/. Embryol. exp. Morph. 88, 71-83 (1985)
71
Printed in Great Britain © The Company of Biologists Limited 1985
The fate of larval chondrocytes during the
metamorphosis of the epibranchial in the salamander,
Eurycea bislineata
P. ALBERCH, G. A. LEWBART AND EMILY A. GALE
Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts
02138, U.S.A.
SUMMARY
The metamorphosis of the epibranchial cartilage, a skeletal component of the hyobranchial
apparatus, in the salamander Eurycea bislineata entails a combination of the reabsorption of a
larval cartilaginous element with the simultaneous genesis of an adult cartilage in the same
place. In this study we focus on the fate of the larval chondrocytes. Two hypotheses are
considered: one, larval cells simply die off during metamorphosis, or, alternatively, they
dedifferentiate and participate in the formation of the adult element.
Thyroxine treatment and experimental tissue manipulation coupled with measurements of
thyroxine levels using radioimmunoassay show that, within 24 h after T4 treatment, larval
chondrocytes in the epibranchials exhibit large autophagocytic vacuoles, disruption of the rough
endoplasmic reticulum, abnormally shaped mitochondria, abundance of lysosomes and nuclear
degeneration, all symptoms of the onset of cell death. In conclusion, evidence from light
microscopy, TEM and SEM show that the larval chondrocytes in response to rising levels of
thyroid hormones undergo a process of lysosomal autophagocytosis and do not participate in the
formation of adult structures.
INTRODUCTION
Since the early experimental work by Gudernatsch (1912), Allen (1918, 1925)
and Etkin (1932) (see also reviews by Dodd & Dodd, 1976, and Gielbert &
Frieden, 1981), amphibian metamorphosis has been perceived as the differential
response of the various larval tissues to global cues, usually fluctuating levels of
hormones. Some tissues can undergo dramatic rearrangement, or even complete
degeneration, while others remain essentially unchanged. As part of an ongoing
study of the organization of cartilage metamorphosis in amphibians, we report
some results addressing the fate of larval chondrocytes in skeletal elements which
degenerate during metamorphosis. There are two possible hypotheses: one, the
chondrocytes in the degenerating larval element dedifferentiate, return to a
mesenchymal condition, and participate in the development of adult structures.
Alternatively, larval cells, in response to some global cue, undergo cell death.
Key words: Eurycea, thyroxine, cartilage, metamorphosis.
72
P. ALBERCH, G. A. LEWBART AND E. A. GALE
R
Ch
L
_
Bp
Fig. 1. Diagram illustrating the morphology and developmental fates at metamorphosis of the larval (A) and adult (B) hyobranchial apparatus. Stippling in (A)
indicates that the elements disappear during metamorphosis; cross hatching indicates
the derivatives of the central basal plate, while vertical hatching indicates remodelling
of the ceratohyals. Heavy stippling illustrates the elements appearing de novo in the
adult. Bbi, Basibranchial 1; Bp, Branchial plate; Cblf Ceratobranchial 1; Cb2,
Ceratobranchial 2; Ch, Ceratohyal; e1} larval epibranchial 1; e2, larval epibranchial 2;
e3, larval epibranchial 3; E, adult epibranchial; L, Lingual cartilage; M, Maxilla; Ptn,
Premaxilla; R, Radials; U, Urohyal.
Our study has focused on the metamorphosis of the hyobranchial apparatus of
the salamander Eurycea bislineata (Fig. 1), and, in particular, on what is perhaps
the most puzzling event of this system: the simultaneous degeneration of the larval
first epibranchial cartilage and the formation of the adult epibranchial in almost
the same place (Figs 2 and 3).
