escape from genomic imprinting at the mouse t-associated

Copyright 0 1991 by the Genetics Society of America

Escape From Genomic Imprinting at the Mouse T-associated maternal eflect (Tme) Locus

Jen-Yue Tsai and Lee M. Silver

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014 Manuscript received June 3, 199 1

Accepted for publication August 28, 199 1

ABSTRACT Genomic imprinting occurs at the paternally inherited allele of the mouse T-associated maternal

effect (Tme) locus. As a consequence, maternal transmission of a functional Tme gene is normally required for viability and individuals that receive a Tme-deleted chromosome (PP or pb? from their mother die late in gestation or shortly thereafter. Here we report that a rearranged paternally derived chromosome duplicated for the Tme locus can act to rescue animals that have not received a maternal copy of the Tnae locus. Unexpectedly, all rescued animals display an abnormal short/kinky tail phenotype. Somatic transfer of genomic imprinting between homologs by means of a transvection- like process between paired Tme and T loci is proposed as a model to explain the results obtained.

T WO independently arising deletion mutations in the vicinity of the T locus on mouse chromo-

some I7 (Z'"p and pub') cause an identical dominant, lethal maternal effect (JOHNSON 1974; WINKING and SILVER 1984). Embryos that receive either of these mutant chromosomes from their father proceed normally through development to become viable and fertile adults. In contrast, embryos that receive either mutant chromosome from their mother develop in an apparently normal manner until late in gestation when death and resorption occur between 15 and 19 days postfertilization. During the lethal period, the only morphological abnormalities found are edema and extra toes (JOHNSON 1974; WINKING and SILVER 1984). The direct cause of death is unknown. Other deletion mutations in the T region do not express a lethal maternal effect, nor do any other spontaneous mutations characterized in any mammalian species to date. These results have led to the proposal that a unique T-associated maternal effect ( T m e ) locus must exist in the sub-centimorgan region of overlap between the pp and Pub' deletions, recently measured at less than one megabase in length from long range genomic mapping studies (BARLOW et al. 199 1 , Figure 1).

The term "genomic imprinting" is used to describe the situation in which the phenotype expressed by a gene varies depending on the sex of the parent from which the gamete containing that gene originated (SAPIENZA 1989). The evidence for genomic imprinting in mice is of two types. First, complementary meiotic nondisjunction has been exploited to produce embryos that receive both homologs of whole chromosomes or portions thereof from either their mother or their father (CATTANACH and KIRK 1985). Dra-

Genetics 129: 1159-1166 (December, 1991)

matic effects on development, including outright lethality, have been demonstrated in experiments of this type for chromosomal regions dispersed through- out the genome. Second, in a number of instances, transgenic lines of mice have been found to express their transgene differently according to the parent from which it is inherited (SURANI, REIK and ALLEN

The lethal maternal effect expressed in the offspring of females deleted for T m e could be a consequence of either a defective maternal/embryonic en- vironment or genomic imprinting at the T m e locus. Results obtained in several experiments indicate that the latter is true. First, pp/+ females can carry pP/+ individuals to term normally when the mutant chromosome is provided by the father. Second, the pp deletion can be recovered maternally if both the mutant and wild-type forms of chromosome I 7 are transmitted together from the mother through complementary nondisjunction (WINKING 198 1). These two results demonstrate that the embryonic and maternal environments provided by a pp/+ female are sufficient to support normal development.

Evidence in direct support of genomic imprinting has been obtained in carefully controlled experiments performed by MCGRATH and SOLTER (1984), who were unable to rescue maternally derived p p / + nuclei by placing them into the cytoplasm of enucleated wild- type zygotes. This result demonstrates a nuclear, as opposed to cytoplasmic, origin for the T m e phenotype, and it is consistent with the idea that the maternal and paternal alleles of Tme function differently during normal wild-type development. Finally, in strong support of this model, the Znsulin-like-growth~actor-2- receptor (Zgf"2r) gene has recently been identified as a

1988).

1160 J.-Y. Tsai and L. M. Silver

candidate for Tme based on its mapping to the tLub2- deleted region and the fact that RNA expression from Igf-2r is only detected from the maternally inherited chromosome (BARLOW et al. 199 1). Genomic imprinting in the vicinity of Igf-2r does not extend across several closely linked genes and appears to be localized to within a 700-kb region (BARLOW et al. 1991 ; No- ZAKI, IWAKURA and MATSUSHIRO 1986).

