Download Programmed cell death during amphibian metamorphosis

Seminars in Cell & Developmental Biology 16 (2005) 271–280
Review
Programmed cell death during amphibian metamorphosis
Keisuke Nakajima, Kenta Fujimoto, Yoshio Yaoita ∗
Division of Embryology and Genetics, Institute for Amphibian Biology, Graduate School of Science,
Hiroshima University, Higashihiroshima 739-8526, Japan
Available online 11 January 2005
Abstract
The death of different types of cells occurs in regressing or remodeling organs to transform from a tadpole to a frog in both temporally
and spatially regulated manners during amphibian metamorphosis. This morphological change is drastic and visible with the naked eye. This
review summarizes our current understanding of the basic mechanism of the cell death during the metamorphosis. It focuses in particular on
the tail resorption and the remodeling of intestine and skin where programmed cell death is executed by thyroid hormone-signaling through
the cell-autonomous response (suicide) and the degradation of the extracellular matrix (murder).
© 2004 Elsevier Ltd. All rights reserved.
Keywords: Amphibian; Metamorphosis; Programmed cell death; Apoptosis; Thyroid hormone
Contents
1.
2.
3.
4.
5.
6.
7.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Induction of metamorphosis by thyroid hormone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Programmed cell death in regressing tails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. The morphology of dying cells in regressing tails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. The up-regulated genes in thyroid hormone-induced tail regression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Expression patterns of thyroid hormone-induced genes in regressing tails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. The suicide model and the murder model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Programmed cell death in remodeling intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Programmed cell death in remodeling skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Apoptosis-related genes in regressing or remodeling organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Amphibian metamorphosis represents dynamic and systematic changes from a larva to an adult, involving absorption
of larva-specific organs and appearance of adult type organs,
and accompanying the life style change from being aquatic
to being terrestrial. This reorganization is triggered simply
by a surge of thyroid hormone. Thyroid hormone-induced
∗
Corresponding author. Tel.: +81 82 424 7481; fax: +81 82 424 0739.
E-mail address: [email protected] (Y. Yaoita).
1084-9521/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcdb.2004.12.006
271
272
273
273
273
274
274
276
277
277
277
278
metamorphosis is observed also in the development of sea
urchin [1] and fish [2,3]. Furthermore, amphibian metamorphosis might correspond to the human perinatal period, which
is marked by an abrupt increase of thyroid hormone after
birth [4–6]. It is because amphibians and mammals switch
hemoglobin from fetal to adult, and a major serum protein to
albumin, and change the resource of oxygen for respiration to
air from water through gills and from maternal blood through
a placenta during post-embryonic development, respectively.
Higher vertebrates retain the similar sensitivity to thyroid hormone in their developmental history. It is reflected as a key
272
K. Nakajima et al. / Seminars in Cell & Developmental Biology 16 (2005) 271–280
Fig. 1. The developmental morphological changes of a single tadpole during the climax of metamorphosis. A single tadpole of Xenopus laevis is photographed
at the various stages. It is worth noting that the head region, dorsal muscle and a tail decrease in size, as the metamorphosis proceeds.
role of thyroid hormone in brain, nerve connective tissue, and
endocrine maturation [7,8]. In all processes of vertebral postembryonic development, amphibian metamorphosis displays
the most prominent transformation, which includes the typical programmed cell death in regressing or remodeling organs.
2. Induction of metamorphosis by thyroid hormone
Amphibian metamorphosis is composed of three developmental periods, premetamorphosis, prometamorphosis and
climax [9]. In Xenopus laevis tadpole, premetamorphosis extends from hatching up to Nieuwkoop and Faber (NF) stage
54 [10] when the developing thyroid gland begins to secrete thyroid hormone [11]. During prometamorphosis (NF
stage 55–57), endogenous thyroid hormone levels rise in the
plasma, and an orderly succession of morphological changes
occurs, the most obvious of which is the development of
hindlimbs. At the climax (NF stage 58–66), when the concentration of thyroid hormone is at peak levels, tadpoles undergo a rapid metamorphic transition, including the eruption
of frontlimbs from the opercular fold, the shrinkage of head,
the resorption of gills, the remodeling of intestine and skin,
and the absorption of tail in the temporally predetermined
order. Almost all organs of tadpoles change during anuran
metamorphosis. Three types of changes take place, which
are the complete degeneration of larval-specific organs such
as the tail and gills, the development of frog-specific organslike limbs, and the remodeling of the existing larval organs
into their adult forms such as intestine, skin, dorsal muscle
[12] and pancreas [9]. Programmed cell death is observed
in regressing or remodeling organs. Only the middle 75% of
the tadpole body apparently remains without altering the size
of the cranium and transforms to a frog after metamorphosis
(Fig. 1A and B), suggesting that a tail, which is about two
times longer than a body, and the rest of the body are absorbed by programmed cell death. Moreover, programmed
cell death occurs even within the middle 75% of the body,
for example, in dorsal muscle, gills, intestine, skin, pancreas
and so on.
The developmental cell death during metamorphosis is
caused by thyroid hormone [9,13]. Because, firstly, the morphological metamorphic changes correlate with the developmentally increasing levels of the endogenous thyroid hormone in the plasma [11] as described above. Secondly, precocious metamorphosis is observed by thyroid hormonetreatment of the premetamorphic or prometamorphic tadpoles [14], their organs such as tail [15], gills [16] and intestine [17,18], and even a myoblastic cell line derived from
tail [19]. Thirdly, metamorphic changes are suppressed by
the congenital defect of thyroid gland [20], inhibitors of thyroid hormone synthesis [21], the surgical removal of a pituitary gland which secretes thyroid-stimulating hormone [9],
the degradation of endogenous thyroid hormone in the type
III deiodinase-overexpressing transgenic tadpoles [22] and
the blocking of thyroid hormone-signaling in transgenic tadpoles which overexpress a dominant negative form of thyroid
hormone receptor (DNTR) [23]. Even the direct developing
anuran, which hatches as a tiny frog without a tadpole stage,
still undergoes a thyroid hormone-dependent metamorphosis
before hatching [24].
