Insights from Xenopus Genomes

6/05/06 7:34 PM Page 138 Volff J-N (ed): Vertebrate Genomes. Genome Dyn. Basel, Karger, 2006, vol 2, pp 138–153 Insights from Xenopus Genomes N. Pollet, A. Mazabraud Abstract f Laboratoire Développement et Evolution, Université Paris-Sud, Orsay, France Amphibians have been used since the 19th century as vertebrate models for the experimentalist. Since 50 years or so, Xenopus laevis is the most widely used anuran amphibian research organism. However, because it is a pseudo-tetraploid species, its genetics has been lagging behind. Contemporary studies shift their focus to the only Xenopus species known to be diploid, the small African tropical clawed frog Xenopus tropicalis. A complete genome project is undertaken, with genetic and physical mapping going alongside cDNA and genome sequencing. Currently, X. tropicalis is the most distantly related vertebrate species to humans that still exhibits long-range synteny. Much of amphibian genetics can be learned from this genomic undertaking, and could shed light on fascinating biological processes such as embryogenesis, regeneration and metamorphosis. Moreover, Xenopus species are exciting models for the study of gene duplication because new species can evolve through allopolyploidization, a type of genome duplication that can result from hybridization among species. The current genomic resources for Xenopus briefly described here, combined with the practical experimental advantages of this non-mammalian vertebrate model, make it ideally suited for systematic functional genomic studies. Pr oo GDY138.qxd Copyright © 2006 S. Karger AG, Basel The anuran amphibian Xenopus is one of the favorite models of biologists, especially embryologists [1–3]. The embryo can be easily manipulated because it is larger than other vertebrate embryos such as fish and mouse embryos. The entire developmental process occurs externally. This makes the embryo accessible to observation and experimentation at all stages. Moreover embryos are easy to obtain in large quantities (from hundreds to thousands per female) and develop rapidly (organogenesis is completed after 3 days). Early cell fate decisions, body plan patterning and early organogenesis are processes where Xenopus proved to be a good model. Indeed, Xenopus was the first cloned vertebrate animal. Contributions in cell biology and biochemistry include seminal work on chromosome replication, chromatin and nuclear 6/05/06 7:34 PM Page 139 f assembly, control of the cell cycle, in vitro reconstruction of cytoskeletal dynamics, and signaling pathways. Late development at metamorphosis and its hormonal control provides another fruitful research area pertinent to human disease. Xenopus has become an appreciated model for ontogenetic and phylogenetic studies of immunity. Finally, embryos and tadpoles are used in toxicological testing worldwide. We know a lot on Xenopus development, but its genome is still poorly characterized. This is due to its large size. Amphibians are well known as an example of the C paradox, and Xenopus is no exception to this. X. laevis has a long generation time and is pseudo-tetraploid, thereby hindering genetic analyses. X. tropicalis, which is diploid and has a shorter generation time, is attracting increasing interest (fig. 1). In this article, we will review the latest development on Xenopus genomics. We will report to the reader the characteristics of the Xenopodinae subfamily, the status of the current research on genetic, physical and gene mapping, the genome sequence and the repeat content. Finally, we will present the ongoing efforts concerning functional genomics in Xenopus. Pr oo GDY138.qxd Xenopodinae Species In the literature, the terms African clawed frog and Xenopus refer most often to Xenopus laevis laevis. However, the X. laevis group alone includes six morphologically distinct subspecies of which X. laevis laevis is but one. A total of 17 Xenopus and Silurana species have been identified and together compose the Xenopodinae subfamily of Pipidae (tongueless frogs with a principal aquatic life, fig. 1b). Here we refer to the preferred scientific name Xenopus tropicalis and not Silurana tropicalis (the same applies for X. epitropicalis). We strongly suggest to our colleagues and to Journal editors to enforce the use of this same terminology to avoid any confusion. Remarkably, it was established that these