Arrested development in Xenopus laevis tadpoles: how size constrains metamorphosis
Department of Anatomy and Neurobiology, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, B3H 1X5, Canada
* Author for correspondence (e-mail: nikcevic{at}dal.ca)
Accepted 29 March 2004
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Summary |
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Key words: Xenopus laevis, giantism, thyroid gland, growth, development, neoteny
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Introduction |
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The African clawed frog Xenopus laevis is the only anuran species
in which a spontaneous lack of metamorphosis has been documented; on rare
occasions, X. laevis tadpoles with arrested development occur
alongside normal siblings. Although these tadpoles cease developing at
approximately NieuwkoopFaber (NF) developmental stages 5355,
i.e. at early hindlimb bud stages
(Nieuwkoop and Faber, 1956),
their growth continues, and they develop into grossly deformed giants
(Fig. 1). These giant tadpoles
can remain at this stage for several years
(Jurand, 1955
;
Dodd and Dodd, 1976
), without
developing further.
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Giant, non-metamorphosing, tadpoles have occasionally been reported in a
number of European populations within the Rana esculenta complex (see
review in Borkin et al., 1982).
However, this giantism is usually attributed to the fact that R.
esculenta is a hybrid; triploids frequently occur among individuals of
that phenotype, and reach much larger body size than diploids
(Berger and Uzzell, 1977
).
Arrested development in X. laevis has been reported in the
literature at least four times (Toivonen,
1952; Jurand,
1955
; Srebro,
1970
; Dodd and Dodd,
1976
). Our laboratory X. laevis colony has spontaneously
produced two such giants in the last three decades. In surveying other
laboratories that raise X. laevis (cf.
Major and Wassersug, 1998
), we
confirmed that the spontaneous appearance of these giant tadpoles is a very
rare event. However, Xenopus One (Dexter, MI, USA), a commercial supplier of
X. laevis in North America, had 45 live giant tadpoles collected over
a period of 8 years and made these specimens available to us.
Jurand (1955) found that
giant non-metamorphosing X. laevis larvae lack thyroid glands. As a
result, they do not produce the thyroid hormones (TH) that are necessary for
the initiation and completion of metamorphic transformation
(Shi, 2000
). Developmental
stages of all giant X. laevis tadpoles, both in this study and
reported in the literature, range from NF 53 to 56. At these stages there is
little or no TH present in a tadpole's circulation, and differentiation
proceeds independent of hormone concentration.
Non-metamorphosing X. laevis tadpoles show obvious morphological
and behavioural differences compared to normal X. laevis tadpoles of
a corresponding stage (Rot-Nikcevic and
Wassersug, 2003). Giants are on average 4 times longer and up to
50 times more massive than normal tadpoles, and they have scoliosis, with C-
or S-shaped back curvature. Their axial and tail muscle masses are
disproportionately large relative to their body mass. They have difficulty
swimming and holding their position in the water column; instead they
frequently rest on the bottom. In this paper we explore how excessive somatic
growth in giants may affect their subsequent development, and eventually
preclude normal metamorphosis.
Allen (1916,
1918
) showed that removing
thyroid glands in Rana pipiens tadpoles at early stages resulted in
giant, non-metamorphosing larvae, similar to our naturally occurring X.
laevis tadpoles, and the ones described by Jurand
(1955
). Development in
thyroidectomized R. pipiens tadpoles proceeded normally until the
hindlimbs began to form. At this stage tadpoles arrested their development,
their forelimbs never erupted through the skin, and their tails retained
larval proportions. Tadpoles continued to grow into giants. In addition, Allen
(1918
) noted that thyroidless
tadpoles retained the head and intestinal features of larvae, but their gonads
proceeded to differentiate and mature. Gonadal development in thyroidectomized
R. pipiens larvae confirmed that germ cell differentiation is not
under the influence of the thyroid gland, while somatic differentiation and
metamorphosis require thyroid gland induction.
In a previous study we showed that, although naturally occurring giant
X. laevis tadpoles lack endogenous TH, their tail tissue retains its
sensitivity to TH, and triiodotironine (T3)-mediated tailtip
resorption could be induced in vitro
(Rot-Nikcevic and Wassersug,
2003). This suggests that treatment with exogenous T3
in whole animals might induce giants to resume development and eventually
metamorphose. Alternatively, giantism per se could physically inhibit
normal development and metamorphosis in tadpoles. The upper size limit for a
tadpole that can still metamorphose is likely to be species-specific though,
as the largest of all tadpoles, Pseudis paradoxa larvae, for example,
can reach body lengths up to 22 cm
(Emerson, 1988
), and still
have normal obligatory metamorphosis.
In the present study we explore how the disrupted ratio between development and growth in giant X. laevis tadpoles limits their normal development. We examine whether growth in giants is a simple allometric extension of normal growth, and whether increase in visceral size, for example, follows the excessive increase in body length and mass. The fact that giant tadpoles have difficulty swimming, and frequently rest on the bottom, leads us to hypothesize that visceral growth is not proportional to total body growth specifically, the lungs of these giants are probably not large enough to provide buoyancy.
We also hypothesize that, even if treatment with exogenous TH can induce metamorphosis in X. laevis giant larvae, the resorption of their massive tail muscle will take disproportionately longer than the transformation of other parts of their body, particularly the viscera. This might pose a barrier for normal development and metamorphosis of these animals.
