/ . Embryo!. exp. Morph., Vol. 16, 3, pp. 401-430, December 1966
With 2 plates
Printed in Great Britain
401
The thyroid and the
development of the nervous system in
Eleutherodactylus martinicensis:
an experimental study
By ARTHUR HUGHES 1
From the Departments of Zoology of the Universities of Bristol
and of the West Indies
INTRODUCTION
In a recent paper (Hughes, 1965 a) the ontogeny of the peripheral nervous
system in Eleutherodactylus martinicensis was studied by counting the numbers
of constituent fibres in the nerves of the hind limb at several stages of development. Eleutherodactylus is one of the smallest of living tetrapods and such
quantitative studies are aided by the comparatively small numbers of fibres
within its peripheral nerves. The next step in the analysis of the various changes
which occur simultaneously in the normal embryo is to attempt to disengage
them by some treatment which affects the general course or development.
Interference with the endocrine system of the embryo is one obvious line of
investigation, more particularly as a number of events in the development of
the nervous system in other vertebrates have been shown to be influenced by
thyroid hormones.
The action of the thyroid in Eleutherodactylus has already been investigated
by Lynn & Peadon (1955). In this Anuran development is wholly embryonic
and direct; of the metamorphic events at organ level in larval Amphibia, only
the loss of the tail and of the pronephros remains. The loss or retention of the
tail in Eleutherodactylus provides a valuable index of the extent to which the
influence of the thyroid gland has been reduced under experimental treatment,
especially as only limited information can be obtained from the histology of the
gland itself in this animal. In these pages, the results are described of treatment
with an anti-thyroid agent, and of both thyroidectomy and of hypophysectomy
on a number of features of development, mainly with reference to events within
the nervous system. The results obtained are summarized in Table 1.
1
Author's address: The Department of Zoology, The University, Bristol 8, England.
402
A. HUGHES
MATERIALS AND METHODS
The source of the embryos of E. martinicensis and the methods of culturing
and observing them are the same as those used in previous studies (Hughes,
1962, 1964a, b, 1965a, b, c). Unoperated embryos of Eleutherodactylus shed
from their envelopes develop equally well in either water or saline.
Table 1
Hypophysectomy
A
I
Effect of...
PTU
Thyroidectomy
Partial
Full
EVENTS RELATED TO METAMORPHOSIS
None, if operation Slowed
at stage ' b ' or
later
Loss of pronephros Slowed or halted None
None
Loss of Rohon-Beard Slowed or halted Slowed
None
cells
Loss of tail
Slowed
Halted
Slowed
Much slowed
EVENTS UNRELATED TO METAMORPHOSIS
Argyrophilia of
ventral horn cells
Number of nerve
fibres entering
triceps femoris
Loss of innervation of
grafted supernumerary limb
Slight loss
Decreased on
treatment at
stage 'a'
Inhibited
Slight loss
Moderate Complete loss
loss
Decreased if opera- None
Gradual
decrease
tion at stage ' b '
—
—
—
Relatively crude methods of thyroidectomy and of hypophysectomy were
adequate for Eleutherodactylus embryos, which suffered no special inconvenience from mutilation of the oral region. For the former operation the lower
jaw was removed as far back as the border of the third aortic arch, and behind
the zone of the developing thyroid. In all of thirteen embryos submitted to this
operation and subsequently examined, regenerated follicles were seen 6-7 days
later, atypical both in number, size and distribution. The partial absence of the
developing gland, however, in a critical period during the first few days after
operation was effective in preventing atrophy of the tail.
Hypophysectomy was attempted on embryos at 7-9 days (stages ' a ' to ' d '
of Table 2) by excising tissue from the roof of the mouth to a level extending
dorsalwards to the floor of the mid-brain. Of 48 operated embryos, the pituitary
was completely removed in 21. The remainder served as sham-operated controls.
In Eleutherodactylus embryos towards the end of development the various
regions of the pituitary can be recognized, though histological differentiation
within each region has not progressed very far. Where however the organ has
not been completely removed, and some hypophysial tissue is present in distorted
Thyroid and nervous system of E. martinicensis
403
relationships, the region to which it belongs cannot be recognized with certainty, particularly as the constituent cells-are less differentiated than in a normal
embryo. In some examples there was evidently a regenerated anterior lobe.
However, all embryos in which hypophysectomy was incomplete are so described in succeeding pages with no attempt to identify the nature of the
residual tissue.
Table 2. Stages of development o/Eleutherodactylus
martinicensis at 25-30 °C
Stage
Days
(approx.)
a
b
7
7*
c
8-8*
d
e
f
8^—9
10
11
g
h
i
12
13
14
Features
Hind limb spatulate, digits on forelimb outlined
First leg movements, melanophores on ventral surface widely
apart
Leg movements independent of trunk. Melanophores
approach ventrally in mid-line
Retraction reflex in hind limb well marked. Egg tooth
Leg extension. First xanthophores over head
Swims. Head xanthophores show interorbital line, sparse
xanthophores in trunk
Tail shrinks to 2-3 mm. Melanophores reach tail
Tail 1-2 mm
Tail a vestige. Air breathing
These glandular extirpations were performed at later stages of development
than are used for the corresponding operations on larval amphibia (Rugh,
1948), for at comparable stages the embryo of Eleutherodactylus is inaccessible.
In one series of embryos an arm was severed and then grafted near an undisturbed leg, in the same way as in previous studies on supernumerary limbs
(Hughes, 1962, 1964a, b).
On several occasions embryos were treated with thyroxine dissolved in saline
at concentrations of 1:107 or 1:108. The sodium salt of L-thyroxine was used,
supplied by British Drug Houses Ltd.
Rate of development and external appearance
Close observations were made on the rate of development of the experimental embryos. One set of landmarks were provided by their progress in reflex
behaviour (Hughes, 1965b). This can be correlated with changes in external
form as is set out in Table 2. This information supplements, and in some details
corrects, that given in an earlier paper (Hughes, 1962, Table 1), in which the
appearance of the egg tooth is wrongly placed later than that of the first xanthophores. A further well-marked stage is when the xanthophores on the head
form an 'interorbital line' separating a dense anterior zone from a sparser area
404
A. HUGHES
which extended caudally behind the head. Melanophores first appear in the
tail at the time when its shrinkage begins.
No differences were observed between the development of intact embryos of
Eleutherodactylus shed from their envelopes and immersed in either water or
Holtfreter saline. The skin thus appears to be largely impermeable to ions.
In Text-fig. 1 the course or development at 26-30 °C of control embryos
freed from their envelopes at various stages and then immersed in saline is
shown. Allowance is made in this figure for instances when an embryo shows
one feature only of a particular stage (e.g. leg extension without xanthophores).
1
2
3
4
5
6
7
8
Days
Text-fig. 1. Development of control embryos in saline; stages as in Table 2 with
isopleths of tail length of 20 mm and 30 mm respectively.
The rate of development is independent of the stage at which the embryo is
removed from its envelopes. However, in those removed relatively early (at
stages ' a ' or 'b') the tail remains at its maximum size of about 4-0 mm in
length for 3-4 days, while in those animals placed in saline at later stages
(' d'-' e') the tail begins to shrink by the end of the first day of immersion. Thus
in Text-fig. 1, where the dotted lines represent isopleths of tail length, those for
3-0 and 2-0 mm tails respectively bulge obliquely towards the top right of the
figure.
The effect of 0-01 % phenylthiourea (PTU)
Lynn & Peadon (1955) showed that treatment of Eleutherodactylus embryos
with PTU caused a general slowing of development. In larval amphibia, on the
Thyroid and nervous system of E. martinicensis
405
other hand, in which development extends over a much longer time-scale, antithyroid agents not only inhibit metamorphosis but also promote growth of the
larva beyond normal limits by suppressing the 'feed-back' of the thyroid to the
pituitary. This effect was shown with thiourea on Rana pipiens by Gordon,
Goldsmith & Charipper (1943); a similar action is seen with larvae of Xenopus
treated with PTU (Prestige & Hughes, unpublished).
