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358Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00PII: S0168-9525(98)01552-2

Genetic mosaic analysis has long been a powerful toolin the service of geneticists studying a variety of species,and recent developments have permitted mouse geneti-cists to wield it with increasing precision. Althoughchimeras have been used for many years to dissect thephenotypes of spontaneous mouse mutants, such studieswere often limited by the molecular markers available toidentify and follow the mutant contributions. The devel-opment of gene targeting and embryonic stem-cell (EScell) technology has extended the range of methods forproducing mice that are mixtures of wild type and mu-tant cells. In addition, a variety of in situ cell markershave been developed to allow tracing of the distributionof the contributing cell types. It is time, then, to con-sider carefully what kind of information can be gainedfrom analysing genetic chimeras and mosaics and whatstill limits their use in mice.

Mouse chimeras as tools for mosaic analysis of mutantsIt is clear that simply examining the phenotype that

results from an embryonic lethal mutation is not alwayssufficient to identify the primary site of action of theaffected gene, because phenotypes can be very complex.Nor does such examination reveal possible roles for thegene in later lineages, because the mutant phenotype re-flects the earliest requirement for the gene’s function. Aswill be discussed later, new techniques of tissue-specificgene targeting and lineage-specific gene rescue providevery precise tools to help solve these problems but arecurrently very technically demanding. However, the classicmouse chimera, generated by mixing mutant and wild-typecells at preimplantation stages, can yield a surprisingamount of information about when and where a gene isrequired in development (see Box 1).

Chimeras can be made by aggregation at the eight-cellstage or by injection at the blastocyst stage. Which of theresulting tissues are of mixed genotype depends on the

stage of the host embryo and the potency of the donorcells (Fig. 1). Before the advent of molecularly taggedmutations made in ES cells, making chimeras betweenwild-type and homozygous lethal mutants involved theuse of embryos derived from heterozygous crosses asone of the chimeric partners. Thus, only one in four of theresulting chimeras was of the required genotypic combi-nation. This, in addition to the lack of molecular markersto identify the mutant components, placed severe limi-tations on the use of chimeric analysis. Even if a linkedmarker for the mutation was available, it was notstraightforward to distinguish heterozygote–wild-typechimeras from homozygous–wild-type combinations.These limitations can all be overcome by the use ofhomozygous mutant ES cells, which have the potentialto colonize all the primitive ectoderm derivatives inchimeras1 (Fig. 1). Whatever the nature of the mutation,targeted or spontaneous, homozygous ES cells can bemade directly from embryos of a heterozygous cross, pro-vided that a genotyping strategy is available. For targetedmutations, homozygous cells can also be made directlyin culture from the original heterozygous targeted line,by a variety of strategies2. When such lines are used togenerate chimeras, there is no limitation on numbers ofmutant embryos available and all offspring are of therequired genotypic combination, dramatically simplify-ing chimeric analysis of mutants.

Whatever method is used to generate chimeras, theinclusion of independent genetic markers to follow thefate of the two cell components is critical. In manyinstances when making a targeted mutation, theEscherichia coli b-galactosidase gene is introduced intothe locus of interest. This means that mutant cells canbe traced directly in chimeras, which can be very use-ful, but because the marker is not independent of themutation, the absence of expression in a tissue neednot necessarily mean that the mutant cells are alsoabsent. Such a conclusion can only be drawn when anindependent cell marker is used. Over the years a num-ber of such markers have been used, and all have somelimitations (Table 1). The E. coli b-galactosidase gene is

Chimeras and mosaics inmouse mutant analysisJANET ROSSANT (rossant@mshri.on.ca)

ANDREW SPENCE (andrew.spence@utoronto.ca)

As the number of mouse mutants generated by genetargeting continues to grow exponentially, the challenge isnot how to generate a mutant but how to analyse thephenotype. Genes might play multiple roles in developmentand act in cell-autonomous and cell non-autonomousmodes, making phenotypic analysis complex. Geneticmosaic analysis is a powerful tool for dissecting complexgene functions. Classical preimplantation chimeras madebetween mutant and wild-type embryos can answer manyquestions, and new genetic techniques for generatingrestricted genetic mosaicism promise to enhance thefuture power of mosaic analysis in mammals.

