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Chimeras and mosaics in mouse mutant analysis
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enetic mosaic analysis has long been a powerful tool in the service of geneticists studying a variety of species, and recent developments have permitted mouse geneticists to wield it with increasing precision. Although chimeras have been used for many years to dissect the phenotypes of spontaneous mouse mutants, such studies were often limited by the molecular markers available to identify and follow the mutant contributions. The development of gene targeting and embryonic stem-cell (ES cell) technology has extended the range of methods for producing mice that are mixtures of wild type and mutant cells. In addition, a variety of in situ cell markers have been developed to allow tracing of the distribution of the contributing cell types. It is time, then, to consider carefully what kind of information can be gained from analysing genetic chimeras and mosaics and what still limits their use in mice.
Mouse chimeras as tools for mosaic analysis of mutants It is clear that simply examining the phenotype that results from an embryonic lethal mutation is not always sufficient to identify the primary site of action of the affected gene, because phenotypes can be very complex. Nor does such examination reveal possible roles for the gene in later lineages, because the mutant phenotype reflects the earliest requirement for the gene’s function. As will be discussed later, new techniques of tissue-specific gene targeting and lineage-specific gene rescue provide very precise tools to help solve these problems but are currently very technically demanding. However, the classic mouse chimera, generated by mixing mutant and wild-type cells at preimplantation stages, can yield a surprising amount of information about when and where a gene is required in development (see Box 1). Chimeras can be made by aggregation at the eight-cell stage or by injection at the blastocyst stage. Which of the resulting tissues are of mixed genotype depends on the
BOX 1. Glossary Cell-autonomous action The function of the gene is required in the cells that exhibit the original mutant phenotype. In a mosaic, the mutant phenotype occurs only in cells that are genotypically mutant and in all such cells in a mosaic tissue. Cell non-autonomous action The function of the gene is required in cells other than those that exhibit the original mutant phenotype. In mosaics, nonautonomous action of a gene in a given tissue is revealed by the presence of genotypically wild-type cells that exhibit the mutant phenotype, or genotypically mutant cells that are phenotypically wild type. Chimera An organism that consists of cells derived from more than one individual, usually of different genotype. Mosaic An organism that consists of cells of more than one genotype. The strict definition requires that the genotypically different cells all derive from a single zygote. The term mosaic is also used more broadly to describe any organism of mixed genotype, whatever the initial cause. Thus, chimeras would be a subset of mosaics under this looser definition.
Chimeras and mosaics in mouse mutant analysis JANET ROSSANT ([email protected]) ANDREW SPENCE ([email protected]) As the number of mouse mutants generated by gene targeting continues to grow exponentially, the challenge is not how to generate a mutant but how to analyse the phenotype. Genes might play multiple roles in development and act in cell-autonomous and cell non-autonomous modes, making phenotypic analysis complex. Genetic mosaic analysis is a powerful tool for dissecting complex gene functions. Classical preimplantation chimeras made between mutant and wild-type embryos can answer many questions, and new genetic techniques for generating restricted genetic mosaicism promise to enhance the future power of mosaic analysis in mammals. stage of the host embryo and the potency of the donor cells (Fig. 1). Before the advent of molecularly tagged mutations made in ES cells, making chimeras between wild-type and homozygous lethal mutants involved the use of embryos derived from heterozygous crosses as one of the chimeric partners. Thus, only one in four of the resulting chimeras was of the required genotypic combination. This, in addition to the lack of molecular markers to identify the mutant components, placed severe limitations on the use of chimeric analysis. Even if a linked marker for the mutation was available, it was not straightforward to distinguish heterozygote–wild-type chimeras from homozygous–wild-type combinations. These limitations can all be overcome by the use of homozygous mutant ES cells, which have the potential to colonize all the primitive ectoderm derivatives in chimeras1 (Fig. 1). Whatever the nature of the mutation, targeted or spontaneous, homozygous ES cells can be made directly from embryos of a heterozygous cross, provided that a genotyping strategy is available. For targeted mutations, homozygous cells can also be made directly in culture from the original heterozygous targeted line, by a variety of strategies2. When such lines are used to generate chimeras, there is no limitation on numbers of mutant embryos available and all offspring are of the required genotypic combination, dramatically simplifying chimeric analysis of mutants. Whatever method is used to generate chimeras, the inclusion of independent genetic markers to follow the fate of the two cell components is critical. In many instances when making a targeted mutation, the Escherichia coli -galactosidase gene is introduced into the locus of interest. This means that mutant cells can be traced directly in chimeras, which can be very useful, but because the marker is not independent of the mutation, the absence of expression in a tissue need not necessarily mean that the mutant cells are also absent. Such a conclusion can only be drawn when an independent cell marker is used. Over the years a number of such markers have been used, and all have some limitations (Table 1). The E. coli -galactosidase gene is
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8-cell/8-cell
8-cell/ICM
8-cell/ES
Blastocyst/ES
Tetraploid/ES
Primitive endoderm
Epiblast
Trophectoderm Yolk sac
Placenta
FIGURE 1. Tissue contributions in chimeras. The various possible outcomes, in terms of contribution to the three lineages derived from the blastocyst are shown for different types of preimplantation chimeras. The primitive endoderm gives rise to the entire endoderm layer of the yolk sac and the trophectoderm to the trophoblast layers of the placenta, while the epiblast gives rise to the entire embryo as well as some extraembryonic cells. Orange–white combinations are embryo–embryo combinations, green–white combinations are ES cell–embryo combinations. Solid colours, non-mosaic contribution; stripes, mosaic contribution. Tetraploid embryos are generated by electrofusion of the blastomeres of a two-cell embryo.
now the most widely used marker for chimeric analysis because its activity can be detected by a simple histochemical stain in whole embryos and in sectioned material. Ubiquitous expression of the -galactosidase gene had been reported in the ROSA26 (Ref. 3) and other gene trap lines4,5 , making them suitable chimeric partners (Fig. 2a). The new marker system based on the green fluorescent protein (GFP) of jellyfish also has potential for chimeric analysis, especially because it can be observed directly in living cells6,7. Embryos8, ES cells9 and mice10 expressing readily detectable levels of GFP in all cells have been reported. However, GFP activity is not well retained after standard embedding and sectioning9. Lightly fixed vibratome sections, combined with confocal microscopy, can provide reasonable cellular resolution (Fig. 2b).
