(CASPARI and DAVID 194.0, BENNETT 1975), this study was undertaken to char- acterize the Tal mutation by a detailed genetic analysis, to test the possibility.
GENIC INTERACTION CAUSING EMBRYONIC MORTALITY IN THE RAT: EPISTASIS BETWEEN THE Tal AND grc GENES1 DANIEL J. SCHAID, HEINZ W. KUNZ
AND
THOMAS J. GILL 1112
Department of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261 Manuscript received July 16, 1981 Revised copy accepted January 11,1982 ABSTRACT
The autosomal dominant mutant gene, tail anomaly lethal ( T a l ) , of the rat is lethal when homozygous but affects tail morphology (kinks and reduced length) and body weight when heterozygous. There is no apparent sex effect on the expression of Tal. It is incompletely penetrant; has variable expressivity, which is influenced partly by its genetic background; and is not linked to the major histocompatibility complex (MHC) . The heterozygous Tal gene and the homozygous grc genes, which are linked to the MHC and affect body size and fertility, interact to cause intrauterine death a t a time between implantation (five to seven days post-fertilization) and 15 days of gestation. This interaction shifts the time of death from the immediate postnatal period when the homozygous grc genes act to the time during gestation when the homozygous Tal gene would cause death. This description of lethal epistatic interaction in the rat appears t o be the first report of this phenomenon in mammals.
HE Tal gene causes a tail anomaly (HOSHINO, ODAand KAMEYAMA 1979) characterized by a short, kinked tail; it is inherited as an autosomal dominant. It was discovered in teratological studies using trypan blue (HOSHINO, ODAand KAMEYAMA 1979), which induces gross skeletal malformations (GILLMAN, GILBERT and GILLMAN1948; GUNBERG 1956; WARKANY, WILSONand GEIGER1958; HOSHINO 1971). The homozygotes for the gene died at approximately 10 days of gestation; and rats with tail defects, which are heterozygous for Tal, appeared to have body weights lower than those of normal-tailed rats. At least 70 inherited conditions can involve the skeleton to some extent (GRUNEBERG 1963), and more than 26 mutant genes or groups of genes affect the tail in mice (Mouse News Letter 1977). The best-studied genes are those of the T / t complex (SNELL1968; BENNETT1975), which are also thought to influence the genetic organization of the major histocompatibility complex (MHC) and KLEIN1975; GOTZE (SNELL1968; ARTZT and BENNETT1975; HAMMERBERG 1977). In the rat, only 12 genes other than Tal are known to affect the skeleton (ALTMAN and KATZ1979), and only one of these-stub, which is now extinctaffects the tail. Efforts to find a T system affecting tail morphology in the rat have failed (DUNNet al. 1942; ROBINSON1965). However, the recessive genes (grc) of the growth and reproduction complex (Grc),* which are linked to the 1 2
Thi, woik was supported by grant HD 08662 from the National Institutes of Health. 1U whom correspondence should be addressed
Genetics 100: (115432 April, 1982
616
D. J. SCHAID, H. W. K U N Z A N D T. J. G I L L I11
MHC, cause death between birth and weaning in approximately 25% of homozygotes; and they have some analogy with the t-recessive genes of the mouse (GILLand KUNZ 1979; KUNZet al. 1980). Spina bifida was proposed to be the T / t analog in humans: it was found to be linked to the MHC in some studies (AMOSet al. 1975) but not in others (BOBROWet al. 1975; DE BRUYEREet al. 1977). Since Tal is phenotypically similar to the T / t and Ki genes of the mouse (CASPARI and DAVID194.0, BENNETT1975), this study was undertaken to characterize the Tal mutation by a detailed genetic analysis, to test the possibility of linkage between Tal and RTI, the major histocompatibility complex of the rat, and to study the possibility of genic interaction between Tal and grc. MATERIALS A N D METHODS
Rats: The nomenclature, genotypes and phenotypes of the strains of rats used in this study and of the test animals produced by various crosses are shown i n Table 1. The Tal mutation originated in Wistar-derived rats and is maintained on the BDIX background; i.e., it is carried as the congenic BDIX.Tal strain (HOSHINO, ODA and KAMEYAMA 1979). The BDIX.Tal animals were obtained from the National Institues of Health, and all other animals were from our colony a t the University of Pittsburgh School of Medicine. The growth and reproduction complex carried b y some of the strains (Table 1) consists of genes causing small body size (dw-3) and small, infertile testes, or reduced reproductive capacity i n females ( f t ) (GILL and KUNZ 1979; KUNZet al. 1980). The r l l strain was derived from a recombinant and has the genotype RTI.ALB2 grc+ (KUNZet al. 1980). In all crosses, the female is written first and the male, second. All animals were fed rat chow and water ad libiturn, and they were kept in a conventional colony with the lights on for 12 hr and the humidity maintained at 50-60%.