However, the fact that X. laevis can live for several years as
larvae, without metamorphosing, raises the question of whether larval
reproduction is possible in tadpoles, and if so, why neoteny, although common
in urodeles, has never evolved in anurans. Hayes
(1997b) suggested that
decoupling of the hormonal requirements for metamorphosis and reproduction
allowed neoteny to evolve several times in urodeles. The fact that
thyroidectomized R. pipiens larvae and naturally thyroidless X.
laevis tadpoles show advanced gonadal development without metamorphosis
(Allen, 1918
) suggests that
larval reproduction at least in terms of maturation of sperm and egg
production may be possible in anurans. Here, we investigate
limitations other than hormonal requirements that may hinder the evolution of
neoteny in anurans.
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Materials and methods |
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Morphology Dissection and measurements
Animals were killed using MS 222, and staged
(NieuwkoopFaber, 1956)
prior to dissection. Total body length, tail length, pleuroperitoneal cavity
length, maximum head width and height, maximum tail fin height and tail muscle
height, were measured on intact animals using a digital caliper. The volume of
intact animals was measured by water displacement in tubes of precisely known
volumes.
Transverse cuts were made at the vent and throat, and along the midline,
and the skin flaps pulled to the sides to expose the viscera. The alimentary
tract was dissected away, and its intestinal volume measured by displacement
of water in notched pipette tips using Hamilton gas syringes graduated to 0.01
ml. Maximum length and width of the liver, heart, spleen, gallbladder, kidneys
and gonads were measured in situ, using an ocular micrometer. Fat
bodies were dissected out and their volume measured by water displacement. The
gallbladder was too small to measure its volume reliably, therefore we used
the equation for the volume of a spheroid,
V=4/3[(L+W)/4]3, where L
and W represented gallbladder length and width, respectively
(Frankenberg and Werner,
1992
).
The lungs were first outlined in situ using a camera lucida, and their area measured in NIH Image (NIH Image for Macintosh 1999, Version 1.62, National Institute for Mental Health). Both the lungs and the gonads were then dissected out and embedded in paraffin, cut in 4 µm cross-sections, and stained with Hematoxylin and Eosin or Masson's Trichrome. After all the viscera were dissected away, the volume of eviscerated animals was measured.
A cross-section of axial muscle from one giant and one normal X. laevis tadpole was traced using a camera lucida and area was measured in NIH Image.
Animals were preserved in 10% formalin. For all giant tadpoles, lungs, intestine and gonads (if present) were preserved.
One normal tadpole, two giant tadpoles and one giant tadpole exposed to
T3-treatment (see below) were cleared and double-stained for bone
and cartilage using Alizarine and Alcian Blue, following the protocol of
Hanken and Wassersug
(1981).
Sample sizes for the gallbladder volume and the lung area in giant tadpoles are smaller than the full sample size, due to the destruction during dissection of these organs in a few specimens.
Hormonal treatment
Five normal and five giant X. laevis tadpoles of the same stage
were reared in two separate 10 l tanks, with exogenous T3 added to
a 3 nmol l1 concentration. Five normal tadpoles reared in a
separate tank without added hormone were used as a control group, as well as
25 giant tadpoles.
Prior to hormonal treatment animals were staged, and their body mass, volume, total body length, body width, tail length, tail fin and muscle height measured. Every 2 weeks the animals were anaesthetised in MS 222, staged and measured. During the first 2 weeks of treatment, water samples from both experimental tanks were drawn and T3 concentrations assayed every other day, at the IWK Health Center in Halifax, NS, USA. Once a week half of the water in the tanks was changed, and fresh hormone added. This proved to be frequent enough to maintain a constant T3 concentration.
The same experiment was repeated with another set of five giant tadpoles exposed to 1 nmol l1 exogenous T3, to test for the possible effects of hormone dosage.
Statistical analyses
The analyses were run in STATISTICA (STATISTICA for Windows 1997, Version
5.0, StatSoft, Inc.). All body measurements were log-transformed. To control
for the overall effect of body size, measurements were regressed to total body
length, snoutvent length (SVL) or total body volume. An
analysis of covariance (ANCOVA) was used to test for differences in relative
body measurements among normal and giant tadpoles. Differences in the slopes
of the regression lines were analysed using the Test for Parallelism
(STATISTICA), to determine whether growth in giants is a simple allometric
extension of normal growth.
Differences in external body measurements in animals before and after T3 treatment were tested using analyses of variance (ANOVA). Visceral measurements in the 10 giant tadpoles exposed to hormonal treatment were compared to corresponding measurements in 25 control giants not exposed to T3.
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Results |
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Visceral morphology
The most obvious qualitative differences between giant and normal tadpoles
were in the lungs, fat bodies, gonads and the coiling of the intestine.
All normal X. laevis tadpoles had functional, inflated lungs. However, the majority of giants, i.e. 17 out of 25 tadpoles (68%), had small and, to varying degrees, solidified lungs (Fig. 2A). Only eight giant tadpoles (32%) had well-developed and inflated lungs (see Fig. 2B). A significant relationship between the total body mass, or volume, and the lung area was not found.
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The lungs of normal tadpoles were not septate (Fig. 3A). The lung structure in giant tadpoles with inflated lungs (Fig. 3B) resembled that of normal tadpoles, i.e. most of the lung was air-filled space, but had more septa. Solid lungs, however, showed a large number of septa with thick layers of smooth muscle tissue, and little space left in the lung sacs to be filled with air (Fig. 3C). The wall of solid lungs consists of a thin lung epithelium, a thick smooth muscle layer with abundant collagen, and numerous melanocytes. At higher magnification, thick bands of collagen were visible within the septa of solidified lungs in giants (Fig. 3D). In our years of experience with giant X. laevis tadpoles we have never seen individuals that were negatively buoyant and resting on the bottom swim to surface and gulp air.
