/ . Embryo!, exp. Morph. Vol. 21, 2, pp. 219-33, April 1969
219
Printed in Great Britain
The effect of cold on hind-limb growth and lateral
motor column development in Rana pipiens
By R. S. DECKER 1 AND J. J. KOLLROS 1
From the Department of Zoology, University of Iowa
A quantitative correspondence between nerve centers and their peripheral
area of innervation has long been inferred from comparative and pathological
studies. Many investigations have shown that by varying the mass or number of
peripheral structures (1) a hypoplasia or hyperplasia, or (2) a hypotrophy or
hypertrophy of their corresponding nerve centers results, indicating that a
developmental relationship exists between that periphery and its innervating
center. These changes in nerve cell number and size have been demonstrated in a
variety of sites, including the spinal cord and spinal ganglia of chicks and
amphibians (Piatt, 1948; Hamburger, 1955, 1958; Weiss, 1955).
In addition to control of nerve cell number and size by the periphery, thyroid
hormone has been demonstrated to affect, directly and indirectly, lateral motor
column (LMC) cell number and size in the frog, Rana pipiens (Beaudoin, 1955,
1956; Kollros & Race, 1960; Race, 1961). Reynolds (1963) has also shown that
thyroxine in high concentrations can initiate a precocious loss of motor cells in
small tadpoles. In R. pipiens, therefore, the final number and size of the LMC
cells may depend upon both thyroid hormone and the presence of the limb,
whereas in chick embryos only the peripheral field seems to control motor horn
development (Hamburger, 1958).
The present research was undertaken to analyse the role of the peripheral
field in the development of the LMC of R. pipiens larvae exposed to varying
degrees of hypothermia, since cold has been shown to enhance growth while
retarding metamorphosis (Huxley, 1929; Etkin, 1955, 1964; Kollros, 1961).
This study involved an examination of the effect of hypothermia on (1) the
growth of R. pipiens larvae, with special attention directed towards the development of the hind limbs, and (2) LMC cell number, size and possible asynchronies
produced between limb and LMC stage. The results are interpreted in the light
of current knowledge of periphero-central effects, the role of thyroxine in limb
growth and spinal cord maturation and the possible indirect role of hypothermia
in inhibiting thyroid hormone release.
1
Authors' address: Department of Zoology, University of Iowa, Iowa City, Iowa 52240,
U.S.A.
15
JEEM2I
220
R. S. DECKER & J. J. KOLLROS
MATERIALS AND METHODS
An adult R.pipiens, collected in Wisconsin, was artificially induced to ovulate,
and the eggs were fertilized, utilizing the method described by Rugh (1934).
When the tadpoles of this single clutch developed to stage 25 (Shumway, 1940),
they were divided into groups of 30 and placed in large pans containing 4 litres
of aerated tap water, kept at 24-26 °C. When the larvae reached stage V (larval
stages according to Taylor & Kollros, 1946) they were regrouped for continuous
exposure to colder temperatures. Stage V was selected for the transition since the
lateral motor column cells are segregated from the adjacent mantle layer at this
time, and can be counted readily without confusion with adjacent mantle cells
(Beaudoin, 1955; Reynolds, 1963). Only those animals which had exhibited
identical growth rates were chosen for the studies. Density of the larvae was
maintained at ten per pan in order to establish growth independent of crowding
influences (Adolph, 1931). The temperatures used were: Group I, 22±1 °C;
Group II, 18 ±1 °C; Group III, 14 ±1 °C; Group IV, 10 ±1 °C; Group V,
6 ± 1 °C. Animals were fixed in Bouin's fluid at stages X, XIII, XVII and XX.
Control animals at stage V were also fixed. Just prior to fixation, measurements
were made of total length, body length and hind-limb length. Hind-limb volumes
were estimated for stages X and XIII larvae (using the formula for a cylinder,
V = 77r2h) and for stages XVII and XX larvae (using the formula for a cone,
V = -377T2h). The lumbo-sacral areas of the spinal cords were removed from the
fixed animals, dehydrated, embedded in paraffin and sectioned serially at 10 /.c.