The observations of Lynn & Peadon on embryos of Eleutherodactylus treated
with this substance have been confirmed in the present experiments, in which
the development of thirty-nine embryos submitted to the action of PTU was
1
2
3
4
5
6
7
8
Days
Text-fig. 2. Development of embryos in saline containing 01 % PTU; stages as in
Table 2 with isopleths of tail length as in Text-fig. 1.
observed (Text-fig. 2). The slope of the time-development curve for treated
embryos decreased with time, and by the fourth or fifth day there is little or no
further progress in development. The stage at which this arrest occurs depends
upon that at which the embryo was placed in PTU; embryos at stage ' a ' are
halted somewhere between stages ' d ' and ' e ' . Xanthophores appear, but the
legs show little or no extensor thrust, and the tail shows only the first signs of
decrease in size. In embryos immersed in PTU from stage ' d ' onwards, the
extensor reflex appears, and they are subsequently able to maintain continuous
swimming, but the shrinkage of the tail is halted at a length of about 1-0 mm,
although the rate of the earlier phases of tail reduction are normal.
As was described by Lynn & Peadon (1955), melanin is lost from pigment
406
A. HUGHES
cells under the influence of PTU. Such embryos become paler than the corresponding saline controls on the second day of treatment. The extent of the
reaction is similar in embryos treated at any stage between ' a ' and ' e ' .
The effect of thyroidectomy. The general retardation of development in
Eleutherodactylus with PTU shows some differences from the effects of removal
of the thyroid gland. When this operation is performed at stage ' d' the subsequent development of the embryos continues at a normal pace (Text-fig. 3);
operated at stage ' b ' , however, while the development of pigmentation and of
leg behaviour proceed normally for the next 48 h, the embryo retains the tail at
Text-fig. 3. Development of thyroidectomized embryos in saline; stages as in
Table 2 with isopleths of tail length as in Text-fig. 1. Embryos operated at stage 'd'
reach stage ' i ' with complete loss of tail; those operated at stage ' b ' are halted in
development. One such is treated with thyroxine on 4th day (arrow) and loses
tail within the next 3 days. All embryos regenerate follicles.
full length even though some thyroid follicles regenerate. There is thus a critical
period, at or near stage ' c ' , when the presence of the thyroid is necessary for
development to reach the juvenile stage. In such embryos the addition of 1:108
thyroxine 4 days after the operation results in shrinkage of the tail at the normal
rate.
The effect of hypophysectomy. The effect on an Anuran larva of extirpation
of the pituitary was first shown by Smith (1916) in Rana boylei. He demonstrated
a hypoplasia of the thyroid gland and a reduction in the general rate of growth.
The action of the amphibian pituitary on the thyroid gland is still imperfectly
Thyroid and nervous system ofH. martinicensis
407
understood, however, for ' the control of thyrotropic hormone production and
its release by the tadpole pituitary is as yet a mystery' (Kollros, 1961).
In the present experiments on Eleutherodactylus, development was slowed in
all embryos in which removal of the pituitary was attempted, even though the
40
30
20
10
40
30
20
10
J3
So
g 40
VS 30
H
20
10
40
30
20
10
I
I
2
I
I
4
I
l
6
I
l
l
I
I
I
I
I
l
I
l
l
l I
8 10 12 14 16 18 20
Days
Text-fig. 4. Lengths of tail with time in (a) saline control embryos; (b) embryos
with incomplete hypophysectomy, treated with thyroxine; (c) untreated incompletely
hypophysectomized embryos; (d) completely hypophysectomized embryos. All at
stage ' b ' to ' c ' at day 0.
whole or part of the gland was present. The initial slowing of development may
thus be attributed to the eifects of operative interference, in which disturbance of
the pituitary stalk may be partly responsible. These embryos, however, showed
a special vigour in their limb movements.
There were however differences to be observed between those in which the
pituitary was wholly removed and those in which some or all of the gland
408
A. HUGHES
remained. In the former, the tail remained largely constant in length for periods
up to 17 days after operation. In others, however, a slow decline in its length
was observed ten or more days after the operation (Text-fig. 4). After fixation
and sectioning these animals were found to have a pituitary, even though there
was much disturbance at the base of the mid-brain, often with damage to the
optic chiasma. In three embryos which were fixed 18-25 days after the operation,
a small undifferentiated group of hypophysial cells was found, which suggested
some degree of regeneration. The tail remained at constant length in only one
of these three embryos.
During the course of observation from day to day, thirteen embryos became
lighter in colour than normally, and in them melanophores were seen to have
contracted to small round dots. In all of these it was afterwards found that the
pituitary was wholly absent and the effect on the pigment cells could be
ascribed to the absence of the melanophore-stimulating hormone (Barrington,
1964). In two others which showed a partial contraction of the melanophores,
some regenerating pituitary cells were present. The earliest stage at which
melanophore contraction was observed was in an embryo at stage 'e', 2 days
after operation.
Eight hypophysectomized embryos were treated with thyroxine at a concentration of 1:107. Five died, but in the three survivors the tail began to dwindle
1-3 days after the operation at rates much greater than were seen in other animals of this group with no treatment with the hormone. In all three of these
embryos some hypophysial cells were subsequently found. In them, however,
the loss of the tail began much earlier than in any other embryo with a reduced
pituitary (Text-fig. 4b, c). Two of these three embryos showed some contraction
of melanophores during life.
The length of the foot
An index of the growth of the embryo is given by the length of the foot,
measured from the tarso-metatarsal joint of the tip of the second digit. In the
normal embryo, from stage *f' to the hatching period, this increases from 1-0 to
about 1-8 mm. The experimental embryos were measured daily and thus it was
possible to detect any inaccuracy in individual values, as on occasions when
movement of the embryo interfered with measurement. In Text-fig. 5 averaged
data are given for a group of 3 or 4 individuals under three sets of circumstances,
namely (a) unoperated embryos developing in saline, and embryos after complete (b) and incomplete (c) removal of the pituitary. There is a clear distinction
between the two latter categories in this respect. In some partially hypophysectomized embryos which were fixed 14-25 days after operation, well after
normal embryos of the same age had hatched, the foot increased in length
beyond that of the normal embryonic range. It is possible that here the differences caused by complete removal of the pituitary are due directly to the action
of a hypophysial growth hormone. In the three partially hypophysectomized
Thyroid and nervous system of E. martinicensis
409
embryos which were treated with thyroxine it did not seem that growth of the
foot was thereby increased.
After thyroidectomy, the growth of the foot is much slowed down, equally
in embryos which retained the tail and in those where it atrophied. Treatment of
thyroidectomized embryos with thyroxine did not make any appreciable
difference in this respect. In embryos immersed relatively early in PTU-saline,
and where development was halted at stage ' e ' , the length of the foot was below
that of a normal embryo of the same stage.
10
13
14
Text-fig. 5. Growth in length of foot with time in (a) average of four saline control
embryos; (b) average of three incompletely hypophysectomized embryos; (c) average
of four embryos with complete hypophysectomy.
The thyroid gland
Lynn & Peadon (1955) have described the effect of treatment with PTU on
the histology of the thyroid in Eleutherodactylus embryos. After 4 days the
height of the follicular epithelium is increased. Hypertrophy of the gland is seen
at 5 days, with the disappearance of much of the colloid from the follicles. Later,
the follicular cells become highly vacuolated. If an embryo is transferred from
PTU to water the normal condition of the thyroid is regained within a short time.
The present observations confirm these results of Lynn & Peadon. All groups
of embryos treated with PTU save one showed the effects described by these
authors. Again, the normal appearance of the gland reappeared on cessation of
the treatment. Colloid reappeared in the glands of embryos treated first with
410
A. HUGHES
PTU for 5 days, and then for 2-3 days with both PTU and thyroxine. Lynn &
Peadon also found that thyroxine added to the water in which embryos were
developing caused a flattening of the follicular epithelium, and an increase in
the amount of stored colloid. In the present experiments, however, no constant
differences of this kind resulted from treatment with exogenous thyroxine, but
the whole gland in such embryos seemed somewhat smaller than normal.
