Somatic and germline mosaicism ininterspecific chimaeras between Mus musculus and
Mus caroli
By J. ROSSANT*1 AND V. M. CHAPMAN2
From the Department of Biological Sciences, Brock University, St. Catharinesand the Department of Molecular Biology, Roswell Park Memorial Institute,
Buffalo
SUMMARY
Detailed analysis of mosaicism in interspecific chimaeras between Mus musculus and Muscaroli revealed that cells of the two species could coexist and interact normally in all tissuesstudied. No selection occurred against M. caroli cells during gestation of chimaeras in the M.musculus uterus, but some tissue-specific differential growth of M. musculus and M. carolicells occurred during postnatal development. Similar effects have, however, been reported ininterstrain M. musculus chimaeras. The similarity between inter- and intraspecific chimaericgrowth patterns supports the use of this interspecific system as a model for analysing celllineage relationships during development.
INTRODUCTION
There is a greater degree of molecular, biochemical and cellular divergencebetween species than within a species. Experimental use of such species dif-ferences to follow cells in a clonal analysis of cell lineages has been attemptedusing interspecific chimaeric combinations. The utility of these interspecificcombinations depends upon the compatibility of their growth patterns and, in thecase of mammals, the ability of cells of a foreign species to thrive in the uterineenvironment of the host mother.
Chimaeras between the chick and the quail have been widely used to followcell lineages in birds, utilizing nuclear differences between the two species as anin situ cell marker (Le Douarin, 1980). Early attempts to use antigenic dif-ferences between the rat and the mouse to develop an interspecific cell markersystem in mammals achieved more limited success. In general, the rat-cellcontribution could be detected in midgestation but was not readily detectable by
1 Author's address: Dept. of Biological Sciences, Brock University, St. Catharines, Ontario,Canada.
2 Author's address: Dept. of Molecular Biology, Roswell Park Memorial Institute, Buffalo,N.Y., U.S.A.
* To whom reprint requests should be sent.
194 J. ROSSANT AND V. M. CHAPMAN
the time of birth (Gardner & Johnson, 1973; 1975). More recently, viable inter-specific chimaeras of mammals have been produced between Mus musculus andMus caroli by blastocyst injection (Rossant & Frels, 1980) and embryo aggrega-tion techniques (Rossant, Mauro & Croy, 1982a). These chimaeras can be ex-ploited for clonal analysis of cell lineages by using an in situ marker system basedupon repetitive DNA differences between the two species (Siracusa et al., 1982;Rossant, Vijh, Siracusa & Chapman, 19826). This marker system shows manyproperties of an ideal cell marker (McLaren, 1976).
M. caroli and M. musculus are much more closely related species than the ratand the mouse and a preliminary report on coat colour mosaicism in adultchimaeras and internal tissue mosaicism in preterm foetuses (Rossant & Frels,1980) suggested that the M. musculus uterine environment did not exert anyselective effect against M. caroli cells in the chimaeras. However, since it isessential to establish the validity of the interspecific chimaera system as a modelfor normal embryogenesis, we present here a more detailed analysis of somaticand germ-line mosaicism in perinatal and adult interspecific chimaeras whichshows that the patterns of mosaicism were essentially similar to those observedin intraspecific chimaeras.
MATERIALS AND METHODS
Interspecific chimaeras between M. caroli and M. musculus were produced byblastocyst injection (Rossant & Frels, 1980) or embryo aggregation (Rossant etal., 1982a), as described previously. Some chimaeras were identified by thepresence of eye pigmentation at birth and were killed and dissected into varioustissues for quantitative GPI analysis (Peterson, Frair & Wong, 1978). Adultchimaeras were identified by coat colour mosaicism, since M. caroli is agouti andthe Ha/ICR strain of M. musculus used is albino, and by double-bandedelectrophoretic phenotype for isozymes of glucose phosphate isomerase (GPI)in the blood. Ha/ICR mice were homozygous for the b allele at the Gpi-1 locus,while M. caroli produced only one GPI isozyme indistinguishable from the Aisozyme of M. musculus. Adult female chimaeras were continuously paired withM. musculus or M. caroli partners, and any litters were recorded. Seven femalechimaeras were artificially inseminated with M. caroli sperm as describedelsewhere (West et al., 1977). Twenty-two adult male chimaeras (aged 6 monthsto 1 year) were killed and various organs were dissected and prepared for quanti-tative GPI analysis.
Any hybrid offspring of test matings could be recognized by coat colour whenchimaeras were mated to M. musculus and by hybrid GPI phenotype in anycross. Male hybrids were test mated to M. musculus females. When the hybridswere sacrificed or died, histological sections of gonads were prepared and stainedwith haematoxylin and eosin.