Dissecting lineage-specific gene function in development Chimeric animals produced by mixing pluripotent cells at preimplantation stages generally show very widespread contributions from the donor cells and extensive mixing of cells of the two genotypes within a tissue. This property of mouse chimeras means that mutant effects can 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 action of a gene without any a priori assumptions based on expression pattern of the gene. If rescue of the mutant phenotype in chimeras correlates with the exclusion of mutant 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, die in mid-gestation with failure to close the anterior neural folds. Evidence that the phenotype results from defects in head mesenchyme, and not in the neural tissue, came from showing that normal neural-tube closure occurred in chimeras with very high percentages of mutant cells in the neural folds, provided that there were very few mutant cells in the head mesenchyme11. Similarly, the complex 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 mutant cells are specifically excluded from these tissues in phenotypically rescued chimeras12. The competition between mutant and wild-type cells in a chimera can also reveal phenotypic effects not detectable in the mutant embryos themselves. For example, in chimeras, platelet-derived growth factor receptor mutant cells are highly impaired in their ability to contribute to all muscle cell lineages, although no obvious defect is seen in the development of these lineages in the purebred mutants13. As well as defining the primary site of action of a mutation, making embryonic chimeras with mutant and wild-type cells often allows rescue of early embryo lethality, and permits analysis of the phenotypes of mutant cells in later lineages. For example, ␣4 integrin null embryos die early in gestation, but viable chimeras with widespread mutant contributions can be generated after injection of mutant ES cells into blastocysts. In these chimeras, mutant cells were excluded from the T- and B-cell lineages, indicating a novel role for integrin
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TABLE 1. Properties of cell markers
Ubiquitous Neutral Cell autonomous Detectable in intact embryos Detectable in sections Single-cell resolution Simple detection system Detectable in living cells
DNA RFLPsa
GPI isozymesb
Species-specific satellite DNAc
Multicopy transgene
-Gal
GFP
⫹ ⫹ ⫹ No No No ⫹ No
⫹ ⫹ ⫹ No No No ⫹ No
⫹ ? ⫹ ? ⫹ ⫹ No No
⫹ ⫹ ⫹ ? ⫹ ⫹ No No
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ Partial
? ⫹ ⫹ ⫹ ? ⫹ ⫹ ⫹
aDNA polymorphisms between strains or between mutant and wild-type cells can be used to quantitate mosaicism in a given tissue 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 multicopy sequences are used. Species-specific satellite DNA probes can be used in interspecific mouse chimeras41, or a high-copy number globin transgenic strain can be used as a chimeric partner42.
signalling in lymphopoiesis14, but they were present in the myocyte lineage, disproving the proposed requirement for ␣4 integrin in myogenesis15. Similarly, chimeric analysis of two mutations that were shown to block embryonic hematopoiesis, Flk1 and Scl (Tal1) showed that mutant cells were also absent in the definitive hematopoietic cells of the adult16,17, implicating the same genetic pathways in the two different lineages. Further definition of multiple roles of a given gene in different tissues would certainly be aided by the ability to restrict mutant-cell contributions to different lineages. The restricted early potential of ES cells provides a special opportunity to distinguish whether early lethality of a mutant is due to defects in embryonic or extraembryonic lineages. Mutant ES cells introduced into wild-type embryos produce conceptuses in which epiblast lineages are mixed mutant and wild type, while the trophectoderm and primitive endoderm derivatives are entirely wild type (Fig. 1). The reverse situation can be achieved by introduction of wild-type ES cells into mutant blastocysts. Aggregation or injection of tetraploid embryos with ES cells produces the most extreme separation of mutant and wild-type contributions, because the tetraploid cells are excluded from the embryonic lineages and the ES cells are excluded from the extraembryonic lineages of trophoblast and primitive endoderm18 (Fig. 1).
Diploid or tetraploid chimeras have been used to show that a number of mutants that have severe defects in the developing embryo at gastrulation have primary defects in the extraembryonic lineages. For example, the block to gastrulation and mesoderm formation seen in embryos lacking either the HNF4 transcription factor19 or SMAD4, a signal transduction molecule involved in TGF/ BMP signalling20, can be rescued by providing wild-type extraembryonic lineages by tetraploid aggregation. ‘Reverse’ chimeras, in which wild-type ES cells are incorporated into mutant blastocysts, have shown that extraembryonic tissues of certain mutants can impose a mutant phenotype on wild-type embryo cells. For example, chimeras in which the epiblast is mixed but the trophoblast and primitive endoderm lineages are mutant in either the type I activin receptor ACTR1B (Ref. 21), or the potential downstream-signalling molecule SMAD2 (Ref. 22), produce relatively normal yolksac structures but no tissues of the embryo proper. This suggests that signalling events within the extraembryonic lineages initiate the programme leading to normal development of the dorso-anterior structures of the embryo. A more confined role for extraembryonic lineages in patterning anterior head structures was suggested by separate recent chimera experiments with embryos mutant for nodal, a TGFrelated signalling molecule5 and for the anteriorly expressed transcription factor, OTX2 (Ref. 23). Accumulating evidence from many sources has pointed to the importance of primitive endoderm as a source of signals for patterning the early embryo24,25, and chimeric analysis of mutants has allowed a definitive test of this hypothesis. Obviously, for resolving lineageFIGURE 2. Examples of visualization of chimerism in tissue sections. (a) Section of specific roles of a gene, it would be 9.5-day-old ROSA26–wild-type chimera, with -galactosidase expression revealed by very helpful to be able to bias the X-gal staining (courtesy of B. Ciruna). (b) Vibrotome section of ureteric region of adult contribution of mutant cells into other kidney in GFP–wild-type chimera, with GFP activity revealed by fluorescence later lineages as well. The so-called microscopy (courtesy of K. Hadjantonakis). blastocyst complementation assay, TIG SEPTEMBER 1998 VOL. 14 NO. 9
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REVIEWS first developed for analysis of mutant effects in the lymphocyte lineages26 has the potential to achieve this. In this approach, mutant cells are introduced into host embryos that are genetically impaired in their ability to make certain lineages. In resulting chimeras, those lineages derive entirely from the donor cells while others are of mixed origin. Rag2-deficient hosts can be used for populating the lymphocyte lineage with donor cells26, aphakia mutants for the lens27, and presumably this approach could be extended to other lineages with the use of appropriate mutant hosts. However, whatever kind of preimplantation chimeras are made, they all have serious limitations for dissecting gene function at all conceivable stages of development, because an early role for a gene in a given lineage will result in exclusion of mutant cells from that lineage throughout succeeding stages of development, precluding any meaningful conclusion about later roles. Mosaics produced by tissuespecific or inducible-gene targeting will circumvent this limitation. Chimeras can be a first-line tool to define lineage effects that can be further analysed using more complex genetic strategies.