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In normal tadpoles the alimentary tract is arranged in a spiral, and fills most of the pleuroperitoneal cavity. In giant tadpoles the classic coiling pattern was not present; i.e. the intestine made random left and right loops, with few coils and no consistent pattern for intestinal packing.
Fat bodies were present in 15 giant tadpoles (60%), and in most cases (80%) the fat bodies were large and occupied most of the visceral cavity. They were bright yellow in colour, with numerous finger-like projections entwined among the other viscera (Fig. 4). Fat bodies were absent in all normal tadpoles of the same stage.
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None of the normal tadpoles had developed gonads. However, many giants had
advanced gonadal differentiation. Out of 25 giant tadpoles, 13 had
well-developed gonads. Out of these 13 tadpoles, 12 were females and their
ovaries were full of eggs (Fig.
5A). The largest oocytes in giant tadpoles were at stage IV
(Dumont, 1972); the animal and
vegetal hemispheres were differentiated, oocytes had yolk, and oocyte size was
on average 600 µm. Consistent left/right asymmetry (handedness) typical of
anurans (Kendal 1938; cited in Viertel and
Richter, 1999
) was not found in the size of ovaries (see also
Malashichev and Wassersug,
2004
). One giant tadpole was male, with well-developed testes.
Histological analyses showed that sperm was present in its testes
(Fig. 5B). Interestingly, this
only male giant came from our Xenopus colony almost three decades
ago. The 12 females all originated from the Xenopus One breeding colony.
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Morphometric analyses
The volume of intact and eviscerated tadpoles and the volume of their
internal organs are presented in Table
2. Giant tadpoles had greater variance in all measured
morphological traits than did normal tadpoles.
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External morphology
Differences in tail length between giant and normal tadpoles, when total
body length was controlled for, were not significant (ANCOVA,
P=0.108). Also, there were no significant differences in the slopes
of the regression lines (P=0.942). Therefore, the relationship
between the increase in the tail length with increase in total body length was
similar in the two groups (see Fig.
6). The test for parallelism for head length, head width, and tail
fin and tail muscle height, revealed homogeneity of slopes
(P>0.05) as well, suggesting that all these traits retain their
normal allometric relationships. Similar results were obtained when the head
length and width were regressed to SVL instead of total body length.
Although differences between giant and normal tadpoles in the length of the
pleuroperitoneal cavity relative to total body length were significant
(ANCOVA, P=0.020), with giant tadpoles having a longer
pleuroperitoneal cavity than normal tadpoles, the regression line slopes were
the same (Fig. 6).
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When total body length was accounted for, giant tadpoles had significantly larger body mass than normal tadpoles (ANCOVA, P=0.004). However, the regression of body mass on total body length revealed no differences in slopes (P=0.103), indicating that the increase in body mass with the increase in total body length had the same growth trajectory in both groups (Fig. 6).
Eviscerated body volume relative to intact body volume was significantly greater for giant tadpoles than normal ones (ANCOVA, P=0.047). In other words, the somatic (non-visceral) tissue in giant X. laevis tadpoles represents a significantly larger portion of the total body volume than in normal tadpoles. The axial muscle mass in our giant tadpoles was disproportionately enlarged. The cross-sectional area of axial muscle tissue was 16 times larger in a representative giant than in a normal tadpole of the same stage (Fig. 7), whereas body length was only 2.5 times greater. Therefore, the axial muscle mass was approximately 2.5 times larger than would be expected if it scaled proportionately as the square of body length.
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Visceral morphology
Although the alimentary tract in giant tadpoles was not coiled as in normal
X. laevis tadpoles, there were no significant differences between the
groups in intestinal volume relative to total body volume (ANCOVA,
P=0.461).
Giant tadpoles had longer livers relative to their total body length than normal tadpoles (ANCOVA, P=0.0001). Also, the difference between the regression line slopes was significant (P=0.022), showing a slower increase in liver length with the increase in total body length in giant compared to normal tadpoles (Fig. 6). The same results were obtained for liver length relative to SVL. No significant differences were found in liver width relative to total body length between groups.
We compared lung area against total body volume in three groups: normal tadpoles (with inflated lungs), giant tadpoles with inflated lungs, and giant tadpoles with solidified lungs. Relative lung area was significantly different in all groups (ANCOVA, P<0.001). Giants with inflated lungs had the largest lung area relative to body volume, followed by normal tadpoles, and lastly by giants with solidified lungs. Slopes of the regression lines showing the relationship between lung area and total body volume (Fig. 8), significantly differed only between normal tadpoles and giants with solid lungs (P=0.047), but were homogenous in normal tadpoles and giants with inflated lungs (P=0.832), as well as in giants with solid lungs and giants with inflated lungs (P=0.147). In giant and normal tadpoles with inflated lungs, the lung area increased with an increase in body volume, following the same pattern. However, in giant tadpoles with solid lungs, the regression line's slope suggests a faster increase in lung area with body volume, when compared to normal and giant tadpoles with inflated lungs.
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Heart length and width relative to total body length were both significantly larger in giant tadpoles (ANCOVA; P=0.02 and P<0.001, respectively) than normal ones. Similar results were obtained when heart length and width were regressed to SVL. However, differences in the regression lines' slopes in the two groups were not significant for either heart length or width (length, P=0.139; width, P=0.614). Thus, giants have relatively larger hearts, but the increase in heart size with increasing body size follows the same pattern in giant and normal tadpoles.
