Seminars in Cell & Developmental Biology 17 (2006) 146–153

Review

Genetic and genomic prospects for Xenopus tropicalis research

Samantha Carruthers, Derek L. Stemple ∗Vertebrate Development and Genetics, The Morgan Building, Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1HH, UK

Available online 20 January 2006

Abstract

Research using Xenopus laevis has made enormous contributions to our understanding of vertebrate development, control of the eukaryotic cellcycle and the cytoskeleton. One limitation, however, has been the lack of systematic genetic studies in Xenopus to complement molecular and cellbiological investigations. Work with the closely related diploid frog Xenopus tropicalis is beginning to address this limitation. Here, we review theresources that will make genetic studies using X. tropicalis a reality.© 2005 Elsevier Ltd. All rights reserved.

Keywords: Xenopus laevis; Xenopus tropicalis; Genetic; Mutant

C

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1462. Genome Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

2.1. Genome sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1472.2. BAC libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1472.3. Ensembl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

3. Expressed sequence tags (ESTs), full-length cDNAs and microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483.1. cDNA libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483.2. ESTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483.3. Clustering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483.4. Full-length cDNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

4. Annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1494.1. JGI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1494.2. Sanger Institute . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

5. Mutant screens and mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505.1. Inbreeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505.2. Chemical mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505.3. Insertional mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505.4. Gamma-ray mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1525.5. Genetic map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

1. Introduction

∗ Corresponding author. Tel.: +44 1223 496857; fax: +44 1223 496802.E-mail address: ds4@sanger.ac.uk (D.L. Stemple).

For over 50 years researchers have been absorbed by thecellular, molecular and developmental biology of the Xenopusembryo. Due to the external development, large size and robust-ness of the embryos they are easily manipulated and have many

1084-9521/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.semcdb.2005.11.009

S. Carruthers, D.L. Stemple / Seminars in Cell & Developmental Biology 17 (2006) 146–153 147

well established techniques for studying gene function [1]. Manygene activities have been described by means of gain of functionexperiments and the consequential effect on the expression pat-terns of other characterised genes. Although gain-of-functionexperiments have been extremely informative, often enablingone to distinguish between direct and indirect effects of geneactivity, there has been no systematic way to ensure that the nor-mal activity of the over-expressed gene is being reported. Thiscaveat is especially important if the ubiquitously over-expressedgene has a spatially and/or temporally restricted pattern ofexpression in the embryo. One of the most notable technicalinnovations for Xenopus researchers has been the introductionof a reliable loss-of function method using antisense morpholinooligonucleotides (MOs) [2,3]. By microinjection of MOs into azygote of early cleavage-stage embryos the translation, or splic-ing of specific mRNAs can be disrupted. There are, however,some drawbacks to MO experiments. For example, maternalproteins cannot be knocked down and therefore residual activitymay be present. Moreover, the effect of MO knockdown is lim-ited to early development and in many situations the specificityof the MO effect can be difficult to demonstrate [4,5]. This is alsocomplicated in Xenopus laevis, where the allotetraploidy of thespecies often requires several MOs to be simultaneously injectedto ensure paralogues of a given gene are disrupted. Although X.laevis has a well-established utility for embryological, molec-ular and cell biological experiments, it is genetically unwieldyo1t1Mapap

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bly provides a basis for gene discovery, investigations into geneorganisation, and the identification of candidate genes duringpositional cloning.

