J. Embryo/, exp. Morph. Vol. 46, pp. 215-225, 1978
215
Printed in Great Britain (gi Company of Biologists Limited 1978
Biochemical changes in developmentally
retarded Xenopus laevis larvae
I. The lens crystallin transition
By M. J. DOYLE 1 AND N. MACLEAN 2
From the Department of Biology, University of Southampton
SUMMARY
Premetamorphic tadpoles of Xenopus laevis reared in water containing 001 % propylthiouracil are developmentally retarded and metamorphosis is prevented. When uncrowded,
they continue to grow to a giant size. Moderate crowding leads to a slower rate of growth.
Thus morphologically premetamorphic tadpoles were produced with lens diameters appropriate to either normal premetamorphic, climactic or post-metamorphic animals. The lens
crystallins of such tadpoles have been separated by immunoelectrophoresis and polyacrylamide gel electrophoresis. The crystallin pattern was correlated with lens diameter rather
than with body stage. Giant retarded larvae possessed crystallin patterns identical to those
of normal adults. The thyroid antagonist propylthiouracil is therefore unable to prevent
the lens crystallin transition, the beginning of which is normally temporally correlated
with metamorphosis.
INTRODUCTION
Morphological differentiation of the amphibian lens into the primary fibre
cells of the nucleus and the secondary fibres of the cortex is accompanied by a
transition in soluble lens proteins. Such proteins are separable by electrophoresis.
These proteins, the crystallins, are classified as alpha, beta and gamma on a
basis of decreasing molecular weight and electrophoretic mobility.
In several amphibian species the gamma crystallins are the earliest detectable
in the embryonic lens (Takata, Albright & Yamada, 1964; McDevitt, Meza &
Yamada, 1969; McDevitt & Brahma, 1973). The transition to beta and alpha
continues through metamorphosis so that in Xenopus laevis Brahma & Bours
(1972) found the concentration of beta crystallins to be doubled and that of
alpha crystallins to be increased by a factor of 15 after metamorphosis.
Clayton (1970) has suggested that the adaptive value of the vertebrate
crystallin transition might be in maintaining appropriate refractile properties
as lens diameter increases in line with general body growth. Polansky & Bennett
(1973) have studied the crystallins of Rana catesbeiana tadpoles induced to
1
Author's address: Department of Chemistry, Florida State University, Tallahassee,
Florida 32306, U.S.A.
2
Author's address: Department of Biology, Medical and Biological Sciences Building,
Bassett Crescent East, Southampton SO9 3TU, U.K.
216
M.J.DOYLE AND N.MACLEAN
metamorphose precociously with exogenous thyroxine. We have adopted the
opposite approach, namely to block the synthesis or release of endogenous
thyroid hormones with a goitrogen and to study the crystallins of developmentally arrested larvae.
Maclean & Turner (1976) found that Xenopus laevis tadpoles, treated with
the goitrogen propylthiouracil, were not only developmentally retarded in
premetamorphosis but continued healthy growth for at least 2 years to reach
gigantic proportions (90 mm length as compared with a normal 40 mm).
Moderate crowding decreases the growth rate of larval amphibians (Rose, 1960).
In the present paper a combination of goitrogen treatment and moderate
crowding was used to produce Xenopus larvae of similar developmental stages
but varying in lens diameter. The development of the adult lens crystallin
transition was not prevented by goitrogen treatment and the degree of differentiation was correlated with lens diameter rather than with developmental
stage, thus supporting Clayton's hypothesis.
MATERIALS AND METHODS
Fertilized eggs were obtained by injecting pairs of Xenopus with human
chorionic gonadotrophin (Billett & Wild, 1975). The embryos and larvae were
reared in aged tap water. At stages 50-51 (Nieuwkoop & Faber, 1956) tadpoles
were placed in 0-01 % (0-59 mM) aqueous solutions of 6-«-propyl-2-thiouracil
(Sigma). Both control and treated tadpoles were fed Complan (Glaxo-Farley
Foods). The development of treated larvae was severely retarded at stages
54-56 but the tadpoles continued healthy growth, to giant size, for at least
15 months.
Two groups of tadpoles were reared to study the effect of lens diameter on
crystallin pattern. In one group ('large giants') the larvae were reared singly or
in small groups (about 2-51. solution per tadpole) while in the other ('stunted
giants') crowding was increased to about 0-41. solution per animal. The
'stunted giant' tadpoles were healthy and grew larger than non-goitrogen
treated controls of similar developmental stage but were much smaller than the
'large giant' group. The differences in lens size between the groups was determined on the basis of their lens diameters.