Eurycea bislineata is a lungless salamander (family Plethodontidae) abundantly
represented along the streams of the eastern United States. Eurycea and its allies
are characterized by a complex life cycle in which the hyobranchial skeleton is not
only morphologically different between the larva and the adult but it is involved in
different functions. Eurycea larvae are adapted to an aquatic existence. The larval
hyobranchial skeleton (Fig. 1A), and its associated musculature, is involved in
branchial respiration and suction feeding. In primitive (generalized) adult
salamanders, as well as frogs, the hyobranchial skeletal elements and muscles are
the dominant effectors of pulmonary ventilation (De Jongh & Gans, 1969;
Severtsov, 1972), but in the lungless plethodontid salamanders, including Eurycea,
the adult hyobranchial apparatus forms a highly sophisticated feeding mechanism
involving tongue projection (Fig. IB) (see Lombard & Wake, 1976, 1977; Wake,
1982; Ozeti & Wake, 1969; Regal, 1966; Severtsov, 1972; Thexton et al. 1977).
Thus, the hyobranchial skeleton has to undergo significant changes during
Fate of larval chondrocytes
73
metamorphosis to adapt to its new function in a terrestrial environment. The work
of Smith (1920; reviewed in Wilder, 1925) provides a fairly detailed histological
analysis of the transformations involved in the development and metamorphosis of
the hyobranchial apparatus in Eurycea bislineata. Our observations have confirmed
Smith's conclusions summarized in Fig. 1. As mentioned above, and the subject of
this paper, Smith's (1920) major discovery was that the adult epibranchial is not
homologous to the larval first epibranchial (some authors, e.g. Romer & Parsons
(1977, p. 212), refer to the epibranchials as ceratobranchials; here we follow Smith
& Wilder's nomenclature). Fig. 3 clearly illustrates the degenerating larval
cartilage, next to the forming adult element. We present the results of experiments
that show that in response to raising levels of thyroid hormones the chondrocytes
of the first larval epibranchial undergo cell death and autophagocytosis.
MATERIALS AND METHODS
Animals
Over 250 specimens of Eurycea bislineata from Massachusetts and New Hampshire were used
in this study. Larvae were maintained at a density no greater than 5-7 animals per litre of
Pm
Fig. 2. Drawing from a cleared and stained specimen undergoing metamorphosis.
Note the simultaneous presence of the adult and larval epibranchials. The radials (R)
are starting to condense and the urohyal has become detached from the basal plate and
has begun to undergo endochondral ossification. The maxillary bone (M) has started to
ossify at that stage. Abbreviations as Fig. 1.
74
P. ALBERCH, G. A. LEWBART AND E. A. GALE
\
4
Fig. 3. Photo of the gill arches (ventrolateral view) of the cleared and stained specimen
illustrated in Fig. 2 (squared area). The adult epibranchial (E) is developing next to the
larval first epibranchial (ei). The matrix of e1 is degenerating (arrow). Larval
epibranchials 2 (e2) and 3 (e3) do not appear affected yet.
50 % Holtfreter's solution with 0-1 g I"1 MgSO4.7 H2O added. Adults were kept in plastic boxes
with moist paper towels.
Microsurgery
While still in a premetamorphic condition, 22 larvae were anaesthetized using a 1:500
concentration of ethyl m-aminobenzoate and prepared for microsurgery which was
accomplished using finely sharpened dissecting scissors and watchmaker forceps. Open
branchial clefts assured premetamorphosis and allowed for easy access to the underlying
epibranchials in the hyoid apparatus. Depending on the specimen, portions of the left first
epibranchial were removed to serve as controls. After the operation the animals were placed
directly into separate bowls containing ice-cold modified Holtfreter's to which streptomycin and
penicillin had been added. We experienced a 100% postoperative survival rate. After 24 h at
4°C, L-thyroxine (3,3',5,5'-tetraiodo-L-thyronine) (Sigma Co., St. Louis, Mo.) was added to
the Holtfreter's solution in the concentration of 5 x 10~ 8 M. Animals were then returned to the
15-18°C room where they remained for periods ranging from 24h to 18 days. Bowls were
cleaned and fresh T4 solution was added daily. At predetermined times the animals were again
prepared for surgery as above. The corresponding right epibranchial was removed and placed in
fixative. Following surgery, animals were sacrificed and fixed in 10% neutrally buffered
formalin, to be subsequently prepared for histology to confirm that the correct epibranchial had
been removed.