TABLE 1 Strain nomenclature and characteristics Strain or cross
Grc
RT1.B
BIL/1 rll
BIL YO NBR BDIX.Ta2 BY* BY1+ BYl2f BY2+ BTK$ BTN$
RT1.A
Tal
+/+ +/+ +/+ +/+
+/+ +/+ +/+ +/+ +/+ +/Tal +/+
Tal/+
(BIL/I x Y0)FI hybrid. +$* From (BIL/I x YO)F2 hybrid. (BY1 x BDIX.Tal)FI hybrid: K, kinked tail and N, normal tail. * The nomenclature used for the growth and reproduction complex is as follo'ws: T h e symbol Grc will be used to refer to the complex as an entity in the same way in which MHC is used to refer to the major histocompatibility complex. Genes in the complex are referred io .by tlie symbol grc: the w i l d - t y p alleles will be indicated by grc+. The specific genes that have been identified in the complex are d w a r f 3 ( d w - 3 ) and alteled fertility (ftj (GILL and Kuixz 1SiD; KUNZ et n l . 1980).
61 7
Tal A N D grc INTERACTION
Body and gonad wrighls: Newborn rats were counted at birth and again at weaning (three weeks), at which time they were sexed. Body weights were measured on a Torbal PL-1 balance a t 10-day intervals beginning a t day 30. The gonads of 60-70 day old rats were removed, trimmed, fixed in 10% formalin for 10 days and weighed on a Mettler H-20 balance. The animals were weighed immediately prior to autopsy to calculate the gonad-body weight ratio. Characreriztion of tails: The tail morphology was characterized as normal or kinked in rats at weaning, and the severity of the kinking at 60 days of age was scored as slight, moderate or severe (Figure 1 ) . The length of the tail was measured from the base of the spine to the tip of the tail, and the length of the spine was measured from the occiput of the skull to the base of the spine. A tail index was calculated as tail length/spinal length. The degree of penetrance of the Tal gene was calculated according to ROBINSON(1971): Penetrance (a)=
( R + 1)a and Rn
- aCR (1-a)
Variance
+ 11
Rn where, a = number of affected rats, (n-a)= number of normal rats, R ratio of a: (n-a) and n = the total number of rats.
= expected segregation
k'
n
A
i
FIGURE I.-Adult
B
C
D
male animals demonstrating the variable effect of the heterozygous Tu1 gene on tail morphology: A, normal; B, slight; C, moderate and D, severe.
618
D. J. SCHAID, H. W. K U N Z A N D T.
J. GILL I11
Serological methods: The RT1 .A antigens were serotyped by the Ficoll hemagglutination method as described by KUNZet al.(1977). Prenatal mortality: Ovulating females were caged with males, and vaginal smears were performed daily until the first appearance of sperm in the smear. That day was counted as day zero of gestation and the males were removed. Ovulation was determined by the morphology of the epithelial cells in vaginal smears stained with filtered Wright stain (Harleco, Gibbstown, N.J.) and filtered Giemsa stain (Fisher Scientific Co., Fair Lawn, N.J.). Prenatal mortality was assessed by counting the number of live and dead fetal implants on day 15 of gestation in both uterine horns of sacrificed females. Embryonic mortality was confirmed histologically. RESULTS
Characterization of Tal: Lethality of homozygotes for Tal was suggested by TOSHINO, ODAand KAMEYAMA (1979) to explain the observed segregation of Tal among offspring from kinky-tailed parents, both of which were heterozygous for Tal. The segregation analysis of the data from this study is consistent ODAand KAYEMA (1979) (Table 2) and supports the with that from HOSHINO, hypothesis of Tal/Tal lethality. An increase in embryonic mortality in [Tal/ Tal+ x Tal/Tal+] matings after 10 days of gestation also suggests that Tal/Tal homozygotes die prenatally (HOSHINO, ODAand KAYEMA 1979). The phenotypic effects of Tal are tail trunkation and kinks and a decreased body weight compared t o normal-tailed rats. The expressivity of the autosomal dominant Tal gene was highly variable: the degree of tail trunkation ranged from severe to approximately normal in length, the number of kinks per tail varied from one to six and the amount of kinking ranged from barely perceptible to complete hooks and corkscrews (Figure 1 ) . The presence of Tal in the heterozygous state caused a consistent decrease in the mean body weight of kinkytailed BDIXaTal rats compared to that of normal-tailed BDIX.Tal rats (Figure 2 ) . Other abnormalities associated with the BDIX.Ta1 strain observed in this study are club foot (incidence of 2/250) and malocclusion of the upper and lower incisors (incidence of 10/250). They occurred independently of each other and of the presence of kinked tails. The transmission of Tal showed the characteristics of incomplete penetrance: decreased number of affected offspring from that expected and skipped generations. Further evidence for incomplete penetrance is given by the pedigree