The simplest interpretation of the data obtained to date is that as a consequence of genomic imprinting, the paternally derived Tme allele is either inactive or expressed at a level below detection in normal wild- type embryos. Thus, in embryos where the maternal allele has been deleted, little or no Tme gene product will be expressed and death will ensue.

Although it now appears from the work of BARLOW and her colleagues that the paternal Tme allele is actually silent rather than expressed at a reduced level, this was not apparent at the time the experiments described in this paper were initiated. On the con- trary, the fact that the mutant phenotype was not visible until late in development led us to speculate that expression from the paternal Tme gene might be reduced, rather than eliminated. If this were the case, we reasoned that it might be possible to rescue maternally deficient embryos from death by providing them with two doses of a paternally derived Tme gene. This experiment can be accomplished readily through the use of available mutant forms of chromosome 17 that are duplicated for the chromosomal region that con- tains Tme. The results obtained and reported here were unexpected. The rescue of maternally deficient embryos was accomplished, but not through a simple gene dosage mechanism. The association of the rescued phenotype with new developmental abnormalities raises the possibility of a novel genetic mechanism by which escape from genomic imprinting may be possible.

MATERIALS AND METHODS

Mice and chromosomes: All animals were bred in the mouse facility at Princeton University. The derivation and structure of the Tt"', pub' and P5 chromosomes is dia- grammed in Figure l and described in more detail elsewhere (Committee for Chromosome 17 1991). The allelic consti- tutions of all variant forms of chromosome 17 used in this study are shown in Table 1A. th4'/t'"*' females were ener- ated by an outcross between Tt"r'/l'"b2 males and 8'/F4' females from a closed colony. Tt0"/Y5 males were generated in two steps: Tt"rL/t'ub2 females were crossed to inbred AKR/ J males for the production of Ttn"/+ males who were mated to females that carried the Y5 chromosome. All Tt0"/Y5 males used in this study carried a AKR/J Y chromosome. Gene dosage and tail phenotypes associated with all chromosome 17 genotypes are indicated in Table 1 B.

Molecular markers and DNA analysis: Probes for four mouse loci were used in the analysis described here. Wild- type and t alleles at the D17Leh48 and DI7Leh89 loci were detected as Tag1 restriction fragment length polymorphisms

(RFLPs) upon hybridization to the Tu48 (Fox et al. 1985) and Tu89 clones (BUCAN et al. 1987) respectively. Alterna- tive wild-type alleles at the Tcp-lob locus were distinguished by a Tag1 RFLP with the pTcr12 clone (SCHIMENTI et al. 1988). Alternative forms of the Mus musculus Y chromosome were distinguished by a EcoRV RFLP with the pY2 clone (LAMAR and PALMER 1984). All DNA analyses were performed by standard Southern blot protocols.

RESULTS

An unexpected phenotypic class of animals born to mothers that carry Pb2: We designed a genetic protocol to determine whether either of two Tme- duplicated chromosomes-Tt"' or tae5 (Figure 1 , Table 1 A)-could confer paternal rescue upon embryos that had received the Tme-deleted tlrb2 chromosome from their mother. An additional feature designed into this protocol was the presumed ability to distinguish rescued animals at birth according to tail phenotype, which is controlled by alleles at the closely linked T locus (Table 1B). Ttor'/tlub2 females were mated to Ttor1/Pe5 males with the expected phenotypic outcomes presented in Table 2. Since chromosomal segregation from Ttor1/tae5 males is Mendelian, the genotypes of fertilized embryos should be divided equally among the four classes listed. However, homozygosity for Ttor' causes early embryonic lethality, and as such, Ttorl/Ttor' embryos will never survive to birth. If rescue of maternally deficient Tme embryos does not occur, only one of the four genotypes-Tto"/tae5-will allow survival to birth in the form of tailless offspring; Ttor'/t'ub2 and Pe5/tlub2 individuals will die late in gestation from the maternal effect. In contrast, if rescue is conferred by either Tto" or P5, one would also expect to observe the birth of animals with normal tail lengths.