Six specimens were used as controls, i.e., one larval epibranchial was removed but they were
not placed in thyroxine but preserved at intervals corresponding to the treated specimens. In
those cases no changes in the morphology of the remaining larval epibranchials were observed.
Various skeletal elements were removed from naturally metamorphosing individuals to
compare their cellular morphology with T4-treated individuals.
Fate of larval chondrocytes
75
Histology
All specimens used in the experiments were either cleared with trypsin and KOH and stained
in toto with Alcian blue 8GX (for cartilage matrix) and alizarin red S (for calcium deposits)
(Hanken & Wassersug, 1981), or embedded in paraplast and sectioned serially at 10/mi. The
sections were stained with modified tricromate Heidenhain's Azan technique (Baldauf, 1958).
In addition, to study the process of normal metamorphosis, 214 specimens at various stages of
metamorphosis were cleared and stained and 28 were serially sectioned.
All the skeletal elements that had been removed in vivo were placed immediately into 3-0 %
glutaraldehyde in 0-lM-sodium cacodylate buffer, pH 7-3, with 7% sucrose. The tissues were
left in this fixative for 2 h at 4°C. After rinsing in a series of cacodylic buffers for an hour at room
temperature, the tissues were postfixed in 2% OsO4 in 0-lM-cacodylic buffer for l h at room
temperature and then stored in buffer at 4°C until embedding. Tissues were dehydrated in
ethanol, infiltrated with propylene oxide and embedded in Epon 812 after Luft (1961).
Transmission and scanning electron microscopy
Ultrathin sections were obtained using a Sorvall MT2B ultramicrotome; sections were stained
with uranyl acetate and lead citrate. 1/wm sections were also obtained and, after heat fixing to
glass slides, were stained for ten seconds with 0-5 % toluidine blue in saturated sodium borate.
Thin sections were examined and photographed in either a Zeiss 9-S2 or an RCA EMU-4
transmission electron microscope.
For scanning electron microscopy, specimens, fixed as indicated above, were bisected using a
razor blade, dehydrated in ethanol, and critical-point dried using a Tousimis SamDri PVT.3
drier. Specimen stubs were then sputter coated with gold-palladium and viewed and
photographed using an AMR AY 1000 scanning electron microscope.
T3 and T4 radioimmunoassay
Radioimmunoassay (RIA) techniques to measure T3 and T4 levels in plasma serum were
modified after Larsen (1976) and Regard etal. (1978). Slight modifications of the procedure were
required to increase the sensitivity of the assay when dealing with small serum volumes. 2jul
aliquots of Eurycea serum were used for the T4 assay, while 5 jul aliquots were used in the T3
assays. No serum from different animals was pooled, and assays were run in duplicate (T3) and
triplicate (T4) unless prevented by limitations in the volume of blood available from a given
specimen (the amount of plasma serum that can be obtained from a single Eurycea ranges from 5
to 30 jul). The reported results are the average of the three counts. Adult Eurycea serum was used
for the standard curve for the T4 assay while serum from the newt Notophthalmus viridescens was
used in the T3 assays. The serum from adults of these two species was found to contain levels of
T3 and T4 below the sensitivity of our assay. Levels of non-specific binding proteins were tested
for every individual and used in the calculation of true % binding. In general, the difference
between the non-specific binding levels of the standard animals and the experimental animals
was negligible. Using these procedures, the minimum detectable levels were estimated to be
0-13 fig 100 ml" 1 for T4 and 40 ng 100 ml"1 for T3.
