Model organisms have been used extensively to dissect fundamental biological mechanisms at the genetic, cellular, tissue, and organ system levels. The resulting information forms our basic understanding of the biological processes underlying human health and disease. In turn, translational research relies on model organism studies to form, refine, and test hypotheses for treatment strategies prior to clinical trials.
The frog Xenopus laevis, with its unique combination of experimental tractability and relationship with humans (both tetrapods), has a long history for in vivo studies. No other model system has contributed to so many fields, from molecular, cell, developmental, stem cell, and systems biology to ion channel physiology, neurophysiology, and toxicology. Although the recently duplicated X. laevis genome complicates genetic and some genomic approaches, X. tropicalis is diploid and retains many of the advantages of X. laevis, enabling additional investigative avenues. These highly flexible Xenopus models enhance our ability to address biomedical questions applicable across tetrapods, with particular relevance for human biology and disease.
X. Laevis: A Historical Perspective
X. laevis has been an important model system for biomedical science since the early 20th Century. The history of its introduction into the laboratory is fascinating and recently reviewed (Gurdon and Hopwood, 2000). First imported to Europe from South Africa as a novelty in the late 19th Century, these frogs survived and bred readily in laboratory conditions, becoming a model system for vertebrate reproductive physiology in the 1930s. In response to elevated hormone levels in pregnant women's urine, frogs lay eggs, forming the basis of the first human pregnancy test.
After World War II, Xenopus' fecundity and year-round breeding caught the attention of developmental biologists. In Hubrecht, Peter Nieuwkoop's pioneering microsurgery experiments demonstrated fundamental embryonic tissue interactions, including the induction of mesoderm by endoderm (Gerhart, 1999; Nieuwkoop, 1973), and his developmental staging series is still in use (Nieuwkoop and Faber, 1994). Serendipitous discovery of the anucleolate (0-nu) frog mutant in the laboratory of Michail Fischberg (Elsdale et al., 1958) provided a critical genetic tool for discriminating host from donor in nuclear transplant experiments. Although the totipotency of somatic nuclei is a cornerstone of our current understanding of biology, this was not well accepted at the time, and these pioneering nuclear transplant experiments with Xenopus were the first to demonstrate this concept. A fully differentiated gut nucleus could give rise to a normal, viable, fertile frog (Gurdon et al., 1958), predating mammalian cloning (Wilmut, 2003) by some 50 years. Xenopus continues to contribute to stem cell biology, with recent work showing that frog oocytes can reprogram adult mammalian somatic nuclei to a totipotent state (Byrne et al., 2003).
At the very outset of the molecular era, Xenopus led the transformation of biomedical research (Fraser and Harland, 2000), in part because few resources were required for radical advances. Seminal discoveries included: (1) location and behavior of ribosomal RNA genes (Brown and Dawid, 1968; Brown and Gurdon, 1964; Gall, 1968), (2) first isolation of a eukaryotic gene (Birnstiel et al., 1966; Brown et al., 1971), (3) mitochondrial DNA and its maternal inheritance (Dawid, 1966), (4) nuclear targeting sequences (De Robertis et al., 1978), and (5) eukaryotic transcription and translation of injected nucleic acids (Brown and Gurdon, 1977; De Robertis and Mertz, 1977; Gurdon et al., 1971).
Xenopus facilitated these discoveries because material could be obtained in sufficient quantity for biochemical purification, and microinjection of nucleic acids or other substances was technically simple. In the 1980s and 1990s, many key vertebrate transcription factors and signaling pathway genes were isolated using biochemistry and clever expression cloning approaches in Xenopus, identifying important developmental mechanisms that are common across vertebrates. For example, the studies of mesoderm induction led to the discovery of key-secreted signaling factors, including many TGF-β family members, and gave embryonic roles to the fibroblast growth factors for the first time (Kimelman and Kirschner, 1987; Rosa et al., 1988; Slack et al., 1987; Smith, 1987; Smith et al., 1990; Weeks and Melton, 1987). Work in Xenopus identified the critical role for wnt signals in axis formation (McMahon and Moon, 1989; Heasman et al., 1994) and that the signals from Spemann's organizer that induce neural tissue and pattern mesoderm are secreted antagonists of growth factors [reviewed by (Harland and Gerhart, 1997)]. Xenopus continues to produce insights into critical signaling pathways (Cordenonsi et al., 2007; Cruciat et al., 2010; Dupont et al., 2009; Fuentealba et al., 2007; Taelman et al., 2010; Zeng et al., 2005).
