/. Embryol. exp. Morph. Vol. 47, pp. 169-177, 1978
Printed in Great Britain © Company of Biologists Limited 1978
169
Allophenic mice in cleft-palate investigations
By K . M I L L E R
From the Department
SULIK
1
A N D R. L. A T N I P
of Anatomy, University of Tennessee
for the Health Sciences
Center
SUMMARY
T l W h (Rb (5; 19)lWh), an albino strain of mice not previously used in the investigation
of cleft palate (CP), is homozygous for a (5; 19) Robertsonian translocation which provides
conspicuous chromosomal markers. Preliminary studies in this laboratory revealed that CP
is induced in all the offspring of TlWh females treated with 2-5 mg of cortisone acetate on
days 11-14 of pregnancy and that palatal closure is accomplished at a later morphological
and chronological age than in C57BL/6J strain mice. The latter strain had a CP frequency
of 42 % under similar experimental conditions.
Eight- to 16-cell embryos were aggregated in the following combinations: TlWh <-» T l Wh,
C57BL/6J «-> C57BL/6Jand T l W h <-> C57BL/6J. The resulting allophenic blastocysts were
transferred to pseudopregnant C57BL/6J recipients which were given the above cortisone
treatment and killed on the 18th day of pregnancy. CP was found in all of the 48 T l W h <->•
T l W h fetuses, 4 (20 %) of 20 C57BL/6J <-» C57BL/6J fetuses and in 17 (26-2 %) of 65 TlWh
<-> C57BL/6J fetuses. Analysis of chromosome preparations from the palates of the TlWh
C57BL/6J fetuses demonstrated that there is a positive correlation between the presence
of CP and the percentage of susceptible cells in the palate (P < 002). Other data suggest a
possible growth advantage in favor of C57BL/6J cells in the palates of TlWh <-> C57BL/6J
allophenic mice.
INTRODUCTION
Production of allophenic mice permits the combination within one individual
of cells from mouse strains of high and low susceptibility to cortisone-induced
CP. Analyses of these individuals demonstrate how the coexistence of such cells
in varying proportions affects clefting.
The percentage of cells from each component strain of allophenic animals
can be determined only if some cell marker is present. T l W h , an albino mouse
strain homozygous for a 5; 19 Robertsonian translocation that provides
conspicuous chromosomal markers, exhibits cortisone-induced CP in all of
the offspring of females treated with 2-5 mg cortisone acetate on days 11 through
14 of pregnancy (Miller & Atnip, 1977). T l W h strain embryos were combined
with those of the C57BL/6J strain, which have a cortisone-induced CP fre­
quency of 42 % in this laboratory, a normal karotype and black pigmentation.
1
Author's address: Department of Anatomy, Georgetown University, 3900 Reservoir
Road, N.W., Washington, D'.C. 20007, U.S.A.
170
K. M. S U L I K A N D R. L. A T N I P
MATERIALS A N D
METHODS
C57BL/6J mice were obtained from the Jackson Memorial Laboratory.
Breeding pairs of T l W h mice were donated by Dr B. J. White. Lights were on
in the animal room from 6.00 a.m. to 6.00 p.m. daily. Temperature was kept
at 72-75 °F. Mice were maintained on a diet of Purina Mouse Chow and
water ad libitum.
T l W h and C57BL/6J female mice were placed with fertile males overnight
and checked for vaginal plugs the next morning (day 0). At 1.00-3.00 p.m. of
day 2 the pregnant females of both strains were killed by cervical dislocation.
Egg handling, fusion and transfer techniques used involved modifications of
techniques developed by Mintz (1971), Wegmann (1973, personal communi­
cation) and Dunn (1974, personal communication). C57BL/6J mice on day 2
of pseudopregnancy were used as recipients for T l W h <-> T l W h , C57BL/6J <-•
C57BL/6J, and T l W h ^ C57BL/6J embryos.
Doses of 2-5 mg cortisone (cortisone-acetate, Merck, Sharp and Dohme)
were injected into the musculature of the thigh of recipient females at 1.002.00 p.m. of gestational days 11 throught 14. Pregnant mice were killed on
gestational day 18 by cervical dislocation. Each uterus was removed and the
location and number of resorptions and fetuses noted. Each embryo was
examined under a dissecting microscope to determine sex and to look for
gross external malformations; the external genitalia were classified as male or
female by noting the distance between the anus and genital tubercle (Kalter,
1965). The body cavity was opened and the gonads observed as another means of
determining the sex of each fetus.
