MOLECULAR MECHANISM OF FROG METAMORPHOSIS
, Head, Section on Molecular Morphogenesis
Biswajit Das, PhD, Research Fellow
Liezhen Fu, PhD, Staff Scientist
Maria Rosaria Fiorentino, PhD, Visiting Fellow
Rachel Heimeier, PhD, Visiting Fellow
Smita Mathew, PhD, Visiting Fellow
Hiroki Matsuda, PhD, Visiting Fellow
Xuedong Wang, PhD, Visiting Fellow
Teresa Washington, PhD, Postdoctoral Fellow
Alexis Oetting, BS, Postbaccalaureate Fellow
Mechanism and function of TR during development
We have proposed a dual-function model for TR during frog development; the model posits that the heterodimers between TR and RXR (9-cis retinoic acid receptor) bind to target genes constitutively in vivo. 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. By using a transgenic approach, we had previously shown that TR is both necessary and sufficient for the metamorphic effects of TH; the non-genomic action of TH, while it exists, plays a minor role, if any, during this postembryonic process. Thus, metamorphosis provides the first known example of TR directly and sufficiently mediating the developmental effects of TH in individual organs by regulating target gene expression in these organs.
To investigate whether unliganded TR also plays a role in premetamorphic development by repressing TH target gene expression, as predicted by our dual-function model, we designed a dominant negative form of the TR-binding nuclear receptor corepressor (dnN-CoR), which contains only the receptor-interacting domain of N-CoR. We first confirmed its dominant negative activity by showing that dnN-CoR competes away the binding of endogenous N-CoR to unliganded TR and relieves unliganded TR–induced gene repression in the frog oocyte transcription system. Next, we overexpressed dnN-CoR under the control of the heat shock–inducible promoter in tadpoles through transgenesis and analyzed its effect on gene expression and development. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed significant derepression of TH-response genes in transgenic animals. More important, transgenic tadpoles developed faster than wild-type siblings, with an acceleration that shortened the 30-day experiment by as much as seven days. These data, which provide in vivo evidence of the presence and role of unliganded TR-induced gene repression in physiological settings, strongly support our dual-function model, which hypothesized that unliganded TR represses TH-response genes in premetamorphic tadpoles to regulate the progress of development.
In addition, changes in the metamorphic complexity of various organs and even in the tissues of a single organ argue for the presence of diverse gene regulation programs under the control of TR. While many genes regulated by TH have been isolated through subtractive screens and other means over the years, these limited analyses revealed little information about whether or how genes associated with various cellular processes, such as cell cycle regulation and apoptosis, are coordinately regulated during metamorphic transformations in different tissues. Knowledge of systematic gene regulation will help identify not only molecular markers but also important cellular pathways or critical genes and thus facilitate future mechanistic studies of metamorphic processes. Recent advances in Xenopus genomic tools make it possible to probe the gene regulation profiles associated with metamorphic processes. First, we used a commercial large-oligo (60-mer) array for Xenopus laevis in order to analyze genome-wide gene expression changes associated with TH-induced intestinal remodeling. We isolated RNA samples from premetamorphic Xenopus laevis tadpole intestines after 0, 1, 3, and 6 days of TH treatment, which induced successive cell death and proliferation essential for intestinal remodeling. Using the set of 21,807 60-mer oligonucleotide probes representing over 98 percent of the Unigene clusters, we found that 1,997 genes were differentially regulated 1.5-fold or more during treatment. They were clustered into four temporal expression profiles: transiently up- or downregulated and late up- or downregulated. Gene Ontology (GO) categories most significantly associated with the clusters included proteolysis, cell cycle, development, and transcription on the one hand, and electron transport and metabolism on the other. Importantly, temporal changes in the GO categories correlated well with tissue transformations. For example, proteolysis genes were upregulated while cell cycle genes were downregulated during early stages of intestinal remodeling when cell death was the predominant event. Later, as cell proliferation occurred, the cell cycle genes were upregulated. Our findings provide not only a molecular description of the gene regulation pathways associated with different metamorphic processes but also a framework for future analysis of the molecular mechanisms governing intestinal metamorphosis.
Buchholz DR, Heimeier RA, Das B, Washington T, Shi Y-B. Pairing morphology with gene expression in thyroid hormone-induced intestinal remodeling and identification of a core set of TH-induced genes across tadpole tissues. Dev Biol 2006;303:576-90.
