/ . Embryol. exp. Morph. Vol. 28, 1, pp. 223-234, 1972
Printed in Great Britain
223
A structural analysis of
congenital limb deformities in experimental
hyperthyroid tadpoles
By K. H. SIT 1 AND R. KANAGASUNTHERAM 2
From the Department of Anatomy, University of Singapore
SUMMARY
An experimental model for the study of hyperthyroidism in embryonic development was
achieved in the local anuran larvae, Bufo melanostictus, by treatment with potassium perchlorate at a critical dose and time of embryogenesis.
Phocomely-hemimely and digital deformities of fore- and hind limbs in high percentages
of the population were found associated with such critically stimulated tadpoles. Similar
deformities were reproduced by exogenous treatment with L-thyroxine sodium. The deformities were severely aggravated if the tadpoles had anti-thyroid treatment before critical
stimulation with thyroxine in the sensitive phase of development.
Incoordinate cellular differentiation in the malformed limbs was indicated histologically by
undifferentiated mesenchyme in the distal extremities although the overlying ectoderm had
already formed mature epidermis with specialized skin glands. The differentiated mesodermal
components (muscles, cartilages and connective tissues) more proximally were afflicted with
degenerative changes. Lytic spinal motor neurones and involuted Mauthner's neurones with
precipitate metamorphosis were accompanying features. Embryonic thyrotoxicosis is discussed in relation to congenital deformities of limbs.
INTRODUCTION
While various embryonic abnormalities are related to hypothyroidism (Kalter
& Warkany, 1959; Woollam & Millen, 1960; Saxen &Rapola, 1969; Jost, 1963),
gross digital and limb deformities have hitherto not been directly correlated
with embryonic hyperthyroidism, although very recently a Negro family with
familial hyperthyroidism was reported to be afflicted with digital clubbing
(acropachy) and shortening (Wilroy & Etteldorf, 1971). The present paper
analyses the structural changes accompanying critical thyroxine stimulation at
a sensitive phase of limb morphogenesis and the resultant picture of deformities.
1
Author's address: Department of Anatomy and Embryology, University College London,
Gower Street, London WC1 6BT, U.K.
2
Author's address: Department of Anatomy, Faculty of Medicine, University of Singapore,
Singapore 3.
224
K. H. SIT AND R. KANAGASUNTHERAM
MATERIALS AND METHODS
Bufo melanostictus tadpoles were obtained by ovulation induction and
artificial insemination (Wong & Sit, 1971). On hatching, they were divided
into batches of 500 and placed in pails of 2-5 1. capacity on the laboratory bench
at room temperature (24-27 °C). Tadpoles were fed a patent staple food called
TetraMin (Tetra Werke, West Germany). This diet gave a uniform rate of
growth in the pails, with very low mortality.
Dose-response experiments were conducted with 1-8 mM concentrations of
potassium perchlorate (AnalaR) from the 5th day of development. Timeresponse experiments were conducted from the 5th, 10th, 15th, 20th and 25th
days of development with 1 mM potassium perchlorate. Effects of L-thyroxine
sodium B.P. were tested from the 10th day of development using a concentration of 10~ 7 M and also gradually increasing concentrations from 10~10 to
10~7 M. Further experiments were conducted by initial treatment with potassium
perchlorate in the first week of development before stimulation with L-thyroxine
in the second week. Effect of potassium iodide (AnalaR) with concentrations up
to 1 mM was also tested. Each of these drugs was introduced into the pails of
pond water after the daily clean-up. Controls were cultured under identical
conditions except for the omission of the drugs. Experiments were stopped
after 5 weeks and the results analysed by microdissections and histochemistry.
The histochemical methods used were Romeis' Kresazan (Duthie & Leach,
1950) and periodic acid-Schiff (PAS) in combination with alcian blue (pH 0-3)orange G (Purves, 1966). The Mauthner's neurone was stained by Gomori's
methanamine silver (Bourne, 1960) after overnight chromation, and periodic
acid-Schiff after oxidation in 0-3 % permanganate-sulphuric acid mixture as
used for pituitary cytology. Methylene blue-molybdate was used for metachromatic staining (Sit, Wong, Ng & Chin, 1972). Tert-butyl alcohol was the
dehydrating agent in the methylene blue-molybdate technique. Specimens for
Kresazan staining were Susa-fixed, while the rest were fixed in Bouin's fluid.
Diagrammatic profiles of the thyroid follicles were traced from camera lucida
drawings of 10 jum histological sections.