Both spleen length and width relative to total body length were greater in giant tadpoles than normal ones (ANCOVA; length, P=0.006; width, P=0.001). The relationship between the increase in spleen size and increase in total body length was similar in giant and normal tadpoles, since the regression slopes did not differ significantly (length, P=0.681; width, P=0.260). Similar results were obtained when spleen measurements were regressed to SVL.
Gallbladders in both normal and giant tadpoles were transparent spherical sacs. Giant tadpoles had larger gallbladders than normal tadpoles relative to total body volume (ANCOVA, P=0.047), but the regression lines' slopes did not differ (P=0.149).
Relative kidney length and width (left and right) were larger in giants than in normal tadpoles, with no significant differences in regression slopes. Left/right asymmetry in the size of kidneys was not present in either giants (ANOVA, length, P=0.619; width, P=0.646), or in normal tadpoles (ANOVA, length, P=0.875; width, P=0.464).
T3-induced metamorphosis in giants
Adding T3 to the water housing the tadpoles induced metamorphic
changes in both normal and giant X. laevis tadpoles. Hormone-induced
metamorphosis proceeded faster in normal tadpoles than in giants, and after a
3-week exposure to 3 nmol l1 T3, all normal
tadpoles had transformed into froglets. During the same period of time, giant
tadpoles developed only to the stage when forelimbs emerge (stage 58). After
an additional week of T3 treatment, all giant tadpoles reached
stage 62, when both fore- and hindlimbs are developed, and the tail starts
resorbing. The developmental and morphological response in giants was the same
in 1 nmol l1 T3. In water containing both 1 and 3
nmol l1 T3, all giant tadpoles died during the
fifth or sixth week, upon reaching stage 63 (determined by limb and cranial
morphology). During transformation the head of giants significantly narrowed,
and their body mass decreased. However, the tails did not reach stage 63,
since they had not started resorbing at that time. Total body length, as well
as tail length, did not change. The only change in the tail induced by
T3 treatment was a significant decrease in tail fin and tail muscle
height. A giant tadpole that underwent T3 treatment, and
subsequently developed forelimbs, is shown in
Fig. 9. Developmental stages
and external body measurements of giants at the beginning (Day 0) and after 4
weeks (Day 28) of treatment are given in
Table 3, for both 1 nmol
l1 and 3 nmol l1 T3
concentrations.
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While some external morphological characters showed metamorphic changes under T3 treatment, visceral morphology did not change significantly. The only significant difference found between groups of giant tadpoles before and after T3 treatment was in gallbladder volume (ANCOVA, P=0.0004). The average gallbladder volume in giant tadpoles not exposed to T3, was 11.87±1.72 mm3 (range 2.2535.09). In giant tadpoles that had been exposed to exogenous T3 for 4 weeks, gallbladder volume was 1.23±0.19 mm3 (range 0.241.94), i.e. reduced to a tenth its former size.
Double-staining for bone and cartilage revealed substantial metamorphic changes in the head of T3-treated giant tadpoles after 4 weeks. These include the normal resorption of the branchial baskets, shortening and reorientation of the palatoquadrate, and extensive elongation of Meckel's cartilage. Many of the dermal bones have started to ossify. The anlage of the adult teeth are now present in the upper jaw. The changes we observed in the head are completely consistent with the normal metamorphic processes for X. laevis at NF stage 63.
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Discussion |
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All external morphological characters that we measured in giants retained normal allometric relationships, except for pleuroperitoneal cavity length. Disproportionate increase in pleuroperitoneal cavity size is an apparent adjustment to the increase in visceral volume, primarily associated with advanced development of the gonads and hypertrophy of fat bodies. The increase in total body volume, which is due to an enlarged axial muscle mass, has a strong effect on giants' morphology and behaviour.
Huang and Brown (2000a)
noted that transgenic frogs overexpressing growth hormone (GH) typically
develop skeletal abnormalities, such as disproportionately large heads and
feet, as well as deformed vertebral columns. This suggests that scoliosis in
our giant X. laevis tadpoles could result from the excessive growth
of the tadpole's body; i.e. enlarged axial muscles could exert excessive and
unbalanced loads on the vertebral column and notochord, forcing them to
curve.
The increased axial muscle of giants would also contribute to the tadpoles'
negative buoyancy. In addition, the majority of giants had solid lungs, with a
relative lung area smaller than in giants with inflated lungs. We suspected
that the enlarged fat bodies of giants, by occupying most of the
pleuroperitoneal cavity space, might prohibit proper lung development.
However, such a relationship was not found, as small, solid lungs were present
in tadpoles both with and without fat bodies. The negative buoyancy in giants,
caused primarily by their excessive mass, likely limits proper lung
ventilation and leads to their partial solidification. Pronych and Wassersug
(1994) showed that the lungs
of X. laevis tadpoles that have been denied access to air were
substantially smaller and frequently showed anomalies, such as partial
solidification.
Atkinson and Just (1975)
showed that during later metamorphic stages of normal tadpoles there is an
increase in the number and size of septa, which potentially increases the
surface area of respiratory epithelium. The increased number of septa in the
inflated lungs of giant tadpoles in our study compared to normal tadpoles'
lungs suggests that the lungs of these giants underwent metamorphic
transformation far beyond the development of the tadpole as a whole. However,
in the solidified lungs of giants the tissue proliferation is so abundant, and
the number of septa extending into the lumen of the lung so extensive, that
the luminal volume is compromised. The faster increase in lung area with
increasing body volume that we observed in giant tadpoles with solid lungs,
compared to normal tadpoles and giants with inflated lungs, may be
compensatory growth in response to the reduced functionality of the solidified
lungs.
