Molecular mechanism of thyroid hormone receptor function during metamorphosis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

DISCOVERED A UNIQUE ROLE OF TR IN REGULATING NOTOCHORD RESORPTION DURING XENOPUS METAMORPHOSIS. Tail resorption during anuran metamorphosis is perhaps the most dramatic tissue transformation that occurs during vertebrate development. Like all other process during metamorphosis, tail resorption is controlled by TH. Earlier studies in highly related anuran species Xenopus laevis and Xenopus tropicalis have shown that TR plays a necessary and essential role for metamorphosis. Of the two known TR genes in all vertebrates, TR is highly expressed during both premetamorphosis and metamorphosis while TR expression is low in premetamorphic tadpoles but highly upregulated as a direct target gene of TH during metamorphosis. In addition, the two TR genes have different temporal regulation patterns in different organs during development. These suggest that the two TRs have different functions during metamorphosis. Indeed, gene knockout studies by us and others have shown that TR is not essential for metamorphosis but controls metamorphic timing and rate of metamorphosis progression during early metamorphosis. TR knockout, however, has no effect on metamorphic timing or early metamorphosis, but significantly delays late metamorphosis, particularly tail resorption. Homozygous TR knockout tadpoles become tailed frogs well after sibling wild type ones complete metamorphosis. Most noticeably, in TR-knockout tadpoles, an apparently normal notochord is present in the tail as late as 3 days after the initiation of tail shortening (stage 62), while in wild-type and TR-knockout tadpoles, the tail notochord disappears in about 1 day. We have investigated how tail notochord resorption is regulated by TR. We show that TR is selectively very highly expressed in the notochord compared to TR. We have also discovered differential regulation of several matrix metalloproteinases (MMPs), which are known to be upregulated by TH and implicated to play a role in tissue resorption by degrading the extracellular matrix. In particular, MMP9-TH and MMP13 are extremely highly expressed in the notochord compared to the rest of the tail. In situ hybridization analyses show that these MMPs are expressed in the outer sheath cells and/or the connective tissue sheath surrounding the notochord. Our findings suggest that high levels of TR expression in the notochord specifically upregulate these MMPs, which in turn degrades the ECM, leading to the collapse of the notochord and its subsequent resorption during metamorphosis. REVEALED THE GENE EXPRESSION PROGRAM UNDERLYING TAIL RESORPTION DURING THYROID HORMONE-DEPENDENT METAMORPHOSIS OF THE ORNAMENTED PYGMY FROG MICROHYLA FISSIPES. Studies on amphibian metamorphosis have been largely focused on the two highly related species Xenopus laevis and Xenopus tropicalis. However, adult X. laevis and X. tropicalis animals remain aquatic. This contrasts with most other anurans that truly changes from being aquatic to terrestrial during metamorphosis, which more closely mimics the postembryonic development in mammals. This makes important to study metamorphosis in a truly terrestrial frog species. In this regard, the anuran Microhyla fissipes offers a number of advantages as an alternative model for developmental and genetic studies. We have made use of the advances in sequencing technologies to investigate the gene regulation profiles underlying the tail resorption program during metamorphosis in M. fissipes. We first used single molecule real-time (SMRT) sequencing to obtain 67, 939 expressed transcripts in M. fissipes. We next identified 4,555 differentially expressed transcripts (DETs) during tail resorption by using Illumina sequencing on RNA samples from tails at different metamorphic stages. Bioinformatics analyses revealed that a number of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and Gene Ontology (GO) terms associated with tail resorption were enriched. Our findings suggest that tail resorption during M. fissipes and X. laevis shares many of the same programs. Future investigations on function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian tail resorption and adaptive evolution from aquatic to terrestrial life. Furthermore, analysis of the M. fissipes model, especially, on the changes in other organs associated with the transition from aquatic to terrestrial living, should help to reveal important mechanistic insights governing mammalian postembryonic developments.