effects of the lethal yellow (a*) mutation in mouse ... · lethal mutations which also exhibit...

Development 109, 683-690 (1990)Printed in Great Britain © T h e Company of Biologists Limited 1990

683

Effects of the lethal yellow (A*) mutation in mouse aggregation chimeras

GREGORY S. BARSH*, MICHAEL LOVETT and CHARLES J. EPSTEIN

Department of Pediatrics and Department of Biochemistry and Biophysics, University of California, San Francisco, California, 94143, USA

•Present address: Department of Pediatrics, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford,California 94305, USA

Summary

The Ay allele is a recessive lethal mutation at the mouseagouti locus, which results in embryonic death aroundthe time of implantation. In the heterozygous state, Ay

produces several dominant pleiotropic effects, includingan increase in weight gain and body length, a susceptibi-lity to hepatic, pulmonary and mammary tumors, and asuppression of the agouti phenotype, which results in ayellow coat color. To investigate the cellular action of Av

with regard to its effects upon embryonic viability andadult-onset obesity, we generated a series of aggregationchimeras using embryos that differ in their agouti locusgenotype. Embryos derived from Ay/axAy/a matingswere aggregated with those derived from A/Ax A/Amatings, and genotypic identification of the resultantchimeras was accomplished using a molecular probe atthe Emv-15 locus that distinguishes among the three

different alleles, Ay, A, and a. Among 50 chimeras, 25analyzed as liveborns and 25 as 9.5 day embryos, 29were a/a*-+A/A and 21 were Ay/a+->A/A. The absence ofAy/Ay<r+A/A chimeras demonstrates that Ay/Ay cellscannot be rescued in a chimeric environment, and therelative deficiency of Ay/a*-+A/A chimeras suggests that,under certain conditions, Ay heterozygosity may par-tially affect cell viability or proliferation. In the 25liveborn chimeras, Ayja*^AJA animals became obese asadults and a/a+-*A/A animals did not. There was nocorrelation between genotypic proportions and rate ofweight gain, which shows that, with regard to its effectson weight gain, Ay heterozygosity is cell non-auton-omous.

Key words: agouti locus, chimera, mouse, obesity.

Introduction

Mutations that interfere with embryonic viability areimportant tools for understanding developmentalmechanisms. In mammals, however, mutagenesis isrelatively inefficient and large numbers of embryoscannot be screened expeditiously. Instead, the assump-tion is often made that mutations that affect differenttypes of tissue identify genes that control fundamentalaspects of embryonic development. Consequently,much attention has been focused on existing recessivelethal mutations which also exhibit pleiotropism.

The lethal yellow allele (Ay) at the mouse agoutilocus, one of the longest recognized and most intensi-vely studied recessive lethal mutations (Castle andLittle, 1910; Cuenot, 1908; Silvers, 1979), is associatedwith a multitude of pleiotropic effects. In the hetero-zygous state, Ay alters the normal back and forth switchbetween eumelanin and phaeomelanin granule forma-tion by follicular melanocytes so that only phaeomela-nin granules are formed, and mice that carry Ay (withwild-type alleles at the albino and brown loci) areentirely yellow. In addition, heterozygosity for Ay

produces increased somatic growth and obesity (Castle,1941; Danforth, 1927), a predisposition to spontaneous

and chemically induced mammary gland, pulmonary,hepatic and skin tumors (Deringer, 1970; Heston, 1942;Heston and Deringer, 1947; Heston and Vlahakis, 1961;Vlahakis and Heston, 1963), premature infertility(Granholm and Brock, 1981; Granholm et al. 1986) andglucose intolerance (Carpenter and Mayer, 1958; Hel-lerstrom and Hellman, 1963). In cases where tested, thelatter effects, like those of Ay on coat color, aredominant over the effects of all other agouti locusalleles (Silvers, 1979).

Embryos homozygous for Ay do not survive muchbeyond implantation, but the exact time and site ofaction of J4y-mediated recessive lethality is contro-versial. A multitude of morphologic abnormalities havebeen described in embryos derived from matings be-tween Ay heterozygotes (Brock and Granholm, 1979;Calarco and Pederson, 1976; Cizadlo and Granholm,1978a; Cizadlo and Granholm, 1978b; Eaton andGreen, 1962; Eaton and Green, 1963; Granholm andJohnson, 1978; Pedersen, 1974), but without an inde-pendent marker, genotypic identification of AyJAy

homozygotes has always been presumptive. In addition,only one study has formally tested if abnormalities in aparticular cell lineage or tissue are responsible forsubsequent embryonic death. Papaioannou and

684 G. S. Barsh, M. Lovett and C. J. Epstein

Gardner (1979) performed a numerical analysis ofreciprocal blastocyst injection experiments in whicheither the inner cell mass component or the hostblastocyst component was expected to contain Ay/Ay

homozygotes 25 % of the time. The results suggestedthat an Ay/Ay inner cell mass could be rescued by acontrol blastocyst but not vice versa, pointing to a cell-autonomous defect of the trophectoderm.