In all animals in which hypophysectomy was attempted the follicular epithelium became progressively flattened, though no general differences could be
detected between those in which the pituitary was wholly removed and those
in which some residual tissue was present. Empty follicles were observed in
some operated embryos of both groups.
In animals which were thyroidectomized and fixed from 6-7 days after operation, thyroid follicles regenerated. These were small in number but abnormally
large in size and were seen both in those embryos in which the tail atrophied and
in those in which it remained. In the latter group, although the regenerated
follicles appeared too late to influence the tail, some internal changes were
arrested, as described below.
The pronephros
Lynn & Peadon (1955) found in Eleutherodactylus that PTU inhibited the
normal degeneration of the pronephros, the loss of which is among the few
events at organ level which is controlled by thyroid hormones.
In embryos taken from their envelopes and then fixed, the first signs of
degeneration of the pronephros are seen at stage ' e ' , which is characterized by
the kick reflex of the legs and the first appearance of xanthophores over the
body. The tail begins to shrink a day or so later. A slight vacuolation of the
pronephric tubules is seen, with the vascular spaces of the surrounding mesenchyme dilated with blood. In other embryos at stage ' e ' , degeneration may be
further advanced, with a flattening of the tubular epithelium, the accumulation
of cell debris in the lumen, and the appearance of necrotic cells around the
tubules. Often the pronephroi on both sides of the embryo are not in the same
phase of degeneration. This second phase is reached in all embryos in which the
tail has been reduced to 2 mm in length. Before the tail has entirely disappeared
the site of the pronephros is recognizable only as an area of reticular connective
tissue. In embryos which have been developing in saline the disappearance of
the pronephros is more advanced relative to that of the tail, which may still be
at its full size when the pronephros already contains necrotic cells.
When PTU has been added to the saline in which an embryo develops, the
extent to which the pronephros degenerates, as with the general development
of the embryo, depends on the age at which the treatment began. With those
embryos placed in PTU relatively late (from ' d ' onwards) and which subsequently reach the juvenile stage, the disappearance of the pronephros bears the
same relation to that of the tail as in a control embryo. When treatment begins
Thyroid and nervous system ofE. martinicensis
411
at stages ' a ' to ' c ' and the tail remains unresorbed, some pronephric tubules
become dilated, but no further changes are seen.
In embryos thyroidectomized at stage ' b ' and in which development is
arrested 3-4 days later with little or no shrinkage of the tail, degeneration of
the pronephros nevertheless proceeds, and only small lengths of tubular epithelium remain intact. The regenerated follicles in these embryos must produce
enough thyroid hormones to affect the pronephros, but not the tail.
In hypophysectomized embryos, degeneration of the pronephros is again
seen without shrinkage of the tail. Here the vacuolation of the epithelial cells is
especially marked, equally where the pituitary was entirely removed and in
those with some residual hypophysial tissue. Vacuolization is seen as early as
the second day after operation. The tubules subsequently break down at varying
rates, which show a slight correlation with the presence or absence of the
pituitary, and among the former group with decrease in size of the tail. In all
embryos of this group only reticular traces of the pronephros remain by the
13th to 14th day after operation.
The Rohon-Beard cells
In Eleutherodactylus the life-history of the Rohon-Beard (RB) cells, the
primary sensory neurones of the trunk, is somewhat different from that in
larval Amphibia, where these cells develop and function long before the appearance of the dorsal root ganglia, and are superseded by them at a stage well before
metamorphosis—in Xenopus at about stage 49, when the limb buds are still
very small (Hughes, 1957). In Eleutherodactylus, on the other hand, the spinal
ganglia appear before the RB cells (Hughes, 1959), and the latter do not vanish
until the tail dwindles.
In Text-fig. 6 the spinal cord with its RB cells is reconstructed in a series of
embryos of Eleutherodactylus. Each RB cell is represented in these reconstructions. Fifteen embryos were studied in this way. The reconstructions include
the first 100 JLI of the tail region of the cord in which the RB cells are the last to
disappear. Text-fig. 6a-c represent the normal course of events from stages ' d '
to ' g ' in embryos taken from their envelopes and then fixed. By the time that
the tail has begun to dwindle the number is already halved and continues to
decrease while the tail degenerates further. In normal embryos the loss of the
RB cells thus proceeds paripassu with the degeneration of the pronephros. This
synchrony is maintained in embryos which have been treated with PTU. If
thyroidectomy is performed early enough to prevent loss of the tail, the
disappearance of RB cells begins some 7 days later. It proceeds to completion with loss of the tail in similar embryos treated with thyroxine (Text-fig.
6g, h).
In all 'hypophysectomized' embryos, decrease in the length of the tail and
in the number of RB cells is closely related to the presence of residual pituitary
tissue. The full number of RB cells may be maintained for 10 days after a com-
412
A. HUGHES
plete hypophysectomy, and more than half may still be present a further 7 days
later (Text-fig. 6d, e). In two such examples the pronephros was already in an
advanced stage of degeneration.
In all 'hypophysectomized' embryos in which the length of the tail decreased
and in which some pituitary cells were found, the RB cells were confined to
the tail region of the cord. In the embryo in which the period between operation
(0 \*
(g)
CO
f\
D
Text-fig. 6. Diagrammatic reconstructions from medulla to tail (upper border
offigure)of neural tube in embryos to show Rohon-Beard cells, (a) Normal embryos
at stage 'd' (Table 2); (b) at stage ' e ' ; (c) at stage 'g'; (d) 10 days after complete
hypophysectomy; (e) 17 days after complete hypophysectomy; (/) 10 days after
partial hypophysectomy; (g) 7 days after thyroidectomy at stage ' b ' , degenerating
cells in black; (/?) 7 days after thyroidectomy at stage ' b ' , with thyroxine on last
3 days. The tail was shrinking in embryos (c) and (/) and was lost in (h).
and fixation was longest, namely 25 days, the tail remained constant in length,
even though there was evidence of a regenerated anterior lobe. RB cells were
found only in the tail region of the cord.
The degenerative changes which have been described in the tail, the pronephros, and in the RB cells are all related to metamorphosis on the greatly
Thyroid and nervous system of E. martinicensis
413
reduced scale of this event in Eleutherodactylus, and in all thyroid hormones
are concerned. Below, the effects of their withdrawal on several features of the
development of the nervous system which are unrelated to metamorphosis are
described.
The ventral horn cells {the lateral motor column)
Between stages ' c ' and ' d ' of the present series in the normal ontogeny of
E. martinicensis, some ventral horn cells are differentiating rapidly. The linear
dimensions of the nucleus enlarge by about 30%, the contents of the nucleus
become sparser, with one nucleolus within a largely clear sap. The distinction
between developing ventral horn cells and those of the mantle layer becomes
very clear. At the same time the cell enlarges as a whole. Hitherto visible cytoplasm has been restricted to polar caps but now the nucleus is surrounded by an
evident layer of cytoplasmic material with marked expansions at the poles. The
development of the lumbar ventral horn is at first in advance of that of the
brachial horn.
From the beginning, the cell axon reacts to silver as to a lesser extent do the
polar caps of cytoplasm. As the cell enlarges the argyrophilic material within
the cell assumes a fibrous appearance against an unstained background. It is
certain that these 'neurofibrillae' are greatly altered by fixation (Hughes, 1954)
and by accretion of silver metal on whatever was their original form in the
living cell. Yet they represent some component of the neuronal cytoplasm in
which changes are seen in the course of development and which is susceptible
to endocrine influences (Plate 1, fig. A). In the maturing neurone the course of
the main bundle of neurofibrillae is usually to one side of the nucleus from where
they sweep dorsally into the dendritic processes of the cell, which branch and
extend widely.
