J. Embryol. exp. Morph. 73, 135-149, 1983
135
Printed in Great Britain © The Company of Biologists Limited 1983
Axial abnormalities following disturbed growth in
Mitomycin C-treated mouse embryos
By B. C. GREGG 1 AND M. H. L. SNOW 1
From the MRC Mammalian Development Unit, London
SUMMARY
Primitive-streak and early-organogenesis-stage mouse embryos were treated with
Mitomycin C (MMC) by intraperitoneal injection of pregnant females. Skeletal preparations
of newborn pups were made and the axial skeleton examined. The treated animals showed a
high incidence of:- (1) changed vertebral numers, (2) malformation of the vertebral column,
(3) changed rib numbers and (4) rib abnormalities. These skeletal disturbances tend to be
located more posteriorly with later MMC treatment. There is regional variation in the susceptibility of vertebrae to abnormal development. Extra elements may occur in any region of the
spinal column and several unique combinations of numbers are reported. The possible origin
of these abnormalities and their similarity to some human syndromes is discussed. It is suggested that they may be the consequence of altered growth profiles in interacting tissues during
the restorative growth following MMC treatment.
INTRODUCTION
Recently Snow & Tarn (1979) have studied the immediate response of the
mouse embryo to a single intraperitoneal injection of the DN A alkylating agent,
Mitomycin C (MMC) at 6-5 days post coitum (d.p.c.) or 7d.p.c, (i.e. early- to
mid-primitive-streak stages). Extensive cell death followed by a reduced cell
proliferation rate occurs within a few hours of administration and can be
mimicked by exposure to MMC for less than 1 h in vitro (Tarn, 1980). As a result
of these two factors the embryo has, on average, only 10-15 % of the normal
number of cells 12 h after injection; the majority of dead cells have disappeared.
The embryo undergoes restorative growth (Snow, 1982) and by 13-5d.p.c,
although foetal weight is still significantly low, foetuses appear to be morphologically normal.
However, during the period of restorative growth from 8d.p.c. to 10d.p.c,
morphogenesis is disturbed in a complex way; individual tissues and organ systems appear to be affected to different extents. Somitogenesis in MMC-treated
embryos has been described by Tarn (1981). Somitogenesis starts on time
(8 d.p.c.) but occurs at a progressively slower rate than normal up to somite
number 25-30; thereafter the rate of somitogenesis in MMC-treated embryos is
maintained while that in normal embryos declines, so that somite 45 is formed
1
Authors' address: MRC Mammalian Development Unit, Wolfson House, 4 Stephenson
Way, London NW1 2HE, U.K.
136
B. C. GREGG AND M. H. L. SNOW
at approximately the same chronological age in treated and normal mice.
Development of organ systems within the embryo appears to be uncoordinated
during this period of restorative growth (Snow, Tarn & McLaren, 1981). As well
as disturbed morphogenesis the growth profiles of tissues and organs in MMCtreated embryos are altered, to differing degrees in different systems. This paper
describes the skeletal defects which are present in newborn mice which were
treated with MMC during primitive-streak stages and early organogenesis. It is
suggested that the defects may be a consequence of abnormal tissue interactions
during the restorative growth period.
The MMC-induced skeletal abnormalities are very similar to the Klippel-Feil,
and Wildervanck syndromes of vertebral dysostoses occurring in man (Beighton,
1978; Salmon & Lindenbaum, 1978). The Wildervanck syndrome in particular
is associated with deafness and retraction of the eyeball; ocular defects have been
found in MMC-treated mice (Snow & Tarn, 1979) and the development of the
inner ear is abnormal in a manner that might be expected to result in deafness
(Deol, unpublished observations). It is possible that the similarity of these syndromes is a reflection of common mechanisms in their production.
