/. Embryol. exp. Morph. 73, 193-205, 1983
193
Printed in Great Britain © The Company of Biologists Limited 1983
Somatic and germline mosaicism in
interspecific chimaeras between Mus musculus and
Mus caroli
By J. ROSSANT* 1 AND V. M. CHAPMAN 2
From the Department of Biological Sciences, Brock University, St. Catharines
and the Department of Molecular Biology, Roswell Park Memorial Institute,
Buffalo
SUMMARY
Detailed analysis of mosaicism in interspecific chimaeras between Mus musculus and Mus
caroli revealed that cells of the two species could coexist and interact normally in all tissues
studied. No selection occurred against M. caroli cells during gestation of chimaeras in the M.
musculus uterus, but some tissue-specific differential growth of M. musculus and M. caroli
cells occurred during postnatal development. Similar effects have, however, been reported in
interstrain M. musculus chimaeras. The similarity between inter- and intraspecific chimaeric
growth patterns supports the use of this interspecific system as a model for analysing cell
lineage relationships during development.
INTRODUCTION
There is a greater degree of molecular, biochemical and cellular divergence
between species than within a species. Experimental use of such species differences to follow cells in a clonal analysis of cell lineages has been attempted
using interspecific chimaeric combinations. The utility of these interspecific
combinations depends upon the compatibility of their growth patterns and, in the
case of mammals, the ability of cells of a foreign species to thrive in the uterine
environment of the host mother.
Chimaeras between the chick and the quail have been widely used to follow
cell lineages in birds, utilizing nuclear differences between the two species as an
in situ cell marker (Le Douarin, 1980). Early attempts to use antigenic differences between the rat and the mouse to develop an interspecific cell marker
system in mammals achieved more limited success. In general, the rat-cell
contribution could be detected in midgestation but was not readily detectable by
1
Author's address: Dept. of Biological Sciences, Brock University, St. Catharines, Ontario,
Canada.
2
Author's address: Dept. of Molecular Biology, Roswell Park Memorial Institute, Buffalo,
N.Y., U.S.A.
* To whom reprint requests should be sent.
194
J. ROSSANT AND V. M. CHAPMAN
the time of birth (Gardner & Johnson, 1973; 1975). More recently, viable interspecific chimaeras of mammals have been produced between Mus musculus and
Mus caroli by blastocyst injection (Rossant & Frels, 1980) and embryo aggregation techniques (Rossant, Mauro & Croy, 1982a). These chimaeras can be exploited for clonal analysis of cell lineages by using an in situ marker system based
upon repetitive DNA differences between the two species (Siracusa et al., 1982;
Rossant, Vijh, Siracusa & Chapman, 19826). This marker system shows many
properties of an ideal cell marker (McLaren, 1976).
M. caroli and M. musculus are much more closely related species than the rat
and the mouse and a preliminary report on coat colour mosaicism in adult
chimaeras and internal tissue mosaicism in preterm foetuses (Rossant & Frels,
1980) suggested that the M. musculus uterine environment did not exert any
selective effect against M. caroli cells in the chimaeras. However, since it is
essential to establish the validity of the interspecific chimaera system as a model
for normal embryogenesis, we present here a more detailed analysis of somatic
and germ-line mosaicism in perinatal and adult interspecific chimaeras which
shows that the patterns of mosaicism were essentially similar to those observed
in intraspecific chimaeras.
MATERIALS AND METHODS
Interspecific chimaeras between M. caroli and M. musculus were produced by
blastocyst injection (Rossant & Frels, 1980) or embryo aggregation (Rossant et
al., 1982a), as described previously. Some chimaeras were identified by the
presence of eye pigmentation at birth and were killed and dissected into various
tissues for quantitative GPI analysis (Peterson, Frair & Wong, 1978). Adult
chimaeras were identified by coat colour mosaicism, since M. caroli is agouti and
the Ha/ICR strain of M. musculus used is albino, and by double-banded
electrophoretic phenotype for isozymes of glucose phosphate isomerase (GPI)
in the blood. Ha/ICR mice were homozygous for the b allele at the Gpi-1 locus,
while M. caroli produced only one GPI isozyme indistinguishable from the A
isozyme of M. musculus. Adult female chimaeras were continuously paired with
M. musculus or M. caroli partners, and any litters were recorded. Seven female
chimaeras were artificially inseminated with M. caroli sperm as described
elsewhere (West et al., 1977). Twenty-two adult male chimaeras (aged 6 months
to 1 year) were killed and various organs were dissected and prepared for quantitative GPI analysis.