During the later stages of embryonic life not all ventral horn cells have
reached this stage. At the caudal pole of the ventral horn all the cells are relatively young. This disparity among the ventral horn cells of developing Anura
suggests that new cells are being recruited to the ventral horn as others degenerate.
In comparing embryos developing under different conditions with respect to
the degree of differentiation of ventral horn cells, one difficulty is that the depth
of impregnation with silver is not constant from slide to slide. One can, however,
reject any specimen in which the fibres of the white matter and of the peripheral
nerves are not deeply stained, and in the remainder it is possible to compare
the most advanced cells with the others.
The early stages of differentiation of ventral horn cells can be accelerated by
adding thyroxin to the medium in which the embryos are developing. This effect
has already been described for tadpoles of Rana pipiens by Beaudoin (1956) and
by Reynolds (1963). In Eleutherodactylus, 1:107 thyroxin administered between
stages ' b ' and ' c ' results in a general increase in the size of the nuclei of cells
in the ventral horn. Treatment with PTU sufficiently early to arrest general
26
J E E M 16
414
A.HUGHES
development at stage ' f or before retards their differentiation. Some effect was
seen as the result of thyroidectomy.
After hypophysectomy there are marked effects within the ventral horn. The
differentiation of the nuclei of the ventral horn cells is retarded and the extent to
which these neurones stain with silver is greatly reduced by complete extirpation of the pituitary. As early as 2 days after operation a difference can be seen
between embryos with full and with partial removal of the gland. In one embryo
with full hypophysectomy the loss of argyrophilia was complete 6 days after
operation. Of eleven embryos in which the pituitary was wholly removed, all
showed a loss of argyrophilia in the ventral horn cells (Plate 1, fig. C). In ten of
these embryos melanophores were seen contracted during life.
This failure to stain on silver impregnation is confined to the cell body of the
neurone, distal to which the axon is as black as in the normal embryo. There is
an intermediate condition in which the poles of the cell take up silver, with no
impregnation near the nucleus. In those embryos in which some pituitary cells
still remained, the degree of reduction of argyrophilia was variable (Plate 1,
fig. B); some were similar in this respect to embryos with complete hypophysectomy. In the three embryos which were partially hypophysectomized and
subsequently treated with thyroxine, the degree of argyrophilia in ventral horn
cells was similar to that of normal embryos, and was greater than that of comparable partially hypophysectomized embryos untreated with the hormone.
In embryos which were treated with PTU or were thyroidectomized, there
were signs of a loss of argyrophilia among ventral horn cells; those neurones
which were most differentiated showed a normal impregnation, while many of
the remainder showed little affinity for silver.
The primary motor neurones of the cord, which innervate the axial musculature, are similar to the ventral horn cells in reduction of argyrophilia after
complete hypophysectomy. In contrast to the effect of the operation on motor
neurones, those cells of the dorsal root ganglia which are fully differentiated
EXPLANATION OF PLATES
All figures are from sections through embryos of Eleutherodactylus, stained by Palmgren's
method. The horizontal line in each figure represents 10/*.
PLATE 1
Fig. A. Part of transverse section of spinal cord in lumbar of saline control embryo near
hatching stage, to show ventral horn cells, fully argyrophilic.
Fig. B. Part of transverse section of spinal cord of embryo incompletely hypophysectomized
for 14 days, to show loss of argyrophilia in ventral horn cells.
Fig. C. Part of transverse section of spinal cord embryo completely hypophysectomized
for 16 days, to show entire loss of argyrophilia in ventral horn cells.
Figs. D, E. Rohon-Beard cell in same section as in fig. C, to show retention of argyrophilia after complete hypophysectomy. D is at level of nucleolus; E at level of axon.
J. Embryol. exp. Morph., Vol. 16, Part 3
PLATE 1
I *
111!
f
D
A. HUGHES
J. Embryol. exp. Morph., Vol. 16, Part 3
PLATE 2
H
A. HUGHES
facing p. 415
Thyroid and nervous system of E. martinicensis
415
continue to take up silver even when the adjacent motor cells within the cord
have lost all reactivity. Rohon-Beard cells within the cord also retain their
affinity for silver (Plate, 1, figs. D, E).
The numbers of ventral horn cells
In normal development, parallel changes are seen in the numbers of cells
in both brachial and lumbar ventral horns (Hughes, 1962, 1965a). About
a thousand neuroblasts are present in each horn at 7-8 days of development,
and this number is reduced to some 400 cells at the hatching stage. The decline
in numbers is most rapid during the tenth and twelfth days, at stages ' e ' to T
of the present series. Degenerating cells are then common in the ventral horns.
The thyroid has apparently little or no influence in this decrease in the number
of ventral horn cells for in embryos thyroidectomized at stage ' b ' , early enough
to prevent any shrinkage of the tail during the subsequent 6 or 7 days, the
numbers of ventral horn cells were similar to those of the normal juvenile.
Addition of thyroxine to the saline in which such embryos were developing
made no difference in this respect.
Again, addition of thyroxine to unoperated embryos was without effect. In
embryos treated with 1:107 thyroxine from stages ' a ' to ' e ' , ventral horn cell
numbers were similar to those of the corresponding control embryos in saline,
even though the differentiation of the individual neurones was accelerated.
Treatment with thyroxine at the same concentration prolonged as far as the
hatching stage was without effect. In embryos treated with PTU from stage ' a '
for 8 days, and arrested in development at stage ' d ' from the fourth day, the
number of ventral horn cells was again that of a normal juvenile, though there
PLATE 2
Fig. F. Section through sciatic nerve near entry into limb of saline control embryo near
hatching stage. There are about 1000 fibres within the nerve, the largest about 20/i in
diameter.
Fig. G. Section through sciatic nerve near entry into limb of embryo thyroidectomized for
7 days, with retention of tail. There are about 500 fibres within the nerve, all less than 1 [i
in diameter. The larger objects within the nerve are impregnated Schwann cells.
Fig. H. Section through sciatic nerve near entry into limb of embryo thyroidectomized for
7 days, and treated with thyroxine for last three, with loss of tail. There are about 840 fibres
in the nerve, the largest of which are just over 1 fi in diameter.
Fig. I. Oblique section through nerve to triceps femoris muscle of normal embryo at kicking
stage. The largest fibres are about 1-0/t in diameter.
Fig. J. Transverse section of nerve to triceps femoris muscle of embryo treated with 0-01 %
PTU for 8 days, and halted in development before kicking stage. The largest fibres in the
nerve are about 0-7 n in diameter.
Fig. K. Oblique section through nerve to triceps femoris muscle of embryo treated for
4 days with 0-1 % PTU and then transferred for a further 4 days to thyroxine-saline. Development advanced to the kicking stage, and some fibres in the nerve enlarged to a diameter of
1-6/*.
26-2
416
A. HUGHES
were indications of further differentiation of new cells in other embryos similarly treated for the first 4 days and then transferred either to saline or to saline
with 1:108 thyroxine. During this second phase of treatment these embryos
resumed development as far as stage T .
The changes in numbers of ventral horn cells in hypophysectomized embryos
are shown in Text-fig. 7. The operation was performed at stage ' e ' at a time
when 700-800 cells are present in the ventral horn. Although the further development of those embryos in which the pituitary had been wholly removed did not
900 i-
• Control, at stage of operation
800 _
-•
o Full hypophysectomy
Ij
c
Incomplete hypophysectomy
c
o (e) Incomplete hypophysectomy + thyroxine
_c
o
o
o
u
•O 600 -
itra
700 _
-
©
Regenerated pituitary, tail length constant
>
9
Regenerated pituitary, tail shrinkage
°o
500 c
Ventral root fibres S,—S,j
o
O
«
c
°O
400 -
O
o
c
©
©
C
300 -
|e®
o
Jl® I
200 -
e
0
O
100
1 1 1 1 1 1 i
i
1
1 1
1 1 1 1 1
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 23 24 25
Days after operation
Text-fig. 7. Numbers of cells in lumbar ventral horns of embryos operated as
indicated, plotted against time after operation. In five examples the total number
of lumbar ventral root fibres is also counted.
progress beyond stage 'f, yet the number of ventral horn cells was found to
diminish to levels even below those of the normal juvenile. This decrease was
also seen in those embryos where a pituitary was present, either through incomplete removal at the time of operation, or by regeneration. There was no
correlation between the number of ventral horn cells and the size of the tail, for
no difference can be seen between those embryos in which it remained at its
original length and those where a sudden decrease took place in the last few
days before fixation. In those hypophysectomized embryos which were subsequently treated with thyroxin it appears that treatment with the hormone had
no effect on the numbers of ventral horn cells.