These and similar experiments with recessive lethalmutations have been complicated by an inability todistinguish cells derived from homozygous embryos. Toovercome these difficulties and to understand better therole of recessive lethal agouti mutations in normaldevelopment, we have used molecular markers thatidentify different agouti alleles to analyze aggregationchimeras between embryos of different agouti geno-types. Restriction fragment length polymorphisms(RFLPs) at the Emv-15 locus, closely linked to agouti(Copeland et al. 1983; Lovett et at. 1987; Siracusa et al.19876), distinguish between cells that contain zero, oneor two doses of the Ay mutation and have allowed us tocompare the genotypic proportions in each chimerawith two components of the pleiotropic phenotype -embryonic viability and increased weight gain. We nowreport that recessive lethal effects of Ay/Ay homozygo-sity cannot be rescued in a chimeric environment andthat adult-onset obesity in Ay heterozygotes is a noncell-autonomous process.

Materials and methods

Mouse strains and mutationsThe strains, C57BL/6J-/l>r and BALB/cJ, of agouti genotypesAy/a and A/A, respectively, were obtained from The JacksonLaboratory, Bar Harbor, Maine. Random bred CD-I females(Charles River Laboratories) were used as uterine fostermothers.

Embryo manipulation and production of aggregationchimerasAfter superovulation, embryos from Ay/axAy/a crosses wereharvested at 1.5 days post coitum at the 2- to 4-cell stage andplaced in microdrop culture for 24 h, after which mostembryos had undergone a single round of division. 4- to 8-cell-stage embryos from A/Ax A/A crosses were then harvestedat 2.5 days post coitum and aggregated with stage-matchedembryos from the Ay/axAy/a cross. (This protocol allowedthe most efficient generation of embryos matched for cleavagestage, due to interstrain variability in the rate of in vitropreimplantation development.) Embryo recovery, dis-sociation, aggregation and culture were all as describedpreviously (Cox et al. 1984).

Identification of chimera genotypes and determinationof genotypic proportionsInsertion of the Emv-15 provirus is very closely associatedwith Ay (Siracusa et al. 1987a). Furthermore, two alternativehaplotypes defined by RFLPs in genomic DNA surroundingthe insertion site are closely associated with the a or A alleles(Lovett et al. 1987; Siracusa et al. 19876). A probe immedi-ately 5' to the genomic insertion site of Emv-15, P0.5(Fig. 1A), can therefore distinguish between all three alleles,

Ay, a, or A, after digestion with Hindlll. This probe detects a2.6 kb Hindlll fragment from the A allele, a 3.8 kb HindUlfragment from the Ay allele, and a 5.4 kb Hindlll from the aallele. Therefore, AyJAy<^AlA chimeras will contain the2.6 kb and 3.8 kb fragments, a/a<-+A/A chimeras will containthe 2.6kb and 5.4kb fragments, and Ay/a*-*A/A chimeraswill contain all three fragments, 2.6kb, 3.8kb, and 5.4kb,with the latter two present in equal abundance. The geneticdistance between Emv-15 and Ay is <0.2cM (upper 95%confidence limit; Siracusa et al. 1987a), and the geneticdistance between Emv-15 and a is 0.6±0.4cM (Siracusa et al.1989). Therefore, although there is some question whetherthe Ay mutation is located at precisely the same genetic mapposition as A and a (Siracusa et al. 1987a; Wallace, 1965), thelikelihood of incorrectly identifying a particular agouti alleledue to recombination between the agouti locus and Emv-15 isextremely small.

DNA from embryos or from tail, brain, liver and kidneysamples of liveborn animals was digested with Hindlll,subjected to electrophoresis through 1% agarose, and trans-fered to a nylon membrane by capillary action. The P0.5probe was radiolabeled by random priming and hybridizationin the presence of 10% dextran sulphate was performedaccording to standard procedures. For the 9.5 day embryos,50% of the entire sample was used per lane. After an 8 dayexposure, 55 of 69 embryos produced a detectable signal, butin 29 cases, chimeric genotype could not be establishedunequivocally (see RESULTS), because of a low recovery ofDNA. For the samples from liveborn chimeras, 5-10 micro-grams of DNA was used per lane. Chimeric genotypes werereadily apparent in every case after a 1-2 day autoradio-graphic exposure, and densitometric measurement of eachallele was used to estimate directly zygotic proportions.

Results

Generation and identification of adult chimerasAnimals heterozygous for the lethal yellow and non-agouti alleles were intercrossed to produce a populationexpected to contain 25 % a/a homozygotes, 50 % Ay/aheterozygotes, and 25 % Ay/Ay homozygotes. Indi-vidual embryos from this group were aggregated withdevelopmentally matched BALB/cJ (A/A) embryos.In the first set of experiments, 25 liveborn animals wereobtained from 89 chimeric embryos transferred intopseudopregnant mothers. Genomic DNA was re-covered from a segment of each animal's tail, and aSouthen blot was prepared after digestion with Hindlll.Hybridization with the P0.5 probe produced the patternof fragments expected for Ay/a++A/A chimeras in 11animals and the pattern of fragments expected fora/a*-+A/A chimeras in 14 animals (Table 1). No ani-mals produced the pattern expected for Ay/Ay++A/Achimeras (X2=16.2, ^=0.003 for difference from a1:2:1 distribution).