In those fetuses derived from T l W h <->C57BL/6J fusions, tissue from the
palates and a small piece of skin (0-5 cm ) from the dorsal midbody region were
removed and prepared for tissue culture. The heads, without the mandibles,
were placed in Bouin's fixative, preserving the eyes for photography.
Tissues were cultured for chromosomal analysis by a technique used in the
laboratory or D r R. L. Summitt for skin and amnion, which was based upon
modifications of the technique of Nadler & Gerbie (1970). Skin and palatal
tissues were minced and initially grown under a coverslip in Ham's F10 with
18% fetal calf serum in 1 % antibiotic antimycotic solution (Grand Island
Biological Co.). Cells were subcultured into a 250 ml Falcon plastic flask, then
harvested for chromosome preparations just prior to cellular confluency. The time
elapsing from the date of explantation to the date of harvest averaged 3 weeks.
Metaphase spreads, up to approximately 100-110 from each tissue which
were of good quality and appeared to have at least 37 chromosomes were noted
for the presence or absence of the marker chromosome and photographed. For
examination and counting, the negatives were projected by a photographic
enlarger onto white paper. Only those spreads with at least 37 chromosomes
and only those tissues from which at least ten acceptable spreads were found
were included in the data.
2
<J
A.
$
<
:
(?)*
<?
A
No. CP
$
CP
(%)
TlWh <-* TlWh
23
1
0
0
0
21
(1000)
26
12
1
1
9
2
C57B1/6J^C57B1/6J
1
6
(20-0)
TlWh<->C57Bl/6J
—
7
1
0
3
Solid black eyes
0
9
(200)
1
0
—
2
0
Solid white eyes
0
0
(1000)
—
1
5
10
4
Mixed eyes
0
22
(22-8)
27
Total
(26-2)
1
17
7
0
31
9
* (?) indicates ambiguous phenotypic sex, probably resulting from the presence of
CP, Cleft palate; CLP, cleft lip ;and palate ; Resorp., resorptions
Strain
No. of 1
mothers ( ? ) *
No. closed
palates
A
0
0
6*
0
0
(00)
(00)
CLP
(%)
No.
28
8
Resorp
(36-8)
(290)
(%)
Resorp.
0
0
0
0
1
0
0
1
—
—
0
(50)
—
—
0
(00)
0
—
—
(00)
0
24
(26-7)
(1-5)
both male and female component cells.
0
0
(?)
No . CLP
Table 1. Cortisone-induced cleft-palate frequency in allophenic mice
•to
3
172
K. M. SULIK AND R. L. ATNIP
RESULTS
As was found among the fetuses of naturally pregnant TlWh females all
48 live TlWh<-> TlWh fetuses which developed in a C57BL/6J uterine environment had cleft palates (CP) (Table 1). Twenty live fetuses from C57BL/6J
<-> C57BL/6J fusions included four with CP, i.e. 20 % CP, a figure less than
the control rate of 42% CP for C57BL/6J fetuses in their natural mothers.
Seventeen (26-2%) of 65 TlWh <->- C57BL/6J fetuses in cortisone-treated
recipients had CP. The presence of both pigmented and unpigmented cells in
the eyes demonstrated that cells from both strains were present in 42 of the 65
TlWh <-> C57BL/6J fetuses. Twenty fetuses from the TlWh • > C57BL/6J
fusion had solid black eyes and three had solid white eyes. Of the 42 fetuses
with mixed eyes, 22-8 % had CP, compared with 100 % for the three fetuses
with pure albino and 20 % for the 20 fetuses with pure black eyes.
The TlWh <-> C57BL/6J fetuses were broken down into smaller groups on
the basis of eye pigmentation by choosing seven 'standard' eyes with the percentage of pigmentation graded in approximately equal steps from pure albino
to pure black (Fig. 1). A photograph of each eye was compared with those of
the standards and each mouse assigned to one of the seven groups (Table 2).
In most instances both the right and left eye of each mouse could be assigned to
the same group. In a few cases one eye differed by one step from the other.