RESULTS
Dose-response experiments demonstrated that 1 mM potassium perchlorate
was the optimum concentration for suppressing limb bud growth and maturation when given from the 5th day of development. However, the time-response
experiments (Fig. 1) showed that the same concentration of potassium perchlorate when administered from the 10th and 15th day of development respectively, produced accelerated maturity with precipitate metamorphosis as shown
by the emergence of forelimbs; resorption of tail, gills, labial teeth, labial folds;
and differentiation of skin, nervous system and gut. This stimulation gave rise
Limb deformities in hyperthyroid tadpoles
225
Time-response with potassium perchlorate 1 m.\i
100 r-
• = K P 0 5 d.iv
• = * K P i 10 day
• = * K P2 15day
80 -
- K P , 20dav
= KP 4 25 day
4 = Control
o
t
60 h.
/
\
\
5
*
40 h
p
£•
•
-1
\
"»0
-
D—
0 -
V
/
•04m-
Limb bud
Paddle
A
A
4—
>\
Foot
Early
Mctamorphic Late
mctamorphic
metamorphic
Larval stages
*Limb malformations present
Fig. 1. Time-response experiments with 1 mM potassium perchlorate. Treatment
initiated from 5th day (KP0), 10th day (KP^, 15th day (KP2), 20th day (KP3) and
25th day (KP4) of larval development. Experiments were stopped at 39th day of
development. Staging of the tadpoles was according to Taylor & Kollros (1946). The
majority of control tadpoles were in paddle and foot stages, while all tadpoles in the
lOth-day group (KPX) and 15th-day group (KP2) had reached metamorphosis already.
Such critically stimulated tadpoles had severe limb deformities in nearly half of the
population in their respective pails.
to tadpoles which became stunted with severe limb deformities in over 40 % of
the population. At this particular phase of development, L-thyroxine stimulation
at a concentration of 10~7 M also produced similar limb deformities and stunting
(Fig. 2). When the tadpoles were first inhibited by treatment with potassium
perchlorate from the 5th day of development and then stimulated with 10~7 M
L-thyroxine from the 10th day, limb deformities increased markedly to about
95%. The main deformities found were gross shortening and deformities of
limb appendages with adactyly, syndactyly and brachydactyly (Fig. 3). But
after third week of development, the same thyroxine challenge could not produce
any deformities. Potassium iodide when administered alone did not accelerate
the maturity of the tadpoles nor did it give rise to any deformities; the resultant
larvae therefore resembled the controls.
Histochemistry
One mM potassium perchlorate given from the 5th day of development produced severe thyroxine deficiency in the tadpoles with marked degranulation of
the pituitary blue-violet or brown-violet basophils (thyrotrophs) (Fig. 4B)
whereas the controls showed dark-staining basophils in a resting state (Fig. 4 A).
There were no significant changes in the pituitary in hyperthyroid tadpoles.
15
E M B 28
226
K. H. SIT AND R. KANAGASUNTHERAM
i.-Thyroxine and potassium iodide effects
100
*
80
j_
=*T 2 10
M
{ =*T, 10 /KC104
., = T ,
=
7
7
D = K I 1 mM
i = Control
10-10M
60
Limb bud
Paddle
Foot
Early
Metamorphic Late
metamorphic
metamorphic
Larval stages
*Limb malformations present
Fig. 2. L-Thyroxine and potassium iodide effects. 1\: 1 mM potassium perchlorate
treatment from 5th day of larval development before stimulation with 10~7 M
L-thyroxine from 10th day of development. T2: 10~7 ML-thyroxine treatment from
10th day of development. T 3 : 1 0 ~ 1 0 M L-thyroxine treatment from 10th day of
development and increasing concentrations to 10~7 M in the 5th week of development.
KI: 1 mM potassium iodide treatment from 10th day of development. Experiment
stopped at 39th day of development. T\ had 95-4 % gross limb deformities,
while T2 had 40-3 % gross limb deformities.
The tadpoles which were given perchlorate treatment during the critical
period of development (10th—15th days) showed enlarged thyroid glands with
follicular proliferation (cf. Figs. 5 A, 6). There was marked vascular congestion
with follicles mostly emptied of colloid. The follicular epithelium was tall with
big pale-staining nuclei (Fig. 4C). On the other hand, tadpoles with exogenous
thyroxine treatment had small thyroids with follicles distended with acidophile
colloid and lined by flattened cuboidal epithelium (Fig. 4D). The controls
showed features intermediate between the two extremes.
The deformed limbs in the critically induced precocious tadpoles showed
differentiated muscles and cartilages more proximally whereas the distal
extremities were blastematous (Fig. 4E) in spite of the fact that these tadpoles
had reached metamorphosis. The blastematous stumps were surprisingly
covered by well-differentiated stratified epithelium of three layers with big skin
glands. This contrasted with the truly young limbs in normal immature tadpoles
where the overlying epithelium was only two cells thick with no skin glands and
lacked specialization except for the apical thickening. More proximally, where
differentiation had taken place, the skeletal muscles showed focal degeneration
with hyaline change and fatty infiltration. Occasionally adjacent muscles showed
basophilia with tightly packed nuclear rows composed of pale-staining, elongated
Limb deformities in hyperthyroid tadpoles
227
Fig. 3. Critically stimulated thyrotoxic tadpoles at metamorphic climax. (A) Phocomelic left hind limb. (B) Tridigital right and adactylous left forelimb with phocomely.
