Comments on the Relationship of Inbred Strains to the Genus Mus

Michael Potter

National Cancer Institute
National Institutes of Health
Bethesda, Maryland

It is difficult with existing data to trace the origins of the vast majority of inbred strains of Mus musculus to feral or commensal wild populations. There are some clear examples where wild mice were introduced into existing colonies. Abbie Lathrop and Leo Loeb, in 1918 ( 1), for example obtained wild mice from Vermont and Michigan and bred them to other mice in the famous collection maintained at Granby, Massachusetts. Lathrop and Loeb, in 1915, imported mice from Europe and purchased others from dealers and made many matings between different stocks ( 2). They supplied mice to the Bussey Institute and were thus a source of the ancestors of some of the inbred strains of today. For example, male C52 and females C57 and C58 in Little's laboratory at Cold Spring Harbor were descended from mice raised at the Granby mouse farm. The lathrop colony then may be the American mouse melting pot, a possibility that adds further obscurity to tracing the origins of inbred strains. The records from other sources documenting the domestication of wild mice are also lacking. Keeler ( 3), in his classic monograph on the history of the laboratory mouse, cites extensive evidence of repeated domestications of mice in both Europe and Asia over a period of several centuries. C.C. Little was apparently also involved in importing mice from distant geographic localities into laboratory colonies ( 4, 5). Mouse fanciers no doubt added to the mixing of mice of different origins and, to complicate matters even more, there were exchanges of fancy mice between Europe and Asia in the last century. All this indicates the ancestors of many of the present day strains of mice may have originated from different geographic areas and no doubt from different subspecies.

Subspecies mixing can then bring together genes that have evolved away from each other over an extensive period of evolutionary time. While such a process may not affect viability under the conditions of domestication, it could lead to unnatural combination of characters. Recent evidence indicates that C-type RNA (oncornaviruses or retroviruses) are integrated into the mouse genome at different chromosomal sites in different inbred strains ( 6). If this process is recent, i.e., occurs post-speciation or is continually occurring, then mixing of genomes could also bring together novel virus genomes.

The almost hopeless mixing of ancestors of most of the existing inbred strains today makes it difficult to trace the origins of specific genes or gene complexes. It will be of value to those interested in evolutionary problems to establish new inbred strains from taxonomically identified subspecies of Mus musculus. A number of potentially interesting stocks have been introduced into the laboratory from the wild and these should be very useful sources of new inbred strains. M.E. Wallace of the University of Cambridge has established stocks from Peru, San Francisco, and Skokholm Island ( 7). A number of strains of Mus musculus molossinus have been established in Japan ( 8). And in the United States, Mus musculus molossinus (imported by the author from Kyushu) has been inbred at The Jackson Laboratory (T. Roderick, personal communication) and partially inbred at the National Cancer Institute. In addition, Mus musculus castaneus from Bangkok and Mus musculus musculus from Denmark (see Chapman, this volume) have been established and some inbreeding is in progress.

Other species in the genus Mus that are close relatives of Mus musculus add another dimension to the understanding of the species. The taxonomy of the genus Mus is still incomplete, and based mainly on morphology. The greatest concentrations of Mus are in Southeast Asia, Malaysia, New Guinea, and Central and East Africa ( 15). The ancestors of these two species concentration probably originated in Southeast Asia, and migrated into new regions, e.g., Africa and island populations in the East Indies occurred millions of years ago. Now, parallel diversifications make the study of the relation of the species more complex.

Insights into relationships between evolving species in the genus Mus and subspecies of Mus musculus are being provided by advances in karyology, DNA hybridization, and the comparative primary and antigenic structure of homologous proteins. The species in the genus Mus may offer unusual opportunities. The discovery of Robertsonian fusion in the pygmy mice of Africa (the superspecies Mus leggada) by Matthey ( 9, 10) and in Mus poschiavinus from the Swiss Alps by Gropp and colleagues ( 11) have revealed that species are developing in these areas.

Greater insight into the interrelationships of the various species of Mus will no doubt evolve from the newer morphologic methods of chromosome identification. Knowledge of the relationships between species in Africa and Asia has proceeded rapidly from the work of Matthey and Petter ( 12), Petter and Genst ( 13), Jotterand ( 14), and Missone ( 15). New interest is developing in the speciation of the genus Mus in Europe (see Marshall and Sage in this volume).

