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Molecular Mechanism of Frog Metamorphosis

Yun-Bo Shi, PhD, Head, Section on Molecular Morphogenesis
Biswajit Das, PhD, Research Fellow
Liezhen Fu, PhD, Staff Scientist
Kenta Fujimoto, PhD, Visiting Fellow
Rachel Heimeier, PhD, Visiting Fellow
Smita Mathew, PhD, Visiting Fellow
Hiroki Matsuda, PhD, Visiting Fellow
Kazuo Matsuura, MD, PhD, Visiting Fellow
Job Sterling, BS, Postbaccalaureate Fellow
Guihong Sun, PhD, Visiting Fellow

This laboratory investigates the molecular mechanisms of amphibian metamorphosis. The control of this developmental process by thyroid hormone (TH) offers a unique paradigm 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 of primarily a single layer of larval epithelial cells. During metamorphosis, it is transformed, through specific cell death and selective cell proliferation and differentiation, into an organ with 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 to (1) study the developmental function of TH receptors (TRs) and the underlying mechanisms in vivo and (2) identify and functionally characterize genes that are critical for postembryonic organ development in vertebrates.

Roles of cofactors in gene regulation by TR

Based on TR expression profiles and TR's molecular properties, we previously proposed a dual function model for TR during frog development; that is, the heterodimers between TR and RXR (9-cis retinoic acid receptor) bind to target genes 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, either from endogenous synthesis during development or by exogenous addition to the rearing water of premetamorphic tadpoles, TR/RXR heterodimers activate TH-inducible genes to initiate metamorphosis. Our studies over the past several years have shown that TR is both necessary and sufficient for the metamorphic effects of TH. Thus metamorphosis provides the first demonstration that TR mediates directly and sufficiently the developmental effects of TH. Furthermore, we showed that TR regulates metamorphic timing by recruiting corepressor complexes to target genes in premetamorphic tadpoles to prevent precocious metamorphosis. During metamorphosis, TR needs to recruit coactivator complexes containing SRC3 (steroid receptor coactivator 3)/p300 to target genes for their activation and metamorphosis. These findings demonstrate that specific cofactor complexes play critical roles in the developmental function of a nuclear receptor in vertebrates.

The SRC/p300 complexes also contain the methyltransferase PRMT1, which has been implicated in TR function in mammalian cell culture studies. Thus, to further investigate the role and mechanisms of the SRC/p300 complexes in development, we cloned and characterized Xenopus laevis PRMT1. By using intestinal remodeling during Xenopus laevis metamorphosis for in vivo molecular analysis, we showed that PRMT1 expression is upregulated during metamorphosis when both TR and TH are present. We then demonstrated a role of PRMT1 in TR-mediated transcription by showing that PRMT1 enhances transcriptional activation by liganded TR in the frog oocyte transcription system and is recruited to the TH response element (TRE) of the target promoter in the oocyte as well as to endogenous TREs during frog metamorphosis. Surprisingly, we found that PRMT1 is only transiently recruited to the TREs in the target during metamorphosis and observed no PRMT1 recruitment to TREs at the climax of intestinal remodeling when both PRMT1 and TH are at peak levels. With regard to mechanism, we showed that overexpression of PRMT1 enhances TR binding to TREs both in the frog oocyte model system and during metamorphosis. More importantly, transgenic overexpression of PRMT1 enhanced gene activation in vivo and accelerated both natural and TH-induced metamorphosis. These results thus indicate that PRMT1 functions transiently as a coactivator in TR-mediated transcription by enhancing TR-TRE binding and further suggest that PRMT1 has tissue-specific roles in the regulation of the rate of metamorphosis.

Regulation and function of matrix metalloproteinases during frog metamorphosis

Matrix metalloproteinases (MMPs) are Zn²⁺-dependent proteases capable of cleaving various proteins of the ECM (extracellular matrix). The ECM not only provides the 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 and in diverse pathological conditions such as arthritis, wound healing, and cancer metastasis. However, it has been difficult to investigate the roles of MMPs in mammals due to the lack of good models and the relatively subtle phenotypes in limited tissues in knockout mice. Metamorphosis in frogs such as the South African frog (toad) Xenopus laevis 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 the associated mechanisms during development. First, the tadpole's organization is simple and its metamorphic transformations have been well characterized. The intestine in an herbivorous tadpole is very long but structurally simple, consisting predominantly of a single layer of epithelial cells. During metamorphosis, the larval epithelial cells undergo degeneration through programmed cell death or apoptosis. Concurrently, cells of the adult epithelium, connective tissue, and muscles proliferate. Eventually, the adult cells differentiate to form a multi-folded epithelium surrounded by elaborate connective tissue and muscle, thus producing an organ resembling mammalian adult intestine. Second, it has been well documented that the ECM that separates the epithelium from the connective tissue, i.e., the basal lamina, undergoes drastic remodeling during metamorphosis. Third, we and others have shown that intestinal remodeling can be reproduced in organ cultures or even in primary cell cultures, making it easy to manipulate the ECM and its remodeling. Finally, a number of MMP genes have been shown to be upregulated by TH during intestinal remodeling in Xenopus laevis. Our long-term goal is to investigate whether and how these MMPs function in tissue remodeling during metamorphosis.