= loxP
loxP
+Cre
=
×
×
×
×
Inefficient ubiquitous Cre
Heart-specific Cre
Ubiquitous inducible Cre
Tissue-specific inducible Cre
+ inducer
+ inducer
Local viral introduction of Cre
FIGURE 3. Generation of mosaics by Cre excision. Various possible outcomes are visualized from crossing a founder mouse, homozygous for a ‘floxed’ allele of the gene of interest, with various Cre-expressing mice. The examples shown generate mutant cells in a wild-type background. If the loxP sites in the gene surround an inactivating insertion in the 5⬘ region of the gene, then Cre excision will generate wild-type cells in a mutant background (not shown).
Production of mosaics Until recently, genetic mosaics have been more important in studies of flies and worms than in mice. However, new techniques of targeted mutagenesis have opened new possibilities for generating mutant clones in otherwise wild-type mice (or vice versa). Tissue-specific gene targeting can be achieved by a combination of homologous recombination and site-specific recombination, using the Cre recombinase28 to excise chromosomal DNA between two loxP-recognition sites. Standard gene-targeting approaches are used to introduce two loxP sites into the gene of interest, such that excision will cause a null mutation, or rescue a defective gene. Mice homozygous for this insertion are then crossed with mice expressing the Cre recombinase from an appropriate tissue-specific promoter, generating tissuespecific mosaicism. Such mosaics can be very useful in defining the lineage specificity of a particular mutant phenotype, in a complementary manner to chimeric analysis. For example, disruption of the RXR␣ nuclear receptor leads to cardiac failure in mid-gestation. However, this defect does not result from defective RXR␣ function in cardiomyocytes, as had been predicted. Homozygous mutant cardiomyocytes can develop normally in embryos showing ventricular cardiac musclespecific ablation of RXR␣ (Ref. 29), as well as in chimeric embryos between homozygous mutant ES cells and wild-type embryos30.
Inducible expression of Cre at different times in development, 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 excision might not occur in all cells of a lineage, it would be useful to incorporate some means of generating an independent cell-autonomous marker to allow recognition of cells in which the excision event has occurred, for example by ensuring that the excision event also inactivates expression of a ubiquitous reporter. The possibility that Cre-mediated excision might not occur in all cells can be turned to advantage as a method for generating mosaic tissues, with much greater possibilities for manipulating the time and place of establishing mosaicism within the embryo than can be achieved with chimeric techniques (Fig. 3). General mosaicism, very similar to that seen in chimeras, has been produced by a cre transgene that is inefficiently expressed in the early embryo, for example32. It should also be possible to modulate the expression of other cre transgenes so as to produce restricted clones of cells. Local introduction of viral vectors expressing Cre has considerable potential for producing highly regulated mosaicism, with the possibility of following the fate of single cell clones. A recent demonstration of the power of this technique was the production of clonal adenomas after introduction of an adenovirus expressing Cre
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REVIEWS into the intestine of mice carrying a conditional ‘floxed’ allele of the APC gene33. The preceding experiments all rely on intrachromosomal excision at the locus of interest to produce mosaicism. In Drosophila, site-specific recombination with the FLP recombinase has also been used to generate mosaics, but the usual approach here relies on interchromosomal recombination between two recognition sites positioned close to the centromere on homologous chromosomes. Inducible expression of the recombinase in animals heterozygous for a mutation in a gene carried on that chromosome produces mitotic recombinant clones that are homozygous for the mutation34,35. An important advantage of this approach is that a chromosome arm carrying a recognition site near the centromere can be used to produce mosaics for most loci mapping to that arm. It is also relatively easy to include genetic markers for later analysis of the mutant clones. Developing analogous tools for the mouse would be a major undertaking because of the larger chromosome number. However, the power of the clonal approach to mutant analysis in Drosophila makes it worth considering its possible application to the mouse.
