DEMONSTRATED THAT ANIMALS LACKING TR COMPLETE METAMOPRHOSIS AROUND THE SAME AGE AS THE WILD TYPE SIBLINGS DO. By using the TALEN technology, we have generated X. tropicalis animals lacking any functional TR and observed that TR knockout animals are surprisingly able to complete metamorphosis at a similar age as the wild typing siblings. Carefully analyses during development, however, revealed that the TR knockout animals initiated metamorphosis at a younger age and with a smaller size but progressed more slowly through metamorphosis. The wild type siblings initiated metamorphosis at an older age but progressed more quickly, eventually completing metamorphosis around the same time as the knockout ones. As the TR knockout animals initiated metamorphosis at a smaller size, they were also smaller at the end of metamorphosis. These findings are consistent with our earlier studies with TR knockdown animals and reveal a critical role of endogenous TR both in mediating the metamorphic effect of TH during metamorphosis and in preventing precocious initiation of metamorphosis when TH is absent. They thus provide direct evidence to support the dual function model for TR in Xenopus development that we proposed over a decade ago based on gene expression and in vitro function studies. DEVELOPED A SIMPLE AND EFFICIENT METHODS TO VISULAIZE AND QUANTIFY THE EFFICIENCY OF CHORMOSOMAL MUTATIONS FROM GENOME EDITING. Genome editing with designer nucleases such as TALEN and CRISPR/Cas enzymes has broad applications. Delivery of these designer nucleases into organisms induces various genetic mutations including deletions, insertions and nucleotide substitutions. Characterizing those mutations is critical for evaluating the efficacy and specificity of targeted genome editing. While a number of methods have been developed to identify the mutations, none other than sequencing allows the identification of the most desired mutations, i.e., out-of-frame insertions/deletions that disrupt genes. During our studies on gene editing in Xenopus development, we developed a simple and efficient method to visualize and quantify the efficiency of genomic mutations induced by gene-editing. Our approach is based on the expression of a two-color fusion protein in a vector that allows the insertion of the edited region in the genome in between the two color moieties. We have shown that our approach not only easily identifies developing animals with desired mutations but also efficiently quantifies the mutation rate in vivo. Furthermore, by using LacZ and GFP as the color moieties, our approach can even eliminate the need for a fluorescent microscope, allowing the analysis with simple bright field visualization. Such an approach will greatly simplify the screen for effective gene-editing enzymes and identify the desired mutant cells/animals. DEVELOPED AN EFFICIENT, SIMPLE, AND NON-INVASIVE PROCEDURE FOR GENOTYPING AQUATIC AND NON-AQUATIC LABORATORY ANIMALS. Various animal models are indispensible in biomedical research. Increasing awareness and regulations have prompted the adaptation of more humane approaches in the use of laboratory animals. With the development of easier and faster methodologies to generate genetically altered animals, convenient and humane methods to genotype these animals are important for research involving such animals. To facilitate genotyping of gene-edited Xenopus animals, we developed skin swabbing as a simple and noninvasive method for extracting genomic DNA from tadpoles and frogs for genotyping. This method is highly reliable and suitable for both immature and adult animals of not only frogs but also mice. Our approach thus allows a simpler and more humane approach for genotyping vertebrate animals. REVEALED THE DIRECT TRANSCRIPTIONAL REGULATION OF HISTIDINE AMMONIA-LYASE 2 GENE BY THYROID HORMONE RECEPTOR IN THE DEVELOPING ADULT INTESTINAL STEM CELLS. Our early studies in Xenopus laevis have shown that intestinal remodeling involves complete degeneration of the larval epithelium and de novo formation of adult stem cells through dedifferentiation of some larval epithelial cells. We have further discovered that the histidine ammonia-lyase (HAL, also known as histidase or histidinase)-2 gene is strongly and specifically activated by TH in the proliferating adult stem cells of the intestine during metamorphosis, implicating a role of histidine catabolism in the development of adult intestinal stem cells. To determine the mechanism by which TH regulates the HAL2 gene, we have carried out bioinformatics analysis and discovered a putative TH response element (TRE) in the HAL2 gene. Importantly, we show that this TRE is bound by TH receptor (TR) in the intestine during metamorphosis. The TRE is capable of binding to the heterodimer of TR and 9-cis retinoic acid receptor (RXR) in vitro and mediate transcriptional activation by liganded TR/RXR in frog oocytes. More importantly, the HAL2 promoter containing the TRE can drive TH-dependent reporter gene expression to mimic endogenous HAL2 expression in transgenic animals. Our results suggest that the TRE mediates the transcriptional activation of HAL2 gene by TH in the developing adult intestinal stem cells during metamorphosis.
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