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 2006;145:1-19.
Buchholz DR, Paul BD, Shi Y-B. Gene-specific changes in promoter occupancy by thyroid hormone receptor during frog metamorphosis: implications for developmental gene regulation. J Biol Chem 2005;280:41222-8.
Sato Y, Buchholz DR, Paul BD, Shi Y-B. A role of unliganded thyroid hormone receptor in premetamorphic development in Xenopus laevis. Mech Dev 2007;124:476-88.
Stewart D, Tomita A, Shi Y-B, Wong J. Chromatin immunoprecipitation for studying transcriptional regulation in Xenopus oocytes and tadpoles. Methods Mol Biol 2006;322:165-81.
Roles of cofactors in gene regulation
TR regulates gene transcription by recruiting cofactors to target genes. In the presence of TH, TR can bind to coactivators while unliganded TR binds to corepressors. Even though many biochemical and molecular studies have been carried out on cofactors, little is known about whether and how they participate in gene regulation by TR in different biological processes in vivo. We therefore investigate how TR uses different cofactors in the context of development in various organs.
Among corepressors, we have studied the role of N-CoR and SMRT (silencing mediator of retinoid and TRs) in gene repression by TR in premetamorphic tadpoles. We showed previously that both are expressed and, more important, bound to TH-response genes in premetamorphic tadpoles. Furthermore, they are released from the target genes upon TH treatment of premetamorphic tadpoles or during natural metamorphosis. Our studies with dnN-CoR, as summarized above, demonstrated in vivo a role of corepressor complexes in gene repression by unliganded TR during premetamorphic tadpole development.
To investigate coactivation, we used the chromatin immunoprecipitation (ChIP) assay to show that, upon TH treatment of premetamorphic tadpoles and during natural metamorphosis, TR recruits Xenopus coactivator SRC3 (steroid receptor coactivator 3) to target genes in a gene- and tissue-dependent manner. Furthermore, we generated transgenic tadpoles expressing a dominant negative form of SRC3 (F-dnSRC3) and observed that the 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. Our studies thus demonstrated that coactivator recruitment, aside from corepressor release, is required for TH function in development. We have shown, for the first time, that a gene-regulation pathway underlying specific developmental events and controlled by a nuclear receptor is coactivator-dependent.
The above studies demonstrated the importance of coactivators in TR function during metamorphosis; however, there are several other TR-binding coactivators. The F-dnSRC3 in the above study would block the binding to TR of not only SRC3 (and the highly related cofactors SRC1 and SRC2) but also that of other coactivators. Thus, it remains unclear whether SRCs (SRC1–3) play an essential role in metamorphosis. To explore such a possibility, we analyzed the in vivo role of the histone acetyltransferase p300, which is a component of SRC-coactivator complexes. A ChIP assay revealed that p300 is recruited to TH-responsive promoters, thereby implicating p300 in TR function. Furthermore, we generated a dominant negative form of p300, F-dnp300, that contained only the SRC-interacting domain of p300 and thus would interfere with the function of only SRCs but not that of other TR-binding coactivators. Transgenic tadpoles overexpressing F-dnp300 also displayed arrested or delayed metamorphosis. Molecular analyses of the transgenic animals showed that TR recruited F-dnp300, displaced endogenous p300, and inhibited the expression of TH-responsive genes. Our results, which suggest that p300 and/or its related protein CBP is an essential component of the TR-signaling pathway in vivo, support the notion that p300/CBP and SRC proteins are part of the same critical coactivator complexes in vivo during postembryonic development.
The SRC/p300 complexes also contain methyltransferases, one of which, CARM1, has been implicated in TR function in mammalian cell culture studies. To investigate further the role and mechanisms of the SRC/p300 complexes in development, we cloned the Xenopus CARM1 and obtained two alternative splicing forms, CARM1a and CARM1b. Both isoforms are expressed throughout metamorphosis, consistent with a role during development. Surprisingly, transcriptional analysis in Xenopus oocyte revealed that overexpression of CARM1b had little effect on TR-mediated transcription, although, as expected, CARM1a enhanced gene activation by liganded TR. ChIP assays showed that liganded TR recruited both endogenous CARM1a and overexpressed CARM1a and CARM1b to a target promoter. However, less liganded TR bound to the target promoter when CARM1b was overexpressed—overexpression that was accompanied by a slight reduction in histone methylation at the promoter. These results suggest that CARM1 may play a role in TR-mediated transcriptional regulation during frog development and that alternative splicing regulates the enzyme’s function.