RESULTS
Table 1 summarizes the number of specimens treated, the number of days of
exposure of the animal to thyroxine (in all cases the specimens had the left first
epibranchial removed, to be used as a control, prior to immersion of the specimen
in the thyroxine solution), as well as the recorded serum levels of L-thyroxine (T4)
and L-triiodothyronine (T 3 ). Albeit limited by the low number of observations,
the RIA data on thyroid hormone levels indicates that the T 4 in the external
76
P. ALBERCH, G. A. LEWBART AND E. A. GALE
environment becomes rapidly incorporated by the organism. Larval specimens do
not have measurable levels of T3 or T4 in their serum (Alberch & Gale,
unpublished data). However, as early as 24h after immersion in the T4 solution,
we record high levels of T4 in the serum (12-18 pg^P 1 ). The T4 serum
concentration was found to be even higher at 48 h (25-32 pg/il" 1 ), before
decreasing and levelling off to levels ranging between 1-5 to 8-5pgfA~l. All
specimens analysed exhibited detectable levels of T4. One assay of T 3 was run after
24 h of exposure to exogenous T4 showing no detectable levels of the T 3 in the
serum, indicating that if T4 was present in the serum it had not either been
transformed into the T3 form or triggered the production of T 3 by the thyroid yet.
After this initial lag relative to T4 levels, T 3 exhibits a similar pattern, which is
characterized by an initial rise to high levels (Spg/A"1 at 3 days) followed by a
decline and subsequent levelling off.
Some changes in the morphology of the chondrocytes were observed as early as
24 h after immersion in T4. These changes became more accentuated 48 h after T 4
treatment (Fig. 4). The treated larval chondrocytes appear highly vacuolated, with
deformed nuclei (Fig. 4A,B), in contrast to the normal appearance exhibited by
the chondrocytes in the control side (Fig. 4C). Unlike the larval chondrocytes, the
perichondral cells do not appear to be negatively affected by the thyroxine
treatment (Fig. 4A). In fact, autoradiographic labelling with [3H]thymidine
indicates that perichondral cells respond to the treatment by increasing their rate
of cell proliferation (Alberch & Gale, in prep.). As exposure to thyroxine
continues, the signs of larval chondrocyte degeneration increase. Fig. 5 illustrates
some chondroblasts prior to T4 treatment and a cell after 11 days of treatment. The
described cellular changes occur prior to any signs of metamorphosis in external
morphology. It also appears that the process of cell death is autonomous, in the
sense that cells began undergoing autophagocytosis while still in their lacunae.
Matrix degeneration and tissue reabsorption occur much later in the process. In
our studies, particularly after the fourth day of T 4 treatment, all the cells in the
Table 1. Summary of specimens treated (N), length of exposure to exogenous T4
(d. T4) and recorded serum levels of T3 and T4
dX^
N
[T3] (pg^r 1 )
[T4] (pg Ml"1)
1
2
3
4
5
7
10
11
14
18
4
5
2
6
1
3
1
1
1
2
< 0-5(1)*
3(2)
12-18 (2)
25-32 (2)
1-5 (1)
1(1)
0-6-1 (2)
7-5 (1)
8-5 (1)
8-5 (1)
* Number between parentheses indicates number of specimens with RIA measurements.
Fate of larval chondrocytes
11
larval epibranchial exhibited some degree of vacuolization. After 7 days, and in
many cases earlier, a majority of the chondrocytes were dead, i.e., the nucleus had
degenerated and the cytoplasm was effectively absent.
The sequence of morphogenetic events triggered by exogenous T4 treatment
closely resembles the naturally occurring metamorphic events. This conclusion is
B
Fig. 4. Cells from the right (A,C) and left (B) first epibranchial of the same specimen
(Th 151). (B) Normal chondrocytes prior to T4 treatment. (A and C) 48 h after
treatment the cells look heavily vacuolated (v) and perichondral activity is present
(arrow in A). This specimen had 25 pg fil'1 of T4 in its serum when preserved after 48 h
of treatment. Bars equal to: A, 10/mi; B, 5/mi; C, 5jum.
