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MOLECULAR mechanismS OF FROG METAMORPHOSIS
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Yun-Bo
Shi, PhD, Head,
Section on Molecular Morphogenesis Tosikazu
Amano, PhD, Visiting Fellow Liezhen
Fu, PhD, Visiting Fellow Takashi
Hasebe, PhD, Visiting Fellow ShaoChung
V. Hsia, PhD, Visiting Fellow Hiroki
Matsuda, PhD, Visiting Fellow Bindu
Diana Paul, PhD, Visiting Fellow Yukiyasu
Sato, MD, PhD, Visiting Fellow Akihiro
Tomita, MD, PhD, Visiting Fellow Daniel Buchholz, PhD, Adjunct Scientist |
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The laboratory
explores molecular mechanisms in amphibian metamorphosis. The control of this
developmental process by thyroid hormone (TH) offers a unique paradigm in
which to study gene function in postembryonic organ development. During
metamorphosis, different organs undergo vastly different changes. Some, like
the tail, undergo complete resorption while others, such as the limb, are
developed de novo. The majority of the larval organs persist through
metamorphosis but are dramatically remodeled to function in a frog. For
example, tadpole intestine in Xenopus laevis is a simple tubular
structure consisting mostly of a single layer of primary epithelial cells.
During metamorphosis, it is transformed, through specific cell death and
selective cell proliferation and differentiation, into an organ of a multiply
folded adult epithelium surrounded by elaborate connective tissue and
muscles. The wealth of knowledge from past research and the ability to
manipulate amphibian metamorphosis both in vivo, by using transgenesis
or hormone treatment of whole animals, and in vitro in organ cultures
offer excellent opportunities to study the developmental function of thyroid
hormone receptors (TRs) and the underlying mechanisms in vivo and to
identify and functionally characterize genes that are critical for
postembryonic organ development in vertebrates. Function
of TR during development Buchholz, Tomita, Shi Based
on earlier studies, we proposed a dual-function model for TR during frog
development: the heterodimers between TR and RXR (9-cis retinoic acid
receptor) activate gene expression during metamorphosis when TH is present
while, in premetamorphic tadpoles, they repress gene expression to prevent
metamorphosis, thus ensuring a proper tadpole growth period. Such a model
argues that transcriptional activation by TR is essential for frog
metamorphosis. To test such a hypothesis, we previously adapted the
sperm-mediated transgenic method to generate transgenic animals expressing a
dominant negative TR (dnTR). Phenotypic analysis indicated that dnTR
overexpression inhibits TH-induced metamorphosis. Molecular studies revealed
that dnTR specifically blocks the expression of TH response genes by
competing for binding to TH target promoters with endogenous wild-type TRs, leading
to the retention of corepressors N-CoR and SMRT and to continued histone
deacetylation even in the presence of TH. These studies thus show that gene
activation is necessary for metamorphosis. Given
that TH is also known to have nongenomic effects and that, in the mouse, TH
deficiency causes distinct phenotypes compared with TR-knockout animals, we
were interested in determining whether gene activation by TR is sufficient
for TH-dependent metamorphosis. For this purpose, we developed a dominant positive
mutant TR (dpTR). In the frog oocyte transcription system, dpTR bound to a
T3-responsive promoter and activated the promoter independently of T3.