Recent progress in studying Southeast Asian mice has been stimulated largely through the efforts of Joe T. Marshall who trapped, identified, and sent breeding stocks of different Mus species from Thailand to various workers in the U.S. The work of Joe Marshall has greatly enriched our biological source of information on the relatives of Mus musculus by providing closely related but genetically isolated species such as Mus caroli and Mus cervicolor. Mus caroli and Mus cervicolor do not breed with Mus musculus nor with each other. All three species have 40 telocentric chromosomes ( 16). A study of the relationship of enzymes, immunoglobulins, hemoglobins, and histocompatibility antigens in these three species should provide great insight into the origins of M. musculus. Mus cervicolor and Mus caroli have provided new type of retroviruses (see Callahan, this volume). A classification of the genus Mus in Southeast Asia, based on extensive field trips and taxonomic studies, has been published by Joe Marshall ( 16).

A list of species in the genus Mus is given in Table 1 based on descriptions of Marshall ( 16), Matthey ( 10), Matthey and Petter ( 12), Petter and Genst ( 13), and Jotterand ( 14). Since the author is not a systemitist, this list is presented to provide and indication of the size of the genus, and not as a proposed classification.

Our interest in the Mus subspecies problem emerged in studies on immunoglobulin allotype genetics. Following the discovery of Kelus and Moor-Jankowski ( 17) that there were strong antigenic differences associated with heavy chains gene products (allotypes) in different strains of mice, Herzenberg ( 18) and Lieberman and Dray ( 19) described a number of Ig allotypic markers in mice. Using myeloma proteins, it was possible to assign some of these antigens to specific C_H genes. This study soon revealed that the IgC_H (there are now eight different C_H genes in the mouse) were closely linked together on one chromosome ( 18, 20, 21). Allelic forms of six of the genes are now known ( 34). Genetic studies indicated that the C_H genes were closely linked together and that crossing over was not observed in samples of about 2,000 progeny ( 20, 21). Because of this close linkage and lack of crossing over, we decided to look at wild mouse allotypes for evidence of recombinations. We found a possible case in a wild mouse population trapped in Kitty Hawk, North Carolina, where two markers always found separately in inbred strains were in the same haplotype and probably the same structural gene (IgC_HG2a) ( 22) in these wild mice. In this study of wild mice we obtained specimens from many different sources throughout the U.S. and even some mice from Europe ( 22, 23). Among the strong allotype markers that have subsequently been shown to be alleles of IgG2a we did not find the strong allelotype "2" (G²) in U.S. or European samples. (The "2" marker is found in strains C57BL, HR, LP, SJL/J, SM, and STR/1.) The possibility that the "2" marker might be associated with Asian mice was suggested by available circumstantial evidence.

First, it is a well documented fact that the Japanese waltzing mouse (a fancy mouse) was imported into Europe from Asia probably during the last century ( 3). Japanese waltzing mice were used in a number of laboratories in the U.S. in the early part of this century. Lathrop and Loeb ( 1) had Japanese waltzing mice in their colony and mated these mice to various other stocks and strains. Japanese waltzing mice were smaller than the usual laboratory mice and were suspected of being a different subspecies. They were first thought to be Mus wagneri (the wild mouse from Northern Asia, China, Tibet, etc.). Schwarz ( 24), however, in 1942 argued that these mice were not of Chinese but were in fact of Japanese origin and hence derived from the wild M. m. molossinus (the common Mus musculus species in Japan). Through the kindness of Dr. Hiroshi Kobayashi of the University of Hokkaido, the author contacted Professor Fusanori Hamajima from the Department of Parasitology, Kyushu University. Professor Hamajima had made extensive studies of the natural habitats of the native Japanese rice-field mouse, Mus musculus molossinus ( 25). These mice live in straw stacks in barns or other human constructions in the winter and migrate into the rice paddies in the summer where they live in the banks of the paddy fields. They have a restricted breeding season in the spring and autumn ( 25). Professor Hamajima very graciously trapped 11 mice and sent them to me in Bethesda where my wife and I bred them in the basement of our home before they were introduced into the laboratory. The original shipment which arrived in 1967 contained 10 very small light black agouti mice, several of which had snowy white bellies, and one large mouse. The identity of the large mouse was never established and all of the matings were made with the small mice. These mice have bred vigorously. They had the singular honor of being imported to Bar Harbor, Maine, where they were introduced into The Jackson Laboratory by Dr. Thomas Roderick. Roderick et al. ( 26) studied the ratio of the weight of the brain to the spinal cord in inbred strains of mice and M.m. molossinus and found that M. m. molossinus has the highest value.