78
P. ALBERCH, G. A. LEWBART AND E. A. GALE
Fate of larval chondrocytes
79
based on the histological and ultrastructural analysis of over 70 naturally
metamorphosing animals. In Fig. 6 we illustrate a representative example of an
advanced stage of normal metamorphosis, where the adult epibranchial (E) is
undergoing chondrogenesis, while the cells in the larval element (e) appear highly
vacuolated. Note that there are still no obvious signs of degeneration in the matrix
of the larval element. TEM photographs show the typical chondroblastic
morphology of the cells forming the adult element (Fig. 7). These cells are
characterized by long cytoplasmic processes, extensive rough endoplasmic
reticulum as well as matrix vesicles, which suggests active synthesis of extracellular
matrix components. At this early stage of chondrogenesis the ECM is still sparse.
In contrast, the chondrocytes from the larval epibranchial exhibit clear signs of cell
degeneration and death (Fig. 8). Many autophagic vacuoles and lysosomes,
coupled with the absence of an organized rough endoplasmic reticulum and the
presence of swollen mitochondria, are all signs of autophagocytosis. Many other
larval cells, both in experimental and in normally metamorphosis animals, showed
>.
Fig. 6. Cross section of a naturally metamorphosing specimen illustrating the larval
epibranchial {e±) and the adult epibranchial (£) undergoing chondrogenesis. Arrow
shows the position of the hypertrophic chondrocyte photographed in Fig. 8. Bar equal
tolOjum.
Fig. 5. (A) Normal larval chondrocyte in SEM. cm, cartilage matrix; /, lacuna; c,
cartilage cell. (B) Larval chondrocytes that have recently divided and are secreting
extracellular matrix {eon). (C) Cross section of a normal larval chondrocyte; n,
nucleus. (D) Cross section of a larval chondrocyte exposed to T4 for 11 days. Note
vacuoles (v). Bars equal to 5jum.
80
P. ALBERCH, G. A. LEWBART AND E . A. GALE
Fig. 7. TEM of a chondroblast from Fig. 6. n, nucleus; rer, rough endoplasmic
reticulum; ecm, extracellular matrix; cp, cytoplasmic process. Bar equal to
condensations of nuclear heterochromatin as well as degeneration of the nucleus,
also suggestive of cell death.
CONCLUSIONS
Our results unequivocally show that the fate of the larval chondrocytes of the
first epibranchial cartilage in Eurycea is cell death. We have no evidence that the
larval cells can dedifferentiate and participate in the formation of the adult
cartilage. These results support the view of development as an irreversible process
of restriction of fate.
Alberch & Gale (in prep.) have measured, using radioimmunoassay techniques
(RIA), the levels of T 3 and T4 during the normal metamorphosis of Eurycea. In
agreement with previous studies (e.g. see Larras-Regard et al. 1981 and review by
White & Nicoll, 1981), neither the larvae nor the adults have any detectable levels
of these thyroid hormones. Metamorphosis in amphibians is characterized by a
rapid increase of T 3 and T4 in the serum. During the metamorphosis of Eurycea,
Alberch & Gale (in prep.) recorded serum levels ranging from 2 to lSpgjUl"1 for
T 4 and from 0-5 to 2pgJul~1 for T3. Animals exposed to exogenous levels of T 4
exhibited an initial surge of endogenous serum T4 that was almost three times the
concentration measured in normal metamorphosis. This initial concentration of
25-32 pgjUl"1 decreased to levels more in the normal range (7-5 to SSpgfil'1) in
Fate of larval chondrocytes
81
the subsequent days of treatment with T4 (see Table 1). These results indicate that
the exogenous hormones penetrate into the tissues of the organisms as early as
during the first 24 h. This observation is consistent with the results reported by
Robinson et al. (1977). These authors, studying how much of the T4 enters the tail
tip of the metamorphosing anuran Xenopus in vitro, found that the amount of T4 in
the tail tips increased during the initial 24 h to subsequently remain constant.