Transgenic expression of dpTR under the control of a heat
shock–inducible promoter in premetamorphic tadpoles led to precocious
metamorphic transformations. Molecular analyses showed that dpTR induced
metamorphosis by specifically binding to known T3-target genes, leading to
increased local histone acetylation and gene activation, similar to T3-bound
TR during natural metamorphosis. Our experiments indicated that the
metamorphic role of T3 operates predominantly, if not exclusively, through
genomic action of the hormone. They further show, for the first time, that TR
alone can directly mediate the developmental effects of T3 in individual
organs by regulating target gene expression in these organs. Stewart D, Tomita A, Shi Y-B, Wong J. Chromatin
immunoprecipitation for studying transcriptional regulation in Xenopus
oocytes and tadpoles. In: Liu J, ed. Xenopus Protocols: Cell Biology and
Signal Transduction. Roles
of cofactors in gene regulation by TR Hsia, Matsuda, Paul,
Sato, Tomita, Shi TR
regulates gene transcription by recruiting cofactors to target genes. In the
presence of TH, TR can bind to coactivators while the unliganded TR binds to
corepressors. Despite the completion of several biochemical and molecular
studies on such cofactors, little is known about whether and how the
cofactors participate in gene regulation by TR in different biological
processes in vivo. Our focus is to investigate how TR uses different
cofactors in the context of development in various organs. To
study the role of cofactors in TR function during metamorphosis, we chose
several that, based on studies in tissue culture cells and in vitro,
are likely to be involved in development. Thus, we have begun to study p300
and SRC, showing that Xenopus p300, SRC2, and SRC3 are expressed
during metamorphosis. Using the frog oocyte system that we had established
earlier for studying TR function in the context of chromatin, we recently
demonstrated, by using a chromatin immunoprecipitation (ChIP) assay, that TR
can recruit SRC3 to a target promoter. Furthermore, we generated a dominant
negative form of SRC3 (dnSRC3) that can be recruited by TR, thus blocking the
recruitment of wild-type SRC and gene activation. Similar studies on p300
suggest that it is also recruited by TR and that a dominant negative p300 can
also inhibit gene activation by TH in the frog oocyte. To determine the
cofactor’s roles in vivo, we are employing the ChIP assay to
analyze the recruitment of both SRC3 and p300 to endogenous TH-response genes
during development. Furthermore, by using sperm-mediated transgenesis, we
obtained preliminary evidence to show that transgenic tadpoles expressing
dnSRC3 are resistant to TH-induced as well as natural metamorphosis. We are
conducting molecular studies to determine the underlying mechanisms. Among
the corepressors, we have been studying the role of N-CoR (nuclear receptor
corepressor) and SMRT (silencing mediator of retinoid and thyroid receptors)
in gene repression by TR in premetamorphic tadpoles. We have shown previously
that both are expressed and, more important, bound to TH-response genes in
premetamorphic tadpoles. The corepressors’ association with TH-response
genes is released upon treatment of the tadpoles with TH, indicating that
binding to TH-response gene promoters is attributable to recruitment by
unliganded TR. More recently, we have shown that the corepressors are also
released during natural metamorphosis when the endogenous TH level rises.
Both N-CoR and SMRT are known to exist in histone
deacetylase–containing complexes in mammals. Among the other proteins
in the complexes is TBLR1 (transducin beta–like protein 1–related
protein). We have previously shown that frog TBLR1 is expressed in tadpoles and
forms complexes with both N-CoR and SMRT, at least in the frog oocyte. To
investigate TBLR1’s involvement in TR function during development, we
again made use of the frog oocyte system. We showed that unliganded TR
interacts with TBLR1 in vivo. Using the ChIP assay, we demonstrated
that unliganded TR recruits TBLR1, together with N-CoR and/or SMRT, to its
target promoter, leading to histone deacetylation and gene repression in
chromatin. Furthermore, a dominant negative N-CoR that contains only the TBLR1-interacting
domain was able to block TBLR1 interaction with TR and derepress the
promoter. The results argue that TBLR1 or related factors are required for
transcription repression by unliganded TR. Finally, we showed that TBLR1 is
expressed throughout frog development and is associated with endogenous
TH-target promoters in premetamorphic tadpoles. More important, the
association is reduced or eliminated upon treatment of the tadpoles with TH
to induce metamorphosis or during natural metamorphosis when TH levels are
high, suggesting that the release of TBLR1-containing corepressor complexes
is one of the mechanisms by which TH response genes are activated during
metamorphosis. Paul BD, Shi Y-B. Distinct expression profiles of
transcriptional coactivators for thyroid hormone receptors during Xenopus
laevis metamorphosis. Cell Res 2003;13:459-464. Sachs LM, Jones P, Havis E, Rouse N, Demeneix BA, Shi Y-B. N-CoR
recruitment by unliganded thyroid hormone receptor in gene repression during Xenopus
laevis development. Mol Cell Biol 2003;22:8527-8538. Tomita A, Tomita A, Regulation
and function of TH response genes during TH-induced tissue remodeling Amano, Buchholz, Fu,
Hasebe, Shi; in collaboration with Ishizuya-Oka In an
effort to identify genes important for postembryonic development, we isolated
many TH response genes during metamorphosis. We have recently characterized
one of them as a novel gene (ID14) expressed in tadpoles and adults of
Xenopus laevis. ID14 encodes a 315–amino acid
protein that has a signal peptide and a nidogen domain. Even though several
genes have a nidogen domain, ID14 is not the homolog of any known
gene. ID14 is a late thyroid hormone–regulated gene in the
tadpole intestine, and its expression in the intestine does not begin until
the climax of metamorphosis, correlating with adult intestinal epithelial
differentiation. In contrast, ID14 is expressed in tadpole skin and
tail throughout postembryonic development and is not regulated by thyroid
hormone. In situ hybridization revealed that this putative
extracellular matrix (ECM) protein is expressed in the epithelia of the
tadpole skin and tail and in the intestinal epithelium after metamorphosis.