A total of 56 live-born offspring were counted from eight different Ttor'/t'"*' females mated with three different Tt0",/tae5 males. Thirty offspring were fully viable and tailless, and presumed to have a genotype of Tto"/tQe5 (Table 2). Ten offspring had normal tails and the edematous phenotype characteristic of Tme- deficient fetuses. DNA analysis indicated that this class of animals had indeed inherited the maternal tLub2 chromosome and either the Ttor' or tQe5 chromosome from their father. In all cases, these animals died within 24 hr.

The third live-born phenotypic class was completely unexpected-16 offspring were observed with short or kinky blunt-end tails normally characteristic of heterozygous T/+ animals that carry only a single dose of the T gene (Tables 1B and 2). Five additional animals of this unexpected class have been generated in a separate cross in which t""/Pb2 females were mated to Ttor'/tQe5 males. All members of this class from both crosses survived to adulthood and, of those tested, several proved fertile. Most members of this class had

Genomic Imprinting at the Tme Locus 1161

A

B

FIGURE 1 .-Schematic representations of variant forms of chromosome 17. (A) Deletion mapping of the Tme locus. The relative positions of marker loci along the proximal region of the chromosome are shown below (the D17Leh48 and D17Leh89 loci are indicated as 48 and 89, respectively). The extent of the deletions associated with three variant forms of the chromosome (Tt"', Pb2 and r'p) is indicated above the line. The Tme locus must map within the Pb2-deleted region. (B) Com- parison of wild-type and complete t haplotype homologs. The boxes with large arrows signify the presence of an inversion polymorphism called [Zn(l7)2] between the two chromosomes. The breakpoints associated with the recombination events that produced the partial t haplotypes Tt"', t'"b2 and PC' are indicated. T o a first order of resolution, Ttff" and Pb2 appear to be reciprocal products of a nearly identical crossing-over event. (C) The relative structures of Tt"", pb2 and PC'. Each of these chromosomes has an inverted duplication of a portion of the genomic material from the inversion polymorphism [ h ( f 7)2] shown above (Committee for Chromosome 17 1991). The orientation of DNA relative to wild-type and t haplotype chromosomes is indi-

lub2 0 48' & 2: Tcp-lobt cated by the arrows.

I 7 ,

TABLE 1 TABLE 2

Alleles, genotypes and phenotypes

A. Alleles at marker loci and Tme dosage associated with chromosome 17 homologs

Tme Chromosome Leh48 T (dosage) Tcp-lob Leh89

~

Wild-type (+) + + 1 l o r 2 + Complete t t t 1 t t Tt0" t del 2 1 +

t,+ 0 t t t"<5 t t,T""" 2 2 + p 7 3 t t 1 t t

t 1 t +

~ ~~~~~

t fubZ +

th4Y 1

€3. Locus-by-locus genotype and expected T locus tail phenotype for each

Genotype Leh48 T Tme Tcp-lob Leh89 Tail length chromosome 17 combination described in the text

+I+ +I+ +I+ +I+ +I+

+,+I+,+ +,+I- +,+/- +,+I+ +/-

Normal Normal Normal Short Tailles Tailless Normal Normal Tailless Normal

In (B), a null allele is indicated with a "-," and a generic wild- type allele is indicated by a "+." Alleles in each locus-by-locus genotype are oriented according to the full chromosome genotype shown at the left of each line.

an extra toe, which is a phenotypic abnormality associated with mutant Tme-deleted fetuses (JOHNSON

1974; WINKING and SILVER 1984) (Figure 2). As the mice aged, the extra toes degenerated. In addition,

Expected and observed phenotypes of offspring from the experimental cross

Corresponding phenotypes

Genotype r e sue cue Observed Expected if no Expected with res-

TtO"lTt"" 0 0 0 Tto"ltae3 100% tailless 33% tailless 30 tailless tlubZITt"' 0 t1ubZlt"'5 0

The maternally inherited chromosome is indicated first within the genotypes listed. Phenotypes listed in the box are associated with either of the two genotypes t'"b2/Tt"' or / t . All mice with normal tails listed in the observed column died within 24 hr; the number indicated ( I O ) is a minimum estimate since some may have perished prior to the initial observation of litters. The results shown were accumulated with three different males mated to a total of 8 different females. All mating combinations produced offspring in each of the three phenotypic classes listed.

a A priori, it was not possible to distinguish the genotypes of animals that expressed the two observed phenotypes in the box; each animal could have had either a t'"bZ/Tt"" genotype or a tfub2/ toe' genotype. A subset of these animals were distmguished by DNA analysis as described in the text.

other abnormalities were observed, including many urogenital defects such as testicular teratomas, ambig- uous genitalia with imperforate vagina (Figure 2B), and vaginocolonic fistulae leading to the passage of feces through the vagina (Figure 2C).