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. CRC Press, 2006;305-19.
Matsuda H, Paul BD, Choi CY, Shi Y-B. Contrasting effects of two alternative splicing forms of coactivator-associated arginine methyltransferase 1 on thyroid hormone receptor-mediated transcription in Xenopus laevis. Mol Endocrinol 2007;21:1082-94.
Paul BD, Buchholz DR, Fu L, Shi Y-B. SRC-p300 coactivator complex is required for thyroid hormone induced amphibian metamorphosis. J Biol Chem 2007;282:7472-81.
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;280:27165-72 (JBC Paper of the Week).
Paul BD, Fu L, Buchholz DR, Shi Y-B. Coactivator recruitment is essential for liganded thyroid hormone receptor to initiate amphibian metamorphosis. Mol Cell Biol 2005;25:5712-24.
Regulation and function of the matrix metalloproteinase stromelysin-3 during TH-induced tissue remodeling
We previously identified several TH-response genes encoding metalloproteinases (MMPs) during intestinal metamorphosis. MMPs are Zn2+-dependent proteases capable of cleaving various proteins of the extracellular matrix (ECM). The ECM not only provides essential physical support within an organism but also influences cell fate during developmental and pathological processes. Upregulation of MMP genes has been observed in many developmental processes such as limb morphogenesis as well as in diverse pathological conditions such as arthritis, wound healing, and metastasis. However, it has been difficult to investigate the roles of MMPs in mammals owing to the lack of good models and the relatively subtle phenotypes in limited tissues in knockout mice. The activation of MMP genes by TH and the observed ECM remodeling during metamorphosis suggest that MMPs participate through ECM remodeling. Frog metamorphosis affects essentially all organs in a tadpole within a short developmental period and thus offers an excellent opportunity to investigate the in vivo functions of MMPs and associated mechanisms during development.
Expression and organ culture studies have led us to propose that, in the metamorphosing intestine, the MMP stromelysin-3 (ST3) is directly or indirectly involved in ECM remodeling, which in turn influences cell behavior. Transgenic studies have now shown that precocious overexpression of ST3, rather than a catalytically inactive mutant, leads to premature apoptosis in the intestinal epithelium of premetamorphic tadpoles, complementing our organ culture studies, which show a requirement for ST3 in TH-induced apoptosis during intestinal metamorphosis.
To understand the mechanism by which ST3 affects tissue remodeling, we used a yeast two-hybrid screen to isolate the 37 kd laminin receptor (LR)—a cell surface receptor for the ECM protein laminin—as a likely substrate for the enzyme. LR binds to ST3 and can be cleaved by ST3 at two sites in vitro. Through peptide sequencing, we determined that the two cleavage sites are located in the extracellular domain between the transmembrane domain and laminin binding sequence. Other MMPs cleave LR in the region that is C-terminal to the laminin-binding sequence, suggesting that LR cleavage solely by ST3 alters cell-ECM interaction. Expression studies showed that LR is expressed in the intestinal epithelium of premetamorphic tadpoles. During intestinal metamorphosis, LR is downregulated in the apoptotic epithelium and concurrently upregulated in the connective tissue; however, it exhibits little expression in the proliferating adult epithelium. Toward the end of metamorphosis, adult epithelial cells begin to express LR as they differentiate. Furthermore, LR is cleaved during intestinal remodeling, when ST3 is highly expressed, or in intestine of premetamorphic transgenic tadpoles overexpressing ST3. These 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, and 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, thereby mediating the effects of ST3 on cell fate and behavior during development and pathogenesis.
Earlier studies have shown that the ST3 gene is upregulated within hours of TH treatment and that its upregulation occurs even in the presence of protein synthesis inhibitors. Given that temporal regulation of MMP expression is an important factor in determining the function of MMPs during tissue remodeling, we have been interested in determining if ST3 is indeed directly regulated by TR at the transcriptional level. We have now identified a strong TH response element (TRE) in the first intron of the Xenopus ST3 gene. TR/RXR heterodimers bind to TRE both in vitro and in vivo. Furthermore, TH induces strong activation of the promoter through the intronic TRE. Surprisingly, while the unliganded TR/RXR was able to recruit corepressors to the promoter, it had little repressive effect on the promoter in vivo. The results show that ST3 is a direct target of TH and that the intronic TRE mediates the inductive effect of TH. The results also suggest that promoter context plays an important role in gene repression by unliganded TR.