Thyroid hormone regulates transcription by binding to heterodimers formed with thyroid hormone receptors [25,26]
and 9-cis retinoic acid receptors (RXR) [27–29]. There are
two TR␣ [30] and two TR␤ genes in Xenopus laevis [31]
due to its tetraploidy [32]. The TR␣ mRNA level in a whole
tadpole increases throughout premetamorphosis, is maximal
by prometamorphosis, and falls after the climax of metamorphosis. On the other hand, TR␤ mRNA is barely detectable
K. Nakajima et al. / Seminars in Cell & Developmental Biology 16 (2005) 271–280
during premetamorphosis, rises in parallel with endogenous
thyroid hormone, reaches a peak at the climax, and drops after it. Thyroid hormone treatment up-regulates TR␤ mRNA
by about 20-fold during premetamorphosis [33]. In general,
organs have higher TR mRNA at times when they are most
actively transforming during metamorphosis, that is, the limb
bud at NF stages 52–56 and the tail at NF stage 60 and later
[33–36]. As to TR proteins [37], TR␣ increases to NF stage
52 in the tail and head region. Even though TR␣ mRNA gradually increases during metamorphosis, TR␣ protein remains
constant, suggesting strongly that post-transcriptional events
control the ultimate levels of TR␣ protein. In contrast, there
is no detectable TR␤ protein until NF stage 52, and both TR␤
mRNA and protein rise along with the increase in endogenous thyroid hormone, reaching a maximum at the climax of
metamorphosis.
3. Programmed cell death in regressing tails
A tadpole tail, which is about two times longer than a
body, disappears completely in several days. This dramatic
resorption of the tadpole tail has attracted a good deal of
attention as an experimental system for a century.
3.1. The morphology of dying cells in regressing tails
The physiologically dying cells adopt one of at least three
different morphological types: “apoptotic”, “autophagic” and
“non-lysosomal vesiculate”. The first type of dying cell degenerates without any detectable role being played by its own
lysosomes, but its fragments are destroyed in the secondary
lysosomes of other cells. The second type is destroyed to a
greater extent within its own lysosomes. And, the third type
is destroyed without lysosomes playing any detectable role
[38].
The electron microscopic study of epidermis in regressing tail revealed the aggregation of chromatin near the nuclear
membrane and cytoplasmic condensation of epithelial cells,
the fragmentation of the nuclei and the formation of compact apoptotic bodies, which are characteristic of apoptosis.
The histological changes of dissolution of muscle cells are
the condensed cytoplasm and the peripheral aggregation of
condensed chromatin, which are typical of apoptosis, too.
However, deletion of striated muscle fibers in tadpole tail appears to be a modification of classical apoptosis, in which
the dilatation and confluence of sarcoplasmic reticulum lead
to the longitudinal clefts between myofibrils, followed by
fragmentation of the cytoplasm into a number of apoptotic
bodies with well preserved cross-striations of myofibrils [39].
Several reports also support the observation that the space between the myofibrils within the muscle cells is occupied by
tightly packed swollen and proliferated vesicles of sarcoplasmic reticulum, and the myofibrils become separated from one
another by enlargement of vesicles [40–42]. These resulting
muscle apoptotic bodies are engulfed by the macrophages,
273
which are probably derived from mesenchymal cells between
muscle fibers of myomeres [40]. Degradation of apoptotic
bodies is presumably brought about by the action of lysosomal enzymes in macrophages.
Autophagy also occurs in the regressing tadpole tail, although autophagic vacuoles are difficult to distinguish from
ingested apoptotic bodies that do not contain nuclear remnants, and no evidence shows that it plays a major part in
the regressive process [39]. Autophagic vacuoles are occasionally observed between the myofibrils, and degenerating
muscle cells include degraded myofilamentous tissue, suggesting some roles of autophagy in regressing tail [42].
3.2. The up-regulated genes in thyroid hormone-induced
tail regression
Thyroid hormone induces involution of isolated tail tips
of Xenopus laevis tadpole in an organ culture [15], which
requires RNA and protein synthesis, suggesting that the thyroid hormone-induced proteins play a pivotal role in tissue resorption [43]. This idea is consistent with a report that thyroid
hormone receptors are ligand-dependent transcription factors
to regulate target gene expression through thyroid hormoneresponse elements [44].
Thyroid hormone-induced resorption needs about a 2-day
lag period before visible regression in tail tip culture of NF
stage 51/52 tadpoles in the presence of 50–500 nM 3,3 ,5tri-iodo-l-thyronine, active form of thyroid hormone (T3)
[45]. Little or no change is observed in organ culture of tails
which are amputated from NF stage 54 tadpole pretreated
with 100 nM T3 for 4-, 8-, or 24-h, while those from 48h treated tadpoles undergo extensive resorption even in the
absence of thyroid hormone. These results suggest that the
complete program of gene regulation for tail absorption is
conferred to tail tip between 24 and 48 h after the addition of
thyroid hormone.
PCR-based subtractive hybridization has been carried out
to isolate up- and down-regulated genes in regressing tails
treated with thyroid hormone by the laboratory of Dr. D.D.
Brown [34]. A probability analysis by Poisson distribution
predicts 25 and 35 response genes up-regulated in 24 and 48 h
in the presence of 100 nM T3, respectively. The up-regulated
genes in the tail 48 h after thyroid hormone treatment include all genes induced in the first 24 h. During spontaneous
metamorphosis, mRNA from nineteen of twenty genes upregulated at 48 h reaches a high level at the climax of metamorphosis (NF stage 60), and remains high as a tail is undergoing resorption (NF stage 63). These genes are not regulated
dramatically during normal limb development, and their mRNAs are much less abundant in limb compared to tail. The
up-regulated genes encode transcription factors, proteinases,
the extracellular matrix and receptors, type III iodothyronine
5-deiodinase and so on [46].
Expression of the up-regulated genes falls into two kinetic
profiles. The direct response genes have a lag of from 2 to
4 h before their mRNA levels begin to increase, reach a peak
274
K. Nakajima et al. / Seminars in Cell & Developmental Biology 16 (2005) 271–280
at 8–12 h, and remain elevated. The mRNA transcribed from
the delayed genes starts rising 12–24 h after thyroid hormone
induction and reaches a maximum during the second day [46].
3.3. Expression patterns of thyroid hormone-induced
genes in regressing tails
The regression of amputated tails requires at least 48-h
pretreatment of tadpole with thyroid hormone, which shows
that delayed response gene expression is necessary for tail regression [34]. Expression of thyroid hormone-induced genes
during tail resorption has been characterized by in situ hybridization [47]. The observation that the highest expression
of TR␤ localizes to fibroblasts that strongly induce the delayed response gene expression (collagenase-3 and serine
dipeptidyl peptidases), implies that TR␤ which is induced by
thyroid hormone [33,48] may activate the delayed response
genes which causes tail regression. This hypothesis is supported by two reports that a TR␤-selective ligand, GC-1, efficiently induces resorption of tadpole tissues such as the gills
and tail, and has less effect on the growth of adult tissues such
as hindlimbs [49], and that TR␣ mRNA is expressed at high
levels in gills, intestine and muscles, but TR␤ mRNA is not
detected in thyroid hormone-resistant neotenic salamander,
Necturus, which cannot be induced to metamorphose even
with large doses of exogenous thyroid hormone [50].