BOX 1. Glossary

Cell-autonomous actionThe function of the gene is required in the cells that exhibitthe original mutant phenotype. In a mosaic, the mutantphenotype occurs only in cells that are genotypically mutantand in all such cells in a mosaic tissue.

Cell non-autonomous actionThe function of the gene is required in cells other than thosethat exhibit the original mutant phenotype. In mosaics, non-autonomous action of a gene in a given tissue is revealed bythe presence of genotypically wild-type cells that exhibit themutant phenotype, or genotypically mutant cells that arephenotypically wild type.

ChimeraAn organism that consists of cells derived from more thanone individual, usually of different genotype.

MosaicAn organism that consists of cells of more than one geno-type. The strict definition requires that the genotypically dif-ferent cells all derive from a single zygote. The term mosaicis also used more broadly to describe any organism of mixedgenotype, whatever the initial cause. Thus, chimeras wouldbe a subset of mosaics under this looser definition.

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now the most widely used marker for chimeric analysisbecause its activity can be detected by a simple histo-chemical stain in whole embryos and in sectionedmaterial. Ubiquitous expression of the b-galactosidasegene had been reported in the ROSA26 (Ref. 3) andother gene trap lines4,5 , making them suitable chimericpartners (Fig. 2a). The new marker system based on thegreen fluorescent protein (GFP) of jellyfish also haspotential for chimeric analysis, especially because it canbe observed directly in living cells6,7. Embryos8, EScells9 and mice10 expressing readily detectable levels ofGFP in all cells have been reported. However, GFPactivity is not well retained after standard embeddingand sectioning9. Lightly fixed vibratome sections, com-bined with confocal microscopy, can provide reason-able cellular resolution (Fig. 2b).

Dissecting lineage-specific gene function indevelopment

Chimeric animals produced by mixing pluripotentcells at preimplantation stages generally show very wide-spread contributions from the donor cells and extensivemixing of cells of the two genotypes within a tissue. Thisproperty of mouse chimeras means that mutant effectscan be analysed in multiple lineages in one animal.Thus, when a mutation results in a complex phenotype,chimeric analysis can reveal the primary sites of actionof a gene without any a priori assumptions based onexpression pattern of the gene. If rescue of the mutantphenotype in chimeras correlates with the exclusion ofmutant cells from a certain tissue, the function of the

gene is probably required in that tissue. For example,embryos mutant for the transcription factor, Twist, diein mid-gestation with failure to close the anterior neuralfolds. Evidence that the phenotype results from defects inhead mesenchyme, and not in the neural tissue, camefrom showing that normal neural-tube closure occurred inchimeras with very high percentages of mutant cells inthe neural folds, provided that there were very few mu-tant cells in the head mesenchyme11. Similarly, the com-plex eye and nasal phenotypes in Pax6 (Smalleye)mutants can be shown to be the result of defects in lens,retinal epithelium and nasal epithelium, because mutantcells are specifically excluded from these tissues in pheno-typically rescued chimeras12. The competition betweenmutant and wild-type cells in a chimera can also revealphenotypic effects not detectable in the mutant embryosthemselves. For example, in chimeras, platelet-derivedgrowth factor receptor mutant cells are highly impairedin their ability to contribute to all muscle cell lineages,although no obvious defect is seen in the developmentof these lineages in the purebred mutants13.

