Studies of Mice with a Balanced Complement of 36 Chromosomes Derived

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 69, No. 10, pp , October 1972 Studies of Mice with a Balanced Complement of 36 Chromosomes Derived from F1 Hybrids of Tlih and TIAld Translocation Homozygotes (Robertsonian translocations/mouse hybrid nondisjunction/quinacrine mustard banding/giemsa banding) BEVERLY J. WHITE, JOE-HIN TJIO, LISA C. VAN DE WATER, AND CLARE CRANDALL Laboratory of Experimental Pathology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland Communicated by Theodore T. Puck, June 29, 1972 ABSTRACT F, hybrids with 38 chromosomes, including single T1Wh and TIAld translocations, resulted when mice homozygous for the Robertsonian translocations T1Wh and TiAld were crossed. Meiotic studies of the hybrids showed two trivalents, indicating nonhomology of the T1Wh and TIAld chromosomes. The hybrids had frequent (25%) unbalanced meiotic metaphase II complements; one trisomic mouse resulted from six F1 crosses. The F, crosses also produced one mouse with 36 chromosomes homozygous for both T1Wh and TIAld, as well as mice with balanced polymorphic complements of 37, 38, 39, and 40 chromosomes. By crossing the F2, a homogeneous line with 36 chromosomes was established. The line is phenotypically normal, fertile, and has balanced meiotic metaphase II complements. Analysis of the chromosomes of these mice with quinacrine mustard and the Giemsa-banding technique confirmed that T1Wh and TIAld consisted of chromosomes 5;19 and 6;15, respectively. Crosses between this line and other existing translocation stocks may produce new strains of mice with even further reduction in chromosome number. Accumulation of Robertsonian translocations, a possible evolutionary mechanism in the wild, can be studied in the laboratory. F1 hybrids from certain crosses are also an important model for human translocation carriers; both have similar meiotic abnormalities and often have aneuploid offspring. Four different Robertsonian translocations have now been described in mice (1-4). Mice homozygous for these translocations carry two biarmed chromosomes and have a total chromosome number of 38, while normal mice have 40 acrocentric chromosomes. The autosomes involved in most of these translocations have been identified by analysis of specific banding patterns after staining with quinacrine mustard. However, some of the chromosome numbers have been reassigned after correlation of banding patterns with length measurements (5). The TlWh translocation chromosome (TW) is formed by fusion of autosomes 5 and 19 (6), while the T163H translocation involves chromosomes 9 and 19 (5, 7, 8). The TIAld translocation chromosome (TA) consists of autosomes 6 and 15 (8). The two arms of the THEM translocation have been tentatively identified as chromosomes 8 or 9 and 16 or 17 by length measurements (4). Reported here are studies of progeny from crosses between TlWh and TlAld homozygotes. The purposes of the study were to confirm the identity of the TW and TA translocation chromosomes, to study meiotic disjunction of the F1 (TW/ Abbreviations: TA and TW, TlAld, and TlWh translocation chromosomes, respectively; M I, meiotic metaphase I; M II, meiotic metaphase II TA/+) hybrids, and to study the F2 offspring for balanced and unbalanced complements. One trisomic mouse was found, and one homozygous (TW/TW TA/TA) mouse was produced. By selecting progeny from certain crosses, a phenotypically normal and fertile line of mice with 36 chromosomes was established. METHODS Animals. Mice homozygous for TlAld (TA/TA) were obtained from Dr. A. L6onard, Centre d'etude de l'energie Nucl6aire, Mol, Belgium. TlWh homozygotes (TW/TW) were from stock maintained by our laboratory. Eight F1 litters from crosses between (TW/TW) females and (TA/TA) males were used for meiotic studies and F1 crosses. 41 F2 progeny were karyotyped, and certain F2 mice were crossed to derive additional (TW/TW TA/TA) mice (Table 1). 