We compared the coat color of each animal to itsgenotypic composition as determined by analysis of theEmv-15 RFLPs. Because the C57BL/6J-/F andBALB/cJ strain carry different albino alleles (C57BL/63-Ay is C/C and BALB/cJ is c/c), as well as differentagouti locus alleles, it was necessary to consider thecellular action of both genes to interpret properly theresultant coat color patterns. The agouti locus acts

Avla JI AVla

4 AIA x AIA

4 AVla -AlA

ala -AIA

A AYIAY-AIA

ia P0.5 probe

* 1 Hhdlll 1 absent in a

Fig.. 1. Generation and identification of lethal yellow and non-agouti chimeras. (A) Breeding strategy, possible outcomes and map of the Emv-15 locus as described in the text. The asterisk indicates a polymorphic HindIII site present on chromosomes that carry the AY and A alleles, but not present on chromosomes that carry the a allele. (B) An autoradiogram of tail DNA from a series of liveborn chimeras after digestion with HindIII and hybridization to the P0.5 probe. The first two lanes are A/A and a/a control samples as indicated. The 3.8kb fragment, seen exclusively in DNA from the Ahallele, contains approximately 450bp of cellular DNA joined to 3.35 kb of proviral DNA from Emv-15. DNA from the A and a alleles does not contain Emv-15, and the 2.6 kb and 5.4 kb fragments result from the presence or absence, respectively, of a polymorphic HindIII site. (C) Appearance of two chimeric mice. Coat color patches correspond to melanocyte rather than hair follicle clones.

Cellular action of the lethal yellow (Ay) mutation 685

Table 1. Generation of lethal yellow and non-agoutichimeras

No. of aggregates transferred"No. recoveredNo. genotypedChimera genotypeAy/a++A/AAy/aa/a+-*A/Aa/aAy/Ay++A/AAy/Ay

A A

Experiment I.Adult chimeras

892525

110

140000

Experiment II.9-day embryo

chimeras

1576954*

91

13200

29

"Excluding transfers to females that failed to become pregnant.b Fifteen of the embryos failed to produce a detectable

hybridization signal, but, as a group, did not exhibit any readilyapparent morphologic differences from the remainder of the 9.5day embryos.

within cells of the dermal papilla to influence thebehavior of overlying follicular melanocytes, but aminimum level of tyrosinase activity, controlled by the clocus, is required to generate phaeomelanin or eumela-nin (Silvers, 1961). In addition, the patch size ofmelanocyte clones is larger than the patch size of hairfollicle clones. Because the albino (c) mutation abo-lishes tyrosinase activity, we expected that any patch ofmelanocytes that had originated from a BALB/cJ{A c/A c) progenitor would appear albino, regardless ofthe agouti locus genotype (- C/- C) of the underlyingdermal papillae cells. Likewise, some hair follicles thatcontained C57BL/6J-/t-v melanocytes (-C/-C) wouldbe expected to produce agouti hairs if the underlyingdermal papilla had originated from a BALB/cJ (A cjA c) progenitor.

In accord with these predictions, the observed coatcolor patterns included patches that were phenotypi-cally albino, yellow or non-agouti, although, asexpected, the latter two types were never seen in asingle animal (Fig. 1C). In contrast, there were no largepatches of agouti. However, close inspection revealedfrequent but scattered agouti hairs in most animals.Animals that contained any yellow patches were scoredas Ay/a++A/A, and animals that contained any non-agouti patches were scored as a/a<-*A/A. Six animalswere entirely albino and therefore could not be ident-ified on the basis of coat color alone, but in theremainder, genotypic identification inferred from Emv-15 RFLP analysis agreed with that predicted by coatcolor.

Generation and identification of 9.5 day embryochimerasFailure to obtain the Ay/Ay++A/A genotype in any ofthe 25 adult animals suggested that this class of chim-eras was not surviving to term. To characterize furtherthe developmental potential of Ay/Ay cells, we gener-ated a second set of chimeras for analysis midway

through gestation. As before, individual 4- to 8-cellembryos from an intercross between Ay/a hetero-zygotes were aggregated with A/A embryos at a similarstage. Chimeric embryos were recovered at 9.5 daysgestation, and constituent genotypes were determinedby analysis of the Emv-15 RFLPs. Each embryo andattached yolk sac were dissected free of maternaldecidua, but, because we wished to recover as muchDNA as possible, no attempt was made to separateembryonic and extraembryonic components. In the 69embryos recovered after 157 transfers, there was aspectrum of developmental stages from early neuralfold with 5-10 visible somites to early limb bud with20-30 somites. Enough DNA was recovered to gener-ate a hybridization signal in 54 cases, but in 29 of thesethe only visible DNA fragment was 2.6 kb, indicative ofthe A/A genotype. Of the remaining 25 embryos, 10were Ay/a*Â/A or Ay/a and 15 were a/a*Â/A or a/a(Table 1).

Distribution of chimeric genotypesAs with the adult animals, none of the 9.5 day embryosgenerated the pattern expected for Ay/Ay<r->A/A chim-eras. Furthermore, in both the adults and the embryos,the observed ratio of Ay/a*+A/A to a/a+Â/A chim-eras was distorted relative to the Mendelian expectationof 2:1 (P=0.0001 assuming non-viability of Ay/Ay++A/A chimeras) (Table 1). Because the amounts of embryoDNA are limiting, we estimate that the minimumgenotypic contribution that can be detected in the 9.5day embryos is 20%. Therefore, the existence of threegroups of embryos with an apparently single genotypiccontribution (1 Ay/a, 2 a/a, and 29 A/A embryos) canbe explained in two ways: either they represent aggre-gates that failed to form chimeras at the morula stage,or they are actually chimeras in which the contributionof one of the genotypes is too small for detection. Bothexplanations could contribute to the altered ratio ofAy/a*r+A/A to a/a<r*A/A chimeras.