Slides were stained with Ehrlich's acid haemotoxylin and light green.
Cell counts of the LMC were made in the mid-region of the lumbo-sacral
cord, counting 20 consecutive sections on the right and left sides of each animal;
additionally, total cell number determinations (counting every fifth section,
right and left) were made for each animal of the group. Inasmuch as the LMC
cells are larger than glial cells or adjacent cells of the mantle, and in later stages
very much larger, it is unlikely that cells other than those appropriate for the
LMC itself were counted. The cell counts were expressed as the mean cell
number per column per section for each side, or as total motor cell number per
column per animal. Cells were counted only when one or two nucleoli were seen
within their nuclei. The cell number figures are in every instance, except for the
'corrected' column of Table 2, the crude or uncorrected numbers. Two nucleoli
per cell are seen so infrequently as to require no obvious correction; in any
event they would be found about as frequently in one stage as in another, and
thus the stage-to-stage comparisons would not be affected. If the thickness of
enumerated objects is significant in relation to section thickness, a correction is
demanded (Abercrombie, 1946), whereby the counted number is reduced. Since
nuclei were counted only when a nucleolus was present, it is the thickness of
nucleoli which is critical. These were shown by Beaudoin (1955) to vary from
0-8 ii at stage V to about 3-5 JLL at stage XX. Thus, in order to get true values of
Cold effect on motor column
221
counts, the apparent counts would need to be reduced for each stage as follows:
V, 7 %; X, 16 %, XIII, 20 %, XVII, 24 %; XX, 26 %. These are presumably
the maximum corrections which are applicable. Failure to identify the small
'end' sections of nucleoli, or the displacement of a nucleolus by the sectioning
knife into one or another of two adjacent sections, as apparently happens on
occasion, would reduce the value of the necessary correction.
110 r-
100
90
80
-
70
60
50
• Group I
o Group II
A Group III
o Group IV
A Group V
40
20 40 60 80 100 120 140 160 180 200 220 240 260
Age in days at a given temperature
Fig. 1. A comparison of the mean larval length of R. pipiens tadpoles exposed to
different temperatures. Each point represents the mean length, in mm, for the indicated mean age of each larval group.
At least 75-125 nuclei per column per side per animal were also drawn with
the aid of a camera lucida. These cells were measured with a polar planimeter
and the planimeter units were converted to square microns. The nuclear
volumes were also computed, utilizing the formula for a prolate spheroid as the
representative shape of an LMC cell nucleus (after Beaudoin, 1955). In order to
avoid bias, all the nuclei in a given section that were counted were drawn and
measured. The data on larval growth, cell counts and cell sizes were expressed
with standard error, and each group was compared to the other, using Student's 'f' test and regression analysis to determine significant differences
between groups.
15-2
XIII
X
XIII
XVII
XX
X
XIII
XVII
XX
X
XIII
XVII
XX
X
XIII
XVII
'' With standard error.
Group V
at6°C
Total
Group IV
at 10 °C
Group III
at 14 °C
Group II
at 18 °C
V
Controls
at 25 °C
Group I
at 22 °C
X
Stage
fixed
Temperature
treatment
8
9
8
8
8
6
7
6
6
6
6
6
6
6
7
4
4
111
4
animals
No.