Xenopus species form a polyploidy series, from the diploid (2n) X. tropicalis to the dodecaploid (12n) X. ruwensoriensis (fig. 1b). These different levels of ploidy are due to interspecific hybridization events that took place over a period ranging from 30 to 55 million years ago [4]. Hence all but one Xenopus species are allopolyploids. Xenopodinae thus provide an excellent animal system to investigate polyploidization and the fate of duplicated genes, also called allogenes since they derive from an allopolyploidization event. In X. laevis, some genes are present at the diploid state, with one individual expressing the two alleles, while others have been conserved as duplicated ‘allogenes’ with various degrees of divergence, generally less than 10% [5]. Duplicated genes may have distinct expression Insights from Xenopus Genomes 139 GDY138.qxd 6/05/06 7:34 PM Page 140 Danio rerio (Zebrafish) Oryzias latipes (Japanese medaka) Tetraodon nigroviridis (Spotted green pufferfish) Takifugu rubripes (Tiger pufferfish or ‘Torafugu’) Homo sapiens (Human) Pan troglodytes (Common chimpanzee) Macaca mulatta (Rhesus macaque) Mus musculus (House mouse) Rattus norvegicus (Norway rat) Canis familiaris (Domestic dog) Felis catus (Domestic cat) Equus caballus (Horse) Sus scrofa (Domestic pig) Bos taurus (Domestic cattle) Ovis aries (Domestic sheep) Gallus gallus (Domestic fowl) Xenopus laevis (African clawed frog) Xenopus tropicalis (Tropical clawed frog) Paleozoic 400 Mesozoic 300 a 200 Cen. 100 0 1 2 3 4 0 Haploid genome mass (pg) f Million years ago Pr oo X. I. laevis victorianus poweri sudanensis petersi X. gilli X. largeni X. vestitus X. wittei X. fraseri X. pygmaeus X. ruwensoriensis X. amieti X. andrei X. boumbaensis 30–50 MY X. clivii X. borealis X. muelleri X. longipes X. tropicalis b ploïdy: 2N!20 X. epitropicalis 4X ! 36;40 8X !72 12X !108 Fig. 1. Xenopus species and ploidy. a Genome evolution in craniates, and taxonomy of X. laevis and X. tropicalis. Divergence and genome size in pg are shown. It is assumed that 1 pg of DNA is equivalent to 1 Gpb. Adapted from [58]. b The Xenopodinae subfamily and its phylogeny according to [4]. Pollet/Mazabraud 140 6/05/06 7:34 PM 0 5,000 Page 141 10,000 15,000 20,000 0 X. tropicalis slug 2,000 Repeat 1 Exon 1 Exon 2 4,000 6,000 Exon 3 8,000 X. laevis slug " Repeat 2 10,000 Repeat 3 12,000 14,000 X. laevis slug # Repeat 4 f 16,000 18,000 20,000 Pr oo GDY138.qxd X. tropicalis slug X. laevis slug " X. laevis slug # Fig. 2. Xenopus slug genes. A dot-matrix comparison of X. tropicalis slug with X. laevis slug " and # allogenes using the dotter software is represented. A sliding window of 22 bp was used, scores were computed using a $5/%4 matrix and pixel were drawn with grey levels calculated as a function of the score. A threshold corresponding to 66% identities was used. Location of exons are shown on the right, as well as the presence of repeats found only in X. tropicalis (repeat 1), only in X. laevis slug " (repeat 3) or in both X. laevis allogenes (repeat 2 and 4, which differs in size). The scale in bp is shown on the axis. patterns [6] and there is no reliable quantitative estimate on duplicate gene retention at the genomic scale. The Major Histocompatibility Complex (MHC) is one of the most intensely studied cases of gene duplication fate in Xenopus species and has been shown to be functionally diploid in most Xenopus species with the exception of X. ruwenzoriensis [7]. Another example is the gene slug characterized in both X. tropicalis and X. laevis where two allogenes coexist. This case shows that the divergence between each allogene in X. laevis is equivalent to the divergence between each allogene and the X. tropicalis gene [8]. This observation is confirmed by similar comparisons at a larger scale using EST data (M. Gilchrist, unpublished observations). Specific tandem repeat combinations are found in both species at this locus (fig. 2). This tells us that the ancestor of X. tropicalis is not one of the two parental species that lead to the X. laevis group. Insights from Xenopus Genomes 141 6/05/06 7:34 PM Page 142 Xenopus tropicalis f X. tropicalis presents the same advantages as the X. laevis species but it is diploid and has a smaller genome (1.7 vs. 3.1 Gbp, fig. 1a), and a shorter generation time (5 and 9 months for males and females, respectively [2, 9]). Thus, scientists interested in amphibian