Although Xenopus' embryos are effective in vivo cauldrons for deconstructing signaling pathways and tissue interactions, its oocytes provide excellent cell-free extracts. These egg extracts can recapitulate all DNA transactions associated with cell-cycle progression and DNA damage, including DNA replication, chromosome segregation, DNA repair, and checkpoints. The ability of egg extracts to support cycling between mitosis and interphase in vitro led to fundamental discoveries in cell division, identifying meiosis maturation promoting factor (Wasserman and Masui, 1976) and regulation of cell-cycle progression by degradation of cyclins (Murray and Kirschner, 1989). This system was the first to demonstrate nuclear and chromosome assembly in vitro (Lohka and Masui, 1983). More recently, egg extracts helped make seminal discoveries in interphase nuclear transport, postmitotic nuclear envelope reassembly, and the mitosis-specific function of nuclear lamin B in regulating spindle morphogenesis (Kalab et al., 2006; Lohka and Masui, 1983; Ma et al., 2009; Tsai et al., 2006). As Xenopus cell-free extracts also support synchronous assembly and activation of origins of DNA replication, they have been instrumental in characterizing biochemical functions of the prereplicative complex (Tsuji et al., 2008; Xu et al., 2009). Extracts have also helped to understand DNA damage responses and check point pathways, including identification of components of signal transduction pathways activated in response to DNA double-strand breaks, replication fork stalling, or DNA interstrand crosslinks (Ben-Yehoyada et al., 2009; MacDougall et al., 2007; Raschle et al., 2008).
Intact Xenopus oocytes have also been key for membrane channel and receptor studies, including the first electrophysiological analysis of cloned membrane channels and receptors (Kusano et al., 1977). Xenopus oocytes enable rapid assays of channel and transporter protein activities because they correctly process the proteins, insert them into the cell membrane, and can be cultured for days (Musa-Aziz et al., 2010; Sobczak et al., 2010). This experimental approach has led to important discoveries in the membrane channels, receptors, and transporters of nervous, cardiac, auditory, and nephric systems. In 2008 alone, more than 50 publications used Xenopus oocytes to analyze cardiac-specific ion channels.
Finally, Xenopus embryos are used in powerful in vivo screens for small-molecule functions, described in detail in Wheeler and Liu (2012). Examples include identification of new small-molecule inhibitors of blood vessel growth, relevant to neovascular growth of tumors (Ny et al., 2008; Kalin et al., 2009). Small-molecule screens using Xenopus egg extracts have isolated novel inhibitors of proteasome-mediated protein degradation and DNA repair enzymes (Dupre et al., 2008; Landais et al., 2009). A sensitive high-throughput assay based on Xenopus transgenic reporter lines was able to detect heavy metal pollution (Fini et al., 2009); similar screens are applicable to a variety of ecotoxicology and public health issues.
Xenopus Studies are Relevant for Understanding Human Disease
The basic cell, molecular, and developmental mechanisms uncovered in Xenopus generally apply to other vertebrates and provide invaluable insight into human disease processes. As can be inferred from the long and far from inclusive list above, examples abound, and the case of noggin is illustrative. Noggin was initially identified as a determinant in axis formation in Xenopus and subsequently shown to be associated with human skeletal abnormalities and hearing loss (Gong et al., 1999; Mangino et al., 2002). The brisk pace of basic biology discovery in Xenopus will continue to provide new insights into human disease.
