/. Embryol. exp. Morph. Vol. 25, 3, pp. 339-345, 1971
Printed in Great Britain
339
Effect of temperature differentials
upon reconstitution of embryonic primordia
in Ambystoma
By JEAN PIATT 1
From the Department of Anatomy,
School of Medicine, University of Pennsylvania
SUMMARY
Eggs of Ambystoma maculatum in early stages of development were separated into two
groups and maintained at 5 and 14 °C, respectively. In one series of animals comprising both
temperature groups, the right otic vesicle was extirpated at stage 27. In another series the right
forelimb disc was extirpated at stage 30. Following operation, animals of both groups were
kept at 18 °C. Frequency and extent of reconstitution were compared between the two
temperature groups.
The 5° group was superior to the 14° group in the number of ears and forelimbs reconstituted. The extent of ear reconstitution and the initial quality of forelimb reconstitution were
also superior in the 5° group. Statistical analysis of the data indicates that the difference
between the two temperature groups is highly significant in both series.
It is concluded that a slower pre-operative rate of development, caused by hypothermia,
enhances the regenerative capacity of both ear and forelimb fields in the salamander embryo.
INTRODUCTION
This paper is concerned with an analysis of the regenerative capacity of ear
and forelimb fields in salamander embryos subjected to different temperatures
prior to removal of their respective primordia. The impetus for such a study arose
from certain observations concerning relative frequencies of ear reconstitution in
Ambystoma embryos in which the otic vesicle had been extirpated for the purpose
of eliminating the vestibular ganglion (Piatt, 1969, 1971). It was noted that the
ear was reconstituted in a number of cases and, apparently, more often in
animals raised at lower pre-operative temperatures. Also, Yntema (1950) has
reported that pre-operative temperatures are critical in affecting the results of ear
induction from foreign ectoderm in Ambystoma. Both of these observations
taken together are suggestive. It was decided therefore to undertake a series of
controlled experiments to test the thesis that differences in pre-operative temperature, resulting in different rates of development, have a significant effect on
reconstitution of the ear in Ambystoma. Similar experiments on the forelimb
1
Author's address: Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A.
340
J. PIATT
were also performed because extirpation and transplantation of amphibian
limbs has been a favourite field of study since the beginning of experimental
embryology. Also, it was important to ascertain whether temperature differentials affect the reconstitution of the ear alone or whether the effect is common to
other organ fields. The results of these experiments provide statistically valid
evidence that the rate of embryonic development prior to ablation of the primordium is an important factor in determining the potency of regenerative
power, irrespective of the level of differentiation attained by the organ rudiment
at the time of its removal.
MATERIALS AND METHODS
Temperature treatment. Eggs of Ambystoma maculatum from stages 1-9 were
separated into two groups and maintained continuously at 5 °C and 14 °C,
respectively, till operation. Following operation, all animals were maintained at
18 °C for the duration of the experiment. Each clutch of eggs was divided equally
between the two temperature groups and the paired series thus formed were
identified by clutch as well as by temperature. In this way a check was provided
against possible differences in results between older and younger clutches and
possible similarities within each paired series.
Ear experiments. The right otic vesicle was extirpated from embryos in both
temperature groups when stage 27 was reached. It should be stated at this time
that the size and organizational aspect of the vesicles were the same in the two
groups. Great care was exercised to ensure that no more than the otic vesicle
was removed and that no cells of the vesicle remained adherent to the brain wall.
All animals were fixed at stage 46 and the entire head was serially sectioned.
Slides were studied for the number of cases within each temperature group
showing development of ear epithelium and the extent of the reconstitution.
Forelimb experiments. The right forelimb disc was extirpated from embryos in
both temperature groups when stage 30 was reached. Extreme care was taken to
ensure that only that portion coextensive with somites 3-5 was removed and that
the dorsoventral diameter of the disc was also the same in every case. All animals
were examined at stage 46 for the presence of a right forelimb and the degree of
development attained. Most of the animals in both temperature groups were then
killed and preserved for future reference. The remaining animals were allowed to
grow until they were approximately 35 mm in length, 30 days from the date of
operation. These older animals consisted of lots of eight - four with and four
without limbs, derived from each operational set in both temperature groups.
The purpose in raising these animals beyond stage 46 was twofold: (1) to
ascertain whether animals without limbs at the end of the embryonic period
might yet develop them later, (2) to determine whether subsequent growth of
reconstituted limbs might differ between the two temperature groups.
Temperature and reconstitution
341
RESULTS
Post-operative rate of development
Animals in both temperature groups reached stage 46 in the same period of
time within their respective series - approximately 15 days in the ear experiments
and 14 days in the forelimb experiments. The time allotment for reconstitution,
therefore, was the same in each temperature group. Some observers have
reported that early retardation at low temperatures is partially or wholly offset by
a greater than normal acceleration in development once the animals are returned
to higher temperatures (Twitty & Elliott, 1934; Buchanan, 1938; Ryan, 1941).