Mosaicism in Mus chimaeras 195
RESULTS
Somatic tissue mosaicism in neonatal chimaeras
Out of 88 live births to date, 68 (77 %) have proved to be interspecificchimaeras. The results of GPI analysis of five foetuses delivered just prior toterm have been previously reported (Rossant & Frels, 1980) and, in Table 1,these data are presented along with data from GPI analysis of five additionalneonatal chimaeras. The mean contribution from M. caroli cells was 42 %. Thisis similar to the mean M. caroli contribution of 48 % found in five chimaerasanalysed at 9-5 days p.c. There was some variation in mosaicism between dif-ferent organs of a given chimaera, but every chimaera showed mosaic GPIphenotype in all tissues analysed. Despite the variation observed, the correlationcoefficients between the M. caroli contribution to a given tissue and to thechimaera as a whole were all statistically significant (P< 0-05). Examination ofthe mean M. caroli contribution to each organ or tissue analysed did not revealany tissue in which there was a marked predominance of one parental type or theother. The mean M. caroli contributions to individual tissues or organs did notdiffer significantly from the overall mean contribution of 42 %. The absence ofany tissue-specific colonization or selective growth was confirmed by determin-ing whether the M. caroli contribution to a given tissue was higher or lower thanthe mean M. caroli contribution to the chimaera. If the contributions of the twoparental types were not subject to tissue-specific selection pressures, one would
Table 1. Mosaicism in neonatal chimaeras as revealed by GPI analysis
predict an equal probability of the M. caroli contribution to a given tissue beingabove or below the chimaera mean. Table 3A shows that the results supportedthis prediction; no statistically significant skewing from a 50:50 ratio was obser-ved.
Somatic tissue mosaicism in adult chimaeras
Table 2 summarizes the results of GPI analysis of 22 adult chimaeras. Themean contribution from M. caroli cells was 28 %, compared with 42 % in neo-natal chimaeras. Correlation coefficients between M. caroli contributions toindividual tissues and the chimaera mean were not as high as those calculated forneonatal chimaeras, but they were all statistically significant at P<0-01.Examination of the mean M. caroli contributions to particular organs or tissuesrevealed that these were not always close to the overall mean M. caroli
Mosaicism in Mus chimaeras 197contribution of 28 %. In particular, M. caroli contributions to liver and kidneywere consistently low and contributions to muscle were consistently high. Ananalysis of the relative proportion of chimaeras with M. caroli contributionsgreater than the chimaera mean for specific tissues revealed that the M. carolicontribution to liver and kidney was significantly reduced (Table 3B). Bycontrast, the M. caroli contribution to skeletal muscle was significantly greaterthan the average M. caroli contribution to all tissues (Table 3B).
Table 3. Tissue-specific variation in mosaicism in neonatal and adult chimaeras
A. Neonatal chimaerasgut liver lung heart muscle carcass
No. of M. caroli 5 7 7 4 7 8contributions abovechimaera mean
No. of M. caroli 6 4 4 7 4 3contributions belowchimaera mean
X2 of difference from 0-0 0-36 0-36 0-36 0-36 1-4550:50 ratio*
B. Adult chimaerasblood lung brain kidney liver muscle heart spleen
No. of M. caroli 13 7 15 2 3 17 4 4contributions abovechimaera mean
No. of M. caroli 8 14 7 20 19 5 5 5contributions belowchimaera mean
f of difference from 0-76 1-71 2-23 13-lt 10-231 5-50$ 0-0 0-050:50 ratio*
* Yates correction used,t significant at P<0-01X significant at P < 0-02
Coat colour and eye pigment mosaicism was also evident in all of the adultchimaeras and the patterns of mosaicism were very similar to those observed inM. musculus chimaeras (Fig. 1).
Germline mosaicism in adult chimaeras
Germline mosaicism was assessed by test breeding female chimaeras, whichwere likely to be XX<->XX, rather than males, which might be XX**XY sexchimaeras producing sperm of only the XY genotype (McLaren, 1976). A major-ity of chimaeras born was male (44/68), suggesting that most XX<-»XY
198 J. ROSSANT AND V. M. CHAPMAN
Fig. 1. Adult interspecific chimaeras between M. musculus and M. caroli, aged 9months.
chimaeras were, indeed, phenotypically male. One hermaphrodite, with normalovary and uterus on one side and aspermic testis and vas deferens on the other,was found.