To address the possibility that Ay/a cells, in chimericcombinations with A/A, might exhibit a generalizeddisadvantage relative to a/a cells, we examined thefrequency distribution of genotypic proportions in tailtissue from the two types of adult chimeras. Measuringthe relative intensities of the 2.6 kb, 3.8 kb, and 5.4 kbHindlll fragments allowed us to estimate the pro-portion of Ay/a or a/a cells in the tail of each Ay/a*-+A/A or a/a<r+A/A chimera. (In the samples from the adultchimeras, enough DNA could be applied to a singlewell to detect a chimeric contribution of 5 % -10 %, butdue to limiting amounts of DNA, a similar analysiscould not be applied to the 9.5 day embryo chimeras.)Although small differences would not have beendetected without applying more sensitive techniques toa larger population of animals, the frequency distri-butions of the proportions of Ay/a or a/a cells do notshow gross differences between the two types of chim-era (Fig. 2). Although these results do not exclude thepossibility that Ay/a cells are less effective than a/a cellsin contributing to the inner cell mass following embryoaggregation, they do suggest that any possible disadvan-


40 60 BO% a/a or AVa cells

100

A/A

A/A

Fig. 2. Genotypic proportions in a/a<^*A/A and Ay/a<^>A/A chimeras. In the 25 liveborn chimeras, the proportion ofa/a or Ay/a cells in a tail sample was estimated bycomparing the densitometric signal(s) from the 5.4kbfragment or the 5.4kb and 3.8kb fragments to the signalfrom the 2.6 kb fragment. The minimum genotypiccontribution detectable in these experiments is 5 %, whichrepresents 0.5 micrograms of genomic DNA. Each symbolrepresents a single measurement from one animal.

tage of Ay/a cells relative to a/a cells does not affect cellproliferation or viability in a generalized fashion, i.e atall stages of pre- and post-natal life.

Adult-onset obesity in liveborn chimerasIncreased somatic growth and adult onset obesity aretwo of the most visible pleiotropic effects associatedwith the Ay allele. A weight difference between yellowand non-yellow littermates first becomes apparent at4-6 weeks of age and increases, more so in females thanmales, over the next 6-12 months. Central to under-standing the underlying physiologic mechanism iswhether increased lipid deposition in Ay/a adipocytes iscaused by expression of the /Fgene within the adipo-cytes or within the environment in which they reside,i.e. whether /^'-mediated obesity is cell-autonomous.The populations of a/a<-+A/A and Ay /a<ÂJA animalswith different degrees of chimerism provided the op-portunity to investigate this question. We expectedthat, if Av-mediated obesity were cell-autonomous, (1)Ay/a*-+A/A animals with a low proportion of Ay/a cellswould exhibit a weight gain similar to a/a<-*A/Aanimals; (2) Ay /aÂ/ A animals with a high proportionof Ay/a cells would exhibit a rate of weight gain similarto Ay/a animals; and (3) the proportion of Ay/a cells inAy/a+-*A/A chimeras would be related linearly to rateof weight gain.

Growth records indicated that, by 6 weeks of age, therange of weights was significantly different between thetwo types of adult chimeras (20.2±1.36g for a/a<r->A/Aanimals; 26.2±2.75g for Ay/a<Â/A animals;P=0.0001). This difference became increasingly appar-ent over the next 12-14 weeks (Fig. 3). We calculatedthe rate of weight gain in each animal as a linearregression between 10 and 20 weeks of age and com-pared the frequency distributions of the two types ofchimeras (Fig. 4). Rates of weight gain were similar

80 100Age (days)

• a/a

•A*/ aA/AA/A

Fig. 3. Growth curves of a/a<-+A/A and Ay/a<-+A/Achimeras. Animals were fed a standard diet with a fatcontent of 6.5 % and were weighed weekly. Each curverepresents a single animal.

1

£.i

• 3

I

I

• I

I

II

(I

I i I

female

OOO i i1 1

male

o

i

O0 .05 .10 15 20 25 30

Growth rate (g/day; 10-20 weeks)

# a/a -QAy/a -

|—• A/A*—- A/A

Fig. 4. Rates of weight gain of a/a<-*A/ A and Ay/a++A/Achimeras. The plotted value represents a linear regressionof weight vs age between 10 and 20 weeks. Each symbolrepresents a single animal.

between female and male Ay/a++A/A chimeras, butthe difference relative to a/a+*A/A chimeras wasgreater in females then in males. Although the pro-portion of Ay/a cells in tails from Ay/a<-+A/A animalsvaried from 20% to 90% (Fig. 2), the distribution ofrates of weight gain did not show a similar degree ofvariation. Instead, the distributions in both types ofchimeras exhibited a similar degree of variation, whichsuggested that rates of weight gain in Ay/a<r+A/Aanimals with a low proportion of Ay/a cells wereindistinguishable from those with a high proportion ofAy/a cells.

To address this question more directly, we comparedgenotypic proportion to rate of weight gain in bothtypes of chimeras. Although we could not directly


20 30 40

%a/a50

or A'/a60 70

cells80

O «D •A A

<>•

90

tall

brain

kidney

liver

100

Fig. 5. Relation of rates of weight gain to genotypicproportions in a/a*+A/A and Ay/a*-*A/A chimeras. Eachsymbol represents a single measurement from tissue, tail,brain, kidney, or liver DNA from an adult chimera,estimated densitometrically as in Fig. 2. The symbols aredisplaced along the abscissa by 2-5 percentile units in caseswhere multiple values overlapped each other.

measure genotypic proportions in the isolated adiposetissue compartment, we examined liver, kidney andbrain DNA from most of the chimeric animals inaddition to the tail DNA sampled previously. We foundno correlation between genotypic proportion and rateof weight gain for either Ay/a<r+A/A or aja*^AJAchimeras, regardless of the tissue examined (Fig. 5).Assuming that genotypic proportions in fat do not varysystematically from those in solid organs, a minimalproportion of Ay/a cells in Ay/a<-*A/A animals issufficient to produce a maximal effect on rate of weightgain, and therefore, adult-onset obesity associated withAy appears to be mediated by a cell non-autonomousmechanism.

