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体上皮干细胞发育至关重要，但对热带爪蟾变态过程中成体肠的形态发生却不是必需的。我们最近培育了 TR 双敲除 (TRDKO) 热带爪蟾动物，并报道 TR 对于完成变态至关重要。此外，TRDKO蝌蚪在最终死亡之前停滞在变态的顶峰。为了研究 TRDKO 引起的潜在缺陷，我们分析了野生型和 TRDKO 动物变态高峰期的肠道。我们发现 TRDKO 肠道在自然变态过程中缺乏幼虫上皮细胞死亡和成体干细胞形成/增殖。有趣的是，TRDKO蝌蚪肠道过早形成了类似成人的上皮褶皱和肌肉发育。此外，对变态前TRDKO蝌蚪进行TH处理未能诱导肠道发生任何变态变化。此外，RNA-seq 分析显示，TRDKO 改变了生物途径中许多基因的表达，例如 Wnt 信号传导和细胞周期，这可能是通过去除两个 TR 基因而抑制幼虫上皮细胞死亡和成体干细胞发育的基础。我们的数据表明，配体的TR是幼虫上皮细胞变性和成体干细胞形成所必需的，而未配体的TR可以防止早熟的成体组织形态发生，例如肠道中的平滑肌发育和上皮折叠。 在热带爪蟾成年肠干细胞发育和增殖过程中，甲状腺激素直接激活线粒体裂变过程 1 (MTFP1) 基因转录。 TH 通过 TR 调节靶基因表达发挥作用。因此，TH 靶基因的鉴定和表征对于理解 TH 如何在变态过程中调节成体肠道干细胞发育至关重要。我们之前已经在热带爪蟾肠变态过程中鉴定了许多候选TR靶基因，这一过程涉及大多数幼虫上皮细胞的凋亡变性和成体上皮干细胞的从头发育。 这些假定的 TR 靶基因包括线粒体裂变过程 1 (Mtfp1)，一种核编码的线粒体基因。我们最近的研究表明，当成体上皮干细胞发育和增殖发生时，Mtfp1 基因表达在自然变态和 TH 诱导的变态过程中在肠道中达到峰值。 此外，Mtfp1 在第一个内含子内包含一个 TH 响应元件，该元件与 TR 结合，介导变态过程中 TH 诱导的局部组蛋白 H3K79 甲基化和肠道中 RNA 聚合酶的募集。此外，我们证明 Mtfp1 启动子可以在体内重建的青蛙卵母细胞系统中被 TH 激活，并且这种激活依赖于内含子 TRE。这些发现表明 TH 通过内含子 TRE 直接激活 Mtfp1 基因，并且 Mtfp1 反过来通过影响线粒体裂变过程促进成体肠干细胞发育/增殖。 蛋白质精氨酸甲基转移酶 1 调节成年小鼠成年肠中的细胞增殖和分化。成体干细胞在成体器官生理学以及组织修复和再生中发挥着重要作用。尽管人们对各种成体干细胞的特性和功能已经了解很多，但它们在哺乳动物中的发育机制仍然知之甚少。早期研究表明，成年小鼠肠道干细胞的形成发生在出生后的最初几周内，即血浆 TH 水平较高的胚胎后时期。此外，TH 信号传导缺陷会导致成年小鼠肠道缺陷，包括干细胞所在的肠隐窝细胞增殖减少。我们早期的研究表明，蛋白精氨酸甲基转移酶 1 (PRMT1)（一种 TR 共激活因子）在小鼠肠道成熟过程中高度表达，类似于其在肠道变态过程中的上调。此外，我们之前已经表明 PRMT1 对于非洲爪蟾变态过程中成体肠道干细胞的发育和/或增殖很重要。为了确定 PRMT1 在成体干细胞发育中是否具有保守作用，我们通过免疫组织化学分析了 PRMT1 的表达，并研究了 PRMT1 在肠上皮中组织特异性敲除的效果。我们发现PRMT1在增殖的转运扩增细胞和隐窝基底干细胞中高表达。通过使用条件敲除小鼠系，我们证明了肠上皮中 PRMT1 的表达对于成年小鼠肠道的发育至关重要。令人惊讶的是，特异性去除肠上皮中的 PRMT1 导致成体肠隐窝变长，细胞增殖增加。此外，上皮细胞沿隐窝-绒毛轴迁移和绒毛上的细胞死亡也增加。此外，隐窝内杯状细胞增多，潘氏细胞减少，而隐窝基底干细胞数量保持不变。考虑到 PRMT1 在 TH 信号传导中的作用以及 TH 对肠道发育的重要性，我们的发现 PRMT1 敲除增加细胞增殖是令人惊讶的，并表明 PRMT1 可能调节除 TH 信号传导之外的途径来影响肠道发育和/或体内平衡。从而影响成人的细胞增殖和上皮细胞更新。