(C) Brachypodium in phocomelic left hind limb. (D) Hemimelic hind limbs with
brachypodia. (E) Left monodigital and right bidigital (fork) hemimelic forelimbs.
(F) Brachydactylous hind limbs. Scales: 1 mm.
nuclei in pairs, triplets or quadruplets where contiguous nuclei had tightly
opposed nuclear membranes with long beady chromatin material (Fig. 4F).
The cartilaginous shafts were almost devoid of matrix with hypertrophied
chondroblasts. Metachromatic staining was very poor with pale nuclei. But by
15-2
228
K. H. SIT AND R. KANAGASUNTHERAM
Limb deformities in hyperthyroid tadpoles
229
contrast, the staining was unusually intense at the ends of the shafts (Fig. 4J).
A similar picture was given by the alcian blue stain. The fibrous connective
tissue often stained strongly with PAS in contrast to the controls where the
staining was weak or negative.
The spinal cord showed chromatolysis of the motor neurones which
occasionally exhibited karyolysis (Fig. 4G). In the hind brain, the pair of
giant Mauthner's neurones had involuted, whereas in the controls they were
prominent with selective staining by Gomori's methanamine silver and oxidation-PAS technique (Fig. 4H, I). It was also observed that the gonads showed
hypertrophy (Fig. 4K) in those tadpoles which were stimulated by potassium
perchlorate or thyroxine when administered during the critical developmental
phase, whereas control tadpoles showed only early gonadal promordia (Fig. 4L).
Fig. 4. Histological sections are 10 /mi thick.
(A) Pituitary gland of control tadpole. At top half of picture, blue-violet to brownviolet basophils (thyrotrophs) in a resting state (arrow). Acidophils with homogenous red cytoplasm at bottom half of picture. Kresazan. Scale: 20/mi.
(B) Pituitary gland in thyroxine deficient tadpole. Marked degranulation of thyrotrophs with vacuolization and hyaline change in cytoplasm (arrows). Nuclei enlarged.
Kresazan. Scale: 20/tin.
(C) Thyroid hyperplasia with hyperactivity in perchlorate-induced tadpole. Marked
vascular congestion. Completely evacuated acini with tall epithelium and big palestaining nuclei. Kresazan. Scale: 20/mi.
(D) Thyroid gland in tadpole with exogenous L-thryoxine treatment. Extended
follicles with low epithelium and acidophile colloid. Kresazan. Scale: 20 /mi.
(E) Malformed limb in thyrotoxic tadpole. Cartilage and muscles differentiated
proximally but only blastema and epidermis at extremity. Kresazan. Scale: 100/mi.
(F) Striated muscles with hyaline degeneration in thyrotoxic tadpole. Adjacent basophilic muscle showed elongated nuclear triplet with long beady chromatin material.
Kresazan. Scale: 20/mi.
(G) Ventral horn of spinal cord in thyrotoxic tadpole. A pair of karyolytic motor
neurones with chromatolysis (arrow). Methylene blue-molybdate. Scale: 10 /mi.
(H) Hind brain in control tadpole. Selective staining of Mauthner's neurone.
Gomori's methanamine silver for glycogen. Scale: 20 um.
(I) Hind brain in control tadpole. Selective staining of Mauthner's neurone.
Oxidation-alcian blue-PAS-orange G. Scale: 20 jim.
(J) Forelimb in thyrotoxic tadpole. Almost unstained midshaft cartilage, but
unusually intense metachromasia at ends of cartilage. Methylene blue-molybdate.
Scale: 200/mi.
(K) Gonad in thyrotoxic tadpole. Advanced differentiation with enlargement
(cf. (L)). Kresazan. Scale: 50/mi.
(L) Gonad of control tadpole. Early gonad primordium (arrow). Kresazan. Scale:
50 /mi.
230
K. H. SIT AND R. K A N A G A S U N T H E R A M
Fig. 5
Fig. 6
Fig. 5. Diagrammatic profiles of normal lateral lobes of control thyroid glands in
(A) limb-bud stage, (B) paddle stage, (C) foot stage with marked distension of
follicles but no follicular proliferation, (D) early metamorphosis stage. Note the
smaller follicles and the increase in numbers. (E) Late metamorphosis stage.
Fig. 6. Diagrammatic profiles of lateral lobes of thyroid glands in (A) critical
potassium perchlorate-induced metamorphosis (note marked follicular proliferation,
(B) exogenous L-thyroxine-induced metamorphosis (follicles are long and distended).
