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

干细胞是具有自我更新能力以及分化成专门细胞类型的能力的未分化细胞。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”新概念的基础。直到最近才确定，尽管果蝇（Drosophila）的寿命只有45-60天，但在分化的成体组织中含有干细胞。 例如，如在哺乳动物中，肠上皮中的旧的或受损的分化细胞不断脱落和丢失，并且这些细胞的替换取决于存在于整个肠上皮中的肠干细胞（ISC）群体的活性。 有趣的是，许多调控ISC的基因和机制在果蝇和哺乳动物之间是保守的。 使用果蝇的研究有几个优势，包括强大而复杂的遗传技术，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。在果蝇中肠中，ISC分裂产生一个保留ISC身份的子细胞和第二个被称为肠母细胞（EB）的定向子细胞。 EB分化发生而没有进一步的细胞分裂，EB可以成为吸收性肠上皮细胞（EC）或分泌性肠内分泌细胞（EE）。 我的NSERC资助的研究涉及调查由基因后见之明编码的C2 H2型锌指转录因子的功能。 我们最近发现，后见之明在所有中肠细胞类型中表达。 然而，与分化的EC相比，ISC、EB和EE的后见之明水平较低。 此外，我们还发现RNAi介导的后见之明的缺失导致所有ISC的丧失，并且引人注目的是，增加后见之明的水平对于EC分化是必要的和足够的。该建议概述了实验，以阐明后见之明在ISC和分化的EC中的作用。 例如，为什么ISC维护需要后见之明？当ISC“丢失”时，它们会发生什么？ 后见之明在成熟分化的内皮细胞中起什么作用？ 除了解决这些问题，我们还提出了实验，以更好地了解后见之明的功能，这是果蝇同源的人Ras反应元件结合蛋白-1（RREB-1）。 有一些证据表明，后见之明被磷酸化修饰，我们建议调查后见之明功能可能会受到这种修饰的影响。 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，而且可能最终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. **