Abstract
Purpose :
During mammalian retinal development, programmed cell death occurs in large waves in a spatiotemporal fashion to generate functional retinas. In zebrafish, comparably smaller waves have been observed and are thought to represent fine-tuning of developing retinal tissue. It is appreciated that tissue resident macrophages clear apoptotic cells, however, specific roles for macrophage populations in cell survival/death and clearance during retinal development in zebrafish have not been documented. Further, apparent levels of programmed cell death during zebrafish retinal development are based on fixed tissue samples. The purpose of this study was to determine the effects of macrophage depletion and to document clearance of apoptotic cells by macrophage populations during zebrafish retinal development.
Methods :
We used an inducible nitroreductase-mediated system to specifically deplete macrophages/microglia followed by analysis of retinal tissue using confocal microscopy. To directly address clearance of apoptotic cells in real-time, we used live confocal imaging of transgenic zebrafish with mpeg1-driven fluorescent transgene together with apoptotic cells (labeled by acridine orange (AO) or a transgenic reporter of apoptosis). Mpeg1+ cells were also imaged in XOPS:mCFP transgenic embryos, in which developing rods die due to a toxic transgene. Time-lapse movies were quantitatively analyzed.
Results :
Depletion of mpeg1+ cells resulted in increased numbers of apoptotic cells in the retina compared to controls (n=3-5, p=4.4x10-7). In normal developing retinas with intact mpeg1+ populations (n=6), mpeg1+ cells were directly observed to sense and phagocytose cell bodies prior to AO incorporation, and engulfed AO+ cells underwent dynamic movements (avg. 1.5 μm/min, displacement of 5-35 μm) as mpeg1+ cells continued active migration, with the duration of AO+ signal lasting an average of 45 min. In XOPS:mCFP retinas, distribution of mpeg1+ cells was spatially altered in vivo prior to detection of apoptosis in fixed tissue.
Conclusions :
These findings indicate rapid sensing and clearance of apoptosis-fated neurons by macrophage populations during zebrafish retinal development, suggesting that levels of apoptosis in developing zebrafish retinal tissue may be greater than previously appreciated, and that mpeg1+ cells are the main cell type responsible for such clearance.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.