2.1. Genome sequencing

In 2002 the JGI started sequencing the X. tropicalis genomewith the aim of reaching 8× coverage of the predicted1.7 Gb genome [11]. The genomic DNA used to generate thesequence came from a seventh generation inbred Nigerianline from the stock of Robert Grainger, University of Virginia(http://faculty.virginia.edu/xtropicalis/). The JGI employed awhole genome shotgun sequencing strategy, generating librariesof differing insert sizes sequenced in both directions to producepaired end sequences. The shotgun sequence has been assem-bled into contigs and larger scaffolds using paired end sequenceinformation from the larger fosmid and bacterial artificial chro-mosome (BAC) clones. The present assembly, V4.1, comprisesapproximately 22.5 million paired end-sequencing reads. It hasan average coverage of 7.65×, is approximately 1.5 Gb andis expected to be the final draft assembly (http://genome.jgi-psf.org/Xentr4/Xentr4.info.html). This assembly contains about28,000 JGI predicted gene models whereas Ensembl predicts24,405 genes from assembly V3.0, with difference arising mostlikely from accounting of splice variants rather than assemblyversion. These gene numbers are comparable in number to the X.lcasotmls

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wing to its allotetroploid genome and long generation time of–2 years [6,7]. By contrast, Xenopus tropicalis is very amenableo genetic studies, with a simpler diploid genome comprising0 haploid chromosomes and a 4–6 month generation time [8].oreover, despite the relatively small size of X. tropicalis eggs

nd embryos, standard protocols developed for X. laevis are com-letely transferable to X. tropicalis. Indeed, many X. laevis genesre so closely related to X. tropicalis that in situ hybridisationrobes are often interchangeable [9].

The acceptance of X. tropicalis as a standard model organismor vertebrate genetics will depend strongly on the availabilityf genetic, genomic and molecular biological resources. Thisas recognised by a gathering of Xenopus researchers at a USational Institutes of Health (NIH) Workshop held in March000 [10]. As a result, with the support of the NIH, the Jointenome Institute (JGI) and the Wellcome Trust, the past 5ears have seen the unprecedented development of resourcesor X. tropicalis research. The sequencing of the X. tropicalisenome by the JGI is well underway and there are an abundancef cDNA libraries and corresponding expressed sequence tagsEST) sequences available. Furthermore, the first mutant linesave been identified and with the aid of inbred strains of X. tropi-alis and the generation of a meiotic, genetic linkage map usingimple sequence repeat (SSR) markers, mutated genes can beeadily mapped.

. Genome Project

One major goal for the community was to sequence the X.ropicalis genome, which is about half the size of the mousend a similar size to the zebrafish genome. A genome assem-

aevis Unigene cluster set. Most importantly, 95% of full-lengthDNAs map to the V4.1 assembly, indicating that this is a goodssembly. The genome assembly can now be improved by theequencing of BAC clones, targeted gap closure, incorporationf physical and genetic mapping information and gene annota-ion. On a note of caution, it is difficult to assess the degree of

is-assembly, as no independent comparator, such as a geneticinkage map, is detailed enough now to verify the publishedhotgun assemblies.

.2. BAC libraries

At least three large insert BAC libraries exist for X. tropicalis.or the genome sequencing effort, one library was generated at

he Children’s Hospital Oakland Research Institute (CHORI;ttp://bacpac.chori.org/libraries.php) CHORI-216 and anotherSB-1, at the Institute for Systems Biology, Seattle WA. Ahird library was constructed at Keio University School of

edicine [12]. The CHORI library was generated using DNArom the same X. tropicalis line that was used to create themall insert libraries for the JGI shotgun genome sequenc-ng, hence BAC sequencing provides further data that cane integrated into the genome assembly. As part of a com-unity sequencing program the Stanford Human Genomeenter, in collaboration with the JGI, are producing full-

ength sequence for 422 BAC clones (http://www-shgc.stanford.du/datarelease/Xenopustropicalis.data.html http://genome.jgi-sf.org/sfo/Xenopus tropicalis.html).