Genome-wide identification of Xenopus matrix metalloproteinases

Our earlier expression studies and those of others found that most of the MMPs that have been analyzed are regulated either directly or indirectly by TH in at least some organs/tissues to varying degrees. Thus, it is of interest to determine whether other MMPs are also regulated by TH and whether various MMPs have distinct functions during metamorphosis in different organs/tissues. To find out, we carried out a genome-wide analysis of MMP genes in both X. laevis and the closely related species X. tropicalis using a bioinformatic approach by making use of the genome sequence information for X. tropicalis and cDNA sequences available for X. laevis and tropicalis genes from the NIH Frog Initiatives Program. We examined X. laevis and X. tropicalis ESTs and genomic sequences for MMPs and obtained likely homologs for 20 out of the 25 MMPs known in higher vertebrates. Four of the five missing MMPs, i.e., MMP8, MMP10, MMP12, and MMP27, were all encoded on human chromosome 1 and the other missing MMP, MMP22 (a chicken MMP), was also absent from the human genome. In addition, we discovered several novel MMPs that appear to be derived from unique duplications over evolution and are present in the genomes of both Xenopus species. Our results suggest that MMP genes undergo dynamic changes during evolution. It will be of interest to investigate whether MMP expression and functions during vertebrate development are conserved. The sequence information reported here should facilitate such an endeavor in the near future.

Differential regulation of cell type specific apoptosis by stromelysin-3

The MMP stromelysin-3 (ST3) is induced by TH and its expression correlates with cell death during metamorphosis. We previously showed that ST3 is both necessary and sufficient for larval epithelial cell death in the remodeling intestine. To investigate the roles of ST3 in other organs and especially in different cell types, we analyzed the effect of transgenic overexpression of ST3 in the tail of premetamorphic tadpoles. We observed that ST3 expression, in the absence of T3, caused significant muscle cell death in the tail of premetamorphic transgenic tadpoles. However, only relatively low levels of epidermal cell death were induced by precocious ST3 expression in the tail in contrast to what takes place during natural and T3-induced metamorphosis. This cell type–specific apoptotic response to ST3 in the tail suggests distinct mechanisms regulating cell death in different tissues. Furthermore, our analyses of the laminin receptor, an in vivo substrate of ST3 in the intestine, suggest that laminin receptor cleavage may be an underlying mechanism for the cell type–specific effects of ST3.

Analysis of the gene expression programs underlying the temporal and tissue-dependent transformations during metamorphosis

The complexity of metamorphic changes in different organs argues for the presence of distinct gene regulation programs regulated by TR. Knowledge of this systematic gene regulation will help identify not only molecular markers but also important cellular pathways or critical genes for future mechanistic studies. Thus, we have begun to use the recently developed Xenopus laevis cDNA array to analyze genome-wide gene expression changes associated with TH-induced intestinal remodeling. Our initial analysis of animals treated with TH for varying times has provided a molecular description of the gene regulation pathways associated with various metamorphic processes in the intestine.