Discussion

Genetic mosaics and chimeras are valuable tools forstudying the relationship of cellular genotype to aparticular developmental phenotype. In the mouse, thetiming of X inactivation, primary sex determination,and the regulation of the switch from alphafetoproteinto albumin are most directly observed in the wholeanimal, and in each case the analysis of aggregation orblastocyst injection chimeras has played a central role inunderstanding the underlying physiologic mechanisms(Burgoyne et al. 1988; Gardner et al. 1985; Vogt et al.1987).

We have applied the analysis of aggregation chimerasto the study of the pleiotropic effects of the lethalyellow allele. In 25 liveborn chimeras, none contained acomponent from Ay/Ay homozygotes, which demon-strates that with regard to fetal viability, the effects ofthe Ay gene cannot be rescued in a chimeric environ-

ment. Neither was Ay/Ay tissue detectable in a series of25 embryonic chimeras, which suggests that the effectsof Ay on cell viability are manifest by 9.5 days ofgestation. Finally, measurements of weight gain andcomparison of these measurements to genotypic pro-portions in Ay/a<r->A/A chimeras suggests that theeffects of Ay on rate of weight gain are mediated by acell non-autonomous mechanism.

A variety of morphologic abnormalities have beendescribed in embryos derived from heterozygous Ay

intercrosses, including an increased frequency ofexcluded blastomeres and ultrastructural abnormalitiesin cleavage stage embryos (Calarco and Pederson, 1976;Pedersen, 1974), reduced growth and development ofinner cell mass and trophectoderm cultures in vitro(Papaioannou, 1988), decreased inner cell mass numberand delayed hatching in peri-implantation blastocysts(Cizadlo and Granholm, 1978a; Granholm and John-son, 1978), and reduced trophectoderm differentiationand cell death in post-implantation blastocysts (Eatonand Green, 1962; Eaton and Green, 1963). In one of thefew studies that directly addressed tissue specificity ofAy homozygous lethality, inner cell mass cells derivedfrom Ay/acxAy/ac matings were injected into normal(random bred) blastocysts (Papaioannou and Gardner,1979). The recovery of 10.5 day chimeric embryos wasnot significantly different from that observed in acontrol experiment in which blastocysts were derivedfrom Ay/aexac/ac matings. Although these resultssuggested that AyJAy cells can survive to at leastmidgestation in a normal tissue environment, genotypicidentification of these cells was not then possible, andwe have not detected any Ay/Ay cells in our series ofchimeras. Because Ay/Ay trophectoderm cells arelikely to have been included in Ay/Ay++A/A aggre-gation chimeras but not in the more defined environ-ment of Ay/Ay++A/A blastocyst injection chimeras,one explanation for the absence of Ay/Ay cells in ourchimeras is that even a small number of these cells inthe trophectoderm is sufficient to prevent post-implan-tation development. Alternatively, if Ay/Ay cells werepresent but too infrequent to be detected in our 9.5 dayembryos, Ay/Ay++A/A chimeras would have contribu-ted to the apparently non-chimeric A/A group. How-ever, all of the liveborn chimeras contained detectablecomponents from two zygotes, and therefore we expectthat any 9.5 day Ay/Ay*^A/A chimeras would not havesurvived until term. At present, we cannot say whetherhomozygosity for Ay in inner cell mass, trophectoderm,or both tissues is responsible for embryonic death, butan analysis of blastocyst injection chimeras usingRFLPs at the Emv-15 locus should allow this questionto be addressed definitively.

The number of chimeras that contained a componentfrom Ay/a embryos was approximately 50 % less thanexpected, given that Ay heterozygosity has no effect onviability in standard breeding experiments. One expla-nation is that Ay heterozygosity affects cell proliferationor viability at specific periods of embryogenesis, andthat a proportion of chimeric embryos die due to adevelopmental mismatch between the Ay and non-/4r


components of the chimera. This effect might well bemasked in a normal (non-chimeric) embryo, in whichthere is no developmental mismatch and a tremendousregulative capacity in response to cell death duringearly development (Snow et al. 1981). Such an effectmay have contributed to the relative excess of appar-ently non-chimeric A/A 9.5 day embryos, although, asargued above, a putative class of Ay/Ay*-*A/A andAy/a*+A/A embryos in which the Ay/Ay or Ay/acomponent was non-detectable is unlikely to havesurived until term. Which chimeras exhibit such adevelopmental mismatch could depend on the randomcontribution of A/A and Ay/a cells to a particular pre-or peri-implantation lineage with a small number offounder cells. For example, in chimeras in which theAy/a component constitutes the majority of trophecto-derm, a developmental mismatch between trophecto-derm and inner cell mass might prevent subsequentgrowth and differentiation of extraembryonic tissues,the chimera would appear non-chimeric at 9.5 daysgestation, and would not survive until term. Alterna-tively, if the Ay/a component of the chimera is, bychance, excluded from trophectoderm, the embryomight not be susceptible to the effects of a developmen-tal mismatch and would not manifest altered genotypicproportions in most adult tissues. This speculativeseries of mechanisms constitutes a single explanationfor both the excess of non-chimeric 9.5 day embryosand the reduced number of Ay/a*-*A/A chimerasamong both 9.5 day embryos and liveborn animals.However, a larger series of chimeras along with anextensive analysis of genotypic proportions in differenttissues will be necessary to allow these issues to beinvestigated more thoroughly.