genome characteristics preferred to decipher the genome of X. tropicalis. Fortunately, most of the work done previously on X. laevis is directly exploitable on X. tropicalis [9]. However, some gene products are not as conserved as others and the recent identification of the olfactory binding protein is a good example since only 54% of amino acids are similar between both species [10]. Some nucleic probes for homologous genes may even not cross-hybridize between the two species. Since X. tropicalis is diploid it has the potential to become a better model in developmental genetics than X. laevis (pseudotetraploid) and zebrafish (duplicated genome). Important genomic resources are underway: physical and genomic map, cDNA collections and genome sequencing (see below Xenopus genomics). Pr oo GDY138.qxd Xenopus Karyotypes Tymowska and collaborators have extensively studied the karyotypes of twelve Xenopus species (e.g. [11]). The 3.1-Gbp haploid genome of X. laevis is divided into 18 chromosomes. Seven groups of metacentric and acrocentric chromosomes from 135 to 249 Mbp in size can be defined from the observation of a X. laevis mitotic karyotype. The nucleolar organizer is located on chromosome 12. Upon examination of X. laevis BrdU-banded chromosomes at the prophase stage, 630–650 early and late replicating bands can be identified [12]. The 1.7-Gbp haploid genome of X. tropicalis is divided among 10 chromosomes. The pair number 5 contains the nucleolar organizer region, and the pair number 6 harbors a heterochromatic region. The karyotypes of X. tropicalis and X. epitropicalis (2N ! 40) contain one special chromosome that is not present in any other Xenopus species. It is the smallest chromosome, it is submetacentric and has been classified as the number 7. This chromosome would have translocated by non-reciprocal tandem-like rearrangement on a different chromosome to reduce the diploid karyotype of the X. tropicalis ancestor to 2N ! 18 (compared to 2N ! 20 in the X. laevis ancestor). Amphibian males are homogametic (Z/Z), like birds but unlike mammals, while the females are heterogametic (Z/W [13]). Studies on the expression of the gene for H-Y cell surface antigen have confirmed heterogamety in X. laevis females [14]. Pollet/Mazabraud 142 6/05/06 7:34 PM Page 143 The giant nucleus, or germinal vesicle (GV) of Xenopus oocytes is ideal for both biochemical and cell biological studies. It contains the so-called lampbrush chromosomes, giant structures in which actively transcribing genes are visible by conventional light microscopy. Xenopus lampbrush chromosome morphology allows the identification of bivalents individually [15]. However, the correspondence between lampbrush and mitotic chromosomes is unknown and awaits molecular markers. Xenopus Genomics f Genetic Mapping Graf published a provisional genetic map for X. laevis in 1989 [16]. It included 28 loci, and one mutation (periodic albinism), placed on 10 linkage groups out of the 18 chromosome pairs. Sex was found to be determined by alleles at one locus. The numbering of chiasmata indicates that the mean number of crossing-over occurring during meiosis in the female germ-line is 33.4 [15]. The female genetic map is estimated to be 1670 cM long. Given an estimated 3.1-Gbp haploid genome, we can estimate that 1 cM is roughly equivalent to 1.8 Mbp. A first genetic map for X. tropicalis localized 51 AFLP and 2 isozyme markers. They were assigned to 13 multipoint linkage groups on a map of the maternal strain, whereas 9 AFLP markers from the paternal strain are assigned to 3 linkage groups on a separate map [17]. A second genetic map is constructed by the group of Amy Sater using microsatellite markers (both tri- and tetranucleotide repeats) identified from each scaffold of the genomic sequence. Currently, it is made of 345 markers distributed on 14 linkage groups. The genetic size of the map is 560 cM (http://tropmap. biology.uh.edu/) and it is nicely integrated with the genome sequence. These maps will be useful to study sex determination in amphibians and to map mutants. These linkage maps have been built using a cross between animals issued of one nearly inbred line (Nigerian) and an outbred one. In the future, several inbred lines will be necessary to ease the genetic mapping of mutants. Pr oo GDY138.qxd Physical Mapping Traditional in situ hybridization was successful in locating different