With regard to the overall rate of development, as measured by attainment of
stage 46, there was no evidence that such was the case in the present experiments.
There may well have been acceleration in development in the 5° group during the
first few days of post-operative life, for no comparison between groups was made
during that period. In any event, whatever minor differences in developmental
rate may have occurred, these were not reflected in the total post-operative
time-span and, if present, can be attributed only to the effects of pre-operative
temperature differentials.
Ear reconstitution
The respective number of cases in which the ear was reconstituted in the two
temperature groups differed markedly. Table 1 summarizes the essential data
upon which an analysis of the difference between the two groups is based. In 309
animals maintained at 5 °C prior to ablation of the otic vesicle, the ear was
Table 1. Frequency of ear and forelimb reconstitution in the 5 and 14° C groups
Ear reconstitutiorii
Temperature
groups (°C)
No. of
animals
Average time
before
operation (h)
5
14
309
350
total 659
849
183
5
14
135
193
total 328
912
212
Total
160 (51-77%)
100(28-57%)
X2 = 360
Small
(40-350/0
Large
(360-700/*)
129(80-62%)
84 (84-0%)
31 (19-37%)
16(16-0%)
Forelimb reconstitution
101 (74-81 %)
103 (53-36%)
X2 = 14-6
reconstituted in 160 animals or 51-77 % of all cases. The average time spent at
5 °C for all the series within this group was 849 h, with extremes of 714 and 1003 h
depending upon the stage at which temperature differentials were initiated. In
350 animals maintained at 14 °C the ear was reconstituted in 100 animals or
28-57 % of all cases. The average time spent at 14 °C was 183 h, with extremes
of 137 and 216 h. Statistical analysis of these data indicates that the difference
23
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25
342
J. PIATT
between the two temperature groups is highly significant (P <^ 0-001). That
portion of the ear field which remained after ablation of the otic vesicle retained
its capacity for ear replacement to a greater degree in those embryos whose early
development proceeded at a slower rate, despite the fact that the visible differentiation of the vesicle was the same in both groups.
All of the reconstituted ears in both temperature groups were abnormally
formed and the great majority were small, undifferentiated vesicles. Degree
of differentiation is difficult to assess quantitatively, especially in very reduced
and grossly abnormal ears (Piatt, 1962, 1968). However, with hardly any
exception, the better-differentiated ears were also larger. Therefore the relative
development of the reconstituted ears is roughly but effectually a function of size
and is so evaluated in this study. Table 1 summarizes the respective number of
small and large ears in each temperature group. It may be seen that the percentage
of small ears in the 14° group is roughly 4 % more than in the 5° group, and 4 %
less in the large ear class. The differences are small but consistently favor the 5°
group with regard to ear size. Furthermore, if the number of reconstituted ears
is analysed separately for each of the two size classes, the difference between the
5° and 14° groups is statistically significant in both instances: small ears,
P < 0-001, x2 = 29-2; large ears, P < 0-001, f = 13-1. These data and their
analyses indicate: (1) a greater percentage of large ears in the 5° group than in
the 14° group, (2) statistically, a significantly greater regenerative potential for
both small and large ears in the 5° group than in the 14° group.
There is no consistent similarity in the respective number of reconstituted ears
among paired series in which temperature differentials were initiated at the same
stage of development; on the contrary, among some of the paired series there is a
great difference in outcome between clutches. Because the number of paired
series is relatively small, the results of such comparisons are inconclusive, but it
would appear that differences between clutches affect the capacity for ear
reconstitution to a greater extent than do differences in developmental age at the
initiation of temperature differentials, at least up to stage 9 in development.
Forelimb reconstitution
Table 1 summarizes the essential data upon which an analysis of the difference
between the two temperature groups is based. In 135 animals maintained at 5 °C
prior to ablation of the forelimb disc, the limb was reconstituted in 101 animals
or 74-81 % of all cases. The average time spent at 5 °C for all the series within
this group was 912 h, with extremes of 861 and 1051 h, depending upon the stage
at which temperature differentials were initiated. In 193 animals maintained at
14 °C the forelimb was reconstituted in 103 animals or 53-36 % of all cases. The
average time spent at 14° was 212 h, with extremes of 164 and 259 h. Statistical
analysis of these data indicates a significantly greater potential for forelimb
reconstitution in the 5° group than in the 14° group (P < 0-001). As in the case of
reconstitution of the ear described above, a slower rate of development results in
Temperature and reconstitution
343
a greater capacity for replacement of the missing primordium than when
development has proceeded at a faster pace, despite the fact that the visible
differentiation of the limb disc at the time of ablation was the same in both
groups.