As well as defining the primary site of action of amutation, making embryonic chimeras with mutant andwild-type cells often allows rescue of early embryolethality, and permits analysis of the phenotypes of mu-tant cells in later lineages. For example, a4 integrin nullembryos die early in gestation, but viable chimeras with widespread mutant contributions can be generatedafter injection of mutant ES cells into blastocysts. Inthese chimeras, mutant cells were excluded from the T- and B-cell lineages, indicating a novel role for integrin

Yolk sac

8-cell/8-cell 8-cell/ICM 8-cell/ES Blastocyst/ES Tetraploid/ES

EpiblastPrimitive

endoderm

Trophectoderm

Placenta

FIGURE 1. Tissue contributions in chimeras. The various possible outcomes, in terms of contribution to the three lineages derived fromthe blastocyst are shown for different types of preimplantation chimeras. The primitive endoderm gives rise to the entire endodermlayer of the yolk sac and the trophectoderm to the trophoblast layers of the placenta, while the epiblast gives rise to the entire embryoas well as some extraembryonic cells. Orange–white combinations are embryo–embryo combinations, green–white combinations areES cell–embryo combinations. Solid colours, non-mosaic contribution; stripes, mosaic contribution. Tetraploid embryos are generatedby electrofusion of the blastomeres of a two-cell embryo.

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signalling in lymphopoiesis14, but they were present inthe myocyte lineage, disproving the proposed require-ment for a4 integrin in myogenesis15. Similarly, chimericanalysis of two mutations that were shown to blockembryonic hematopoiesis, Flk1 and Scl (Tal1) showedthat mutant cells were also absent in the definitivehematopoietic cells of the adult16,17, implicating thesame genetic pathways in the two different lineages.

Further definition of multiple roles of a given gene indifferent tissues would certainly be aided by the abilityto restrict mutant-cell contributions to different lineages.The restricted early potential of ES cells provides a spe-cial opportunity to distinguish whether early lethality ofa mutant is due to defects in embryonic or extraembry-onic lineages. Mutant ES cells introduced into wild-typeembryos produce conceptuses in which epiblast lineagesare mixed mutant and wild type, while the trophecto-derm and primitive endoderm derivatives are entirely wildtype (Fig. 1). The reverse situation can be achieved byintroduction of wild-type ES cells into mutant blastocysts.Aggregation or injection of tetraploid embryos with EScells produces the most extreme separation of mutantand wild-type contributions, because the tetraploidcells are excluded from the embryonic lineages and theES cells are excluded from the extraembryonic lineagesof trophoblast and primitive endoderm18 (Fig. 1).

Diploid or tetraploid chimeras have been used toshow that a number of mutants that have severe defectsin the developing embryo at gastrulation have primarydefects in the extraembryonic lineages. For example,the block to gastrulation and mesoderm formation seen in embryos lacking either the HNF4 transcriptionfactor19 or SMAD4, a signal transduction molecule in-volved in TGFb/ BMP signalling20, can be rescued by pro-viding wild-type extraembryonic lineages by tetraploidaggregation.

‘Reverse’ chimeras, in which wild-type ES cells areincorporated into mutant blastocysts, have shown thatextraembryonic tissues of certain mutants can impose amutant phenotype on wild-type embryo cells. Forexample, chimeras in which the epiblast is mixed butthe trophoblast and primitive endoderm lineages aremutant in either the type I activin receptor ACTR1B(Ref. 21), or the potential downstream-signalling mole-cule SMAD2 (Ref. 22), produce relatively normal yolk-sac structures but no tissues of the embryo proper. Thissuggests that signalling events within the extra-embryonic lineages initiate the programme leading tonormal development of the dorso-anterior structures ofthe embryo. A more confined role for extraembryoniclineages in patterning anterior head structures was sug-gested by separate recent chimera experiments with

embryos mutant for nodal, a TGFb-related signalling molecule5 and forthe anteriorly expressed transcriptionfactor, OTX2 (Ref. 23). Accumulatingevidence from many sources haspointed to the importance of primitiveendoderm as a source of signals forpatterning the early embryo24,25, andchimeric analysis of mutants hasallowed a definitive test of thishypothesis.