The unexpected short/kinky tail mice have inherited pb2 from their mother: Probes for two DNA loci that flank the t haplotype region of chromosome 17- D17Leh48 and DI7Leh89-were used to determine if the unexpected class of animals carried a complete

1162

I

J.-Y. Tsai and L. M. Silver

-7

I

i FIGURE 2.--Gross morphological

phenotypes expressed by rescued Tp'/t'"% animals. (A) Normal TIO"/ 8" female generated by paternal transmission of the Pb2 chromosome; (B)-(F) Examples of Tl""/~"tn animals generated by maternal transmission of the t'"'' chromosome. (B) Fe- male with an imperforate vagina; (C) female with compacted feces in the vagina, as a consequence of a vaginocolonic fistula; (D) and (E) examples of Brachury-like tail; (F) extra toe on front right paw (indicated with ar- row).

copy of the Pb2 chromosome. At both loci, Pb2 carries an allele which is distinguishable from those associated with each of the other two forms of chromosome I7 present in the two parental genotypes used in the original cross (Figure 1C). The results obtained demonstrate that all offspring with short tails carry Pb2 alleles at both D l 7Leh48 and D l 7Leh89.

Since we had not previously observed maternal inheritance of Pb2 in our mouse colony, it was impor- tant to confirm that the Pb2 chromosomes present in the offspring described here were indeed derived in this manner rather than by some form of laboratory error. Confirmation was accomplished through an analysis of the Y chromosome. As a consequence of the maternal effect, all other Pb2-containing animals in our laboratory must be derived by strict paternal descent from the original Pb2-carrying mutant mouse found in the laboratory of H. WINKING in Lubeck, Germany (WINKING 1981). Therefore, all of these

animals must share the same Y chromosome deriving from the original founder. In contrast, the class of maternally derived td2-carrying individuals should have inherited the AKR/J form of the Y chromosome present in the Ttor1/P5 fathers (see MATERIALS AND METHODS).

A Y chromosome-specific probe was used to analyze DNA from various Pb2-carrying animals sampled over an 8-year period in our colony in comparison with the newly derived t'ub2-carrying individuals described in this report. As shown in Figure 3, the Y chromosome present in the newly derived individuals is clearly distinguishable from that shared by all previously generated Pb2-carrying mice and identical to that observed in AKR/J males. This result demonstrates a maternal origin for the Pb2 chromosome present in the unexpected class of mice that carry a short/kinky tail. To distinguish this class of maternally inherited P b 2 chromosomes from those inherited through the


M 1 2 3 4 5 6 7 a b c d e f g ~ )Ir

-A-

- L -

FIGURE 3.-Comparison of Y chromosomes from animals that carry the t'"'' haplotype. DNA samples were digested with EcoRV, separated by agarose gel electrophoresis, blotted, and probed with pY2 (LAMAR and PALMER 1984). A restriction fragment length polymorphism was observed between the Y chromosomes present in the inbred strain AKR/J (lane 1) and f'"'-carrying males in our colony (lane 2). An AKR/J-specific fragment is labeled "A," a f'"*'- specific fragment is labeled "L." Lanes 3-7 represent five independent f'"bz-carrying males taken from our colony over an 8-year period. Lanes a, b and c represent the three TP'/P5 males used in the experimental cross. Lanes d-g represent male t'"'mcarrying offspring from this cross. The marker lanes are labeled (M).

paternal line, we will use the symbol pb2m. Although the short/kinky tail mice can have one of

two genotypes-Tp1/pb2m or YS/pb2m-it was not possible to discriminate between these genotypes directly by DNA analysis with currently available probes. However, we have analyzed a marker gene (Tcp-lob) linked within one centimorgan to the duplicated region as a first estimate of comparative transmission (Figure 1C). In an analysis of 1 1 mice from the short tail class, 10 were found to have the marker allele linked to the paternal Ttor' chromosome, and one was found to have the marker allele linked to the paternal Pes chromosome.