The immunologic allotypic analysis of the M. m. molossinus mice proved to be most interesting ( 23). First, we found the missing "2" marker in these mice. Second, all of the other allotypic markers associated with the IgC_H complex locus of the C57BL were also identified. Third and most unusual was the presence of a marker associated with IgG2a of the BALB/c mouse, the G^1,6,7,8. Immunoelectric studies indicated that the Ig carrying G^1,6,7,8 and G² were on different molecules, indicating the M. m. molossinus (Kyushu) locus carried an extra gene.

The novel IgC_H locus could then have evolved by unequal crossing over. This complex haplotype has not been found in any other inbred strain.

We have studied another marker in M. m. molossinus that has proved to be equally extraordinary and this is the electrophoretic pattern of the major urinary protein complex (MUP). As has been known for many years ( 27), mice excrete a large amount of highly acidic proteins in the urine (0.5 to 1.5 mg/ml). These proteins have a molecular weight of 17,900 and exhibit considerable electrophoretic heterogeneity ( 28). There is a sex dimorphism in the pattern so that males and females of the same strain can often be distinguished ( 28). The sex pattern can be reversed by gonadectomy and treatment with appropriate hormones. The mouse urinary proteins (MUP) are produced in the liver ( 29). Their exact function has not yet been determined although some recent evidence by Ruopslahti and colleagues ( 30) suggests MUP may be a nuclear non-histone protein. Two major phenotypic forms of one of the components controlled by the MupA locus, the 1 and 2 components, are apparently allelic variations ( 28). All of the inbred strains so far studied are either of the "1" type (the most common) or the "2" type. When M. musculus molossinus urine was electrophoresed, a completely new type was seen that had much slower mobility than any component seen in the inbred strains ( 31) ( Figure 1). Mus musculus castaneus trapped by Joe Marshall was also studied and these mice have the same new slow component and still another new type. Dev et al. ( 32) have compared the chromosomes of M. m. molossinus with C57BL by Quinacrine (Q) and Centromeric, heterochromatin (C) banding patterns. They found no differences in Q banding but very different C banding patterns. The latter were much greater than found among inbred strains ( 33) providing further evidence of genetic distance between M. m. molossinus and laboratory mice. Genetic differences between subspecies may be more extensive than simply an accumulation of allelic forms. Subspecies could differ from each other, by having structurally different complex loci. The variation of IgC_H ( 23), MUP ( 31), brain:spinal cord weight ratios ( 26) and C-banding ( 32, 33) suggests this possibility. Retrovirus integration ( 6) is yet another basis for a difference, however, retrovirus loci have not yet been mapped in subspecies such as M. m. molossinus.

The remarkable advances that are being made in the descriptive genetics of the laboratory mouse make it very desirable to understand this species as it naturally evolved. The inbred strains of mice that are in common use today are probably derived from several subspecies backgrounds and thus cannot be used to trace gene origins. The construction of new inbred strains, or the use of stocks from taxonomically identified subspecies from different geographic locations might provide a useful source of information on the origin of alleles and complex genetic loci. This information should allow us to better understand the flexibility of a single genome throughout millions of years of evolution. The systematics of the living species of the genus Mus are still incomplete. Many exciting new advances have been made in the last 25 years, promising that we shall better understand these fascinating animals. This is a time when species of Mus and subspecies of Mus musculus are being adapted to the laboratory from many parts of the world.

Some unusual findings with the IgC_H complex locus and the MUP complex locus in Mus musculus molossinus (Kyushu) were summarized.