Larvae and adults were anaesthetized with MS 222 (Sandoz) and the lenses
removed and cleared of extra lenticular tissue. Lens diameters were measured
with the graticule of a stereo microscope. Pooled lenses were homogenized in
small volumes (less than 100 /A) of ice-cold amphibian ringer, pH 7-4 (Rugh,
1962) with glass homogenizers. Homogenates were centrifuged at 500 g for
20 min and the total protein content of the supernatant crystallin extract was
estimated (Lowry, Rosebrough, Farr & Randall, 1951) using bovine serum,
albumin as a standard. Dithiothreitol to a final concentration of 3 mM was
added to crystallin solutions after protein determination.
Lens crystallin transition in retarded Xenopus larvae
217
Polyacrylamide disc gels of 7% acrylamide were prepared according to
Davis (1964) using a tris-glycine/tris-HCl discontinuous buffer system. The
specific gravity of samples was increased with sucrose before application to the
stacking gel. The running buffer was tris-glycine, pH 8-3 (Davis, 1964). Both
running and gel buffers contained dithiothreitol (3 HIM). Gelling was catalysed
with ammonium persulphate and persulphate ions were electrophoresed from
the gels by pre-running for \ h at 3 mA/gel (Mitchell, 1967). Samples were concentrated in the 2-5 % acrylamide stacking gels by 10 min running at \ mA/gel.
The final separation took \\ h at 3 mA/gel. Gels were stained with 1 % naphthalene black 12B in 7% acetic acid and destained with the same solvent.
An antiserum was prepared in New Zealand White rabbits against a crystallin
solution from pooled adult Xenopus lenses. Immunoelectrophoresis was performed on glass microscope slides with 1 % Litex HSA agarose (International
Enzymes) in a tris-EDTA-boric acid buffer, pH 9-0 (Aronsson & Gronwall,
1957). Two [A of crystallin solution (each adjusted to 6 mg/ml total protein)
was applied per antigen well and electrophoresis proceeded for 1 h at constant
current with an initial potential difference of 180 V. After 24 h in the presence
of the antiserum the slides were washed with saline and distilled water, air
dried and stained with 1 % naphthalene black 12B in 7% acetic acid.
RESULTS
Normal lens crystallin transition
A typical adult lens crystallin polyacrylamide gel separation (Fig. 1A)
contains 11 bands. About 90% of lens soluble protein is crystallin (Clayton,
1970).
Bands 2 and 3 sometimes overlapped, as did bands 4 and 5. The largest
changes in relative concentration during larval development were in bands 6, 8
and 11, the latter being the most anodal. Bands 6 and 8 were the most prominent
bands in adult crystallin gels but were scarcely visible in control tadpole gels.
This is in agreement with Manwaring (1972).
Samples were also separated simultaneously on polyacrylamide slab gels. The
slab ensured that the identification of individual bands in different samples was
correct but resolution was poorer than with discs.
Crystallin precipitin arcs obtained in immunoelectrophoresis were classified
as alpha, beta and gamma (Figs. 2 and 3). Proteins from pooled slices of bands
6 and 8 were eluted from polyacrylamide gels by reference to gels stained with
the rapid Coomassie brilliant blue method of Allen, Spicer & Zehr (1976). When
re-electrophoresed on polyacrylamide gels each eluate appeared as a single
band which was precipitated in the beta position by immunoelectrophoresis.
Band 11 was precipitated in the alpha position.
When adult and normal stage-54 and -57 tadpole crystallin solutions (all
adjusted to 6 mg/ml total protein) were compared (Fig. 2A, B, D) the alpha arc
218
M. J. DOYLE AND N. MACLEAN
i (A) y (B> fj ( o 11 (D) |
Origin
Fig. 1. Polyacrylamide gel electrophoresis of lens crystallins from, (A) an adult
female, (B) a stage-55 propylthiouracil-treated giant tadpole, (C) pool of two stage-55
tadpoles propylthiouracil-treated for only 8 weeks (non-giant) and (D) pool of six
normal stage-53 tadpoles. Bands 6, 8 and 11 (marked with arrows) increased most
markedly in relative concentration during normal development and in giant tadpoles
(gei E;.
was visible only on adult slides. There was also an increase in the number of
beta and gamma antigens precipitated after metamorphosis.
The crystallin transition in developmentally retarded larvae
When propylthiouracil-treated uncrowded tadpoles (Fig. 1B) were compared
with normal controls (Fig. 1A, D) it was found that polyacrylamide bands
6, 8 and 11 had increased to near adult proportions in the former. Immunoelectrophoresis slides prepared with treated tadpole crystallins were almost
identical to those prepared from adult crystallins. Unlike slides from normal
untreated tadpoles of similar Nieuwkoop & Faber stage, the alpha arc was
always present in uncrowded treated tadpoles (Figs. 2E, G). Thus thyroid
blockage does not prevent the lens crystallin transition.
