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: 8351233
• 负责人: Yun-Bo Shi
• 金额: $ 100.54万
• 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/8351233
• 关键词: Active Biological Transport; Adult; Affect; African; Alleles; Amino Acid Transporter; Amino Acids; Amphibia; Apoptosis; Basal lamina; Biological Metamorphosis; Branched-Chain Amino Acids; C-terminal; Cell Culture Techniques; Cell Differentiation process; Cell Nucleus; Cell Proliferation; Cell surface; Cells; Cessation of life; Chimeric Proteins; Collaborations; Complement; Complex; Connective Tissue; Cytoplasmic Tail; Development; ES Cell Line; Embryo; Embryonic Development; Epithelial; Epithelial Cells; Epithelium; Erinaceidae; Exhibits; Extracellular Domain; Extracellular Matrix; Food Processing; Future; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Heat-Shock Response; Integrins; Intestines; Knock-out; Knockout Mice; Laboratories; Life; Light; Maintenance; Mediating; Mus; Muscle; Mutant Strains Mice; National Institute of Diabetes and Digestive and Kidney Diseases; Nutrient; Oocytes; Organ; Organ Culture Techniques; Physiological; Plasma Cells; Play; Population; Primary Cell Cultures; Process; Proliferating; Protein-Arginine N-Methyltransferase; Proteins; Rana; Recombinants; Regulation; Reporting; Role; Signal Transduction; Site; Staging; Stem Cell Development; Stem cells; Structure; Tadpoles; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Transgenic Organisms; Transmembrane Domain; United States National Institutes of Health; Wound Healing; Xenopus; Xenopus laevis; absorption; adult stem cell; embryonic stem cell; extracellular; implantation; in vivo; interest; intestinal epithelium; knockout gene; mouse development; mutant; overexpression; promoter; receptor; receptor expression; reconstitution; research study; stem cell division; stem cell niche; tissue regeneration; toad; zebrafish development

EPITHELIAL-CONNECTIVE TISSUE INTERACTIONS INDUCED BY THYROID HORMONE RECEPTOR ARE ESSENTIAL FOR ADULT STEM CELL DEVELOPMENT IN THE XENOPUS LAEVIS INTESTINE. We have previously shown that the adult intestinal stem cells originate from differentiated larval epithelial cells in the Xenopus laevis intestine. To determine whether TH signaling in the epithelium alone is sufficient for inducing the adult stem cells, we have performed tissue recombinant culture experiments by using transgenic Xenopus laevis tadpoles that express a dominant positive TH receptor (dpTR) under a control of heat shock promoter. Wild-type (Wt) or dpTR transgenic (Tg) larval epithelium (Ep) was isolated from the tadpole intestine, recombined with homologous or heterologous non-epithelial tissues (non-Ep), and then cultivated in the absence of TH but with daily heat shocks to induce transgenic dpTR expression. Adult epithelial progenitor cells expressing sonic hedgehog became detectable on day 5 in both the recombinant intestine of Tg Ep and Tg non-Ep (Tg/Tg) and that of Tg Ep and Wt non-Ep (Tg/Wt). However, in Tg/Wt intestine, they did not express other stem cell markers such as Musashi-1 and never generated the adult epithelium expressing a marker for absorptive epithelial cells. Our results indicate that, while the adult progenitor cells may be predetermined in the larval epithelium, TR-mediated gene expression in the surrounding tissues other than the epithelium is required for them to develop into adult stem cells, suggesting the importance of TH-inducible epithelial-connective tissue interactions in establishment of the stem cell niche in the amphibian intestine. PROTEIN ARGININE METHYLTRANSFERASE 1 (PRMT1) PLAYS AN EVOLUTIONARILY CONSERVED ROLE IN ADULT INTESTINAL STEM CELL DEVELOPMENT DURING POSTEMBRYONIC DEVELOPMENT. As indicated above, TH action in the larval epithelium can induce some larval epithelial cells to dedifferentiate into adult progenitor cells via a cell autonomous