Cell-autonomous versus non-autonomous action A common goal of genetic mosaic or chimeric analysis is to determine whether a gene functions cellautonomously or non-autonomously, information that cannot be reliably gained by any other means. In mammalian chimeras, cell-autonomous action can be recognized in two ways. The first is to correlate a mutant cellular property with mutant genotype. For example, in chimeras, Fgfr1 (Refs 36, 37) and Brachyury (Ref. 4) mutant cells accumulate at the posterior of the embryo instead of passing through the primitive streak, whereas wild-type cells are not affected. This phenotype is much more apparent in the competitive situation of a chimera than in the homozygous mutants, and it suggests some cell-autonomous role for both molecules in controlling cell behaviour in the streak. The second is to observe that mutant cells are excluded from a given structure, a more common finding than behavioural change of the sort described above in the highly regulative mammalian embryo, where selection against mutant cells can take place throughout development and even in some adult tissues. A cell-autonomous requirement is apparent whatever the proportion of mutant and wild-type cells in a tissue: mutant cells might be entirely excluded from the affected tissue, or they might exhibit a mutant phenotype within the tissues to which they contribute. On the other hand, if the mutant cells are defective in a non-autonomous function, the presence of wild-type cells can rescue the mutant phenotype, provided that the mutant cell contribution to tissues that require the function is low. At high contributions in the critical tissues, mutant cells might impose a mutant phenotype on surrounding wild-type cells. Is it really necessary to go through the rigour of making and analysing chimeras to make conclusions about cell autonomy? People are often tempted to infer the mode of action of a gene from its site of expression and the nature of its gene product. If gene expression is detected only in cells that exhibit a defect in the mutant animal, then it seems reasonable to propose a cell-
autonomous mode of action. Similarly, if gene expression is seen only in cells other than those defective in the mutant, this might suggest a cell non-autonomous role for the gene. However, such suggestions depend on the accuracy and sensitivity of determining gene expression, and life is rarely so simple nor gene expression so conveniently restricted. Mosaic analysis is the critical test for informed guesses about where a gene’s expression is functionally significant. Again, the analysis of Fgfr1 provides an example. Somites fail to form in Fgfr1 mutants and Fgfr1 is expressed in the presomitic mesoderm precursors, suggesting that it plays an autonomous role in somite specification. However, Fgfr1 mutant cells were able to contribute to somites in chimeras, albeit at low levels, disproving an absolute cell-autonomous requirement. Rather, it appears that the problems of the Fgfr1 mutant cells in the primitive streak prevent them from contributing to the somites. Other genes might be very widely expressed, and yet give a very specific phenotype, precluding even educated guesses about their mode of action in the absence of mosaic data. Even when the expression pattern of a gene appears to correlate cleanly with the mutant phenotype, mosaic analysis can yield surprises. For example, the transcription factor, MASH2, is confined in its expression in the placenta to the two trophoblast layers, the spongiotrophoblast and the labyrinthine trophoblast. Mutant embryos show defective placental development, with absent spongiotrophoblast and defective labyrinth, consistent with a cell-automous role for MASH2 in both tissues38. However, chimera analysis showed that Mash2 mutant labyrinthine trophoblast could develop normally, provided that the spongiotrophoblast was wild type39. Thus, MASH2 appears to act autonomously in the spongiotrophoblast but non-autonomously with respect to the labyrinth, showing that the same gene can behave differently with respect to different cellular phenotypes. The nature of a gene product also has some bearing on whether it is likely to act in a cell-autonomous or non-autonomous manner but, as the previous example illustrates, it is not a secure criterion. Receptors, signaltransducing molecules and transcription factors, although acting within a cell, might play their key roles in a given process by regulating, directly or indirectly, the production of extracellular molecules that affect the fate of other cells. They can appear to act autonomously if the readout is the production of such a molecule, but non-cell autonomously if the read-out is the induced change in cell fate. Conversely, although one might imagine that mutations in secreted molecules would always act cell non-autonomously, this would not be true if the molecule acted solely in an autocrine manner or its diffusion were sufficiently restricted. Thus, only mosaic or chimeric analysis can provide the critical test par excellence for autonomy of gene action.
The future The tools of preimplantation chimeric analysis have been around for a long time in mammalian experimental embryology and have now been refined to the point where they can, and should, be used as part of the routine phenotypic analysis of any developmental mutation. With the addition of new techniques for generating
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Acknowledgements We thank J. Pearce and D. Dufort for useful discussion. J.R. is an MRC Distinguished Scientist and an HHMI International Scholar.
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22 Waldrip, W.R. et al. (1998) Cell 92, 797–808 23 Rhinn, M. et al. (1998) Development 125, 845–856 24 Bouwmeester, T. and Leyns, L. (1997) BioEssays 19, 855–863 25 Beddington, R. and Robertson, E. (1998) Trends Genet. 14, 277–284 26 Chen, J. et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4528–4532 27 Liegeois, N.J., Horner, J.W. and DePinho, R.A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1303–1307 28 Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5166–5170 29 Chen, J., Kubalak, S.W. and Chien, K.R. (1998) Development 125, 1943–1949 30 Tran, C.M. and Sucov, H.M. (1998) Development 125, 1951–1956 31 Porter, A. (1998) Trends Genet. 14, 73–79 32 Betz, U.A., Vosshenrich, C.A., Rajewsky, K. and Muller, W. (1996) Curr. Biol. 6, 1307–1316 33 Shibata, H. et al. (1997) Science 278, 120–123 34 Dang, D.T. and Perrimon, N. (1992) Dev. Genet. 13, 367–375 35 Xu, T. and Rubin, G.M. (1993) Development 117, 1223–1237 36 Deng, C. et al. (1997) Dev. Biol. 185, 42–54 37 Ciruna, B.G. et al. (1997) Development 124, 2829–2841 38 Guillemot, F. et al. (1994) Nature 371, 333–336 39 Tanaka, M., Gertsenstein, M., Rossant, J. and Nagy, A. (1997) Dev. Biol. 190, 55–65 40 Chapman, V.M., Whitten, W.K. and Ruddle, F.H. (1971) Dev. Biol. 26, 153–158 41 Rossant, J., Vijh, M., Siracusa, L.D. and Chapman, V.M. (1983) J. Embryol. Exp. Morph. 73, 179–191 42 Lo, C.W., Coulling, M. and Kirby, C. (1987) Differentiation 35, 37–44
J. Rossant and A. Spence are in the Department of Medical Genetics and Microbiology, Medical Sciences Building, 1 King’s College Circle, University of Toronto, Toronto, Ontario, Canada M5S 1A8. J. Rossant is also affiliated with the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5.