Lens crystallin transition in retarded Xenopus larvae
(A)
(B)
(Q
(D)
(E)
(F)
(G)
(H)
Fig. 2. Immunoelectrophoretic precipitation of lens crystallin antigens by a rabbit
anti-adult Xenopus total lens crystallin serum. (A) Adult female, (B) normal stage 57,
(C) normal stage 57, (D) normal stage 54, (E) large giant tadpoles stage 54/55, (F)
normal adult female, (G) large giant tadpoles stage 54/55, and (H) normal stage 57.
All crystallin solutions were adjusted to 6 mg/ml total protein. Arrows mark
crystallin arcs which appeared after metamorphosis in untreated animals.
219
220
M.J.DOYLE AND N.MACLEAN
Table 1. Lens diameters of normal Xenopus laevis compared with those of
propylthiouracil-treated 'stunted giant' and'large giant' tadpoles by the ' ? ' test
Lens diameter,
Stage
mm {n —)
48-49
0-47 (8)
0-52 (20)
0-51 (20)
0-53 (24)
0-55 (18)
0-66 (38)
0-68 (16)
0-73 (28)
0-77 (26)
0-85(16)
0-91 (4)
50
51
52
53
54
55
56
57
58
59
Toadlet
Adult
Stunted
giants
(st. 52-55)
Large giants
(st. 53-56)
1-02(14)
2-00(16)
0-82 (31)
1-08(16)
Stunted giant
tadpoles
(stages 52-55)
Large giant
tadpoles
(stages 53-56)
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
**
NS
NS
***
***
* **
**•
***
** *
—
NS
***
***
***
—
NS, Lens diameters of propylthiouracil-treated larvae not significantly different from
controls.
Significant differences: *** P < 0001; ** P < 002.
'«', Sample size.
The correlation between lens diameter and lens crystallin transition
It was noted in preliminary work with polyacrylamide gels that tadpoles
treated with goitrogen for short periods (about 8 weeks) had not grown markedly
larger than controls and still possessed a tadpole-like complement of crystallins
(Fig. 1C). Thus it was decided to study the transition in connexion with lens
size to test Clayton's hypothesis.
The normal growth in lens diameter is shown in Table 1. When the two
groups of goitrogen-treated tadpoles, 'large' and 'stunted', were compared
statistically with respect to lens diameter it was found that large giants (stages
53-56) had lenses appropriate in size to untreated toadlets whereas the stunted
group (stages 52-55) had lenses identical in size to untreated tadpoles of stages
58-59. The difference between the lens diameters of stunted and large giant
tadpoles was highly significant.
Lens crystallin solutions from untreated tadpoles and adults were compared
with those from the two treated groups by immunoelectrophoresis. The' stunted'
and 'large giant' crystallins are compared directly in Fig. 3E and F.
Fig. 3A-D show the 'stunted giant' crystallin antigens precipitated to be
Lens crystallin transition in retardedXenopus
larvae
221
Origin
(A)
7
(B)
I
a
t
(C)
(D)
(E)
(F)
Fig. 3. Immunoelectrophoretic precipitation of lens crystallin antigens. (A) Stunted
giant tadpoles stage 53/54, (B) adult female, (C) stunted giant tadpoles stage 53/54,
(D) normal stage 57, (E) stunted giant tadpoles stage 53/54, and (F) large giant tadpoles stage 54/55. All crystallin solutions were adjusted to 6 mg/ml total protein.
Arrows mark crystallin arcs which appeared after metamorphosis in untreated
animals.
unlike those of adults but like normal stage-54 or stage-57 tadpoles in that the
alpha arc is absent and that only single beta and gamma crystallin arcs were
precipitated.
Fig. 2E and F shows that crystallins precipitated in 'large giant' extracts were
identical to those from normal adults. The alpha arc was present in both cases.
The 'large giant' group was morphologically in stage 53-56, but the crystallin
pattern was clearly more advanced than those of normal stage-57 lenses.
Although both groups of tadpoles were in premetamorphosis and had been
treated for similar times with propylthiouracil, the precipitin patterns are clearly
different. Thus it appears that lens crystallin patterns are related to lens diameter
222
M.J.DOYLE AND N.MACLEAN
rather than to developmental stage of gross morphology. 'Large giant' tadpoles
whose lenses were appropriate in size to normal toadlets produced an adult-like
pattern. 'Stunted giant' lenses which were similar to normal lenses of stages
58-59 had a premetamorphic crystallin pattern.
