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Alex Yuan, Rose DiCicco, Brent A Bell, Shiming Luo, Bela Anand-Apte, Brian D Perkins; Misdirected migration of retinal progenitors following photoreceptor injury in zebrafish. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4583. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In zebrafish, focal laser can generate injury to the outer nuclear layer (ONL). Rapid retinal regeneration takes place with restoration of the ONL within 2-3 weeks. In 100% of lesions, cell nuclei can be found in the inner plexiform layer (IPL) up to 15 weeks following injury. In this study, we used bromodeoxyuridine (BrdU) to label proliferating cells and tracked their migration following injury.
An OCT-guided laser was used to ablate photoreceptors in zebrafish. One or 3 days after injury, zebrafish were labeled with 0.25 µmol/g body weight of BrdU (intraperitoneal). Eyes from injected fish were then analyzed by confocal microscopy to determine the pattern of BrdU labeling and presence of BrdU positive cells at intervals up to 14 days following injury. To determine the population of proliferating cells, we also performed immunohistochemistry using anti-PCNA antibody at similar intervals up to 14 days following injury.
Proliferating cells labeled with PCNA on day 1 were found diffusely in all layers of the retina. Proliferating cells labeled with BrdU on day 1 and sectioned on day 3 displayed a similar diffuse pattern. A greater number of proliferating cells were labeled by BrdU and PCNA on day 3 following laser injury. On day 3, proliferating cells labeled by PCNA were found primarily in the inner nuclear layer (INL). Proliferating cells labeled by BrdU on day 3 were found primarily in the INL and ONL by day 4, with fewer in the INL and more in the ONL by day 7. Although a majority of cells labeled with BrdU either remained in the INL or migrated to the ONL, a significant portion of BrdU positive cells were also found in the IPL and ganglion cell layers (GCL). In contrast, PCNA labeling revealed the majority of proliferating cells remained in the INL and ONL but not in the IPL.
Following ONL injury, retinal progenitors in the INL proliferated then migrated predominantly to the ONL where aggregates of microglia rapidly migrated. However, some of these progenitors appeared to migrate towards the IPL and GCL. Many of the misplaced nuclei found in the IPL were labeled by BrdU injected on day 3 following injury, suggesting a migration of retinal progenitors to the IPL. These IPL cells, however were not positive for PCNA suggesting they have lost their stem cell properties and no longer proliferated. Future work to identify the expression profiles of these cells are under way.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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