In all embryos in which hypophysectomy was attempted development was
Thyroid and nervous system ofE. martinicensis
417
largely arrested 4 days after the operation and remained at the same stage for
periods up to 3 weeks. At this time, control embryos had reached juvenile stages.
While in the operated embryos the numbers of ventral horn cells ultimately fall
to levels below that of a normal juvenile, the rate of loss is less than in unoperated controls.
In normal development the decrease in the population of ventral horn cells is
accompanied by an increase in the number of ventral root fibres to which they
give rise. The proportion of developing neurones which send an axon into
a lumbar ventral root rises from about one in five to a nearly equal ratio
(Hughes, 1965a). In embryos with either complete or partial hypophysectomy
there was no indication of any rise in the number of lumbar ventral root fibres
with time.
Peripheral changes in numbers of nerve fibres
In the development of embryos taken from their envelopes and then fixed it
has been shown that at a time when the tail first shows signs of shrinkage the
number of fibres in many nerves within the leg is at a maximum. That this is
a general feature of this phase of development is suggested by the fact that the
embryo is then often larger than before this stage or during the subsequent day
before emergence from the envelope. In embryos developing in saline this peak
in the numbers of nerve fibres is less clear. Here it is not seen in terminal branches
in the thigh; in the main sciatic nerve at the entry into the limb it is somewhat
obscured (Text-fig. 8), but is still recognizable distally at the bifurcation into
tibial and peroneal nerves.
Why there should be this difference between embryos developing under these
two circumstances is obscure. It is possible that the small volume of fluid within
the egg envelope may contain some growth-promoting substance, secreted by
some organ or tissue, which is diluted below its effective range when the embryo
is in saline. It is certainly true that the rapid decline on the further side of the
normal peak occurs after the envelope has been ruptured. The addition of
thyroxine to unoperated embryos developing in saline makes no difference in
this respect.
Observations of the effects of thyroidectomy and of hypophysectomy on the
number of fibres in peripheral nerves have been made at three points; high in
the sciatic trunk (Plate 2, figs. F, G, H), in the Ramus profundus anterior which
supplies the triceps femoris muscle (Plate 2, figs. I, J, K), and in the crural
cutaneous. The two latter are the largest nerves of their respective kinds in
the leg.
In the sciatic nerve at the point of entry into the limb, no differences are seen
in the number of fibres between embryos with full and partial hypophysectomy
(Text-fig. 8). In those thyroidectomized at stage ' b ' , early enough to prevent
loss of the tail, the corresponding numbers of fibres fall well below this range,
and are low enough to suggest that there may be some actual loss of fibres in
418
A. HUGHES
the days following the operation. Embryos of this group treated with thyroxine
4 days before fixation show a marked increase in this respect to levels within the
range of saline controls, thus providing a quantitative demonstration of the
influence of thyroxine on the outgrowth of fibres of the peripheral nervous
system.
1100 i—
_
1000 -
®
_
900 -
©
800
•
°©
oo
US
700
O
on
O
-
®® 0
• Control embryo,
at stage of operation
O
1
-
©
©
°
®
®
O Full hypophysectomy
o
©
©
o
600
Partial
hypophysectomy
°
® Saline control
50C _•
A
** Thyroidectomy
A
400 ^ Thyroidectomy +
thyroxine
_
300 -
1 1 1 1 1 1 1 1 1 1 1 1
2 3 4 5 6 7 8 9 10 11121314151617 18
01
Days after operation
1
f
1
g
1
h
1
i
1
Saline controls
Text-fig. 8. Numbers of fibres in the sciatic nerve at the point of entry into the
limb in embryos operated as indicated, plotted against time after operation, together with corresponding data for four stages of saline control embryos.
The ratio of number of nerve fibres to muscle volume is plotted for the triceps
femoris under various conditions in Text-figs. 9-11. At stages ' c ' and ' d ' there
are 40-50 fibres in the nerve which supplies the triceps, and the volume of the
muscle is from 0-01 to 0-02 mm3. In normal development, the number of nerve
fibres and the volume of the muscle increase markedly during stages ' g ' and ' h ' .
The variance for both parameters is then high, although the ratio between them
is less variable. By stage T , when the embryo hatches, both quantities drop
sharply, the number of nerve fibres to a value little above that of the embryo at
stage ' c ' or 'd', and the volume of the muscle to 0-06-0-09 mm3. The ratio
between these quantities then becomes less than at stages ' g ' or ' h ' . From this
time the muscle grows without increase in the number of innervating fibres, for
this remains at the same level in an adult animal. The number of fibres in
Thyroid and nervous system ofE. martinicensis
419
cutaneous nerves, on the other hand, increases greatly during post-embryonic
growth.
The newfibreswhich enter the nerve before stage 'g' are all small in calibre,
as are those which are lost between stages ' h ' and 'i'. These changes suggest
that in normal development there is a peripheral turnover of nerve fibres at
1 1 0 i—
105
Control taken from envelope
100
_
Control in saline
95
90
\ J
Saline control+ thyroxine
85
80
75
S 70
o
2 65
<5
"I 60
s
50 r
l°l
»UIH)~MV7
sr\
45
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
001 aO2 003 004 0-05 006 007 008 009 010 011 012 013 014 015 016 017 018
Volume of muscle (mm3)
Text-fig. 9. Numbers of fibres in the nerve to the triceps femoris plotted against
volume of muscle. The stage of each embryo at fixation is shown.
stages after that of the first movements of the limb. It seems likely that during
development there is both an ingrowth of newfibresinto a peripheral nerve and
a concomitant loss. The two processes vary separately in rate during development, and, after the peak in numbers, loss overtakes gain.
The changes in muscle volume seem more obscure. There may be some turnover in the constituent musclefibresbut it seems that the increase in volume of
the triceps femoris is partly due to intake of water, for in sections it can be seen
that the individual fibres at stages 'g' and 'h' are then proportionately larger
in cross-section, with the myofibrils separated by clear spaces.
420
A. HUGHES
In embryos freed from their envelopes and which are developing in saline, the
triceps femoris shows no peaks either in the number of nerve fibres which enter
it or in the volume of the muscle itself (Text-fig. 9). The effects of exogenous
thyroxine in these respects on such embryos is variable. Some are slightly
retarded in development and in them the number of nerve fibres which enter
o
54
53
52
O O
51
O
50
O
49
O
48
O
47
<*
O
AL
O
<u 4 o
In
O
0
Normal embryo, stage C
O
Saline control
•° 45
<D 44
«2£ 43
S
co 41
|
Saline with PTU
40
S 39
£
Saline with PTU, then
saline with thyroxine
38
37
36 b-d)
35
34 h-
(b-d)
(b-d)
33 32
31
30
001
002
003
004
I
005
I
006
|
007
L
008
009
0-10
011
012
Volume of muscle (mm3)
Text-fig. 10. Numbers of fibres in the nerve to the triceps femoris, plotted against
volume of muscle, mainly for embryos treated with 0-01 % PTU during the stages
of development as indicated. The data for saline control embryos of Text-fig. 9
are given for comparison.
the triceps femoris is below normal. Deprivation of thyroid hormones also
reduces the number of nerve fibres which enter the muscle. It falls to between
25 and 30 in embryos thyroidectomized late in stage ' b ' , which is sufficiently
early to prevent loss of the tail. This decrease is proportionately greater than
that seen in the main sciatic nerve. If exogenous thyroxine is added to such
operated embryos 4 days later at stages ' e ' to 'f, the tail subsequently disappears and the number of fibres in the R. profundus anterior rises to near that
of saline controls (Text-fig. 11). When thyroidectomy is postponed until stage' d'
the operation leads to no loss of fibres.