These libraries have additional utility. They can be used forargeted gap closure in the genome assembly; this involves BACequencing with primers designed at the end of scaffolds. The

148 S. Carruthers, D.L. Stemple / Seminars in Cell & Developmental Biology 17 (2006) 146–153

BACs are also an excellent resource for promoter cloning toinvestigate the genetic regulation of gene expression as manypromoter elements are disseminated over long distances of morethan 100 kb and these are likely to be contained in a singleBAC. In this light, X. tropicalis is amenable to the creationof transgenic lines to study elements that control specific geneexpression by inserting a reporter, such as a fluorescent proteingene, into a BAC by homologous recombination, then generat-ing a transgenic individual [13–15]. A recently published studyshows a modified X. tropicalis BAC used to create a transgenicX. laevis line in which green fluorescent protein is placed underthe control of the aristaless-related homeobox (Arx) gene [16].Given the established X. tropicalis transgenic technique it shouldbe straightforward to use this method to generate BAC transgenicX. tropicalis lines. While the method can be used to study con-trol of gene expression using transgenic reporters, they can alsobe used to express other gene products, such as dominant nega-tive proteins or GAL4, in the same temporal and spatial patternas the endogenous gene. Despite the increasing availability ofgenomic and cDNA sequence the identification of the Xenopushomologue of a gene can sometimes be problematic. Recently aBAC library was been used to solve such problem in the isola-tion of a Xenopus homologue of PKD1, a gene responsible forautosomal dominant polycystic kidney disease type 1 in humans[17].

2

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so that you can find your gene again as these do not changebetween assemblies, although scaffolds will.

3. Expressed sequence tags (ESTs), full-length cDNAsand microarrays

3.1. cDNA libraries

For some time now cDNA libraries representing a widevariety of developmental stages and adult tissues have existedfor both X. laevis and X. tropicalis. Recently, however, thenumber of new cDNA libraries for both Xenopus species hasincreased significantly; over the past 5 years the number of X.laevis libraries has quadrupled, while the number of X. tropicalislibraries has increased by eleven fold (Table 1; http://www.ncbi.nlm.nih.gov/UniGene/lbrowse2.cgi?AXID=8364). Sev-eral X. tropicalis cDNA libraries from a variety of developmentalstages have been used for gene cloning and for gain of functionscreens to isolate new gene activities [20,21].

3.2. ESTs

The increased availability of good cDNA libraries hasallowed for a significant increase in the cDNA end-sequence.Community contributions of cDNA libraries combined withttAm(nEaure

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.3. Ensembl

All of the sequences generated by the genomics projectseed to be readily and easily accessible to the community. Toacilitate this, in November 2004 the European Bioinformat-cs Institute and Wellcome Trust Sanger Institute released annsembl database for X. tropicalis based on the third genomessembly V3.0 from JGI ([18,19]; http://www.ensembl.org/enopus tropicalis/index.html). Ensembl is a system that pro-uces and maintains automatic annotation on selected eukaryoticenomes. The web-based Ensembl interface allows free accesso a comprehensive database of genome sequence and annota-ion data for 19 species with more species being added regularly19]. Ensembl allows the user to zoom into any point of theenome and analyse it in detail. The region of interest can beound by a blast of a given gene sequence or by text searchor the gene name. Once zoomed into a genomic region alethora of information is available, including aligned X. trop-calis and X. laevis cDNAs and mRNAs, Emsembl predicted

RNAs, and orthologous proteins and Unigene sets. The inter-ace is interactive and clicking on any component will providehe option of retrieving the corresponding sequence. In addi-ion, links to pages where alternative transcripts, intron/exonnformation, protein sequences and top protein BLAST hitsre provided. This amount of information would have pre-iously taken a lot of tedious BLAST searching to accumu-ate and now it is available in seconds. Ensembl is routinelypdated and should incorporate the next JGI assembly. Old ver-ions of Ensembl are available on the Ensembl archive, suchs http://feb2005.archive.ensembl.org/Xenopus tropicalis. It isseful to note Ensembl gene ids (e.g. ENSXET00000000645)

he efforts of several major sequencing centres has lead tohe generation of an enormous number of Xenopus ESTs.ccording to the NCBIs dbEST there are now more than aillion (1,038,272) X. tropicalis and almost half a million

473,289) X. laevis EST sequences (Table 1; http://www.ncbi.lm.nih.gov/dbEST/dbEST summary.html). The number ofSTs available for X. tropicalis is in third place, with only Mousend Human having more ESTs. These sequences are extremelyseful for gene discovery and morpholino design and the cor-esponding cDNA clones can be used to generate probes forxpression pattern analysis and gain of function studies.

