Regulation of stem cell development during tissue remodeling

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

UPREGULATION OF PROTOONCOGENE SKI BY THYROID HORMONE IN THE INTESTINE AND TAIL DURING XENOPUS METAMORPHOSIS. TH affects frog metamorphosis through TH receptor (TR)-mediated regulation of TH response genes, where TR forms a heterodimer with RXR (9-cis retinoic acid receptor) and binds to TH response elements (TREs) in TH response genes to regulate their transcription. To study how TH regulates intestinal stem cell development and/or proliferation, we have previously identified many putative direct TH response genes in Xenopus tropicalis tadpole intestine by using ChIP (chromatin immunoprecipitation)-on-chip assays. Among them is the proto-oncogene Ski, which encodes a nuclear protein with complex functions in regulating cell fate. We have now shown that Ski is upregulated in the intestine and tail of premetamorphic tadpoles upon TH treatment and its expression peaks at stage 62, the climax of metamorphosis. We have further discovered a TRE in the first exon that can bind to TR/RXR in vitro and mediate TH regulation of the promoter in vivo. These data demonstrate that Ski is activated by TH through TR binding to a TRE in the first exon during Xenopus tropicalis metamorphosis, implicating a role of Ski in regulating cell fate in this process. LIVER DEVELOPMENT DURING XENOPUS TROPICALIS METAMORPHOSIS IS CONTROLLED BY TH-ACTIVATION OF WNT SIGNALING. Many mammalian organs and tissues including erythrocytes mature into their adult forms during postembryonic development when plasma TH level peaks, resembling amphibian metamorphosis. TR mutations/deletions can cause hematopoietic dysfunction, suggesting that TH plays a role in erythropoiesis during development. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis as a model to study TH function during postembryonic development. Our analyses of TRDKO tadpoles during metamorphosis revealed that TRDKO tadpoles exhibited characteristics similar to human iron deficiency anemia. As the liver is the hematopoietic organ, our finding suggests a defect in liver development in TRDKO tadpoles. We analyzed liver metamorphosis in wild type and TRDKO tadpoles and found that wild type liver metamorphosis involved increased cell proliferation, hepatocyte hypertrophy, and activation of urea cycle gene expression, a key feature of adult/mature liver in vertebrates. Interestingly, TRDKO liver had developmental defects such as reduced cell proliferation and failure to undergo hepatocyte hypertrophy or activate the expression of urea cycle genes. To reveal the molecular pathways regulated by TH during liver remodeling, we performed RNA-seq analysis and found that TH activated canonical Wnt pathway in the liver. Particularly, Wnt11 was activated in both fibroblasts and hepatic cells, and in turn, likely acted to promote stem cell development and/or proliferation and maturation of hepatocytes. Our findings also resemble those from studies on liver regeneration in mammals. Thus, analyses of liver metamorphosis have the potential to bring new insights on not only how TH regulates liver development but also potential means to improve liver regeneration. L-TYPE AMINO ACID TRANSPORTER 1 (LAT1) IN HYPOTHALAMIC NEURONS IN MICE MAINTAINS ENERGY AND BONE HOMEOSTASIS. To regulate cellular processes, TH has to be actively transported into cells and this process is mediated by several different types of transporters. One of our previously identified TH-response genes in Xenopus intestine, LAT1, encodes the light chain of a heterodimeric system L type of TH transporter, which also transports several amino acids. Interestingly, LAT1 is highly upregulated at the climax of metamorphosis in tadpole intestine, coinciding with the formation and rapid proliferation of adult intestinal stem cells. We also found out that LAT1 was also highly expressed in the mouse intestine during the neonatal period when mouse intestine matured into the adult form, a process that appears also to involve TH-dependent formation and/proliferation of adult intestinal stem cells. Through a collaborative study, we generated a mouse line with the LAT1 gene floxed, which allows conditional knockout of LAT1 upon expression of the Cre recombinase. We are currently analyzing the effect of LAT1 knockout specifically in the mouse intestine by expressing Cre under the control of the intestinal epithelial specific villin promoter. In addition, through another collaborative study, we discovered LAT1 expression in hypothalamic neurons, which regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). Importantly, we found that LAT1 in hypothalamic leptin receptor (LepR)-expressing neurons was important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.