The October issue of TIG is a special issue that coincides with the American Society of Human Genetics meeting, and includes the following articles: Use of isolated inbred human populations for identification of disease genes by V.C. Sheffield, E.M. Stone and R. Carmi
Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits by J.R. Lupski
The VHL tumour suppressor gene paradigm by W.G. Kaelin, Jr and E.R. Maher
Protein precipitation: a common etiology in neurodegenerative disorders? by A. Kakizuka
Genetics and human reproduction by A. McLaren
Genetics of programmed cell death in C. elegans: past, present and future by M.M. Metzstein, G.M. Stanfield and H.R. Horvitz
Putting the genome on the map by J.M. Bridger and W.A. Bickmore
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enetic mosaic analysis has long been a powerful tool in the service of geneticists studying a variety of species, and recent developments have permitted mouse geneticists to wield it with increasing precision. Although chimeras have been used for many years to dissect the phenotypes of spontaneous mouse mutants, such studies were often limited by the molecular markers available to identify and follow the mutant contributions. The development of gene targeting and embryonic stem-cell (ES cell) technology has extended the range of methods for producing mice that are mixtures of wild type and mutant cells. In addition, a variety of in situ cell markers have been developed to allow tracing of the distribution of the contributing cell types. It is time, then, to consider carefully what kind of information can be gained from analysing genetic chimeras and mosaics and what still limits their use in mice.
Mouse chimeras as tools for mosaic analysis of mutants It is clear that simply examining the phenotype that results from an embryonic lethal mutation is not always sufficient to identify the primary site of action of the affected gene, because phenotypes can be very complex. Nor does such examination reveal possible roles for the gene in later lineages, because the mutant phenotype reflects the earliest requirement for the gene’s function. As will be discussed later, new techniques of tissue-specific gene targeting and lineage-specific gene rescue provide very precise tools to help solve these problems but are currently very technically demanding. However, the classic mouse chimera, generated by mixing mutant and wild-type cells at preimplantation stages, can yield a surprising amount of information about when and where a gene is required in development (see Box 1). Chimeras can be made by aggregation at the eight-cell stage or by injection at the blastocyst stage. Which of the resulting tissues are of mixed genotype depends on the
BOX 1. Glossary Cell-autonomous action The function of the gene is required in the cells that exhibit the original mutant phenotype. In a mosaic, the mutant phenotype occurs only in cells that are genotypically mutant and in all such cells in a mosaic tissue. Cell non-autonomous action The function of the gene is required in cells other than those that exhibit the original mutant phenotype. In mosaics, nonautonomous action of a gene in a given tissue is revealed by the presence of genotypically wild-type cells that exhibit the mutant phenotype, or genotypically mutant cells that are phenotypically wild type. Chimera An organism that consists of cells derived from more than one individual, usually of different genotype. Mosaic An organism that consists of cells of more than one genotype. The strict definition requires that the genotypically different cells all derive from a single zygote. The term mosaic is also used more broadly to describe any organism of mixed genotype, whatever the initial cause. Thus, chimeras would be a subset of mosaics under this looser definition.
Chimeras and mosaics in mouse mutant analysis JANET ROSSANT ([email protected]) ANDREW SPENCE ([email protected]) As the number of mouse mutants generated by gene targeting continues to grow exponentially, the challenge is not how to generate a mutant but how to analyse the phenotype. Genes might play multiple roles in development and act in cell-autonomous and cell non-autonomous modes, making phenotypic analysis complex. Genetic mosaic analysis is a powerful tool for dissecting complex gene functions. Classical preimplantation chimeras made between mutant and wild-type embryos can answer many questions, and new genetic techniques for generating restricted genetic mosaicism promise to enhance the future power of mosaic analysis in mammals. stage of the host embryo and the potency of the donor cells (Fig. 1). Before the advent of molecularly tagged mutations made in ES cells, making chimeras between wild-type and homozygous lethal mutants involved the use of embryos derived from heterozygous crosses as one of the chimeric partners. Thus, only one in four of the resulting chimeras was of the required genotypic combination. This, in addition to the lack of molecular markers to identify the mutant components, placed severe limitations on the use of chimeric analysis. Even if a linked marker for the mutation was available, it was not straightforward to distinguish heterozygote–wild-type chimeras from homozygous–wild-type combinations. These limitations can all be overcome by the use of homozygous mutant ES cells, which have the potential to colonize all the primitive ectoderm derivatives in chimeras1 (Fig. 1). Whatever the nature of the mutation, targeted or spontaneous, homozygous ES cells can be made directly from embryos of a heterozygous cross, provided that a genotyping strategy is available. For targeted mutations, homozygous cells can also be made directly in culture from the original heterozygous targeted line, by a variety of strategies2. When such lines are used to generate chimeras, there is no limitation on numbers of mutant embryos available and all offspring are of the required genotypic combination, dramatically simplifying chimeric analysis of mutants. Whatever method is used to generate chimeras, the inclusion of independent genetic markers to follow the fate of the two cell components is critical. In many instances when making a targeted mutation, the Escherichia coli -galactosidase gene is introduced into the locus of interest. This means that mutant cells can be traced directly in chimeras, which can be very useful, but because the marker is not independent of the mutation, the absence of expression in a tissue need not necessarily mean that the mutant cells are also absent. Such a conclusion can only be drawn when an independent cell marker is used. Over the years a number of such markers have been used, and all have some limitations (Table 1). The E. coli -galactosidase gene is
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8-cell/8-cell
8-cell/ICM
8-cell/ES
Blastocyst/ES
Tetraploid/ES
Primitive endoderm
Epiblast
Trophectoderm Yolk sac
Placenta
FIGURE 1. Tissue contributions in chimeras. The various possible outcomes, in terms of contribution to the three lineages derived from the blastocyst are shown for different types of preimplantation chimeras. The primitive endoderm gives rise to the entire endoderm layer of the yolk sac and the trophectoderm to the trophoblast layers of the placenta, while the epiblast gives rise to the entire embryo as well as some extraembryonic cells. Orange–white combinations are embryo–embryo combinations, green–white combinations are ES cell–embryo combinations. Solid colours, non-mosaic contribution; stripes, mosaic contribution. Tetraploid embryos are generated by electrofusion of the blastomeres of a two-cell embryo.
now the most widely used marker for chimeric analysis because its activity can be detected by a simple histochemical stain in whole embryos and in sectioned material. Ubiquitous expression of the -galactosidase gene had been reported in the ROSA26 (Ref. 3) and other gene trap lines4,5 , making them suitable chimeric partners (Fig. 2a). The new marker system based on the green fluorescent protein (GFP) of jellyfish also has potential for chimeric analysis, especially because it can be observed directly in living cells6,7. Embryos8, ES cells9 and mice10 expressing readily detectable levels of GFP in all cells have been reported. However, GFP activity is not well retained after standard embedding and sectioning9. Lightly fixed vibratome sections, combined with confocal microscopy, can provide reasonable cellular resolution (Fig. 2b).