of
11-4 ±2-1
25-3 ±2-6
42-8 ±2-3
49-7 ±1-8
21-6 ±1-9
37-4 ±2-2
56-3 ±2-6
66-2 ±3-1
40-5 ±2-3
86-4 ±3-1
131-7±4-8
187-9 ±4-2
67-2 ±2-7
132-6±3-6
234-2 ±6-8
118-7 ±10-6
261-4±15-7
Mean age
in days
after
stage V*
57-7 ±2-84
64-2 ±3-47
77-5 ±3-67
71-2±3-86
64-3 ±2-61
74-2 ±3-02
89-7±416
77-6 ±3-94
68-7 ±3-11
88-3 ±4-36
95-7 ±5-02
84-7 ±5-35
74-8 ±4-87
89-3 ±3-66
99-8 ±302
67-4 ±4-78
77-3 ±5-37
38-3 ±3-02
Mean
length of
larvae
(mm)*
15-3± 1-12
20-4 ±1-43
25-3 ±1-37
23-7 ±1-07
17-9+ 1-31
23-6 ±1-67
28-6 ±1-43
25-8 ±1-29
21-0 ±1-81
27-3 ±2-10
32-7 ±1-69
28-6 ±1 01
27-2 ±1-35
32-1 ±1-39
33-1 ±1-27
23-9 ±2-67
30-4 ±2-95
14-2±1-14
Mean body
length
(mm)*
2-1 ±0-35
4-1 ±0-75
12-4 ±0-85
36-2 ±1-11
2-4 ±0-41
6-3 ±0-32
16-5 ±0-37
39-8 + 101
2-8 ±0-21
7-6 ±0-34
19-9 ±0-41
43-3 ±1-27
3-8 + 0-31
12-4 ± 0-64
23-9 ±0-69
2-1 ±0-87
5-2 ±0-75
1-1 ±0-27
(mm)*
Mean
hind-limb
length
Table 1. Effects of hypothermia on larval growth in R. pipiens.
0-7 ±0-21
1-7 ±0-42
10-8±7-21
1161 ±14-32
1 0 ±0-34
3-2 ±0-75
25-8 ±6-63
137-4 ±15-64
1-6 ±0-57
6 0 ±0-89
46-8±3-12
249-7 ±18-2
3-9 ±1-24
13-8 ±2-07
79-2 ±8-75
0-6 ±0-22
2-2 ±0-34
Mean
hind-limb
volume
(mm 3 )*
1-74
1-87
1-94
2-02
1-70
1-89
1-96
2-05
1-75
1-85
1 91
1-95
1-70
1-78
1-98
1-82
1-54
1-70
Ratio of
TL.BL
014
0-21
0-49
1-53
013
0-27
0-58
1-54
013
0-28
0-61
1 51
014
0-38
0-72
009
017
007
Ratio of
HLL.BL
on
o
r
r
o
'
Op
tn
?o
o
o
m
50
/>*
223
Cold effect on motor column
RESULTS
I. General effects of hypothermia on growth and metamorphosis ofR. pipiens
The colder the water in which the larvae were kept, the greater was the total
length and limb length achieved by animals in each group, except for those
maintained at 6 °C (Table 1; Fig. 1). An examination of tail length to body
length (TL:BL) and hind-limb length to body length (HLL:BL) ratios confirms
45
u
50
40
• Control
• Beaudoin, 1955
• Group I
o Group II
A Group III
D Group IV
A Group V
"45
40 35
35
H 30
25
30
25
20
20
15
_ 15
I 10
c 10
v ix
X
XIII
J
XVII
Stage of development
Fig. 2
I
L
XX
• Group I
Group II
A Group III
° Group IV
A Group V
0
I
20
I
I
I
i
i
i
I
I
I
60
100 140 180 220 260
Age in days at a given temperature
Fig. 3
Fig. 2. A comparison of LMC cell number in the mid-region of the lumbo-sacral
spinal cord of R. pipiens tadpoles exposed to different temperatures. Each point
represents the mean cell number per section per column for the indicated larval
stage. The values indicated by (•) are after Beaudoin (1955).
Fig. 3. Regression lines of the calculated mean LMC cell number against the age of
the larvae. Lines A, B, C and D represent stages X, XIII, XVII and XX, respectively.
that the pattern of larval growth is comparable to that demonstrated by control
larvae at 25 °C, indicating that the relative growth rates are approximately
normal (Etkin, 1964; Table 1). Only the larvae of Group V demonstrated a different pattern, with both the HLL:BL and TL:BL ratios differing substantially
from the ratios expressed by the warmer groups. In Group V the changes in
total length and hind limb lengths appeared to be greatly inhibited rather than
stimulated by 6 °C treatment.