repeat families in X. laevis chromosomes [18]. By means of FISH, IgH was mapped to the long arm of pair 1 and both MHC and Xenopus non classical class I genes (XNC) loci were mapped to the same chromosome, from the 12–18 group [19]. The c-SRC1 (v-src sarcoma viral oncogene homolog) locus was mapped to another chromosome from the same group as the MHC [20]. Using the same technique, both malate dehydrogenase 2 allogenes were mapped: Mdh2a to Insights from Xenopus Genomes 143 6/05/06 7:34 PM Page 144 f chromosome q3 and Mdh2b to chromosome q8 [21]. Even though somatic cell hybrids have been obtained in X. laevis, they have not been extensively used for chromosomal mapping [22]. Large insert genomic libraries based on BAC clones have been constructed for both X. laevis and X. tropicalis. The group of Shimizu obtained a 6 genome equivalent BAC library from X. tropicalis sperm DNA [23]. Shizen Qin at the Institute for Systems Biology has made a BAC library of 75-kbp mean insert size and 6.8& coverage. The DNA was isolated from erythrocytes of a female X. tropicalis (Nigerian strain), partially digested using HindIII or MboI and cloned in pBeloBAC11. The BAC-PAC resource of the Children Hospital Oakland Research Institute (CHORI) has produced a 175-kbp mean insert size library in the pTARBAC2.1 vector that covers 6.6 times the genome (CHORI-216). The DNA was isolated from erythrocytes of a male X. tropicalis (Nigerian ‘N7’ strain, i.e. in the 7th generation of inbreeding) and partially digested using EcoRI/EcoRI methylase before cloning. However, the screening for 215 loci of this CHORI-216 BAC library showed either an over- or an under-representation (20 loci with more than 20 clones, 75 loci without positive clones; http://tropicalis.berkeley.edu/ home/genomic_resources/bac/bac_bias.html). A second library has been made using an MboI partial digest and the pTARBAC1.3 vector that showed less bias since 70% of the previously not represented loci in the CHORI-216 library gave positive results. An X. laevis library was made at CHORI BAC-PAC resource using EcoRI/EcoRI methylase digestion and the pBACGK1.1 vector. The mean insert size is 144 kbp and the estimated coverage is 9.8&. It has not been extensively characterized by screening for defined loci and unfortunately, for now there is no physical mapping project for X. laevis. All these libraries are available from this BAC-PAC resource (http://bacpac.chori.org/). The Washington University Genome Sequencing Centre has fingerprinted over 268,224 clones from 2 X. tropicalis libraries and produced 147,072 endsequences as well. Some (about 500) of these BACs are being shotgunsequenced in the framework of the ENCODE project, or by the Joint Genome Institute. Finishing of these BACs proves to be difficult due to the nature of the repetitive elements present (fig. 3). Nevertheless, the development of a Xenopus tropicalis physical map is important to ensure a long-range continuity of the genomic sequence, eventually at the chromosomal level. Pr oo GDY138.qxd Gene Mapping: X. laevis and X. tropicalis cDNA Sequencing Both X. laevis and X. tropicalis cDNA sequences (ESTs and full-length cDNAs) have been massively produced with an international support. The major contributors are the NIH and Department of Energy’s Joint Genome Institute in the U.S.A., the Sanger Institute and Wellcome Trust/Cancer Research UK Gurdon Institute in the U.K., the NIBB and NIG in Japan, the CNRS and Génoscope in Pollet/Mazabraud 144 6/05/06 7:34 PM Page 145 f VegT CH216-139C6 0 Pr oo GDY138.qxd 100 Zic3 CH216-1C3 "-Globin CH216-11K6 200 kb Fig. 3. Repeat content of individual X. tropicalis BAC clones. Sequences of three different BACs isolated from the VegT, Zic3 and "-globin loci were analyzed using the Miropeats software with a stringent threshold of 500 to highlight long stretches of conserved repeats. Each BAC is drawn as a horizontal line. Each repeat is drawn as a black rectangle, and similar DNA sequences are connected by curves. The top BAC is nearly devoid of tandem repeats, the bottom BAC is mildly composed of such repeats and the middle BAC proves to be filled of tandem repeats, rendering its sequencing very difficult to finish. This situation proves to be not so uncommon. Insights from Xenopus Genomes 145 6/05/06 7:34 PM Page 146 f France and the RZPD and DKFZ in Germany. The collection of X. tropicalis ESTs is currently