A critical period after which deprivation of thyroxin has no effect can similarly be demonstrated by treatment of the embryo with PTU (Text-fig. 10). It
Thyroid and nervous system of E. martinicensis
I
|
\ f
Embryo at stage of operation
O
o
o
Thyroidectomized
Thyroidectomized + thyroxine
421
Control in saline
Full hypophysectomy
Partial hypophysectomy
O O
•
MHO
O
•
^
0
0
•
-D
0
O
O
001
002
003
0-04
005
006
007
008
009
Volume of muscle (mm3)
Text-fig. 11. Numbers of fibres in the nerve to the triceps plotted against volume
of muscle. For hypophysectomized embryos the number of days between operation and fixation is given. The thyroidectomized embryos were those operated at
stage ' b ' and fixed 7 days afterwards, which lost their tails only on treatment with
thyroxine. The data for saline control embryos of Text-fig. 9 are given for comparison.
422
A.HUGHES
was mentioned above that immersion in PTU saline early in stage ' b ' usually
results in an arrest of development between stages ' d ' and ' e \ In such embryo
after 8 days of treatment, the number of nerve fibres which enters the triceps
femoris is about 35 (Text-fig. 10). No change is seen if after the first 4 days in
PTU-saline the embryo is then transferred to saline without PTU, or even into
saline containing thyroxine. On the other hand, if embryos are immersed in
PTU-saline from stage *c' or ' d ' they can continue development nearly to the
end of the embryonic period and then show little or no difference from saline
controls in respect to the volume of the triceps femoris or the number of nerve
fibres which enter the muscle. With thyroidectomy the critical period comes
later in stage *b' than with treatment with PTU which necessarily acts more
slowly in the removal of thyroxine from the tissues of the embryo.
Embryos which had been 'hypophysectomized' subsequently fell into two
groups with respect to changes in the volume of the triceps femoris and the
number of nerve fibres which entered it (Text-fig. 11). In one there was growth
of the muscle, but the number of nerve fibres steadily decreased with time after
operation, though at a rate somewhat slower than that seen after thyroidectomy.
These were the embryos in which there was no pituitary and in which the tail
remained constant in length. The only exception was the oldest, fixed 25 days
after operation, in which a regenerating anterior lobe was found, but where the
tail nevertheless remained unchanged. In this embryo the number of nerve
fibres which entered the muscle is among the lowest of all. In all embryos of
this group the volume of the muscle increased steadily with time after operation.
In the second group, which consists of embryos in which some pituitary
tissue was found, and where the tail decreased in length in those fixed ten or
more days after the operation, the number of fibres which entered the triceps
femoris was within the range of saline controls (45-55). Those partially hypophysectomized embryos which were treated with thyroxine fall also into this group.
The effects of thyroidectomy and of hypophysectomy which have just been
described presumably concern both motor and proprioceptor fibres in the nerve
to the triceps femoris. As with turnover in normal development, fibres of small
diameter are lost, while those of larger calibre remain, although the R. profundis anterior is the only muscular nerve which has been studied systematically
in the course of this work. Comparison suggests however that while this nerve
is specially sensitive to thyroid hormones, in others finer fibres also tend to
disappear after thyroidectomy.
In order to test whether the exteroceptive fibres of cutaneous nerves show any
corresponding effect, the number of fibres in the crural cutaneous has been
counted under various conditions, and compared with the length of the foot,
which can be used as an index of the general growth of the hind limb (Text-fig. 12).
For a complete investigation it would be necessary to measure the area of skin
surface supplied by each cutaneous nerve, the difficulties of which would be
formidable.
Thyroid and nervous system ofE. martinicensis
423
Only in embryos thyroidectomized relatively early is there no correlation
with growth in length of the foot, when the number of nerve fibres apparently
remains the same as at the time of operation, though without actual decrease.
In such embryos treated with thyroxine 3 days before fixation the number of
fibres in the crural cutaneous increases to levels similar to those of saline
controls. In both groups of 'hypophysectomized' embryos, as in controls, the
numbers of fibres in the crural cutaneous increases with length of the foot, but
no general differences in this respect are seen between these three groups of
embryos within the range of lengths of foot common to all.
72
70
68
66
64
62
60
58
56
54
52
50
48
46
«« 44
O Embryo at stage of operation
o Full hypophysectomy
« Partial hypophysectomy
A Thyroidectomy
©
o
2«
-
A Thyroidectomy + thyroxine
• Saline control
„
c
o
£ 40
I 38
§ 36
Z 34
32
30
28
26
24
22
20
18
16
c o
o
•
c
•
A
A
09
10
1-1
I
1
I
1-2
12
1-3
1-4
1-5
I
i
1-6
16
17
18
I
19
i
20
i
21
Length of foot (mm)
Text-fig. 12. Numbers of fibres in the crural cutaneous nerve under various conditions plotted against length of foot. The thyroidectomized embryos were those
operated at stage ' b ' and fixed 7 days afterwards, which lost their tails only on
treatment with thyroxine.
The calibre of fibres in muscular nerves
In Hughes (1965 a) mention is made of the enlargement of the diameter of
individual nerve fibres which occurs towards the end of development in Eleutherodactylus. Further observations have shown that in the R. profundus
anterior this increase in calibre is associated with the onset of function of the
triceps femoris. In a limb paralysed by deafferentation of the cord the fibres
in this nerve remain relatively small in diameter (Hughes, 1965 c).
424
A. HUGHES
In normal embryos up to stage ' d' when the leg can retract but not extend,
no fibre in the nerve reaches a diameter of more than half a micron, and most
are considerably smaller. At stage ' e ' , however, when the leg is first able to kick
and to use this muscle, the calibre of the largest fibres exceeds 1-0/* (Plate 2,
fig. I). This increase in diameter begins at the entry of the nerve into the muscle
and progresses proximally with time. The group of relatively large fibres may
include both motor axons and proprioceptive fibres. Gray (1957) has shown that
in the nerve to an adult frog muscle, spindle afferents are among the larger
fibres present.
If embryos are treated with 0-1 % PTU at stage ' a ' , then development slows
down before they acquire the kick reflex and the leg shows little or no signs of
extension. In such embryos fixed after 8 days of treatment no fibres in the nerve
to the triceps femoris were found to be as large as a micron in diameter (Plate 2,
fig. J). After 4 days of immersion in PTU saline, similar groups of embryos were
transferred to saline without PTU, or to saline containing 1:107 thyroxine. In
such embryos the kick reflex appeared 2 days after the cessation of treatment
with PTU, and after a further 2 days progressed as far as stage 'g', with the
first signs of decrease in length of the tail. After fixation, fibres up to 1-5 /JL in
diameter were found in the nerve to the triceps femoris (Plate 2, fig. K).
In a further experiment, a group of embryos were treated with PTU saline
for 5 days from stage 'a'. In them, xanthophores characteristic of stage ' e '
appeared, but the action of the leg did not progress beyond the withdrawal
reflex. These embryos were then divided into groups, in one of which treatment
with PTU was continued, while in another 1:107 thyroxine was added to the
PTU. After a further 2 days the extension reflex of the leg appeared in all
embryos of the second group, while it was doubtful whether there was any
trace of leg extension in the former. Two other groups of embryos were transferred from PTU to water, or to water with thyroxine. In embryos given the
hormone during this second phase of treatment mortality was very high, but in
one which survived for 2 days in PTU-thyroxine with the appearance of leg
extension there was enlargement of some nerve fibres to the triceps femoris.
