Regulation of stem cell development during tissue remodeling

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

THYROID HORMONE RECEPTOR IS ESSENTIAL FOR LARVAL EPITHELIAL APOPTOSIS AND ADULT EPITHELIAL STEM CELL DEVELOPMENT BUT NOT ADULT INTESTINAL MORPHOGENESIS DURING XENOPUS TROPICALIS METAMORPHOSIS. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals and reported that TR is essential for the completion of metamorphosis. Furthermore, TRDKO tadpoles are stalled at the climax of metamorphosis before eventual death. To investigate the underlying defects due to TRDKO, we analyzed the intestine at the climax of metamorphosis in the wild type and TRDKO animals at the climax of metamorphosis. We showed that TRDKO intestine lacked larval epithelial cell death and adult stem cell formation/proliferation during natural metamorphosis. Interestingly, TRDKO tadpole intestine had premature formation of adult-like epithelial folds and muscle development. In addition, TH treatment of premetamorphic TRDKO tadpoles failed to induce any metamorphic changes in the intestine. Furthermore, RNA-seq analysis revealed that TRDKO altered the expression of many genes in biological pathways such as Wnt signaling and cell cycle that likely underlay the inhibition of larval epithelial cell death and adult stem cell development caused by removing both TR genes. Our data suggest that liganded TR is required for larval epithelial cell degeneration and adult stem cell formation whereas unliganded TR prevents precocious adult tissue morphogenesis such as smooth muscle development and epithelial folding in the intestine. THYROID HORMONE DIRECTLY ACTIVATES MITOCHONDRIAL FISSION PROCESS 1 (MTFP1) GENE TRANSCRIPTION DURING ADULT INTESTINAL STEM CELL DEVELOPMENT AND PROLIFERATION IN XENOPUS TROPICALIS. TH functions by regulation target gene expression through TRs. Thus, identification and characterization of TH target genes are essential toward understanding how TH regulates adult intestinal stem cell development during metamorphosis. We have previously identified many candidate TR target genes during Xenopus tropicalis intestinal metamorphosis, a process that involves apoptotic degeneration of most of the larval epithelial cells and de novo development of adult epithelial stem cells. Among these putative TR target genes is mitochondrial fission process 1 (Mtfp1), a nuclear-encoded mitochondrial gene. We recent studies showed that Mtfp1gene expression peaked in the intestine during both natural and TH-induced metamorphosis when adult epithelial stem cell development and proliferation took place. Furthermore, Mtfp1 contained a TH-response element within the first intron that was bound by TR to mediate TH-induced local histone H3K79 methylation and RNA polymerase recruitment in the intestine during metamorphosis. Additionally, we demonstrated that the Mtfp1 promoter could be activated by TH in a reconstituted frog oocyte system in vivo and that this activation is dependent on the intronic TRE. These findings suggest that TH activates Mtfp1 gene directly via the intronic TRE and that Mtfp1 in turn facilitate adult intestinal stem cell development/proliferation by affecting mitochondrial fission process. PROTEIN ARGININE METHYLTRANSFERASE 1 REGULATES CELL PROLIFERATION AND DIFFERENTIATION IN ADULT MOUSE ADULT INTESTINE. Adult stem cells play an essential role in adult organ physiology and tissue repair and regeneration. While much has been learnt about the property and function of various adult stem cells, the mechanisms of their development remain poorly understood in mammals. Earlier studies suggest that the formation of adult mouse intestinal stem cells takes place during the first few weeks after birth, the postembryonic period when plasma TH levels are high. Furthermore, deficiency in TH signaling leads to defects in adult mouse intestine, including reduced cell proliferation in the intestinal crypts, where stem cells reside. Our earlier studies have shown that protein arginine methyltransferase 1 (PRMT1), a TR coactivator, is highly expressed during intestinal maturation in mouse, resembling its upregulation during intestinal metamorphosis. Furthermore, we have previously shown that PRMT1 is important for adult intestinal stem cell development and/or proliferation during Xenopus metamorphosis. To determine if PRMT1 has a conserved role in adult stem cell development, we analyzed the expression of PRMT1 by immunohistochemistry and studied the effect of tissue-specific knockout of PRMT1 in the intestinal epithelium. We showed that PRMT1 was expressed highly in the proliferating transit amplifying cells and crypt base stem cells. By using a conditional knockout mouse line, we demonstrated that the expression of PRMT1 in the intestinal epithelium was critical for the development of the adult mouse intestine. Specific removal of PRMT1 in the intestinal epithelium resulted in, surprisingly, more elongated adult intestinal crypts with increased cell proliferation. In addition, epithelial cell migration along the crypt-villus axis and cell death on the villus were also increased. Furthermore, there were increased Goblet cells and reduced Paneth cells in the crypt while the number of crypt base stem cells remained unchanged. Our finding that PRMT1 knockout increases cell proliferation is surprising considering the role of PRMT1 in TH-signaling and the importance of TH for intestinal development, and suggests that PRMT1 likely regulates pathways in addition to TH-signaling to affect intestinal development and/or homeostasis, thus affecting cell proliferating and epithelial turn over in the adult.