.3. Clustering

The EST sequences have been utilised independently byhe Gurdon Institute (http://informatics.gurdon.cam.ac.uk/nline/xt-fl-db.html), NCBI (http://www.ncbi.nlm.nih.gov/Uniene/UGOrg.cgi?TAXID=8364) and Sanger Institute (http://ww.sanger.ac.uk/cgi-bin/Projects/X tropicalis/sang est db

earch) to assemble EST clusters. These are groups of sequenceshat represent unique genes. Sequences that do not fit intony cluster are designated as singletons. The Gurdon Institute

able 1omparison of X. laevis vs. X. tropicalis cDNA and EST resources in 2002 [51]nd 2005 (data from dbEST; http://www.ncbi.nlm.nih.gov/dbEst/index.html)

Year X. laevis X. tropicalis

DNA libraries2002 42 62005 176 66

ST sequences2002 ∼215000 ∼1600002005 473289 1030804

S. Carruthers, D.L. Stemple / Seminars in Cell & Developmental Biology 17 (2006) 146–153 149

Table 2EST cluster comparison of two small four librarya and two comprehensive ESTclustering projects

Sequences clustered Clusters Singletons

Gurdon InstituteFour EST libraries

19713 15748Sanger Institute 17773 25764NCBI Unigene #26

All ESTs24866 7893

Gurdon Institute 39730 41512

a Egg, gastrula, neurula and tadpole.

and Sanger Institute have separately clustered ESTs fromfour libraries and have identified similar numbers of clusters(Table 2). In contrast NCBI periodically clusters the entire X.tropicalis EST database (http://www.ncbi.nlm.nih.gov/dbEST/)as well as mRNA sequences (Table 2). The Gurdon Insti-tute is also undertaking a large-scale clustering project of982,489 ESTs (Table 2). However, this project is ongoingso the numbers may alter significantly as the clustering isoptimised (Mike Gilchrist, personal communication). Thereis a notable variation in the number of clusters generated bythese four projects which no doubt reflects differences in theirmethodology and stringency during cluster assembly as wellas the number of sequences used to build the clusters. Theorigin of the sequences will also no doubt significantly alterthe number of clusters assembled as the Sanger Institute hasused only sequences from libraries of four early developmentalstages whereas NCBI has clustered sequences from a muchwider variety of libraries (hence the approximately 7000 extraclusters). There are a large number of singletons remaining inall the clustering projects and these are thought to be sequencesrepresenting rarely expressed genes, which may be of greatbiological interest. The combination of these clustering projectsprovides a staggering amount of information and all threeInstitutes provide easily searchable web interfaces. The GurdonInstitute presents additional information along with the cluster.For example, the predicted start and stop codons are highlightedaN(colbe

3

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neurula cDNA’s for use in targeted gain of function screens.These cDNA have been sequenced by the Sanger Insti-tute (http://informatics.gurdon.cam.ac.uk/online/xt-fl-db.html;[21,23,24]). The full-length cDNAs facilitate the production ofincredible tools such as micoarrays that can provide a wealth ofgene expression information in one experiment that would havepreviously taken months to accumulate by in situ and/or RT-PCRstudies. Many groups have used X. laevis microarrays and the useof these has been described in detail including a design processfor making custom arrays [25–27]. Microarrays can be used tostudy both global gene expression patterns [28–30] and to inves-tigate expression in a more focused way [31–35]. Furthermore,it has been shown that X. laevis and X. tropicalis microarraysare virtually interchangeable as they work across species [36,37].The abundance of full-length cDNA’s available for Xenopus laysthe groundwork for a wealth of microarray experiments in thefuture. Microarrays can be used to complement existing gainof function and knock down experiments by enabling the rapididentification of altered gene expression and also to identifygene expression changes in mutant lines to compliment candi-date gene approaches when mapping mutants. Having namesassigned to these full-length sequences will be extremely usefulfor speeding up analysis of microarray data.