Eight chimaeric females were mated successfully with M. musculus males and34 litters, containing 206 offspring, were produced. Three of the femalesproduced interspecific hybrids among their offspring (Fig. 2A). The breedingrecords of these chimaeras are shown in Table 4.
Fifteen hybrids were produced, which represented 11-6 % of the offspring ofthese three females and 7-3 % of all offspring produced. The remaining fivefemales may also have been capable of producing hybrids but they producedfewer litters.
No chimaeric female mated successfully with a M. caroli male. In an attemptto produce this reciprocal cross, seven chimaeras were artificially inseminatedwith M. caroli sperm. None of the females produced offspring. Two females weresacrificed at term and one contained 2 or 3 resorbed embryos. The remaining fivewere mated with M. musculus males and four produced M. musculus offspring.
Hybrid breeding
All 15 hybrids were phenotypically and chromosomally male and showed ahybrid GPI band in electrophoresis (Fig. 2B). The mice were much larger thaneither parental species and had a tendency to become obese with increased age.Several were mated with M. musculus females and copulation and vaginal plugswere observed. However, no females became pregnant and no sperm weredetected in vaginal smears or in epididymal squashes. All testes were small and
contained no signs of spermatogenesis (Fig. 2C). The testis tubules were linedwith a single layer of cells which were presumed to be Sertoli cells; no meioticfigures were observed.
DISCUSSION
The utility of our newly developed in situ interspecific cell marker system(Siracusa et al., 1982; Rossant et al., 19826) for following cell lineages in mousedevelopment depends on demonstrating that M. caroli and M. musculus cells cansuccessfully coexist and interact normally in chimaeric mice. Several pieces ofevidence suggest this. Interspecific M. caroli *+ M. musculus chimaeras resem-bled intraspecific chimaeras morphologically at all stages of development; noabnormalities of morphogenesis occurred. Also, no selection occurred againstM. caroli cells in chimaeras during uterine development, despite the fact thatintact M. caroli embryos die in the M. musculus uterus around 10-15 days ofdevelopment (Frels, Rossant & Chapman, 1980). We have previously shownthat the presence of M. musculus trophoblast can protect M. caroli foetal cellsfrom the M. musculus uterine environment (Rossant et al., 1982a) and theanalysis of mosaicism presented here suggests that this protection is complete.Neonatal chimaeras showed a mean M. caroli contribution of 42 %, which makesthe M. caroli <-» M. musculus chimaeric combination a more balanced one thanmany M. musculus strain combinations (Mullen & Whitten, 1971). A similarmean M. caroli contribution was observed in chimaeras at 9-5 days of gestation,indicating that there was no overall selection against M. caroli embryo cellsduring pregnancy. There was also no evidence for any tissue-specific selectionacting against M. caroli cells. Mosaicism in individual tissues (Table 1 & 3A)correlated well with overall mosaicism in each mouse.
There is also some evidence that M. musculus and M. caroli cells behavenormally within a given tissue and do not sort according to species type. Thepatterns of coat and eye pigmentation observed were similar to those observedin intraspecific chimaeras (Mintz, 1967; West & McLaren, 1976). Also, hybridGPI isozyme was observed in all chimaeric muscle samples, showing thatmyotubes could form by fusion of cells of the two species. Preliminary resultswith the new cell marker (Rossant et al., 19826) revealed fine-grained mosaicismin liver and brain samples as observed with other markers in intraspecificchimaeras (West, 1976; Dewey, Gervais & Mintz, 1976; Oster-Granite & Gear-hart, 1981).
Fig. 2. (A) Litter of two M. musculus <-» M. caroli interspecific hybrids and one M.musculus produced by mating an interspecific chimaeric female with M. musculus.(B) GPI phenotype of hybrid offspring. Lane 1 = BB control; Lane 2 = hybrid ABGPI; Lane 3 = A A control. (C) Section of testis of interspecific hybrid male showingabsence of spermatogenesis. Grid bar = 50jum.
