DEMONSTRATED A REQUIREMENT FOR T3-INDUCED SONIC HEDGEHOG PARACRINE SIGNALING DURING ADULT INTESTINAL DEVELOPMENT. We have demonstrated that T3 activates the transcription of the Sonic hedgehog (Shh) gene directly at the transcriptional level during intestinal metamorphosis and that exogenous Shh promotes cell proliferation in intestinal organ cultures. To determine the role of Shh during metamorphosis, we treated premetamorphic tadpoles with Shh inhibitor cyclopamine and observed that it specifically inhibited the expression of Shh response genes Snai2 and Twist1. More importantly, cyclopamine reduced the proliferation of both developing adult stem cells in the epithelium and cells in the other intestinal tissues at the climax of metamorphosis, leading to delayed/incomplete remodeling of the intestine at the end of metamorphosis. We further revealed that both Snai2 and Twist1 were strongly upregulated specifically in the connective tissue during intestinal metamorphosis, suggesting that Shh signals the connective tissue to promote stem cell proliferation and the formation of the adult intestine. Consistently, we found that Shh receptor Patched (Ptc)-1 and the signaling protein Smoothened (Smo), as well as the downstream transcription factors Gli1, Gli2, and Gli3, were all transiently up-regulated in the mesenchymal tissues, but not the epithelium, where Shh was induced by T3, during intestinal metamorphosis. Finally, we showed in intestinal organ cultures that overexpression of Shh enhanced the expression of Ptc-1, Smo and Glis even in the absence of T3, indicating that Shh regulates the components of its own pathway during intestinal remodeling. Thus, Shh signaling induced by T3 is required for adult intestinal stem cell proliferation and functions via a paracrine mechanism. REVEALED AN ESSENTIAL ROLE OF SYSTEM L1 AMINO ACID/THYROID HORMONE TRANSPORTER DURING MOUSE DEVELOPMENT. The frog model offers a unique opportunity to identify and functionally characterize novel genes that are involved in the development of the adult intestine, particularly the adult stem cells. Interestingly, in all vertebrates, the formation/maturation of the adult intestine takes place around the time when plasma T3 levels are high. T3 or TR deficiency in mouse leads to abnormal intestinal morphology and a decrease in stem cell proliferation in the adult, and liganded TRα1 regulates stem cells during mouse intestinal maturation and homeostasis. Furthermore, we have shown that many genes with peak levels of expression at the climax of metamorphosis are upregulated during mouse intestinal maturation. Finally, our earlier studies suggest a conserved role of PRMT1 in stem cell development across vertebrates. Thus, we hypothesize that the formation of adult intestinal stem cells utilize conserved mechanisms, including regulation by T3 and the involvement of conserved T3 target genes during vertebrate development. To test this, we have initiated studies to take advantage of the ability to generate conditional knockout mice to investigate the developmental roles of the mouse homologs of the novel stem cell genes that we have discovered. One of the genes, LAT1, that we previous discovered as a T3 response gene in the intestine during metamorphosis encodes the catalytic subunit (light chain) of the heterodimeric System L1 amino acid transporter. Interestingly, when LAT1 was co-expressed with the heavy chain (CD98hc) of the transporter, it mediated the intracellular uptake of T3 and more importantly, enhanced transcriptional activation by TR in the presence of T3. Thus, LAT1 is activated by T3 and in turn may function to enhance the effect of T3 on adult intestinal development. We have since shown that targeted disruption of the CD98hc gene to specifically inactivate its ability to form a functional transporter with LAT1 led to mouse embryonic lethality, suggesting that LAT1 transporter activity is critical for organogenesis in mouse. In addition, we generated a mouse line with Floxed LAT1 gene, which when crossed with a mouse expressing the Cre recombinase, will lead to the inactivation of LAT1 gene. When it was crossed with mice expressing Cre driven by a global promoter, we observed that homozygous LAT1 knockout animals were embryonic lethal, consistent with the findings on CD98hc mutant animals. By using this Floxed line in a collaborative study, we found that LAT1 functioned as a metabolic checkpoint for T cell immune responses by regulating intracellular supply of large neutral amino acids. In particular, the System L transport activity in T cells was found to be regulated by exposure to pathogen through T cell antigen receptor (TCR) and/or inflammatory cytokines such as interleukin 2 (IL-2). LAT1 in turn regulates amino acid transport to coordinate the metabolic re-programming that is essential for T cell differentiation. These findings suggest that LAT1 affects diverse developmental processes.
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