4. Annotation

mX

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nd sequences are aligned with protein BLAST hits. TheCBI Unigene collection offers digital differential display

DDD; http://www.ncbi.nlm.nih.gov/UniGene/info ddd.html)omparison between libraries. This exploits the informationbtained from clustering to discover the number of times eachibrary is represented in a specific cluster. The information cane compared among the different libraries to identify genesnriched at specific stages or in particular organs.

.4. Full-length cDNAs

In addition to the clustering, many of the cDNA libraries arelso being analysed to find libraries with a high probability ofontaining full-length inserts for the NIH Xenopus Gene Col-ection full-length sequencing project (http://xgc.nci.nih.gov/;22]). This is generating another mass of sequencing datand presently consists of 8062 X. laevis and 2940 X. trop-calis full open reading frame clones. The Gurdon Instituteas also created a unique full-length set of 7000 cDNA’srom four libraries and a smaller set of 2500 gastrula and

Gene annotation takes advantage of the wealth of gene infor-ation available from other species to assign names to predicted. tropicalis genes and is underway by JGI and Sanger Institute.

.1. JGI

The JGI have used their automated annotation pipeline,hich takes the most homologous known gene to assign arovisional gene name to a sequence. This is then manuallynspected and amended where appropriate (http://genome.jgi-sf.org/Xentr4/Xentr4.info.html).

.2. Sanger Institute

The Sanger Institute arranged an annotation jamboree in July005, which brought together 30 scientists from the Xenopusommunity who volunteered to participate in a 5-day intensiveanual annotation session at the Sanger Conference Centre. The

im was to annotate the full-length cDNA’s from the Gurdonnstitute collection (Egg, Gastrula, neurula and tadpole) plus allther X. tropicalis published sequences as of June 2005. Muchreparation was necessary to gain maximum benefit from thisamboree. Accordingly, the approximately 8500 cDNA’s wererranged into families by means of their protein domain similar-ty and protein BLAST hit information. Each gene family wasnnotated by a one person; thereby reducing the chance of over-ap between annotators and promoting the chance of being ableo distinguish between closely related family members, whichould be annotated together. A total of 6855 sequences wererouped into families and the remainder were left to annotatendividually. Gene names were appointed according to human

150 S. Carruthers, D.L. Stemple / Seminars in Cell & Developmental Biology 17 (2006) 146–153

gene nomenclature, when no human name was available a genewas usually called novel protein plus some information aboutprotein domains and/or similarity to genes from other species.When a Xenopus name was available and it was different to thehuman name the Xenopus name was recorded as an alias. Atthe jamboree 4302 clones were annotated, the remaining clonesare presently being finished off at the Sanger Institute largelyby the team 71 HAVANA (Human And Vertebrate Analysis aNdAnnotation) group (http://www.sanger.ac.uk/HGP/havana/). Outof 6074 annotated clones 92% are full length, with identified startand stop codons (Jennifer Harrow, personal communication). Ofthese, 3153 had a known human gene name and 1444 are poten-tially novel genes, many with homologues in species other thanhuman. This information will have been recorded even thoughthe gene was called novel. The number of genes with knownnames plus the novel genes does not add up to the total number ofgenes annotated as there were a large number of duplicates anno-tated. It is apparent that the jamboree assigned names to a largeproportion of genes that have not previously been characterisedin Xenopus and as such the jamboree has produced a profusionof new information for the community. On completion the datawill be made available by publication, re-submission to the Emblnucleotide sequence database (http://www.ebi.ac.uk/embl/) viathe third party annotation route, integration into Ensembl,and incorporation into the existing X. tropicalis EST website(http://www.sanger.ac.uk/Projects/X tropicalis/). Annotation isipm

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rapid identification of mutants as it circumvents the require-ment to raise a second generation for heterozygote crosses until aknown mutation is found. This method has revealed 42 potentialmutant phenotypes of which 10 have currently been confirmedas genetically heritable recessive mutations. These screens haveestablished the viability of genetic investigations in X. tropicalisand have led to the discovery of some fascinating mutations.