Driven by high-throughput sequencing (HTS), human genetics has entered a new era creating an additional opportunity for Xenopus to make an impact on disease. HTS enables geneticists to identify sequence variants in disease samples within weeks rather than years. However, human genomes are riddled with sequence variants, only a subset are disease causing. A major challenge is deciphering which variations are relevant to disease.
Xenopus' rapid gene function assays provide a key to this challenge. A study of patients with heterotaxy [a human condition affecting patterning along the left–right (LR) axis] and congenital heart disease identified rare copy number variations that affected numerous genes, none of which had been previously implicated in LR patterning. After first rapidly screening Xenopus orthologs of these genes for expression in critical embryonic structures, strong candidates were quickly knocked down by injection of antisense morpholino oligonucleotides (MOs) in Xenopus embryos. Most knockdowns recapitulated the disease phenotype, identifying novel genes involved in cardiac patterning and providing functional correlation of these loci to human disease (Fakhro et al., 2011). Genome-wide associations that identify noncoding sequences rather than genes are even more challenging, but in the case of colorectal cancer, a noncoding variant that appears to alter the expression of smad7 was confirmed using Xenopus transgenics (Pittman et al., 2009).
Although these new approaches help test functional effects of certain human mutations in vivo, Xenopus is also contributing to tissue-engineering approaches. Xenopus embryos provide abundant totipotent cells that can be transformed into a host of different tissues, including heart, kidney, and the eye, where engineered tissues have even been shown to be functional (Asashima et al., 2009; Lan et al., 2009; Viczian et al., 2009).
Progress in Developing Genomic and Genetic Resources in Xenopus
Many of these important contributions were accomplished without the aid of community-wide resources common nowadays in model systems. Early advances in Xenopus reflected a remarkable cottage industry, with each laboratory generating its own “genomic” resource, often cDNA libraries, and exploiting it in creative ways. However, to further advance basic and translational research using Xenopus, large-scale genomic resources were needed. Genome sequencing in mammals created a comprehensive catalogue of vertebrate genes, critical for transcriptomics and proteomics (Lander et al., 2001; Waterston et al., 2002). To augment gain-of-function mRNA injection screens, Xenopus researchers constructed the first sequenced, annotated, full-length, nonredundant cDNA libraries (Klein et al., 2002; Voigt et al., 2005). The success of the cDNA/EST sequencing projects led the desire for Xenopus researchers to build additional resources to benefit both the Xenopus community and the broader biomedical community.
In the USA, much of the funding for biomedical research is provided by the National Institutes of Health (NIH). However, as resource building is rarely hypothesis driven, such grants generally fare poorly in the review process. Resource grants can be more positively reviewed if the community makes a clear statement about what resources are needed especially if that community is an integral part of the biomedical research program. These authoritative statements, or White Papers, must document the community's biomedical discoveries and impact on human health, justify the resources needed to accelerate research, and prioritize. White Papers can significantly influence the NIH, which has a strong incentive to facilitate the research of a community whose goals are aligned with its own mission. The Xenopus community submitted White Papers to the NIH in 2002, 2006, 2009, and more recently in 2011 (see http://www.xenbase.org/community/xenopuswhitepaper.do). Below is an outline of the resources that have received NIH support after being highlighted in Xenopus White Papers.
Sequencing Xenopus Genomes: X. tropicalis
With support from the Xenopus community and facilitation from the NIH, the US Department of Energy's Joint Genome Institute began to sequence the frog genome in 2002. X. tropicalis was chosen rather than X. laevis because of the expense and anticipated difficulties in assembling the larger duplicated genome of X.laevis. In 2010, a draft genome assembly was published (Hellsten et al., 2010). This sequence is extremely useful for creating complete gene sets, defining transcriptomes/proteomes, identifying cis-regulatory elements, analyzing chromatin-immunoprecipitation (ChIP-seq) experiments, and for genome comparisons across species. However, the published genome is clearly a draft and improvements would further enable these experiments as well as other high-priority resources such as the ORFeome, X. laevis genome assembly, and X. tropicalis genetics (see below). Plans for improvement are underway and detailed elsewhere in Gilchrist (2012).