Any hybrid offspring of test matings could be recognized by coat colour when
chimaeras were mated to M. musculus and by hybrid GPI phenotype in any
cross. Male hybrids were test mated to M. musculus females. When the hybrids
were sacrificed or died, histological sections of gonads were prepared and stained
with haematoxylin and eosin.
195
Mosaicism in Mus chimaeras
RESULTS
Somatic tissue mosaicism in neonatal chimaeras
Out of 88 live births to date, 68 (77 %) have proved to be interspecific
chimaeras. The results of GPI analysis of five foetuses delivered just prior to
term have been previously reported (Rossant & Frels, 1980) and, in Table 1,
these data are presented along with data from GPI analysis of five additional
neonatal chimaeras. The mean contribution from M. caroli cells was 42 %. This
is similar to the mean M. caroli contribution of 48 % found in five chimaeras
analysed at 9-5 days p.c. There was some variation in mosaicism between different organs of a given chimaera, but every chimaera showed mosaic GPI
phenotype in all tissues analysed. Despite the variation observed, the correlation
coefficients between the M. caroli contribution to a given tissue and to the
chimaera as a whole were all statistically significant (P< 0-05). Examination of
the mean M. caroli contribution to each organ or tissue analysed did not reveal
any tissue in which there was a marked predominance of one parental type or the
other. The mean M. caroli contributions to individual tissues or organs did not
differ significantly from the overall mean contribution of 42 %. The absence of
any tissue-specific colonization or selective growth was confirmed by determining whether the M. caroli contribution to a given tissue was higher or lower than
the mean M. caroli contribution to the chimaera. If the contributions of the two
parental types were not subject to tissue-specific selection pressures, one would
Table 1. Mosaicism in neonatal chimaeras as revealed by GPI analysis
%M. . caroli GPI
leg
Chimaera No.
gut
liver
lung
heart
24*
25*
26*
27*
28*
29*
1
2
3
4
5
16
25
52
38
87
11
78
60
27
81
31
17
10
25
23
81
48
17
34
26
39
52
36
75
17
88
55
33
71
51
65
27
33
54
37
55
64
31
45
33
58
4
15
67
58
29
33
93
7
32
89
69
33
38 ±2
49 ±7
43 ±7
36 ± 6
46 ±8
49 ±7
0-63
0-84
0-83
0-84
0-88
0-81
Mean ±S.E.
Correlation coefficient
with chimaera mean
6
*From Rossant & Frels, 1980.
t Hybrid GPI band also observed in all samples.
musclef
4
83
57
47
36
56
carcass
21
73
45
38
11
65
Mean
±S.E.
19 ±5
71 ±9
57 ±5
25 ±7
25 ±4
46 ±6
54 ±6
31 ±4
72 ±5
34 ±9
30 ±4
42 ±6
196
J. ROSSANT AND V. M. CHAPMAN
Table 2. Mosaicism in adult chimaeras as revealed by GPI analysis
% M. caroli GPI
Chimaera No.
2
3
4
5
6
9
10
11
12
15
16
17
25
13'
2'
1'
4'
3'
11'
8'
12'
10'
leg
Mean
Blood Lung Brain Kidney Liver musclet Heart Spleen ±S.E.
0
19
34
30
12
63
—
38
36
50
38
3
45
49
37
71
22
28
23
8
21
23
0
0
7
10
17
45
41
42
30
37
33
7
50
12
14
48
—
5
19
41
20
48
0
7
43
18
43
33
44
27
60
42
0
0
5
35
23
12
33
5
17
12
21
10
32
14
19
27
21
9
6
1
13
2
0
0
2
7
3
45
38
4
5
50
2
5
32
21
18
20
51
5
18
3
16
17
6
6
20
10
12
64
70
71
68
14
69
32
75
70
27
21
80
52
42
51
57
60
0-82
0-71
0-46
0-76
0-71
l±0.0
5±3
18 ±7
18 ±5
18 ±6
43 ±8
45 ±6
31 ±10
36 ±10
34 ±7
42
34 ±9
18
12 ±4
43
46 ±6
42 37 ±7
33
59
24 25 ±3
23
36
35
48 37 ±6
29
39
38 39 ±8
28
13 20 ±5
18
29
13
30 23 ±4
29
4 25 ±8
39
50
43
41 34 ±7
60
45
50
29 34 ±7
31 ± 4 25 ± 4 36 ±3 14 ± 2 16 ±4 44±5 31 ± 4 30 ± 5 28 ±3
Mean
Correlation
coefficient with
chimaera mean 0-69
0-66
0-79
t Hybrid GPI band also observed in all samples.
predict an equal probability of the M. caroli contribution to a given tissue being
above or below the chimaera mean. Table 3A shows that the results supported
this prediction; no statistically significant skewing from a 50:50 ratio was observed.