Stromelysin-3 and the delayed response proteinases including collagenase-3 and serine dipeptidyl peptidases are
induced in a layer of subepidermal fibroblasts, adjacent to
muscle, but not in the tail muscle. These enzymes are also
up-regulated in myotendinous junctions to which the muscle
fibers are attached. These observations lead to an idea that the
thyroid hormone-induced secretion of extracellular matrixdegrading proteinases from subepidermal fibroblasts results
in degeneration of myotendinous junctions, which detaches
muscle cells from the extracellular matrix and induces their
programmed cell death. This is called a murder model. On
the other hand, since in situ hybridization analysis shows very
low expression of proteolytic enzymes in epidermal cells, it
is suggested that they commit suicide (a suicide model) [47].
3.4. The suicide model and the murder model
Based on the observation that thyroid hormone induces the
increase of the collagenase activity [51] in explants of tadpole tail fin [52] and gills [16] and the concomitant decrease
of their sizes, it has been proposed that thyroid hormoneinduced collagenase production is involved in the remodeling of collagen in these organs. The expression patterns of
the thyroid hormone-induced genes in regressing tails suggest that the delayed response proteinases induce muscle cell
death by proteolysis of the extracellular matrix, that is, the
murder model [47]. This process is supported by the phenomenon “anoikis” which is programmed cell death caused
by disruption of the interactions between normal epithelial
cells and the extracellular matrix [53–55].
To characterize the mechanism of tail muscle cell death
and to facilitate in vitro analysis, we have established a myoblastic cell line derived from NF stage 57 tadpole tail of
Xenopus laevis [19]. This cultured cell line dies in response
to 10 nM of T3 and exhibits positive TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and internucleosomal DNA cleavage, indicating apoptotic cell death. A half-day incubation with T3 is not sufficient
to provoke cell death, but a day of T3 treatment reduces cellular survival, which means that a lag time is between half a
day and a day. The condition needed to induce the death of
the cultured myoblastic cells is in contrast with the requirement (more than 2 days in the presence of 50–500 nM T3) for
the resorption of the tail tip of NF stage 51–54 tadpoles [45].
Firstly, these differences may be due to the tadpole stage.
The myoblastic cell line is derived from NF stage 57, just
before the climax, whereas tail tips in the organ culture are
prepared from NF stage 51–54. The sensitivity of isolated
tail tips to thyroid hormone increases with development, as
evidenced by a shortened lag period before the onset of regression [56]. The myoblastic cell line seems to keep the
characteristics of NF stage 57. Secondary, it reflects the difference of the myoblastic cell death and tail resorption. The
process of thyroid hormone-induced tail regression is composed of multiple steps including degradation of extracellular
matrix, spinal cord, vessels and notochord, death of muscle
cells and many types of cells, the migration of macrophages,
and absorption of cell debris.
The suicide model of muscle cell death is strongly supported by the cell-autonomous death of the myoblastic cell
line induced by thyroid hormone in the absence of subepidermal fibroblasts, which release extracellular matrix-degrading
proteinases. Since the myoblastic cell death cannot be promoted by addition of a condition medium incubated with the
cultured cells in the presence of T3 for 2 days, thyroid hormone appears not to work through a paracrine mechanism
by which cells secrete death-inducing proteins, for example, extracellular matrix-degrading proteinases to kill each
other [19]. It is possible that the cell-autonomous death of
the myoblastic cells might be an in vitro artifact due to the
absence of the extracellular matrix in the cell culture condition. This possibility is excluded by the elegant experiment
using a transgenic tadpole, which carries a gene of a dominant
negative form of the thyroid hormone receptor under the cardiac actin promoter, and overexpresses DNTR only in muscle
cells [57]. DNTR binds to the thyroid hormone response elements but not to thyroid hormone, and prevents transcription
of thyroid hormone-induced genes by the wild-type TR in
mammalian cells [26,58] and Xenopus tadpoles in vivo [59].
At 1 week after fertilization, control and transgenic tadpoles
are treated for an additional week with thyroid hormone to induce precocious metamorphosis. DNTR-overexpressing tail
muscle cells of 2-week old transgenic tadpoles are protected
from thyroid hormone-induced death, although tails shorten.
The tail muscle cells avoid the suicide, too, in NF stage 62
transgenic tadpoles, which always die just before drastic tail
K. Nakajima et al. / Seminars in Cell & Developmental Biology 16 (2005) 271–280
275
Fig. 2. Comparison of the murder model and the suicide model. Green and yellow squares indicate DNTR-expressing and non-transfected muscle cells,
respectively, and white rectangles mean subepidermal fibroblasts. Thyroid hormone (light blue ovals) induces these cells to release the death factor or the
extracellular matrix-degrading protease (orange circles) outside cells in the murder model or to produce suicide protein (blue circles) in the suicide model. The
basement membrane (red region) between muscle cells is dissolved by the protease secreted by subepidermal fibroblasts and muscle cells.
regression. Even this transgenic experiment cannot deny a
possibility that tail muscle cells murder each other by thyroid hormone-dependent releasing of soluble factor. Indeed,
the tail-derived myoblastic cell line secretes a gelatinase in
response to thyroid hormone with the induction of cell death,
raising a possibility that thyroid hormone induces muscle
cells to produce and release a gelatinase for the degradation
of the basement membrane between muscle cells [60].
To clarify the underlying mechanism of the death in tail
muscle cells, we have performed both the transfection of the
myoblastic cell line and the DNA injection into tail muscle
with DNTR expression construct and a reporter gene, followed by the examination of the viability of reporter geneexpressing cells in a condition in which programmed cell
death is induced [60]. The principle of our experiment is
schematically drawn in Fig. 2. In the presence of thyroid
hormone, the thyroid hormone-signaling pathway should be
repressed only in DNTR-overexpressing cells, but not in the
non-transfected surrounding cells, which are the majority.