Genome-wide annotation of intergenic regions of X. tropicalis is also underway. Transcriptionally permissive and repressive histone modifications have been mapped using a combination of ChIP-seq and RNA-seq (Akkers et al., 2009). The Veenstra laboratory (Radboud Univ.) is now analyzing eight different histone modifications and DNA methylation during five different stages of development, identifying enhancers and defining functional interactions with promoters. A full discussion of this study is presented by Bogdanović et al. (2012).
Sequencing Xenopus Genomes: X. laevis
Sequencing the X. laevis genome became feasible with rapid progress of HTS. Although the X. tropicalis assembly was highly useful for many applications, the Xenopus community needed X. laevis sequence where species-specific sequence is essential: such as analysis of mass spectrometry data from biochemical purifications, site-specific studies of DNA replication or repair, and analysis of chromatin immunoprecipitation. Release of an initial X. laevis draft assembly is expected early in 2012, with multiple centers in the USA and in Japan contributing. There is considerably excitement over the X. laevis genome assembly because initial expectations were modest and have been surpassed. In fact, a de novo assembly is possible even with the short reads inherent to HTS and does deconvolute the genome duplication (Dan Rokhsar and Richard Harland, personal communication). Additional details of the X. laevis genome projects are detailed in Gilchrist (2012).
Reducing the time from gene identification to analysis is a pressing need for the Xenopus community. In Xenopus, a myriad of high-throughput assays lead to gene discovery, and the same set of transcripts can be analyzed in a host of different ways rapidly. However, often the rate-limiting step is identifying full-length clones and transferring them into appropriate expression vectors with or without protein tags that are in the correct frame.
The Xenopus ORFeome project, led by Todd Stukenberg (Univ. Virginia) and David Hill (Dana-Farber), will clone full-length transcripts from nearly all genes into Gateway® (Invitrogen) compatible vectors. The Gateway system allows efficient shuttling of inserts to various “destination” vectors, maintaining the reading frame to permit creation of a host of fusion proteins using an identical recombination step. The ORFeome will comprise a complete set of nonredundant cDNA clones for high-throughput expression cloning, a powerful gene discovery strategy in Xenopus. The ORFeome also allows sets of genes to be rapidly fused to fluorescent, epitope, and purification tags for large-scale functional analysis [for further details, see Gilchrist, 2012.
Xenbase: The Xenopus Database
Major model systems are supported by organism-specific databases, providing access to gene annotations, references, gene expression resources, reagents, protocols, and community information. Using his own laboratory resources, Peter Vize then at the University of Texas—Austin, created Xenbase to collect gene expression data and plasmid information. Although helpful to the Xenopus community, its scope was necessarily limited. However, with the onset of genome and large-scale EST data sets, the need for Xenbase expansion became urgent (Bowes et al., 2008). With NIH support under the joint supervision of Peter Vize (Univ. Calgary) and Aaron Zorn (Cincinnati Children's Hospital), Xenbase has evolved into a critical resource for all aspects of Xenopus research. In particular, Xenbase creates and updates gene pages that organize and store a plethora of gene specific information and present it in an easy-to-use format. It currently features its own genome browser with the latest X. tropicalis (v7.1) genome assembly and annotation as well as chromatin modification data. Xenbase collects and stores gene expression data, not only information collected by large in situ screens, but also by mining Xenopus publications. Xenbase collects information on reagents, protocols, and Xenopus researchers, and stores large data sets for easy access from its FTP site, and includes a Wiki to facilitate communication within the community. Various text-mining tools scour the literature to populate gene pages and provide search tools for users. Xenbase also acts as a useful interface for the stock centers, providing information on availability of lines and reagents. As new genomic resources are created, Xenbase will remain critical as a simple, efficient interface accessing expanding sets of Xenopus data.
