Regulation of stem cell development during tissue remodeling

组织重塑过程中干细胞发育的调节

• 批准号: 10459127
• 负责人: Yun-Bo Shi
• 金额: $ 97.32万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10459127
• 关键词: Adult; Affect; Animals; Apoptosis; Apoptotic; Basal lamina; Biological; Biological Metamorphosis; Birth; Cell Culture Techniques; Cell Cycle; Cell Death; Cell Differentiation process; Cell Proliferation; Cells; Cessation of life; Complement; Connective Tissue; DNA-Directed RNA Polymerase; Data; Defect; Development; Embryo; Epithelial; Epithelial Cells; Excision; Extracellular Matrix; Food Processing; Gene Expression; Genes; Genetic Transcription; Goals; Goblet Cells; Histones; Homeostasis; Immunohistochemistry; Intestinal Hormones; Intestines; Introns; Knock-out; Knockout Mice; Life; Ligands; Mammals; Mediating; Methylation; Mitochondria; Morphogenesis; Mus; Muscle; Muscle Development; Nuclear; Oocytes; Organ; Organ Culture Techniques; Paneth Cells; Pathway interactions; Physiological; Physiology; Plasma; Play; Primary Cell Cultures; Process; Proliferating; Property; Protein-Arginine N-Methyltransferase; Rana; Regulation; Reporting; Role; Smooth Muscle; South African; Stem Cell Development; Structure; System; Tadpoles; Technology; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Up-Regulation; Vertebrates; Villus; WNT Signaling Pathway; Xenopus; Xenopus laevis; adult stem cell; base; cell motility; conditional knockout; epithelial stem cell; hormonal signals; hormone response element; in vivo; intestinal crypt; intestinal epithelium; intestinal maturation; knock-down; knockout animal; nutrient absorption; premature; prevent; promoter; protein expression; reconstitution; recruit; stem cell division; stem cells; tissue regeneration; tissue repair; toad; transcriptome sequencing

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.

甲状腺激素受体对于幼虫上皮细胞凋亡和成年上皮干细胞的发育至关重要，而在Xenopus tropicalis metamorphosis期间不是成人肠道形态发生。我们最近产生了TR双基因敲除（TRDKO）爪蟾动物，并报告TR对于完成变态至关重要。此外，在最终死亡之前，Trdko Tadpoles在变态的高潮处停滞不前。为了研究由于TRDKO引起的潜在缺陷，我们在野生型变质的高潮和Trdko动物的高潮时分析了肠道。我们表明，在自然变态过程中，Trdko肠缺乏幼虫上皮细胞死亡和成年干细胞的形成/增殖。有趣的是，Trdko Tadpole肠肠结构过早地形成了成人样的上皮褶皱和肌肉发育。此外，对术前TRDKO t的治疗未能诱导肠道的任何变质变化。此外，RNA-seq分析表明，TRDKO改变了许多基因在Wnt信号传导和细胞周期等生物学途径中的表达，这可能是抑制幼虫上皮细胞死亡的抑制和由去除两个TR基因引起的成人干细胞发育的抑制。我们的数据表明，配体TR是幼虫上皮细胞变性和成年干细胞形成所必需的，而无配合的TR则阻止了早熟的成年组织形态发生，例如平滑肌发育和肠中的上皮折叠。 甲状腺激素直接激活线粒体裂变过程1（MTFP1）基因转录期间在成年肠干细胞发育和热带爪蟾的增殖过程中。调节靶基因表达通过TRS的功能。因此，TH靶基因的鉴定和表征对于了解TH在变形过程中如何调节成人肠道干细胞的发育至关重要。我们先前曾在热带肠道肠道变态中确定许多候选TR靶基因，这一过程涉及大多数幼虫上皮细胞的凋亡变性以及成年上皮干细胞的从头发展。 在这些假定的TR靶基因中，有线粒体裂变过程1（MTFP1），一种核编码的线粒体基因。我们最近的研究表明，在成年上皮干细胞发育和增殖时，在天然和TH诱导的变态过程中，MTFP1GENE表达在肠道中达到峰值。 此外，MTFP1在第一个内含子中包含一个TH响应元件，该内含子被TR绑定，用于介导TH诱导的局部组蛋白H3K79甲基化和RNA聚合酶在变质过程中肠道中的诱导。此外，我们证明了MTFP1启动子可以在体内重组的青蛙卵母细胞系统中激活TH，并且这种激活取决于内含子TRE。这些发现表明，TH直接通过内含子TRE激活MTFP1基因，而MTFP1反过来又通过影响线粒体裂变过程来促进成人肠道干细胞的发育/增殖。 蛋白精氨酸甲基转移酶1调节成年小鼠成年肠的细胞增殖和分化。成年干细胞在成人器官生理学以及组织修复和再生中起着至关重要的作用。尽管已经了解了各种成年干细胞的特性和功能，但在哺乳动物中，其发育的机制仍然很少了解。较早的研究表明，成年小鼠肠道干细胞的形成发生在出生后的头几周，即血浆TH水平高的胚胎后期。此外，TH信号的缺乏会导致成年小鼠肠的缺陷，包括减少了干细胞驻留的肠道隐窝中的细胞增殖。我们的较早研究表明，蛋白精氨酸甲基转移酶1（PRMT1）是TR共激活剂，在小鼠肠道成熟过程中高度表达，类似于其在肠道变质过程中的上调。此外，我们以前已经表明，PRMT1对于爪蟾变态过程中成人肠道干细胞的发育和/或增殖很重要。为了确定PRMT1在成年干细胞发育中是否具有保守的作用，我们通过免疫组织化学分析了PRMT1的表达，并研究了PRMT1在肠上皮中的组织特异性敲除的影响。我们表明，PRMT1在增殖的放大细胞和隐窝碱干细胞中高度表达。通过使用条件基因敲除小鼠系，我们证明了PRMT1在肠上皮中的表达对于成年小鼠肠的发展至关重要。肠上皮中PRMT1的特异性去除导致令人惊讶的是，成年肠密含量更高，细胞增殖增加。此外，沿着隐窝磁膜轴的上皮细胞迁移和绒毛上的细胞死亡也增加了。此外，地下室中的杯状细胞增加并减少了Paneth细胞，而隐窝碱干细胞的数量保持不变。考虑到PRMT1在TH信号中的作用以及TH对肠道发育的重要性的作用，我们的发现增加了细胞增殖，这令人惊讶，并表明PRMT1可能调节途径，除了影响肠道发育和/或体内稳态外，还会影响细胞扩散和上皮的跨越成人。