/ . Embryol. exp. Morph. Vol. SI, pp. 217-225, 1979
Printed in Great Britain © Company of Biologists Limited 1979
217
Factors regulating the degree and extent of
experimental chimaerism in the mouse
By EWA T. MYSTKOWSKA, 1 WACtAW OZDZENSKF
AND ANNA N1EMIERKO 1
From the Laboratory of Experimental Embryology, Institute of
Obstetrics and Gynaecology, Medical Academy, Warsaw and the
Department of Embryology, Zoological Institute, University of Warsaw
SUMMARY
The degree of chimaerism of the coat, bone marrow and cornea in mouse chimaeras
obtained by aggregation of an albino inbred embryo of A strain with an AxCBA-T6T6
hybrid was investigated. Out of 2.1 individuals born, 18 were chimaeras. Statistically significant correlation has been demonstrated between chimaerism of the coat, bone marrow and
the cornea. The contribution of the two components appears to be affected by two factors:
first, variable participation of the two components in the embryonic ectoderm of the chimaeric
egg cylinder, and second, the rate of proliferation of the two cell lines.
A photometric method is described which permits the quantitative evaluation of coat
chimaerism. The method is sensitive and reliable.
INTRODUCTION
When two cleaving embryos are aggregated, the initial proportion of cells of
the two components in the chimaeric embryo is 1:1, and one might expect that
the resulting individual would also consist of a mixture of both components in
equal proportion. However, usually the opposite situation is true: in the
majority of chimaeric individuals cells derived from one embryo predominate
and some animals are not chimaeras at all. The aim of this study was to analyse
what factors may be responsible for this situation and also whether or not there
is a correlation between the degree of chimaerism of various tissues.
MATERIAL AND METHODS
The material consisted of 21 individuals obtained by aggregation of two
8- to 16-cell embryos, one from the A inbred strain and the other from a cross
between A females and CBA-T6T6 males. All eggs were spontaneously ovulated.
1
Authors' address: Laboratory of Experimental Embryology, Institute of Obstetrics and
Gynaecology, Medical Academy, 00-315 Warsaw, Karowa 2, Poland.
2
Authors' address: Department of Embryology, Zoological Institute, University of
Warsaw, 00-927 Warsaw, Poland.
218
E. T. MYSTKOWSKA AND OTHERS
Fig. 1. Schematic drawing of photometric device. (A) Measured object, (B) source
light, (C) lens, (D) sliding selenium photocell, (E) microammeter, (F) focusing
screen.
The procedure of combining the eggs and culturing them to the blastccyst stage
has been described before (Mystkowska & Tarkowski, 1970). The recipients
were Swiss albino females mated with Swiss albino vasectomized males. Transplantation of chimaeric blastocysts was performed in the morning (9.00-12.00)
of the fourth day of pseudopregnancy (day of vaginal plug = first day). The
recipients were killed on the 20th day of pregnancy and the embryos were
removed by Caesarian section and placed with foster mothers that had littered
2-4 days earlier.
Nine chimaeras were killed on the 20th day after birth and the remaining ones
after attaining sexual maturity.
The skins were taken off and stretched on a piece of styrofoam to make them
flat. The degree of chimaerism of the coat was photometrically determined on
the basis of intensity of the reflected light. The measurement was done in a dark
room by the device shown in Fig. 1. The measured object was lighted up by two
frosted bulbs (75 W) fed by an equalizer. The distance between the lens and
measured object was adjusted for measuring an area of 2 cm in diameter. Nine
measurements were taken of each large skin (from adult animals) and four of
the small ones (from young animals). The arrangement of the measured areas
on large and small skins is illustrated in Fig. 2A and B. The mean value of
these measurements (nine of the big skin and four of the small one) characterizes
Degree and extent of chimaerism in the mouse
219
Fig. 2. The arrangement of the measured areas on large (A) and on small (B) skins.