224
R. S. DECKER & J. J. KOLLROS
II. The effects of hypothermia on the development of the
lateral motor column (LMC)
With each decrease in temperature the mean motor-cell count per section
increased in stage-for-stage comparisons (Table 2; Figs. 2, 3). Total cell counts
of the entire LMC were also computed, and indicated increases with each
168
162
156
150
144
138
132
126
120
114
108
102
• Group I
Group II
^ Group III
o Group IV
A Group V
• Beaudoin, 1955.
0
96
90
84
78
• Group I
° Group II
A Group III
a
Group IV
A Group V
• Beaudoin, 1955
72
66
XIII
XVII
Stage of development
Fig. 4
XX
XIII
XVII
XX
Stage of development
Fig. 5
Fig. 4. A comparison of the nuclear cross-sectional area of the LMC cells in the
mid-region of the lumbo-sacral cord of R. pipiens tadpoles exposed to different
temperatures. Each point represents the mean value for 75-125 nuclei per column
per animal for each indicated larval stage.
Fig. 5. A comparison of the nuclear volumes of the LMC cells in the mid-region of
the lumbo-sacral cord of R. pipiens tadpoles exposed to different temperatures.
Each point represents the mean value for 75-125 nuclei per animal for each indicated
larval stage.
decrease in temperature (Table 2). Nuclear area and volume, on the other hand,
were unchanged in response to a decrease in temperature (Figs. 4, 5). When these
data were compared with those of Beaudoin (1955), it was seen that there are
no differences in cell sizes, whereas cell numbers were significantly higher than
those of Beaudoin.
Cold effect on motor column
225
A. Group I, maintained at 22 ± 1 °C
The number of motor cells found in the LMC of animals kept at this temperature was greater than the number found by Beaudoin (1955) in all stages examined except for stage XX (Table 2). Measurements of nuclear area and volume
at equivalent stages of larval development indicate that no differences exist
between Beaudoin's data and the nuclear sizes of the LMC cells in Group I
(Table 2).
B. Group II, maintained at 18 ± I °C
Tadpoles of this group had mean cell numbers that were also greater than
those of Beaudoin (1955). A comparison of mean cell numbers between Groups I
and II failed to show any significant differences for stages X and XVII, while
those for stages XIII and XX were slightly significant (P < 005). An examination of mean nuclear size (Table 2) failed to show any significant differences
between Beaudoin's data and that of Group I except marginally for one stage,
XVII. Nuclei of Group I and of Beaudoin were both larger than for Group II
at this stage (P < 005).
C. Group III, maintained at 14 ±1 °C
The larvae of Group III possess still higher mean cell counts, stage-for-stage,
than do the two previous groups and Beaudoin's controls (Table 2). While
motor cell number was not significantly different between Groups II and III,
changes were apparent between Groups I and III. Differences for stages XIII
and XX were significant at the 1 % level, while those for stages XVII and XX
were only slightly significant (P < 005). No apparent differences in mean
nuclear size could be detected by comparing Group III to any of the groups
kept at higher temperatures.
D. Group IV, maintained at 10 ±1 °C
In this group of tadpoles the mean cell number for each developmental stage
was still greater than that found in any previous group; a comparison of Group I
and Group IV indicated that cell counts at all stages of development were significantly different from each other (P < 001). No significant differences in
motor cell number were detected between Groups III and IV, and an examination of cell number difference between Groups II and IV revealed a significant
difference only for stage XVII (P < 001). Differences in nuclear size were
found to be marginally significant between stages XIII and XVII of Groups II
and III as against Group IV (P < 0-05).