1. Lathrop, A.E.C., and Loeb, L. (1918). J. Exp. Med. 28: 475.

2. Lathrop, A.E.C., and Loeb, L. (1919). J. Exp. Med. 22: 713.

3. Keeler, C.E. (1931). The Laboratory Mouse: Its Origin, Heredity, and Culture. Harvard University Press, Cambridge.
See also MGI.

4. Schwartz, E., and Schwartz, H.K. (1943). J. Mammology 24: 59.

5. Green, C.W. (1930). Am. Nat. 64: 540.

6. Rowe, W.P., Hartley, J.W., and Bremner, T. (1972). Science 178: 860.
See also MGI.

7. Wallace, M.E. (1971). Environ. Pollut. 1: 175, Elsevier, England.

8. Nishimura, M., Kondo, K., Nakamura, H., and Watanabe, T. (1973). In Experimental Animals Volume 22. Suppl. Proceedings of the ICLA Asian Pacific Meeting of Laboratory Animals, Tokyo and Inuyama.

9. Matthey, R. (1966). Revue Suisse Zool. 73: 585.

10. Matthey, R. (1970). Revue Suisse Zool. 77: 625.
See also PubMed.

11. Gropp, A., Winking, H., Zech, L., and Muller, H. (1972). Chromosoma 39: 265.
See also MGI.

12. Matthey, R., and Petter, F. (1968). Revue Suisse Zool. 75: 461.
See also PubMed.

13. Petter, F., and Genst, H. (1970). Mammalia 33: 118.

14. Jotterand, M. (1972). Revue Suisse Zool. 79: 287.
See also PubMed.

15. Missone, X. (1969). Ann. Musee Royal Afr. Central 172: 1.

16. Marshall, J.T., Jr. (1977). Bull. Am. Mus. Natl. Hist. 158, Art. 3: 177.

17. Kelus, A., and Moor-Jankowski, J.K. (1961). Nature 191: 1405.
See also PubMed.

18. Herzenberg, L.A. (1964). Cold Spring Harbor Symp. Quant. Biol. 29: 455.
See also MGI.

19. Lieberman, R., and Dray, S. (1964). J. Immunol. 93: 584.
See also PubMed.

20. Potter, M., and Lieberman, R. (1967). Cold Spring Harbor Symp. Quant. Biol. 32: 187.

21. Potter, M., and Lieberman, R. (1967). Adv. Immunol. 7: 91.
See also PubMed.

22. Lieberman, R., and Potter, M. (1966). Science 154: 535.
See also PubMed.

23. Lieberman, R., and Potter, M. (1969). J. Exp. Med. 130: 519.
See also PubMed.

24. Schwarz, E. (1942). Science 95: 46.

25. Hamajima, F. (1962). Science Bull. of the Faculty of Agriculture, Kyushu Univ. 20: 61 (in Japanese).

26. Roderick, T.H., Wimer, R.E., Wimer, C.C, and Schwartzkroin, P.A. (1973). Brain Res. 64: 345.
See also MGI.

27. Finlayson, J.S., and Baumann, C.A. (1958). Am. J. Physiol. 192: 69.
See also PubMed.

28. Finlayson, J.S., Potter, M., and Runner, C.C. (1963). J. Natl. Cancer Inst. 31: 91.
See also MGI.

29. Finlayson, J.S., Asofsky, R., Potter, M., and Runner, C.C. (1965). Science 149: 981.
See also PubMed.

30. Ruoslahti, E., Engvall, E., Jalanko, H., and Comings, D.E. (1977). J. Exp. Med. 146: 1054.
See also PubMed.

31. Finlayson, J.S., Potter, M., Shinnick, C.S., and Smithies, O. (1974). Biochem. Genetics 11: 325.
See also PubMed.

32. Dev, V.G., Miller, D.A., Tantravahi, R., Schreck, R.R., Roderick, T.H., Erlanger, B.F., and Miller, O.J. (1975). Chromosoma 53: 335.
See also PubMed.

33. Miller, D.A., Tantravahi, R., Dev., V.G., and Miller, O.J. (1976). Genetics 84: 67.
See also PubMed.

34. Lieberman, R. (1978). Springer Seminars in Immunopathol. 1: 7.
See also MGI.