Investigating the function of Hindsight/RREB-1 in the intestinal stem cell lineage of Drosophila

干细胞是未分化的细胞，既有自我更新的能力，也有分化为特化细胞类型的能力。了解成体干细胞的调节是一个非常活跃的研究领域，使用干细胞治疗损伤或疾病的可能性代表了“再生医学”的新概念的基础。*直到最近才被证实，尽管果蝇(果蝇)的寿命只有45-60天，但干细胞储存在分化的成体组织中。例如，与哺乳动物一样，肠道上皮中陈旧或受损的分化细胞不断脱落和丢失，这些细胞的更替取决于遍布肠道上皮的一群肠道干细胞(ISCs)的活动。有趣的是，许多调控ISCs的基因和机制在果蝇和哺乳动物之间是保守的。利用果蝇进行研究有几个优势，包括强大而复杂的基因技术，这些工具现在可以应用于有关干细胞功能和行为的基本问题。*在果蝇的中肠，ISCs分裂产生一个保留ISC身份的子细胞和第二个固定的子细胞，称为肠母细胞(EB)。EB的分化不需要进一步的细胞分裂，EBS可以成为吸收的肠细胞(ECs)或分泌性的肠内分泌细胞(EES)。我的NSERC资助的研究包括研究C2H2型锌指转录因子的功能，这种转录因子是由基因后见之明编码的。我们最近发现，后见之明在所有类型的中肠细胞中都有表达。然而，与不同的EC相比，ISCS、EBS和EES的后见之明水平较低。此外，我们还发现，RNAi介导的后见之明的枯竭会导致所有ISCs的丧失，而且，引人注目的是，提高后见之明的水平对于EC的分化既是必要的，也是充分的。*这一建议概述了实验，以阐明后见之明在ISCs和分化的EC中的作用。例如，为什么ISC维护需要事后诸葛亮？当ISC“迷失”时，他们会发生什么？后见之明在成熟分化的ECs中的作用是什么？除了解决这些问题，我们还建议进行实验，以更好地了解后见之明的功能，后见之明是人类RAS反应元件结合蛋白-1(RREB-1)的果蝇同源物。有一些证据表明，后见之明会被磷酸化改变，我们建议研究这种改变对后视力功能的影响。RREB-1与胰腺癌、甲状腺癌和结肠癌有关--在果蝇中对其同源物的基础研究不仅将促进对RREB-1在人类中功能的理解，而且最终可能有助于开发治疗这些癌症的新方法。**

Stem cells are undifferentiated cells that have the ability of self-renewal as well as the capacity to differentiate into specialized cell types. Understanding the regulation of adult stem cells is a very active area of research, and the possibility of using stem cells to treat injury or disease represents the foundation of the new concept of "regenerative medicine".***It has only recently been established that the fruit fly (Drosophila), despite having a lifespan of only 45-60 days, houses stem cells within differentiated adult tissues.  For example, as in mammals, old or damaged differentiated cells in the gut epithelium are constantly shed and lost, and the replacement of these cells depends on the activity of a population of intestinal stem cells (ISCs) that reside throughout the gut epithelium. Interestingly, many genes and mechanisms regulating ISCs are conserved between Drosophila and mammals. Research using Drosophila has several advantages, including powerful and sophisticated genetic techniques, and these tools can now be applied to fundamental questions regarding the function and behaviour of stem cells.*** In the Drosophila midgut, ISCs divide to produce one daughter cell that retains the ISC identity and a second committed daughter cell known as an enteroblast (EB). EB differentiation occurs without further cell division, and EBs may become either absorptive enterocytes (ECs) or secretory enteroendocrine cells (EEs). My NSERC-funded research has involved investigating the function of the C2H2 type Zinc-finger transcription factor encoded by the gene hindsight. We recently found that hindsight is expressed in all midgut cell types. ISCs, EBs, and EEs, however, are associated with lower levels of Hindsight than differentiated ECs. In addition, we have found that RNAi-mediated depletion of Hindsight results in the loss of all ISCs, and, strikingly, that increasing the level of Hindsight is both necessary and sufficient for EC differentiation.*** This proposal outlines experiments to elucidate the role of Hindsight in ISCs and differentiated ECs. For example, why is Hindsight required for ISC maintenance? What happens to ISCs as they are "lost"? And what is the function of Hindsight in mature differentiated ECs? In addition to addressing these questions, we also propose experiments to better understand the function of Hindsight, which is the Drosophila homologue of human Ras Responsive Element Binding protein-1 (RREB-1). There is some evidence that Hindsight is modified by phosphorylation, and we propose investigating how Hindsight function might be affected by such modification. RREB-1 has been implicated in pancreatic, thyroid, and colon cancer - and basic research of its homologue in the Drosophila will not only advance the understanding of RREB-1 function in humans, but may ultimately contribute to developing novel approaches for treating these forms of cancer. **