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，通过使用一种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，当引入细胞（或动物）时，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脊索细胞存活是必要的。据推测，Brachyury在脊索瘤（脊索瘤）引起的癌症形成中起着核心作用，这一工具也可能为设计治疗这些非常难以治疗的癌症的新疗法提供动力。我们将该shRNA构建体导入转基因小鼠，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零突变（T+/-）杂合的小鼠胚胎中，这种敲除构建体的早期激活会再现零胚胎致死表型，同时失去前肢水平的尾轴。T野生型背景的敲除产生较弱的表型，在后肢芽水平失去身体轴。现在我们已经证明Brachyury可以通过这种方法有效地从细胞和动物中去除，我们正在利用它来研究Brachyury在脊索形成、原始条纹迁移产生主体中胚层和肢体发育过程中调节生长和细胞命运的正常功能。Brachyury表达的激活被认为是脊索瘤发生的必要条件，脊索瘤是在脊索残余中产生的癌症。我们开发的用于研究Brachyury功能的基因敲除工具将有助于验证这一假设，并揭示该基因促进肿瘤形成的可能机制。我们还利用鸡的遗传策略分析了Gnot （noto）和Brachyury的功能，发现这两个因子可能在调节胚胎细胞增殖中起互补作用。与小鼠不同的是，小鸡Gnot （Noto）不仅在脊索中表达，也在肢体中表达。有趣的是，我们之前在鸡和小鼠胚胎中发现Brachyury在肢体中表达，基于遗传学研究，我们提出Brachyury在肢体芽的生长中发挥积极作用，通过调节顶端外胚层脊的信号传导促进肢体芽的生长。利用鸡胚胎的错误表达方法，我们还获得了另一种调节脊索形成的转录因子Gnot/noto的功能的新见解。与Brachyury/T正调控肢体生长相反，Gnot/noto抑制肢体生长。这似乎也可以通过调节顶端外胚层脊功能来降低来自这个特化外胚层的FGF信号的数量。我们现在正在利用cDNA微阵列分析Gnot （Noto）对全球基因表达的影响，以确定Gnot/ Noto的其他下游靶点（如中胚层中的直接靶点）。这些研究将揭示Gnot/noto效应对肢体发育的功能机制，并可能为其在脊索发育中的作用提供新的见解，有助于开发新的潜在的脊索瘤治疗干预措施。