Our analysis of rates of weight gain in livebornchimeras shows that Ay/a<r->A/A animals with a smallproportion of Ay/a cells in tail, liver, kidney or brain,have growth characteristics indistinguishable from ani-mals with a large proportion of Ay/a cells. In earlierstudies directed at understanding the mechanism of Ay-mediated obesity, means other than aggregation chim-eras have been used to alter the environment ofheterozygous Ay cells. Experiments in which parabioticconnections were established between Ay/A and a/aanimals suggested that a circulating substance from theformer animal could lead to increased body length inthe latter (Wolff, 1963). More recently, reciprocaladipose tissue transplantation experiments betweenAy/a and A/A animals have suggested that fat cell sizeand proliferation are a function of cellular environmentrather than cellular genotype (Meade et al. 1979). Thus,several lines of evidence now point to a cell non-autonomous mechanism underlying ^-induced obes-ity, but the primary physiologic changes are still un-known. Obesity associated with Ay is not caused by adecrease in activity or an increase in nutrient intake(Carpenter and Mayer, 1958; Cizadlo and Granholm,1976; Fenton and Chase, 1951; Hollifield and Parson,1957) and is not prevented by pituitary or adrenalablation (Jackson et al. 1976; Plocher and Powley,1976). Although altered thermoregulation has been

described in fat yellow mice as well as in other geneticobesity syndromes (Cizadlo et al. 1977; Herberg andColeman, 1977), these changes are likely to be second-ary effects of increased body fat.

Cell-autonomous behavior of a lethal mutation isoften taken to mean that the normal gene controls anessential intracellular process. Conversely, rescue of amutant phenotype may imply that the normal genecodes for a diffusible product. These conclusions arevalid, however, only in situations in which the mutationbeing tested is amorphic or null. For example, given aparticular gene that codes for a secreted protein,expression of a mutated coding sequence might blocksecretion of that protein, leading to progressive intra-cellular accumulation and eventual cell lysis. In thiscase, the mutant cells could not be rescued and cell-autonomous death would indicate erroneously that thenormal gene product is part of an essential intracellularprocess. If, on the other hand, a different mutationentirely prevented expression of the secreted protein,mutant cells would survive if the normal protein wasprovided via a chimeric environment. In this case, cellnon-autonomy would indicate correctly that the normalgene coded for a diffusible product. It is not known ifthe effects of Ay on recessive lethality are due toinactivation, altered expression, or overexpression of anormal gene. However, we have previously reportedthat a 75 kb deletion is closely associated with theradiation-induced non-agouti lethal allele, a1 (Barshand Epstein, 1989), and a is lethal in combination withAy (Lyon et al. 1985). To this extent, ,4-v-mediatedrecessive lethality may represent a null mutation, andthe normal agouti gene product (or at least the oneaffected by Ay and a1) may indeed be part of an essentialintracellular process. In this case, the effect of Ay/Ay orclja1 cell loss on chimeric embryo survival woulddepend on the time of cell death. Prior to implantation,extensive cell death may be compensated by the regu-lative capacities of the mammalian embryo (Snow et al.1981), but after gastrulation, significant cell death islikely to be incompatible with embryonic survival.Analysis of aggregation chimeras with a1 and otherradiation-induced recessive lethal agouti mutations isnow possible and will help to resolve these questions.

Mutations induced by radiation frequently affectmultiple transcriptional units, in which case the pleio-tropic effects of a particular mutation are more likely tosignify the proximity of one gene to the next, ratherthan a single gene that affects multiple types of tissue(Russell, 1989). There are two agouti alleles withpleiotropic effects other than recessive lethality, Ay andAvy (viable yellow), and both arose spontaneously(Silvers, 1979). Animals heterozygous for Avy exhibitvariable expression of both yellow coat color andobesity, and variation in expression of one phenotype isdirectly correlated with variation in expression of theother (Wolff and Pilot, 1973; Wolff et al. 1986). Further-more, like the effect of Ay on obesity and femaleinfertility (Granholm and Dickens, 1986), suppressionof the phaeomelanin to eumelanin switch is cell non-autonomous (Silvers, 1961), supporting the possibility


that these phenotypes are mediated by the same oroverlapping transcriptional units. However, althoughallelomorphic series for the agouti locus exist in mostmammalian orders, Mus musculus is the only species inwhich agouti locus mutations are also known to affectgrowth and viability (Searle, 1968). In addition, interac-tion and recombination between agouti locus alleleshave suggested a complex genetic structure for the locus(Russell etal. 1963; Wallace, 1954; Wallace, 1965). It ispossible, therefore, that different transcriptional unitsmediate one or more of the pleiotropic effects associ-ated with Ay. Resolution of these questions will requireisolation of agouti locus transcripts, a goal now clearlyin sight.

We thank Sandra Smith for preparing the adult chimeras,Elaine Carlson for preparing the 9.5 day embryo chimeras,Teodosia Zamora for excellent technical assistance, and DrVirginia Papaioannou for helpful discussions. This work wassupported by research grant HD-03132 from the NationalInstitute of Child Health and Human Development. G.S.B.was supported by a postdoctoral research training grant fromthe National Institute of General Medical Sciences (GM-07085).