Somatic tissue mosaicism in adult chimaeras
Table 2 summarizes the results of GPI analysis of 22 adult chimaeras. The
mean contribution from M. caroli cells was 28 %, compared with 42 % in neonatal chimaeras. Correlation coefficients between M. caroli contributions to
individual tissues and the chimaera mean were not as high as those calculated for
neonatal chimaeras, but they were all statistically significant at P<0-01.
Examination of the mean M. caroli contributions to particular organs or tissues
revealed that these were not always close to the overall mean M. caroli
Mosaicism in Mus chimaeras
197
contribution of 28 %. In particular, M. caroli contributions to liver and kidney
were consistently low and contributions to muscle were consistently high. An
analysis of the relative proportion of chimaeras with M. caroli contributions
greater than the chimaera mean for specific tissues revealed that the M. caroli
contribution to liver and kidney was significantly reduced (Table 3B). By
contrast, the M. caroli contribution to skeletal muscle was significantly greater
than the average M. caroli contribution to all tissues (Table 3B).
Table 3. Tissue-specific variation in mosaicism in neonatal and adult chimaeras
A. Neonatal chimaeras
lung
heart
muscle
gut
liver
No. of M. caroli
contributions above
chimaera mean
No. of M. caroli
contributions below
chimaera mean
5
7
7
4
7
8
6
4
4
7
4
3
X2 of difference from
50:50 ratio*
0-0
0-36
0-36
0-36
0-36
1-45
B. Adult chimaeras
kidney liver muscle heart
blood
lung
brain
13
7
15
2
3
17
4
4
8
14
7
20
19
5
5
5
0-76
1-71
2-23
13-lt
10-231 5-50$
0-0
0-0
No. of M. caroli
contributions above
chimaera mean
No. of M. caroli
contributions below
chimaera mean
f of difference from
50:50 ratio*
carcass
spleen
* Yates correction used,
t significant at P<0-01
X significant at P < 0-02
Coat colour and eye pigment mosaicism was also evident in all of the adult
chimaeras and the patterns of mosaicism were very similar to those observed in
M. musculus chimaeras (Fig. 1).
Germline mosaicism in adult chimaeras
Germline mosaicism was assessed by test breeding female chimaeras, which
were likely to be XX<->XX, rather than males, which might be XX**XY sex
chimaeras producing sperm of only the XY genotype (McLaren, 1976). A majority of chimaeras born was male (44/68), suggesting that most XX<-»XY
198
J. ROSSANT AND V. M. CHAPMAN
Fig. 1. Adult interspecific chimaeras between M. musculus and M. caroli, aged 9
months.
chimaeras were, indeed, phenotypically male. One hermaphrodite, with normal
ovary and uterus on one side and aspermic testis and vas deferens on the other,
was found.
Eight chimaeric females were mated successfully with M. musculus males and
34 litters, containing 206 offspring, were produced. Three of the females
produced interspecific hybrids among their offspring (Fig. 2A). The breeding
records of these chimaeras are shown in Table 4.
Fifteen hybrids were produced, which represented 11-6 % of the offspring of
these three females and 7-3 % of all offspring produced. The remaining five
females may also have been capable of producing hybrids but they produced
fewer litters.
No chimaeric female mated successfully with a M. caroli male. In an attempt
to produce this reciprocal cross, seven chimaeras were artificially inseminated
with M. caroli sperm. None of the females produced offspring. Two females were
sacrificed at term and one contained 2 or 3 resorbed embryos. The remaining five
were mated with M. musculus males and four produced M. musculus offspring.
Hybrid breeding
All 15 hybrids were phenotypically and chromosomally male and showed a
hybrid GPI band in electrophoresis (Fig. 2B). The mice were much larger than
either parental species and had a tendency to become obese with increased age.
Several were mated with M. musculus females and copulation and vaginal plugs
were observed. However, no females became pregnant and no sperm were
detected in vaginal smears or in epididymal squashes. All testes were small and
1
2
3
4
5
6
Litter
No.
1
2
2
1
1
2
2
1
3
1
1
0
Ave. litter size == 3-6
% hybrids = 36-4
0
1
2
1
0
1
0
0
0
0
0
0
Female No. 7
No. M. musculus
No. hybrids
female
male
male
female
1
2
3
4
5
6
7
8
Litter
No.
