Genetic interactions among targets of master regulator genes as drivers of complex behavior in Drosophila intestinal stem cells

项目摘要 几个主调节因子（MR）基因已经在跨器官的组织干细胞中被表征， 物种然而，它们仍然是治疗操作的不良候选者，因为它们高度 多效性，影响数百个目标，并在许多器官系统中运作。因此，只有A 更好地理解较少的多效性下游MR靶点如何协调干细胞增殖， 自我更新和分化将释放干细胞在再生医学中的全部潜力。的 该项目的长期目标是绘制MR基因及其 靶向肠干细胞（ISCs），使用果蝇作为模型系统。 果蝇的ISC不对称分裂，产生一个新的ISC和一个姐妹ISC，后者将成为一个新的ISC。 吸收性肠上皮细胞（EC）或分泌唾液酸的肠内分泌细胞（EE）。具体假设 推动这一提议的是，MR靶通路之间的交叉调节相互作用可能导致非- 当它们同时被操纵时，对ISC行为的线性、不可预测的结果。为了验证这一 假设，CAP，Klaroid和Indy，ISC MR基因的三个实验验证的靶标 和STAT将单独或在ISC内使用诱导型Gal 4/UAS系统组合操作。 将通过以下方式评估其单独与联合操作对肠道稳态的影响： 三种不同但互补的方法。在目标1中，将使用免疫荧光显微镜 比较ISC的数量、形态、有丝分裂率和分化潜能， 细胞类型标记（ISC的esg-GFP，EB的Su（H）活化，EC和EE的Pdm 1和Pros染色， 分别）。通过ImageJ和CellProfiler进行的自动图像分析将用于分析多个 每组图像，允许对数据进行稳健的统计分析。在目标2中，荧光活性报告基因 将用于比较单与双MR靶点操作对关键ISC信号的影响 转导途径（EGFR、Notch、Wnt、STAT和JNK）。在目标3中，寿命和肠屏障 完整性（Smurf）测定将用于比较单个与组合MR靶点的效果 操作对化学损伤后肠组织再生能力的影响， 病原体感染这些研究目标可能会产生证据，挑战人们普遍认为的 前提是联合治疗只能改善结果，这是由于 积极的影响。如果是这样的话，这个项目将对我们干细胞的概念方法产生重大影响 用于再生医学此外，该项目是专门为参与 大量来自生物医学研究背景的学生，满足了另一个 国家卫生研究院的一项重要使命：使科学劳动力多样化，从而促进创新，提高 提高研究质量，增强公众对科学的信任和投资。

PROJECT SUMMARY Several master regulator (MR) genes have been characterized in tissue stem cells across organs and species. However, they remain poor candidates for therapeutic manipulation because they are highly pleiotropic, affecting hundreds of targets and operating across many organ systems. Therefore, only a better understanding of how less pleiotropic downstream MR targets coordinate stem cell proliferation, self-renewal and differentiation will unlock the full potential of stem cells in regenerative medicine. The long-term goal of this project is to map the regulatory landscape established by MR genes and their targets in intestinal stem cells (ISCs), using the fruit fly Drosophila melanogaster as a model system. Drosophila ISCs divide asymmetrically, giving rise to a new ISC and a sister that will become an absorptive enterocyte (EC) or a hormone-secreting enteroendocrine cell (EE). The specific hypothesis driving this proposal is that cross-regulatory interactions between MR target pathways can lead to non- linear, unpredictable outcomes on ISC behavior when they are manipulated simultaneously. To test this hypothesis, CAP, Klaroid and Indy, three experimentally validated targets of the ISC MR genes Escargot and STAT, will be manipulated alone or in combination within ISCs using an inducible Gal4/UAS system. The effect of their individual vs. combined manipulations on intestinal homeostasis will be assessed via three separate but complementary approaches. In Aim 1, immunofluorescence microscopy will be used to compare ISC number, morphology, mitotic rate, and differentiation potential, based on well-established cell type markers (esg-GFP for ISCs, Su(H) activation for EBs, Pdm1 and Pros staining for ECs and EEs, respectively). Automated image analysis through ImageJ and CellProfiler will be used to analyze multiple images per group, allowing a robust statistical analysis of the data. In Aim 2, fluorescent activity reporters will be used to compare the effect of single vs. dual MR target manipulations on key ISC signal transduction pathways (EGFR, Notch, Wnt, STAT and JNK). In Aim 3, lifespan and intestinal barrier integrity (Smurf) assays will be used to compare the effect that individual vs. combined MR target manipulations have on the regenerative capacity of intestinal tissue following chemical injury or pathogenic infection. These research aims may generate evidence that challenges the widely held premise that combination therapies can only improve outcome due to additive complementation of positive effects. If so, this project will have a significant impact on our conceptual approach to stem cell manipulation for regenerative medicine. In addition, this project was specifically designed to engage a large number of students from underrepresented backgrounds in biomedical research, satisfying another important mission of the NIH: to diversify the scientific workforce, and thus foster innovation, improve research quality and enhance the public trust and investment in science.