shown in Figure 3, in which phenotypically normal animals produced off spring with tail anomalies: hence, they were heterozygous for Tal without showing its effects. The exact determination of the degree of penetrance was not feasible, since testmating of all normal-tailed rats would have been required. Estimates were made by comparing the observed and expected numbers of animals with kinked tails (ROBINSON 1971) ;the results are shown in Table 2. Linkage between Tal and RTI: The BY1 and B I L strains were mated with BTK F1 hybrid animals to test the possibility of linkage between Tal and R T I . These strain combinations were chosen because the kinky-tailed offspring which resulted from the matings were often severely affected. The BTK rats, which received RTld and Tal from the same parent (Table l), were backcrossed into BY1 females or into BIL males and females. The phenotypic marker used for
619
Tal AND grc INTERACTION e
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A N D T. J. G I L L 111
FIGURE 2.-Growth curves of kinky-tailed (M) and normal-tailed ( 0 ) offspring from reciprocal crosses between kinky-tailed (K) and normal-tailed (N) parents of the BDIX.Tal strain. The error bars represent the standard deviation, and they are omitted on the inside of the curves for the sake of clarity.
the MHC was the class I antigen controlled by the RT1.A locus. Out of 82 [BY1 X BTK],29 [BIL X BTK] and 118 [BTK X BIL] testcross offspring, no kinky-tailed RT1.AL/lrats were observed, but all other classes of off spring were present (Table 3 ) . This finding is highly significant (x2 = 47.0053;P < O.OOl), since approximately ?&, (20.50) of the [BY1 X BTK] testcross offspring, % (3.63) of the [BILx BTK] testcross offspring and 1/8 (14.75)of the [BTKx BILj testcross off spring were expected to be kinky-tailed and RTI.AZIz,giving an expected total of 39 out of the 229 rats observed. The lack of RT1.Az homozygotes with kinky tails suggested that the presence of the Tal gene in animals with this genotype and its linked grc genes was lethal.
Tal AND grc INTERACTION
BY1
BI L
621
BDIX.Tal
b
FIGURE 3.-Pedigree showing the variable penetrance of Tal. Circles are males, the squares, females and the diamonds, sex not distinguished. Solid symbols are the animals with affected tails, and the open symbols are phenotypically normal animals. The parentheses under the symbols contain the number of animals in each group. Animals a and b were phenotypically normal, but some of their offspring and some of the (a x BIL) offspring had tail anomalies. Thus, these animals are heterozygous for Tal but did not show any of its effects.
Because the Tal gene displayed incomplete penetrance and phenotypically normal animals could carry it, this hypothesis was tested by determining whether any viable RTI.Az homozygotes carried the Tal gene. Nine female offspring from the above testcrosses which were RTI.Az homozygotes and normal-tailed were testmated with normal-tailed males of the NBR strain to determine whether these females were carriers of Tal. Seven of the females gave birth, and all of the offspring were normal-tailed. Five of these females gave birth to a minimum of six offspring, further proof that they were not carriers of Tal. Since the gene coding for RTI-Az in all three testcrosses originated from the BIL/I strain, which carries the growth and reproduction complex; since grc and Tal both affect body weight and development, it was postulated that the lack of kinkytailed RTI.Az homozygous rats was due to interaction between the heterozygous Tal gene and the homozygous grc genes causing prenatal death (see below). To avoid having to correct the segregation ratios for the missing kinky-tailed RTI.AZI1rats and the decreased viability of the normal-tailed RTI.AZI1rats so that the segregation pattern of RTI-A could be examined, the BTK F1 hybrids were reciprocally crossed with the NBR strain. The off spring were either RTI A'/' and grc/grc+ or RTI.AIIdand gl;c+/grc+, and all were viable (Table 3 ) . The
622
D. J. SCHAID, H. W. KUNZ A N D T. J. GILL I11
TABLE 3 Relationship between RTI .A and tail phmotypes and degree of tail trunkation (tail index) in uarious crosses Cross
Tail'
Y
type
(F
W
BY1
x BTK
BIL
x BTK
N K N
K BTK x BIL NBR
x BTK
BTK
x
rll
NBR
x BTK
BTK x r l l
N K N K N
K N K N K
1/1
R T I . A genotype I/d l/n
27 39 0 21 4 3 0 1 1 13 17 0 14 18 13 17 9 11 11 13 9
21 17 11 5 13 14 16
4 2 26 24
n/d
3 2 11 13
Mean tail index+ l/d l/n
n/d
1.08
1.12 0.44
1.13 0.36
1.13 0.87
1.13 0.87
I/I
1.08 0.43
eo
* N, normal tail and K, kinked tail.
t The standard deviations of the mean tail indices varied from 41.2% of the mean in normal-
tailed animals and from 5-52% of the mean in kinky-tailed animals.