5.2. Chemical mutagenesis

Diverse approaches to chemical mutagenesis screens arein progress. These are classic forward genetics, gene focusedreverse genetics and phenotype focused reverse genetics. Incollaboration with Lyle Zimmerman, National Institute forMedical Research (NIMR, London) we have used ethylni-trosourea (ENU) mutagenesis of mature sperm to in vitro fer-tilise wild-type eggs and generate a library of mutation carryingX. tropicalis frogs. A forward genetic screen of these muta-genised frogs using a two-generation gynogenetic approachhas uncovered recessive mutations in heterozygous females.Using this method we have identified at least 76 putative muta-tions that produce a variety of developmental phenotypes (Godaet al., manuscript in preparation; Fig. 1 , for an interactiveversion see http://www.nimr.mrc.ac.uk/devbiol/zimmerman/enu phenotype/). The assembled genome sequence and geneticmap will be essential for the subsequent identification of inducedm

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mportant for genetics as it enables studies of gene clusters, com-lements microarray studies and will assist positional cloning ofutants by conveying information on candidate genes.

. Mutant screens and mapping

The principal reason for establishing X. tropicalis as a modelrganism was the insufficiency of X. laevis as a genetic model.lthough inbreeding in X. laevis has led to the identification ofutations [38] the complexity of its genome is not amenable to

ystematic genetic investigations. There are a number of ongoingenetic screens using different approaches to identify mutations.

.1. Inbreeding

Inbreeding the X. tropicalis Nigerian strain led to the identifi-ation of three recessive embryonic lethal mutations that displayhe expected Mendelian ratios when heterozygous carriers arerossed [39]. In this study the authors show the isolation ofouble and triple heterozygous carriers of these mutations andurther show that it is relatively straightforward to demonstratehat these are independently segregating mutations consistentith the expected Mendelian ratios. This is in part due to the

arge number of individuals available in a single clutch of X.ropicalis embryos. By inbreeding the authors were also able tosolate a strain of X. tropicalis that is free from the background

utations. Another group have carried out a gynogenetic screenn four wild X. tropicalis populations [40]. This entails in vitroertilisation of eggs (from wild lines) with UV-irradiated spermollowed by high-pressure treatment to prevent polar body for-ation resulting in gynogenetic diploid embryos. This allows

utations.A reverse genetic screen is also in progress, this is based on

method called TILLING (Targeting Induced Local LesionsN Genomes; [41–44]). In this approach we design primers toCR amplify exons of particular genes from genomic DNArom the library mutation carrying individuals (Fig. 2 ). PCRroducts are then sequenced and computationally analysed toetect point mutations. Thus far this project has identified 5onsense mutations in early exons of different genes and we areurrently attempting to recover these from frozen sperm (Godat al., manuscript in preparation). These screens are intendeds a resource for the community, thus mutant lines will be dis-ributed freely and gene requests are welcome for the TILLINGcreen. Another screen is using ENU spermatagonial mutage-esis and a cold shock method of gynogenesis similar to thatreviously described for fish [45]. The screen is designed todentify mutations of interest by visual inspection of phenotypesirectly and/or by in situ or transgenic gene expression analysis.o date a number of mutants have been identified and 8 haveeen confirmed genetically (Robert Grainger, personal commu-ication). Mutations that do not fit the criteria will be distributedo other groups with correlated interests.