According to the murder model, all cells should be killed by
soluble factors such as extracellular matrix-degrading proteinases that are synthesized by thyroid hormone-responsive
non-transfected cells including subepidermal fibroblasts. In
the suicide model, the minority of cells in which the thyroid hormone signaling is interrupted by the overexpres-
sion of DNTR should survive, whereas the non-transfected
cells die.
A result that the transfected myoblastic cells which overexpress DNTR survive in the presence of thyroid hormone,
demonstrates that the cell line commits suicide, and excludes
a possibility of cell death by murder. Fig. 3A shows the process of muscle cell death during the metamorphosis in a tail
injected with a GFP expression construct. Muscle cell death
starts at NF stage 58 when the endogenous T3 increases
abruptly and the climax of metamorphic changes begins.
GFP-expressing muscle cells are almost extinguished at NF
stage 64 when a tail resorbs to less than one-third of body in
length. The overexpression of DNTR protects muscle cells till
NF stage 61, but only delays and cannot inhibit cell death perfectly after NF stage 62 when tail regression begins (Fig. 3B).
Since the similar death of DNTR-overexpressing cells is observed even in a tail injected with six times more DNA of
DNTR expression construct, the death is not due to an insufficient amount of DNTR protein to compete endogenous TR.
Taken together, tail muscle cells undergo DNTR-sensitive
cell death (suicide) which can be inhibited completely by
blocking thyroid hormone signaling till NF stage 61 before
tail shortening, and then execute DNTR-resistant cell death
(suicide and murder) which is only delayed even by the overexpression of DNTR and finally leads to the death of 80–90%
276
K. Nakajima et al. / Seminars in Cell & Developmental Biology 16 (2005) 271–280
Fig. 3. The process of tail muscle cell death during amphibian metamorphosis. Green rhomboids represent transfected muscle cells in a tail injected
with control vector (A) or DNTR expression construct (B), while yellow
ones are non-transfected muscle cells. At NF stage 63, the notochord (blue)
is degenerated, and muscle cells change the shape.
of cells in NF stage 62–64 when a tail regresses rapidly. It
is tempting to speculate that thyroid hormone induces proteolytic enzymes to degrade the extracellular matrix and notochord, which causes losing of the cell–cell and cell–matrix
interactions and relaxing the cell tension. This loss of the cell
interaction and cell tension may be causative of cell death by
murder.
4. Programmed cell death in remodeling intestine
As anurans transform from a omnivorous tadpole to a carnivorous frog by metamorphosing, the long small intestine,
which consists of predominantly a single tubular layer of
larval epithelium with very little connective tissue or muscle, reduces in its length by about 90% [61] and is replaced
with the adult type of complex organ comprised of a multiply folded epithelium with elaborate connective tissue and
muscle, accompanied by the programmed cell death of larval
epithelium [62,63]. Both apoptotic bodies derived from larval epithelial cells and intraepithelial macrophage-like cells
suddenly increase in number around the beginning of spontaneous metamorphic climax (NF stages 59–61) and decrease
after NF stage 62, when large macrophage-like cells with the
apoptotic bodies appear in the lumen [64]. The remodeling
of the small intestine can be reproduced in an in vitro organ
culture system in the presence of thyroid hormone [17,18].
In this organ culture using the small intestine of NF stage
57, apoptotic cells or bodies are first detected on the second day of cultivation, while the number of macrophage-like
cells begins to increase on the same day and reaches its maximum around the third day when the larval epithelial cells
rapidly reduce in number. These observations imply that the
degeneration of larval epithelial cells occurs by apoptosis and
involves heterolysis by macrophages [64].
Thyroid hormone induces the intestinal epithelial cells
from NF stage 57/58 tadpoles to undergo apoptosis in a primary culture system as in an organ culture, suggesting that the
apoptotic event is cell autonomous [65]. This degeneration
of larval epithelium can be inhibited by using plastic culture
dishes which are coated with the extracellular matrix such
as laminin, collagen type IV, and fibronectin, components of
the basement membrane. However, the adult epithelial cells
from NF stage 64 tadpole intestine also undergo cell death
just like larval cells from NF stage 57/58 in the presence of
thyroid hormone, indicating that the removal of the extracellular matrix and the underlying mesenchyme in a primary
culture system alters their cell fate to the same one as larval
cells.
PCR-based subtractive differential screening has isolated
22 up-regulated genes and a single down-regulated gene using intestines of NF stage 52–54 tadpoles treated with 5 nM
T3 for 18 h [66]. When intestines of NF stage 52–54 tadpoles are treated with thyroid hormone for a week to induce
intestinal remodeling in an organ culture, the same expression patterns of these up-regulated genes are observed as
those found during spontaneous metamorphosis. Both TR␤
and stromelysin-3 are up-regulated by thyroid hormone as
direct-response genes. Their mRNAs peak in expression at
the climax (NF stage 60), decrease by NF stage 66 and remain
very low in frog gastrointestinal tract, while, in an organ culture, they increase to a peak level after 2 or 3 days of thyroid
hormone induction, and fall after 5–6 days.
In situ hybridization analysis reveals that TR␤ genes are
expressed both in the larval intestinal epithelial cells prior to
their apoptotic degeneration and in the proliferating cells of
adult epithelium, connective tissue and muscles, and downregulated upon the differentiation of these adult cells. The
observation that the expression of TR␤ genes increases before cellular death or proliferation implies that thyroid hormone signaling promotes it, depending on the cell types [67].
Stromelysin-3 mRNA is first detectable in larval fibroblasts
near the muscular layer around the beginning of the climax
(NF stage 58), then increasing in amount throughout connective tissue, and localized in fibroblasts just beneath the
epithelium by NF stage 61, when massive cell death takes
place in the larval epithelium, and adult epithelial cells proliferate rapidly. Thereafter, stromelysin-3 mRNA gradually
decreases and is no longer detected after NF stage 63. The
immunohistochemical analysis using anti stromelysin-3 antibody also confirms the same temporal and spatial expression pattern of stromelysin-3 protein as that of stromelysin3 mRNA [68]. The transient expression of stromelysin-3
mRNA and protein in fibroblasts under the epithelium and
K. Nakajima et al. / Seminars in Cell & Developmental Biology 16 (2005) 271–280
basement membrane is in good temporal accordance with
the thickening and folding of the basement membrane, the
apoptosis of the larval epithelial cells, and the proliferation
of adult epithelial cells [69,70].
