Mechanisms governing stem cell coordination by the niche

项目摘要/摘要

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.