E. Group V, maintained at 6 ±1 °C
When these larvae were compared to all other groups of animals at stages X
and XIII, the differences were found to be significant with respect to their
4
—
—
—
—
—
—
—
4
5
4
4
4
4
5
4
4
4
4
4
4
4
5
4
4
71
V
V
IX
XI
XIII
XVII
XIX
XXI
X
XIII
XVII
XX
X
XIJI
XVII
XX
X
XIII
XVII
XX
X
XIII
XVII
X
XIII
Controls (25 °C)
Beaudoin (1955) at
approx. 25 °C
47-8± 211
51-1 ± 1-13
38-8 ±1-08
17-7 + 0-97
121 ±0-42
6-8 ±0-38
7-9 ±0-45
8-3 ±0-65
29-3 ±1-04
15-5 ±0-89
13-5 ±0-67
8-3 ±0-65
32-4 ±1-15
18-2 ±0-98
14-3 ±0-59
10-9 ±0-42
33-0 ±1-07
22-4 ±1-02
18-4 ±0-93
13-7±0-71
36-6± 114
21-3 ±1 01
20-3 ±0-96
41-2 ±3-23
29-212-99
7327 ±285
—
—
—
—
—
—
—
4898 ±176
3474 ±196
2526 ±211
2243 ±114
5435 ±227
3786 ±235
2983 ±183
2536 ±107
6042 ± 247
4702 ± 200
3887 ±163
3621 ±123
6443 ±281
5839 ±142
4688 ±123
6921 ±314
6081 ±247
Uncorrected
6814
—
—
—
—
—
—
—
4114
2779
1920
1660
4565
3029
2267
1877
5075
3762
2954
2680
5412
4671
3563
5814
4865
Corrected
Mean cell count per
column per animal*
* With mean ± standard error.
t Nuclear volume computed using the formula for a prolate spheroid, V = 4/3 TTab2, with standard error.
Total
Group V (6C C)
Group IV (10 °C)
Group III (14 °C)
Group II (18 °C)
Group I (22 °C)
No. of
animals
Stage
fixed
Temperature
treatment
Mean cell
count per
column per
section*
58-4 ±2-61
62
61
105
105
131
143
167
77-3 ±2-73
101-2 + 3-13
126-8 ±3-47
151-1 ±5-26
80-8 ±2-84
99-6 ±3-62
110-7 ±4-62
147-7 ±5-76
82-2 ±2-95
103-8 ±3-21
119-4 ±3-02
155-1 ±5-67
84-3 ±2-76
1J1-7 + 3-88
127-5 ±5-26
67-2 ±2-15
91-5 ± 3 0 2
a r e a * (JLC2)
Mean nuclear
Table 2. Effects of hypothermia on LMC cell number and nuclear area and volume
253 ±14-7
270
282
710
725
996
1148
1475
483 ±15-6
654 ±23-7
974 ±43-7
1297 ±57-8
503 ±21-3
627 ±29-2
772 ±37-9
1203 ±49-3
509 ±28-4
668 ±31-2
823 ±42-1
1312±52-1
521 ±30-2
781 ±39-2
975 ±59-5
359 ±12-4
549 ±23-2
Mean nuclear
volume (/A3) *t
J
in
/••>i
r&
O
w
&
C
o
m/"*
?°
226
Cold effect on motor column
227
larger cell number (P < 0-001). When nuclear size measurements were examined, however, the nuclei were found to be significantly smaller (P < 005)
than in the cells of the previous groups (Table 2).
F. Controls, fixed at stage V
These animals, maintained at 25 °C until being fixed at stage V, showed, on
average, 7327 cells in each lumbo-sacral motor column. The mean cell counts
per section and nuclear sizes (Table 2) were close to those published by Beaudoin
(1955). In Groups I-V, all average cell counts, per section or per column, were
smaller than at stage V, and in all instances nuclear sizes were greater than at
stage V.
DISCUSSION
The present study indicates that differences in the environmental temperature
affect LMC cell numbers and larval growth, while not greatly affecting LMC
cell size. These external effects are expressed in three ways: (1) for each decrease
in temperature the period of time necessary to attain a given developmental
stage increases; (2) for each decrease in temperature a larger body length and
total length is achieved for each stage; (3) for each decrease in temperature a
greater hind limb length is seen for' each stage, but with little variation in the
HLL: BL ratios for stages X and XX, but with considerably greater values for
such ratios at stages XIII and XVII.