the third largest one in dbEST, with 1,038,272 ESTs and X. laevis is 11th with 473,289 ESTs listed. A collection of 34,282 cDNAs partial sequences from a urodele, the axolotl Ambystoma mexicanum, may prove quite useful for amphibian comparative genomics [24]. To sequence potential full-length (FL) clones, the Xenopus Gene Collection used the Mammalian Gene Collection pipeline and produced FL cDNAs for 7,810 X. laevis and 2,825 X. tropicalis genes [25]. Candidate clones to promote the project are available and await further sequencing capacity. The Wellcome Trust/Cancer Research UK Gurdon Institute assembled a collection of full-length cDNAs for X. tropicalis in expression vectors, suitable for functional assays of individual or pooled clones [26] (http://informatics.gurdon. cam.ac.uk/online/xtfl-db.html). An annotation workshop was held in August 2005 at the Sanger Institute with the goal of analyzing all the 8,572 full-length cDNAs (4,173 from Sanger Institute, 3,397 from XGC and 1,002 from other sources). This enabled to identify that 6,207 (72%) have a complete CDS. A total of 5,004 gene names were attributed, 1,705 being potentially novel (34%). The shortest cDNA is 110 bp long (TGas048h09), the longest is 8,372 bp long (BC095906) and the mean length is 1,767 bp. The annotation results will be incorporated in the next release of the ENSEMBL annotation of the X. tropicalis genome (http://www.ensembl.org). The German Resource Center RZPD developed a sequence-verified, nonredundant cDNA clone set for X. laevis on the basis of NCBI Unigene Clustering, each clone representing one cluster. The set can be used to study gene expression of almost 13,000 Xenopus transcripts. In summary, cDNA sequencing efforts in both X. laevis and X. tropicalis proceeded at a very rapid pace. Still these cDNA collections are incomplete. In X. tropicalis, the annotation of the assembly in its version 4.1 shows that 45% of the gene models are lacking cDNA representation. Pr oo GDY138.qxd Genome Sequencing The genome of an F6 and an F7 inbred Nigerian female X. tropicalis was sequenced at 8& depth using a whole genome shotgun approach by the Joint Genome Institute from the U.S.A. Department of Energy. About 22.5 millions of paired-end reads from 3 kbp, 8 kbp plasmid libraries as well as fosmid and BAC libraries have been obtained. The mean sequence coverage is 7.65& and the clone coverage is 44.9&. The JGI assembler JAZZ was used to build a genome assembly of approximately 1.5 Gbp, with a total length of contigs of 1.35 Gbp. The assembly in its version 4.1 contains 19,501 scaffolds. Nearly half of the genome covered by the assembly is contained in 272 scaffolds of at least 1.56 Mbp length. Given the coverage of the genome in terms of sequence and clone coverage, the assembly is a bit disappointing. In comparison, the chicken genome Pollet/Mazabraud 146 6/05/06 7:34 PM Page 147 f assembly displays a better long-range continuity. This situation is mostly due to inherent difficulties raised by large amounts of repeated DNA, much of it arranged in long stretches (fig. 3). Such sequences include Xenopus-specific repeats of differing sizes, from 2 to more than 5 kbp. Frequently, in comparing the BAC sequences and the shotgun assembly, these regions are collapsed in the assembly. The JGI annotation pipeline predicted approximately 28,000 gene models. Only 55% of these have support from available X. tropicalis and X. laevis EST and cDNA data. On the other hand, an estimated 5% of cDNA sequences are not mapped on the genome assembly. The average gene length is 16.5 kbp and the average transcript length is 1300 nt, with the average protein containing 409 amino acids. There are approximately 6.5 exons per gene averaging 200 bp each with an intron spacing of 2.8 kbp. The ENSEMBL annotation pipeline is used on the same assembly, and it is not uncommon to have different gene models between the JGI and the ENSEMBL genome browser. The comparison of this genomic sequence with those of other vertebrates highlights a good conservation in synteny with human and chicken genome (fig. 4). Thus, X. tropicalis is the most distantly related vertebrate species to the human species that still exhibits long-range synteny. In conclusion, finishing efforts are required to obtain a better continuity of the X. tropicalis genome. In the future, sequencing the X. laevis genome should be considered