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细胞中转录因子brachyury/T的产生，方法是使用慢病毒产生针对brachyury的shRNA，当被引入细胞(或动物)时，导致brachyury/T的丢失。这个新的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。有人假设，短距离在脊索(脊索瘤)引起的癌症的形成中起着核心作用，这一工具也可能为设计新的疗法来治疗这些非常难以治疗的癌症提供动力。我们已经将该shRNA导入转基因小鼠，并利用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。我们已经证明，在短臂缺失突变(T+/-)杂合子的小鼠胚胎中，这种敲除结构的早期激活会复制零胚胎致命表型，并使体轴尾端丧失到前肢水平。现在我们已经证明了使用这种方法可以有效地从细胞和动物中去除短毛蛋白，我们正在利用它来研究短毛蛋白在脊索形成过程中调节生长和细胞命运的正常功能，并与它在原始条纹中的作用进行比较。短缩蛋白的激活被认为是脊索瘤发生的关键，脊索瘤是发生在脊索残留部位的癌症。我们开发的用于研究短臂功能的基因敲除工具使我们能够检验这一假说，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脊索瘤治疗干预措施提供新的见解。令人惊讶的是，我们的结果表明，短缩功能对于维持脊索细胞的命运是必不可少的，但对于脊索祖细胞的生存和增殖是必不可少的。目前正在审查一份报告这些结果的手稿。我们还在开发一种脊索瘤的遗传小鼠模型，该模型基于脊索谱系中激活的规范Wnt通路和p53肿瘤抑制因子的失活。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杂基因(TSC1，2)的突变等位基因的影响，这些基因在人类中被认为是脊索瘤的易感基因，而在小鼠中则不是，这表明其他因素在TSC相关脊索瘤的发病机制中也是必要的。我们还计划使用shRNA转基因方法在这个小鼠脊索瘤模型中下调brachyury的表达，以评估brachyury灭活作为治疗这种肿瘤的方法的可能性。

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.