22 Progeny from these F2 crosses were then karyotyped. For crosses of F1 males and females with nontranslocation-bearing animals, AKR/J mice from the Jackson Laboratories, Bar Harbor, Me. were used. Chromosome Preparations. Meiotic metaphase I(M I) and II (M II) cells from testicular preparations (3) from 29 F1 males were analyzed. All progeny were karyotyped from 72-hr suspension cultures of spleen removed without killing the animal (3). Direct preparations were used to karyotype fetuses of days gestation from the crosses of F1 with AKR/J mice. Similar preparations of 15-day fetuses were used to determine the banding patterns of the TW and TA chromosomes with quinacrine mustard and the Giemsa-banding differential staining techniques. The method of Caspersson et al. (9) was used for quinacrine mustard studies. For Giemsabanding, the acetic-saline-giemsa technique of Buckland et al. (10) was used. Karyotypes were arranged according to the Committee on Standardized Genetic Nomenclature for Mice (5). RESULTS Progeny of crosses between (TW/TW) females and (TA/TA) males 51 Mice resulted from eight matings. The average litter size at birth was 6.4 (Table 1), and litter size ranged from four to nine. Karyotypes from F1 spleen cultures showed 38 chromosomes, including the submetacentric TW and TA translocations (Fig. la). Analysis of 722 M I cells from 29 F1 males consistently showed 15 autosomal bivalents, an XY bivalent, and

2 2758 Genetics: White et al. TABLE 1. Progeny from crosses between T1 Wh and TiAld No. of mice Average karyotyped Total litter Chro- No. of no. of size at Fe- mosome Translocation litters mice birth Male male no. chromosomes 38(TW/TW) 9 X 38(TA/TA) ' 8 Fi TW/+ TA/+ 38(TW/+ TA/+) 9 X 38(TW/+ TA/+) CP 6 F TW/TW TA/TA TW/+ TA/TA TW/TW TA/ TW/+ TA/ TW/TW +/ /+ TA/TA TW/+ +/ /+ TA/ /+ +/ TW/+ TA/+* 37(TW+ TA/TA) 9 X 37(TW/+ TA/TA)e TW/TW TA/TA 2 37 TW/+ TA/TA /+ TA/TA 36(TW/TW TA/TA) X 37(TW/+ TA/TA) e TW/TW TA/TA TW/+ TA/TA 38(TW/TW +/+) 9 X 38(TW/+ TA/+) e TW/TW TA/+ 38 TW/TW +/ TW/+ TA/ TW/+ +/+ 37(TW/TW TA/+).9 X 37(TW/+ TA/TA)e TW/TW TA/TA 2 37 TW/TW TA/ TW/+ TA/+ TA and TW = T1 Ald and TlWh translocation chromosomes, respectively. *Trisomy'19. two trivalents (Fig. 2a). One trivalent consisted of the TW chromosome associated with the autosomes homologous to its short and long arms, and the other included the TA chromosome paired with its homologues. If the two trivalents undergo balanced disjunction independently of each other, the following types of M II complements would occur in equal numbers: (1) cells with a chromo- TABLE 2. Metaphase II complemnts of 29(TW/+ TA/+) males No. of trans- No. of Chromo- location chromo- Cells counted some chromo- some no. somes arms Number Percent * * * Total * Balanced complements total 74.8%. 4e *At c 44.1 qw 1 u (% b 0 07 CC FIG.1. (a) Spleen metaphase of an F1 38(TW/+ TA/+) male showing the TA and the TW submetacentrics (arrows). (b) Metaphase from a newborn female with 39 chromosomes, 41 chromosome arms, and trisomy (arrows) for number 19. some number of 18 including TW and TA, (2) cells with a count of 19 including TW, (3) those with a count of 19 including TA [in practice, types (2) and (3) usually could not be distinguished], and (4) cells with 20 acrocentrics. The actual counts deviated from this (Table 2), and while the expected balanced complements predominated (74.8%, see also Fig. 3a, b, and c), many of the cells (25.2%) appeared to be the results of nondisjunction (Fig. 3d). It is therefore theoretically possible for the F1 to produce zygotes with duplication or deficiency for one or more of the chromosomes involved in the translocations (chromosomes 5, 6,15, or 19). Results of other crosses The average size of the six F2 litters was 7.2 (range 3-10). 41 of 43 F2 survived and were karyotyped (Table 1). According to the M II analysis, balanced progeny with a polymorphic series of 36, 37, 38, 39, or 40 chromosomes should be found. The karyotype 38(TW/+ TA/+) should be most frequent and karyotypes 36(TW/TW TA/TA), 40(+/+ +/+), 38- (TW/TW +/+), and 38(+/+ TA/TA) least frequent. The actual results (Table 1) were consistent with this, although there were fewer animals of karyotype 37(TW/TW TA/+) than expected. One nonviable female F2 mouse had 39 chromosomes, including TW and TA (Fig. lb). The number of chromosome arms was 41, and trisomy for chromosome 19 was present. In this instance, trisomy was apparently the result of nondisjunction of the TW trivalent chromosomes. The results of F2 crosses are shown in Table 1. Most of the stock with 36 chromosomes resulted from a cross between a (TW/TW TA/TA) female and a 37(TW/+ TA/TA) male. Animals of karyotype 36(TW/TW TA/TA) were phenotypi-