BTK animals were also reciprocally crossed with the recombinant r l l strain, which has a genetic background similar to BY1 except that it is grc+ (Table 1). Kinky-tailed RTI.Az/L offspring were observed in the expected proportions (%) among the [BTK x r l l ] offspring (x2= 2.7816; 0.05 < P < 0.10) and among the [rll x BTK] offspring (x2 = 0.1701; 0.50 < P < 0.70) (Table 3 ) . Thus, Tal and RT1.A were not interacting in a detrimental manner. Segregation analysis of the [NBR x BTK] testcross offspring showed that the RT1.A genotypes and tail phenotypes segregated independently, since the number of offspring with a recombination between RTI.A and Tal was approximately equal to the number of parental-type offspring (Table 4). Disturbed segregation of the RT1.A genotype and tail phenotype: The segregation of RTZ A genotypes in the [BTK x NBR] testcross offspring was significantly distorted in males, yet it fitted the expected ratio in females (Table 5 ) . There was also a distortion of tail phenotypes in females of this cross. The reciprocal [NBR x BTK] testcross offspring showed no such differences in either sex. The litters from the [BTK x NBR] testcross did not show preweaning death, so the cause of the distortion in genotypes and tail phenotypes was not due to postnatal selection. Since homozygotes for grc could not be produced by this cross, these distortions were not caused by Tal and grc interacting lethally. Segregation of R T I - A gefiotypes was also significantly distorted in both the [BIL X BTK] and [BTK X BIL] testcross offspring (Table 5 ) . Since kinkytailed RTI.A1I1rats were expected to occur among the offspring from both of
Tal AND grc INTERACTION
623
TABLE 4 Test for linkage between RT1 and Tal in the (NBR Segregating marker
Genotype or phenotype'
RT1.A
1/1 l/d N
Tal
Males
RT1.A from Tal
12 18 20 10 16 14
19 20 18 21
recombinant parental
Deviation from expected 1 :1 ratio+
Number of animals Females Total
19 20
K
x BTK) testcross offspring
31 38 39 30 34 35
0 0 0 0 0 0
* 1 and d are RT1.A genotypes. N, normal tail and K, kinked tail are Tal phenotypes.
t +,significant at P < 0.05 and 0, not significant.
these crosses, yet were not observed, the total number of RTl.AZI1rats was decreased, and this loss altered the ratio of RTI-A genotypes. Exclusion of the RT1.A1/loffspring from the analysis of the data from the [BIL x BTK] cross showed the expected Mendelian segregation of the remaiEing genotypes in the proportions 1 RTI.A1Id: 1 RTI.Al/": 1 RT1.AnId.By contrast, the RT1.A segregation was still significantly altered in female [BTK x BIL] offspring even after RTl.A'/' rats were excluded from the analysis. Segregation for RTI-A in the TABLE 5 Segregation of RTI .A genotypes and tail phenotypes in mrious crosses RT1.A genotypes Cross
NBRxBTK BTKx NBR
Sex
t*
M F t
B I L x BTK BTKx BIL
t*
M F t
BY1 xBTK rll
x
BTK
Experted ratio
Deviation from expected+ K
1/1
l/d
31
38
1:l
16 10 26
6 10 16
1:l 1:l 1: 1
4
14
6
5
1:l:l:l
+s
15
10 3 13
18 13 31
24 26 50
13 11
1:l:l:l 1:l:l:l 1:l:l:l
0 +$
24 27 51
l/n
n/d
24
t*
27
55
1:1
BTK
t*
22
27
1:l
x rll
t*
22
36
1:l
0
+
30
39
1:l
9
13 15 28
1:l 1:l 1:l
+
14
1:l
0
41 26 67
1:l 1:l 1:l
0 0
5
0 0
14
+t
Tail phenotypes Deviation Expecfed from N ratio expected:-
0 0
+'I 21
61
1:l
0
25
24
1:l
0
0
29
29
1:l
0
* NO significant differences between males and females, and the numbers are for the total population studied (t). t P 5 0.05 represents significant deviation: 0, no deviation and deviation. 0 If the (1/1) genotype is excluded, there is no deviation from a 1:1: 1 ratio (P 0.05). $ T h e deviation is still signlficant if the ( 1 / E ) genotype is excluded in both the total postweaning population and in the population of litters with no preweaning death. 11 If the (1/1) genotype is excluded, there is no deviation from a 1:l ratio (P 0.05). 1' If the kinky-tailed rats are excluded, there is no deviation from a 1:1 ratio (P 0.05).