DISCUSSION
Immersion in solutions of the anti-thyroid compound propylthiouracil
arrested the development of Xenopus larvae in premetamorphosis. Nevertheless
mortality was not increased and healthy growth was not prevented. A recent
cell-free in vitro study (Taurog, 1976) has confirmed earlier reports that, in
mammals, propylthiouracil inhibits the peroxidase-catalysed iodination of
protein and tyrosine. In the present work the thyroid glands of treated larvae
became goitrous, indicating that thyroid stimulating hormone was released in
abnormal quantities in response to lack of circulating thyroid hormone. MiraMoser (1972) noted histological criteria typical of surgically thyroidectomized
animals in the pituitaries of propylthiouracil-treated Bufo bufo larvae. Following
Etkin's (1966) evidence that hypothalamic differentiation is stimulated by
thyroxine, Goos (1968) found the process to be inhibited by propylthiouracil in
Xenopus.
Thus propylthiouracil is well established as a potent goitrogen in Amphibia,
and was an ideal compound with which to study the effect of thyroid blockage
on the lens crystallin transition.
Uncrowded tadpoles arrested in premetamorphosis had lens diameters
similar to those of normal post-metamorphic toadlets and their crystallin
transition was not arrested.
Polansky & Bennett (1973) showed that, in Rana catesbeiana, lens diameter
increases significantly during natural and thyroxine-induced metamorphosis.
However, no change could be detected between the cellulose acetate electrophoretic patterns of crystallins from control tadpoles and tadpoles which had
undergone thyroxine-induced metamorphosis. Study of crystallins from normal
tadpoles, froglets, young adults and mature adults suggested the transition to
be a gradual one. It involved an increase in the relative concentration and
mobility of alpha crystallins, a decrease in the relative concentration of gamma
crystallins, and an increase in the relative concentration of beta crystallins, and
was, therefore, similar to that observed in Xenopus.
In the present work, lens size continued to increase in uncrowded developmentally retarded Xenopus suggesting that thyroid hormones do not control
lens growth directly. It is known that lens size and fibre differentiation have
important extralenticular control mechanisms (Coulombre & Coulombre, 1969).
However, the lens growth associated with metamorphosis takes place during a
period when overall body length is decreasing as the tail regresses, and is part
of an allometric process in which the proportions of the head become more
Lens crystallin transition in retarded Xenopus larvae
223
adult-like. In the absence of metamorphosis, lens diameter appeared to be
related to body size in that crowded goitrogen-treated tadpoles, whose overall
growth was slower than that of uncrowded specimens, also exhibited slower
lens growth. Thus it is unlikely that lens growth was a process initiated by
thyroid hormones before the onset of propylthiouracil treatment, or that the
process was controlled by extremely low thyroid hormone release which may
have persisted in the presence of the goitrogen.
Similar arguments apply to the crystallin transition which was correlated
with lens diameter irrespective of gross developmental stage or age. As previously mentioned, alpha and beta crystallins increase in relative proportion
during development at the same time that lens diameter increases (Brahma &
Bours, 1972; Polansky & Bennett, 1973). These classes of lens protein have also
been shown to be the last detectable in the early development of the amphibian
lens (McDevitt, 1972). Thus the increase in concentration of alpha and beta
crystallins with increasing lens size might be expected as the proportion of
post-embryonic to embryonic lens tissue in whole lens homogenates rises with
larger lenses.
This argument is supported by Polansky & Bennett's (1970) finding that the
electrophoretic pattern exhibited by the crystallins of adult Rana catesbeiana
lens cortex is unlike that of the adult lens nucleus. The cortex contained proportionately more alpha crystallin. However, the adult lens nucleus crystallins
were similar to those of the tadpole whole lens with gamma and beta crystallins
predominating.
As no sudden change in crystallin electrophoretic pattern occurs at metamorphosis (Polansky & Bennett, 1973) and as the present work has shown the
transition to occur irrespective of developmental stage there is no evidence for
thyroidal control of this aspect of lens differentiation. This contrasts with other
ocular tissues such as the eyelid, nictitating membrane, cornea and extrinsic
ocular muscles, which in Rana pipiens, metamorphose to forms thought to be
adaptive to terrestrial life. In these tissues Kaltenbach (1953) obtained evidence
for direct and local thyroxine control. The crystallin transition which also occurs
in such non-metamorphosing vertebrates as birds and mammals (Soloman, 1965)
appears to have a different control mechanism.
The evidence of Maclean & Turner (1976) that the haemoglobin transition
in giant Xenopus tadpoles is not directly linked to developmental stage is thus
paralleled by our work with crystallins. Additional work on haemoglobin and
other proteins in these giant tadpoles has now been completed and will be
published in a later paper.
We are pleased to acknowledge the interest shown by Drs S. C. Turner and F. S. Billett.
Financial support was provided by the Natural Environment Research Council and the
University of Southampton.
224
M.J.DOYLE AND N.MACLEAN
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