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

项目总结 几个主调节(MR)基因已经在跨器官的组织干细胞中得到了表征 物种。然而，它们仍然是治疗操作的糟糕候选者，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 更好地理解下游多效性较低的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。这个 这个项目的长期目标是绘制出吉恩斯先生和他们的 以果蝇黑腹果蝇为模型系统的肠道干细胞(ISCs)靶点的研究。 果蝇的ISCs不对称分裂，产生了一个新的ISC和一个将成为 可吸收的肠细胞(EC)或分泌激素的肠内分泌细胞(EE)。具体的假设 推动这一提议的是MR靶通路之间的交叉调控相互作用可以导致非 当它们同时被操纵时，ISC行为的线性、不可预测的结果。为了测试这一点 假说，CAP，Klarid和Indy，ISC MR基因的三个实验验证靶点 和STAT，将在ISCS内使用可诱导的GAL4/UAS系统单独或组合操作。 他们单独和联合操作对肠道内环境平衡的影响将通过以下方式进行评估 三种独立但相辅相成的方法。在目标1中，将使用免疫荧光显微镜 比较ISC数量、形态、有丝分裂率和分化潜能 细胞类型标记物(ISCs的ESG-GFP，EBS的SU(H)激活，ECs和EES的Pdm1和Pros染色， )。通过ImageJ和CellProfiler进行的自动图像分析将用于分析 每组图像，允许对数据进行可靠的统计分析。在Aim 2中，荧光活动记者 将用于比较单MR目标和双MR目标操作对关键ISC信号的影响 转导途径(EGFR、Notch、Wnt、STAT和JNK)。在目标3中，寿命和肠道屏障 完整性(SMurf)分析将用于比较单独和联合MR靶点的效果 手法对化学性损伤后肠组织再生能力的影响 病原体感染。这些研究目的可能会产生证据，挑战人们普遍持有的 前提是联合治疗只能通过相加补充才能改善结果 积极的影响。如果是这样的话，这个项目将对我们研究干细胞的概念方法产生重大影响。 再生医学的手法。此外，这个项目是专门设计的，目的是让 大量来自生物医学研究背景不足的学生，满足了另一个 NIH的重要使命：使科学劳动力多样化，从而促进创新，改进 提高科研质量，增强公众对科学的信任和投入。

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.