Role of Brachyury in regulating notochord development and neoplasia

我们最近开发了一种新的方法来控制转录因子Brachyury/T在细胞中的产生，通过使用产生针对Brachyury的shRNA的慢病毒，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是脊索细胞生存所必需的。据推测，Brachyury在脊索（脊索瘤）引起的癌症的形成中起着核心作用，这种工具也可能为这些非常难以治疗的癌症设计新的疗法提供动力。我们将该shRNA构建体导入转基因小鼠，并采用Cre-lox技术在特定时间选择性地激活其在不同胚胎组织中的表达。我们已经表明，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再现了无效胚胎致死表型，其中体轴尾侧至前肢水平丢失。现在，我们已经表明，Brachyury可以有效地从细胞和动物使用这种方法，我们正在使用它来研究Brachyury在调节生长和细胞命运在脊索形成过程中的正常功能，与其在原始条纹中的作用相比。已经提出Brachyury表达的激活对于脊索瘤（脊索残留物中出现的癌症）的发生是必不可少的。我们开发的用于研究Brachyury功能的基因敲除工具使我们能够验证这一假设，并揭示该基因可能促进肿瘤形成的可能机制，也可能为开发潜在的治疗干预提供新的见解。令人惊讶的是，我们的研究结果表明，Brachyury功能是必不可少的维持脊索细胞的命运，但不可或缺的生存和增殖的脊索祖细胞。目前正在审查报告这些结果的手稿。我们还开发了一种基于激活的经典Wnt通路结合脊索谱系中p53肿瘤抑制因子失活的脊索瘤遗传小鼠模型。这样的遗传模型将使我们能够引入和评估结节性硬化症复合体基因（TSC 1，2）的突变等位基因的影响，已知这些基因在人类中易患脉络膜炎，但在小鼠中则不然，这表明其他因素在TSC相关脉络膜炎的发病机制中也是必要的。我们还计划使用shRNA转基因方法来敲低该小鼠脉络膜模型中的短尾畸形表达，以评估短尾畸形失活作为治疗该肿瘤的方法的潜力。

We 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 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. 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, in comparison to its role in primitive streak. 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 have allowed us to test this hypothesis and unravel the possible mechanisms by which this gene may promote tumor formation, and may also give new insights on developing potential therapeutic interventions for chordoma. Surprisingly, our results indicate that Brachyury function is essential for maintaining notochord cell fate, but dispensible for the survival and proliferation of notochord progenitors. A manuscript reporting these results is ccurrently under review. We are also developing a genetic mouse model for chordoma based on activated canonical Wnt pathway in conjunction with p53 tumor suppressor inactivation in the notochord lineage. Such a genetic model will allow us to introduce and evaluate the effects of mutant alleles for tuberous sclerosis complex genes (TSC1,2) which are know to predispose to chordoma in humans, but not in mice, suggesting other factors are also necessary in the pathogenesis of TSC-related chordoma. We also plan to use the shRNA transgene approach to knock-down brachyury expression in this mouse chordoma model, to assess the potential of brachyury inactivation as an approach in treating this tumor.