The maternally inherited t'*2m chromosome is indistinguishable from the normal 9 chromosome: To further understand the phenomenon of maternal

transmission of Pb2m, it was critical to determine whether the pb2m chromosome had been altered in any way through this process. To answer this question, pb2m chromosomes were analyzed in subsequent generations for the expression of five independent phenotypes that are characteristic of the standard pb2 chromosome. These are (1) suppression in trans of the tail-shortening phenotype caused by mutations at the T locus (WINKING and SILVER 1984, Table IB); (2) expression of the Tcd-2, Tcd-3' and Tcr' alleles with high levels of transmission ratio distortion in trans configuration with a chromosome that carries Tcd-I' and Tcd-4' (SILVER, LUKRALLE and GARRELS 1983; SILVER and REMIS 1987); (3) failure to complement the tW7' lethal mutation which maps in the vicinity of Tme (SARVETNICK et al. 1986); (4) failure to complement the pbl lethal mutation (WINKING and SILVER 1984); and (5 ) reexpression of the Tme maternal effect.

Analysis of the first three phenotypes was accomplished within the context of a single cross between TP1/pb2m males and TIP7' females. If the pb2m chromosome has the complete set of pb2 alleles involved in transmission ratio distortion, it will be transmitted at a very high ratio in this cross. If not, the transmission of t'"b2rn will be reduced, and transmission of the TP" chromosome will be observed in the form of tailless Tto'1/t"7' offspring. If kub2m is able to complement P7', an equal number of Pb2/tW7' and pb2/T animals will be born. If complementation fails, only pb2/T animals will be observed. The results obtained were that 27 of the 28 offspring born had normal tails, indicating a transmission ratio for pb2m of 96%, identical to that observed in previous studies (SILVER, LUKRALLE and GARRELS 1983). DNA analysis was performed on 14 of the offspring with normal tails, and all were found to have a genotype of pb2/T rather than pb2/tw7' (data not shown). This result demonstrates the failure of pb2m to complement the P" lethality ( P < 0.001), and in addition, it shows that the pb2m chromosome can still suppress the T locus effect on tail phenotype.

Tto"/kub2m males were also mated to T/pbl females to test complementation with the t'*' lethal mutation. Ten offspring were obtained with normal tails and were subjected to DNA analysis; all were found to have a pb2/T genotype (date not shown). This result demonstrates the failure of pb2m to complement the j u b l lethal mutation ( P < 0.01).

Finally, another cross was performed with Tf"/ m females mated to wild-type +/+ males to deter-

mine whether the lethal maternal effect would be reexpressed in generations subsequent to the passage of pb2 through the female germ line. The results were unequivocal; all 31 offspring obtained from these crosses had a short tail, indicating maternal transmis-

p b 2


sion only of the T-containing chromosome and not of tlub.2

DISCUSSION

The Tto"Jt'ubzm genotype can express two dramat- ically different phenotypes: Females that carry the variant Tme-deleted pb2 form of chromosome 17 are normally unable to transmit this chromosome to viable offspring. Previous investigations indicated that Tme- deficient fetuses could succumb to death during a broad window of time extending from 15 to 19 days postfertilization (JOHNSON 1974; WINKING and SILVER 1984). On rare occasions, however, maternal-Tme- deficient individuals produced in standard crosses have survived to birth; such newborns typically express the characteristic Tme mutant phenotypes- edema and extra toes-and die within 24 hr (our unpublished observations).

In the experimental cross reported here, two phe- notypically distinct classes of live-born maternal-Tme- deficient individuals were observed. The first class of individuals were indistinguishable from the short- lived animals found sporadically in standard pbz and Thp colonies. The simplest explanation for the observed increase in the frequency of these mutant individuals is that modifying genes present in the paternal genetic background have acted to move the window of death forward by 1 or 2 days; the live- born, but short-lived, mutants would simply represent the tail end of this delayed window.

The second class of live-born maternal-Tme-deficient individuals have escaped lethality and typically survive into long-lived adults. Maternal transmission of the tlub2 chromosome to viable offspring has not been observed previously, and hence, it must be a consequence of the particular paternal genetic contribution provided in the experimental cross described in this report.