Xenopus Stock Centers
The growth in genomic and genetic applications and proliferation of transgenic and mutant frog strains created a need for stock centers to facilitate efficient utilization of these precious resources. Two of these have now been established: the European Xenopus Resource Center at the University of Portsmouth, United Kingdom, and most recently the National Xenopus Resource (NXR) at the Marine Biology Laboratory at Woods Hole, USA. Xenopus has a long tradition at the Marine Biology Laboratory, which hosts the renowned Embryology and Physiology courses, both of which use Xenopus extensively. The resources available from each center are described in detail elsewhere by Pearl et al. (2012).
X. tropicalis Genetics
Although most of the Xenopus community uses X. laevis, the fast-breeding diploid X. tropicalis has been adopted for genetic analysis. Several different screens for heritable defects in X. tropicalis were performed, identifying many different phenotypes (Goda et al., 2006; Grammer et al., 2005; Noramly et al., 2005). Cloning the mutant genes (Abu-Daya et al., 2009; Geach and Zimmerman, 2010) relied on the genomic resources described above, as well as on a meiotic map (Wells et al., 2011). Finally, reverse genetic resources are being developed to identify carriers of mutations in user-selected genes (Goda et al., 2006 and see Abu-Daya et al., 2012).
Development of Future Resources Essential to Support Xenopus
Over 60 members of the Xenopus community met at the MBL in October 2011 to discuss additional resources for supporting Xenopus as a major model for biomedical research. These resources were divided into two categories, Immediate Needs and Essential Resources, and prioritized.
The community identified three pressing needs: (1) novel technologies for implementing loss of function in Xenopus, (2) proteomic resources, and (3) novel transgenic strategies. Although MOs can knock down gene functions in Xenopus, alternatives that avoid limitations of MOs (toxicity, lack of tissue or temporal control, cost) are needed. Possibilities include siRNA (Lund et al., 2011) and genetic methods including zinc finger nucleases (Young et al., 2011), TALENS (Huang et al., 2011), and TILLING (Abu-Daya, 2012), as well as further developing forward genetics. Second, with the availability of genome sequence, proteomic databases are needed to deconvolute mass spectrometry data from biochemical analyses. Finally, new and more efficient transgenic methods would be extremely valuable for F0 enhancer analysis as well as for generating transgenic lines. Some of these new technologies are described by Fish et al., Nedelkovska and Robert, and Zuber et al. (2012). With Xenopus stock centers now operating in both the United Kingdom and the USA, transgenesis can be fully exploited to create, maintain, and distribute transgenic lines.
The Xenopus genomes have significantly facilitated Xenopus research, and ongoing improvement of these resources will have a long and lasting impact; many essential resources are therefore genomics related. A high priority for the Xenopus community is intensive annotation of the genome. Gene modeling and preliminary annotation of most genes are available through Xenbase; however, many gene models are incorrect or missing. Genome improvements will help, but careful annotation is also essential especially to facilitate system-level analyses. Finally, noncoding annotation is also critical and currently incomplete. Xenbase needs continued support and expansion especially as more and more of these genomics data become available. Other needed genomic improvements include information on chromatin modifications, nascent transcriptome sequencing, and additional BAC libraries for X. laevis and X. tropicalis. Genetics will benefit from continued genomics support, including hapmaps and SNP sequencing that will facilitate additional forward genetic screening and analysis of mutants.
The Xenopus community also identified education needs. Both new and established Xenopus investigators would benefit from training to explore new fields or methods. To this end, support was expressed for updating and expanding the existing Cold Spring Harbor manual (Sive et al., 2000) into a more encompassing FrogBook, continued provision for the introductory Cold Spring Harbor Xenopus course, and minicourses focusing on specialized topics at the NXR.
Finally, protein detection remains a challenge for the Xenopus community. Although a plethora of antibodies bind mammalian proteins, a much smaller and more unpredictable subset actually detect orthologous proteins in Xenopus. Generating antibodies to Xenopus proteins would be very useful in many aspects of Xenopus research, and obtaining these antibodies or alternative novel technologies to detect epitopes is an important concern.