DISCUSSION
The second week of larval development presented a labile phase of differentiation in the limb buds and thyroid glands of Bufo melanostictus tadpoles.
Relative iodine deprivation from perchlorate treatment (Pitt-Rivers, 1967) had
apparently resulted in thyroid hyperplasia with hyperfunction (Oakeson &
Lilley, 1957; Albright, Heninger & Larson, 1965). The dramatic consequence of
precipitate metamorphosis (Taylor & Kollros, 1946; Kollros & Kaltenbach,
1952; Weber, 1967; Eaton & Frieden, 1969) with severe limb malformations in
high percentages of experimental animals were recorded. Similar effects were
reproduced with 10~7 M L-thyroxine treatment (thyrotoxicosis factitia) when
given from the same critical second week of development. This dosage was
critical too since a lower concentration of L-thyroxine (10~10 M) given during the
2nd week of development with gradual concentration increases to 10~7 M in
the 5th week, had not produced any deformities although development was
accelerated (Fig. 2). It was significant that when growth of the limbs was first
Limb deformities in hyperthyroid tadpoles
231
retarded by anti-thyroid treatment in the first week of development and then
critically exposed to thyroxine in the second week, limb deformities increased
markedly in severity and numbers affecting nearly every tadpole in the pail! On
the other hand, after 3 weeks of development with differentiation of cartilage
and muscles setting in, the same concentration of thyroxine could no longer elicit
any deformities. It was there concluded that before the blastematous limb bud
had sufficiently differentiated there was a short period in the development when
persistent thyroxine stimulation was incompatible with normal limb morphogenesis. Such a period corresponded to Waddington's epigenetic crisis when a
developing organ became delicately vulnerable to teratogens.
The deformed limbs of these precocious tadpoles sometimes appeared as mere
stumps (Fig. 3). At the distal ends of these stumps there were evidences of uncoordinated differentiation with the overlying ectoderm (skin) apparently well
differentiated, but the underlying mesoderm presented as mesenchyme only.
Although the more proximal parts of these limbs had differentiated mesodermal
tissues, they were not altogether normal, as indicated by the frequently occurring
degenerate muscles with occasional attempts at regeneration (Godman, 1958;
Gutmann & Zelena, 1962), and degenerate cartilaginous shafts (Fell, 1956). The
unusually intense metachromatic and alcian blue staining at the ends of the limb
cartilages were probably due to excessive accumulation of mucopolysaccharides
which had been reported in thyrotoxic mucoid degeneration (Murray &
Symington, 1967).
In the deformed tadpoles, the intense PAS-staining of fibrous tissues could be
due to a fibrinoid change in the collagen as described by Walter & Israel (1967).
This corroborates well the changes in connective tissues from high thyroxine
action as reported by Moltke (1957), Jorgensen (1963), Gross (1964), Davidson
(1964) and Malt & Speakman (1965). It is noteworthy that abnormalities in
the connective tissues had been incriminated as the underlying cause of congenital limb defects in experimental lathyrism (Gross, Lapiere & Tanzer, 1963;
McCallum, 1965; McManus, 1968).
Allowing for normal cataclysmic neuroblast degeneration (mostly in cervical
and lumbar spinal levels), persistence of lytic motor neurones in the brachial
and pelvic spinal levels might be the result of retrograde neuronal degeneration
caused by thyrotoxic myopathy in both limbs (Tweedle, 1971). Persistence of
axonal degeneration (Cavanaugh, 1951) would reduce the normal trophic effect
of nerves on limb development and regeneration (Litwiller, 1938; Singer,
Rzehak & Maier, 1967; Schmidt, 1968; Goss, 1969). The Mauthner's neurones
being connected by reflex arcs to the lytic spinal neurones would be expected to
show transneuronal chromatolysis, if the degeneration observed had not been
the direct effect of high thyroxine action and precocious metamorphosis (Weiss
& Rossetti, 1951; Eayrs, 1964; Hughes, 1968).
In conclusion, it appeared that limb deformities associated with critical
thyroxine stimulation during the short sensitive phase (Waddington, 1966) of
232
K. H. SIT AND R. KANAGASUNTHERAM
embryonic development could be the result of thyrotoxic changes in the mesodermal tissues (muscles, cartilages and connective tissues) with uncoordinated
differentiation (Inman, 1941; Zwilling, 1956, 1964; Saunders & Fallon, 1966;
Wolfe, 1966). The situation was probably aggravated by the precocious loss of
major regenerative capacity (Bullough, 1967) from precipitate metamorphosis
and reduction of neuronal trophic influence.
We thank the University of Singapore and Lee Foundation for a grant to meet the cost
of the colour reproduction, and Miss Magaret Sim for her expert histological assistance. We
are grateful to Dr Ruth Bellairs for encouragement and reading the manuscript.
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(Manuscript received 29 September 1971, revised 20 February 1972)