Novel features of Drosophila hematopoiesis uncovered by long-term live imaging

Stem cells are subject to continuous regulation to ensure that the correct balance between stem cell differentiation and self-renewal is maintained. The dynamic and ongoing nature of stem cell regulation, as well as the complex signaling microenvironment in which stem cells are typically found, means that studying them in their endogenous environment in real time has multiple advantages over static fixed-sample approaches. We recently described a method for long-term, ex-vivo, live imaging of the blood progenitors in the Drosophila larval hematopoietic organ, the Lymph Gland (LG). This methodology has allowed us to analyze multiple aspects of fly hematopoiesis, in real time, in a manner that could not be carried out previously. Here, we describe novel insights derived from our quantitative live imaging approach. These insights include: the identification of extensive filopodia in the progenitors and description of their morphology and dynamics; visualization and quantitative analysis of JAK/STAT signaling in progenitors by the simultaneous tracking of thousands of vesicles containing internalized Domeless receptors; quantitative analysis of the location, morphology, and dynamics of mitochondria in blood progenitors; long-term tracking of patterns of cell division and migration of mature blood cell in the LG; long-term tracking of multiple cell behaviors in the distal committed progenitors; analysis of Ca²⁺ signaling of blood progenitors in the secondary lobes of the LG. Together, these observations illustrate the power of imaging fly hematopoiesis in real time and identify many previously undescribed processes and behaviors in the LG that are likely to play important roles in the regulation of progenitor differentiation and self-renewal.