Table 2. Relative development of reconstituted forelimbs at stage 46
after treatment at different temperatures
5°C
Stages of limb
development
37
38
39
40
41
42
43
44
45
Abnormal
14 °C
K
f
Cases
%
2
2
3
5
12
27
25
15
8
2
101
1-98
1-98
2-97
4-95
11-88
26-73
24-75
14-85
7-92
1-98
\
A
Cases
11
8
15
9
11
27
15
3
2
2
103
0/
/o
10-67
7-76
14-56
8-73
10-67
26-21
14-56
2-91
1-94
1-94
Table 2 summarizes the data on the comparative development of the reconstituted forelimbs at stage 46 - technically the termination of the embryonic period.
In both temperature groups reconstitution ran the entire gamut of development
from stage 37 through stage 45 forelimbs; none attained stage 46 in development
as did the left, unoperated forelimb. The distribution of the different stages of
forelimb development, however, varied markedly and consistently between the
two groups. The percentage of reconstituted limbs in less advanced stages of
development (37-40) was consistently greater in the 14° group; total percentages
were: 5° group, 11-88 %; 14° group, 41-72 %. The percentage of limbs that had
attained more mature stages of development (41-45) was consistently greater in
the 5° group; total percentages were: 5° group, 86-13 %; 14° group, 56-29 %.
Both groups peaked at stage 42 with practically equal percentages of cases.
These results demonstrate that not only is there a significantly greater number of
forelimbs reconstituted in embryos whose early development has been retarded
but that replacement of the missing part is accelerated and development is more
precocious, following the initial appearance of the limb bud.
The eventual development of the reconstituted forelimbs was approximately
the same in both groups. That is to say, regardless of their relative development
at stage 46, 30 days after ablation of the limb disc all limbs were well formed and
approximately normal in size, except two abnormal cases in each group. Despite
the fact that a greater number of limbs were slightly smaller than normal in the
14° group than in the 5° group at the end of 30 days, the difference in size was so
23-2
344
J. PIATT
minor that full parity with the normal, left forelimb would assuredly have been
attained had the animals been allowed to continue their growth. All of the
animals in which no right forelimb was discernible at stage 46 remained limbless
at the termination of the experiment in both temperature groups.
The range of early developmental stages at which temperature differentials
were initiated in the limb experiments is less than that in the ear experiments.
Consequently, comparison within the same stage or between stages is even less
conclusive than in the ear experiments. The evidence, however, points in the same
direction as in the ear experiments. That is, within the range of stages employed,
differences between clutches appear to be more effective in producing variability
in results than do differences between stages.
DISCUSSION
As early as 1924 Huxley pointed out the importance of distinguishing between
'invisible determination' and 'visible differentiation' in embryonic development,
and Needham (1933) in a review article called attention to the fact that various
consequent processes in ontogenesis may be separated from each other in time.
Since then a number of investigators have accumulated considerable evidence
that differences in rate of development in poikilothermal animals result in
differences in subsequent differentiation, growth or initiation of function. The
reader is referred to earlier papers (Piatt, 1968, 1969, 1971) for a brief review of
the literature on this subject and for additional evidence that the rate of development may affect subsequent differentiation. The present study furnishes related
evidence that embryonic regenerative potentials are also influenced by temperature differentials applied at early stages of development.
No satisfactory explanation can be offered at this time to explain the superior
regenerative capacity of ear and forelimb fields in animals whose early development has been retarded by hypothermia. It would appear, though, that prolonged
development tends to maintain the pleuripotency of the cells which constitute
ear and limb fields. In other words, their 'invisible determination' is less
advanced relative to their 'visible differentiation' than when development has
not been retarded. This appears to be the most plausible assumption in the
present instance to explain the results obtained. Actually, on the generally
accepted proposition that degree of cellular determination and embryonic age
are correlative functions, one would have anticipated a greater regenerative
potential in the 14° group than in the older 5° group because of the shorter
developmental period of the former.
The practical implication of these experiments is plain. Experiments concerned
directly or indirectly with the reconstitution of embryonic parts in amphibians
necessitate control of developmental temperatures prior to operation. Ten Cate
(Woerdeman & Raven, 1946, pp. 33-38), Woerdeman (1950) and Jacobson (1958)
have demonstrated the expediency of temperature control for comparison of data
Temperature and reconstitution
345
on lens induction and the results of the present study indicate that problems
dealing with the reconstitution of embryonic primordia must take into account
the pre-operative thermal environment as well as the amount of tissue removed
and the stage of development.
This research was supported by grant 1 RO1-NS08112-02 from the U.S. National Institutes of Health.
BUCHANAN,
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{Manuscript received 19 August 1970)