In the cold the LMC cell counts are all reduced below the initial values established at stage V, but are nonetheless graded with both temperature and stage
in such a way that with temperature held constant there is a decline in cell
numbers in successive stages, and when comparing individual stages the cell
counts are greater the colder the environment. On the simple notion that
adjustments in LMC cell populations ordinarily come about by progressive
cell loss, it can be suggested that cold differentially affects body growth and the
cell loss mechanisms, such that in the cold, for any stage, cell loss rate is reduced, and the degree of this reduction varies directly with the degree of temperature decrease. If, however, regulation of LMC cell populations in R. pipiens
is on the same basis as reported by Hughes (1961) in Xenopus laevis, in which
cell recruitment into the LMC proceeds even while cell degeneration is going on,
but at a slower rate than the cell degeneration, then it would be necessary to
postulate that the cold differentially affects the cell recruitment and the cell loss
mechanisms, favoring the rate of recruitment more than the rate of cell loss, thus
accounting for the progressive lessening of cell number reductions with progressive drops in temperature. In this regard it is worth noting that such differential
influences upon cell migration to the LMC and cell degeneration within the
LMC have been reported in X. laevis by Hughes & Fozzard (1961) following
X-irradiation. They also reported a stage-dependent variation of the influence
of X-irradiation.
228
R. S. DECKER & J. J. KOLLROS
In this study subjection to cold was begun at stage Y, and preliminary data
of J. J. Kollros & H. Kaung (unpublished) indicate that no or very few LMC
cells are produced by cell division after stage V. The modifications in LMC
numbers must be accounted for by either the reduction of cell degeneration
tendencies, or by enhancement of the capacity of cells still in the mantle layer to
migrate into the LMC and differentiate as LMC motor cells, or by some combination of these two alternatives. The question must still be asked, how does the
cold bring about the larger cell numbers, stage for stage? Is the larger cell
10 4 i-
10d,=
£
== 10 3
10-
103
102
• Group
° Group
A Group
a Group
±Group
• Group I
o Group II
AGroup III
a Group IV
A Group V
I
II
III
IV
V
XIII
XVII
Stage of development
Fig. 6
XX
XIII
XVII
Stage of development
XX
Fig. 7
Fig. 6. Ratios of the total LMC cell number per column to estimated hind-limb volume
of tadpoles exposed to different temperatures.
Fig. 7. Ratios of the total LMC nuclear volume per column to estimated hind-limb
volume of tadpoles exposed to different temperatures.
number a response to the larger than normal limb? Is thyroid hormone less
effective in the cold than at warm temperatures in initiating cell degeneration?
Is the larger cell number a reflexion of a decreased thyroxine concentration in
the blood at the lower temperatures ?
Many investigations have demonstrated that hyperplasia of motor and sensory centers follows an increase in the mass of the periphery to be innervated
(review in Piatt, 1948). In the present study the increase in the periphery—
the hind limb—which is manifested increasingly with successive decreases in
Cold effect on motor column
229
temperature, is associated with cell numbers in the LMC which also increase
with decreases in temperature, compared to the work of Beaudoin at about
25 °C. There is, thus, a positive correlation between LMC cell number and
limb size, even though limb size grows, stage by stage, and cell number falls.
The values are quite different for the various experimental groups in the early
stages, but at stage XX the values come very nearly together (Fig. 6). Since this
stage XX relationship is independent of temperature, it is possible that the mass
of limb musculature might control the number of motor cells which are retained,
and that as stage XX is approached the degree of this control is progressively
established.