5
6
2
4
3
5
5
7
0
1
1
0
0
0
0
0
Ave. '.litter size = 9-9
%> hybrids == 2-5
7
5
0
3
8
5
6
4
0
0
0
0
0
0
0
0
Female No. 18
No. hybrids
No. M. musculus
female
female
male
male
0
3
2
2
1
3
1
1
1
2
1
4
2
0
2
2
2
4
0
0
0
1
1
0
0
0
2
0
0
1
0
0
0
0
0
0
0
0
0
0
Female No. 22
No. hybrids
No. M. musculus
male
male
female
female
Ave. litter size = 3-7
% hybrids == 13-5
1
2
3
4
5
6
7
8
9
10
Litter
No.
Table 4. Breeding records ofM. musculus «-» M. caroli chimeras producing hybrid offspring
Co
§
C5
in
S
a55
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200
J. ROSSANT AND V. M. CHAPMAN
X..
2A
if.
*.
;
s **'
Fig. 2
Mosaicism
in Mus chimaeras
201
contained no signs of spermatogenesis (Fig. 2C). The testis tubules were lined
with a single layer of cells which were presumed to be Sertoli cells; no meiotic
figures were observed.
DISCUSSION
The utility of our newly developed in situ interspecific cell marker system
(Siracusa et al., 1982; Rossant et al., 19826) for following cell lineages in mouse
development depends on demonstrating that M. caroli and M. musculus cells can
successfully coexist and interact normally in chimaeric mice. Several pieces of
evidence suggest this. Interspecific M. caroli *+ M. musculus chimaeras resembled intraspecific chimaeras morphologically at all stages of development; no
abnormalities of morphogenesis occurred. Also, no selection occurred against
M. caroli cells in chimaeras during uterine development, despite the fact that
intact M. caroli embryos die in the M. musculus uterus around 10-15 days of
development (Frels, Rossant & Chapman, 1980). We have previously shown
that the presence of M. musculus trophoblast can protect M. caroli foetal cells
from the M. musculus uterine environment (Rossant et al., 1982a) and the
analysis of mosaicism presented here suggests that this protection is complete.
Neonatal chimaeras showed a mean M. caroli contribution of 42 %, which makes
the M. caroli <-» M. musculus chimaeric combination a more balanced one than
many M. musculus strain combinations (Mullen & Whitten, 1971). A similar
mean M. caroli contribution was observed in chimaeras at 9-5 days of gestation,
indicating that there was no overall selection against M. caroli embryo cells
during pregnancy. There was also no evidence for any tissue-specific selection
acting against M. caroli cells. Mosaicism in individual tissues (Table 1 & 3A)
correlated well with overall mosaicism in each mouse.
There is also some evidence that M. musculus and M. caroli cells behave
normally within a given tissue and do not sort according to species type. The
patterns of coat and eye pigmentation observed were similar to those observed
in intraspecific chimaeras (Mintz, 1967; West & McLaren, 1976). Also, hybrid
GPI isozyme was observed in all chimaeric muscle samples, showing that
myotubes could form by fusion of cells of the two species. Preliminary results
with the new cell marker (Rossant et al., 19826) revealed fine-grained mosaicism
in liver and brain samples as observed with other markers in intraspecific
chimaeras (West, 1976; Dewey, Gervais & Mintz, 1976; Oster-Granite & Gearhart, 1981).
Fig. 2. (A) Litter of two M. musculus <-» M. caroli interspecific hybrids and one M.
musculus produced by mating an interspecific chimaeric female with M. musculus.
(B) GPI phenotype of hybrid offspring. Lane 1 = BB control; Lane 2 = hybrid AB
GPI; Lane 3 = A A control. (C) Section of testis of interspecific hybrid male showing
absence of spermatogenesis. Grid bar = 50jum.
202
J. ROSSANT AND V. M. CHAPMAN
Thus, the evidence accumulated so far suggests that the growth patterns of M.
musculus and M. caroli cells are sufficiently compatible to produce chimaeras
that can serve as models for normal embryogenesis. However, patterns of
mosaicism in adult chimaeras, aged six months or older, were harder to reconcile
with this contention. The mean contribution by M. caroli cells fell from 42 % in
neonatal chimaeras to 28 % in adult chimaeras, suggesting that M. musculus cells
tended to overgrow the M. caroli cells. This is perhaps not surprising since adult
M. musculus are much larger than M. caroli (West, Frels & Chapman, 1978) and
chimaeras were closer to M. musculus in size. Overgrowth by M. musculus cells
did not occur in all tissues. Skeletal muscle, for example, showed a similar mean
M. caroli contribution to that found in neonatal chimaeras, while liver and
kidney showed very low contributions from M. caroli cells. Similar differential
growth of cells of the two component genotypes has, however, been reported to
occur in tissues of chimaeras made between different inbred strains of M.