Table 1. Interdependence between current intensity and
blackness of measured field
Relation of white area
to black area in the
measured field
Value of the current
intensity
4/0
3/1
2/2
1/3
0/4
75-4
57-1
39-7
22-3
5-1
(M)*
Percentage contribution
of white area in the
measured fieldf
100
740
49-2
24-5
0
* Mean value of 10 measurements.
t The percentage contribution of the white area was calculated from the formula:
(C-B)x 100
A-B
'
where A is measurement of white field; B, measurement of black field; C, measurement of
the investigated field.
the amount of light reflected by the skin. The measurement was performed by
using a selenium photocell in which the changes of current intensity are directly
proportional to the changes in the degree of blackness of the examined area
(see Table 1).
The intensity of reflected light may be changed by different arrangements of
220
E. T. MYSTKOWSKA AND OTHERS
Table 2. The measurements of reflected light
Mean value of
reflected light
Skin
(M)
±S.D.
Albino pattern
1
2
3
4
5
6
7
8
9
10
84-8
57-8
56-2
53-4
46-8
40-3
35-8
30-4
291
280
25-4
1-1
0-8
0-8
11
0-7
1-4
0-8
1-6
0-7
0-8
0-9
11
240
0-6
12
13
14
15
16
17
18*
Agouti patternf
22-1
20-9
16-4
15-2
12-3
10-6
9-9
13-8
10
1-2
0-7
1-6
1-8
0-9
0-5
0-8
* Used as anagouti pattern.
f Not used.
the skins, so the arrangement of the skins in each series of measurements was
the same but was changed in successive series (the head side directed to the left
or right). Measurements were repeated ten times and mean value was accepted
(Table 2). The value of the standard deviation shows that the results are repeatable. As points of reference two skins of albino and agouti individuals
were measured beside the chimaera skins. As agouti skin was lighter than the
darkest chimaera skin, the latter was used as an agouti pattern.
The amount of albino in each skin was calculated from the formula:
A /o
(z-jQxlQQ
x-y
'
where A % is percentage of albino, z, value for chimaera skin, x, value for
albino skin, y, value for agouti skin.
The method presented here does not reveal individual hairs of the opposite
colour, so it cannot be used for estimating chimaerism when one component is
vestigial. In such a case chimaerism can be detected by sight. But unlike the
subjective rough estimation, the photometric method enables one to range
skins objectively according to the amount of one component in the coat, and to
evaluate quantitatively the contribution of the two components to the fur.
221
Degree and extent of chimaerism in the mouse
EMB
5r
222
E. T. MYSTKOWSKA AND OTHERS
Table 3. Contribution of albino and hybrid components in coat,
bone marrow and cornea
No. of chimaera
Contribution of
albino component
in coat
(%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
64
62
58
49
41
35
27
26
24
21
19
16
15
9
7
3
1
0
% of metaphase plates
without marker in
bone marrow
(total no. scored)
68 (50)
71 (21)
65 (20)
-(0)
74 (50)
57 (63)
38 (50)
25 (20)
27 (52)
60 (50)
38 (50)
22 (50)
19(31)
15 (20)
15(20)
38 (50)
4(26)
1 (100)
% of metaphase plates
without marker
in cornea
(total no. scored)
59 (74)
-(0)
— (0)
-(0)
43 (61)
17 (21)
31 (65)
~(0)
9(46)
3(67)
29 (44)
2(43)
8(38)
-(0)
-(0)
16(51)
-(0)
9(22)
Karyological preparations were made from the bone marrow of all chimaeras
according to the method of Ford (1966) and from the cornea (only sexually
mature individuals) according to the method of Fredga (1964). The number of
mitotic plates with and without T6 marker chromosome was determined.
The number of hybrid cells was compared with that of genetically homozygous
cells in the whole material by the chi-square test, separately for the bone marrow
and cornea. The correlation between chimaerism of coat, bone marrow and
cornea was calculated by the product moment correlation.
RESULTS
Out of 21 individuals, 18 showed overall chimaerism. These were ranged
according to the decreasing amount of the albino component in the coat (Fig. 3).