+,
>
>
>
624
D. J. SCHAID,
H.
W. KUNZ A N D T.
J. G I L L I11
male offspring was normal in this cross before and after the RTZ-Az/z rats were excluded. Since removal of the RTI.AZIzrats from consideration in this segregation analysis corrected f o r the RT1.A genotype distortion caused by the lethal interaction of Tal and homozygous grc, an influence other than the Tal-grc interaction must cause the altered RT1.A genotype ratios in the female rats of the [BTK x BIL] cross. This influence was not preweaning death, since litters from the [BTK x BIL] testcross that did not have preweaning death still demonstrated distortion of RT1.A genotypes among female offspring after the RTZ A’/’ rats were excluded. The [BY1 x BTK] offspring showed disturbed segregation for RT2.A (Table 5 ) , but elimination of kinky-tailed rats from the analysis led to the expected Mendelian segregation ratio. Therefore, the distorted segregation of the RT1.A genotypes was due to the lack of kinky-tailed RTI.Az/zanimals. The RT1.A genotypes of the offspring from reciprocal crosses between BTK and rll segregated as expected (Table 5 ) . Tail phenotypes failed to segregate as expected in [BTK x BIL] males and in [BY1 x BTK] males and females, since there was a deficit of kinky-tailed animals in both cases (Table 5 ) . However, when these populations were corrected for the missing kinky-tailed RTZ.Az/zanimals, the segregation of tail phenotypes was normal. The offspring from [NBR X BTK], [BIL X BTK], [rll X BTK] and [BTK x r l l ] testcrosses segregated as expected for kinky-tails. Znteraction between Tal and grc: The lack of kinky-tailed RT1.A”’ offspring from the testcrosses [BY1 x BTK], [BTK x BIL] and [BIL x BTK] was thought to result from interaction between the heterozygous Tal gene and the homozygous grc genes; therefore, the interactions between heterozygous Tal and homozygous grc and between heterozygous Tal and heterozygous grc were examined in greater detail. The former interaction could be deduced by absence of an expected class of offspring, and the latter interaction could be tested by quantitative measurements, since these rats are viable (KEMPTHORNE 1969; MATHERand JINKS1971). The phenotypic parameters of homozygous grc are reduced body weight in both sexes; male sterility; and reductions in testicular weight, female reproductive capacity, and viability (GILL and KUNZ 1979; KUNZet al. 1980). The parameters of heterozygous Tal are incomplete penetrance, trunkated tail, tail kinks and a slightly reduced body weight in both sexes (HOSHINO, ODAand KAMEYAMA 1979; this study). Quantitative measurements of the tail index, number of kinks per tail, body weight and gonad weight were made on offspring from the crosses [BTK x BIL] and [NBR x BTK] to test for interaction between heterozygous Tal and heterozygous grc. Mean body weights were compared by the two-tailed Student t-test; all other comparisons were made by the one-tailed Mann-Whitney U-test. The grc genes in these crosses were inherited from either the BTK or the BIL parent. The criterion for homozygous grc in the offspring of the [BTK x BIL] cross was the RTI.A1/lgenotype, and that f o r heterozygous grc was RT2-A1/-.In the offspring of the LNBR x BTK] cross, the R T Z S A ~animals /~ were heterozygous for
Tal A N D grc INTERACTION
625
grc, since the NBR strain [ R T I . A L / ' is ] grc+. In both crosses these parameters were compared between the progeny that were kinky-tailed and grc/grcf and those that were kinky-tailed and grc+/grc+. In all comparisons, no significant differences (P > 0.05) were found between the two classes of animals. Thus, there is no interaction between heterozygous Tu1 and heterozygous grc. The expected proportion of lethal Tal/Tal homozygotes from the heterozygous mating [TaZ/Tal+ X Tal/Tal+] is 25%, and HOSHINO, ODAand KAMEYAMA (1979) speculated that the homozygotes are absent because the embryos die at 10 days of gestation. Reanalysis of their data (Table 6) showed that the pooled estimate of prepatal mortality for days 8 and 9 of gestation in the heterozygous cross was 10.8%, while an estimate for 10 days of gestation in the same cross was 39.4%. The control cross with normal animals, [Tal+JTaZ+X Tal+/Tal+], gave a mortality rate of 12.5% by using the pooled data for days 8 to 20 of gestation. Thus, the excess mortality in the heterozygous cross was 26.9%, which would be expected if the Tal homozygotes died in utero. Since the effects of Tal are exerted at approximately 10 days of gestation, the lethal interaction between heterozygous Tu2 and homozygous grc may also occur at approximately the same time. This hypothesis was tested by observing the mortality rates of embryos at 15 days of gestation ir, females of the matings [BY1 XBTK] and [ r l l X BTK] . This time was chosen because dead and viable embryos could easily be distinguished if a lethal interaction occurred at approximately 10 days. Since 25% of the [BY1 X BTK] offspring would be heterozygous for Tal and homozygous for grc, while none o f the [rll X BTK] offspring would be. the prenatal mortality rate of [BY1 x BTK] embryos should exceed that of [ri 1 x BTK] embryos by approximately 25% if a lethal interaction between Tal and homozygous grc occurred. The prenatal mortality rates observed for these crosses are shown in Table 6. If it is assumed that the 8.7% prenatal mortality rate in the [rll X BTK] cross is the rate that would be observed in the [BY1 X BTK] cross if heterozygous Tal and homozygous grc were not interTABLE 6