since much has to be learned from the comparison of these two related genomes. Pr oo GDY138.qxd A Panorama of Xenopus Repetitive Elements In the 1970s, seminal dissociation-reassociation studies showed that 20–30% of X. laevis genomic DNA is composed of reiterated sequences. More than 20 distinct repeats have been cloned and sequenced but they represent only 20% of this repetitive DNA and 5% of the genome. The most abundant elements were mapped to the genome (satellite DNA, rDNA, tRNA genes). Xstir is the most highly repetitive tandem sequence in the Xenopus genome with an estimated 1 million copies per haploid genome [27]. It is highly dispersed in the genome and mainly organized in small tandem arrays but some elements are found as inverted repeats in MITE of the piggyBac T2-group (Xstir-TIR). A possible reason for the amplification of tandem repeats in Xenopus is the presence of a rolling-circle amplification activity during oogenesis and early development. The satellite 1 sequence, also referred to as the oocyte activation in Xenopus (OAX) sequence and Repetitive HindIII monomer 2, is a highly repetitive 741 bp DNA that exists as dispersed tandem clusters and represents 1.35% of the Xenopus genome [18]. Satellite DNAs are often confined to the heterochromatin present around centromeric or telomeric regions. Satellite 1 is Insights from Xenopus Genomes 147 _8 Tn 8 _1 Gg Gg 7 _1 6 _1 Gg Gg _14 _15 Gg 3 Gg_1 Gg_11 Gg_12 Gg_10 Tn_18 Gg_9 Tn_19 Gg_8 Gg_7 Tn_21 Hs_X _8 Hs 9 _ 10 Hs_ 11 Hs_ Hs b Z g_ G _1 Gg 2 _ Gg _3 Gg _4 Gg _5 Gg 6 Gg_ f Hs_14 Hs_13 Hs_12 Hs_15 Hs_16 Hs_17 Hs_ 18 Hs _19 Hs _ Hs 20 _2 1 _2 2 _6 Hs 7 _ Hs W Scaffold 1 original scale a Hs_ Y Hs _1 Hs _ Hs 2 _3 Hs _4 Hs _5 Hs g_ X. tropicalis 1 0 _1 Tn 11 _ Tn 2 _1 Tn _13 Tn _14 Tn _15 Tn 16 Tn_ Tn_17 Tn_20 G Scaffold 1 normalized scale _9 Tn_3 Xt_1 Tn_1 Tn_2 Gg_28 Tn 9 _1 Chromosome 8 normlized scale Tn_ 4 Tn _5 Tn _ Tn 6 _7 Chromosome 8 original scale GGA8 Gg_27 Page 148 Gg_26 7:34 PM 0 _2 Gg 21 _ Gg 2 _2 Gg _23 Gg 24 Gg_ 6/05/06 Gg GDY138.qxd Pr oo Fig. 4. Example of synteny conservation in the Xenopus genome. Orthologous genes were identified in X. tropicalis and either chicken, human or Tetraodon fish by computing the best reciprocal alignments of gene products. The genomic location of each orthologous gene was extracted and analyzed in their chromosomal context. Portions exhibiting conserved synteny were identified and displayed in linear plots (a) or rosetta windows (b). The results concerning X. tropicalis scaffold 1 (XTR1) are shown. In a, the conservation of synteny with chicken chromosome 8 (GGA8) is obvious and documents a breakpoint and an inversion. In b, each scaffold or chromosome is represented as an arc. Orthologous genes on syntenic segments are connected by lines. Tn: Tetraodon nigroviridis, Hs: Homo sapiens, Gg: Gallus gallus. Conservation of synteny is observed with human chromosome 1. The situation with fish is much more complicated (with the courtesy of Frédéric Brunet, Laboratoire de Biologie Moléculaire de la Cellule, Lyon, France). believed to derive from a transfer RNA (tRNA) gene and it is found specifically expressed in somites during early development [28]. The identification of all X. tropicalis genome reiterated segments is an important component of genome analysis as it can shed light on the evolution of amphibian species. Most interspersed repeats are transposable elements and in Xenopus species it is possible that the increase of genome size concomitant to the serial polyploidization facilitated their expansion. Thus we can hypothesize the presence of active transposons or the signatures of their dynamics in Xenopus. Tc1-mariner superfamily of DNA transposons is very widespread in animal genomes and multiple lineages of Tc1 related transposable elements were identified in Xenopus genome. For most of these TLEs, copies containing an intact transposase ORF suggest that these elements may still be active [29]. Overall, the five X. tropicalis TLE lineages identified represent at least 1% of the genome. A Mariner-like element (MLE) within the genome of a poikilotherm vertebrate was reported in both X. laevis and X. tropicalis [30]. It was found to Pollet/Mazabraud 148 6/05/06 7:34 PM Page 149 f belong to the irritans subfamily of MLEs showing that these elements are present in chordate genomes including most craniates for 750 Myr and that their main