MATERIALS AND METHODS
Randomly bred Q-strain mice were used. The experimental mice received a
single intraperitoneal injection of 100 ^g Mitomycin C (Sigma, London) in
0-25 ml 0-9 % NaCl between 6-5 days and 8-5 days post coitum (d.p.c). This
period ranges from the formation of the primitive streak: 50 % of embryos have
a primitive streak at 6-5 days (Snow, 1977), to early somite stages (the average
somite number at 8-5 d.p.c. is 6). Foetuses were left to develop to term, but if
they had not been born by 19d.p.c. they were removed by Caesarian section.
Pups were sexed and weighed. Cleared skeletal preparations were made using
a modification of the differential staining technique for cartilage and bone
described by Inouye (1976). Skinned and eviscerated pups were fixed in 95 %
ETOH for 3 days, defatted in absolute acetone for 24 h, and stained in the
acidified 0-015 % Alcian blue/0-005 % Alizarin red in 70 % ETOH of Inouye
(1976) for 2-3 days. The stained carcasses were macerated in 20% glycerol
containing 1 % KOH for about 2 days, care being taken not to overdo this step
which would result in skeletons falling to pieces. They were then cleared through
a graded series of glycerol and stored in 100 % glycerol to which a small crystal
of Thymol was added. A control group of mice which had not been treated with
Mitomycin C, was carried through this procedure. Each skeleton was examined
under a dissecting microscope. The axial skeleton was scored for the numbers
of vertebrae in each region of the presacral vertebral column (i.e. cervical,
thoracic and lumbar). Malformed vertebrae were scored according to their
position along the axis, including sacral and caudal regions. Statistical analysis
of the data was done using the Chi-squared test, or a Student t test.
Axial abnormalities after mitomycin C
137
RESULTS
As can be seen from Table 1, the mean weights of MMC-treated pups were
significantly lower than normal at 19d.p.c. Mean litter sizes of MMC-treated
mice were also lower than normal. The appendicular skeleton appeared to be
normal after MMC treatment at these stages; no evidence of fused elements or
polydactyly was found in any of the treatment groups. A low incidence of other
abnormalities has been reported as a result of MMC treatment (Snow & Tarn,
1979). The overall incidence of abnormality of the vertebral column and ribs was
over 85 % in animals treated at 7-5, 8 or 8-5 d.p.c. and 70 % after treatment at
6-5 d.p.c. (Fig. 1). Pups were classified as abnormal if they had extra or reduced
numbers of elements or if any element had an abnormal structure. A low
frequency of 'floating' cartilaginous elements at the posterior end of the rib cage
was observed in the control group (10 %), and in all the treatment groups (6-5
day (8%), 7-5 day (13%), 8 day (6%) and 8-5 day (16%)). These 'floating'
elements resemble the costal part of a rib and were positioned as such. The
significance of these elements is not clear and they are not considered in this
study. The vertebrae adjacent to the 'floating' elements were classified as lumbar
and not thoracic as the element does not articulate with the vertebra. In the
following analysis of the variation in the numbers of elements the right and left
sides of the body are considered independently, the number of elements on each
side was scored separately.
1. Variation in the number of presacral vertebrae
Q-strain mice show little natural variability in the number of presacral
vertebrae (p.s.v.). This region is most commonly composed of 7 cervical, 13
thoracic and 6 lumbar vertebrae. In the control group of 66 pups two had
vertebra 27 incorporated into the sacrum on one side only, i.e. the sacrum was
asymmetrical and there were thus 6/7 lumbar vertebrae. Only one pup had 14
thoracic vertebrae, with attached ribs, instead of the normal 13. After MMC
Table 1. The mean weights and litter sizes at 19 d.p. c. of control and MMC-treated
pups
Day of treatment
(d.p.c.)
Total number of
foetuses
(No. litters)
Mean litter size
(± I s .E.)
Control
6-5
7-5
8
8-5
66(6)
106 (12)
89 (10)
77(9)
60(7)
11.0 (± 0-45)
8-8 (± 0-49)
8-9 (± 0-86)
8-6 (± 0-73)
8-9 (± 1-82)
Mean weight*
(±1S.E.)
