Yun-Bo Shi, PhD, Head, Section on Molecular Morphogenesis
Biswajit Das, PhD, Research Fellow
Liezhen Fu, PhD, Staff Scientist
Maria Rosaria Fiorentino, PhD, Visiting Fellow
Takashi Hasebe, PhD, Visiting Fellow
Rachel Heimeier, PhD, Visiting Fellow
Smita Mathew, PhD, Visiting Fellow
Hiroki Matsuda, PhD, Visiting Fellow
Yukiyasu Sato, MD, PhD, Visiting Fellow
Daniel Buchholz, PhD, Postdoctoral Fellow
Teresa Washington, PhD, Postdoctoral Fellow
Alexis Oetting, BS, Postbaccalaureate Fellow

We are exploring molecular mechanisms in amphibian metamorphosis. The control of metamorphosis 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, such as the tail, undergo complete resorption while others, such as the limb, develop de novo. Most larval organs persist through metamorphosis but, in a frog, they undergo dramatically remodeling. For example, tadpole intestine in Xenopus laevis is a simple tubular structure largely consisting of a single layer of primary epithelial cells. During metamorphosis, the intestine 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 an excellent opportunity (1) to study the developmental function of thyroid hormone receptors (TRs) and underlying mechanisms in vivo and (2) to identify and functionally characterize genes critical for post-embryonic organ development in vertebrates.

Function of TR during development

Buchholz, Das, Paul, ¹ Washington, Shi

We have proposed a dual-function model for TR during frog development, that is, the heterodimers between TR and RXR (9-cis retinoic acid receptor) activate gene expression during metamorphosis when TH is present. In premetamorphic tadpoles, the heterodimers repress gene expression in the absence of TH to prevent metamorphosis, thus ensuring a proper tadpole growth period. When TH is present from either endogenous synthesis during development or exogenous addition to the rearing water of premetamorphic tadpoles, TR/RXR heterodimers activate TH-inducible genes to initiate metamorphosis. Using the sperm-mediated transgenic approach, we previously generated transgenic animals expressing either a dominant negative TR, which does not bind to TH and functions as a constitutive repressor, or a dominant positive TR, which activates TH-response genes without requiring TH. Our phenotypic and molecular analyses led us to conclude that the metamorphic role of TH occurs predominantly, if not exclusively, through genomic action of the hormone mediated by TR. Non-genomic action of TH, while it exists, plays a minor role, if any, during this post-embryonic process. We have thus shown for the first time that TR mediates directly and sufficiently the developmental effects of TH in individual organs by regulating target gene expression in these organs.

To investigate how TR differentially regulates different genes in various organs/tissues during metamorphosis, we used the chromatin immunoprecipitation (ChIP) assay to examine TR binding to promoters in vivo. We analyzed TRβ and TH/bZIP (TH-responsive basic leucine zipper transcription factor), two target genes with TH response elements (TRE) in their promoters. Using an antibody that recognizes both TRα and TRβ, we found that TR binding to the TRβ promoter is constitutive. Surprisingly, TR binding to the TH/bZIP promoter increases dramatically after TH treatment of premetamorphic tadpoles and during metamorphosis. Using an antibody specific to TRβ, we found that TRβ binding increases at both promoters in response to TH, likely owing to a TH-induced increase in TRβ expression. In vitro biochemical studies have revealed that TRs bind to TH/bZIP TRE with four-fold lower affinity than to TRβ TRE. Our data show that only high-affinity TRβ TRE is occupied by limiting levels of TR during premetamorphosis and that lower-affinity TH/bZIP TRE becomes occupied only when overall TR expression is higher during metamorphosis, thereby providing the first in vivo evidence to suggest that tissue- and gene-specific regulation of TR target gene expression may occur through tissue- and developmental stage-dependent regulation of TR levels, which is likely to be a critical mechanism for coordinating development in different organs during post-embryonic development.

Roles of co-factors in gene regulation by TR

Buchholz, Heimeier, Matsuda, Paul, ¹ Sato, Shi

TR regulates gene transcription by recruiting co-factors to target genes. In the presence of TH, TR can bind to co-activators while the unliganded TR binds to co-repressors. Many biochemical and molecular studies have investigated such co-factors. Much less is known, however, about whether and how the co-factors participate in gene regulation by TR in different biological processes in vivo. We investigate how TR uses different co-factors in the context of development in various organs.

Among co-repressors, we have studied the role of N-CoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid receptors) in gene repression by TR in premetamorphic tadpoles. We have shown that, in premetamorphic tadpoles, both N-CoR and SMRT are expressed and, more important, bind to TH-response genes and thus bring to the promoters other components, such as TBLR1 (transducin beta-like protein 1-related protein), of the histone deacetylase-containing complexes. Furthermore, TH treatment of premetamorphic tadpoles leads to the release of TBLR1, together with N-CoR and/or SMRT, and to increased histone acetylation and gene activation. The results support the notion that TBLR1-containing N-CoR/SMRT deacetylase complexes or related complexes are required for transcription repression by unliganded TR in tadpoles and that their release is one of the mechanisms by which TH response genes are activated during metamorphosis. To investigate the role of such repression in tadpole development, we generated a dominant negative N-CoR containing only the TR-interacting domain and introduced it into developing animals through transgenesis. We are conducting phenotypic and molecular analyses to determine the effects of the transgene.