Furthermore, a function-blocking antibody against
stromelysin-3 inhibits the thyroid hormone-induced apoptosis of the larval epithelium, thickening of the basement
membrane, and the invasion of the adult epithelial primordia into the connective tissue in an organ culture, although
prolonged culturing eventually leads to the degeneration of
larval epithelium and remodeling of extracellular matrix. This
suggests that the role of extracellular matrix remodeling by
stromelysin-3 lies mainly at tissue transformations but is not
the determining effector for cell fate [68].
5. Programmed cell death in remodeling skin
When an anuran tadpole metamorphoses from an aquatic
larval to a terrestrial frog life, the conversion of the larval
thin skin into the adult stratified skin is one of the important changes to adapt to the dry environment. The larval skin
consists of one or two layers of cuboidal cells (basal skein
cell layers) which are overlain by an outer squamous epithelium (apical cells) and lying on a thin basement membrane,
a thicker collagenous lamella, and an adjacent single layer of
subepithelial fibroblasts. At a middle larval stage, NF stage
55, small larval basal cells emerge in the basal skein layer.
The skin drastically transforms into adult one during the climax of metamorphosis, NF stage 60–64, larval basal cells
differentiating into adult basal cells, and the outer two cell
layers, apical and skein cells, dying and sloughing [71–73].
The morphological study of regressing tail skin suggests
that tail epidermal cells undergo apoptosis as mentioned
above [39]. Moreover, the immunohistochemical analysis
with active caspase-3 antibody reveals that the dying cells
are observed also in the tadpole body skin during the climax of metamorphosis, restricted to the first two layers of
the epidermal cells and often highly vacuolated, a characteristic of autophagy [74]. The purified tail epidermal cells do
not proliferate in the presence of thyroid hormone and detached themselves from the dish within 5 days, implying the
tail epidermal cells degenerate by the direct action of thyroid
hormone [75].
A larval keratin is expressed from late embryogenesis
throughout tadpole life in the apical and skein cells of
the epidermis. Since this larval keratin promoter drives the
same expression as the endogenous larval keratin, a transgene containing DNTR gene downstream of this promoter
blocks the thyroid hormone-signaling specifically in those
two outer layers of prepared transgenic tadpole skin [74].
They develop normally except for retaining a larval epidermis over the developing adult epithelium. This observation
that DNTR expression protects larval epidermal cells from
thyroid hormone-induced cell death indicates that the larval
apical and skein cells are direct targets of thyroid hormone,
suggesting the suicide model.
277
6. Apoptosis-related genes in regressing or
remodeling organs
Many morphological and biochemical studies have
demonstrated that apoptosis mainly mediates programmed
cell death in the resorption of the larval-specific organs and
the remodeling of the larval organs into their adult forms during the amphibian metamorphosis regardless of whether cell
death takes place according to the suicide model or the murder model, although autophagic cell death is also observed
in tail [39,42] and skin [74]. Caspase and Bcl-2 families play
an important role in apoptosis [76,77].
Xenopus laevis tadpole expresses mRNAs encoding
caspase-1, -2, -3, -6, -7, -8, -9, and -10 during the metamorphosis. These caspase mRNAs increase in regressing tail
and remodeling intestine [19,78,79], but not in a tadpole tailderived cultured cell line, which is induced to die by thyroid
hormone, showing that the induction of caspase expression
is dispensable to cell death [78]. However, the activation of
caspase-3, -6 and -7 is observed in apoptotic tail-derived cells
[78], regressing tail [57,60,79] and remodeling dorsal skin
[74].
The expression of Xenopus Bcl-XL is modulated insignificantly in regressing tail during natural or thyroid hormoneinduced metamorphosis [80], although it can inhibit cell death
of neurons [81] and tail muscle cells [60,79] during the spontaneous metamorphosis. A study on the expression profile of
Bax mRNA [82] suggests that Bax is induced in regressing
tail and thyroid hormone-treated tail, leading to muscle cell
death [93].
7. Conclusions and perspectives
Programmed cell death reaches a peak level in dorsal skin,
intestine and tail muscle around NF stage 60 [74], 61 [68] and
62 [60], respectively, when the endogenous T3 concentration
in plasma is at the highest level [11]. The larval epithelium
in skin degenerates by suicide, while the larval epithelium in
intestine is suggested to die by the combination of suicide and
murder. Muscle cells in tail die by suicide before tail regression (NF stage 61), and then are executed completely by both
the suicide and murder mechanisms as a tail is shortening.
In all cases thyroid hormone induces apoptotic cell death.
In the suicide model, each cell directly responds to thyroid
hormone, and expresses some proteins, which induce to kill
itself. On the other hand, in the murder model, the surrounding cells are suggested to be induced by thyroid hormone to
secrete extracellular matrix-degrading proteinases which disrupt the cell–cell and cell–matrix interactions, leading to the
loss of cell’s anchorage and apoptosis regardless of whether a
dying cell responds to thyroid hormone or not. Apoptosis by
murder corresponds to anoikis [53,54], the process of which
is analyzed intensively in mammals. The mammalian mechanism of anoikis may apply to the amphibian cell death by
murder.
278
K. Nakajima et al. / Seminars in Cell & Developmental Biology 16 (2005) 271–280
The molecular mechanism of apoptosis by suicide during
metamorphosis seems to remain poorly elucidated in spite of
the extensive PCR-based subtractive hybridization screening
[34]. There are three typical examples of apoptotic pathways.
Firstly, in mammals, the binding of the death receptor such
as Fas to its death factor forms oligomers of receptors, which
bind to FADD (Fas-associated protein with death domain).
Then, procaspase-8 associates with the receptor-FADD complex, resulting in the cleavage of procaspase-8 and the sequential activation of caspases to apoptosis [83]. T-cell receptor
signal induces the expression of the membrane-bound death
factor (Fas–ligand) and death receptor (Fas), the interaction
of which on the cell surface causes apoptosis in a single T
hybridoma cell [84,85]. It is possible that tail muscle cells die
through the expression and binding of the death ligand and receptor. The cDNAs encoding caspase-8 [78], death receptors
[86,87], and FADD [94] have been cloned in Xenopus laevis.