In contrast to the growth of the lungs, the growth of most of the viscera
in giants keeps up with the increase in total body size. However, the fact
that the biliary system and intestine in giants do not follow the same growth
trajectory could be a limitation to the tadpoles' normal transformation and
viability. Interestingly, in giants the typical coiling pattern in the
intestine was absent, and only irregular loops were present. Kemp
(1951) concluded that
intestinal coiling in anuran larvae results from elongation of the intestine
within a visceral cavity that is not spacious enough to permit development as
a straight tube or a single loop. By experimentally increasing the coelomic
cavity in Hyla regilla embryos, Kemp
(1946
) induced the development
of an intestine that was nearly straight for most of its length, terminating
in a short, irregularly coiled region. The giant larvae in our study had
significantly larger pleuroperitoneal cavities compared to normal tadpoles,
but the intestine volume relative to total body volume did not differ, which
might explain the absence of typical gut coiling in giants.
A left-right asymmetry in the size of the kidneys was not found, although
generally, larval kidneys are asymmetrical in size and shape
(Nodzenski et al., 1989;
Malashichev and Wassersug,
2004
).
Of special interest to us were the hypertrophied fat bodies in the giants,
and the complete absence of fat bodies in normal tadpoles. It has been shown
that hypophysectomy of normal X. laevis tadpoles augments the total
body lipid content, and over the long term fat bodies may become so enlarged
that they occupy most of the abdominal cavity
(Dodd and Dodd, 1976).
Treatment of tadpoles with adrenocorticotropin (ACTH, secreted by the
pituitary gland), lowers total body lipids, especially those of the fat
bodies, and also increases lipase activity in the fat bodies
(Dodd and Dodd, 1976
). It
remains unclear if the lack of a thyroid gland in giant tadpoles and therefore
production of TH, possibly affects the pituitary production of ACTH by
abolishing negative feedback to the pituitary gland.
Our most remarkable finding was the advanced gonadal development in giant
tadpoles. It is still an ongoing debate as to how TH affects gonadal
differentiation. Some researchers claim that gonadal differentiation is not
under TH control (see review by Hayes,
1997b). However, Hayes
(1997a
) suggested that TH
might be required for testicular development in X. laevis, since the
administration of goitrogen thiourea to X. laevis larvae blocked TH
production and resulted in a skewed sex ratio, i.e. 100% females. Our results
show that gonadal development is not affected by the absence of TH the way the
rest of the body is. Moreover, both male and female gonads were developed in
our athyroid X. laevis tadpoles. In both sexes the gonads were
mature, with sperm present in testes, and eggs in ovaries. Oocytes in giant
tadpoles have yolk, which is normally produced in the liver
(Duellman and Trueb, 1986
),
thus proving that the giants' reproductive systems are mature (if not
functional). This is in contrast to previous findings
(Wangh and Schneider, 1982
)
that without TH X. laevis is unable to synthesize vitellogenin and
produce eggs. It is thought that estrogens from the ovary induce vitellogenin
synthesis and secretion by the liver only after the liver has been exposed to
TH (Hayes, 1997b
). There is a
slim possibility that our giant tadpoles had ectopic thyroid tissue that we
failed to find, and thus may have produced some hormone that influenced liver
function. However, if this were true, we would expect to see more metamorphic
changes in giant tadpoles, and their development to proceed beyond NF stage
56.
Allen's work on thyroidectomized Rana pipiens larvae (Allen,
1916,
1918
), also showed that gonadal
development is advanced in both sexes in the absence of TH. We propose that TH
are not needed for gonadal differentiation in X. laevis.
However, the fact that the only male in our study was from a different
population than all the females, suggests that other factors may be
influencing sex determination. For example, Hayes et al.
(2002) have reported that
extremely low concentrations of the commonly used pesticide atrazine
demasculinizes X. laevis tadpoles. Some antioxidants, such as
bisphenol A, have also been shown to induce feminisation in X. laevis
tadpoles as well (Levy et al.,
2004
). We do not have data, though, showing high levels of these
pesticides in the water used to raise tadpoles at Xenopus One.
Ogielska and Kotusz (2004)
pointed out that the somatic stage of a tadpole often does not reflect its
actual age; in fact age, rather than somatic stage, seems to be crucial for
gonadal development. Chang and Hsu
(1987
) compared two groups of
Rana catesbeiana tadpoles; the tadpoles did not differ in somatic
stage, but did differ in age and size (older tadpoles were larger). Older
tadpoles had more advanced differentiation of ovaries. The giant tadpoles in
our study were up to 8 years old, which may have provided the time necessary
for gonadal development beyond what would be expected for their NF
developmental stage.
The advanced gonadal differentiation in our larval X. laevis
raises the possibility of neoteny in anurans. In one sense, Allen
(1918) had artificially
produced neoteny in an anuran; i.e. although his tadpoles could not reproduce,
they had differentiated gonads in the larval body. However, neotenic tadpoles
have never been found, and several researchers have hypothesised why.
Wassersug (1975
), for example,
speculated that space for adult genitalia, and particularly for storage of
eggs in the female, is only made at metamorphosis by the extension of the body
cavity, when the pelvic girdle and urostyle elongate. Our results show,
however, that giant tadpoles have longer pleuroperitoneal cavities relative to
body size than normal tadpoles, which may allow for enlarged viscera,
including gonads, without full metamorphosis of the pelvis. Indeed, the giant
tadpoles in our study were able to develop large ovaries full of eggs within
their coelomic cavities.