1-308 (±
1-076 (±
1-191 (±
1-077 (±
1-046 (±
0-017)
0-017)
0-014)
0-023)
0-021)
* Students t-test, all treated groups are significantly different from the control P < 0-001.
138
B. C. GREGG AND M. H. L. SNOW
98%
96%
86%
70%
14%
control
6-5
7-5
8-5
Day p.c. of MMC treatment
Fig. 1. Incidence of pups with vertebral or rib abnormalities after MMC treatment
at 6-5, 7-5, 8 and 8-5d.p.c..
treatment at all stages the incidence of these minor skeletal variations was drastically increased and there was a trend towards an increased number of both
thoracic vertebrae and p.s.v.. The number of cervical vertebrae was reduced or
increased in a small proportion of pups (Table 2). Two animals treated at
7• 5 d. p. c. and one at 8• 5 d. p. c. had 8 cervical vertebrae. Three other animals with
8 cervical vertebrae were found in a pilot study; as these litters were not sexed
they are not considered in this report. After treatment at7-5,8or8-5d.p.c. over
65% of animals had 14 thoracic vertebrae (Table 2). Treatment at 8-5d.p.c.
produced 15 thoracic vertebrae in 3 % of animals. In addition following treatment at 6-5,7-5 or 8d.p.c.a small percentage of pups had only 11 or 12 thoracic
vertebrae. The number of p.s.v. in offspring of MMC-treated mice showed a
similar trend (Table 2). 2 1 % of 7-5-day-treated, and 3 3 % of 8-day-treated
animals had greater than the normal number of p.s.v. whereas following treatment at 8-5 d.p.c. 80 % had 27 p.s.v. and 1 % had 28 p.s.v.. A low percentage had
less than 26p.s.v. after treatment at 6-5, 7-5 or 8d.p.c.
Decreased numbers of elements in the cervical or thoracic regions were usually
associated with vertebral fusion but were not obviously the result of such fusions.
Reduced numbers, (4,5 or 6), of cervical vertebrae were always associated with
such fusions, but in seven pups treated at 6-5 d.p.c. and one pup treated at
7-5 d.p.c. in which the numbers of thoracic vertebrae were reduced, no evidence
for such fusions was seen. It is possible that this reduction is the consequence of
Control
6-5
7-5
8
8-5
MMC treatment
(d.p.c.)
2
—
—
1
_
—
4
5
—
4
—
100
92
98
96
99
—
2
—
1
Number of cervical
vertebrae
5
6
7
8
1
1
1
—
8
3
3
—
99
75
29
28
31
1
17
67
69
66
—
—
—
3
Number of thoracic
vertebrae
11 12 13 14 15
1
—
1
—
22
1
—
1
—
1
2
1
—
9
2
4
—
99
81
75
61
19
1
6
21
33
80
Number of presacral
vertebrae
23 24 25 26 27
—
—
—
1
28
Table 2. Percentage incidence of different numbers of cervical, thoracic and presacral vertebrae after MMC treatment
I
3
3
140
B. C. GREGG AND M. H. L. SNOW
complete and perfect fusion of vertebrae, but no other evidence for this was
found. The seven pups in the 6-5-day group each had 12 perfect thoracic
vertebrae and 7 cervical vertebrae. The pup in the 7-5-day group had 12 perfect
thoracic vertebrae and 8 cervical vertebrae. 5 instead of 6 lumbar vertebrae were
present at a high frequency after MMC injection at 7-5 d.p.c. and at a lower
frequency after treatment at 6-5, 8 and 8-5 d.p.c. This reduction in lumbar
vertebrae was very rarely associated with vertebral fusion, only being the case
in severely malformed animals in which the number of elements in two or more
regions of the vertebral column were reduced. Excluding severely malformed
pups, animals with 5 lumbar vertebrae generally had 14 (or 15) thoracic
vertebrae; there were four exceptions, two in the 6-5-day group and two in the
7-5-day group. One pup in the 6-5-day group had a normal number of cervical
and thoracic vertebrae (7,13), the other had 7 cervical and only 12 thoracic
vertebrae. Both animals in the 7-5-day group had 8 cervical and 13 thoracic
vertebrae. Overall 53 % of pups with 14 thoracic vertebrae had 5 lumbar and
47% had 6 lumbar vertebrae. These observations are summarized in Fig. 2.