On the activator side, we have used a ChIP assay to show that TR recruits the Xenopus co-activator SRC3 to target genes in a gene- and tissue-dependent manner, both upon TH treatment of premetamorphic tadpoles and during natural metamorphosis. In addition, we generated transgenic tadpoles expressing a dominant negative form of SRC3 (F-dnSRC3). The transgenic tadpoles exhibited normal growth and development throughout embryogenesis and premetamorphic stages. However, transgenic expression of F-dnSRC3 inhibited essentially all aspects of TH-induced metamorphosis as well as natural metamorphosis, leading to delayed or arrested metamorphosis or the formation of tailed frogs. Molecular analysis revealed that F-dnSRC3 functioned by blocking the recruitment of endogenous co-activators to TH-target genes without affecting co-repressor release, thereby blocking the TH-dependent gene regulation program responsible for tissue transformations during metamorphosis. Our studies thus demonstrate that, aside from co-repressor release, co-activator recruitment is required for TH function in development. Our work also provides the first example of a specific co-activator-dependent gene regulation pathway by a nuclear receptor underlying specific developmental events. Using a similar approach, we determined that also liganded TR recruits the co-activator p300 to TH target promoters. We generated a dominant negative form of p300 that contained only the SRC-binding domain of p300, which is capable of inhibiting activation of a reporter gene by liganded TR in vivo. Through transgenesis, we showed that the dominant negative p300 is also capable of inhibiting gene activation by TR in developing animals and thus prevents metamorphosis, demonstrating a critical role of SRC-p300 complexes in TR-mediated metamorphosis. To investigate further the role of co-activator complexes, we are studying the role of methyltransferases PMRT1 and CARM1 (PMRT4), which are able to bind to SRC proteins in vitro as well as in tissue culture cells.

Involvement of matrix metalloproteinases during TH-induced tissue remodeling

Amano, ¹ Fu, Hasebe, Mathew, Shi; in collaboration with Ishizuya-Oka

We have previously identified several TH-response genes that encode matrix metalloproteinases (MMPs) during intestinal remodeling. Expression and organ culture studies led us to propose that the MMP stomelysin-3 (ST3) is directly or indirectly involved in extracellular matrix (ECM) remodeling, which in turn influences cell behavior. We generated transgenic animals expressing ST3 or a catalytically inactive mutant under the control of a heat-shock-inducible promoter. Heat shock treatment of premetamorphic tadpoles leads to overexpression of wild-type or mutant ST3 in all organs without visible morphological changes in the tadpoles. Analyses of the intestine showed that overexpression of wild-type but not mutant ST3 causes premature apoptosis in the tadpole epithelium, consistent with results from our earlier organ culture studies. Electron-microscopic studies revealed that the apoptosis is accompanied by drastic remodeling of the basal lamina or the ECM that separates connective tissue from epithelium in the intestine. Taken together, our results suggest that ST3 directly or indirectly modifies the ECM, which in turn facilitates cell fate changes and tissue morphogenesis during metamorphosis.

Toward understanding the mechanism by which ST3 affects tissue remodeling, we used a yeast two-hybrid screen to isolate the 37 kD laminin receptor precursor (LR) as a likely substrate. LR binds to ST3 in vitro and can be cleaved by ST3 at two sites distinct from those where other MMPs cleave; the sites are located in the extracellular domain between the transmembrane domain and laminin binding sequence, suggesting that LR cleavage by ST3 alters cell-ECM interaction. Expression studies have shown that LR is cleaved when ST3 is highly expressed either during intestinal remodeling or in premetamorphic intestine of transgenic tadpoles overexpressing ST3. Thus, LR is likely a physiological substrate of ST3 and plays a role in cell fate determination and tissue morphogenesis, in part through its cleavage by ST3. Interestingly, ST3 cleavage sites in LR are conserved in human LR. Furthermore, high levels of LR are known to be expressed in tumor cells, which are often surrounded by fibroblasts expressing ST3. Thus, LR may be a conserved substrate of ST3; the fact that its cleavage by ST3 may alter cell-ECM interactions suggests that LR might play a role in mediating the effects of ST3 on cell fate and behavior during development and pathogenesis.

In addition to ST3, several other MMPs are upregulated during metamorphosis, although most, such as gelatinase A (GelA), are not direct TH-response genes. Like GelA but unlike ST3, most MMPs require extracellular activation by cleavage of the propeptide from the secreted pro-enzyme. It has been shown that the membrane-type MMP-1 (MT1-MMP) participates in the activation of GelA, suggesting that these MMPs may function together in development. Indeed, we found that Xenopus laevis MT1-MMP is also upregulated in the intestine and tail when both organs undergo metamorphosis. Within the organs, MT1-MMP and GelA are co-expressed in connective tissues during both natural and thyroid hormone-induced metamorphosis, although MT1-MMP, but not GelA, is also expressed in the longitudinal muscle cells of the metamorphosing intestine. Thus, it is likely that MT1-MMP and GelA function together in ECM degradation or remodeling during metamorphosis and that MT1-MMP plays additional, GelA-independent roles in the development of the adult longitudinal muscle in the intestine.

¹ former Postdoctoral Fellow

COLLABORATOR

Atsuko Ishizuya-Oka, PhD, Nippon Medical School, Kawasaki, Kanagawa, Japan

For further information, contact shi@helix.nih.gov.