However, typical anuran amplexus is not possible in tadpoles since their
forelimbs are covered by the operculum, or, if already emerged at later
stages, not large enough to embrace another individual. Tadpoles would need a
way of proximating their sperm and eggs to ensure fertilisation, which would
require major changes in their behaviour. Alternatives to amplexus do exist in
anurans. For example, in the family Dendrobatidae adult frogs use cloacal
apposition to transfer sperm (Dendrobates granuliferus,
Crump, 1972; Dendrobates
pumilis, Limerick, 1980
).
But, extrusion of eggs and sperm may still not be possible in tadpoles, simply
because premetamorphic anurans lack the musculoskeletal features necessary to
raise the pleuroperitoneal pressure to forcefully eject materials from their
body cavity (see Naitoh et al.,
1989
; Wassersug,
1996
,
1997
). Obviously, for neoteny
to occur in anuran larvae, some major behavioural and morphological changes
beyond gonadal differentiation are necessary. In sum, the gonadal
differentiation seen in our giant X. laevis tadpoles is necessary,
but not sufficient, for neoteny.
The fact that tadpoles fail to metamorphose due to a lack of TH raises the question of whether athyroid giants can resume their development and eventually transform into normal frogs if given TH. Our treatment of giants with exogenous T3 indicates that the arrested development of giant tadpoles is not due to an absence of thyroid hormone receptors (TR).
Xenopus laevis has at least two types of receptor isoforms,
TR and TRß (Yaoita et al.,
1990
). TR
in tadpoles is expressed constitutively and is
present before the appearance of TH (Eliceiri and Brown, 1999). TRß
responds to TH and its expression follows the rise of TH concentrations in
cells (Yaoita et al., 1990
;
Wang and Brown, 1993
; Eliceiri
and Brown, 1999). During metamorphosis growing limbs have high TR
and
low TRß levels. However, during the climax of spontaneous metamorphosis
TRß is upregulated in tails and the TRß protein is more abundant
than TR
(Eliceiri and Brown, 1999). Our treatment with exogenous
T3 resulted in further growth and development of hindlimbs, as well
as the eruption of forelimbs and loss of tail fin in giant Xenopus
tadpoles, which suggests that, most probably, both TR
and TRß are
present in our giants. Significant decreases in tail fin and muscle height, a
decrease in body mass, narrowing of the head, and a change in the shape of the
mouth were all observed. These are features associated with normal
metamorphosis in tadpoles. Total body length and tail length, however,
remained unchanged, and all giant tadpoles died at NF stage 63, which is the
stage when tail shrinkage occurs in normal tadpoles
(Nieuwkoop and Faber,
1956
).
X. laevis tadpoles that retain their tails have been observed
several times before (Kinoshita and
Watanabe, 1987; Elinson et
al., 1999
; Huang and Brown,
2000b
). The `tailed frogs' produced by Huang and Brown
(2000b
), were transgenic
X. laevis tadpoles overexpressing PRL. These animals developed to NF
stage 63 with a long thin tail. The muscle resorbed and the notochord
collapsed, but the fins did not resorb. In fact the tadpoles continued to grow
in the PRL-overexpressing tailed frog. Tailed frogs were also produced by
treatment with goitrogen methimazole
(Elinson et al., 1999
), and in
transgenics overexpressing type III deiodinase that encodes a
T3-inactivating enzyme (Huang
et al., 1999
). In all these cases though, the tails are small
compared to ours and lack the typical chevron-shaped blocks of fast muscle.
They do, however, retain the longitudinal slow muscle cords. It should be
noted that those tails look very different from the ones found in our
tadpoles. Ours are the opposite: the excessive muscle mass was retained,
although the fins are lost with T3 induced metamorphosis.
None of these `tailed frogs', however, have a tail like that of
Ascaphus truei, which is a copulatory organ only present in the males
(Stephenson and Verrell,
2003). Our giant X. laevis tadpoles, in contrast, still
maintained fast muscle chevrons in their tails after T3 treatment,
suggesting that there are several different scenarios for tail retention in
Xenopus after metamorphosis. Huang and Brown
(2000b
) showed that transgenic
tadpoles overexpressing PRL develop into frogs whose tails are filled with
collagen and not muscle. Interestingly, their tails are resistant to
T3 treatment. Since our giant tadpoles lack TH that would
counteract the effects of PRL, it is possible that their tails have excessive
collagen; that, in turn, might make them resistant to shrinkage under
T3 treatment.
The retention of a tail of any sort in a post-metamorphic anuran may reflect the atavistic fact that the amphibian ancestor of modern anurans surely had a tail. The fact that these tails show up in Xenopus may reflect the more generalized nature of this pipoid frog compared to other anurans. Alternatively it may simply be due to the fact that scientists work more with Xenopus than other anuran species.
No substantial metamorphic changes in viscera were detected in T3-treated giant tadpoles, except for the drastic decrease in gallbladder volume. (The reason for this extreme reduction in gallbladder size with metamorphosis is unclear. In fact, the function of the large gallbladders typical of tadpoles remains unexplored.)
We propose that giant tadpoles might be incapable of resorbing the massive axial musculature in their tails and backs. As well, they may not be capable of metamorphosing their viscera at the same rate as the rest of their body. All this, as well as small and solidified lungs, may preclude normal metamorphosis in giant X. laevis tadpoles.
The increased somatic growth in giants has interesting implications to
phenotypic plasticity in tadpoles. It has been demonstrated that tadpoles
alter their morphology when exposed to increased competition, i.e. competition
induces tadpoles to have larger bodies and smaller tails (Relyea,
2002a,b
).