Prenatal mortality among [Tal/Tal+
Esperiment
Mating type
HOSHINO. ODA and KAMEYAMA Tal (1979)
x
Tal
-X-
Tal+
Tal+
Tal+
Tal+
XTali- Tal+ This paper
BY 1 x BTK rll
x BTK
Tal/Tal+] offspring during gestation Gestation (days)
No. of No. of female No. of dead Mortality parents implants implants (%)
8-9* 10 12-20*
12 6 19
I20 66 184
13 26 80
10.8 39.4 43.5
8-9* 10 12-20'
10
13
5
91 46 126
11 4 18
1&.1 8.7 14.3
7 6
46 69
12 6
26.1 8.7
15 15
* The mortality rates were pooled since they were approximately the same for each of the days in the given range.
626
D. J. SCHAID, H. W. KUNZ A N D T. J. G I L L 111
acting, the excess prenatal mortality rate in the [BY1 x BTK] cross is 17.4%. This rate does not differ significantly from the excess rate expected to occur (i.e., 25%) if heterozygous Tal and homozygous grc interacted in a lethal manner (x2= 1.1932; 0.20 < P < 0.30). I n addition, the proportion of dead implants per female was significantly higher for the [BY1 x BTK] cross than for the [rll x BTK] cross (comparison by a one-tailed Mann-Whitney U-test with U = 8 gave P = 0.04). Thus, the lethal interaction between heterozygous Tal and homozygous grc took place between implantation, which occurs at five to 1980), and 15 days seven days of gestation (BAKER,LINDSEY and WEISBROTH of gestation; it most likely occurred close to the time of action of the homozygous T a l genes (10 days). Litter sizes: The mean litter sizes at birth and at weaning and the sex ratios of the various crosses are summarized in Table 7. The mean litter sizes were approximately the same f o r reciprocal crosses between NBR and BTK, BTK and BIL and r l l and BTK. The [BY1 x BTK] cross had the lowest mean litter size. Since this cross was expected to have a higher proportion of kinky-tailed RTI A ‘1’ offspring than any of the other crosses, the decrease in litter size probably resulted from the interaction of heterozygous Tal and homozygous grc to cause intrauterine death. The ratio of males to females was highest in the offspring from the [NBR x BTK] and [BTK x r l l ] crosses and lowest in the offspring from the [BIL X BTK] cross. Growth of kinky-tailed and normal-tailed offspring: The growth of kinkytailed testcross offspring (Figure 4) was influenced to some extent either by the maternal environment or by cytoplasmic inheritance; these two effects could not be distinguished in this experiment. Animals homozygous for grc were excluded from growth curves A through D, because the effect of the grc on body weight would have distorted the effect of Tal on body weight. In curves A, B, E, F, I and J, Tal was inherited from the male parent, and in curves C, D, G. H, K and L, Tal was inherited from the female parent. The kinky-tailed testcross progeny have lower body weights than the normal-tailed progeny in all cases, and they appear to have relatively lower weights when the Tal gene is transmitted by the mother. TABLE 7 Litter sizes and sex ratios of uarious crosses
Cross
BY1 x BTK BIL x BTK BTK x BIL NBR x BTK BTK x NBR rll x BTK BTK x r l l
No. of mating No. of pairs litters
14 4
15 8 4
6 7
No. of animals born
94 30 133
70 42 53 59
Litter size at birth mean SD
6.71
7.50 8.87 8.75 10.50 8.83 8.43
2.13 0.58
2.10 0.71 3.87 3.97 1.51
No. of animals weaned male female
42 13 65 39 22 27 34
40 16 53 30 20 22 24
Sex
(M:F)
Litter size weaning SD
at
ratlo
mean
1.05 0.81 1.23 1.30 1.10 1.23 1.42
5.86 7.38 7.87 8.63
10.50 8.17 8.29
2.07 0.52 2.77 0.52 3.87
3.37 1.50
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Tal AND grc INTERACTION
I50 100
50
0
20
40
60 0
20
40
60 0 20 AGE (DAYS)
40
60 0
20
40
60
FIGURE 4.-The growth curves of kinky-tailed and normal-tailed ( a ) testcross offspring from reciprocal crosses between normal-tailed and kinky-tailed parents. In growth curves A, B, C and D, animals homozygous for grc (ie., RTIC/Z) were not included so that the effect of heterozygous TaE on growth would not be distorted. The Tal phenotypes of the parental strains are shown in Table 1. The error bars represent the standard deviation, and they are omitted on the inside of the curves for the sake of clarity.