The success of this study also prompted us to ask whether we could use metamorphosis coupled with cDNA array analysis to study whether endocrine-disrupting compounds (EDCs) can affect vertebrate development via the TH signaling pathway. EDCs are exogenous substances that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism, its progeny, or in (sub) populations. Given that TH plays a central role in vertebrate development, growth, and metabolism, adverse effects of EDCs on TH signaling would undoubtedly pose a threat to human and wildlife health. However, the lack of a suitable in vivo model to study EDCs’ effects on TR function in vertebrate development impedes our understanding on whether and how persistent exposure to these bioaccumulative compounds affects human health. As a test case, we analyzed the effect of bisphenol A (BPA) on Xenopus metamorphosis. BPA, a chemical widely used in the manufacture of plastics, is estrogenic and capable of disrupting sex differentiation. Recent in vitro studies have shown that BPA can also antagonize TH activation of TRs. The difficulty in studying uterus-enclosed mammalian embryos has hampered analysis of the direct effects of BPA during vertebrate development. We thus studied the effect of BPA on TH-dependent metamorphosis at both the morphological and molecular level. After four days of exposure, BPA inhibited TH-induced intestinal remodeling in premetamorphic Xenopus laevis tadpoles. Importantly, microarray analysis revealed that BPA antagonized the regulation of most TH-response genes, thereby explaining the inhibitory effect of BPA on metamorphosis. Surprisingly, most of the genes affected by BPA in the presence of TH were TH-response genes, suggesting that BPA predominantly affected TH-signaling pathways during metamorphosis. Our finding that this endocrine disruptor, well known for its estrogenic activity in vitro, functions to inhibit TH-pathways that affect vertebrate development in vivo thus not only provides a mechanism for the likely deleterious effects of BPA on human development but also demonstrates the importance of studying endocrine disruption in a developmental context in vivo.

Regulation of stem cell development during tissue remodeling

Adult stem cells are essential for the development of adult organs and tissue repair and regeneration. The intestine is an excellent organ for studying adult stem cells as the vertebrate intestinal epithelium—the tissue responsible for the food processing and nutrient absorption—is continuously renewed through stem cell division followed by cell differentiation and eventual death of the differentiated cells throughout adult life. Intestinal remodeling during TH-dependent metamorphosis of the Xenopus laevis offers a unique opportunity to study adult stem cell development and proliferation because of the advantages and properties mentioned in the section on MMPs. In addition, while adult epithelium develops de novo during metamorphosis, there are no identifiable stem cells for the adult intestinal epithelium in premetamorphic tadpoles; intestinal metamorphosis thus also offers an opportunity to study the development of adult stem cells. As a first step, we recently determined the origin of the adult intestinal stem cells. For this purpose, we made use of transgenic Xenopus tadpoles expressing GFP for recombinant organ cultures. The larval epithelium isolated from the wild-type or GFP transgenic intestines before metamorphic climax was recombined with homologous and heterologous non-epithelial tissues and cultivated in the presence of TH. In all kinds of recombinant intestines, adult progenitor cells expressing markers for intestinal stem cells such as sonic hedgehog became detectable and then differentiated into the adult epithelium expressing intestinal fatty acid binding protein, a marker for absorptive cells. Importantly, whenever the epithelium was derived from transgenic intestine, both the adult progenitor/stem cells and their differentiated cells expressed GFP, while neither expressed GFP in the wild-type–derived epithelium. Our results have thus provided direct evidence that stem cells that generate the adult intestinal epithelium originate from the larval epithelium, presumably through TH-induced dedifferentiation of larval epithelial cells.

Additional Funding

JSPS (Japan Society for the Promotion of Science) fellowship (2008) to Dr. Kenta Fujimoto (ongoing)

Publications

Fu L, Das B, Mathew S, Shi Y-B. Genome-wide identification of Xenopus matrix metalloproteinases: conservation and unique duplications in amphibians. BMC Genomics 2009 10:81.
Heimeier RA, Das B, Buchholz DR, Shi Y-B. The xenoestrogen bisphenol A inhibits postembryonic vertebrate development by antagonizing gene regulation by thyroid hormone. Endocrinology 2009 150:2964-2973.
Ishizuya-Oka A, Hasebe T, Buchholz DR, Kajita M, Fu L, Shi Y-B. The origin of the adult intestinal stem cells induced by thyroid hormone in Xenopus laevis. FASEB J 2009 23:2568-2575.
Mathew S, Fu L, Fiorentino M, Matsuda H, Das B, Shi Y-B. Differential regulation of cell type specific apoptosis by stromelysin-3: A potential mechanism via the cleavage of the laminin receptor during tail resorption in Xenopus laevis. J Biol Chem 2009 284:18545-18556.
Matsuda H, Paul BD, Choi CY, Hasebe T, Shi Y-B. Novel functions of protein arginine methyltransferase 1 in thyroid hormone receptor-mediated transcription and in the regulation of metamorphic rate in Xenopus laevis. Mol Cell Biol 2009 29:745-757.

Collaborator

Atsuko Ishizuya-Oka, PhD, Nippon Medical School, Tokyo, Japan

Contact

For more information, email shi@helix.nih.gov or visit smm.nichd.nih.gov.

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