References

BARSH, G. S. AND EPSTEIN, C. J. (L989). Physical and geneticcharacterization of a 75-kilobase deletion asssociated with d', arecessive lethal allele at the mouse agouti locus. Genetics 121,811-818.

BROCK, K. T. AND GRANHOLM, N. H. (1979). Histological analysesof lethal yellow mouse embryos (Ay/Ay) at 132h post coitum.Anat. Embryol. 157, 161-166.

BURGOYNE, P. S . , BUEHR, M . , KOOPMAN, P . , ROSSANT, J. AND

MCLAREN, A. (1988). Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX<-*XYchimaeric mouse testes. Development 102, 443-450.

CALARCO, P. G. AND PEDERSON, R. A. (1976). Ultrastructuralobservations of lethal yellow (Ay/Ay) mouse embryos. J.Embryol. exp. Morph. 35, 73-80.

CARPENTER, K. J. AND MAYER, J. (1958). Physiologic observationson yellow obesity in the mouse. Am. J. Physiol. 193, 499-504.

CASTLE, W. E. (1941). Influence of certain coat color mutations onbody size in mice, rats, and rabbits. Genetics 26, 177-191.

CASTLE, W. E. AND LITTLE, C. C. (1910). On a modified Mendelianratio among yellow mice. Science 32, 868-870.

CIZADLO, G. R. AND GRANHOLM, N. H. (1976). Relationship ofactivity and obesity in genetically obese yellow (Ay/a) mice.Proc. S. D. Acad. Sci. 55, 97-99.

CIZADLO, G. R. AND GRANHOLM, N. H. (1978a). In vivodevelopment of the lethal yellow (Ay/Ay) mouse embryo at 105 hpost coitum. Genetica 89-93,

CIZADLO, G. R. AND GRANHOLM, N. H. (1978ft). Ultrastructuralanalysis of preimplantation lethal yellow (Ay/Ay) mouseembryos. J. Embryol. exp. Morph. 45, 13-24.

CIZADLO, G. R., GRANHOLM, N. H. AND WAGERS, R. C. (1977).

Aberrant body temperature regulation in obese yellow (Ay/a)mice. Proc. S. D. Acad. Sci. 56, 159-162.

COPELAND, N. G., JENKINS, N. A. AND LEE, B. K. (1983).

Association of the lethal yellow (Ay) coat color mutation with aecotropic murine leukemia virus genome. Proc. natn. Acad. Sci.U.S.A. 80, 247-249.

Cox, D. R., SMITH, S. A., EPSTEIN, L. B. AND EPSTEIN, C. J.

(1984). Mouse trisomy 16 as an animal model of human trisomy21 (Down syndrome): production of viable trisomy 16<->diploidmouse chimeras. Devi Biol. 101, 416-424.

CUENOT, L. (1908). Sur quelques anomalies apparentes desproportions Mendeliennes. Arch. Zool. exp. Gener. 9, 7-15.

DANFORTH, C. H. (1927). Hereditary adiposity in mice. J. Hered.18, 153-162.

DERINGER, M. K. (1970). Influence of the lethal yellow (/*-*) geneon the development of reticular neoplasms. J. natn. Cancer Inst.45, 1205-1210.

EATON, G. J. AND GREEN, M. M. (1962). Implantation and lethalityof the yellow mouse. Genetica 33, 106-112.

EATON, G. J. AND GREEN, M. M. (1963). Giant cell differentiationand lethality of homozygous yellow mouse embryos. Genetica 34,155-161.

FENTON, P. F. AND CHASE, H. B. (1951). Effect of diet on obesityof yellow mice in inbred lines. Proc. Soc. exp. Biol. med. 77,420-422.

GARDNER, R. L., LYON, M. L., EVANS, E. P. AND BURTENSHAW, M.

D. (1985). Clonal analysis of X-chromosome inactivation and theorigin of the germ line in the mouse embryo. J. Embryol. exp.Morph. 88, 349-363.

GRANHOLM, N. H. AND BROCK, K. T. (1981). Effects of the lethalyellow gene (Ay) on mating preference in C57BL/6J mice. Proc.S. D. Acad. Sci. 60, 24-28.

GRANHOLM, N. H. AND DICKENS, G. A. (1986). Effects ofreciprocal ovary transplantation on reproductive performance oflethal yellow mice (Ay/a; C57BL/6J). J. Reprod. Fen. 78,749-753.

GRANHOLM, N. H., JEPPESEN, K. W. AND JAPS, R. A. (1986).

Progressive infertility in female lethal yellow mice (Ay/a\ strainC57BL/6J). J. Reprod. Fen. 76, 279-287.

GRANHOLM, N. H. AND JOHNSON, P. M. (1978). Enhancedidentification of lethal yellow (Ay/Ay) mouse embryos by meansof delayed development of four-cell stages. J. exp. Zool. 205,327-334.

HELLERSTROM, C. AND HELLMAN, B. (1963). The islets ofLangerhans in yellow obese mice. Metabolism 12, 527-536.

HERBERG, L. AND COLEMAN, D. L. (1977). Laboratory animalsexhibiting obesity and diabetes syndromes. Metabolism 26,59-99.

