We explore 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 post-embryonic organ development. During metamorphosis, organs undergo vastly different changes. Some, like the tail, undergo complete resorption while others, such as the limb, develop de novo. In a frog, most larval organs persist through metamorphosis but are dramatically remodeled. For example, the 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, coupled with the ability to manipulate amphibian metamorphosis both in vivo by using transgenesis or hormone treatment of whole animals and in vitro in organ cultures, offers an excellent opportunity to study the developmental function of thyroid hormone receptors (TRs) and their underlying mechanisms in vivo and to identify and functionally characterize genes that are critical for post-embryonic organ development in vertebrates.
Function of TR during development
Buchholz, Tomita,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 pre-metamorphic tadpoles, the heterodimers repress gene expression in the absence of TH to prevent metamorphosis, thus ensuring a proper tadpole growth period. By using the sperm-mediated transgenic approach, we previously generated transgenic animals expressing a dominant negative TR (dnTR), allowing us to show that gene activation by TR in the presence of TH, in part through the release of co-repressors such as N-CoR and SMRT, is necessary for metamorphosis. In addition, by generating transgenic tadpoles expressing a dominant positive TR under a heat shock-inducible promoter, which activates TH response genes without a requirement for TH, we more recently showed that heat-shock induction of TR expression is sufficient to induce all morphological and gene expression changes associated with TH-induced metamorphosis. Such studies have led us to conclude that TH exerts its metamorphic effect predominantly, if not exclusively, through genomic action of the hormone. Nongenomic action of TH, while it exists, plays a minor role, if any, during this post-embryonic process. The studies also show, for the first time, that TR directly mediates and is sufficient for the developmental effects of TH in individual organs by regulating target gene expression in these organs. Our current research focuses on how TR differentially regulates genes in various organs/tissues during metamorphosis.
Buchholz DR, Paul BD, Fu L, Tomita A, Shi Y-B. Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog. Gen Comp Endocrinol 2005 [Epub ahead of print].
Buchholz DR, Tomita A, Fu L, Paul BD, Shi Y-B. Transgenic analysis reveals that thyroid hormone receptor is sufficient to mediate the thyroid hormone signal in frog metamorphosis. Mol Cell Biol 2004;24:9026-9037.
Roles of co-factors in gene regulation by TR
Buchholz, Heimeier, Hsia, Matsuda, Paul, Sato, Tomita,1 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. While many biochemical and molecular studies of such co-factors have been conducted, much less is known 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 the co-repressors, we have been studying the role of N-CoR (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid receptors) in gene repression by TR in pre-metamorphic tadpoles. We previously showed that both co-repressors are expressed and, more important, bind to TH-response genes in pre-metamorphic tadpoles. Both N-CoR and SMRT are known to exist in histone deacetylase–containing complexes in mammals. Among the proteins in the complexes is TBLR1 (transducin beta-like protein 1–related protein). Using a chromatin immunoprecipitation (ChIP) assay, we demonstrated that unliganded TR recruits TBLR1, together with N-CoR and/or SMRT, to its target promoters in chromatin in pre-metamorphic tadpoles and that TH treatment of pre-metamorphic tadpoles leads to the release of TBLR1, together with N-CoR and/or SMRT, and increased histone acetylation and gene activation. The results support the argument that TBLR1 or related factors are required for transcription repression by unliganded TR in tadpoles and that the release of TBLR1-containing co-repressor complexes is one of the mechanisms by which TH-response genes are activated during metamorphosis.
In addition, our earlier studies showed that the Xenopus co-activator SRC3 is upregulated as a late TH-response gene during natural as well as TH-induced metamorphosis in both the tail and intestine. Using a ChIP assay, we found, surprisingly, that SRC3 is recruited in a gene- and tissue-dependent manner to target genes by TR, both upon TH treatment of pre-metamorphic tadpoles and during natural metamorphosis. In particular, in the tail, SRC3 is not recruited in a TH-dependent manner to its target TRbetaA promoter but is recruited by liganded TR to the TRbetaA promoter in the intestine and to the TH/bZIP (another direct TH target gene) promoter in both the tail and intestine. Furthermore, we generated transgenic tadpoles expressing a dominant negative form of SRC3 (F-dnSRC3). The transgenic tadpoles exhibit normal growth and development throughout embryogenesis and pre-metamorphic stages. However, transgenic expression of F-dnSRC3 inhibits 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 preventing the TH-dependent gene regulation program responsible for tissue transformations during metamorphosis. Our studies thus demonstrate that co-activator recruitment, aside from co-repressor release, is required for TH function in development, further providing the first example of a co-activator–dependent pathway of gene regulation via a nuclear receptor, a pathway that underlies specific developmental events.
