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Molecular Mechanism of Frog Metamorphosis

Yun-Bo Shi, PhD
  • Yun-Bo Shi, PhD, Head, Section on Molecular Morphogenesis
  • Biswajit Das, PhD, Research Fellow
  • Liezhen Fu, PhD, Staff Scientist
  • Kenta Fujimoto, PhD, Visiting Fellow
  • Smita Mathew, PhD, Visiting Fellow
  • Kazuo Matsuura, MD, PhD, Visiting Fellow
  • Job Sterling, BS, Postbaccalaureate Fellow
  • Guihong Sun, PhD, Visiting Fellow
  • Fiona Mitchell, 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, diverse 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 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 consistingprimarilyof 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 critical for postembryonic organ development in vertebrates.

Gene Regulation by TR

Based on TR expression profiles and the receptor's molecular properties, we previously proposed a dual-function model for TR during frog development. We postulated that the heterodimers between TR and RXR (9-cis retinoic acid receptor) bind to target genes in vivo; in premetamorphic tadpoles, they 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 exogenous addition to the rearing water of premetamorphic tadpoles, TR/RXR heterodimers activate TH-inducible genes to initiate metamorphosis. Our studies in the last several years have provided molecular and genetic support for this model. Furthermore, we revealed important roles of corepressor and coactivator complexes in TR action during metamorphosis. To understand the molecular pathways induced by TR during this process, it is critical to identify and characterize the immediate early, direct target genes of T3, i.e., genes regulated by T3 in the absence of new protein synthesis. We took advantage of the ability to easily induce metamorphosis with physiological levels of T3 and to carry out microarray analysis in Xenopus laevisand genome-wide sequence analysis in Xenopus tropicalis. This allowed us to identify 188 genes up-regulated and 249 genes down-regulated by T3 in the absence of new protein synthesis in whole animals. We have provided further evidence to show that these genes contain functional TH response elements (TREs) bound by TR in tadpoles and that their promoters are regulated by TR in vivo. More importantly, gene ontology analysis showed that the direct up-regulated genes are enriched in categories important for transcriptional regulation and protein degradation–dependent signaling processes but not for DNA replication. Our findings thus revealed the existence of interesting pathways induced by T3 at the earliest step of metamorphosis.

Analyzing the gene expression programs underlying temporal transformations during metamorphosis

The complexity of metamorphic changes in diverse organs argues for the presence of different gene regulation programs regulated by TR. Knowledge on 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, a process that involves selective degeneration of the larval epithelium through apoptosis and de novo development of the adult epithelium. Clustering of the expression patterns revealed co-expressed genes involved in essential cell processes such as apoptosis and proliferation. Furthermore, we showed that most of the genes highly induced at metamorphic climax are also upregulated in the mouse intestine around birth, the postembryonic period resembling metamorphosis, supporting conservation of the underlying molecular pathways. Moreover, our genome-wide analysis identified many larval and embryo- and adult-specific genes. Detailed analysis revealed 17 larval stage–specific genes that may represent molecular markers for human colon cancers, while many adult-specific genes are associated with dietary enzymes. This global developmental expression study provides the first detailed molecular description of intestinal remodeling and maturation during postembryonic development, which should help improve our understanding of intestinal organogenesis and human diseases.

Spatio-temporal expression profile of stem cell–associated gene LGR5 in the intestine during thyroid hormone–dependent metamorphosis in Xenopus laevis

The leucine-rich repeat–containing G protein–coupled receptor 5 (LGR5 or Gpr49) is a well-established stem cell marker in the adult mouse intestinal crypt. We have cloned and analyzed the spatiotemporal expression profile of the LGR5 gene during frog metamorphosis. We showed that the two duplicated LGR5 genes in Xenopus laevis and the LGR5 gene in Xenopus tropicalis are highly homologous to the LGR5 in other vertebrates. The expression of LGR5 is induced in the limb, tail, and intestine by TH during metamorphosis. More importantly, LGR5 mRNA is localized to the developing adult epithelial stem cells of the intestine. The results suggest that LGR5-expressing cells are the stem/progenitor cells of the adult intestine and that LGR5 plays a role in the development and/or maintenance of the adult intestinal stem cells during postembryonic development in vertebrates.