In these cases, the two were averaged and the mouse was assigned to the appropriate group. CP rates tended to be lower in those individuals that showed
fewer TlWh cells in their eyes: 3/3 with CP in those with entirely albino eyes;
4/9 with CP in group 2; 3/10 with CP in group 3; 2/8 with CP in group 4; 1/8
with CP in group 5; 1/7 with CP in group 6; and 4/20 with CP in group 7, the
group with solid black eyes.
Because albino cells may have been hard to see in eyes that contained predominantly C57BL/6J cells, chromosomal analysis of the allophenic tissues was
assumed to more accurately indicate actual component proportions. Cell
counts made using a Coulter counter on palatal tissue from 18-day TlWh and
C57BL/6J fetuses indicated similar growth rates in the two strains during the time
elapsing prior to chromosomal preparation. Therefore, the percentage of
mitotic figures from each component strain in cultures of TlWh <-> C57BL/6J
tissues was assumed to represent actual cellular proportions. Cells from the
palatal shelves and/or skin from 44 TlWh <-> C57BL/6J fetuses were cultured
and the chromosomes analyzed (Table 2). Preparations from the palate were
more successful than those from the skin. Two of the three mice with solid
white eyes were analyzed and found to have 100 % TlWh cells in their palatal
tissue. Fourteen of the 20 mice with solid black eyes were analyzed; only two
of the 14 contained the TlWh markers. One of these two mice had only one
spread of 42 chromosomes with one chromosome which looked like a 5; 19
translocation marker; the other had. a significant number (11-4 %) of cells with
markers.
Allophenic mice in cleft-palate investigations
173
Fig. 1. The seven 'standard' eyes used for the assignment of TlWh <-> C57BL/6J
fetuses to groups (Table 2).
Allophenic mice showing mixed cell populations in their eyes almost always
showed mixed cell populations also in cells cultured from palate and skin.
When there was a large proportion of TlWh cells in the eyes, there was usually
also a large proportion of TlWh cells in the palate and skin. However, the percentages of TlWh cells in the palate were in general considerably less than would
have been predicted from the appearance of the eyes, which suggests that TlWh
cells grow more slowly in the palate than in the eye. These results were tested
and confirmed by regression analysis. It was assumed that the eye groups
represent equally spaced percentages of TlWh cells (group 1 with 100 %, to
12
EMB 47
4
3
2
1
Eye pigmentation
groups
18
50
—
103
93
86
19
106
99
104
52
101
106
102
—
107
110
31
26
95
28
16
34
1
2
—
1
2
1
2
3
4
5
1
2
3
1
2
3
1
1
2
3
4
5
6
0
4 (44-0 %)
5
3 (300 %)
7
2 (250 %)
6
Closed
Cleft
Closed
Cleft
Closed
Cleft
Closed
Embryo no.
No.
cells
3 (1000 %)
No. of embryos in group,
(%withCP)
Cleft
Condition of
palate
A
63-6
63-6
—
26-3
—
—
11
22
—
95
—
10
101
—
—
—
—
—
44-2
38-6
21-7
55-9
—
23-4
91
161
154
34-7
71
6-3
00
100
17-8
—
—
—
—
—
—
—
55-4
68-8
51-6
—
190
—
—
65
16
91
—
21
%TlWh
cells
89-3
67-7
51-2
421
37-7
39-4
12 5
A
—
—
No.
cells
Skin cells analyzed
—
—
1000
1000
—
%TlWh
cells
Palatal cells analyzedj
Table 2. Comparison of degree of eye pigmentation with proportions ofTlWh and C57BL/6J cells in palate
and skin of fetuses with closed and cleft palates
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AUophenic mice in cleft-palate investigations
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176
K. M. SULIK AND R. L. ATNIP
group 7 with 0% TlWh cells). By these criteria it was found that the % TlWh
cells in the palate were significantly lower (P < 0-005) than predicted by the
degree of eye pigmentation. Also, of ten mice with mixed cell populations
from which both skin and palatal tissue was analyzed, only one had a smaller
proportion of TlWh cells in the skin than in the palate.
There are other points that should be noted as well. There is a positive correlation between the percentage of TlWh cells in the palate and the occurrence
of CP (probability of zero correlation < 0-02, TV = 42, Spearman's p = 0-376).