Obviously, for resolving lineage-specific roles of a gene, it would bevery helpful to be able to bias thecontribution of mutant cells into otherlater lineages as well. The so-calledblastocyst complementation assay,

TABLE 1. Properties of cell markers

DNA GPI Species-specific Multicopy RFLPsa isozymesb satellite DNAc transgene b-Gal GFP

Ubiquitous 1 1 1 1 1 ?Neutral 1 1 ? 1 1 1Cell autonomous 1 1 1 1 1 1Detectable in intact embryos No No ? ? 1 1Detectable in sections No No 1 1 1 ?Single-cell resolution No No 1 1 1 1Simple detection system 1 1 No No 1 1Detectable in living cells No No No No Partial 1

aDNA polymorphisms between strains or between mutant and wild-type cells can be used to quantitate mosaicism in a giventissue by Southern analysis or PCR.bElectrophoretic variants of GPI provides a very sensitive assay of mosaic contributions to tissues40.cIn situ DNA–DNA hybridization to tissue sections provides single-cell resolution of mosaicism when probes to multicopysequences are used. Species-specific satellite DNA probes can be used in interspecific mouse chimeras41, or a high-copynumber globin transgenic strain can be used as a chimeric partner42.

FIGURE 2. Examples of visualization of chimerism in tissue sections. (a) Section of 9.5-day-old ROSA26–wild-type chimera, with b-galactosidase expression revealed by

X-gal staining (courtesy of B. Ciruna). (b) Vibrotome section of ureteric region of adultkidney in GFP–wild-type chimera, with GFP activity revealed by fluorescence

microscopy (courtesy of K. Hadjantonakis).

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first developed for analysis ofmutant effects in the lymphocytelineages26 has the potential toachieve this. In this approach, mu-tant cells are introduced into hostembryos that are genetically impairedin their ability to make certain lin-eages. In resulting chimeras, thoselineages derive entirely from thedonor cells while others are of mixedorigin. Rag2-deficient hosts can beused for populating the lympho-cyte lineage with donor cells26,aphakia mutants for the lens27, andpresumably this approach could beextended to other lineages with theuse of appropriate mutant hosts.

However, whatever kind of pre-implantation chimeras are made,they all have serious limitations fordissecting gene function at all con-ceivable stages of development,because an early role for a gene ina given lineage will result in exclu-sion of mutant cells from that lin-eage throughout succeeding stagesof development, precluding anymeaningful conclusion about laterroles. Mosaics produced by tissue-specific or inducible-gene targetingwill circumvent this limitation.Chimeras can be a first-line tool todefine lineage effects that can befurther analysed using more com-plex genetic strategies.

Production of mosaicsUntil recently, genetic mosaics have been more

important in studies of flies and worms than in mice.However, new techniques of targeted mutagenesis haveopened new possibilities for generating mutant clones inotherwise wild-type mice (or vice versa). Tissue-specificgene targeting can be achieved by a combination ofhomologous recombination and site-specific recombi-nation, using the Cre recombinase28 to excise chromo-somal DNA between two loxP-recognition sites. Standardgene-targeting approaches are used to introduce twoloxP sites into the gene of interest, such that excisionwill cause a null mutation, or rescue a defective gene.Mice homozygous for this insertion are then crossedwith mice expressing the Cre recombinase from anappropriate tissue-specific promoter, generating tissue-specific mosaicism. Such mosaics can be very useful indefining the lineage specificity of a particular mutantphenotype, in a complementary manner to chimericanalysis. For example, disruption of the RXRa nuclearreceptor leads to cardiac failure in mid-gestation. How-ever, this defect does not result from defective RXRafunction in cardiomyocytes, as had been predicted.Homozygous mutant cardiomyocytes can develop nor-mally in embryos showing ventricular cardiac muscle-specific ablation of RXRa (Ref. 29), as well as inchimeric embryos between homozygous mutant EScells and wild-type embryos30.

Inducible expression of Cre at different times in devel-opment, either ubiquitously or in a tissue-specific manner,would add significantly to the power of this technique(Fig. 3; reviewed in Ref. 31). Also, because Cre excisionmight not occur in all cells of a lineage, it would be use-ful to incorporate some means of generating an inde-pendent cell-autonomous marker to allow recognitionof cells in which the excision event has occurred, forexample by ensuring that the excision event also inacti-vates expression of a ubiquitous reporter.