In the adult, the ID14-encoded protein is found predominantly in the
intestine with weak expression in the stomach, lung, and testis. Its
exclusive expression in adult intestinal epithelial cells makes it a useful
marker for developmental studies and may provide insights into cell-cell
interactions in intestinal metamorphosis and adult intestinal stem cell
maintenance. Among
the other genes regulated by TH during metamorphosis, those encoding matrix
metalloproteinases (MMPs) are of particular interest for functional
investigations. MMPs are extracellular enzymes capable of digesting various
ECM components. Our earlier studies led us to propose that the MMP
stomelysin-3 (ST3) is directly or indirectly involved in ECM remodeling,
which in turn influences cell behavior in the remodeling intestine. This
notion is supported by intestinal organ culture studies in vitro, in
which we showed that ST3 function is important for TH-induced apoptosis of
larval intestinal epithelial cells and the invasion of the proliferating
adult epithelial cells into the connective tissue. To investigate directly
the roles of MMPs in developing animals, we have generated transgenic
tadpoles expressing wild-type ST3 or a catalytically inactive mutant
under the control of a heat shock–inducible promoter. Heat shock at
tadpole stages led to overexpression of wild-type or mutant ST3 in all
organs. However, we observed no visible morphological changes in the tadpoles
for up to a few weeks, although animals expressing wild-type ST3
transgene appeared to grow more slowly. Importantly, analysis of the
intestine showed that overexpression of wild-type but not mutant ST3
caused premature apoptosis in the tadpole epithelium, accompanied by drastic
remodeling of the basal lamina or the ECM that separates the connective
tissue and epithelium in the intestine. Thus, our results suggest that ST3
directly or indirectly modifies the ECM, which in turn facilitates cell fate
changes and tissue morphogenesis during metamorphosis. Current work aims to
analyze the transgenic animals in detail, focusing on the intestine. To
determine how ST3 affects tissue remodeling, we have begun to isolate and
characterize ST3 substrates. As a first step, we investigated the ability of Xenopus
laevis ST3 to cleave an in vitro substrate of mammalian ST3,
the alpha 1-proteinase inhibitor (PI). We analyzed the ability of the Xenopus
laevis ST3 catalytic domain to cleave frog PI. Surprisingly, we found
that ST3 failed to recognize the site in Xenopus PI equivalent to the
major site in human PI that is cleaved by mammalian ST3. Sequence and
mutagenic analyses revealed that multiple substitutions at P2-P3´ positions
between human and Xenopus PI contributed to the failure of Xenopus
PI to be cleaved by ST3. Our studies demonstrated that (A)(G/A)(A)(M)(F/A)(L)
(P3-P3´) is a preferred cleavage site for ST3. We further demonstrated that
mutations in the cleavage sites affected cleavage by ST3 in a manner that
differed from several other MMPs. These findings, together with earlier
reports on ST3, showed that ST3 has substrate specificities that are distinct
from those of other MMPs. Our results further suggest that PI is unlikely to
be a physiological substrate for ST3, at least with regard to evolutionarily
conserved developmental processes. Amano T, Liezhen F, Sahu S, Markey M, Shi Y-B. Substrate
specificity of Xenopus matrix metalloproteinase stromelysin-3. Int
J Mol Med 2004;14:233-239. Buchholz DR, Ishizuya-Oka A, Shi Y-B. Spatial and temporal
expression pattern of a novel gene in the frog Xenopus laevis:
correlations with adult intestinal epithelial differentiation during
metamorphosis. Gene Expr Patterns 2004;4:321-328. Ishizuya-Oka A, Amano T, Fu L, Shi Y-B. Regulation of apoptosis
by extracellular matrix during postembryonic development in Xenopus laevis.
In: Lockshin RA, Zakeri Z, eds. When Cells Die II: a Comprehensive
Evaluation of Apoptosis and Programmed Cell Death. COLLABORATOR Atsuko Ishizuya-Oka, PhD, Department of
Biology,
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