Among the seven mice from group 2, the two with CP had much higher percentages of TlWh cells in their palates than did the five with closed palates.
All 30 mice with closed palates had less than 56 % TlWh cells in their palatal
cell cultures, while the 12 with CP ranged from 100 % to 0 % TlWh cells.
DISCUSSION
Allophenic mice differ widely in the proportions of cells of the two genotypes
among individuals and from tissue to tissue in the same individual. This variability in C57BL/6J <->TlWh fetuses of cortisone-treated females was of value
in studying how the coexistence of susceptible and non-susceptible cell types
in varying proportions affected the incidence of CP.
A direct correlation was found between CP and the percentage of TlWh
cells found in palatal tissues of TlWh <->• C57BL/6J fetuses. That is, when the
percentage of TlWh cells was high, CP was more frequently present than when
very few susceptible cells were found in palatal tissues.
Initially, on the basis of eye pigmentation and the occurrence of CP, there
seemed to be some form of 'rescue' process (Mintz, Custer & Donnelly, 1971)
by the C57BL/6J cells. Although many of the allophenic mice had very high
estimated proportions of TlWh cells in their eyes, the CP frequencies among
them remained low (22-8 % CP in those with both cell types present in the
eye).
The common neural crest origin for the pigment cells and palatal shelf
mesenchyme(Weston, 1970; Johnston, Bhakdinaronk & Reid, 1974; LeLievre &
LeDourain, 1975) would lead one to believe there would be equal proportions of
each cell strain within both tissues. Chromosomal analyses indicated, however,
that the percentage of TlWh cells in the palates was in general consistently
less than in the skin and less than was the apparent proportion of TlWh cells
in the eyes. Such a difference might indicate a possible growth advantage in
favor of C57BL/6J cells in the palates of those mice with high proportions of
TlWh cells elsewhere. A slower palatal fusion in TlWh than in C57BL/6J
mice, possibly resulting from a slower growth rate in vivo by the TlWh palate
cells, was previously shown using a morphological rating system (Miller &
Atnip, 1977). However, in vitro growth analyses suggested similar cellular
growth rates in the two strains. Autoradiographic analyses are presently being
Allophenic mice in cleft-palate investigations
111
conducted to compare in vivo proliferation rates in palatal tissue of TlWh
and C57BL/6J fetuses.
With the data gained from the chromosomal analyses in mind, the idea of
a rescue phenomenon by the presence of a few C57BL/6J cells in predominantly
TlWh mice must be abandoned in favor of the idea of a possible growth
advantage for the C57BL/6J cells in palatal tissue which would account for
low CP frequencies in TlWh <-> C57BL/6J mice.
Acknowledgement is made to the Gerontology Research Center, NICHD, for use of
facilities provided under-its Guest Scientist Program. The authors wish to thank Dr Robert
L. Summitt for his generously given assistance and support. Special thanks is given to John
Karon who provided statistical advice and to Dr M. C. Johnston for his help in preparation
of the manuscript. This research was supported by United States Public Health Service Grant
CM 00202.
REFERENCES
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crest in oral and pharyngeal development. In Fourth Symposium on Oral Sensation and
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Office.
KALTER, H. (1965). Interplay of intrinsic and extrinsic factors. In Teratology: Principles and
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LELIEVRE, C. S. & LEDOUARIN, N. M. (1975). Mesenchymal derivatives of the neural crest:
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MILLER, K. K. & ATNIP, R. L. (1977). TlWh, a promising mouse strain with chromosomal
markers for cleft palate research. Teratol. 16, 41-46.
MINTZ, B. (1971). Allophenic mice of multiembryo origin. In Methods in Mammalian Embryology (ed. J. Daniel, Jr.) pp. 186-214. San Francisco: Freeman.
MINTZ, B., CUSTER, R. P. & DONNELLY, A. J. (1971). Genetic diseases and developmental
defects analyzed in allophenic mice. International Review of Experimental Pathology 10,
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NADLER, H. L. & GERBIE, A. G. (1970). Role of amniocentesis in the intra-uterine detection
of genetic disorders. New England J. Med. 282, 596-599.
WESTON, J. A. (1970). The migration and differentiation of neural crest cells. Adv. Morphog.
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{Received 1 February 1977, revised 20 April 1978)
JOHNSTON,