Use of chimeras to transmit lethal genes in the mouse and to demonstrate allelism of the two X-linked male lethal genes jp and msd.

The uses of experimental chimeras to transmit lethal genes are discussed. A s an example, the X-linked lethal mutation jp was genetically transmitted by a chimeric male. This male produced abnormal offspring when bred t o j p l + and m s d l f females. Thus, allelism of the mutations jp and msd is proven and the symbol for msd should be changed tojpmsd Of the many known X-linked genes in the mouse, about one-third are lethal in males, the lethality occurring before sexual maturity. In general the mapping of these X-linked male-lethal mutations has not been hindered because linkage tests of X-linked loci are made with heterozygous females. However, problems arise when the allelism of two phenotypically identical X-linked genes is in question. To date, the only recourse has been to locate each allele in relation to at least two other X-linked genes. Even then the question remains: when two phenotypically identical genes are shown to be located in the same region of the X-chromosome, are they alleles? Two such X-linked male-lethal mutations are jimpy ( j p ) and myelin synthesis deficiency ( m s d ) (Sidman, Dickie and Appel, '64; Meier and MacPike, "70). Both affect myelin synthesis. Both are located in the same region of the X-chromosome with respect to the genes bent-tail ( B n ) and tabby (Tu), the order being Bn-Ta( jp , m s d ) (Phillips, '54; Eicher, '72). Thus, allelism of j p and m s d is likely but not proven by the standard allelism test. Both j p and m s d have received much attention because a dissection of the mechanism of their action could lead to a better understanding of myelin synthesis. The jimpy defect is similar to the human X-linked trait sudanophilic leukodystrophy (Sidman et al., '64; Wolf and Holden, '69), and genetic homology of one or both of the mouse genes with the human gene is likely. Unfortunately, the primary defect has not been uncovered for either gene. Without knowledge of allelism of j p and m s d , the geneticist and biochemist are unable to tell whether their results pertain to how two alleles affect a given trait or to how two genes affect the same trait. If more X-linked male-lethal mutations are found that also affect myelin synthesis, the genetic and biochemical complications generated would become unruly. An immediate solution to this problem was desirable. We have used the chimeric mouse as the tool to solve the problem of allelism between m s d and j p . We postulated that in chimeric mice with a male phenotype and containing j p / Y cells, enough normal cells ( + /Y or + / + , see Mullen and Whitten, '71) would exist to counteract the lethality of the j p lY genotype and allow j p /Y cells to differentiate in the gonads and form functional j p sperm. If some abnormal female offspring are produced when the chimeras are mated to known m s d / + females, m s d and j p are alleles. Conversely, if only normal females are produced, m s d and j p are mutants at different loci. We report here the genetic results obtained when a chimeric male proven to transmit sperm containing j p was mated 1 This research was supported by NIH Research grants HD 04083 from the National Institute of Child Health and Human Development and NS 09378 from the National Institute of Neurological Diseases and Stroke, by an allocation from NIH Research Support grant RR-05545 ham the Division of Research Resources to The Jackson Laboratory, and in part by the Aaron F. Norman Fund to The Jackson Laboratory. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care. J. EXP. ZOOL., 183: 181-184. 181 182 EVA M. EICHER AND PETER C. HOPPE to the appropriate females to solve the question of allelism of m s d andjp. MATERIALS AND METHODS Animals were obtained from the research or production colonies at The Jackson Laboratory. Tu j p / + + hybrid females, produced by the continuous crossing of Tu j p / + + females to + +/Y (C57BL/6J X CBA/J)FI males, were mated to similar hybrid males. Chimeras were produced by fusing the resulting hybrid zygotes with C3HeB/FeJ inbred zygotes. We speculated that the hybrid embryonic cells would have a greater probability both of contributing to the cellular makeup of the embryo and of producing spermatozoa. Mullen and Whitten (’71) had previously reported that the multicolored male chimeras from unbalanced genotypes produced gametes of the predominant genotype. The technique used for producing the chimeras was that of Mullen and Whitten (‘71). Females in proestrus were selected in the afternoon by the visual appearance of the vagina (Champlin, Dorr and Gates, unpublished) and paired with fertile males. Mating was verified by the presence of a vaginal plug the following morning (day 1) and eight cell ova were flushed from the oviducts on day 3 with Whitten’s medium (‘71). The zona pellucidae were removed in media containing 0.5% pronase (B grade, Calbiochem) and 0.1% polyvinylpyrrolidone (General Aniline and Filter Corp.). The embryos were then washed three times in fresh medium. Two embryos, one embryo of each genotype, were cultured in a small drop of Whitten’s medium in a 60 X 15 mm plastic culture dish (Brinster, ’63) under paraffin oil that had been equilibrated with medium. Contact between the two embryos in each drop was accomplished with the aid of a glass probe. Embryos were examined four hours later to verify or reestablish contiguity. The following day chimeric morulae were transplanted into the uteri of hybrid females produced from the strains C57BWlOWt and SJL/ Wt. These females were housed individually and allowed to deliver their offspring. All male offspring whose coats appeared phenotypically like Tu/ + females, thus known chimeras, were raised and mated to Tu j p / + +, Tu m s d l + +, and Tu +/ + m s d females. When the male offspring began to show the m s d or j p phenotype (between 10 to 12 days), their female littermates were closely observed for similar behavior. RESULTS AND DISCUSSION Four potential chimeric males were tested. Only one proved to carry both Ta and j p . Breeding data of this male are presented in table 1. Since he produced tabby jimpy females in the cross to T u j p l + + females, he was proven to transmit the Tu j p X-chromosome. The proven Tu jp/Y chimera was crossed to Tu m s d / + + females (table 1). He produced eight Tu/Tu jimpy appearing female offspring. In the cross of this same male to Tu +/ + m s d females, there werefive Tu/ + jimpy appearing female offspring. The jimpy phenotype of thejplmsd females was the same as that observed for their msdlY male sibs, for j p / j p females, and for the jp/Y males produced in the earlier crosses. Wolf and Holden (‘69) concluded from their studies of cultured cerebellum of newborn j p / + females that the myelin synthesizing cells do not behave autonomously according to their genotype. Nonautonomy would explain why j p l + females are normal. If j p l j p cells are non-autonomous and j p and m s d are different loci, j p + / + m s d females should be normal. If j p l j p cells are non-autonomous and j p and m s d are alleles, these females should be abnormal. Thus m s d andjp are alleles as predicted by the criterion of the standard allelism test. We propose that the symbol m s d for myelin synthesis deficiency be replaced by the symbol j p m s d to conform with standard nomenclature rules for mice (Staats, ’64). It is of interest that the male chimera appeared phenotypically like a typical TuIY male except for hair On his tail that is missing in the normal Tu/Y male. Furthermore, when young he had tremors typical of a jimpy male. As he grew older, the tremors became less severe until by four weeks of age no tremors were observed. Apparently the myelin deficiency defect, or whatever causes tremors, was USE OF CHIMERAS TO TRANSMIT LETHAL GENES 183