Severely malformed animals in which the numbers of elements in two or more
regions were reduced are not included in Fig. 2. They constitute 3 % of the
6-5-day group, 1 % of the 7-5-day group and 2 % of the 8-day group. Figure 2
shows that the sites of vertebral change tend to shift posteriorly with increasing
age at treatment.
2. Incidence of malformed vertebrae
A low incidence of malformed vertebrae was found in the control group (Fig.
3). In each case the malformation was minor and would have been unlikely to
have decreased the viability of the animal. In MMC-treated pups both the incidence of malformed vertebrae and the severity of the malformation was increased. The malformations found in the controls were all of the same type, i.e.
fusion of the vertebral arches of adjacent vertebrae. A range of types of malformations was found in the MMC-treated groups. These include fusion of
vertebral arches, fusion of vertebral centra, bilateral ossification of centra,
ankylosis of centra, hemivertebrae, jumbling of vertebrae often with loss of
elements and intrapelvic crowding. In this report no differentiation will be made
between the different types of malformation.
Figure 3 shows that regional location of malformed vertebrae was not random
in the MMC-treated groups but was dependent on the time of the MMC injection. As the time of treatment becomes later, there appears to be posteriad shift
in the distribution of malformed vertebrae.
If the pups are separated according to sex it is apparent that overall females
were more sensitive than males to the production of malformed vertebrae, (Fig.
4A). This difference is significant in the posterior thoracic and lumbar regions.
If the different treatment groups are considered separately then the tendency for
females to be more susceptible than males is seen to differ after treatment at
10.
9.
8.
7.
6.
5.
4.
3.
2.
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Cervical
1%
3%
—
13%
—
—
69%
1%
7%
3%
6-5
—
21%
—
46%
1%
1%
27%
1%
2%
7-5
1%
32%
—
37%
—
—
26%
1%
1%
8
21%
57%
1%
11%
—
—
10%
_
—
8-5
Time of MMC treatment
Fig. 2. Presacral vertebral combinations after MMC treatment at 6-5, 7-5, 8 and 8-5 d.p.c.
Notes 1. Loss of cervical vertebra(e); 2. Loss of thoracic vertebra(e); 3. Loss of lumbar vertebra(e); 4. Normal
column; 5. Extra cervical vertebra, loss of thoracic vertebra; 6. Extra cervical vertebra, loss of lumbar vertebra;
7. Extra thoracic vertebra(e), loss of lumbar vertebra; 8. Extra cervical vertebra; 9. Extra thoracic vertebra(e);
10. Extra lumbar vertebra. (Also one pup, produced after MMC at 6-5 d.p.c., with vertebral combination 7 cervical
12 thoracic 5 lumbar which is not included in figure.)
High
Normal
Low
P.s.v. number
t
3
142
B. C. GREGG AND M. H. L. SNOW
Control
4%
6%
2%
0%
6-5 d.p.c.
63%
44% 42%
12% 7%
1% 1%
0%
0%
7-5d.p.c.
52%
b'2%
40%
37%
36%
6% 0 % 0 %
1
8d.p.c.
36% 34%
57%
20% 19% 22% 27%
0% 0%
8-5d.p.c.
80%
64%
18%
24%
36% 30%
42%
24%
n—
CA
10%
CP
TA TM
TP
L
S
T
V.C.