.3. Insertional mutagenesis

The gene trap approach is another method with potentialor generating mutant X. tropicalis and this prospect is beingursued by a number of groups (http://www.gurdon.cam.c.uk/∼amayalab/Flashver/Interests/trop.html; http://faculty.irginia.edu/xtropicalis/methods/methaim4-b.htm; http://www.tjuderesearch.org/depts/pathology/meadlab/index.html). The

S. Carruthers, D.L. Stemple / Seminars in Cell & Developmental Biology 17 (2006) 146–153 151

Fig. 1. Phenotypic classes of mutations recovered from a gynogenetic forward screen. Adult F1 females (see Fig. 2) derived from IVF after in vitro sperm mutagenesiswere screened by gynogenesis. Eggs were squeezed from F1 females and then fertilised using ultraviolet light treated sperm. Without further manipulation the resultanteggs would produce haploid embryos. Instead UV-sperm fertilised eggs were subjected either to a brief period of low temperature, which prevents the first cleavage,or to high hydrostatic pressure which prevents anaphase of meiosis II. Either treatment produces diploid embryos, in which regions of the maternal genome aredriven to homozygosity, uncovering induced mutations. These diploid embryos were scored for mutant phenotypes and shown are the names of classes of phenotypeidentified in such a screen. More detailed phenotype information can be found at http://www.nimr.mrc.ac.uk/devbiol/zimmerman/enu phenotype/.

gene-trap method is especially attractive since a mutated genecan be cloned relatively easily using the insertion site asan initiation point. The objective of a gene trap is to obtainintronic transgene insertions in the genome. For a gene-trap thetransgene consists of a splice acceptor fused to a reporter such

as GFP. In theory the GFP would then be spliced into the middleof an mRNA creating a chimera and hopefully disrupting genefunction. The chimera is expressed under the control of therespective gene promoter and therefore also functions as areporter construct. This approach has been successful in X.

Fig. 2. Pipeline for chemical mutagenesis and identification of induced mutations in Xenopus tropicalis. Isolated testes were treated with ENU in vitro and usedto fertilise eggs of wild-type females in vitro (IVF). Mutagenised sperm are considered to be generation zero (G0). The IVF progeny, family one (F1), were raisedto adulthood. F1 females were used for forward genetic screens (see Fig. 1) and F1 males were sacrificed, their testes were frozen as well as used to generate F2tadpoles. F2 DNA was isolated for reverse genetic (TILLING) screens. In TILLING screens the genomic sequence of a selected gene is used to design nested PCRprimers. Amplicons are sequenced and induced mutations are detected. Mutations can be recovered from frozen testes by IVF.

152 S. Carruthers, D.L. Stemple / Seminars in Cell & Developmental Biology 17 (2006) 146–153

laevis [46,47]. A different insertional mutagenesis approachaims to use transposable elements. In this case double transgenicmale lines carrying a GFP transposon and a tranposase under thecontrol of a testis specific promoter would be used to stimulatetransposon hopping events during sperm development. Crossingthe double transgenics to wild-type females should then revealnew GFP expression patterns in the progeny [48]. Alternativelythe transposon DNA and trasposase mRNA can be co-injectedinto embryos resulting in heritable integration into the genome[49].

5.4. Gamma-ray mutagenesis

Gamma-ray mutagenesis is being employed to producean array of chromosome mutations, small or large deletions,inversions, translocations and other alterations (http://tropicalis.berkeley.edu/home/mission/RMH-Mutagenesis.html). Emb-ryos are assayed for morphological phenotypes as well ascomprehensive in situ screening at various stages. This has leadto the preliminary identification of seven mutant lines that areundergoing further confirmation and characterisation.

5.5. Genetic map

One prerequisite for the transformation of X. tropicalis intoan efficient genetic model is the generation of a genetic link-atoHltegmposrraoltfs

6

maaiiwg

Acknowledgements

We would like to thank Jennifer Harrow at the Sanger Insti-tute for providing annotation statistics prior to publication, MikeGilchrist at the Gurdon Institute for providing current ESTclustering data, Lyle Zimmerman at NIMR for allowing us toreproduce his figure, Robert Grainger for sharing unpublisheddata and Kathy Joubin and Mario Caccamo for comments on themanuscript. S.C. and D.S. are supported by the Wellcome Trustand NIH grant 1 RO1 HD4 2276-01.

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