RNA interference approach to dissect roles of notochord regulators

我们最近开发了一种新的方法来控制 通过使用产生shRNA的慢病毒在细胞中表达转录因子Brachyury/T 针对Brachyury，并且当引入细胞（或动物）时，引起Brachyury的损失。 Brachyury/T.这种新的工具将使我们能够在小鼠实验期间改变Brachyury功能。 了解它如何调节细胞命运和行为，然后确定 它的功能是否是脊索细胞存活所必需的。已经假设 短尾畸形在脊索癌的形成中起着核心作用 （脊索瘤），这一工具也可能为设计新的治疗方法提供动力， 很难治疗癌症。我们已经将这种shRNA构建体引入转基因小鼠中， 并采用Cre-lox技术，使我们能够选择性地激活它的表达， 不同的胚胎组织。我们已经证明，早期激活这种 短尾畸形无效突变杂合的小鼠胚胎中的敲除构建体 （T+/-）复制了无效胚胎致死表型， 前肢水平在T野生型背景上的敲低产生较弱的表型， 在后肢芽水平失去体轴。既然我们已经证明了Brachyury可以 使用这种方法有效地从细胞和动物中去除，我们正在使用它来研究 Brachyury在脊索形成过程中调节生长和细胞命运的正常功能， 原条迁移产生主体中胚层，并在肢发育。的 已经提出Brachyury表达的激活对于Brachyury的发生是必不可少的。 脊索瘤，脊索残留物中产生的癌症。我们现有的基因敲除工具 开发研究Brachyury功能将有助于测试这一假设，并解开 这种基因可能促进肿瘤形成的可能机制。我们也在使用基因 在鸡中分析Gnot（诺托）和Brachyury功能的策略，并发现 这两个因子在调节细胞增殖中可能起互补作用， 胚胎与小鼠不同，鸡Gnot（诺托）在四肢和脊索中表达。 有趣的是，我们以前发现Brachyury在鸡和小鼠的肢体中表达， 胚胎，并根据遗传研究，我们提出，Brachyury发挥积极作用， 肢体芽的生长调节来自顶端外胚叶嵴的信号传导，以促进肢体发育 萌芽生长利用鸡胚中的错误表达方法，我们还获得了新的 对另一种调节脊索形成的转录因子的功能的了解， Gnot/诺托。与Brachyury/T相反，Gnot/诺托正调节肢体生长， 抑制生长。这似乎也发生在调节顶端外胚层嵴功能 来降低这个特化外胚层的FGF信号量。我们现在正在分析Gnot （诺托）对整体基因表达的影响，使用cDNA微阵列鉴定其他下游基因 Gnot/诺托的靶点（如中胚层中的直接靶点）。这些研究将揭示 Gnot/诺托影响肢体发育的功能机制，也可能提供新的见解 研究其在脊索发育中的作用，这可能有助于开发新的潜在治疗方法。 对霍乱的干预。

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.