Deprivation of thyroid hormones either by thyroidectomy or by hypophysectomy in the present experiments had no effect either on the behaviour of
the hind limb or on the calibre of its nerve fibres at the periphery, even in
embryos in which there was no shrinkage of the tail, though some difference
in this respect was seen in the sciatic trunk in thyroidectomized embryos after
treatment with thyroxin. The growth in diameter of individual fibres in nerves
of supply is apparently a separate process from increase in total number, for
the two are affected by different degrees of deprivation of thyroid hormones.
The effect of PTU on the movement of supernumerary limb grafts
In embryos of Eleutherodactylus, if a limb is grafted near to an undisturbed
member the transplant may become innervated, depending on the age of the
Thyroid and nervous system of E. martinicensis
425
embryo at operation (Hughes, 1962). The nerve fibres to the muscles of the
graft are however progressively lost towards the end of the embryonic period
(Hughes, 1964a). Since the withdrawal of innervation may occur at much the
(a)
1
2
3
4
5
6
7
8
9
10 11 12
1
2
3
4
5
6
7
8
9
10 11 12
Transplantation
Graff moves
Fixation
O
Slight movement
Text-fig. 13. Records of movement in supernumerary grafted forelimbs, with stages
of development of host embryo as in Table 2. (a) Eleven untreated embryos;
(b) corresponding embryos treated with 001 % PTU (arrows).
426
A. HUGHES
same time as the atrophy of the tail, it seemed of value to test the effect of
treatment with PTU on embryos bearing supernumerary limb grafts. Accordingly, forelimbs were autografted near hind limbs in a group of embryos, much
as in a previous series of experiments (Hughes, 1964o), except that in the present
group the operation was performed one day later, at 6 days of development.
These grafts when innervated usually show some movement with the nearby
leg on light stimulation. Such observations when repeated from day to day
serve as a means of gauging the degree of innervation of the grafts in the
living animal.
In Text-fig. 13 the history of eleven such grafts is followed over periods of 8-11
days from the time of operation. Movement of the grafts may be seen as early
as 3 days after operation but only in one example was this maintained continuously until the end of the experiment. In others, movement was intermittent
and for much of the time was recorded as 'slight'. The stage of development
of the embryo as a whole was noted and in Text-fig. 13 dotted lines are drawn
through those points on each record at which the embryo reached stages 'd',
' e ' , T and ' g ' respectively. The operation itself results in a slowing of development to a variable extent but only in one instance did the embryo not reach
stage ' f after 11 days of observation. In unoperated embryos developing in
saline these stages succeed each other approximately at daily intervals.
In Text-fig. 13 b the results are shown for seven similar embryos which were
immersed at stage V o r ' d ' i n 0-01 % PTU at either 3 or 4 days after grafting
an arm near a leg, and fixed 7 or 8 days later. The embryos reached stage ' e '
much at the same time as in the untreated group, but none advanced any further.
Embryos without grafts treated with PTU at stages ' c ' or ' d ' reach stage T
with continuous swimming within the same period. The extent of this arrest
of development must therefore be due to the combined effects of the operation
and the treatment with PTU, for, as already mentioned, limb grafting by itself
retards the development of the host embryo. Furthermore, the thyroid glands in
the present series of embryos were found to be much less affected by the PTU
than in corresponding ungrafted embryos, for follicles containing colloid were
still present.
The movement of the grafted arm in treated embryos was from the first
much greater than in those of the corresponding untreated embryos. Once
movement had begun in the supernumerary arm it continued until fixation in
six out of seven of these embryos. Frequently the movement was described in
the experimental record as 'good' or ' + + '.
It seems that there is an inverse relationship between the continued action of
a supernumerary arm and progress in the movement of the nearby normal limb.
A comparable result was seen in a previous experiment (Hughes, 19646) where
the nerve to the adjacent leg was cut at the time of grafting the supernumerary,
with the result that both were innervated by axons from a much reduced number
of regenerated ventral horn cells. Here both the innervation and the develop-
Thyroid and nervous system of E. martinicensis
427
ment of movement in the leg was greatly restricted, yet the action of the supernumerary arm was much greater than if the nearby leg had received a full
nerve supply.
Where a transplanted limb shares in the unreduced innervation of another
member, into which nerve fibres have already penetrated at the time of operation, the graft becomes innervated by axon collaterals which are withdrawn
towards the end of development in untreated embryos. In embryos of the present
series which were treated with PTU, and from which successful silvered preparations were obtained, it is possible to trace these collateral fibres inwards to
the lumbar plexus as far as the region of the spinal ganglia, where there is
always some residual axonal branching in juvenile animals.
It was observed that the fibres to the muscles of a supernumerary arm which
was motile at fixation were very few in number, some five or six at the most.
The fate of these fibres as judged by the behaviour of the graft during life is thus
a sensitive test of any changes in the extent of fibre branching within the limb
plexus. The errors involved in counts of the total number of fibres at various
levels conceal differences of this order.
DISCUSSION
The influence of thyroxine on the developing nervous system of Anuran
amphibians has been studied in several ways. Exogenously administered thyroxine has been shown to hasten certain normal features of development
including some events which occur during metamorphosis, such as the acquisition of the eye retraction reflex (Kollros, 1943). To these we may now add the
disappearance of the Rohon-Beard cells in Eleutherodactylus. Events within
the nervous system unrelated to metamorphosis have been shown to be accelerated by thyroxine, such as the rate of mitosis in the neural tube of larval Rana
tcmporaria (May & Mugard, 1955) and the maturation of ventral horn cells in
R. pipiens (Beaudoin, 1956; Reynolds, 1963). Such immature neurones respond
by increase in nuclear size. The action may be a direct one, as when the hormone
is applied as pellets close to the spinal cord, or indirectly through the mediation
of the periphery when the substance is inserted into the tissues of the limb
(Beaudoin).
Another procedure has been to observe the effect of the hormone on hypophysectomized larvae. Kollros & McMurray (1956) found that in the differentiation of the mesencephalic nucleus of the trigeminal in Rana pipiens the hormone
could fully compensate for the loss of the pituitary. Ventral horn cells, however,
do not develop normally under these conditions. In hypophysectomized larvae
the legs develop only as far as the late limb bud and the ventral horn remains
at the stage with large numbers of small cells. The effect of thyroxine on such
a larva is to promote limb growth as well as the loss of cells from the ventral
horn, without however any concomitant differentiation of the remainder
(Kollros & Race, 1960; Kollros, 1961; Race, 1961). In their experiments, de-
428
A. HUGHES
crease in cell number exhibited a lower threshold of response to thyroxine than
did increase in cell size.
A comparable result emerges from the present observations, for no treatment
of Eleutherodactylus embryos which depressed the activity of the thyroid was
found to reduce the loss of cells from the ventral horn. The differentiation
of the individual cells however was largely inhibited but was resumed after
treatment with thyroxine. In the embryo of Eleutherodactylus, unlike the larval
Anuran, the development of the limbs is not suppressed by thyroidectomy.
The effects of deprivation of thyroxine on developing ventral horn cells in
Eleutherodactylus may thus be ascribed to a direct effect on the hormone.
The only experimental observations on the peripheral effects of deprivation
of thyroid hormones on the development of nerve fibres which have hitherto
been described are those of Eayrs and his colleagues which concern the neuropil
of the cerebral cortex of thyroidectomized infant rats (Eayrs, 1955). The failure
under these circumstances to develop the correlative neural mechanism of
higher centres has far-reaching consequences on the behaviour of these animals,
with striking similarity to mental retardation in the human cretin.
In Eleutherodactylus thyroidectomy at a stage well before the end of embryonic
life is necessary in order to cause any inhibition of the development of the peripheral nervous system. For a cutaneous nerve such treatment results in failure
to increase the number of constituent fibres, while there is an actual decrease
in number within a muscular nerve. This contrast can be explained on the
hypothesis that fibre turnover in the latter is especially active, or is particularly
sensitive to thyroxine. The number of fibres will decrease if loss continues
normally after thyroidectomy, without replacement by newly developed axons.
