RNA interference approach to dissect roles of notochord regulators

RNA 干扰方法剖析脊索调节因子的作用

• 批准号: 7966091
• 负责人: Susan Mackem
• 金额: $ 12.51万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/7966091
• 关键词: Affect; Animals; Apical Ectodermal Ridge; Apoptosis; Behavior; Brachyury protein; Cell Communication; Cell Fate Control; Cell Proliferation; Cell Survival; Cells; Cephalic; Chick Embryo; Child; Chordoma; Development; Developmental Process; Ectoderm; Embryo; Embryonic Development; Fibroblast Growth Factor; Forelimb; Gene Expression; Genes; Genetic; Growth; Hindlimb; Infant; Intercept; Knowledge; Life; Limb Bud; Limb Development; Limb structure; Location; Malignant Neoplasms; Mesoderm; Methods; Modeling; Morbidity - disease rate; Movement; Mus; Muscle; Names; Neoplasm Metastasis; Pathogenesis; Phenotype; Play; Primitive Streaks; Process; Production; Proliferating; RNA Interference; Role; Signal Transduction; Spinal Cord; Structure; Subfamily lentivirinae; Technology; Testing; Therapeutic Intervention; Time; Tissues; Transgenic Mice; Tumor Biology; Vertebral column; base; bone; cDNA Arrays; cancer cell; cell behavior; cell growth; cell motility; cellular targeting; embryo tissue; insight; interest; knock-down; migration; mortality; mouse development; notochord; notochord development; novel; null mutation; skull base; small hairpin RNA; tool; transcription factor; tumor; tumorigenesis; young adult

We have recently developed a novel method to control the production of the transcription factor Brachyury/T in cells by using a lentivirus that produces an shRNA directed against Brachyury and, when introduced into cells (or animals), causes the loss of Brachyury/T. This novel tool will enable us to alter Brachyury function during mouse development to understand how it regulates cell fate and behavior, and then to determine whether its function is necessary for notochord cell survival. It has been hypothesized that Brachyury plays a central role in the formation of cancers that arise from notochord (chordomas), and this tool may also provide the impetus for devising new therapies for these very difficult to treat cancers. We have introduced this shRNA construct into transgenic mice and employed Cre-lox technology to enable us to selectively activate its expression in different embryonic tissues at specific times. We have shown that early activation of this knock-down construct in mouse embryos that are heterozygous for the Brachyury null mutation (T+/-) reproduces the null embryonic lethal phenotype with loss of the body axis caudal to the forelimb level. The knock-down on a T wild-type background produces a weaker phenotype with loss of the body axis at the hindlimb bud level. Now that we have shown that Brachyury can be effectively removed from cells and animals using this approach, we are using it to study the normal function of Brachyury in regulating growth and cell fate during notochord formation, primitive streak migration to produce the main body mesoderm, and in limb development. The activation of Brachyury expression has been proposed to be essential for the genesis of chordomas, cancers that arise in notochord remnants. The gene knock-down tools we have developed to study Brachyury function will help to test this hypothesis and unravel the possible mechanisms by which this gene may promote tumor formation. We are also using genetic strategies in chick to analyze the function of Gnot (noto) and Brachyury and have discovered that these two factors may play complementary roles in regulating cell proliferation in the embryo. Unlike mouse, in chick Gnot (Noto) is expressed in limb, as well as in the notochord. Interestingly, we previously found that Brachyury is expressed in limb in chick and in mouse embryos, and based on genetic studies, we proposed that Brachyury plays a positive role in limb bud outgrowth be regulating signaling from the apical ectodermal ridge to promote limb bud outgrowth. Using a mis-expression approach in chick embryos, we have also gained new insights on the function of another transcription factor that regulates notochord formation, Gnot/noto. In contrast to Brachyury/T, which positively regulates limb growth, Gnot/noto inhibits growth. This appears to also occur by modulating the apical ectodermal ridge function to lower the amount of FGF signaling from this specialized ectoderm. We are now analyzing Gnot (Noto) effects on global gene expression using cDNA microarrays to identify other downstream targets of Gnot/noto (such as direct targets in mesoderm). These studies will uncover the functional mechanism of Gnot/noto effects on limb development, and may also give new insights on its role in notochord development, which could help develop new potential therapeutic interventions for chordoma.

我们最近开发了一种新的方法，通过使用产生针对Brachyury的shRNA并引入细胞（或动物）的shRNA的慢病毒，以控制细胞中的转录因子/T的产生。这个新颖的工具将使我们能够在小鼠发育过程中改变脑臂功能，以了解其如何调节细胞的命运和行为，然后确定其功能是否对于脊索细胞存活是必要的。已经假设Brachyury在由Notochord（Chordomas）产生的癌症的形成中起着核心作用，并且该工具还可能为这些非常困难治疗的癌症制定新疗法提供动力。我们已经将这种shRNA构建体引入了转基因小鼠，并采用了Cre-Lox技术，使我们能够在特定时间有选择地激活其在不同胚胎组织中的表达。我们已经表明，这种敲低构建体在小鼠胚胎中的早期激活，该构建体的杂合无效突变（t +/--）重现了无效的胚胎致死表型，而人体轴尾部损失到前肢级别。野生型背景上的敲低击倒会产生较弱的表型，而在后肢芽级别的身体轴丢失。现在，我们已经证明，可以使用这种方法有效地从细胞和动物中清除了野牛，我们正在使用它来研究义大利在调节固定形成过程中调节生长和细胞命运的正常功能，原始条纹迁移以产生主体中胚层和肢体发育。已经提出，已经提出了臂表达的激活对于诺修（Notochord Remnants）出现的脊柱癌的起源至关重要。我们开发的基因敲除工具来研究跑步功能，将有助于检验这一假设，并揭示该基因可能促进肿瘤形成的可能机制。我们还使用雏鸡中的遗传策略来分析gnot（noto）和Brachyury的功能，并发现这两个因素可能在调节胚胎中细胞增殖方面起互补作用。与小鼠不同，小鸡gnot（noto）在肢体以及脊索中表达。 有趣的是，我们先前发现，在雏鸡和小鼠胚胎中的肢体中表达了胸骨，基于遗传研究，我们提出胸骨在肢体芽的生长中起着积极的作用，是调节从根尖的外皮脊来调节信号传导以促进肢体芽出生。使用雏鸡胚胎中的错误表达方法，我们还获得了对调节脊索形成的另一个转录因子功能的新见解，即gnot/noto。与Brachyury/T相反，该/T阳性地调节了肢体的生长，GNOT/NOTO抑制了生长。这似乎也是通过调节根尖外胚层脊功能以降低该专用外胚层的FGF信号的量来实现的。现在，我们正在使用cDNA微阵列分析对全局基因表达的gnot（noto）效应，以识别gnot/noto的其他下游靶标（例如中胚层中的直接靶标）。这些研究将揭示GNOT/NOTO对肢体发育的影响的功能机制，还可能对其在脊索发展中的作用有了新的见解，这可能有助于开发脊架的新潜在治疗干预措施。