Dissecting lineage-specific gene function in development Chimeric animals produced by mixing pluripotent cells at preimplantation stages generally show very widespread contributions from the donor cells and extensive mixing of cells of the two genotypes within a tissue. This property of mouse chimeras means that mutant effects can 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 action of a gene without any a priori assumptions based on expression pattern of the gene. If rescue of the mutant phenotype in chimeras correlates with the exclusion of mutant 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, die in mid-gestation with failure to close the anterior neural folds. Evidence that the phenotype results from defects in head mesenchyme, and not in the neural tissue, came from showing that normal neural-tube closure occurred in chimeras with very high percentages of mutant cells in the neural folds, provided that there were very few mutant cells in the head mesenchyme11. Similarly, the complex 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 mutant cells are specifically excluded from these tissues in phenotypically rescued chimeras12. The competition between mutant and wild-type cells in a chimera can also reveal phenotypic effects not detectable in the mutant embryos themselves. For example, in chimeras, platelet-derived growth factor receptor mutant cells are highly impaired in their ability to contribute to all muscle cell lineages, although no obvious defect is seen in the development of these lineages in the purebred mutants13. As well as defining the primary site of action of a mutation, making embryonic chimeras with mutant and wild-type cells often allows rescue of early embryo lethality, and permits analysis of the phenotypes of mutant cells in later lineages. For example, ␣4 integrin null embryos die early in gestation, but viable chimeras with widespread mutant contributions can be generated after injection of mutant ES cells into blastocysts. In these chimeras, mutant cells were excluded from the T- and B-cell lineages, indicating a novel role for integrin
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TABLE 1. Properties of cell markers
Ubiquitous Neutral Cell autonomous Detectable in intact embryos Detectable in sections Single-cell resolution Simple detection system Detectable in living cells
DNA RFLPsa
GPI isozymesb
Species-specific satellite DNAc
Multicopy transgene
-Gal
GFP
⫹ ⫹ ⫹ No No No ⫹ No
⫹ ⫹ ⫹ No No No ⫹ No
⫹ ? ⫹ ? ⫹ ⫹ No No
⫹ ⫹ ⫹ ? ⫹ ⫹ No No
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ Partial
? ⫹ ⫹ ⫹ ? ⫹ ⫹ ⫹
aDNA polymorphisms between strains or between mutant and wild-type cells can be used to quantitate mosaicism in a given tissue 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 multicopy sequences are used. Species-specific satellite DNA probes can be used in interspecific mouse chimeras41, or a high-copy number globin transgenic strain can be used as a chimeric partner42.
signalling in lymphopoiesis14, but they were present in the myocyte lineage, disproving the proposed requirement for ␣4 integrin in myogenesis15. Similarly, chimeric analysis of two mutations that were shown to block embryonic hematopoiesis, Flk1 and Scl (Tal1) showed that mutant cells were also absent in the definitive hematopoietic cells of the adult16,17, implicating the same genetic pathways in the two different lineages. Further definition of multiple roles of a given gene in different tissues would certainly be aided by the ability to restrict mutant-cell contributions to different lineages. The restricted early potential of ES cells provides a special opportunity to distinguish whether early lethality of a mutant is due to defects in embryonic or extraembryonic lineages. Mutant ES cells introduced into wild-type embryos produce conceptuses in which epiblast lineages are mixed mutant and wild type, while the trophectoderm and primitive endoderm derivatives are entirely wild type (Fig. 1). The reverse situation can be achieved by introduction of wild-type ES cells into mutant blastocysts. Aggregation or injection of tetraploid embryos with ES cells produces the most extreme separation of mutant and wild-type contributions, because the tetraploid cells are excluded from the embryonic lineages and the ES cells are excluded from the extraembryonic lineages of trophoblast and primitive endoderm18 (Fig. 1).
Diploid or tetraploid chimeras have been used to show that a number of mutants that have severe defects in the developing embryo at gastrulation have primary defects in the extraembryonic lineages. For example, the block to gastrulation and mesoderm formation seen in embryos lacking either the HNF4 transcription factor19 or SMAD4, a signal transduction molecule involved in TGF/ BMP signalling20, can be rescued by providing wild-type extraembryonic lineages by tetraploid aggregation. ‘Reverse’ chimeras, in which wild-type ES cells are incorporated into mutant blastocysts, have shown that extraembryonic tissues of certain mutants can impose a mutant phenotype on wild-type embryo cells. For example, chimeras in which the epiblast is mixed but the trophoblast and primitive endoderm lineages are mutant in either the type I activin receptor ACTR1B (Ref. 21), or the potential downstream-signalling molecule SMAD2 (Ref. 22), produce relatively normal yolksac structures but no tissues of the embryo proper. This suggests that signalling events within the extraembryonic lineages initiate the programme leading to normal development of the dorso-anterior structures of the embryo. A more confined role for extraembryonic lineages in patterning anterior head structures was suggested by separate recent chimera experiments with embryos mutant for nodal, a TGFrelated signalling molecule5 and for the anteriorly expressed transcription factor, OTX2 (Ref. 23). Accumulating evidence from many sources has pointed to the importance of primitive endoderm as a source of signals for patterning the early embryo24,25, and chimeric analysis of mutants has allowed a definitive test of this hypothesis. Obviously, for resolving lineageFIGURE 2. Examples of visualization of chimerism in tissue sections. (a) Section of specific roles of a gene, it would be 9.5-day-old ROSA26–wild-type chimera, with -galactosidase expression revealed by very helpful to be able to bias the X-gal staining (courtesy of B. Ciruna). (b) Vibrotome section of ureteric region of adult contribution of mutant cells into other kidney in GFP–wild-type chimera, with GFP activity revealed by fluorescence later lineages as well. The so-called microscopy (courtesy of K. Hadjantonakis). blastocyst complementation assay, TIG SEPTEMBER 1998 VOL. 14 NO. 9
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REVIEWS first developed for analysis of mutant effects in the lymphocyte lineages26 has the potential to achieve this. In this approach, mutant cells are introduced into host embryos that are genetically impaired in their ability to make certain lineages. In resulting chimeras, those lineages derive entirely from the donor cells while others are of mixed origin. Rag2-deficient hosts can be used for populating the lymphocyte lineage with donor cells26, aphakia mutants for the lens27, and presumably this approach could be extended to other lineages with the use of appropriate mutant hosts. However, whatever kind of preimplantation chimeras are made, they all have serious limitations for dissecting gene function at all conceivable stages of development, because an early role for a gene in a given lineage will result in exclusion of mutant cells from that lineage throughout succeeding stages of development, precluding any meaningful conclusion about later roles. Mosaics produced by tissuespecific or inducible-gene targeting will circumvent this limitation. Chimeras can be a first-line tool to define lineage effects that can be further analysed using more complex genetic strategies.