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;15:150-9.
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;280:27856-65.
Fu L, Tomita A, Wang H, Buchholz DR, Shi Y-B. Transcriptional regulation of the Xenopus laevis stromelysin-3 gene by thyroid hormone is mediated by a DNA element in the first intron. J Biol Chem 2006;281:16870-8.
Ishizuya-Oka A, Shi Y-B. Regulation of adult intestinal epithelial stem cell development by thyroid hormone during Xenopus laevis metamorphosis. Dev Dyn 2007;236:3358-68.
A cooperative role of gelatinase A (GelA) and membrane type-1-MMP (MT1-MMP) during Xenopus laevis development
Amano,5 Hasebe,6 Shi; in collaboration with Ishizuya-Oka
We and others previously reported that, in addition to the MMP gene encoding ST3, several other MMP genes are activated during amphibian metamorphosis. Our findings are consistent with the complex nature of the ECM, which would require many enzymes for its remodeling and degradation during metamorphosis. Interestingly, our earlier Northern blot analysis suggested that, unlike ST3, the MMP GelA appeared to be a late TH-response gene in both the tail and intestine of Xenopus laevis during metamorphosis, suggesting that this MMP plays a different role than does ST3. To investigate the function of GelA, we analyzed its detailed spatial and temporal expression profiles. In addition, we cloned and analyzed Xenopus MT1-MMP, whose mammalian homologue has been shown to participate in the activation of pro-GelA to the active or mature GelA lacking the pro-peptide. We showed that both GelA and MT1-MMP are upregulated in the intestine and tail during metamorphosis and coexpressed in the connective tissues of both organs during natural and TH-induced metamorphosis. In addition, MT1-MMP, but not GelA, is expressed in the longitudinal muscle of the metamorphosing intestine. These results suggest that GelA and MT1-MMP function together in ECM degradation or remodeling during metamorphosis and that MT1-MMP plays additional GelA-independent roles, at least in the development of the adult longitudinal muscle in the intestine.
To investigate the possible cooperative role of the two MMPs, we turned to Xenopus embryogenesis as a model and showed that Xenopus GelA and MT1-MMP are coexpressed during embryogenesis. Co-immunoprecipitation studies indicated that they interact with each other in vivo; we also showed that overexpression of GelA and MT1-MMP together in Xenopus embryos led to the activation of pro–GelA. Furthermore, overexpression of both MMPs caused developmental abnormalities and embryonic death by a mechanism that required the MMPs’ catalytic activity. More importantly, coexpression of wild-type GelA and MT1-MMP led to a cooperative effect on embryonic development that was abolished when the catalytic activity of either MMP was eliminated. Thus, our studies support a cooperative role of the two MMPs during development, at least in part through the activation of pro–GelA by MT1-MMP.
Hasebe T, Fu L, Amano T, Shi Y-B. Evidence for a cooperative role of gelatinase A and membrane type-1 matrix metalloproteinase during Xenopus laevis development. Mech Dev 2007;124:11-22.
Hasebe T, Hartman R, Matsuda H, Shi Y-B. Spatial and temporal expression profiles suggest the involvement of gelatinase A and membrane type 1 matrix metalloproteinase in amphibian metamorphosis. Cell Tissue Res 2006;324:105-16.
Shi Y-B, Fu L, Hasebe T, Ishizuya-Oka A. Regulation of ECM remodeling and cell fate determination by matrix metalloproteinase stromelysin-3 during thyroid hormone-dependent postembryonic development. Pharmacol Ther 2007;116:391-400.
1 Daniel Buchholz, PhD, former Postdoctoral Fellow2 Yukiyasu Sato, MD, PhD, former Visiting Fellow
3 Choel Yong Choi, PhD, former Visiting Fellow
4 Bindu Diana Paul, PhD, former Visiting Fellow
5 Tozikazu Amano, PhD, former Visiting Fellow
6 Takashi Hasebe, PhD, former Visiting Fellow
COLLABORATOR
Atsuko Ishizuya-Oka, PhD, Nippon Medical School, Kawasaki, Japan
For further information, contact shi@helix.nih.gov.