There are two separate pieces of evidence that developing nerve fibres to
muscles are more unstable than those of cutaneous nerves in Eleutherodactylus,
namely the effects of thyroidectomy, and secondly the withdrawal of motor
innervation from supernumerary limb grafts which leaves cutaneous nerves
largely unaffected (Hughes, 1964<z).
SUMMARY
1. The effects of treatment with phenylthiourea and of thyroidectomy and
hypophysectomy on the embryo of Eleutherodactylus are described, mainly
with reference to changes within the nervous system.
2. In Eleutherodactylus the events at organ level during metamorphosis which
are controlled by the thyroid are reduced to loss of the tail and of the pronephros.
The primary sensory neurones of the cord, the Rohon-Beard cells, are also lost
at the same time.
3. The normal loss of cells from the ventral horns during development is not
affected by any treatment to which the embryo has been submitted. Development is retarded by treatment with phenylthiourea, and also by hypophysectomy
even when the pituitary is not wholly removed. In embryos submitted to this
Thyroid and nervous system of E. martinicensis
429
operation there is a loss of affinity for silver in the neurones of the ventral horn,
which is most severe when the pituitary is completely excised.
4. The addition of thyroxine to thyroidectomized embryos has been shown
to increase the number of fibres within a muscular and also a cutaneous nerve.
5. The number of fibres in a muscular nerve may be reduced by thyroidectomy or by hypophysectomy. It is suggested that in muscular nerves the loss
of fibres is unaffected by thyroid hormones but that the accession of new fibres
is sensitive to their action.
6. The movement of supernumerary grafted limbs is much enhanced by
general retardation of development with phenylthiourea.
RESUME
La thyroide et le developpement du systeme nerveux chez
Eleutherodactylus martinicensis: une etude experimental
1. On decrit les effets d'un traitement a la phenylthiouree, et de la thyroidectomie et de l'hypophysectomie, sur l'embryon d!Eleutherodactylus, surtout dans
leurs rapports avec des modifications a l'interieur du systeme nerveux.
2. Chez Eleutherodactylus, au niveau des organes, les phenomenes controles
par la thyroide au cours de la metamorphose se reduisent a la perte de la queue
et du pronephros. Les neurones sensoriels primaires de la moelle epiniere, les
cellules de Rohon-Beard, disparaissent aussi en meme temps.
3. La disparition normale des cellules des cornes ventrales au cours de
developpement n'est affectee par aucun des traitements auxquels l'embryon
a ete soumis. Le developpement est retarde par un traitement a la phenylthiouree, et aussi par l'hypophysectomie, meme quand l'hypophyse n'est pas
completement extirpee. Chez les embryons soumis a cette operation, il se produit
une perte d'affinite pour l'argent dans les neurones de la corne ventrale, plus
importante quant l'hypophyse est completement excisee.
4. L'adjonction de thyroxine a des embryons thyroidectomises augmente le
nombre de fibres dans un nerf musculaire et aussi un nerf cutane.
5. Le nombre de fibres dans un nerf musculaire peut etre reduit par la thyroidectomie ou par l'hypophysectomie. On suppose que, des deux composants
du renouvellement rotatif des fibres dans les nerfs musculaires, la perte de
fibres n'est pas affectee par les hormones thyroiidiennes, mais, l'arrivee de
nouvelles fibres est sensible a leur action.
6. Le mouvement de membres surnumeraires greffes est fortement accru par
un retard general du developpement, avec la phenylthiouree.
I am again grateful to Professor Ivan Goodbody and his colleagues at the Zoology Department of the University of the West Indies for all the help which they continue to provide for
these researches. I am much indebted to my colleague Martin Prestige for frequent discussion of common problems. The expenses of the work were partly met by a grant from the
Medical Research Council.
27
JEEM
l6
430
A. HUGHES
REFERENCES
BARRINGTON, E. J. W. (1964). Hormones and the control of color. In The Hormones, vol. iv,
chapter v, pp. 299-364. Ed. G. Pincus, K. V. Thimann, and K. V. Astwood. New York:
Academic Press.
BEAUDOTN, A. R. (1956). The development of lateral motor columns in the lumbo-sacral
cord in Rana pipiens. II. Development under the influence of thyroxin. Anat. Rec. 125,
247-60.
EAYRS, J. T. (1955). The cerebral cortex of normal and hypothyroid rats. Acta. Anat. 25,
160-83.
GORDON, A. S., GOLDSMITH, E. D. & CHARIPPER, H. A. (1943). Effect of thiourea on the
development of the Amphibian. Nature, Lond. 152, 504-5.
GRAY. E. G. (1957). The spindle and extra-fusal innervation of a frog muscle. Proc. R. Soc.
B, 146, 416-30.
HUGHES, A. (1954). The effect of fixation on neurones of the chick. /. Anat. 88, 192-203.
HUGHES, A. (1957). The development of the primary sensory system in Xenopus laevis
(Daudin). /. Anat. 91, 323-38.
HUGHES, A. (1958). Studies in embryonic and larval development in Amphibia. I. The
embryology of Eleutherodactylus ricordii, with special reference to the spinal cord. J.
Embryol. exp. Morph. 7, 22-38.
HUGHES, A. (1962). An experimental study of the relationships between limb and spinal cord
in the embryo of Eleutherodactylus martinicensis. J. Embryol. exp. Morph. 10, 575-601.
HUGHES, A. (1964 a). The innervation of supernumerary and replacing grafts of limbs in
Eleutherodactylus martinicensis. J. Embryol. exp. Morph. 12, 27-41.
HUGHES, A. (19646). Further experiments on the innervation and function of grafted supernumerary limbs in the embryo of Eleutherodactylus martinicensis. J. Embryol. exp. Morph.
12, 229-45.
HUGHES, A. (1965 a). A quantitative study of the development of the nerves in the hind limb
of Eleutherodactylus martinicensis. J. Embryol. exp. Morph. 13, 9-34.
HUGHES, A. (19656). The development of behaviour in Eleutherodactylus martinicensis
(Amphibia, Anura). Proc. zool. Soc. Lond. 144, 153-61.
HUGHES, A. (1965 c). Some effects of deafferentation on the developing Amphibian nervous
system. /. Embryol. exp. Morph. 14, 75-87.
KOLLROS, J. J. (1943). Experimental studies on the development of the corneal reflex in
Amphibia. II. Localised maturation of the reflex mechanism effected by thyroxin-agar
implants into the hind brain. Physiol. Zool. 16, 269-79.
KOLLROS, J. J. (1961). Mechanisms of amphibian metamorphosis: Hormones. Am. Zoologist
I, 107-14.
KOLLROS, J. J. & MCMURRAY, V. M. (1956). The mesencephalic V nucleus in Anurans.
II. The influence of thyroid hormones on cell size and cell number. /. exp. Zool. 131, 1-26.
KOLLROS, J. J. & RACE, J. (1960). Hormonal control of development of the lateral motor
column in the lumbo-sacral cord in Rana pipiens tadpoles. Anat. Rec. 136, 224.
LYNN, W. G. & PEADON, A. M. (1955). The role of the thyroid gland in direct development
in the Anuran Eleutherodactylus martinicensis. Growth 19, 263-86.
MAY, R.-M. & MUGARD, H. (1955). Action de l'ingestion de poudre de thyroide sur la multiplication cellulaire dans l'encephale des tetards de Rana temporaria. Ann. Endocrin. 16,46-66.
RACE, J. (1961). Thyroid hormone control of development of lateral motor column cells in
the lumbo-sacral cord in hypophysectomized Rana pipiens. Gen. comp. Endocrinol. 1,
322-31.
REYNOLDS, W. A. (1963). The effects of thyroxine upon the initial formation of the lateral
motor column and differentiation of motor neurones in Rana pipiens. J. exp. Zool. 153,
237-50.
RUGH, R. (1948). Experimental Biology. Burgess Publ. Co.
SMITH, P. E. (1916). The effect of hypophysectomy in the early embryo upon the growth
and development of the frog R. boylei. Anat. Rec. 11, 57-64.