(total)
Fig. 3. Incidence of regional location of malformed vertebrae after MMC treatment
at 6-5, 7-5, 8and8-5d.p.c.
Notes: CA = Anterior cervical vertebrae (1, 2 and 3); CP = Posterior cervical
vertebrae (4, 5, 6 and 7); TA = Anterior thoracic vertebrae (8, 9, 10 and 11);
TM = Mid thoracic vertebrae (12, 13, 14, 15 and 16); TP = Posterior thoracic
vertebrae (17, 18, 19, (20 and 21)); L = Lumbar vertebrae; S = Sacral vertebrae;
T = Tail vertebrae; VC = Incidence of malformation of the vertebral column.
Fig. 4. Incidence of regional location of malformed vertebrae in males and females
after MMC treatment. Abbreviations as for Fig. 3. (A) combined total of all treated
groups; (B) 6-5d.p.c.-treated group; (C) 7-5d.p.c.-treated group; (D)
8d.p.c.-treated group; (E) 8-5 d.p.c.-treated group. * Chi-squared test, significantly
different P< 0-01; t significantly different P< 0-05. • = male; • = female.
143
Axial abnormalities after mitomycin C
~2
^
^
I I I in
CA CP TA T M T P
CA CP TA TM
o
o
o
TP
L
S
L
S
v.c.
n
V.C.
CA CP TA TM TP
L
S
V.C.
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V.C.
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ii
I
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I
I
L
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I
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Fig. 4
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144
B. C. GREGG AND M. H. L. SNOW
different stages. The difference is most marked in the 8- and 8-5-day groups (Figs
4D, E), less so in the 7-5-day group (Fig. 4C) and in the 6-5-day group males and
females show an equal tendency for vertebrae to be malformed (Fig. 4B).
Figure 5 shows that individual vertebrae did not show an equal frequency of
40
20
40 •
B
20
% malformation
40
C
20
40
D
20
40
20
Vertebra
12
7 s 9 i o n 12 13 u 1516 17 is iy 2021 22 23 24 2^ 2h 27 2*29.111
Cervical
Thoracic
Lumbar
Sacral
Fig. 5. Incidence of malformation of vertebrae after MMC treatment. (A) combined
total of all treated groups; (B) 6-5 d.p.c.-treated group; (C) 7-5 d.p.c.-treated group;
(D) 8d.p.c.-treated group; (E) 8-5 d.p.c.-treated group.
Axial abnormalities after mitomycin C
145
malformation. Vertebrae 2 and 3 in the cervical region and the vertebrae in the
middle of the thoracic and lumbar regions were more susceptible to malformation than were vertebrae at the thoracic borders, i.e. vertebrae 7 and 8, 21 and
22: these vertebrae showed a low incidence of malformations.
In embryos treated at 8-5 d.p.c. malformation of the sacral vertebrae was often
found in conjunction with intrapelvic crowding, when more than the normal four
sacral vertebrae were present along the length of the ischium.
3. Rib malformations
The highest incidence of malformations of the ribs was found in pups treated
at 7-5 d.p.c. (Fig. 6A). Malformed ribs were usually present as fusion of adjacent
ribs: this may involve two, or occasionally three ribs. In addition 'split ribs' were
also present, in which a 'single' vertebral rib process splits into two costal ribs.
The frequency of malformation was not equal for all ribs (Fig. 6B). There was
a relatively high incidence of fusion of the costal cartilages of ribs 1 and 2, both
ribs ending at a single sternebra. The middle of the rib cage showed a more or
less constant incidence of malformation (i.e. ribs 3-9). Incidence of malformation of ribs 11,12 and 13 was low and ribs 14 and 15, where present, were never
found to be malformed. The incidence of fused ribs was not always equally
distributed between the right and left hand sides (Fig. 6C). In animals treated
with MMC at 7-5 and 8 d.p.c., 80 % and 75 % respectively of rib fusions occurred
on the right-hand side of the body. In addition there was a low incidence of
38%
A
24%
17%
80%
10%
5%
|—|
Control 6-5
53%
75%
44%
7-5
40
6-5
30
number
malformed
7-5
20
10
Rib
1 2 3 4 5 6
7
8
9 10 11 12 13 14
Fig. 6. Rib malformations after MMC treatment at 6-5, 7-5, 8 and 8-5d.p.c. (A)
Incidence of animals with rib fusions or splits; (B) Frequency of fusions or splits of
individual ribs; (C) Percentage of rib fusions and splits on the right hand side of the
body. * Chi-squared test, significantly different P< 0-001; t significantly different
P<0-01.