function. Interestingly, the TR-coactivator, PRMT1, is strongly induced in these progenitor cells. More importantly, our in vivo studies showed that transgenic overexpression of PRMT1 leads to increased population of adult stem cells while knockdown of endogenous PRMT1 reduces the number of stem cells. Additionally, PRMT1 is also upregulated during zebrafish and mouse development in the equivalent postembryonic period when thyroid hormone levels are high and adult intestinal stem cells develop, suggesting that PRMT1 plays an evolutionally conserved role in the development of adult intestinal stem cells. EXTRACELLULAR DOMAIN OF A TH TRANSPORTER SUBUNIT CD98HC IS REQUIRED FOR EARLY MURINE DEVELOPMENT. To regulate gene transcription in the nucleus, TH is taken up from the circulating plasma by cells via active transport. A number of transporters for TH have been identified over the years. We have previously identified one of the TH direct target genes in the metamorphosing intestine as the light chain of a heterodimeric transporter, the L-type amino acid transporter-1 (LAT1) and shown that it is capable of transporting TH into cells. LAT1 complexes with the heavy chain CD98, which is a multifunctional protein CD98 (CD98hc, Slc3a2) that can also associates with integrin β1 through its cytoplasmic and transmembrane domains. It has been shown that the CD98hc-mediated integrin signaling is required for maintenance of ES cell proliferation. CD98hc-null mice exhibit early post-implantation lethality similar to integrin β1-null mice, supporting the importance of its interaction with integrin β1. On the other hand, the extracellular domain of CD98hc interacts with LATs and is essential for appropriate cell surface distribution of LATs. LATs mediate the transport of both thyroid hormone and branched chain amino acids. In this respect, CD98hc may also affect development via these transporters. As indicated above, TH is important for the maturation of the adult intestine in mouse and many TH target genes identified during Xenopus metamorphosis are similarly regulated during mouse intestinal development. To investigate the role of TH in adult intestinal stem cell development, we are interested to make sure of the ability to carry out gene knockout in mouse, which is not possible at the moment in frogs. Thus, in collaboration with Dr. C Dengs laboratory at NIDDK, NIH, we have generated a mutant mouse line from an embryonic stem (ES) cell line (PST080) harboring a mutant CD98hc allele (CD98hcΔ/+). Expression of the CD98hc mutant allele results in ΔCD98hc-β geo fusion protein where extracellular C-terminal 102 amino acids of CD98hc are replaced with β geo. Analyses of PST080 ES cells as well as reconstituted frog oocytes demonstrated that ΔCD98hc-β geo fusion protein preserved its ability to interact with integrin β1 although this mutant protein was hardly localized on the cell surface. These findings suggest that ΔCD98hc-β geo protein can mediate integrin signaling but cannot support TH or amino acid transport through LATs. CD98hcΔ/+ mice were normal. Although some of the implantation sites lacked embryonic component at E9.5, all the implantation sites contained embryonic component at E7.5. Thus, CD98hcΔ/Δ embryos are likely to die between E7.5 and E9.5. Considering that CD98hc complete knockout (CD98hc-/-) embryos are reported to die shortly after implantation, our findings suggest potential stage-specific roles of CD98hc in murine embryonic development. CD98hc may be essential for early post-implantation development by regulating integrin-dependent signaling, while the other function of CD98hc as a component of TH and amino acid transporters may be required for embryonic development at later stages. The availability of this mouse line will allow us to investigate whether TH transport plays a role in intestinal stem cell development in the near future.