3 a FIG. 2. (a) Meiotic metaphase I from an F1 male with 15 autosomal bivalents, an XY bivalent, and two trivalents (arrows). (b) A 36(TW/TW TA/TA) male diakinesis with 18 bivalents. Note two large rings (arrow8) formed by the paired TW and TA chromosomes. cally normal and fertile; the average size at birth of ten (TW/ TW TA/TA) litters was 6.1 (range 2-10). Meiosis of males with 36 chromosomes Cells (1082 M I and 310 M II) from 13 (TW/TW TA/TA) males were analyzed. At M 1, 17 autosom'al bivalents plus an XY bivalent were consistently present. Two large rings were frequent (Fig. 2b), typical of the single large ring of males homozygous for a single Robertsonian translocation (1, 3). Balanced M II cells from (TW/TW TA/TA) males should have a count of 18, including both TW and TA. Of the 310 M II cells studied, 301 were 'apparently balanced (97.1%) and 9 (2.9%) appeared to be damaged during processing, since their counts were less than 18. Cells with counts greater than 18, indicating nondisjunction, were not present. I Mice with 36 Chromosomes 2759 Crossesbetween F,and AKR/J These crosses are described in Table 3. In the six crosses between (TW/+ TA/+) females and AKR/J males, 83.8% of corpora lutea were represented by implants and 82.3% of the total implants appeared to be viable fetuses. A lower proportion of corpora lutea resulted in 'implants (60.0%) and fewer of the total implants appeared viable (69.0%) when the AKR/J females and F1 males were crossed. All fetuses appearing viable were successfully karyotyped, and no unbalanced complements were observed. Our M II studies of (TW/+- TA/+) males indicated that equal numbers of a series of four balanced karyotypes could be expected in a cross with normal mice. The results shown in Table 3 were consistent with this, although there was greater deviation from the expected 1: 1: 1: 1 ratio in the six crosses of the F, females with normal males than in the crosses of AKR/J females with F1 males. Differential staining studies The quinacrine mustard karyotype of a male 36(TW/TW TA/TA) fetus is shown in Fig. 4. The TW chromosomes show banding patterns typical for chromosome 5 (long arm) and 19 (short arm) (5). The banding patterns of the arms of TA are consistent with those described by Miller et al. (8) and by the Committee on Standardized Genetic Nomenclature for Mice (5), for chromosomes 6 (long arm) and 15 (short arm). Giemsa-banded karyotypes of (TW/TW TA/TA) mice (Fig. 5) indicated that in general, the Giemsa- and quinacrine mustard-bands coincide, and confirmed the identity of TW and TA as chromosomes 5; 19 and 6; 15, respectively. In metaphases with prominent heterochromatic staining near the centromeres (C-bands), these regions of both TW and TA appeared double, a finding consistent with the theory that Robertsonian translocations result from centric fusion, without loss of paracentromeric heterochromatin. DISCUSSION Meiotic studies of F1 males from crosses between (TW/TW) females and (TA/TA) males confirmed that TW and TA do not share a chromosome in common. Two trivalents at M I indicated nonhomology of the arms of TW and TA, in contrast to previous meiotic studies of F1 males from crosses between TlWh and T163H homozygotes (6), where the chain quadrivalent at M I showed that TlWh shared a chromosome in common with T163H (number 19). In both cases, meiotic studies were consistent with identification of the chromosomes by differential staining methods (6-8). The significant proportion of unbalanced M II complements in (TW/+ TA/+) F1 males contrasts with the low percentage of such cells in males heterozygous for TlWh or T163H alone TABLE 3. Crosses of (TW/+ TA/+) F1 with AKR/J mice at days gestation F19 F. cr Cross X AKR/J a" X AKR/J 9 Number of crosses 6 6 Total corpora lutea Total resorptions Necrotic fetuses 0 1 Total viable implants Total implants % of total implants viable % of corpora lutea represented by implants Karyotypes 38(TW/+ TA/+) 9 (3d 69) 7 (4e 39) 39(TW/+ +/+) 15 (6d' 9 Q) 7 (4d'3 9) 39 (+/+ TA/+) 8 (4"49 ) 7 (2 5 Q9) 40(+/+ +/+) 19 (10d 9Q9) 8 (4'4 9) Total karyotyped 51 (236' 28 9 ) 29 (146' 15 9 )

4 2760 Genetics: White et al. a C d~~ { s o - e-.rcf C ~~~~~~~d FIG. 3. Meiotic metaphase II cells from F1 with chromosome numbers of: (a) 18 including TW and TA, (b) 19 including one translocation, (c) 20, and (d) 20 including one translocation. I a v~~~~~i FIG. 4. Quinacrine mustard karyotype of a 36(TW/TW TA! FIG. 5. Giemsa-banded karyotype of a 36(TW/TW TA/TA) I] ra) male identifying the TW chromosome as 5;19 and the TA male indicating similarity of the quinacrine and Giemsa-banding a 6;15. patterns.