The possibility that Cre-mediated excision might notoccur in all cells can be turned to advantage as a methodfor generating mosaic tissues, with much greater possi-bilities for manipulating the time and place of establish-ing mosaicism within the embryo than can be achievedwith chimeric techniques (Fig. 3). General mosaicism,very similar to that seen in chimeras, has been pro-duced by a cre transgene that is inefficiently expressedin the early embryo, for example32. It should also bepossible to modulate the expression of other cre trans-genes so as to produce restricted clones of cells. Localintroduction of viral vectors expressing Cre has con-siderable potential for producing highly regulatedmosaicism, with the possibility of following the fate ofsingle cell clones. A recent demonstration of the powerof this technique was the production of clonal adeno-mas after introduction of an adenovirus expressing Cre

loxP loxP

=

=

+Cre

×Inefficient

ubiquitous Cre

×Heart-specific

Cre

×Tissue-specific inducible Cre

Local viralintroduction

of Cre

×Ubiquitous

inducible Cre

+ inducer+ inducer

FIGURE 3. Generation of mosaics by Cre excision. Various possible outcomes are visualizedfrom crossing a founder mouse, homozygous for a ‘floxed’ allele of the gene of interest, withvarious Cre-expressing mice. The examples shown generate mutant cells in a wild-typebackground. If the loxP sites in the gene surround an inactivating insertion in the 59 region ofthe gene, then Cre excision will generate wild-type cells in a mutant background (not shown).

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into the intestine of mice carrying a conditional ‘floxed’allele of the APC gene33.

The preceding experiments all rely on intrachromo-somal excision at the locus of interest to producemosaicism. In Drosophila, site-specific recombinationwith the FLP recombinase has also been used to gener-ate mosaics, but the usual approach here relies on inter-chromosomal recombination between two recognitionsites positioned close to the centromere on homologouschromosomes. Inducible expression of the recombinasein animals heterozygous for a mutation in a gene car-ried on that chromosome produces mitotic recombinantclones that are homozygous for the mutation34,35. Animportant advantage of this approach is that a chromo-some arm carrying a recognition site near the centro-mere can be used to produce mosaics for most locimapping to that arm. It is also relatively easy to includegenetic markers for later analysis of the mutant clones.Developing analogous tools for the mouse would be amajor undertaking because of the larger chromosomenumber. However, the power of the clonal approach tomutant analysis in Drosophila makes it worth consider-ing its possible application to the mouse.

Cell-autonomous versus non-autonomous actionA common goal of genetic mosaic or chimeric

analysis is to determine whether a gene functions cell-autonomously or non-autonomously, information thatcannot be reliably gained by any other means. In mamma-lian chimeras, cell-autonomous action can be recognizedin two ways. The first is to correlate a mutant cellularproperty with mutant genotype. For example, in chimeras,Fgfr1 (Refs 36, 37) and Brachyury (Ref. 4) mutant cellsaccumulate at the posterior of the embryo instead of pass-ing through the primitive streak, whereas wild-type cellsare not affected. This phenotype is much more apparentin the competitive situation of a chimera than in the homo-zygous mutants, and it suggests some cell-autonomousrole for both molecules in controlling cell behaviour inthe streak. The second is to observe that mutant cellsare excluded from a given structure, a more commonfinding than behavioural change of the sort describedabove in the highly regulative mammalian embryo, whereselection against mutant cells can take place throughoutdevelopment and even in some adult tissues.

A cell-autonomous requirement is apparent whateverthe proportion of mutant and wild-type cells in a tissue:mutant cells might be entirely excluded from the affectedtissue, or they might exhibit a mutant phenotype withinthe tissues to which they contribute. On the other hand,if the mutant cells are defective in a non-autonomousfunction, the presence of wild-type cells can rescue themutant phenotype, provided that the mutant cell contri-bution to tissues that require the function is low. Athigh contributions in the critical tissues, mutant cellsmight impose a mutant phenotype on surroundingwild-type cells.