A second possibility of control of motor cell number is suggested by the
studies of Kollros (1956, 1961), who has followed tissue responses of hypophysectomized (and thus effectively thyroidectomized) tadpoles to given concentrations of thyroid hormones at different temperatures. As an example,
rupture of the skin window may occur with thyroxine concentrations of 0-4 jag.j\.
at a temperature of 25 °C, but if the temperature is kept at 15 °C concentrations
as great as l-O/tg./l. are ineffective in bringing about skin window perforation
and emergence of the forelimbs in R. pipiens. Cold, therefore, produces not only
a reduced rate of change for a given hormone dosage, but also a substantial
elevation in the hormone concentration thresholds required to achieve given
metamorphic changes (Kollros, 1961). The cell degeneration mechanisms might
possess similar temperature dependent threshold requirements. It is worth
noting that, since the parent frogs were from Wisconsin, and thus nearer the
northern limit of their range than the middle of their north-south distribution
(Blair et al. 1957), the tadpoles might have been expected to possess response
capacities to cold somewhat different from those of tadpoles derived from
animals originating from warmer, more southern parts of the distribution of
this species.
A third possibility of control of motor cell number also relates to thyroid
hormone concentration, and the way in which this concentration is influenced by
hypothalamic control of thyroid stimulating hormone (TSH) production and
release. The possibility of temperature influences controlling metamorphosis
by way of the hypothalamus was suggested by Etkin (1964). This idea was
supported by the earlier studies of Etkin & Lehrer (1960), in which pituitary
primordia were transplanted ectopically to isolate them from the influence of
the hypothalamus. Such animals had their growth rate increased greatly, while
metamorphosis was inhibited to varying degrees, depending upon the location
of the graft. It has further been shown by Hanaoka (1967) that the hypothalectomized tadpoles of R. pipiens grow to much larger sizes than normal, but that
none of the animals proceeds beyond stage XIX, i.e. not quite to the stage of
forelimb emergence. Studies of Voitkevich (1962) indicate that in the cold
(approximately 10 °C) the accumulation of Gomori-positive neurosecretory
material in the preoptic nucleus of the hypothalamus is favored, apparently
230
R. S. DECKER & J. J. KOLLROS
none of this accumulation being transferred to the anterior pituitary. Voitkevich
(1963) also showed that in the cold the acidophil-basophil ratio indicated an
increase in the number of acidophils when compared to the animals kept
warm, presumably demonstrating that growth hormone is preferentially secreted
over TSH. When the tadpoles were transferred from cold to warm water, the
neurosecretory material was observed to migrate into the adenohypophysis, and
at the same time the acidophil to basophil ratio changed, favoring the elaboration of TSH over that of growth hormone, thus promoting metamorphosis.
Etkin (1964) and Voitkevitch (1963) agree in implying that in the cold the level
of thyroid hormone output decreases with decreasing environmental temperature. Since Kollros & Race (1960) and Race (1961) have demonstrated that
LMC cell numbers are directly dependent upon the level of thyroid hormone
made available to hypophysectomized tadpoles, it is proper to suggest that the
reduction in TSH output which is brought about by hypothermia might be
responsible for lowered thyroid hormone titers in the blood, and thus be indirectly responsible for lateral motor columns with more than the usual number
of motor cells.
Cell sizes, as reflected in the sizes of their nuclei, appeared to be influenced
but little by differences in temperature, except that at the lowest temperature
of 6 °C nuclear sizes were significantly smaller than in animals of the same
stages maintained in warmer water. The nuclear sizes were also very close to
those reported by Beaudoin (1955). Since Beaudoin (1956) demonstrated that
stimulation of limb growth (by implantation of thyroxine-containing pellets
directly into the hind limb) was quickly reflected in increase in LMC cell size,
it may be asked why similar relationships were not shown in this study. The
significant feature appears to be that in the present work comparisons are all
between animals of the same stage, whereas in the limb-growth comparisons of
Beaudoin his stimulated limbs were not only larger, but they had also differentiated more fully, being up to two stages more advanced than the control
limb of the contralateral side, which was used as a control.