= loxP
loxP
+Cre
=
×
×
×
×
Inefficient ubiquitous Cre
Heart-specific Cre
Ubiquitous inducible Cre
Tissue-specific inducible Cre
+ inducer
+ inducer
Local viral introduction of Cre
FIGURE 3. Generation of mosaics by Cre excision. Various possible outcomes are visualized from crossing a founder mouse, homozygous for a ‘floxed’ allele of the gene of interest, with various Cre-expressing mice. The examples shown generate mutant cells in a wild-type background. If the loxP sites in the gene surround an inactivating insertion in the 5⬘ region of the gene, then Cre excision will generate wild-type cells in a mutant background (not shown).
Production of mosaics Until recently, genetic mosaics have been more important in studies of flies and worms than in mice. However, new techniques of targeted mutagenesis have opened new possibilities for generating mutant clones in otherwise wild-type mice (or vice versa). Tissue-specific gene targeting can be achieved by a combination of homologous recombination and site-specific recombination, using the Cre recombinase28 to excise chromosomal DNA between two loxP-recognition sites. Standard gene-targeting approaches are used to introduce two loxP sites into the gene of interest, such that excision will cause a null mutation, or rescue a defective gene. Mice homozygous for this insertion are then crossed with mice expressing the Cre recombinase from an appropriate tissue-specific promoter, generating tissuespecific mosaicism. Such mosaics can be very useful in defining the lineage specificity of a particular mutant phenotype, in a complementary manner to chimeric analysis. For example, disruption of the RXR␣ nuclear receptor leads to cardiac failure in mid-gestation. However, this defect does not result from defective RXR␣ function in cardiomyocytes, as had been predicted. Homozygous mutant cardiomyocytes can develop normally in embryos showing ventricular cardiac musclespecific ablation of RXR␣ (Ref. 29), as well as in chimeric embryos between homozygous mutant ES cells and wild-type embryos30.
Inducible expression of Cre at different times in development, 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 excision might not occur in all cells of a lineage, it would be useful to incorporate some means of generating an independent cell-autonomous marker to allow recognition of cells in which the excision event has occurred, for example by ensuring that the excision event also inactivates expression of a ubiquitous reporter. The possibility that Cre-mediated excision might not occur in all cells can be turned to advantage as a method for generating mosaic tissues, with much greater possibilities for manipulating the time and place of establishing mosaicism within the embryo than can be achieved with chimeric techniques (Fig. 3). General mosaicism, very similar to that seen in chimeras, has been produced by a cre transgene that is inefficiently expressed in the early embryo, for example32. It should also be possible to modulate the expression of other cre transgenes so as to produce restricted clones of cells. Local introduction of viral vectors expressing Cre has considerable potential for producing highly regulated mosaicism, with the possibility of following the fate of single cell clones. A recent demonstration of the power of this technique was the production of clonal adenomas after introduction of an adenovirus expressing Cre
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REVIEWS into the intestine of mice carrying a conditional ‘floxed’ allele of the APC gene33. The preceding experiments all rely on intrachromosomal excision at the locus of interest to produce mosaicism. In Drosophila, site-specific recombination with the FLP recombinase has also been used to generate mosaics, but the usual approach here relies on interchromosomal recombination between two recognition sites positioned close to the centromere on homologous chromosomes. Inducible expression of the recombinase in animals heterozygous for a mutation in a gene carried on that chromosome produces mitotic recombinant clones that are homozygous for the mutation34,35. An important advantage of this approach is that a chromosome arm carrying a recognition site near the centromere can be used to produce mosaics for most loci mapping to that arm. It is also relatively easy to include genetic markers for later analysis of the mutant clones. Developing analogous tools for the mouse would be a major undertaking because of the larger chromosome number. However, the power of the clonal approach to mutant analysis in Drosophila makes it worth considering its possible application to the mouse.