(m)
DISCUSSION
Heterozygotes for the Tal gene showed incomplete penetrance of the phenotypic characteristics of tail truncation, tail kinks and decreased body weight; the penetrance could be influenced by modifying background genes in the BIL and NBR strains. The mean tail index of kinky-tailed males and females in the [NBR x BTK] testcross offspring was 75-80% of that in normal-tailed animals, whereas in the [BTK x BIL] testcross offspring, it was 3 5 4 0 % (Table 3). Therefore, the genetic background of the BIL strain seemed to enhance the exand IVANYI(1974) pression of Tal, at least in regard to tail trunkation. MICKOVA demonstrated that the tail length of brachyury mice heterozygous at the T locus depended upon genetic background: BIO-T mice had significantly shorter mean tail lengths than A-T and C3H-T mice. Other studies also demonstrated that the and expression of the heterozygous brachyury gene (DUNN1942; WHITTMAN
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HAMBURGH 1968) and of other mutant genes (GRUNEBERG 1963) is influenced by their genetic backgrounds. The prenatal lethality of the Tal gene in the homozygous state was inferred ODAand KAMEYAMA (1979) obfrom two lines of evidence. First, HOSHINO, served a 30.7% net prenatal mortality after 10 days of gestation among embryos from crosses in which both parents were heterozygous for Tal (Table 6). Second, the segregation of Tal among offspring from parents heterozygous for Tal fits the ratio of 2 kinky-tailed: 1 normal-tailed animal expected if the Tal/Tal offspring are missing (HOSHINO, ODAand KAMEYAMA 1979; this paper). The lethality of the interaction between heterozygous Tal and homozygous grc was demonstrated by the 17.4% net prenatal mortality which occurred by at least 15 days of gestation in the [BY1 x BTK] cross (Table 6). Death of the embryos probably occurred earlier in gestation, however, because absorption of the dead implants was apparent by 15 days: there were no visible embryos, and the implants consisted of raised nodules of degenerated placental tissue and clotted blood. I n addition, this lethal interaction had to occur after implantation, because death prior to this time would not have been detected as dead uterine implants. Two other homozygous lethal conditions in the rat can be compared with Tal. The autosomal dominant gene restricting hood pattern (H'") causes death in homozygotes within 24 hr after birth, and male heterozygotes are fertile for et al. 1971). Some autosomal reonly a short time after puberty (GUMBRECK cessive mutallts die between birth and approximately 35 days of age (ROBINSON 1980) ; but only stub ( s t ) , which is now extinct, appears to resemble Tal phenotypically. However, homozygotes for st usually died at birth o r shortly thereafter (RATCLIFFE and KING 1941), so the primary action of st probably occurs at a time different from that of Tal. The effect of Tal appears to resemble more closely the effects of the T dominant gene of the T / t complex and of the Ki gene in the mouse (BENNETT1975; KLEIN1975) than that of any other mutant gene in the rat. These genes are linked to the MHC in the mouse, whereas Tal in the rat is not; this need not be critical from a functional point of view. The cause of the disturbed segregation of the RTI-A antigens among the progeny of the [BTK x NBR] and [BTK x BIL] testcrosses (Table 5) is not clear. There may have been a chance deviation from the expected segregation in the [BTK x NBR] testcross offspring population since it had only 42 animals, but the [BTK X BIL] testcross had 118 animals, so chance deviation seems unlikely. Alternatively, selection against a specific antigen may have decreased the number of animals of certain RT1.A genotypes. In both of these crosses, there were fewer heterozygous offspring carrying the RTI.Ad antigen, which was inherited from kinky-tailed F1 hybrid mothers heterozygous f o r RTI.A1/d.By contrast, the reciprocal crosses in which the RTI.Ad antigen was inherited from kinky-tailed F1 hybrid fathers heterozygous for RTZ*A1/d showed no genotypic distortion. Thus, the mechanism of selection may have been by a maternal influence, but a specific cause cannot be inferred from this study. The kinky-tailed off spring from the various crosses had consistently lower body weights than normal-tailed off spring; the differences appeared somewhat
Tal AND grc INTERACTION
629