mode of maintenance has been via vertical transmission. Other DNA transposons identified in X. tropicalis genome include hAT family members (1,723 and other uncharacterized ones related to Hermes, Homer and the Ciona hAT) and Harbinger family members. Among non-LTR retrotransposons, elements belonging to the CR1, L1 (most numerous) and L2 clade are clearly identifiable. Concerning LTR-retrotransposons and endogenous retroviruses, one can find a variety of Gypsy, (the most numerous belonging to the DIRS superfamily), BRIDGE and CATCH3-related elements as well as ERV3-related endogenous retroviruses. Manipulating Xenopus Genome and Gene Expression Molecular genetics makes successful use of injected DNA, mRNA, antibodies or morpholino antisens oligonucleotides into Xenopus embryos. All these methods are essentially transient, however, as the genome is not altered and the injected substance is progressively degraded by cellular processes. The nuclear transplantation approach was adapted to develop a transgenesis technique called ‘Restriction Enzyme Mediated Insertion’ (REMI) [31]. This method produces non-mosaic and stable transgenic animals in F0, but suffers from its technical complexity. In addition, transgenes are inserted as concatemers at one or several chromosomal positions creating damages or insertional mutations into cellular genes. Most recently simpler and more powerful transgenesis methods were developed that use phage phiC31 site-specific integrase [32] and I-SceI meganuclease [33]. Transgenesis in Xenopus has been employed to study gene regulatory regions [34] and to assess the effects of misexpressing wild type or dominantnegative forms of various gene products [31, 35]. Germ-line transmission of transgenes in Xenopus has been demonstrated and several transgenic lines have been generated [36]. Recently, more elaborated tools allowing inducible gene expression [37–39], BAC transgenesis [40] or the use of site-specific recombinase have been developed [41, 42]. Finally, loss-of-function studies using morphant technology has been shown to work efficiently in frog [43]. These approaches extend the utility of Xenopus beyond the earliest stage to any stages during embryogenesis and reinforce its advantages. Pr oo GDY138.qxd Xenopus Functional Genomics With the advent of functional genomics approaches, studies of large-scale gene expression screening were performed in Xenopus [44, 45]. These studies Insights from Xenopus Genomes 149 6/05/06 7:34 PM Page 150 f helped define the notion of synexpression groups that prove to be most useful in deciphering BMP and FGF signaling pathways [46]. Since then more in situ screening was performed in Xenopus and led to a growing collection of embryonic gene expression patterns. In parallel, large-scale functional screening has been performed using gain-of-function experiments by means of mRNA injections [47, 48]. A loss-of-function screen using morpholinos was conducted on a pilot scale and showed the interest of mining gene expression data for such endeavors [49]. The REMI transgenic method was also used to conduct genetrapping experiments, but the difficulty of recovering insertion sites is a significant limitation [50]. RNA interference has been reported to be effective in few cases, however it is not yet the knock-down method of choice for Xenopus [51]. Various microarrays are available, including one developed by Affymetrix and several studies have made use of this resource [52, 53]. Xenopus tropicalis Genetics Currently, X. tropicalis is the best model available to study amphibian genetics, but it is in its infancy. Few laboratories have pioneered genetic studies in X. tropicalis by looking for mutants, either by inbreeding various strains, or by mutagenesis using ENU or gamma-irradiation [54, 55]. Targeted mutagenesis is developed by a couple of laboratories [56]. Pr oo GDY138.qxd Databases The Xenopus community has just recently benefited from the model organism database Xenbase that serves as a hub for all kind of informations (http:// xenbase.org). 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Science 2002;297:1283–1285. f 51 7:34 PM Nicolas Pollet, Laboratoire Développement et Evolution CNRS UMR 8080, Université Paris-Sud 91405 Orsay CEDEX (France) Tel. $33 1 69 15 72 73; Fax $33 1 69 15 68 16, E-Mail Nicolas.Pollet@ibaic.u-psud.fr Pr oo GDY138.qxd Insights from Xenopus Genomes 153