Cell-autonomous versus non-autonomous action A common goal of genetic mosaic or chimeric analysis is to determine whether a gene functions cellautonomously or non-autonomously, information that cannot be reliably gained by any other means. In mammalian chimeras, cell-autonomous action can be recognized in two ways. The first is to correlate a mutant cellular property with mutant genotype. For example, in chimeras, Fgfr1 (Refs 36, 37) and Brachyury (Ref. 4) mutant cells accumulate at the posterior of the embryo instead of passing through the primitive streak, whereas wild-type cells are not affected. This phenotype is much more apparent in the competitive situation of a chimera than in the homozygous mutants, and it suggests some cell-autonomous role for both molecules in controlling cell behaviour in the streak. The second is to observe that mutant cells are excluded from a given structure, a more common finding than behavioural change of the sort described above in the highly regulative mammalian embryo, where selection against mutant cells can take place throughout development and even in some adult tissues. A cell-autonomous requirement is apparent whatever the proportion of mutant and wild-type cells in a tissue: mutant cells might be entirely excluded from the affected tissue, or they might exhibit a mutant phenotype within the tissues to which they contribute. On the other hand, if the mutant cells are defective in a non-autonomous function, the presence of wild-type cells can rescue the mutant phenotype, provided that the mutant cell contribution to tissues that require the function is low. At high contributions in the critical tissues, mutant cells might impose a mutant phenotype on surrounding wild-type cells. Is it really necessary to go through the rigour of making and analysing chimeras to make conclusions about cell autonomy? People are often tempted to infer the mode of action of a gene from its site of expression and the nature of its gene product. If gene expression is detected only in cells that exhibit a defect in the mutant animal, then it seems reasonable to propose a cell-
autonomous mode of action. Similarly, if gene expression is seen only in cells other than those defective in the mutant, this might suggest a cell non-autonomous role for the gene. However, such suggestions depend on the accuracy and sensitivity of determining gene expression, and life is rarely so simple nor gene expression so conveniently restricted. Mosaic analysis is the critical test for informed guesses about where a gene’s expression is functionally significant. Again, the analysis of Fgfr1 provides an example. Somites fail to form in Fgfr1 mutants and Fgfr1 is expressed in the presomitic mesoderm precursors, suggesting that it plays an autonomous role in somite specification. However, Fgfr1 mutant cells were able to contribute to somites in chimeras, albeit at low levels, disproving an absolute cell-autonomous requirement. Rather, it appears that the problems of the Fgfr1 mutant cells in the primitive streak prevent them from contributing to the somites. Other genes might be very widely expressed, and yet give a very specific phenotype, precluding even educated guesses about their mode of action in the absence of mosaic data. Even when the expression pattern of a gene appears to correlate cleanly with the mutant phenotype, mosaic analysis can yield surprises. For example, the transcription factor, MASH2, is confined in its expression in the placenta to the two trophoblast layers, the spongiotrophoblast and the labyrinthine trophoblast. Mutant embryos show defective placental development, with absent spongiotrophoblast and defective labyrinth, consistent with a cell-automous role for MASH2 in both tissues38. However, chimera analysis showed that Mash2 mutant labyrinthine trophoblast could develop normally, provided that the spongiotrophoblast was wild type39. Thus, MASH2 appears to act autonomously in the spongiotrophoblast but non-autonomously with respect to the labyrinth, showing that the same gene can behave differently with respect to different cellular phenotypes. The nature of a gene product also has some bearing on whether it is likely to act in a cell-autonomous or non-autonomous manner but, as the previous example illustrates, it is not a secure criterion. Receptors, signaltransducing molecules and transcription factors, although acting within a cell, might play their key roles in a given process by regulating, directly or indirectly, the production of extracellular molecules that affect the fate of other cells. They can appear to act autonomously if the readout is the production of such a molecule, but non-cell autonomously if the read-out is the induced change in cell fate. Conversely, although one might imagine that mutations in secreted molecules would always act cell non-autonomously, this would not be true if the molecule acted solely in an autocrine manner or its diffusion were sufficiently restricted. Thus, only mosaic or chimeric analysis can provide the critical test par excellence for autonomy of gene action.
The future The tools of preimplantation chimeric analysis have been around for a long time in mammalian experimental embryology and have now been refined to the point where they can, and should, be used as part of the routine phenotypic analysis of any developmental mutation. With the addition of new techniques for generating
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Acknowledgements We thank J. Pearce and D. Dufort for useful discussion. J.R. is an MRC Distinguished Scientist and an HHMI International Scholar.
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22 Waldrip, W.R. et al. (1998) Cell 92, 797–808 23 Rhinn, M. et al. (1998) Development 125, 845–856 24 Bouwmeester, T. and Leyns, L. (1997) BioEssays 19, 855–863 25 Beddington, R. and Robertson, E. (1998) Trends Genet. 14, 277–284 26 Chen, J. et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4528–4532 27 Liegeois, N.J., Horner, J.W. and DePinho, R.A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1303–1307 28 Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5166–5170 29 Chen, J., Kubalak, S.W. and Chien, K.R. (1998) Development 125, 1943–1949 30 Tran, C.M. and Sucov, H.M. (1998) Development 125, 1951–1956 31 Porter, A. (1998) Trends Genet. 14, 73–79 32 Betz, U.A., Vosshenrich, C.A., Rajewsky, K. and Muller, W. (1996) Curr. Biol. 6, 1307–1316 33 Shibata, H. et al. (1997) Science 278, 120–123 34 Dang, D.T. and Perrimon, N. (1992) Dev. Genet. 13, 367–375 35 Xu, T. and Rubin, G.M. (1993) Development 117, 1223–1237 36 Deng, C. et al. (1997) Dev. Biol. 185, 42–54 37 Ciruna, B.G. et al. (1997) Development 124, 2829–2841 38 Guillemot, F. et al. (1994) Nature 371, 333–336 39 Tanaka, M., Gertsenstein, M., Rossant, J. and Nagy, A. (1997) Dev. Biol. 190, 55–65 40 Chapman, V.M., Whitten, W.K. and Ruddle, F.H. (1971) Dev. Biol. 26, 153–158 41 Rossant, J., Vijh, M., Siracusa, L.D. and Chapman, V.M. (1983) J. Embryol. Exp. Morph. 73, 179–191 42 Lo, C.W., Coulling, M. and Kirby, C. (1987) Differentiation 35, 37–44
J. Rossant and A. Spence are in the Department of Medical Genetics and Microbiology, Medical Sciences Building, 1 King’s College Circle, University of Toronto, Toronto, Ontario, Canada M5S 1A8. J. Rossant is also affiliated with the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5.
The October issue of TIG is a special issue that coincides with the American Society of Human Genetics meeting, and includes the following articles: Use of isolated inbred human populations for identification of disease genes by V.C. Sheffield, E.M. Stone and R. Carmi
Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits by J.R. Lupski
The VHL tumour suppressor gene paradigm by W.G. Kaelin, Jr and E.R. Maher
Protein precipitation: a common etiology in neurodegenerative disorders? by A. Kakizuka
Genetics and human reproduction by A. McLaren
Genetics of programmed cell death in C. elegans: past, present and future by M.M. Metzstein, G.M. Stanfield and H.R. Horvitz
Putting the genome on the map by J.M. Bridger and W.A. Bickmore
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