Secondly, from the studies using nematode and mammals,
the induction or the processing of a proapoptotic BH3-only
member of Bcl-2 family is shown to result in the activation
of proapoptotic multidomain members of the same family,
Bax and Bak, which are located on the mitochondria and endoplasmic reticulum, leading to the release of cytochrome c
and other death-promoting proteins from the mitochondria,
and Ca2+ ion from the endoplasmic reticulum in order to activate caspases [76,77]. Thyroid hormone might induce the
expression of a BH3-only protein during the metamorphosis, but there is no report on Xenopus BH3-only protein so
far. Thirdly, during the Drosophila metamorphosis, ecdysone
induces the expression of reaper and hid, and these protein
products physically interact Drosophila inhibitor of apoptosis 1 to block its ability to inhibit the effector caspases, which
culminates in cell death [88–90]. The ablation of diablo/smac,
a functional homolog of reaper and hid in mammals, in mice
indicates that apoptosis can proceed in its absence [91], and
its overexpression does not appear to induce apoptosis in
healthy cells [92]. Although diablo/smac does not seem to
have a potent proapoptotic activity, these three possibilities
must be considered to investigate the molecular mechanism
of the thyroid hormone-induced cell death.
The molecular mechanism of programmed cell death during amphibian metamorphosis should be not only shared in
the resorption of the larval-specific organs and the remodeling of the tadpole organs into their adult forms, but also evolutionarily conserved in the other thyroid hormone-induced
metamorphic systems of sea urchin and fish and the postembryonic transition of higher vertebrates.
References
[1] Chino Y, Saito M, Yamasu K, Suyemitsu T, Ishihara K. Formation of
the adult rudiment of sea urchins is influenced by thyroid hormones.
Dev Biol 1994;161:1–11.
[2] Inui Y, Miwa S. Thyroid hormone induces metamorphosis of flounder larvae. Gen Comp Endocrinol 1985;60:450–4.
[3] Brown DD. The role of thyroid hormone in zebrafish and axolotl
development. Proc Natl Acad Sci USA 1997;94:13011–6.
[4] Stubbe P, Gatz J, Heidemann P, Muhlen A, Hesch R. Thyroxinebinding globulin, triiodothyronine, thyroxine and thyrotropin in newborn infants and children. Horm Metab Res 1978;10:58–61.
[5] Wassenaer AG, Kok JH. Hypothyroxinaemia and thyroid function
after preterm birth. Semin Neonatol 2004;9:3–11.
[6] Tata JR. Gene expression during metamorphosis: an ideal model for
post-embryonic development. Bioessays 1993;15:239–48.
[7] Frieden E. The dual role of thyroid hormones in vertebrate development and calorigenesis. In: Gilbert LI, Frieden E, editors. Metamorphosis. New York: Plenum Press; 1981. p. 545–63.
[8] Flamant F, Samarut J. Involvement of thyroid hormone and its alpha
receptor in avian neurulation. Dev Biol 1998;197:1–11.
[9] Dodd MHI, Dodd JM. The biology of metamorphosis. In: Lofts
B, editor. Physiology of the amphibia. New York: Academic Press;
1976. p. 467–599.
[10] Nieuwkoop PD, Faber J. Normal table of Xenopus laevis. Amsterdam: North-Holland Publishing Company; 1956.
[11] Leloup J, Buscaglia M. La tiiodothyronine, hormone de la metamorphose des amphibiens. CR Acad Sci 1977;284:2261–3.
[12] Nishikawa A, Hayashi H. Isoform transition of contractile proteins
related to muscle remodeling with an axial gradient during metamorphosis in Xenopus laevis. Dev Biol 1994;165:86–94.
[13] Shi YB. Amphibian metamorphosis. New York: John Wiley & Sons;
2000.
[14] Gudernatsch JF. Feeding experiments on tadpoles. Arch Entw Mech
Org 1912;35:457–83.
[15] Weber R. Induced metamorphosis in isolated tails of Xenopus larvae.
Experientia 1962;18:84–5.
[16] Derby A, Jeffrey JJ, Eisen AZ. The induction of collagenase and
acid phosphatase by thyroxine in resorbing tadpole gills in vitro. J
Exp Zool 1979;207:391–8.
[17] Pouyet JC, Hourdry J, Mesnard J. A histological and dynamic study
of the gastric region of Discoglossus pictus larvae, cultured with or
without thyroxine. J Exp Zool 1983;225:423–31.
[18] Ishizuya Oka A, Shimozawa A. Induction of metamorphosis by thyroid hormone in anuran small intestine cultured organotypically in
vitro. In Vitro Cell Dev Biol 1991;27a:853–7.
[19] Yaoita Y, Nakajima K. Induction of apoptosis and CPP32 expression
by thyroid hormone in a myoblastic cell line derived from tadpole
tail. J Biol Chem 1997;272:5122–7.
[20] Rot-Nikcevic I, Wassersug RJ. Tissue sensitivity to thyroid hormone in athyroid Xenopus laevis larvae. Dev Growth Differ 2003;45:
321–5.
[21] Gordon AS, Goldsmith ED, Charipper HA. Effect of thiourea on the
development of the amphibian. Nature 1943;152:504–5.
[22] Huang H, Marsh-Armstrong N, Brown DD. Metamorphosis is inhibited in transgenic Xenopus laevis tadpoles that overexpress type III
deiodinase. Proc Natl Acad Sci USA 1999;96:962–7.
[23] Schreiber AM, Das B, Huang H, Marsh-Armstrong N, Brown DD.
Diverse developmental programs of Xenopus laevis metamorphosis
are inhibited by a dominant negative thyroid hormone receptor. Proc
Natl Acad Sci USA 2001;98:10739–44.
[24] Callery EM, Elinson RP. Thyroid hormone-dependent metamorphosis in a direct developing frog. Proc Natl Acad Sci USA
2000;97:2615–20.
[25] Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans
RM. The c-erb-A gene encodes a thyroid hormone receptor. Nature
1986;324:641–6.
[26] Sap J, Munoz A, Damm K, Goldberg Y, Ghysdael J, Leutz A, et al.
The c-erb-A protein is a high-affinity receptor for thyroid hormone.
Nature 1986;324:635–40.
[27] Yu VC, Delsert C, Andersen B, Holloway JM, Devary OV, Naar AM,
et al. RXR beta: a coregulator that enhances binding of retinoic acid,
thyroid hormone, and vitamin D receptors to their cognate response
elements. Cell 1991;67:1251–66.
K. Nakajima et al. / Seminars in Cell & Developmental Biology 16 (2005) 271–280
[28] Zhang XK, Hoffmann B, Tran PB, Graupner G, Pfahl M. Retinoid
X receptor is an auxiliary protein for thyroid hormone and retinoic
acid receptors. Nature 1992;355:441–6.
[29] Kliewer SA, Umesono K, Mangelsdorf DJ, Evans RM. Retinoid X
receptor interacts with nuclear receptors in retinoic acid, thyroid
hormone and vitamin D3 signalling. Nature 1992;355:446–9.