The paternal chromosome Z 7 carries two doses of the Tme gene: One component of the paternal contribution is a double dosage of a functional Tme gene. Thus, the simplest explanation of our results is that this increased dosage is responsible for the rescue of some individuals from lethality. This simple explanation is unsatisfactory for several reasons. First, although both of the father's chromosomes carry two doses of a functional Tme gene, one-Ttorl-is, at the very least, much more efficient at allowing rescue than the other-tae'. Although both chromosomes are segregated at an equal ratio, 10 of 1 1 rescued animals carried a marker allele that is closely linked (< 1 cM) to Tt""; this result is significantly different from parity with a P value of less than 0.007. The single exception could represent a recombination event between the marker and the duplicated region, or a bonafide Per chromosome that allows rescue with reduced effi-

ciency relative to Tt'". In either case, chromosome 17-specific factors other than Tme duplication must play a role in the escape from lethality.

A second argument against a primary role for gene dosage in phenotype rescue is the evidence from molecular studies showing no detectable paternal expression from a strong candidate for the Tme locus (BAR- LOW et al. 1991). The data indicate that if the paternal allele is expressed, its level must be at least 50-fold below that of the maternal allele. If the paternal allele is expressed at a level just below detectability, then two doses would provide maternally deficient embryos with 4%, rather than 2%, of the Tme product present in normal wild-type embryos. It seems doubtful to us that this would be sufficient to allow rescue to occur, although we cannot rule it out.

A final problem with the gene dosage explanation is that it does not provide an understanding of the developmental abnormalities associated with the rescued phenotype. If rescue occurred simply through the production of a closer-to-normal level of Tme gene product, one would not expect to see an effect on tail length or the urogenital tract.

T locus dosage effects on tail length: Haploid insufficiency at the T locus in animals heterozygous for T""" and wild-type alleles (effectively +/-) causes a short or kinky tail phenotype. However, Tt'r1/t'ub2 and tae5/tlub2 animals carry two or three functional T locus alleles (respectively) leading to the expression of normal tail lengths when pb2 is contributed by the father (Table 1B). A speculative explanation for the appearance of t'"bzm-containing mice with short tails is that during their development as fetuses, the expression of one or more T locus alleles had been abnor- mally repressed. If repression caused an "epigeno- type" (SAPIENZA 1989) of (+/-), the resulting animal would have a short or kinky tail.

Possible somatic pairing and cross-talk between T and Tme loci: A second component of the paternal contribution to rescued animals could provide a mechanism by which rescue would occur in conjunction with the repression of T locus alleles. In particular, the f u b 2 and Tt'" chromosomes each carry large, mu- tually exclusive, inverted duplications of genomic material (Figure IC). If these two chromosomes initiate pairing in homoIogous regions external to the inverted duplications, the two Tme loci present on the paternal Ttor' chromosome could be brought into ap- proximate alignment with the two T loci present on the maternal tlub2 chromosome (Figure 4). If the hypothetical imprinting machinery present on the paternal homolog crossed onto the adjacent region present on the maternal homolog, a de-repression of Tme activity could occur in conjunction with the repression of T locus activity (Figure 4B). A process of this type could be considered formally analogous to "transvec-


(Td BM) kb NOT 8Wkb

PXPLBSGBDII BXeRBSSBD

FIGURE 4.-Transvection model for transfer of genomic imprinting between Tme and T. (A) Low resolution schematic representa- tion of potential homolog alignment in the proximal region of chromosome 17 from TP1/Pbz animals. (B) High resolution repre- sentation of alignment in the distal border region of the In(17)2 inversion in Tto'l/,,bz animals. Pairing can occur in the vicinity of the Tcp-106 locus. The distance between Tme and the inversion border is approximately 600 kb (BARLOW et al. 1991); the distance between the T locus and the inversion border is approximately 800 kb [HERRMANN et al. (1990) and our unpublished results]. The shaded oval represents the hypothetical "imprinting machinery" initially present on the paternally derived Tme locus.

tion," defined as an "allelic interaction that shows dependence on chromosome pairing or allele prox- imity" (JUDD 1988). Zeste-mediated transvection in Drosophila appears to occur by protein-DNA inter- actions across homologs (BIGGIN et al. 1988), and a similar process of allelic cross-talk has been invoked recently by MONK (1990) to explain switches that occur in the genomic imprinting of some transgenic lines of mice.