202 J. ROSSANT AND V. M. CHAPMAN
Thus, the evidence accumulated so far suggests that the growth patterns of M.musculus and M. caroli cells are sufficiently compatible to produce chimaerasthat can serve as models for normal embryogenesis. However, patterns ofmosaicism in adult chimaeras, aged six months or older, were harder to reconcilewith this contention. The mean contribution by M. caroli cells fell from 42 % inneonatal chimaeras to 28 % in adult chimaeras, suggesting that M. musculus cellstended to overgrow the M. caroli cells. This is perhaps not surprising since adultM. musculus are much larger than M. caroli (West, Frels & Chapman, 1978) andchimaeras were closer to M. musculus in size. Overgrowth by M. musculus cellsdid not occur in all tissues. Skeletal muscle, for example, showed a similar meanM. caroli contribution to that found in neonatal chimaeras, while liver andkidney showed very low contributions from M. caroli cells. Similar differentialgrowth of cells of the two component genotypes has, however, been reported tooccur in tissues of chimaeras made between different inbred strains of M.musculus. The particular tissues affected seem to depend on the particular straincombination used. Overgrowth by cells of one parental genotype has been repor-ted to occur in blood cells (Mintz & Palm, 1969; Warner, Mclvor & Stephens,1977; West, 1977), germ cells (Mintz, 1968), pigment cells (Gearhart & Oster-Granite, 1981) and muscle (Peterson, Frair, Rayburn & Gross, 1979), in dif-ferent chimaeric combinations. Thus, tissue-specific differential growth may bea complicating factor in quantitative analysis of clonal precursors in interspecificchimaeric combinations, but this problem is likely to be shared by anyintraspecific marker system unless congenic strains can be used.
M. musculus and M. caroli cells can coexist in the germ line as well as insomatic tissues, as shown by the production of mixed litters of M. musculus andinterspecific hybrid offspring when female chimaeras were mated to M.musculus. A much higher rate of hybrid production was achieved by matingchimaeric females than ever achieved by artificial insemination (West et al.,1911,1978), suggesting that hybrid embryos survive better in chimaeric femalesthan in M. musculus females. This is compatible with the hypothesis that part ofthe reason for poor survival of hybrid embryos in M. musculus females is im-munological rejection of the foetus by the mother (Frels et al., 1980). Evidencethat M. musculus females can mount an immune response against M. caroliembryos has been found (Croy, Rossant & Clark, 1982). Chimaeric femalesshould be tolerant to both species' antigens (Matsunaga, Simpson & Meo, 1980)and should not reject the hybrid foetuses. However, it must be remembered thatthe interspecific hybrids reported here were the result of the reciprocal crossfrom those produced by artificial insemination- mouse hinnies, not mouse mules(West et al., 1978). It is possible, therefore, that fertilization and developmentare more successful when M. musculus is the sperm donor rather than M. caroli.This could not be tested directly by breeding the same chimaeric females to M.caroli males since no successful pregnancies resulted from such pairings. How-ever, the results of a limited series of artificial inseminations of female chimaeras
Mosaicism in Mus chimaeras 203with M. caroli sperm were compatible with the hypothesis; no successful preg-nancies ensued and some resorbing embryos were observed. Four of thesechimaeras later produced M. musculus offspring, showing that they should havebeen capable of producing hybrid progeny. Further investigation of possibledifferences between the reciprocal crosses will be carried out using in vitrofertilization techniques.
All 15 hybrids produced from mating female chimaeras were phenotypicallyand chromosomally male. This constitutes a highly significant skewing of thenormal sex ratio. We do not know the reason for this peculiar sex ratio whichseems to break Haldane's rule which states that when one sex is absent, rare orsterile in the hybrid offspring of two species, that sex is the heterogametic sex(Haldane, 1922). In the reciprocal cross (West etal., 1977,1978) a predominanceof females was reported. All hybrids produced here were sterile, as were thefemales produced by artificial insemination. Karyotypically, both species havethe same number of chromosomes (Marshall, 1977) but there are substantialdifferences in repetitive DNA between M. caroli and M. musculus (Sutton &McCallum, 1972; Rice & Straus, 1973; Siracusa etal., 1982). These differencesmay have prevented normal meiotic pairing of chromosomes followed bydegeneration of germ cells similar to that reported for the mule (Taylor & Short,1973; Chandley et al., 1974). Although it may not be possible to use these hybridsto introduce new genetic polymorphisms directly into the M. musculus gene pool(West et al., 1978), the ability to produce such hybrids in relatively large numberswill be useful for many other studies on the interaction between regulatory andstructural genes of the two species in the same cell.
CONCLUSIONS
Detailed analysis of mosaicism in interspecific M. musculus <-»M. carolichimaeras has shown that there are no properties of these interspecific chimaerasthat are not shared by intraspecific chimaeras, making our new cell markersystem as valid as any marker system developed between different strains of onemouse species. The marker system should be very useful for analysing celllineage relationships, particularly during embryonic development, but should beused with caution in any numerological studies. However, the same caveatshould be applied to any in situ cell marker system, even if developed within onespecies, given the evidence for tissue-specific differential growth in a wide rangeof chimaeric combinations.
This work was supported by the Canadian Natural Sciences and Engineering ResearchCouncil (J.R.) and the General Medical Sciences of N.I.H. (V.M.C.).
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