5 (1, 3). These singly heterozygous males had a single trivalent at M I and no detectable trisomic progeny. Similar studies of TlAld heterozygotes have not been reported. Baranov and Dyban (11) found that male T11EM heterozygotes had only balanced progeny in crosses with normal female mice terminated at 8 days of gestation. However, heterozygous females crossed with normal males produced 14.3% trisomic embryos. The tendency for only female T11EM to have unbalanced offspring is similar to that of humans carrying Robertsonian translocations. Female D;G, translocation carriers have a significantly greater frequency of trisomic offspring than male carriers, while data on families in which G; G and D; D translocations are segregating are not extensive enough to compare the risk of female and male carriers (12). The similarity between the results of studies of T1IEM carriers and human D;G families emphasizes that mice with Robertsonian translocations are an important model for studying the behavior of human translocation chromosomes. Our crosses of (TW/+ TA/+) F, hybrids with normal AKR/J mice were done to determine if females had a higher frequency of aneuploid progeny than males. However, none of the crosses produced aneuploid fetuses. This finding was unexpected because of the trisomic mouse detected in one F, cross and the meiotic studies of (TW/+ TA/+) F, males that demonstrated frequent nondisjunction of trivalents (Table 2). When more than two trivalents are present at M I, such as in the F1 from crosses between Mus musculus X Mus poschiavinus (7 trivalents), more than 50% unbalanced M II complements are observed, trisomic fetuses are found in backerosses, and fertility of the F1 is decreased (13). The single trisomic from our F1 crosses (TW/+ TA/+) 9 X (TW/+ TA/+) d' contrasts with the 12% incidence of trisomy 19 at birth resulting from crosses between F1 mice heterozygous for both TlWh and T163H (6). In the latter case, trisomy was due to regular nondisjunction of quadrivalent chromosomes, while the present instance was related to trivalent nondisjunction. A total of seven mice (2 male, 5 female) homozygous for Mice with 36 Chromosomes 2761 both TW and TA with 36 chromosomes resulted from the crosses shown in Table 1. Like homozygotes for a single Robertsonian translocation with 38 chromosomes (1, 3) they were phenotypically normal, their meiotic studies showed no evidence of nondisjunction, and their fertility was not reduced. Evidently, the translocation chromosomes of the large ring bivalents of such homozygotes are comparable to the chromosomes of normal autosomal bivalents in their consistently balanced mode of disjunction. While Robertsonian fusion has been considered one of several possible evolutionary mechanisms, no obvious phenotypic alteration occurred in mice with the polymorphisms generated by crossing (TW/+ TA/+) F1 hybrids. The studies of Robertsonian polymorphism in the African pigmy-mouse by Matthey (14) demonstrated a similar phenomenon in the wild. The pigmy-mouse chromosome number varies from 18 to 34, while the number of chromosome arms remains 36. This suggests that further polymorphism could be developed in the laboratory mouse by crossing the (TW/TW TA/TA) line with other existing translocation stocks. While the double structure of the centromeric regions of the TW and TA translocations indicated that paracentromeric heterochromatin is intact, there is no objective evidence that significant genetic change occurs in Robertsonian fusion, which presumably requires breaks in the centromeric regions of both involved chromosomes. Further comparison of characteristics of (TW/ TW TA/TA) mice with their parent strains might reveal more about the effects of chromosomal fusion and their role in the evolutionary process. The quinacrine mustard- and Giesmsa-banding patterns of the TW and TA chromosomes reported here are consistent with those described by others. The quinarine mustard- and Giesmsa-bands are similar, indicating that either technique is sufficient to identify specific mouse chromosomes. In addition to identifying linkage groups with certain chromosomes (number 5, LG XIV; number 6, LG XI; number 9, LG II; number 19, LG XII) (5), these techniques can be used to identify specific chromosomal aberrations during gestation and may allow studies of the embryogenesis of anomalies associated with aneuploidy. Crosses of mice from existing translocation stocks can produce mice with various deviations from the normal chromosome complement and can now be used as a system to study human chromosomal rearrangements. 1. 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