An essential and evolutionarily conserved role of protein arginine methyltransferase 1 for adult intestinal stem cells during postembryonic development

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, given that the 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 throughoutvertebrateadult life. Intestinal remodeling during TH-dependent metamorphosis of the South African toad Xenopus laevis offers a unique opportunity to study adult stem cell development and proliferation. In addition, while adult epithelium develops de novo during metamorphosis, there are no identifiable stem cells for the adult intestinal epithelium in premetamorphic tadpoles. Thus, intestinal metamorphosis also offers an opportunity to study the development of adult stem cells. As indicated above, we showed that TR recruits coactivator complexes to control metamorphosis. More recently, we showed that the TR-coactivator PRMT1 (protein arginine methyltransferase 1) is upregulated in a small number of larval epithelial cells and that these cells dedifferentiate to become the adult stem cells. More importantly, our in vivo studies showed that PRMT1 plays an evolutionally conserved role in the development of adult intestinal stem cells. The findings are not only important for the understanding of organ-specific adult stem cell development but also have important implications in regenerative medicine of the digestive tract.

Gene expression atlas for human embryogenesis

In a separate, collaborative cDNA array study, we carried out a genome-wide expression analysis during the fourth–ninth weeks of human embryogenesis, a critical period when most organs develop. About half of all human genes were found to be expressed and 18.6% of the expressed genes were significantly regulated during this important period. We further identified over 5,000 regulated genes, most of which were previously not known to be associated with animal development. This study fills an important gap in mammalian developmental studies by identifying functional pathways involved in this critical but previously unstudied period. It also revealed that the genes involved here are distinct from those expressed during early embryogenesis, which include three groups of maternal genes. Furthermore, we discovered that genes controlling a given developmental process are coordinately regulated. This led us to develop an easily searchable database of this entire collection of gene expression profiles, allowing for the identification new genes important for a particular developmental process/pathway and deducing the potential function of a novel gene. Two examples of our spatiotemporal analyses of the two novel genes demonstrated the validity of the predictions from the database. Such a database should serve as a highly valuable resource for the molecular analysis of human development and pathogenesis.

Distinct function of different MMPs and a requirement for proper levels of MMp activities during development

We previously showed that the MMP stromelysin-3 (ST3) is induced by TH and that its expression correlates with cell death during metamorphosis. We further demonstrated that ST3 is both necessary and sufficient for larval epithelial cell death in the remodeling intestine. On the other hand, ST3 expression, in the absence of T3, causes significant muscle cell death in the tail of premetamorphic transgenic tadpoles, while only relatively low levels of epidermal cell death are induced by precocious ST3 expression in the tail, in contrast to what takes place during natural and T3-induced metamorphosis, when ST3 expression is high. We further provided evidence that these differential effects of ST3 may be mediated by its cleavage of the laminin receptor, an in vivo substrate of ST3. To investigate whether other MMPs also regulate cell fate during metamorphosis, we carried out transgenic studies on another metamorphosis-associated MMP, the membrane-type 1 MMP or MT1-MMP. Interestingly, overexpression of this MMP caused lethality in metamorphosis tadpoles, although little effect on apoptosis has been observed. Thus, different MMPs indeed have distinct roles during metamorphosis.

To inhibit MMP activities in vivo, we carried out a transgenic study in which we overexpressedTIMP2, a tissue inhibitor of MMP, which is also induced during metamorphosis. Surprisingly, overexpression of TIMP2 also caused lethality in metamorphosis tadpoles without affecting apoptosis. On the other hand, crossing the TIMP2 and MT1-MMP transgenic animals rescued this lethal phenotype. Thus, proper balance of MMP activities is critical for metamorphosis.

Additional Funding

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

Publications

  • Das B, Heimeier RA, Buchholz DR, Shi Y-B. Identification of direct thyroid hormone response genes reveals the earliest gene regulation programs during frog metamorphosis. J Biol Chem. 2009;284:34167–34178.
  • 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.
  • Heimeier RA, Das B, Buchholz DR, Fiorentino M, Shi Y-B. Studies on Xenopus laevis intestine reveal biological pathways underlying vertebrate gut adaptation from embryo to adult. Genome Biol. 2010;11:R55, 1-20.
  • Matsuda H, Shi Y-B. An essential and evolutionarily conserved role of protein arginine methyltransferase 1 for adult intestinal stem cells during postembryonic development. Stem Cells. 2010;In press.
  • Yi H, Xue L, Guo M-X, Ma J, Zeng Y, Wang W, Cai J-Y, Hu H-M, Shu H-B, Shi Y-B, Li W-X. Gene expression atlas for human embryogenesis. FASEB J. 2010;24:3341-3350.

Collaborator

  • Atsuko Ishizuya-Oka, PhD, Nippon Medical School, Tokyo, Japan
  • Wenxin Li, PhD, College of Life Sciences, Wuhan University, Wuhan, China

Contact

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

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