146
B. C. GREGG AND M. H. L. SNOW
incompletely developed ribs in all groups, including the control group. This was
usually manifest as a gap in the middle of the most posterior rib. The highest
incidence occurred in the 6-5-day group (6 %). 92 % of gaps occurred in the 14th
rib.
DISCUSSION
A number of teratogenic agents alter the composition of the vertebral column
in mice. The vertebral types produced, the critical periods and the times of peak
sensitivity seem to depend on a number of factors. These include (a) the strain
of mouse (b) the teratogenic agent, and (c) the dose administered. All laboratory
strains of mice show some natural variability in skeletal type, the degree and
localization of this variability depending on the strain (Green, 1941). Russell
(1950) demonstrated that the ease with which the thoracolumbar and lumbosacral borders may be shifted by X-irradiation of the embryo in utero depends
on the natural variability the strain exhibits at the border. Teratogenic insults
which shift these borders seem always to have produced combinations which, if
they are not naturally present in the treated strain of mice, are present in other
mouse strains. Russell (1979) has used the term homeotic shifts for these variations in skeletal type. In the present study a number of pups with 8 cervical
vertebrae were produced and one mouse had 12 thoracic and 5 lumbar vertebrae.
These combinations do not occur naturally in mice, nor have they previously
been produced experimentally. The times of peak sensitivity of shift in the
thoracolumbar and lumbosacral borders produced by MMC treatment agree
with data published by Russell (1950) using X-irradiation. X-irradiation of the
pregnant dam at 50 R or more, like MMC, causes cell death and mitotic delay
in dividing cells.
The difference in the susceptibility of vertebrae to malformation following
treatment with a teratogenic agent has been noted following X-irradiation
(Russell, 1956) and the trend towards a more posterior localization of abnormality with increasing embryonic age has been reported following treatment with
adenine (Fujii, 1970); hypoxia (Murakami & Kameyama, 1963; Ingalls & Curley, 1957); X-irradiation (Murakami & Kameyama, 1964) and ethylurethane
(Takaori, Tanabe & Shimamoto, 1966). Fujii for instance described three peaks
of sensitivity corresponding to treatment at 7-5, 8-5 and 9-5 d.p.c. which
produced abnormality in the cervical, thoracic and lumbar regions respectively.
It is suggested that this merely reflects the developmental stage of the embryos
at the time of treatment. By implication therefore treatment at8-0or9-0d.p.c.
would be expected to produce peaks of malformation straddling the cervicothoracic or thoracolumbar borders. In our 8-day MMC-treated mice that is
clearly not so and since many animals show abnormality in two, and some in all
three regions we would suggest that there is genuine variability between
vertebrae in their susceptibility to damage. Deol (1961) in his description of the
Axial abnormalities after mitomycin C
147
skeleton of the Tail-short (Ts/+) mouse reports a similar variability between
vertebrae in their incidence of malformation. Cervical vertebrae 3 and 4 and
thoracic vertebrae 9, 10 and 11 show the highest incidence of malformation. In
addition Ts/+ mice usually have 14 instead of the normal 13 thoracic vertebrae.
The human syndromes, Klippel-Feil and Wildervanck are characterized by cervical and thoracic fusions. It is of interest in the light of the MMC study, that both
syndromes occur predominantly in females (Salmon & Lindenbaum, 1978;
Beighton, 1978).