There is no reason to expect that the imprinting machinery would move specifically to the T locus on the maternal homolog. However, in prior studies of T-region deletions (such as TtO"), haploid insufficiency at other loci in the vicinity of T showed no detectable effect on phenotype. Nevertheless, abnormal imprinting at nearby loci could provide an explanation for the unusual developmental abnormalities that have been observed here at high frequency (Figure 2).

If the Tme-imprinting machinery could switch homologs in these Pb2/Ttor1 animals, one would expect that it could do the same in somatic cells of wild-type animals. There are currently no data that address this question; a somatic switch in imprinting between wild- type homologs would not have any effect on phenotype.

A somatic transfer of genomic imprinting between a paternal Tme gene and a maternal T locus gene might occur in a stochastic manner in a subset of the embryonic cells present in Ttor1/t1ub2 embryos. How- ever, even a minor contribution of T-haploid insuffi-

cient cells could lead to the birth of "mosaic" animals with altered tails (BENNETT 1978).

One would not expect to observe the short tail phenotype with Ttor1/t'ub2 mice generated through a cross in which Pb2 is provided by the father and Ttor' is provided by the mother because such mice would not carry the Tme-genomic imprinting machinery needed for inter-homolog transfer. This prediction has been borne out in observations of 1053 progeny over the last decade (our unpublished observations). Finally, the partial penetrance of the rescued phenotype observed in this report could be a consequence of both stochastic events as well as unlinked, segregat- ing modifying alleles.

Unexplained observations and complicating factors: The transvection scenario just described works well to explain the results obtained with rescued animals having a Ttori/t1ub2m genotype. However, it is not clear why the tau' chromosome is less efficient at, or incapable of, causing rescue as well. Two explanations can be considered. The first is that the efficiency of somatic pairing in the appropriate region is affected by the size of the duplicated region, which differs by at least sixfold between Ttor' and PC'. The second possibility is that a gene(s) closely linked to the duplicated region has an epistatic effect on the rescued phenotype.

A complicating factor in this analysis is the possibility that expression from the T locus is in-and-of-itself normally subject to epigenetic controls apart from the ones proposed here. For example, a t/t genotype at the T locus normally results in a mouse with a normal tail (Table 1B). However, in two independent situa- tions, the addition to such a genotype of chromosomal regions surrounding null alleles at the T locus leads to the birth of animals with short tails. The first case arises with partial haplotypes similar in structure to tnc' which are duplicated for a region that includes the T locus with one T allele and one Tu'' allele (Table 1, Figure 1, (Committee for Chromosome 17 1991)). Within certain genetic backgrounds, animals homozygous for PC', or the similar haplotype tw7'Jr', express the unexplained short tail phenotype (NADEAU, VARNUM and BURKART 1989; VOJTISKOVA et al. 1976). The second case arises through the experimental production of animals having partial trisomies for the proximal portion of chromosome 17. RUVINSKY and his colleagues (1 99 1) have demonstrated that the T"""/T/T genotype also gives rise consistently to animals with short tails. Finally, a previously defined T locus allele-T"-has been characterized clearly as an antimorph, because it produces a short tail phenotype from a Tc/T+T' genotype (MACMURRAY and SHIN 1988), whereas a T"""/FT' genotype produces a normal tail phenotype (Table 1). Both the unexplained null allele effects and the very existence of an anti-


morphic allele imply that the tail length phenotype expressed by the T locus is not simply a function of gene dosage alone.

Concluding thoughts: Although it is possible to proffer various explanations that bring the accumulated data obtained in line with the proposed model for the rescue of Pb2, none are entirely satisfactory. Nevertheless, the imprinting-transvection model still represents the best current working hypothesis. Clearly, further experiments will be necessary to test and refine, or to rule out, this hypothesis; to determine the effects of genetic background on the results obtained; and to understand the molecular basis of T locus effects on tail phenotype. Irrespective of the precise mechanisms involved, the ability to overcome genomic imprinting in a particular breeding protocol provides an experimental tool with which to study the nature of this enigmatic genetic phenomenon.

This research was supported by grants from the National Insti- tutes of Health to L.M.S. We thank RONI BOLLAG for providing us with additional data on the relative rescue efficiency with the two duplicated chromosomes. We thank all members of the Silver laboratory for fruitful discussions of the results presented in this paper.

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Communicating editor: T. SCHUPBACH

escape from genomic imprinting at the mouse t-associated

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