Finally, since the relationship between LMC cell numbers and mass of the
hind limb appeared to become identical at stage XX, for animals maintained
at various temperatures, and since cell sizes varied but little, it was expected
that a similar relationship might be expressed between total volume of LMC
cells and total mass of the hind limb. These relationships are shown in Fig. 7,
and are as anticipated. Thus, as metamorphic climax approaches the ratio of
motor cell volume per LMC to the hind limb mass becomes constant, and for
those conditions which permit the animal to develop to stage XX (above 10 °C
for R. pipiens) this ratio appears to be independent of temperature.
Cold effect on motor column
231
SUMMARY
1. The present study indicates that differences in environmental temperature
affect larval growth as follows: (1) the lower the temperature, the longer the
larval period; (2) the lower the temperature, the longer the larval size at each
stage; (3) the lower the temperature, generally, the longer the hind limb at each
stage.
2. The hind-limb length to body length and tail length to body length ratios
indicate that in the temperature range of 22-10 °C the rate of larval growth is
slowed, but is not distorted greatly.
3. Greater hypothermia (6 °C) resulted in animals whose total length at a
given stage was less than for those animals maintained at higher temperatures,
rather than greater. Limb proportions relative to body length also failed to
conform with those obtained at higher temperatures.
4. The low temperatures at which the larvae were kept resulted in an increase
in lateral motor column cell number for each stage. Three possible mechanisms
of cell retention are considered: (1) cell retention to innervate a large limb;
(2) reduced sensitivity to the 'cell loss mechanism' to thyroid hormone; (3) decrease in thyroid hormone titer, and thus less cell loss.
5. Despite variations in cell numbers in relation to limb size at early stages,
at different temperatures, at stage XX a constant relationship was obtained
between the ratio of lateral motor column cell number to hind limb volume.
A similar relationship was also obtained, at stage XX, between total lateral
motor column nuclear volume and hind-limb volume.
RESUME
Action dufroid sur la croissance du membre posterieur et le
developpement de la colonne motrice laterale, chez Rana pipiens
1. Les recherches indiquent que des differences de temperature du milieu
affectent comme suit la croissance larvaire: (1) plus la temperature est basse,
plus la periode larvaire est longue; (2) plus la temperature est basse, plus la taille
de la larve a chaque stade est elevee; (3) plus la temperature est basse, plus le
membre posterieur est allonge, en general.
2. Le rapport entre la longueur du membre posterieur et la longueur du corps
et le rapport entre la longueur de la queue et la longueur du corps indiquent que,
dans la gamme des temperatures de 22 a 10 °C, le taux de croissance larvaire
est ralenti mais n'est pas fortement perturbe.
3. Une hypothermie plus forte (6 °C) a produit des animaux dont la longueur
totale a un stade donne etait moindre, plutot que plus grande, que celle
des animaux maintenus a des temperatures plus elevees. Les proportions des
membres par rapport a la longueur du corps n'etaient pas conformes a celles
obtenues a des temperatures elevees.
232
R. S. DECKER & J. J. KOLLROS
4. Les basses temperatures auxquelles on a maintenu les larves ont provoque
un accroissement du nombre de cellules de la colonne motrice laterale a chaque
stade. On considere trois mecanismes possibles de retention cellulaire: (1) retention cellulaire pour innerver un membre plus gros; (2) sensibilite reduite
du 'mecanisme de perte des cellules' envers l'hormone thyroidienne; (3) diminution du titre en hormone thyroidienne, et par suite moindre perte de cellules.
5. En depit des variations du nombre de cellules par rapport a la taille du
membre aux premiers stades, a des temperatures differentes, une relation
constante a ete obtenue au stade XX entre le rapport du nombre de cellules
de la colonne motrice laterale au volume du membre posterieur. Une relation
similaire a ete egalement obtenue, au stade XX, entre le volume nucleaire total
de la colonne motrice laterale et le volume du membre posterieur.
This study was accomplished, in part, while one author (R.S.D.) held a National
Institute of Mental Health traineeship. The research was supported by PHS Research
Grant no. AM 02202 from the National Institute of Arthritis and Metabolic Diseases. One of
us (R.S.D.) is presently a National Institutes of Health Predoctoral Fellow.
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(Manuscript received 30 April 1968)