Mechanisms governing stem cell coordination by the niche

项目概要/摘要 成体干细胞之间的协调对于维持组织稳态和防止 肿瘤的过度生长许多结构，包括毛囊、造血网络和 发育中的卵巢需要严格控制干细胞增殖和协调子细胞 从不同的干细胞谱系产生细胞。在大多数情况下， 这种协调在很大程度上是未知的。利用果蝇的力量 遗传学和建立系统的纵向（20+小时）活成像的干细胞内 我们已经开始揭示控制干细胞的机制， 睾丸的协调性。睾丸的体细胞和生殖系干细胞（GSC）必须 以精确的2：1比例产生子细胞，使生殖细胞有效地分化为精子。 我们的实时成像显示，GSC中修改的胞质分裂程序是 只有在一个GSC子系统与两个子系统正确关联后， 体干细胞谱系。这种改良的胞质分裂程序被控制在两个阶段： 由来自小生境的Jak/STAT信号传导调节的暂停和完成 来自体干细胞的胞质分裂。两个控制点都必须正确执行 或者干细胞胞质分裂失败，形成干细胞肿瘤，生殖细胞不能分化。虽然我们 已经确定了暂停和触发的来源，这些机制 控制GSC胞质分裂的信号仍然未知。在父母补助金中，我们建议审问 生态位信号和体干细胞联合收割机调节的特定机制 使用分子遗传学和延长寿命成像的GSC胞质分裂。在此，我们要求 采购快速和超分辨率激光扫描的行政补充 共聚焦显微镜，这将提高成像能力，质量和数据输出 （生产力）我们的R 01资助的项目。新仪器将使固定的成像 更快的活睾丸，更少的光损伤以及前所未有的分辨率（100 nm 横向，200纳米轴向）。这种增强的分辨率将有助于鉴定改变的F-肌动蛋白 在Aim 1中研究的GSC-子代对之间的细胞间桥结构 家长补助在我们的时间动力学研究中， GSC-子细胞间桥（父母资助的目标2），新的仪器将使 在2D和3D中以前所未有的时空分辨率跟踪ESCRT机器， 具有上级量子效率的探测器。通过使用先进的调制，如 动态增强和自适应图像质量的确定和重建，我们将 能够以更高的分辨率获得高质量的延时数据。这是与 从多个（最多三个）波长获取图像的能力 我们的工作解剖体细胞和生殖细胞之间的复杂相互作用的基本特征 干细胞通过两个群体的活体成像（父母资助的Aim 3）。结果将脱落 对干细胞中小生境调节的胞质分裂修饰的有价值的见解。另外这款 这项工作将提供第一个实时的，高分辨率的分析干细胞的相互作用， 内源性生态位是维持组织稳态所必需的。

Project Summary / Abstract Coordination between adult stem cells is essential to maintain tissue homeostasis and prevent tumorous overgrowth. Many structures, including the hair follicle, hematopoietic network and developing ovary require tight control over stem cell proliferation and coordination of daughter cell production from distinct stem cell lineages. In most cases, the molecular mechanisms orchestrating this coordination are largely unknown. Leveraging the power of Drosophila genetics and establishing a system for longitudinal (20+ hours) live imaging of stem cells within an endogenous niche we have begun to reveal the mechanisms controlling stem cell coordination in the testis. Somatic stem cells and germline stem cells (GSCs) of the testis must generate daughters in a precise 2:1 ratio for germ cells to effectively differentiate into sperm. Our live imaging has revealed a modified cytokinesis program in GSCs as the mechanism to coordinate release of one GSC daughter only after it correctly associates with two daughters of the somatic stem cell lineage. This modified cytokinesis program is controlled at two stages—a pause regulated by Jak/STAT signaling from the niche and a trigger for completion of cytokinesis derived from the somatic stem cells. Both control points must be properly executed or stem cell cytokinesis fails, stem cell tumors form and germ cells fail to differentiate. While we have identified the source of both the pause and trigger, the mechanisms by which these signals control GSC cytokinesis remain unknown. In the parent grant, we propose to interrogate the specific mechanisms by which niche signals and somatic stem cells combine to regulate GSC cytokinesis using molecular genetics and extended live imaging. Here, we request an administrative supplement for the acquisition of fast and super-resolution laser scanning confocal microscopy, which will improve the imaging capabilities, quality and data output (productivity) of our R01-funded projects. The new instrumentation will enable imaging of fixed and live testes faster and with less photodamage as well as unprecedented resolution (100 nm lateral, 200 nm axial). This enhanced resolution will aid in identification of altered F-actin structure at the intercellular bridge between GSC-daughter pairs investigated in Aim1 of the parent grant. In our studies of temporal dynamics in abscission machinery localization to the GSC-daughter intercellular bridge (Aim2 of parent grant), the new instrumentation will enable tracking of ESCRT machinery with unprecedented spatiotemporal resolution in 2D and 3D using detectors with superior quantum efficiency. Through the use of advanced modulations such as dynamic enhancement and adaptive image quality determination and reconstruction, we will be able to acquire high-quality time-lapse data with improved resolution. This is in conjunction with the ability to acquire images from multiple (up to three) wavelengths simultaneously—an essential feature for our work dissecting the complex interactions between somatic and germline stem cells through live imaging of both populations (Aim3 of parent grant). Outcomes will shed valuable insight into niche-regulated cytokinesis modifications in stem cells. In addition, this work will provide the first real-time, high-resolution analysis of stem cell interactions within an endogenous niche that are essential for maintenance of tissue homeostasis.