Is it really necessary to go through the rigour ofmaking and analysing chimeras to make conclusionsabout cell autonomy? People are often tempted to inferthe mode of action of a gene from its site of expressionand the nature of its gene product. If gene expression isdetected only in cells that exhibit a defect in the mutantanimal, then it seems reasonable to propose a cell-

autonomous mode of action. Similarly, if gene expres-sion is seen only in cells other than those defective inthe mutant, this might suggest a cell non-autonomousrole for the gene. However, such suggestions dependon the accuracy and sensitivity of determining geneexpression, and life is rarely so simple nor gene expres-sion so conveniently restricted. Mosaic analysis is thecritical test for informed guesses about where a gene’sexpression is functionally significant. Again, the analysisof Fgfr1 provides an example. Somites fail to form inFgfr1 mutants and Fgfr1 is expressed in the presomiticmesoderm precursors, suggesting that it plays anautonomous role in somite specification. However,Fgfr1 mutant cells were able to contribute to somites inchimeras, albeit at low levels, disproving an absolutecell-autonomous requirement. Rather, it appears thatthe problems of the Fgfr1 mutant cells in the primitivestreak prevent them from contributing to the somites.Other genes might be very widely expressed, and yetgive a very specific phenotype, precluding even edu-cated guesses about their mode of action in the absenceof mosaic data.

Even when the expression pattern of a gene appearsto correlate cleanly with the mutant phenotype, mosaicanalysis can yield surprises. For example, the transcrip-tion factor, MASH2, is confined in its expression in theplacenta to the two trophoblast layers, the spongiotropho-blast and the labyrinthine trophoblast. Mutant embryosshow defective placental development, with absentspongiotrophoblast and defective labyrinth, consistentwith a cell-automous role for MASH2 in both tissues38.However, chimera analysis showed that Mash2 mutantlabyrinthine trophoblast could develop normally, pro-vided that the spongiotrophoblast was wild type39.Thus, MASH2 appears to act autonomously in the spon-giotrophoblast but non-autonomously with respect tothe labyrinth, showing that the same gene can behavedifferently with respect to different cellular phenotypes.

The nature of a gene product also has some bearingon whether it is likely to act in a cell-autonomous ornon-autonomous manner but, as the previous exampleillustrates, it is not a secure criterion. Receptors, signal-transducing molecules and transcription factors, althoughacting within a cell, might play their key roles in a givenprocess by regulating, directly or indirectly, the productionof extracellular molecules that affect the fate of othercells. They can appear to act autonomously if the read-out is the production of such a molecule, but non-cellautonomously if the read-out is the induced change incell fate. Conversely, although one might imagine thatmutations in secreted molecules would always act cellnon-autonomously, this would not be true if the mol-ecule acted solely in an autocrine manner or its diffusionwere sufficiently restricted. Thus, only mosaic or chimericanalysis can provide the critical test par excellence forautonomy of gene action.

The futureThe tools of preimplantation chimeric analysis have

been around for a long time in mammalian experimen-tal embryology and have now been refined to the pointwhere they can, and should, be used as part of the rou-tine phenotypic analysis of any developmental mutation.With the addition of new techniques for generating

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restricted genetic mosaicism, the full power of mosaicanalysis can now be applied to understanding thedetailed mode of action of individual genes, at all stagesof development.

AcknowledgementsWe thank J. Pearce and D. Dufort for useful discus-

sion. J.R. is an MRC Distinguished Scientist and an HHMIInternational Scholar.

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Use of isolated inbred human populations for identification

of disease genesby V.C. Sheffield, E.M. Stone and R. Carmi

The VHL tumour suppressor gene paradigmby W.G. Kaelin, Jr and E.R. Maher

Genetics and human reproductionby A. McLaren

Putting the genome on the mapby J.M. Bridger and W.A. Bickmore

Genomic disorders: structural features of the genome can lead to DNA rearrangements

and human disease traitsby J.R. Lupski

Protein precipitation: a common etiology in neurodegenerative disorders?

by A. Kakizuka

Genetics of programmed cell death in C. elegans: past, present

and futureby M.M. Metzstein, G.M. Stanfield and H.R. Horvitz

The October issue of TIG is a special issue that coincides with the American Society of Human Genetics meeting, and includes

the following articles: