/. Embryol. exp. Morph., Vol. 17, 2, pp. 283-292, April 1967
Printed in Great Britain
283
Genotype and environment
in the determination of minor skeletal variants
and body weight in mice
BY W. L. HOWE 1 & P. A. PARSONS 1
From the Department of Genetics, University of Melbourne
Numerous minor skeletal variants have been described in the mouse (Griineberg, 1963), other small mammals (Berry & Searle, 1963) and in man (Comas,
1960; Brothwell, 1963; Griineberg, 1963). In genetically heterogeneous material
such as man it is very difficult to sort out the factors causing these variants.
However, in experimental animals such as the mouse the use of inbred strains
and crosses derived from them permits a more detailed understanding of these
factors, in particular the relative importance of heredity and environment.
Griineberg (1963) cites numerous references showing very great differences in
the percentage incidence of many minor variants in mice between inbred strains
and in some cases between hybrids. The conclusion is that much of this variation is genetic in origin. Even so, certain environmental factors have been shown
to be of importance, such as maternal age, parity, maternal weight and maternal
diet. Some of these environmental factors may be highly correlated, so that
large amounts of data may be needed to determine the causative factor. For
example, Kalter (1956) found a decrease in the incidence of median cleft palate,
induced by injecting primigravid pregnant mice with cortisone, with increasing
maternal age. However, from multiple regression analyses, he concluded that
maternal weight was the most important factor. Maternal age seemed to play
a role only because it is highly correlated with maternal weight (see also
Parsons, 1964).
There have been few attempts to look at the total variation between strains
or populations in an integrated way. Perhaps the best work has been done by
Berry (1963), who studied variation between populations of wild mice simultaneously for a number of skeletal variants, using a mean measure of divergence
or distinctiveness devised by C. A. B. Smith which is based on the percentage
incidence of all the variants in the populations to be compared.
In the present study, variation between three inbred strains of mice, C57BL,
BALB/c and C3H, and the hybrids between them, is examined for twenty-five
minor skeletal variants using C. A. B. Smith's method. In two of the inbred
1
Authors' address: School of Biological Sciences, La Trobe University, Victoria, Australia.
284
W. L. HOWE & P. A. PARSONS
strains, C57BL and BALB/c, an investigation of the effect of environment is
discussed, there being insufficient data for the C3H strain and the hybrids. The
environmental factors studied were parity, maternal age, litter size, age at death,
whether the mother was lactating while pregnant, and whether the mother was
pregnant while lactating. Sex differences were also considered.
METHOD
The skeletons were prepared by a modification (Searle, 1954 a) of Luther's
(1949) technique for the digestion of non-osseous tissue by the proteolytic
enzyme papain. Scoring was carried out at 60 days of age for all the hybrids
and for about one-half of the inbred strains. The 25 minor skeletal variants are
given in Table 1: 15 of these are variants of the skull, 8 are of the vertebral
column and 2 of the appendicular skeleton.
Table 1. The skeletal variants
Variant
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Reference
A. Variants of the skull
Lower turbinal—premaxilla fusion
Deol (1955)
Interfrontal present
Keeler (1933), Truslove (1952)
Parted frontals
Keeler (1930), Truslove (1952)
Frontal fontanelle
Deol & Truslove (1957)
Preoptic sutures present
Truslove (1954)
Metoptic roots abnormal
Truslove (1954)
Alae palatinae absent
Deol (1955)
Foramen sphenoidale medium present
Deol (1955)
Foramen ovale double
Deol (1955)
Foramen pterygoideum double
Berry (1963)
Foramen infra-ovale double
Berry (1963)
Processus pterygoideus
Deol (1955)
Foramen hypoglossi double
Deol (1955)
Mandibular foramen double
Berry (1963)
Accessory mental foramen
Deol (1955)
B. Variants of the vertebral column: C = cervical, Th = Thoracic and
S = Sacral vertebrae
CII-CVI. Foramina transversaria
Gruneberg (1950)
imperfecta
CIII. Double foramina
Weber (1950)
CIII-CIV. Dystopia of foramina
Gruneberg (1950)
CV-CVII. Th. I. Accessory foramina
Weber (1950)
CVI. Inflexion of tuberculum anterius
Gruneberg (1950)
Gruneberg (1950)
CVI. Absence of tuberculum anterius
Th. II. Variation in the size of the
Gruneberg (1950)
processus spinosus
SI-SII. Fusion of sacral vertebrae
Searle (1954)
C. Variants of the appendicular skeleton
24 Dyssymphysis ischio-pubica
Griineberg (1952), Searle (1954 a)
25 Fossa olecrani perforata
Weber (1950)
Skeletal variants in mice
285
To compute C. A. B. Smith's mean measure of divergence (see Grewal, 1962;
Berry, 1963), the first step is to convert the percentage incidence of each variant
into angular values. This makes variances of the incidence virtually independent
of the incidence. The angular value <f> corresponding to the incidence p is defined
by <fi = sin"1 (1 - 2p) measured in radians, and the variance of 0 in a sample of
n is nearly l/« irrespective of the value of <j). If the actual incidences of a given
variant are px and p2 in two large populations, and the corresponding angular
transformations are <f>Xi and 0 2 , then a measure of divergence between the samples from the populations is taken as
X = (0!-0 2 ) 2
where nx and n2 are the sample sizes from the two populations.
If a number of variants are classified, a mean measure of divergence can be
computed by dividing the sum of the individual measures of divergence for
each variant by the number of variants. In this case it will be SZ/25. This provides a quantitative expression of the separation of the populations. Expressions
for variances are given in Berry (1963) from which the significance of the measures
of divergence for a variant, and the mean measure of divergence for two populations based on a number of variants can be assessed.
The method assumes that the determinants of all the variants have an equal
effect on fitness and can be summed legitimately. Although this is almost
certainly an incorrect assumption, it is hoped that the summation of all differences provides a reasonable estimate of divergence between the populations
concerned, especially in view of some work of Truslove (1961) who found that
the occurrences of nearly all the variants she studied were uncorrelated, indicating that the sensitivity of detection of differences between populations
increases with the number of variants considered.
RESULTS
Variation within strains C57BL and BALBjc
For each environmental factor the mice within an inbred strain were divided
into two groups so that mean measures of divergence (2Z/25) could be computed. The subdivisions were as follows:
Variable
Sex
Parity
Maternal age
Subdivision A
Males
1st and 2nd litters
Low(C57BL < 140 days;
BALB/c < 120 days)
Litter size
Small (1-6 mice)
Age at death
Small (^ 60 days)
Mother lactating while pregnant Yes
Mother pregnant while lactating Yes
Subdivision B
Females
Beyond the 2nd litter
High(C57BL^ 140 days;
BALB/c 2* 120 days)
Large ( ^ 7 mice)
Large (> 60 days)
No
No
286
W. L. HOWE & P. A. PARSONS
Mean measures of divergence are given in Table 2 with standard deviations.
None of the factors has any obvious effect on the incidence of the variants. In
fact for only one factor (age at death in the C 57 BL1 strain) does the mean
measure of divergence exceed its standard deviation.
However, scattered significant measures of divergence were found for certain
variants by themselves, but in view of the number of comparisons made this
would be expected on statistical grounds. Even so, some of these significant
comparisons may be meaningful. For example, the incidence of fusion of the
lower turbinal to the premaxilla and fusion of the two sacral vertebrae increase
markedly in both strains with increasing age at death. Conversely, the frequency
Table 2. Mean measures of divergence (ZX/25) and their standard deviations
for sex and the various environmental factors listed in the text
C57BL strain
BALB/c strain
A.
Factor
(SJT/25)
S.D.
Sex
Parity
Maternal age
Litter size
Age at death
Mother lactating while pregnant
Mother pregnant while lactating
00141
00206
00139
00252
01037
00256
00139
00342
00442
00375
00463
00979
00756
00394
(ZA725)
00210
00254
00080
—00004
00607
00043
00085
S.D.
00365
00421
00267
—
00643
00185
00270
of variants which are due to a lack of fusion (parted frontals and dyssymphysis
ischio-pubica) decreases with increasing age at death. Some of these findings
agree with previous reports. Thus fusions between the first two sacral vertebrae
are more frequent in older mice of strain A (Searle, 1954 c), and Griineberg
(1952) found that the fusion of the ischium and pubis, which is normally completed at the age of 3 or 4 weeks, may be considerably delayed in C57BL mice,
so that the frequency of dyssymphysis ischio-pubica is higher in younger mice.
For those variants which were significantly influenced by maternal age
(metoptic roots abnormal and foramen pterygoideum double in strain BALB/c,
and fusion of the first two sacral vertebrae in strain C57BL), the trend was for a
decrease in frequency with increasing maternal age. This agrees with earlier
work in guinea-pigs and mice where the incidence of polydactyly was shown to
decrease with maternal age (Wright, 1926; Holt, 1948). Furthermore, Searle
(19546) has made the same observation for certain skeletal abnormalities in the
mouse (see also Parsons, 1964, for discussion). However, as pointed out in the
introduction, it may often be difficult to separate maternal age from other highly
correlated factors.
In relatively few cases was a given variant influenced by the same environmental factor in the two strains. Because of the genetic differences between
strains, it may be postulated that given variants in the two strains have different
Skeletal variants in mice
287
patterns of developmental homeostasis. An environmental change may be
sufficient to upset stabilizing mechanisms in the development of a structure in
one strain, but not in the other. Thus the environmental fluctuation may cause a
shift in the underlying distribution of a minor variant in one strain affecting its
incidence, but in the other strain, even if the underlying distribution is affected,
the incidence of the variant remains unaltered. This may also explain why some
variants (e.g. foramen pterygoideum double in the BALB/c strain) are influenced
by a number of different environmental variables in one strain, but by none in
the other.
Comparisons between inbred strains and hybrids
Although significant measures of divergence were obtained for individual
variants for some environmental factors in the two inbred strains just discussed,
mean measures of divergence for the twenty-five variants were not significant
Table 3. Percentage incidence of variants* in inbred strains and
hybrids between them
Hybrids
A
J.UUICU. olLctlLlo
Variant*
C57BL
BALB/c
C3H
C57BL
x BALB/c
1
9
49-5
17-9
5-3
15-3
11-6
1-3
4-5
31-6
98-2
16-3
0-4
900
4-4
45-8
960
99-8
611
71-2
151
72-6
890
21-9
89-7
73-3
98-6
76-9
55-8
14-5
0
76-7
4-7
151
46-5
831
69-8
96-5
10
11
250
3-9
81
12
13
14
15
16
17
18
7-6
74-2
15-5
73-9
18-9
43-1
24-7
4-8
4-8
890
740
19
610
20
21
22
23
24
25
360
28-3
53-8
13-7
29-8
63-7
190
f
" 2
3
4
5
6
7
8
No. of mice
5-2
78-8
46-8
65-3
83-7
01
23-7
67-5
14-7
10
0-4
43-7
10-3
10
0-8
252
11-6
92-5
0
411
74-3
28-0
0
0-7
19-2
110
2-7
29-5
73
.
1-7
1-7
83-7
69-2
41-3
79-7
5-2
38-2
66-7
50-3
4-9
1-2
94-2
0
1-2
43-6
86
BALB/c
xC3H
(
:< C 3 H
9-2
1-3
30
0
42-4
30
27-3
33-3
68-2
78-8
74-2
89-5
&6
49-3
89-4
99-3
750
74-3
2-6
4-6
95-4
54-6
651
980
0
22-3
640
25-1
0
0
22-4
0
0
45-4
76
1-5
'
* The variants correspondingI to numbers 1-25 are described in Table 1.
0
66-7
74-2
45-5
74-2
2-5
530
78-8
49-6
4-7
0
970
0
0
68-2
33
288
W. L. HOWE & P. A. PARSONS
(Table 2). Because of this, it seemed legitimate to include data for mice killed
at all ages, when determining mean measures of divergence between inbred
strains and hybrids. To make sure that this procedure is legitimate, mean
measures of divergence were computed just for those mice killed at 60 days, and
were found to be in complete agreement with the measures of divergence
obtained on the total data.
In Table 3 the percentage incidences of the skeletal variants in the three inbred
strains and their hybrids are given. First, differences between the three inbred
Table 4. Mean measures of divergence and their standard deviations between
inbred strains
C57BL
BALB/c
BALB/c
C3H
1-326 ±0-221
—
1012 ±0-275
0-348 + 0-156
Table 5. Mean measures of divergence and their standard deviations between hybrids and their corresponding parental strains
C57BL
C57BLx BALB/c
BALB/c xC3H
C57BLxC3H
0-754 ±0-226
—
0-703 ±0-316
BALB/c
0-320 ±0141
0119 ±0090
—
C3H
—
0-268 ±0170
0-608 ±0-327
strains will be considered. The difference between the C57BL and BALB/c
strains is greatest (P < 0-01), followed by that between the C57BL and C3H
strains (P < 0-05) (see Table 4). The difference between the BALB/c and C3H
strains is considerably smaller than the other two comparisons. It can be assumed that these various differences are largely genetic in origin, since the diet
was the same for all strains, and the various other non-genetic factors discussed
already have been shown to have little overall effect.
Before seeing how the hybrids fit into this pattern it should be pointed out that
reciprocal hybrids were obtained between C 57 BL and BALB/c, for which the
mean measure of divergence came to 0-073 + 0-138, and between C3H and
BALB/c to 0-060 ± 0-166. This shows that there is no overall significance in the
mean measure of divergence between reciprocal hybrids. Thus all the data for
hybrids between a given pair of inbred strains are combined in the discussions
to follow.
In Table 5 the hybrids between pairs of inbred strains are compared with the
corresponding inbred strains. The C57BLx BALB/c hybrids diverge more
from the C57BL strain than from the BALB/c strain, thus the BALB/c strain
can be said to be 'dominant' to the C57BL strain, with respect to overall incidence of skeletal variants. The BALB/c x C3H hybrids diverge more from the
289
Skeletal variants in mice
C3H strain than from the BALB/c strain, so that the BALB/c strain is dominant
to C3H. Finally, the C57BLxC3H hybrids diverge more from the C57BL
strain than from the C3H strain, so the C3H strain is dominant to C57BL.
This gives an order of dominance for overall skeletal differences between strains
as BALB/c > C3H > C57BL. For certain variants by themselves, overdominance either in a positive or negative direction was found, but this disappears when the information on all twenty-five variants is combined as above.
For some mice of each inbred strain and hybrid, weights at 60 days of age
were obtained (Table 6), in view of suggestions made by Deol & Truslove (1957)
and Griineberg (1963) that many if not most minor skeletal variants are expressions of generalized or localized size variations. A number of interesting observations emerge from Table 6. First, for the means combining sexes, the hybrid
means lie between the inbred strain means as found for mean measures of
Table 6. Mean weights (g) at death of mice killed at 60 days
Males
Inbred strain
or hybrid
C57BL
BALB/c
C3H
C57BLx BALB/c
BALB/c xC3H
C57BLxC3H
Females
Males and Females
No. of
mice
Weight (g)
No. of
mice
Weight (g)
No. of
mice
Weight (g)
64
92
14
41
36
18
20-323 ±2-233
23-995 + 1-757
21-658 + 2-221
23-173 + 2-346
22-508 + 2-134
20-903 + 2-184
60
77
12
45
37
14
17-942+1-808
20-373 + 1-658
18-146 + 1-632
19-288 + 2-234
20-244 ±1-335
18-485 ±1-705
124
169
26
86
73
32
19-132 + 2-027
22184± 1-711
19-902+1-949
21-231+2-287
21-376+1-729
19-694+1-974
divergence (Tables 4, 5). Secondly, there is a similarity between the mean weight
of inbred mice at 60 days and their mean measures of divergence. For both of
these traits, the greatest differences are between the C57BL and BALB/c
strains. The differences between the C57BL and C3H strains are smaller, and
between the C 3 H and BALB/c strains smallest of all. Thirdly, the pattern of
dominance for weight is similar to that for the mean measure of divergence
giving the order of dominance as BALB/c > C3H > C57BL. Considering
sexes alone, minor but unimportant discrepancies occur from this pattern of
results. The similarities between weight and pattern of skeletal variation support
an association between the incidence of many skeletal variants and the size of
structures which are correlated with body weight as suggested above.
DISCUSSION
Berry's (1963) approach to the analysis of skeletal variability is of considerable help in this type of work because it combines information on a number of
variants which if treated in isolation can be classified only as present or absent,
or in the case of bilateral variants as present on both sides of the animal, present
290
W. L. HOWE & P. A. PARSONS
on one side, or absent. In isolation, therefore, skeletal variants classified in this
way are far less informative than the information obtainable on metrical traits
such as height or weight. Berry's (1963) approach forms one attempt at integrating information available from the whole skeleton.
Although there is significant variation within inbred strains for certain variants for some of the environmental factors, there are no significant mean measures of divergence considering all the variants together. In comparison are the
large and significant mean measures of divergence between strains. This shows
that the genotype is more important than the environment in the determination
of skeletal variability. The hybrids lie between the inbred strains from which they
were derived, showing that dominance rather than overdominance is involved in
overall skeletal variability, although some variants by themselves do show overdominance.
It is remarkable that the divergence between strains and hybrids is so closely
paralleled by differences in body weight at 60 days, strongly suggesting that the
incidence of many skeletal variants is closely associated with the size of structures
correlated with body weight. There has been a certain amount of work on the
analysis and classification of physique in man (Tanner, 1964). Simple measures
such as height and leg length can be used but are inefficient, since frequently
they are highly correlated with each other. However, there are several classifications of human form that have been developed, e.g. Sheldon's system of somatotyping based on three extreme forms namely the endomorph, mesomorph and
ectomorph. All persons can be classified according to the degree to which they
approach these extremes. Factor analysis, a branch of multivariate statistical
technique, has also been used in this field. Arguing from the evidence for a genetic
component in the skeletal variability of the mouse, and from the evidence in
man for variations in the incidence of minor skeletal variants, especially of the
cranium, teeth and vertebral column (Griineberg, 1950; Comas, 1960; Brothwell,
1963) in different populations, it would seem likely that some of the skeletal
variants would be associated with the somatotype and would also be under partial
genetic control. Unfortunately it is difficult to test this as precisely in man as has
been done in mice, but this does seem to provide a field for future investigation.
SUMMARY
1. The effects of various environmental factors on skeletal variability as
measured by the incidence of twenty-five minor skeletal variants in two inbred
strains (C57BL and BALB/c) of mice has been studied using a method devised
by C. A. B. Smith. The environmental factors were found to have no significant
overall effect when combining information on all variants, although some variants taken individually are affected significantly by the environment.
2. Large differences in skeletal variability were found between three inbred
stains, which are mainly genetic. Combining the information on the inbred strains
Skeletal variants in mice
291
with the hybrids between them gives an order of dominance for skeletal variability for the three strains as BALB/c > C3H > C57BL.
3. Observations on body weight at 60 days also give this order of dominance,
suggesting an association between the incidence of many skeletal variants and
the size of structures which are correlated with body weight.
RESUME
Genotype et milieu exterieur dans la determination de variations squelettiques
mineures et du poids corporel, chez les souris
1. Au moyen de la methode mise au point par C. A. B. Smith, on a etudie les
effets de divers facteurs du milieu exterieur sur la variabilite du squelette,
exprimee par la realisation de vingt-cinq variantes squelettiques mineures
chez deux lignees endogames de souris (C57BL et BALB/c). Les facteurs du
milieu n'ont pas d'effet general significatif quand on combine Finformation sur
toutes les variantes quoique certaines d'entre elles, prises individuellement,
soient affectees de maniere significative par le milieu exterieur.
2. On a trouve de grandes differences dans la variabilite du squelette, entre
trois lignees endogames, et elles sont sans doute essentiellement genetiques. En
combinant l'information sur les lignees endogames avec celles sur les hybrides
entre elles, on obtient un ordre de grandeur de la dominance pour la variabilite
du squelette, tel que BALB/c > C3H > C57BL.
3. Les observations sur le poids corporel a 60 jours donnent aussi l'ordre de
dominance, suggerant une association entre l'incidence de beaucoup de variantes
du squelette et la taille de structures en rapport avec le poids du corps.
We are grateful to Miss Astrid Fleiss who helped to maintain the stocks of mice.
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(Manuscript received 5 September 1966)