greater when the mother was kinky-tailed (Figures 2 and 4). The size of the kinky-tailed mother may have influenced her ability to rear her young, the greatest impact being on those offspring prone to retarded development, i.e., those carrying the Tal gene. Such an effect on the size of offspring has been observed in mice where an interaction occurs between the maternal environment influenced by body size and the genotype of the implanted embryo (BRUMBY 1960). The Tal and grc genes interact in a unique manner, and no similar situation has been reported in mammals. Interactions between these genes were studied by observing disturbed segregation ratios and by comparing the phenotypic ef1979). fects of the mutant genes separately and in various combinations (BARKER This approach to studying the interaction between Tal and grc has precedence in mice in at least three situations. First, Sd (Danforth's short-tail), an autosomal semidominant gene which is lethal when homozygous, and un (undulated), an autosomal recessive gene, both can affect the vertebral bodies. When heterozygous Sd is combined with homozygous dn, the effects on the vertebrae are much greater than those that occur when either is present alone; this finding 1953). Second, this same type of intersuggests genic interaction (GRUNEBERG action led to reduction in tail length when heterozygous Pt (pintail) and heterozygous Sd (BERRY1960), heterozygous Pt and heterozygous T (brachyury) (BERRY1960) or the dominant and recessive factors of the T / t complex (BENNETT 1975) were combined. Third, additive interaction between genes affecting the same trait occurs with the semidominant X-linked tabby ( T a ) and the autosomal recessive crinkled ( c r ),which individually cause some reduction in tooth size but together cause an even greater reduction in a double heterozygote (SOFAER1979). Therefore, genes affecting the same structure can interact ad1963). ditively (GRUNEBERG Both heterozygous Tal and homozygous grc affect growth and developmental processes; their interaction fits the previously established pattern for epistasis. However, the result of this genic interaction, prenatal lethality, is unique. Also, the interaction shifts the time when the progeny die from the immediate postnatal period for the grc genes alone (GILLand KUNZ 1979; KUNZ et aE. 1980) to the approximate time when the homozygous T a l genes result in intrauterine death (10 days of gestation). Another important consequence of this interaction is that R T I segregation is also affected due to the tight linkage of grc to R T I . The implications of the Tal-grc interaction are threefold. First, it provides a unique system for a variety of studies in developmental genetics. Second, it provides a model system for studying how genic interaction could maintain polymorphism at a locus or group of loci. For example, genes linked to the MHC and similar to grc exist in the mouse (the T / t complex) (KLEIN 1975) ; so they may exist in other species as well. Interactions between them and genes similar to T a l could select against animals homozygous f o r certain MHC alleles and favor the propagation of heterozygotes. Third, it may provide an animal model for the study of spontaneous abortions. Estimates of the incidence of spontaneous abortions among humans have been given variously as 15% (ROTH1963; WARBURTON and FRASER 1964), 2 6 2 9 % (ERHARDT 1963; BIERMANet al. 1965) or 43%
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(MILLERet al. 1980) of all pregnancies. Of these abortions, 60% had a chromo1975) that is usually a trisomy; such somal anomaly (BouB, BouB and LAZAR anomalies are more prevalent in abortions occurring at the earlier stages of gestation (SCHMIDT,NITOWSKY and DAR1976). Neural tube defects are also associated with spontaneous abortions (NISHIMURA 1970), and there is a high incidence of spontaneous abortions in families with children affected by neural tube defects (ALBERMAN, CREASY and POLANI1973; SCHACTER et al. 1979). Even though families with recurrent abortions often have one parent who is the 1977; carrier of a balanced translocation (BYRD,ASKEWand MCDONOUGH et al. 1977; NERIet al. 1980), chromosomal anomalies account for STENCHEVER only a small percentage of all recurrent abortions (CARR1971). Thus, there may be genes in the human which interact epistatically, similar to the interaction between Tal and grc, which can cause intrauterine death and be responsible for a substantial amount of the spontaneous abortions that occur in humans. LITERATURE CITED
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