[30] Brooks AR, Sweeney G, Old RW. Structure and functional expression of a cloned Xenopus thyroid hormone receptor. Nucleic Acids
Res 1989;17:9395–405.
[31] Yaoita Y, Shi YB, Brown DD. Xenopus laevis alpha and beta thyroid
hormone receptors. Proc Natl Acad Sci USA 1990;87:7090–4.
[32] Bisbee CA, Baker MA, Wilson AC, Haji-Azimi I, Fischberg
M. Albumin phylogeny for clawed frogs (Xenopus). Science
1977;195:785–7.
[33] Yaoita Y, Brown DD. A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis. Genes Dev
1990;4:1917–24.
[34] Wang Z, Brown DD. Thyroid hormone-induced gene expression program for amphibian tail resorption. J Biol Chem 1993;268:16270–8.
[35] Wong J, Shi YB. Coordinated regulation of and transcriptional activation by Xenopus thyroid hormone and retinoid X receptors. J Biol
Chem 1995;270:18479–83.
[36] Kawahara A, Gohda Y, Hikosaka A. Role of type III iodothyronine 5-deiodinase gene expression in temporal regulation of Xenopus
metamorphosis. Dev Growth Differ 1999;41:365–73.
[37] Eliceiri BP, Brown DD. Quantitation of endogenous thyroid hormone
receptors alpha and beta during embryogenesis and metamorphosis
in Xenopus laevis. J Biol Chem 1994;269:24459–65.
[38] Clarke PG. Developmental cell death: morphological diversity and
multiple mechanisms. Anat Embryol 1990;181:195–213.
[39] Kerr JF, Harmon B, Searle J. An electron-microscope study of cell
deletion in the anuran tadpole tail during spontaneous metamorphosis
with special reference to apoptosis of striated muscle fibers. J Cell
Sci 1974;14:571–85.
[40] Weber R. Ultrastructural changes in regressing tail muscles of Xenopus larvae at metamorphosis. J Cell Biol 1964;22:481–7.
[41] Watanabe K, Sasaki F. Ultrastructural changes in the tail muscles of anuran tadpoles during metamorphosis. Cell Tissue Res
1974;155:321–36.
[42] Fox H. Aspects of tail muscle ultrastructure and its degeneration in
Rana temporaria. J Embryol Exp Morphol 1975;23:191–207.
[43] Tata JR. Requirement for RNA and protein synthesis for induced
regression of the tadpole tail in organ culture. Dev Biol 1966;13:
77–94.
[44] Glass CK, Franco R, Weinberger C, Albert VR, Evans RM, Rosenfeld MG. A c-erb-A binding site in rat growth hormone gene mediates trans-activation by thyroid hormone. Nature 1987;329:738–41.
[45] Smith KB, Tata JR. Cell death. Are new proteins synthesized
during hormone-induced tadpole tail regression? Exp Cell Res
1976;100:129–46.
[46] Brown DD, Wang Z, Furlow JD, Kanamori A, Schwartzman RA,
Remo BF, et al. The thyroid hormone-induced tail resorption program during Xenopus laevis metamorphosis. Proc Natl Acad Sci
USA 1996;93:1924–9.
[47] Berry DL, Schwartzman RA, Brown DD. The expression pattern of
thyroid hormone response genes in the tadpole tail identifies multiple
resorption programs. Dev Biol 1998;203:12–23.
[48] Kanamori A, Brown DD. The regulation of thyroid hormone receptor
beta genes by thyroid hormone in Xenopus laevis. J Biol Chem
1992;267:739–45.
[49] Furlow JD, Yang HY, Hsu M, Lim W, Ermio DJ, Chiellini G,
et al. Induction of larval tissue resorption in Xenopus laevis tadpoles by the thyroid hormone receptor agonist GC-1. J Biol Chem
2004;279:26555–62.
[50] Safi R, Begue A, Hanni C, Stehelin D, Tata JR, Laudet V. Thyroid hormone receptor genes of neotenic amphibians. J Mol Evol
1997;44:595–604.
279
[51] Gross J, Lapiere CM. Collagenolytic activity in amphibian tissues:
a tissue culture assay. Proc Natl Acad Sci USA 1962;48:1014–22.
[52] Davis BP, Jeffrey JJ, Eisen AZ, Derby A. The induction of collagenase by thyroxine in resorbing tadpole tailfin in vitro. Dev Biol
1975;44:217–22.
[53] Meredith JEJ, Fazeli B, Schwartz MA. The extracellular matrix as
a cell survival factor. Mol Biol Cell 1993;4:953–61.
[54] Frisch SM, Francis H. Disruption of epithelial cell–matrix interactions induces apoptosis. J Cell Biol 1994;124:619–26.
[55] Boudreau N, Sympson CJ, Werb Z, Bissell MJ. Suppression of ICE
and apoptosis in mammary epithelial cells by extracellular matrix.
Science 1995;267:891–3.
[56] Robinson H, Chaffee S, Galton VA. The sensitivity of Xenopus laevis
tadpole tail tissue to the action of thyroid hormones. Gen Comp
Endocrinol 1977;32:179–86.
[57] Das B, Schreiber AM, Huang H, Brown DD. Multiple thyroid hormone-induced muscle growth and death programs during metamorphosis in Xenopus laevis. Proc Natl Acad Sci USA
2002;99:12230–5.
[58] Damm K, Thompson CC, Evans RM. Protein encoded by verbA functions as a thyroid-hormone receptor antagonist. Nature
1989;339:593–7.
[59] Ulisse S, Esslemont G, Baker BS, Krishna V, Chatterjee K, Tata JR.
Dominant-negative mutant thyroid hormone receptors prevent transcription from Xenopus thyroid hormone receptor beta gene promoter
in response to thyroid hormone in Xenopus tadpoles in vivo. Proc
Natl Acad Sci USA 1996;93:1205–9.
[60] Nakajima K, Yaoita Y. Dual mechanisms governing muscle cell
death in tadpole tail during amphibian metamorphosis. Dev Dyn
2003;227:246–55.
[61] Marshall JA, Dixon KE. Cell specialization in the epithelium of
the small intestine of feeding Xenopus laevis tadpoles. J Anat
1978;126:133–44.
[62] Bonneville MA. Fine structural changes in the intestinal epithelium
of the bullfrog during metamorphosis. J Cell Biol 1963;18:579–97.