HESTON, W. E. (1942). Relationship between the lethal yellow (Ay)gene of the mouse and susceptibility to induced pulmonarytumors. J. natn. Cancer Inst. 3, 303-308.

HESTON, W. E. AND DERINGER, M. K. (1947). Relationship betweenthe lethal yellow (Ay) gene of the mouse and susceptibility tospontaneous pulmonary tumors. J. natn. Cancer Inst. 7, 463-465.

HESTON, W. E. AND VLAHAKIS, G. (1961). Influence of the Ay geneon mammary-gland tumors, hepatomas, and normal growth inmice. J. natn. Cancer Inst. 26, 969-982.

HOLUFIELD, G. AND PARSON, W. (1957). Food drive and satiety inyellow mice. Am. J. Physiol. 189, 36-38.

JACKSON, E., STOLZ, D. AND MABTIN, R. (1976). Effect of

adrenalectomy on weight gain and body composition of yellowobese mice (Ay/a). Horm. Metab. Res. 8, 452-455.

LOVETT, M., CHENG, Z. Y., LAMELA, E. M., YOKOI, T. AND

EPSTEIN, C. J. (1987). Molecular markers for the agouti coatcolor locus of the mouse. Genetics 115, 747-754.

LYON, M. F., FISHER, G. AND GLENISTER, P. H. (1985). A recessive

allele of the mouse agouti locus showing lethality with yellow,Ay. Genet. Res. 46, 95-99.

MEADE, C. J., ASHWELL, M. AND SOWTER, C. (1979). Is genetically

transmitted obesity due to an adipose tissue defect? Proc. R.Soc. Lond. B. 205, 395-410.

PAPAIOANNOU, V. E. (1988). Investigation of the tissue specificity ofthe lethal yellow (Ay) gene in mouse embryos. Dev. Genet. 9,155-165.

PAPAIOANNOU, V. E. AND GARDNER, R. L. (1979). Investigation ofthe lethal yellow (Ay/Ay) embryo using mouse chimaeras. J.Embryol. exp. Morph. 52, 153-163.

PEDERSEN, R. A. (1974). Development of lethal yellow (Ay/Ay)mouse embryos in vitro. J. exp. Zool. 188, 307-320.

PLOCHER, T. A. AND POWLEY, T. L. (1976). Effect ofhypophysectomy on weight gain and body composition in thegenetically obese yellow (Ay/a) mouse. Metabolism 25, 593-602.

RUSSELL, L. B. (1989). Functional and structural analyses of mousegenomic regions screened by the morphological specific-locustest. Mutat. Res. 212, 23-32.

RUSSELL, L. B., MCDANIEL, M. N. C. AND WOODIEL, F. N. (1963).


Crossing over within a "locus" of the mouse (abstr.). Genetics48,907.

SEARLE, A. G. (1968). Comparative Genetics of Coat Color inMammals. New York: Academic Press.

SILVERS, W. K. (1961). Genes and the pigment cells of mammals.Science 134, 368-373.

SILVERS, W. K. (1979). The agouti and extension series of alleles,umbrous and sable (ed. W. K. Silvers), pp. 6-28. New York:Springer-Verlag.

SlRACUSA, L. D . , BUCHBERG, A . , CoPELAND, N. G. AND JENKINS,

N. A. (1989). Recombinant inbred strain and interspecificbackcross analysis of molecular markers flanking the murineagouti coat color locus. Genetics 122, 669-679.

SIRACUSA, L. D., RUSSELL, L. B., EICHER, E. M., CORROW, D. J.,

COPELAND, N. G. AND JENKINS, N. A. (1987a). Genetic

organization of the agouti region of the mouse. Genetics 117,93-100.

SIRACUSA, L. D., RUSSELL, L. B., JENKINS, N. A. AND COPELAND,

N. G. (19876). Allelic variation within the Emv-15 locus definesgenomic sequences closely linked to the agouti locus on mousechromosome 2. Genetics 117, 85-92.

SNOW, M. H. L., TAM, P. P. L. AND MCLAREN, A. (1981). On the

control and regulation of size and morphogenesis in mammalian

embryos. In S. Subtelny (ed.), Levels of Genetic Control iiiDevelopment. Cambridge: Cambridge University Press.

VLAHAKIS, G. AND HESTON, W. E. (1963). Increase of induced skintumors in the mouse by the lethal yellow gene (Ay). J. natn.Cancer Inst. 31, 189-195.

VOCT, T. F., SOLTER, D. AND TILGHMAN, S. M. (1987). Raf, a trans-

acting locus, regulates the alpha-fetoprotein gene in a cell-autonomous manner. Science 236, 301-303.

WALLACE, M. E. (1954). A mutation or crossover in the housemouse? Heredity 8, 89-105.

WALLACE, M. E. (1965). Pseudoallelism at the agouti locus in themouse. J. Hered. 56,267-271.

WOLFF, G. L. (1963). Growth of inbred yellow (Aya) and non-yellow (ofl) mice in parabiosis. Genetics 48, 1041-1058.

WOLFF, G. L. AND PTTOT, H. C. (1973). Influence of backgroundgenome on enzymatic characteristics of yellow (Ay/-, Avy/-)mice. Genetics 73, 109-123.

WOLFF, G. L., ROBERTS, D. W. AND GALBRAITH, D. B. (1986).

Prenatal determination of obesity, tumor susceptibility, and coatcolor pattern in viable yellow (A'y/a) mice. The yellow mousesyndrome. J. Heredity 77, 151-158.

(Accepted 22 March, 1990)

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