The reason for the detected initial 'surge' of hormone levels is not clear, since
simple diffusion of the exogenous T4 into the serum could not explain, in the face
of constant exogenous concentrations, the subsequent decrease and 'levelling off
of the hormones to within about one-third of the initial concentration. The
observed T4 pattern could be the result of a combination of diffusion of exogenous
hormone and the onset of endogenous synthesis of T4 by the thyroid followed by a
decline in the plasma levels due to an increase in the rate of T4 binding to larval
tissues. As discussed by Galton (1983) and Larras-Regard etal. (1981), there is no
evidence to suggest that deiodinated T4 is converted into T3 in amphibians.
Robinson & Galton (1976) suggest, based on their in vitro experiments, that T4
has direct biological activity in amphibians. Our data tend to support their
conclusions, since after 24 h of treatment, we observe significant morphogenetic
ecm
Fig. 8. TEM of a larval chondrocyte cell indicated with an arrow in Fig. 6 showing
large vacuoles (v) with cellular debris (d), poorly organized rer and abnormal
mitochondria (m). Many electron dense lysosomes and matrix vesicles are also
apparent. Bar equal to 1 /mi.
82
P. ALBERCH, G. A. LEWBART AND E. A. GALE
responses in the absence of any detectable levels of T3. T 3 exhibited a similar surge
after 3 days of exposure to T4. We favour the hypothesis that tissue binding is the
parameter that regulates the levels of these thyroid hormones in the serum. This is
supported by the observation that specimens that are allowed to metamorphose
immersed in a constant T 4 solution exhibit extraordinarily high concentrations of
T 4 in their serum (25-50 pg/il" 1 ) relative to controls that exhibit undetectable
levels at the end of metamorphosis (Alberch & Gale, unpublished). This pattern
might reflect the continuous diffusion of the exogenous hormone into the organism
coupled with a decrease in the binding affinity of the metamorphosed tissues for
the hormone. Further research is needed to resolve these issues.
In spite of the described differences in hormone levels, the sequence of cellular
changes in experimental animals was in general agreement with observations of
unperturbed metamorphic process. In both cases, larval chondrocytes were undergoing a process of cell death and autophagocytosis while perichondral cells remained healthy. The response of the larval cells to an increase in the thyroxine
plasma levels was very rapid; results were observed as early as 48 h after treatment.
In this paper we have not addressed the issue of the origin of the cells that form
the adult element. Circumstantial evidence from histology as well as ongoing
studies involving autoradiographic labelling and removal of the larval element
prior to metamorphosis indicate that the 'stem' cells that form the adult element
are located either in the perichondrium of the larval element or in the immediately
surrounding connective tissue. This is in agreement with studies on skeletal repair
and endochondral ossification where it has been shown that new cell types are
generated by stem cells located in the perichondrium or periosteum (e.g. see reviews by Hall, 1970 and 1978; and Albright & Misra, 1983). We believe that further
exploration of this peculiar system can provide useful information on the complicated dynamics of cellular differentiation and remodelling of skeletal systems.
We would like to thank Prof. S. Tilley for helping us in the collection of Eurycea. Prof. B. K.
Hall offered comments on the manuscript. Prof. P. R. Larsen, Brigham and Women's Hospital,
gave us expert advice and generously placed his laboratory facilities and equipment at our disposition to run the radioimmunoassay tests. Prof. R. Woollacott allowed us access to his
electron microscope and related equipment. Ed Seling and Karyn Moskowitz assisted with the
SEM, Laszlo Meszoly with the artwork and Catherine McGeary With the preparation of the
manuscript. G. Lewbart thanks William Fowle and Edgar Meyhoefer for technical assistance
and Northeastern University's Marine Science and Maritime Studies Center for making available their equipment and facilities. This research was supported by NSF grant PCM 83-08640.
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DE JONGH,
{Accepted 12 February 1985)