musculus. The particular tissues affected seem to depend on the particular strain
combination used. Overgrowth by cells of one parental genotype has been reported to occur in blood cells (Mintz & Palm, 1969; Warner, Mclvor & Stephens,
1977; West, 1977), germ cells (Mintz, 1968), pigment cells (Gearhart & OsterGranite, 1981) and muscle (Peterson, Frair, Rayburn & Gross, 1979), in different chimaeric combinations. Thus, tissue-specific differential growth may be
a complicating factor in quantitative analysis of clonal precursors in interspecific
chimaeric combinations, but this problem is likely to be shared by any
intraspecific marker system unless congenic strains can be used.
M. musculus and M. caroli cells can coexist in the germ line as well as in
somatic tissues, as shown by the production of mixed litters of M. musculus and
interspecific hybrid offspring when female chimaeras were mated to M.
musculus. A much higher rate of hybrid production was achieved by mating
chimaeric females than ever achieved by artificial insemination (West et al.,
1911,1978), suggesting that hybrid embryos survive better in chimaeric females
than in M. musculus females. This is compatible with the hypothesis that part of
the reason for poor survival of hybrid embryos in M. musculus females is immunological rejection of the foetus by the mother (Frels et al., 1980). Evidence
that M. musculus females can mount an immune response against M. caroli
embryos has been found (Croy, Rossant & Clark, 1982). Chimaeric females
should be tolerant to both species' antigens (Matsunaga, Simpson & Meo, 1980)
and should not reject the hybrid foetuses. However, it must be remembered that
the interspecific hybrids reported here were the result of the reciprocal cross
from those produced by artificial insemination- mouse hinnies, not mouse mules
(West et al., 1978). It is possible, therefore, that fertilization and development
are more successful when M. musculus is the sperm donor rather than M. caroli.
This could not be tested directly by breeding the same chimaeric females to M.
caroli males since no successful pregnancies resulted from such pairings. However, the results of a limited series of artificial inseminations of female chimaeras
Mosaicism in Mus chimaeras
203
with M. caroli sperm were compatible with the hypothesis; no successful pregnancies ensued and some resorbing embryos were observed. Four of these
chimaeras later produced M. musculus offspring, showing that they should have
been capable of producing hybrid progeny. Further investigation of possible
differences between the reciprocal crosses will be carried out using in vitro
fertilization techniques.
All 15 hybrids produced from mating female chimaeras were phenotypically
and chromosomally male. This constitutes a highly significant skewing of the
normal sex ratio. We do not know the reason for this peculiar sex ratio which
seems to break Haldane's rule which states that when one sex is absent, rare or
sterile in the hybrid offspring of two species, that sex is the heterogametic sex
(Haldane, 1922). In the reciprocal cross (West etal., 1977,1978) a predominance
of females was reported. All hybrids produced here were sterile, as were the
females produced by artificial insemination. Karyotypically, both species have
the same number of chromosomes (Marshall, 1977) but there are substantial
differences in repetitive DNA between M. caroli and M. musculus (Sutton &
McCallum, 1972; Rice & Straus, 1973; Siracusa etal., 1982). These differences
may have prevented normal meiotic pairing of chromosomes followed by
degeneration of germ cells similar to that reported for the mule (Taylor & Short,
1973; Chandley et al., 1974). Although it may not be possible to use these hybrids
to introduce new genetic polymorphisms directly into the M. musculus gene pool
(West et al., 1978), the ability to produce such hybrids in relatively large numbers
will be useful for many other studies on the interaction between regulatory and
structural genes of the two species in the same cell.
CONCLUSIONS
Detailed analysis of mosaicism in interspecific M. musculus <-»M. caroli
chimaeras has shown that there are no properties of these interspecific chimaeras
that are not shared by intraspecific chimaeras, making our new cell marker
system as valid as any marker system developed between different strains of one
mouse species. The marker system should be very useful for analysing cell
lineage relationships, particularly during embryonic development, but should be
used with caution in any numerological studies. However, the same caveat
should be applied to any in situ cell marker system, even if developed within one
species, given the evidence for tissue-specific differential growth in a wide range
of chimaeric combinations.
This work was supported by the Canadian Natural Sciences and Engineering Research
Council (J.R.) and the General Medical Sciences of N.I.H. (V.M.C.).
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(Accepted 25 July 1982)
EMB 73