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方法，通过使用慢病毒来控制细胞中转录因子 Brachyury/T 的产生，该慢病毒产生针对 Brachyury 的 shRNA，并且当引入细胞（或动物）时，会导致 Brachyury/T 的丢失。这种新工具将使我们能够在小鼠发育过程中改变 Brachyury 功能，以了解它如何调节细胞命运和行为，然后确定其功能是否对于脊索细胞生存是必要的。据推测，Brachyury 在脊索（脊索瘤）引起的癌症的形成中发挥着核心作用，并且该工具也可能为为这些非常难以治疗的癌症设计新疗法提供动力。我们已将这种 shRNA 构建体引入转基因小鼠中，并采用 Cre-lox 技术，使我们能够在特定时间选择性激活其在不同胚胎组织中的表达。我们已经证明，在 Brachyury 无效突变 (T+/-) 杂合的小鼠胚胎中，这种敲低构建体的早期激活会复制无效胚胎致死表型，并导致前肢水平尾部的体轴丧失。对 T 野生型背景的敲除会产生较弱的表型，并导致后肢芽水平的体轴丧失。现在我们已经证明，使用这种方法可以有效地从细胞和动物中去除 Brachyury，我们正在用它来研究 Brachyury 在脊索形成、原条迁移以产生主体中胚层过程中调节生长和细胞命运的正常功能，以及肢体发育。 Brachyury 表达的激活被认为对于脊索瘤（脊索残余物中出现的癌症）的发生至关重要。我们开发的用于研究 Brachyury 功能的基因敲除工具将有助于检验这一假设并揭示该基因促进肿瘤形成的可能机制。我们还在小鸡中使用遗传策略来分析 Gnot (noto) 和 Brachyury 的功能，并发现这两个因子可能在调节胚胎细胞增殖中发挥互补作用。与小鼠不同，小鸡 Gnot (Noto) 在四肢以及脊索中表达。 有趣的是，我们之前发现Brachyury在小鸡和小鼠胚胎的肢体中表达，并且基于遗传学研究，我们提出Brachyury通过调节来自顶端外胚层脊的信号来促进肢芽生长，从而在肢芽生长中发挥积极作用。利用鸡胚胎中的错误表达方法，我们还对另一种调节脊索形成的转录因子 Gnot/noto 的功能有了新的认识。与 Brachyury/T 积极调节肢体生长相反，Gnot/noto 抑制生长。这似乎也是通过调节顶端外胚层脊功能以降低来自该特殊外胚层的 FGF 信号量来实现的。我们现在正在使用 cDNA 微阵列分析 Gnot (Noto) 对全局基因表达的影响，以确定 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 对肢体发育影响的功能机制，也可能对其在脊索发育中的作用提供新的见解，这可能有助于开发针对脊索瘤的新的潜在治疗干预措施。