甲状腺激素受体诱导的上皮-结缔组织相互作用对于非洲爪蟾肠道中成体干细胞的发育至关重要。我们之前已经证明，成体肠道干细胞起源于非洲爪蟾肠道中分化的幼虫上皮细胞。为了确定仅上皮细胞中的 TH 信号传导是否足以诱导成体干细胞，我们使用转基因非洲爪蟾蝌蚪进行了组织重组培养实验，这些蝌蚪在热激启动子的控制下表达显性阳性 TH 受体（dpTR）。从蝌蚪肠道中分离出野生型（Wt）或 dpTR 转基因（Tg）幼虫上皮（Ep），与同源或异源非上皮组织（non-Ep）重组，然后在没有 TH 的情况下培养，但每天添加热激诱导转基因 dpTR 表达。第 5 天，在 Tg Ep 和 Tg non-Ep (Tg/Tg) 以及 Tg Ep 和 Wt non-Ep (Tg/Wt) 的重组肠中可检测到表达 sonic humility 的成体上皮祖细胞。然而，在Tg/Wt肠中，它们不表达其他干细胞标记物，例如Musashi-1，并且从未产生表达吸收性上皮细胞标记物的成体上皮。我们的结果表明，虽然成体祖细胞可能在幼虫上皮中预先确定，但它们发育成成体干细胞需要在上皮以外的周围组织中TR介导的基因表达，这表明TH诱导的上皮细胞的重要性-两栖动物肠道中干细胞生态位建立过程中结缔组织的相互作用。 蛋白质精氨酸甲基转移酶 1 (PRMT1) 在成人肠干细胞胚胎后发育过程中发挥着进化保守的作用。如上所述，幼虫上皮中的 TH 作用可以通过细胞自主功能诱导一些幼虫上皮细胞去分化为成体祖细胞。有趣的是，TR 共激活因子 PRMT1 在这些祖细胞中被强烈诱导。更重要的是，我们的体内研究表明，PRMT1 的转基因过度表达会导致成体干细胞数量增加，而内源性 PRMT1 的敲除则会减少干细胞的数量。此外，在斑马鱼和小鼠发育过程中，当甲状腺激素水平较高且成体肠道干细胞发育时，PRMT1也在相应的胚胎后时期上调，这表明PRMT1在成体肠道干细胞的发育中发挥着进化上保守的作用。 TH 转运蛋白亚基 CD98HC 的细胞外域是早期小鼠发育所必需的。为了调节细胞核中的基因转录，细胞通过主动转运从循环血浆中摄取 TH。 多年来已确定了许多 TH 转运蛋白。我们之前已经鉴定出变态肠中 TH 直接靶基因之一是异二聚体转运蛋白 L 型氨基酸转运蛋白 1 (LAT1) 的轻链，并表明它能够将 TH 转运到细胞中。 LAT1 与重链 CD98 形成复合物，后者是一种多功能蛋白 CD98 (CD98hc、Slc3a2)，还可以通过其细胞质和跨膜结构域与整合素 β1 结合。研究表明，CD98hc 介导的整合素信号传导是维持 ES 细胞增殖所必需的。 CD98hc 缺失小鼠表现出与整合素 β1 缺失小鼠相似的早期植入后致死率，这支持了其与整合素 β1 相互作用的重要性。另一方面，CD98hc 的胞外结构域与 LAT 相互作用，对于 LAT 适当的细胞表面分布至关重要。 LAT 介导甲状腺激素和支链氨基酸的运输。在这方面，CD98hc 也可能通过这些转运蛋白影响发育。 如上所述，TH 对于小鼠成年肠道的成熟非常重要，并且在非洲爪蟾变态过程中鉴定的许多 TH 靶基因在小鼠肠道发育过程中也受到类似的调节。 为了研究 TH 在成体肠道干细胞发育中的作用，我们有兴趣确保在小鼠中进行基因敲除的能力，而目前在青蛙中这还不可能。 因此，我们与 NIDDK、NIH 的 C Dengs 博士实验室合作，从含有突变 CD98hc 等位基因 (CD98hcΔ/+) 的胚胎干 (ES) 细胞系 (PST080) 中产生了突变小鼠系。 CD98hc 突变等位基因的表达产生 ΔCD98hc-β geo 融合蛋白，其中 CD98hc 的胞外 C 端 102 个氨基酸被 β geo 取代。对 PST080 ES 细胞以及重建青蛙卵母细胞的分析表明，ΔCD98hc-β geo 融合蛋白保留了与整合素 β1 相互作用的能力，尽管这种突变蛋白几乎不位于细胞表面。这些发现表明 ΔCD98hc-β geo 蛋白可以介导整合素信号传导，但不能支持 TH 或氨基酸通过 LAT 的转运。 CD98hcΔ/+小鼠正常。尽管一些植入位点缺乏E9.5的胚胎成分，但所有植入位点都含有E7.5的胚胎成分。 因此，CD98hcΔ/Δ胚胎很可能在E7.5和E9.5之间死亡。考虑到据报道 CD98hc 完全敲除 (CD98hc-/-) 胚胎在植入后不久就会死亡，我们的研究结果表明 CD98hc 在小鼠胚胎发育中具有潜在的阶段特异性作用。 CD98hc 可能通过调节整合素依赖性信号传导对于植入后早期发育至关重要，而 CD98hc 作为 TH 和氨基酸转运蛋白的组成部分的其他功能可能是后期胚胎发育所必需的。该小鼠系的出现将使我们能够在不久的将来研究 TH 转运是否在肠道干细胞发育中发挥作用。