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

主调节基因靶标之间的遗传相互作用作为果蝇肠道干细胞复杂行为的驱动因素

• 批准号: 10629992
• 负责人: Mariano A Loza Coll
• 金额: $ 14.75万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629992
• 关键词: Address; Affect; Automobile Driving; Behavior; Binding; Biological Assay; Biological Models; Biomedical Research; Cell Count; Cell Differentiation process; Cell Therapy; Chemical Injury; Combined Modality Therapy; Complement; Complex; Critical Pathways; DNA; Development; Drosophila genus; Drosophila melanogaster; Enterocytes; Enteroendocrine Cell; Epidermal Growth Factor Receptor; Experimental Models; Family; Fostering; Future Generations; Gastrointestinal Diseases; Gene Targeting; Genetic; Genetic Transcription; Goals; Hormone secretion; Image; Immunofluorescence Microscopy; Individual; Infection; Injury; Intervention; Intestines; Investments; Knowledge; Longevity; MAPK8 gene; Maps; Midgut; Minority; Mission; Mitotic; Molecular; Morphology; Organ; Outcome; Pathogenicity; Pathway interactions; Phenotype; Regenerative Medicine; Regenerative capacity; Regulation; Regulator Genes; Regulatory Pathway; Reporter; Research; Research Personnel; Research Project Grants; STAT protein; Science; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Sister; Snails; Stains; Statistical Data Interpretation; Students; System; Testing; Therapeutic; Tissues; Toxic effect; Underrepresented Students; United States National Institutes of Health; Universities; adult stem cell; automated image analysis; body system; cell behavior; cell type; combinatorial; design; educational atmosphere; gene conservation; genome-wide; improved; improved outcome; in vivo; innovation; intestinal barrier; intestinal homeostasis; model organism; notch protein; novel strategies; premature; prevent; public trust; regeneration function; regeneration potential; self-renewal; side effect; stem cell function; stem cell proliferation; stem cell therapy; stem cells; therapeutic candidate; therapeutic target; therapy development; tissue stem cells; training opportunity; transcription factor; undergraduate student

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.

项目摘要 几种主调节元（MR）基因在器官的组织干细胞中的表征 物种。但是，他们仍然是较差的治疗操作的候选人 多效性，影响数百个目标，并在许多器官系统中运行。因此，只有一个 更好地理解多效性下游MR靶标坐标干细胞增殖的程度 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 该项目的长期目标是绘制MR Genes及其建立的监管景观 肠道干细胞（ISC）中的靶标，使用果蝇果蝇黑色素果蛋白酶作为模型系统。 果蝇ISCS不对称地划分，引起了新的ISC和一个将成为一个姐姐 吸收性肠球菌（EC）或分泌激素的肠内分泌细胞（EE）。特定的假设 推动此建议的是，MR目标途径之间的交叉调节相互作用可能导致非 同时对ISC行为进行线性，不可预测的结果。测试这个 假设，CAP，KLAROID和INDY，三个经过实验验证的ISC MR基因的靶标 和STAT，将使用诱导的GAL4/UAS系统单独或在ISC中进行操纵。 他们的个人与组合操作对肠内稳态的影响将通过 三种单独但互补的方法。在AIM 1中，将使用免疫荧光显微镜 比较ISC数，形态学，有丝分裂率和分化潜力，基于良好的 细胞类型标记（ISC的ESG-GFP，EB，PDM1的SU（H）激活EC和EES的ProS染色， 分别）。通过ImageJ和Cell -Profiler的自动图像分析将用于分析多个 每组图像，允许对数据进行强大的统计分析。在AIM 2中，荧光活动报告家 将用于比较单个与双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AIM 3中，寿命和肠屏障 完整性（蓝精灵）测定将用于比较单个与联合MR目标的效果 操纵具有化学损伤后肠道组织的再生能力 致病感染。这些研究的目的可能会产生证据，以挑战广泛持有的 组合疗法只能由于加性互补而改善结果的前提 积极影响。如果是这样，该项目将对我们的干细胞概念方法产生重大影响 进行再生医学的操纵。此外，该项目是专门设计的 来自生物医学研究中代表性不足背景的大量学生满足了另一位 NIH的重要使命：使科学劳动力多样化，从而促进创新，改善 研究质量并提高公众对科学的信任和投资。