis also presents potential limitations. But another factor mitigating use of these systems for genetics in past times has been the lack of complementary techniques for studying mutants in detail at the molecular level. A more recently introduced model, Xenopus tropicalis, offers an array of new opportunities for genetic studies. Its short generation time and smaller, diploid genome, together with accumulating genomic resources, make X. tropicalis a very attractive model organism for addressing complex issues in modern cell and developmental biology. Here we will introduce the main features of the X. tropicalis system and briefly discuss possible methodologies for isolating developmental mutants for genetic studies.

Transposons or transposable elements (TEs) are segments of DNA that are able to mobilize from one region of DNA to another. TEs serve as powerful genetic tools in invertebrates such as Drosophila and in plants such as maize. With the molecular reconstruction of the salmonid transposon, Sleeping Beauty, it is possible to adapt transposon technology to vertebrate systems, and Xenopus is no exception. In this review, we give a brief history of TEs followed by a description of how TEs are classified and their mechanism of action. We then give an overview of the many uses of TEs as experimental tools. We focus on the transposon system Sleeping Beauty, and describe its use as a genetic tool to facilitate insertional strategies in vertebrates in general, and Xenopus tropicalis in particular.

The most direct approach to study the physiological role of a protein of unknown function (Functional Genomics) is to change its expression pattern in an intact organism and analyze the phenotypic consequences of this manipulation. The introduction of a method to generate stably transgenic Xenopus laevis has paved the way to the use of tissue / cell- and developmental stage-specific promoters allowing to study the physiological function of proteins in a defined set of fully differentiated cells. Whereas stable (over)expression of proteins in Xenopus is now within reach, stable inhibition of protein expression can only be accomplished randomly, by gene trap approaches. We here report our efforts to induce stable RNA interference (RNAi) in X. laevis via transgene-driven expression of inverted repeats. Stable, and muscle- and neuron-specific knock-down of expression of exogenous green fluorescent protein (GFP) reporter was achieved via RNA polymerase II promoter-driven expression of long GFP RNA duplexes. Unfortunately, our attempts to induce RNAi directed against various endogenous targets, based on the use of RNA polymerase II and III promoters, and long and short inverted repeats have not resulted in a reliable protocol for stable, transgene-driven RNAi in Xenopus. In the second part, we present an example of the use of a cell-specific promoter for functional studies. Cell-specific transgene overexpression of a GFP-tagged member of the p24 family thought to be involved in intracellular protein transport was achieved and this manipulation of the intermediate pituitary melanotrope cell had a phenotypic consequence at its physiological target, the skin melanophore. Thus, the traditional experimental advantages of X. laevis combined with the recently developed technique of stable, non-mosaic Xenopus transgenesis make this lower vertebrate an attractive model organism for Functional Genomics.