[63] McAvoy JW, Dixon KE. Cell proliferation and renewal in the small
intestinal epithelium of metamorphosing and adult Xenopus laevis.
J Exp Zool 1977;202:129–38.
[64] Ishizuya Oka A, Shimozawa A. Programmed cell death and heterolysis of larval epithelial cells by macrophage-like cells in the anuran
small intestine in vivo and in vitro. J Morphol 1992;213:185–95.
[65] Su Y, Shi Y, Stolow MA, Shi YB. Thyroid hormone induces apoptosis in primary cell cultures of tadpole intestine: cell type specificity
and effects of extracellular matrix. J Cell Biol 1997;139:1533–43.
[66] Shi YB, Brown DD. The earliest changes in gene expression
in tadpole intestine induced by thyroid hormone. J Biol Chem
1993;268:20312–7.
[67] Shi YB, Ishizuya-Oka A. Autoactivation of Xenopus thyroid hormone receptor beta genes correlates with larval epithelial apoptosis
and adult cell proliferation. J Biomed Sci 1997;4:9–18.
[68] Ishizuya-Oka A, Li Q, Amano T, Damjanovski S, Ueda S, Shi YB.
Requirement for matrix metalloproteinase stromelysin-3 in cell migration and apoptosis during tissue remodeling in Xenopus laevis. J
Cell Biol 2000;150:1177–88.
[69] Patterton D, Hayes WP, Shi YB. Transcriptional activation of the
matrix metalloproteinase gene stromelysin-3 coincides with thyroid
hormone-induced cell death during frog metamorphosis. Dev Biol
1995;167:252–62.
[70] Ishizuya-Oka A, Ueda S, Shi YB. Transient expression of
stromelysin-3 mRNA in the amphibian small intestine during metamorphosis. Cell Tissue Res 1996;283:325–9.
[71] Robinson DH, Heintzelman MB. Morphology of ventral epidermis of Rana catesbeiana during metamorphosis. Anat Rec
1987;217:305–17.
[72] Yoshizato K. Molecular cues in the epithelial–mesenchymal interaction required for the body- and tail-dependent metamorphic transformation of anuran larvel skin. Int Rev Cytol, in press.
280
K. Nakajima et al. / Seminars in Cell & Developmental Biology 16 (2005) 271–280
[73] Suzuki K, Utoh R, Kotani K, Obara M, Yoshizato K. Lineage
of anuran epidermal basal cells and their differentiation potential
in relation to metamorphic skin remodeling. Dev Growth Differ
2002;44:225–38.
[74] Schreiber AM, Brown DD. Tadpole skin dies autonomously in response to thyroid hormone at metamorphosis. Proc Natl Acad Sci
USA 2003;100:1769–74.
[75] Nishikawa A, Yoshizato K. Hormonal regulation of growth and life
span of bullfrog tadpole tail epidermal cells cultured in vitro. J Exp
Zool 1986;237:221–30.
[76] Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell
2004;116:205–19.
[77] Adams JM. Ways of dying: multiple pathways to apoptosis. Genes
Dev 2003;17:2481–95.
[78] Nakajima K, Takahashi A, Yaoita Y. Structure, expression, and
function of the Xenopus laevis caspase family. J Biol Chem
2000;275:10484–91.
[79] Rowe I, Coen L, Le Blay K, Le Mevel S, Demeneix BA. Autonomous regulation of muscle fibre fate during metamorphosis in
Xenopus tropicalis. Dev Dyn 2002;224:381–90.
[80] Cruz Reyes J, Tata JR. Cloning, characterization and expression of
two Xenopus bcl-2-like cell-survival genes. Gene 1995;158:171–9.
[81] Coen L, du Pasquier D, Le Mevel S, Brown S, Tata J, Mazabraud
A, et al. Xenopus Bcl-X(L) selectively protects Rohon–Beard neurons from metamorphic degeneration. Proc Natl Acad Sci USA
2001;98:7869–74.
[82] Sachs LM, Abdallah B, Hassan A, Levi G, De Luze A, Reed
JC, et al. Apoptosis in Xenopus tadpole tail muscles involves Baxdependent pathways. FASEB J 1997;11:801–8.
[83] Nagata S. Apoptosis by death factor. Cell 1997;88:355–65.
[84] Dhein J, Walczak H, Bäumler C, Debatin KM, Krammer PH.
Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature
1995;373:438–41.
[85] Brunner T, Mogil RJ, LaFace D, Yoo NJ, Mahboubi A, Echeverri F, et al. Cell-autonomous Fas (CD95)/Fas–ligand interaction
mediates activation-induced apoptosis in T-cell hybridomas. Nature
1995;373:441–4.
[86] Hutson LD, Bothwell M. Expression and function of Xenopus laevis p75(NTR) suggest evolution of developmental regulatory mechanisms. J Neurobiol 2001;49:79–98.
[87] Tamura K, Noyama T, Ishizawa YH, Takamatsu N, Shiba T, Ito M.
Xenopus death receptor-M1 and -M2, new members of the tumor
necrosis factor receptor superfamily, trigger apoptotic signaling by
differential mechanisms. J Biol Chem 2004;279:7629–35.
[88] Vucic D, Kaiser WJ, Harvey AJ, Miller LK. Inhibition of reaperinduced apoptosis by interaction with inhibitor of apoptosis proteins
(IAPs). Proc Natl Acad Sci USA 1997;94:10183–8.
[89] Wang SL, Hawkins CJ, Yoo SJ, Muller HA, Hay BA. The Drosophila
caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 1999;98:453–63.
[90] Yin VP, Thummel CS. A balance between the diap1 death inhibitor
and reaper and hid death inducers controls steroid-triggered cell
death in Drosophila. Proc Natl Acad Sci USA 2004;101:8022–7.
[91] Okada H, Suh WK, Jin J, Woo M, Du C, Elia A, et al. Generation
and characterization of Smac/DIABLO-deficient mice. Mol Cell Biol
2002;22:3509–17.
[92] Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid
GE, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell
2000;102:43–53.
[93] Sachs LM, Mevel SL, Demeneix BA. Implication of bax in Xenopus
laevis tail regression at metamorphosis. Dev Dyn 2004;231:671–82.
[94] Sakamaki K, Takagi C, Kominami K, Sakata S, Yaoita Y, Kubota HY,
et al. The adaptor molecule FADD from Xenopus laevis demonstrates
evolutionary conservation of its pro-apoptotic activity. Genes Cells
2004;9:1249–64.