The remaining three animals (one albino and two agouti) most probably were
not chimaeras (examined tissues contained only one component) and were not
taken into account in further considerations. In the majority of chimaeras the
hybrid component prevailed in all three tissues. The preponderance of the
albino component (over 50%) in the coat, bone marrow and cornea was
recorded only in three, six and one chimaeras respectively (the numbers of
animals in which these tissues were examined were 18, 17 and 11). A statistically
Degree and extent of chimaerism in the mouse
223
significant (at a 0005 level) preponderance of hybrid cells over the genetically
homozygous ones was found in the bone marrow and the cornea. Chi-square
for bone marrow was 43-82 > 7-88 and for the cornea 146-32 > 7-88. A
correlation was recorded between the chimaerism of the hair, the bone marrow
and the cornea. The coefficient of correlation of product moment is:
(a) chimaerism of the coat and the bone marrow p = 0-853, N = 17 (correlation significant at 0-01 level)
(b) chimaerism of the hair and the cornea p = 0-788, N = 11 (correlation
significant at 0-01 level)
(c) chimaerism of the bone marrow and the cornea p = 0-636, N = 11
(correlation significant at 0-05 level).
DISCUSSION
Tn the overwhelming majority of the chimaeras investigated in this study, the
contribution of the two components to the three tissues examined deviated
from the initial proportion of 1:1. Falconer & Avery (1978) consider that this
phenomenon is due to two successive segregations of cell material which take
place during the early stages of development. Initially this material is divided
between trophoblast and inner cell mass (ICM); the next segregation takes
place when the ICM is divided to form embryonic ectoderm and embryonic
endoderm. It is known that blastomeres of the two aggregated embryos do not
mix together during the formation of the blastocyst, i.e. not before these two
processes begin (Garner & McLaren, 1974). The variable relation between the
axis of a chimaeric blastocyst and the plane separating the two aggregated
embryos may therefore result in the ICM being composed of cells of the two
types in various proportions, or perhaps, in extreme situations, of one type only.
Each proportion of the two components in an individual formed in this way
should be equally probable. Falconer & Avery (1978) quote several bodies of
data in support of this conclusion.
However, if each proportion of the two components is equally likely to occur,
the overall proportion of the two components in a group of chimaeras would
be close to 1:1. In our material the proportion of the two components deviated
markedly from 1:1 and in all the tissues examined we observed a preponderance
of one component. A similar preponderance of one component over the other
is reported by Mintz & Palm (1969) and by Mullen & Whitten (1971). We believe
that this phenomenon arises because of different rates of proliferation of the
cells of the two components. In our material the hybrid cells proliferated faster
than the homozygous ones. A faster rate of proliferation of hybrid cells as
compared to homozygous ones was reported by Krzanowska (1967). Falconer &
Avery take into account the possible role of this process but in the material
described by them it is not as clear, probably because different strains of mice
were used.
224
E. T. MYSTKOWSKA AND OTHERS
If neither component shows any predominance in rate of proliferation, the
composition of the chimaera will be the result of accidental division of the
initial material between the embryonic ectoderm and the other elements that
do not take part in the formation of the embryo. In such a case, a flat distribution
will be observed. But if one of the components has a faster rate of proliferation,
the resultant individual will be composed of relatively more cells from faster
proliferating line than were initially present in its embryonic ectoderm. The
distribution observed in this case will be logarithmic.
These factors operate during organogenesis. However it is possible that the
situation existing after the completion of organogenesis is not final. In some
tissues, changes in the proportion of the components may take place later; for
example Mintz described elimination of one line of cells from the spermatogonia
of chimaeric male mice (Mintz, 1968) and from the red cell population (Mintz &
Palm, 1969).
Our observations show that there is a correlation between the chimaerism of
the coat, bone marrow and cornea. Similarities in the quantitative composition
of various tissues in chimaeric mice were also observed by Wegmann & Gilman
(1970), Gornish, Webster & Wegmann (1972) and Kelly (1975). This phenomenon can be explained on the assumption that, in contrast to the formation
of a blastocyst, formation of the embryonic part of the egg-cylinder and subsequently of the embryo is accompanied by thorough mixing of cells.
We have described a new photometric method which allows for quantitative
estimation of the chimaerism of skins. We hope that this method, which is
repeatable, sensitive and reliable, may be useful not only for chimaera studies
but also for other investigations into coat and skin pigmentation, e.g. in
taxonomy or ecology.
We are grateful to Professor Andrzej K. Tarkowski for his help and valuable criticism
during the course of the work.
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{Received 21 November 1978)
15-3