Larger tadpoles have a larger feeding apparatus (i.e. branchial baskets)
which, in turn, makes them better competitors. Huang and Brown
(2000a
) noted that X.
laevis tadpoles overexpressing GH show a disproportionate increase in
head size due to the increase in gill arches, i.e., their branchial baskets.
It is, thus, possible that stress from competition raises levels of GH, which
could produce larger heads and specifically larger branchial baskets in
tadpoles. So far, tadpoles subjected to stressful environments have been shown
to exhibit elevated hypothalamic corticotropin-releasing hormone content,
which, by stimulating the production of pituitary hormones, leads to the
increase in TH and corticosteroid levels. The pituitary production of GH could
be under similar control. However, this has not yet been investigated.
Conclusions
The growth in giant X. laevis tadpoles represents an allometric
extension of normal development for most traits. However, the lung development
does not keep up with the increase in the total body volume, which evidently
contributes to negative buoyancy, and results in the development of lung
anomalies, such as partial solidification. At the same time, the
pleuroperitoneal cavity is significantly enlarged in giant tadpoles, allowing
for the storage of enlarged viscera and advanced gonadal differentiation.
The advanced gonadal differentiation associated with giantism would in principle allow neoteny; however, giantism constrains development of other organ systems in anuran larvae. Giants may mature reproductively, but are not capable of reproducing. Major behavioural and morphological changes would be necessary for making larval reproduction possible. Urodele larvae, in contrast, are similar to adults, and fewer morphological changes are necessary during maturation and metamorphosis, allowing for multiple evolution of neoteny in urodeles.
Moreover, tadpoles usually inhabit temporary, uncertain habitats with large
fluctuations in resources, in which neoteny would not be adaptive
(Wassersug, 1975). Clearly,
given the collective morphological, behavioural and ecological limitations on
larval reproduction in anurans, neotenic anurans have not and are not likely
to evolve.
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Allen, B. M. (1916). The results of extirpation of the anterior lobe of the hypophysis and the thyroid of Rana pipiens larvae. Science 44,755 -758.
Allen, B. M. (1918). The results of thyroid removal in the larvae of Rana pipiens. J. Exp. Zool. 26,499 -519.
Atkinson, B. G. and Just, J. J. (1975). Biochemical and histological changes in the respiratory system of Rana catesbeiana larvae during normal and induced metamorphosis. Dev. Biol. 45,151 -165.[Medline]
Berger, L. and Uzzell, T. (1977). Vitality and growth of progeny from different egg size classes of Rana esculenta L. (Amphibia, Salientia). Zool. Polon. 26,291 -317.
Borkin, L. J., Berger, L. and Gunther, R. (1982). Giant tadpoles of water frogs within Rana esculenta complex. Zool. Polon. 29,105 -127.
Chang, L.-T. and Hsu, C.-Y. (1987). The relationship between age and metamorphic progress and the development of tadpole ovaries. Proc. Natl. Sci. Counc. B ROC 11,211 -217.
Crump, M. (1972). Territoriality and mating behavior in Dendrobates granuliferus (Anura: Dendrobatidae). Herpetologica 28,195 -198.
Dodd, M. H. I. and Dodd, J. M. (1976). The biology of metamorphosis. In Physiology of the Amphibia (ed. B. Lofts), pp. 467-599. New York: Academic Press.
Duellman, W. E. and Trueb, L. (1986). Biology of Amphibians. New York: McGraw-Hill Book Co.
Dumont, J. N. (1972). Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136,153 -180.[Medline]
Elicieri, B. P. and Brown, D. D. (1994).
Quantitation of endogenous thyroid hormone receptors and ß during
embryogenesis and metamorphosis in Xenopus laevis. J. Biol.
Chem. 269,24459
-24465.
Elinson, R. P., Remo, B. and Brown, D. D. (1999). Novel structural elements identified during tail resorption in Xenopus laevis metamorphosis: lessons from tailed frogs. Dev. Biol. 215,243 -252.[CrossRef][Medline]
Emerson, S. (1988). The giant tadpole of Pseudis paradoxa. Biol. J. Linn. Soc. 34, 93-104.
Frankenberg, E. and Werner, Y. H. (1992). Egg, clutch and maternal sizes in lizards: intra- and interspecific relations in near-eastern Agamidae and Lacertidae. Herpetol. J. 1,7 -18.
Gould, S. J. (1977). Ontogeny and Phylogeny. Cambridge: Harvard University Press.
Hanken, J. and Wassersug, R. (1981). The visible skeleton. A new double-stain technique reveals the native of the `hard' tissues. Funct. Photogr. 16, 22-26.
Hayes, T. B. (1997a). Steroids as potential modulators of thyroid hormone activity in anuran metamorphosis. Amer. Zool. 37,185 -194.
Hayes, T. B. (1997b). Hormonal mechanisms as potential constraints on evolution: examples from the Anura. Amer. Zool. 37,482 -490.
Hayes, T. B., Collins, A., Lee, M., Mendoza, M., Noriega, N.,
Stuart, A. A. and Vonk, A. (2002). Hermaphroditic,
demasculinized frogs after exposure to the herbicide, atrazine, at low
ecologically relevant doses. Proc. Natl. Acad. Sci.
USA 99,5476
-5480.
Huang, H. and Brown, D. D. (2000a).
Overexpression of Xenopus laevis growth hormone stimulates growth of
tadpoles and frogs. Proc. Natl. Acad. Sci. USA
97,190
-194.
Huang, H. and Brown, D. D. (2000b). Prolactin
is not a juvenile hormone in Xenopus laevis metamorphosis.