In all the above studies, and in our data, the vertebrae which become malformed are usually not present at the time of treatment, even in their precursor
somite form. The 8-5 d.p.c. mouse embryo has on average six somites and it has
recently been suggested that a further six are present in the presomitic
mesoderm, as somitomeres (Tam, Meier & Jacobson, 1982, in press). The sacral
and caudal abnormalities associated with teratogenic treatment at 8-5 d.p.c.
involve the products of somites 31 onwards, which in Q-strain mice are not
formed until 10 d.p.c. or later. Similarly the production of ribs on vertebra 21
(formed from somites 25 and 26) can follow MMC treatment at 6-5 d.p.c., some
82 h prior to the formation of the appropriate somites in the treated embryos.
The cellular necrosis caused by MMC is extreme but is cleared away so rapidly
(Snow & Tam, 1979; Tam, 1980), that there seem to be only two mechanisms by
which these abnormalities might arise:
1) The teratogen acts directly on the vertebral primordia causing them to
develop abnormally. These primordia would need to be present at
6-5-7-5d.p.c, or
2) the effect is not direct but leads to a disturbance in epigenetic processes of
later morphogenesis whereby the vertebrae (and ribs) are formed.
The first alternative can probably be eliminated. If a common mechanism
underlies these abnormalities then direct interference with the tissue determined
to form these vertebrae can be ruled out. It is extremely unlikely, but not impossible, that the mosaicism seen within the primitive-streak-stage mouse embryo
(Snow, 1981) is on such a fine scale that small groups of cells specifying lumbar
vertebrae are present at that stage. Even if this were so it is unlikely that the
various teratogenic insults which result in these 'homeotic shifts' (Russell, 1979)
would have precisely the same effect and outcome especially as they are applied
at such different stages of development. Furthermore the existence of many of
these aberrations as normal variations between strains of mice (Green, 1951,
1953; McLaren & Michie, 1956) indicate that they are not necessarily the result
of damage by teratogenic agents. Thus, the second alternative seems the more
likely. Two possible parameters, smallness or changed growth profiles, may
underlie the abnormal development.
Smallness perse is not necessarily associated with skeletal defects, particularly
not with extra elements. The pygmy mouse which has a low birthweight does not
have extra elements (King, 1955). In pigs which show an unusually large variation
148
B. C. GREGG AND M. H. L. SNOW
in the number of ribs, extra ribs are usually associated with extra body vertebrae
and larger pigs (Freeman, 1939). Furthermore, the distribution of the birthweights of control and MMC-treated mice overlap, more than half of the control
mice have birth weights which fall within the MMC-treated range. If the presence
of extra ribs was correlated with smaWn&ss per se then far more than the observed
1-5 % of control mice would be expected to have extra ribs.
In our results there is no correlation between birthweight and rib number in
any of the treatment groups. However, there is a negative correlation between
the number of p.s.v. and birthweight in the 8-5-day group (sigP<0-02). In a
study of C57BL mice which show a high natural variability at the lumbosacral
border, Deol & Truslove (1957) found that sacralization of the 6th lumbar
vertebra was associated with high birthweight. They suggest that there is a tendency for small mice to have higher p.s.v. numbers than large mice. Deol (1961)
however suggests that the skeletal defects in the Tail-short mouse may be a
consequence of the observed growth retardation in which different organs seem
to be retarded to different extents at various stages in development. The
Diminutive mouse which is phenotypically very similar to Tail-short and may
have 15 ribs also shows growth retardation (Stevens & Mackensen, 1958).
Similar differential rates of development of different organ systems are observed
in MMC-treated mice (Snow, Tarn & McLaren, 1981). We therefore suggest that
during the restorative growth period following MMC-treatment when the growth
profiles of tissues and organs are mismatched it is likely that abnormal tissue
interactions result in the observed skeletal defects.
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{Accepted 12 September 1982)