CYTOLOGICAL AND MORPHOLOGICAL ANALYSES REVEAL DISTINCT FEATURES OF INTESTINAL DEVELOPMENT DURING XENOPUS TROPICALIS METAMORPHOSIS. We have been using intestinal remodeling in Xenopus laevis as a model to study how TH regulates metamorphosis via gene regulation by the heterodimers between TR and RXR (9-cis retinoic acid receptor). Recent advances in genome sequencing and genetic tools for functional studies in vivo have made Xenopus tropicalis a superior model for many studies. To establish X. tropicalis intestinal metamorphosis as a model for adult organogenesis, we analyzed the morphological and cytological changes in X. tropicalis intestine during metamorphosis. We observed that in X. tropicalis, the premetamorphic intestine was made of mainly a monolayer of larval epithelial cells surrounded by little connective tissue except in the single epithelial fold, the typhlosole. During metamorphosis, the larval epithelium degenerates and adult epithelium develops to form a multi-folded structure with elaborate connective tissue and muscles. Interestingly, typhlosole, which is critical for adult epithelial development, is present along the entire length of the small intestine in premetamorphic tadpoles, in contrast to X. laevis, where it is present only in the anterior 1/3. TH-treatment induces intestinal remodeling, including the shortening of the intestine and the typhlosole, just like in X. laevis. Our observations indicate that the intestine undergoes similar metamorphic changes the two related species, making it possible to use the large amount of molecular information available on X. laevis intestinal metamorphosis and the genome sequence information and genetic advantages of X. tropicalis to dissect the pathways governing adult intestinal development. GENOME-WIDE ANALYSIS IDENTIFIED DIRECT TARGET GENES OF TR DURING INTESTINAL REMODELING. The direct target genes of TR are the participants immediately downstream of TH signal in the TH-induced metamorphosis. To identify such genes, we have carried out a chromatin-immunoprecipitation (ChIP)-on-chip study on the intestine of premetamorphic X. tropicalis tadpoles treated with or without TH by using a set of microarray chips covering a 8 kb region flanking each putative promoter of 17000 Xenopus tropicalis genes to look for genes bound by TR. Preliminary analyses of the ChIP-on-chip data led to the identification of many putative TR target genes. Among them are the genes encoding iodotyrosine deiodinase (IYD) and Dot1L (Dot1-Like). Dot1L is the homolog of yeast Dot1 gene, originally identified as a disruptor of telomeric silencing in Saccharomyces cerevisiae, and belongs to the family of lysine methyltransferases. To date, Dot1L is the only known methyltransferases that possesses histone methyltransferase activity toward histone H3 lysine (K) 79. IYD is believed to be involved in the recycling of iodine from the byproducts of TH biosynthesis and metabolism: 3-monoiodotyrosine and 3, 5-diiodotyrosine. Our further analyses of these two genes have not only confirmed the binding of TR to their promoter regions in vivo but also revealed the binding sites responsible for their strong induction by TH. Furthermore, expression studies supported their likely role in intestinal metamorphosis. Thus, it is quite likely the other candidate genes identified from the ChIP-on-chip assay are true target genes of TR and participate in metamorphosis as well.

Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2013
Total Cost
$399,475
Indirect Cost
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