Proc. Natl. Acad. Sci. USA
97,195
-199.
Huang, H., Marsh-Armstrong, N. and Brown, D. D.
(1999). Metamorphosis is inhibited in transgenic Xenopus
laevis tadpoles that overexpress type III deiodinase. Proc.
Natl. Acad. Sci. USA 96,962
-967.
Jurand, A. (1955). Zjawisko neotenii u `Xenopus laevis' Daud. Folia Biologica 4, 315-330.
Kemp, N. E. (1946). Regulation in the entoderm of the tree frog Hyla regilla. Univ. Calif. Publ. Zool. 51,159 -183.
Kemp, N. E. (1951). Development of intestinal coiling in anuran larvae. J. Exp. Zool. 116,259 -287.[Medline]
Kinoshita, T. and Watanabe, K. (1987). Collecting and raising. In Embryogenesis and Metamorphosis of Amphibians: Electron Microscopic Observations in Tadpoles (ed. K. Watanabe), pp. 185-200. Nishimura Shoten, Niigata. [In Japanese]
Levy, G., Lutz, I., Krüger, A. and Kloas, W. (2004). Bisphenol A induces feminization in Xenopus laevis tadpoles. Environ. Res. 94,102 -111.[CrossRef][Medline]
Limerick, S. (1980). Courtship behavior and oviposition of the poison-arrow frog Dendrobates pumilis.Herpetologica 36,69 -71.
Major, N. and Wassersug, R. J. (1998). Survey of current techniques in the care and maintenance of the African clawed frog (Xenopus laevis). Contemp. Top. 37, 57-60.
Malashichev, Y. and Wassersug, R. J. (2004). Left and right in the amphibian world: which way to develop and where to turn? BioEssays 26 (in press).
Naitoh, T., Wassersug, R. J. and Leslie, R. A. (1989). The physiology, morphology and ontogeny of emetic behavior in anuran amphibians. Physiol. Zool. 62,819 -843.
Nieuwkoop, P. D. and Faber, J. (1956).Normal Table of Xenopus laevis (Daudin). Amsterdam: North Holland Publishing.
Nodzenski, E., Wassersug, R. J. and Inger, R. F. (1989). Developmental differences in visceral morphology of megaphryine pelobatid tadpoles in relation to their body form and mode of life. Biol. J. Linn. Soc. 38,369 -388.
Ogielska, M. and Kotusz, A. (2004). Pattern of ovary differentiation with reference to somatic development in anuran amphibians. J. Morph. 259, 41-54.[CrossRef][Medline]
Pronych, S. and Wassersug, R. J. (1994). Lung use and development in Xenopus laevis tadpoles. Can. J. Zool. 72,738 -743.[Medline]
Relyea, R. A. (2002a). Competitor-induced plasticity in tadpoles: consequences, cues, and connections to predator-induced plasticity. Ecol. Monogr. 72,523 -540.
Relyea, R. A. (2002b). The many faces of predation: How selection, induction and thinning combine to alter prey phenotypes. Ecology, 83,1953 -1964.
Rot-Nikcevic, I. and Wassersug, R. J. (2003). Tissue sensitivity to thyroid hormone in athyroid Xenopus laevis larvae. Dev. Growth Differ. 45,321 -325.[CrossRef][Medline]
Shi, Y.-B. (2000). Amphibian Metamorphosis: From Morphology to Molecular Biology. Toronto: John Wiley & Sons, Inc.
Srebro, Z. (1970). Neurosecretion in thyroidless Xenopus laevis larvae. Experientia 27,849 -850.
Stephenson, B. and Verrell, P. (2003). Courtship and mating of the tailed frog (Ascaphus truei). J. Zool. Lond. 259,15 -22.
Toivonen, S. (1952). Ein Fall von partieller Neotenie bei Xenopus laevis Daudin und experimentelle Untersuchungen zu seiner kausalen Erklarung. Arch. Soc. Zool. Bot. Fenn. `Vanamo' 6,107 -123.
Viertel, B. and Richter, S. (1999). Anatomy: Viscera and Endocrines. In Tadpoles: The Biology of Anuran Larvae (ed. R. W. McDiarmid and R. Altig), pp.92 -148. Chicago: The University of Chicago Press.
Wakahara, M. (1996). Heterochrony and neotenic salamanders: possible clues for understanding the animal development and evolution. Zool. Sci. 13,765 -776.[Medline]
Wang, Z. and Brown, D. D. (1993). Thyroid
hormone-induced gene expression program for amphibian tail resorption.
J. Biol. Chem. 268,16270
-16278.
Wangh, L. J. and Schneider, W. (1982). Thyroid hormones are corequisites for estradiol 17ß in vitro induction of Xenopus vitellogenin synthesis and secretion. Dev. Biol. 89,287 -293.[Medline]
Wassersug, R. J. (1975). The adaptive significance of the tadpole stage with comments on the maintenance of complex life cycles in anurans. Amer. Zool. 15,405 -417.
Wassersug, R. J. (1996). The biology of Xenopus tadpoles. In The Biology of Xenopus (ed. R. C. Tinsley and H. R. Kobel), pp. 195-211. Oxford: Clarendon Press.
Wassersug, R. J. (1997). Where the tadpole meets the world Observations and speculations on biomechanical and biochemical factors that influence metamorphosis in anurans. Amer. Zool. 37,124 -136.
Yaoita, Y., Shi, Y.-B., and Brown, D. D. (1990). Xenopus laevis alpha and beta thyroid hormone receptors. Proc. Natl. Acad. Sci. USA 87,7090 -7094.[Abstract]