Buchholz DR, Paul BD, Shi Y-B. Chromatin immunoprecipitation for in vivo studies of transcriptional regulation during development. In: Whitman M, Sater AK, eds. Methods in Signal Transduction: Analysis of Growth Factor Signaling in Embryos. Florida: CRC Press, 2005 (in press).
Paul BD, Buchholz DR, Fu L, Shi Y-B. Tissue- and gene-specific recruitment of steroid receptor coactivator-3 by thyroid hormone receptor during development. J Biol Chem 2005;280:27165-27172.
Paul BD, Fu L, Buchholz DR, Shi YB. Coactivator recruitment is essential for liganded thyroid hormone receptor to initiate amphibian metamorphosis. Mol Cell Biol 2005;25:5712-5724.
Tomita A, Buchholz DR, Shi Y-B. Recruitment of N-CoR/SMRT-TBLR1 corepressor complex by unliganded thyroid hormone receptor for gene repression during frog development. Mol Cell Biol 2004;24:3337-3346.
Involvement of matrix metalloproteinases during TH-induced tissue remodeling
Fu, Hasebe, Li, Mathew, Shi; in collaboration with Ishizuya-Oka
We previously identified several TH-response genes that encode matrix metalloproteinases (MMPs) during intestinal remodeling. Furthermore, earlier 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. This notion is supported by 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. We have now provided in vivo evidence for a role of ST3 in regulating ECM remodeling and cell death. We generated transgenic animals expressing ST3 or a catalytically inactive mutant under the control of a heat shock–inducible promoter. Heat shock treatment of pre-metamorphic 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, consistent with our earlier organ culture studies, overexpression of wild-type but not mutant ST3 causes premature apoptosis in the tadpole epithelium. Electron microscopic studies revealed that the apoptosis is accompanied by drastic remodeling of the basal lamina or the ECM that separates the connective tissue and epithelium in the intestine. Together, our results suggest that ST3 directly or indirectly modifies the ECM, which in turn facilitates cell fate changes and tissue morphogenesis during metamorphosis.
To understand the mechanism by which ST3 affects tissue remodeling, we used a yeast two-hybrid screen with mRNAs from metamorphosing tadpole intestine to identify potential substrates of ST3 during development. We thus isolated the 37 kDa laminin receptor precursor (LR) as a likely substrate and showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites distinct from those that other MMPs cleave. Through peptide sequencing, we determined that the two cleavage 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. To investigate if LR is indeed an in vivo substrate of ST3, we analyzed LR’s expression during metamorphosis and found expression in the intestinal epithelium of pre-metamorphic tadpoles. During intestinal metamorphosis, LR is downregulated in the apoptotic epithelium and concurrently upregulated in connective tissue but with little expression in the developing adult epithelium. Toward the end of metamorphosis, as adult epithelial cells differentiate, they begin to express LR. Furthermore, LR is cleaved during intestinal remodeling, when ST3 is highly expressed, or in pre-metamorphic intestine of transgenic tadpoles overexpressing ST3. The results suggest that LR is a physiological substrate of ST3 and plays a role in cell fate determination and tissue morphogenesis, in part through changes in its spatial expression during development and 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, and its cleavage by ST3 may alter cell-ECM interactions, thus playing a role in mediating the effects of ST3 on cell fate and behavior during development and pathogenesis.
Amano T, Fu L, Marshak A, Kwak O, Shi Y-B. Spatio-temporal regulation and cleavage by matrix metalloproteinase stromelysin-3 implicate a role for laminin receptor in intestinal remodeling during Xenopus laevis metamorphosis. Dev Dyn 2005;234:190-200.
Amano T, Kwak O, Fu L, Marshak A, Shi Y-B. The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain. Cell Res 2005;15:150-159.
Fu L, Ishizuya-Oka A, Buchholz DR, Amano T, Shi Y-B. A causative role of stromelysin-3 in ECM remodeling and epithelial apoptosis during intestinal metamorphosis in Xenopus laevis. J Biol Chem 2005;280:27856-27865.
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. New York: John Wiley, 2004;123-141.
Wei L, Shi Y-B. Matrix metalloproteinase stromelysin-3 in development and pathogenesis. Histol Histopathol 2005;20:177-185.
1Akihiro Tomita, MD, PhD, former Visiting Fellow
Collaborator
Atsuko Ishizuya-Oka, PhD, Nippon Medical School, Tokyo, Japan
For further information, contact shi@helix.nih.gov.