Abstract
Purpose :
Microglia (MiG) are established phagocytes. In contrast, Müller glia (MG) have a lesser-known phagocytic role. We previously demonstrated compensatory phagocytosis by MG in absence of MiG (Thiel et al., 2022). We used real-time imaging in living zebrafish larvae to uncover dynamic interactions of MiG, MG, and dying cells, in wild-type and in context of increased rod cell death. We also examined MiG/MG expression patterns of selected phagocytic receptors. We hypothesized the revelation of previously undescribed interactions between glia and dying cells and contextual insight into such dynamics.
Methods :
Real-time imaging was performed using fluorescent confocal microscopy (5 sessions, 8-10 hrs, 3-min intervals). Glial response to apoptotic cells was imaged in wild-type (WT) larvae expressing transgenic reporters for MG, MiG, and apoptotic cells and in the XOPS:mCFP rod degeneration system. We quantified first engagement and final engulfment of dying cells/rods by glial cell type. Phagocytic receptor expression was examined by fluorescent in-situ hybridization in WT and MiG-deficient larvae (n=3-6).
Results :
Surprisingly, during normal retinal development Müller glia first engage 55-80% of dying cells through extension of cellular processes, at times remarkably long and dynamic, for encapsulation and retraction; however, microglia commandeer most of their cargo (50-75%). Ultimately, 52-84% of targets are terminally engulfed by MiG while the rest remain in MG. In the XOPS:mCFP system, conversely, MiG dominate first contact with dying rods (55-75%) and a smaller proportion of MG cargo is transferred to MiG (15-55%). Final engulfment of rods remains dominated by MiG (75-85% vs MG 15-25%). MiG expressed multiple PtdSer receptors (axl, mertka, havcr1) as well as complement receptors itgb2 and itgam.1. In WT retinas MG express low levels of mertka but increase expression of mertka and itgb2 in MiG-deficient retinas, consistent with compensatory phagocytosis.
Conclusions :
Our experiments reveal coordinated interactions between MiG and MG, with MG displaying an active and surprisingly dynamic role in cell clearance for the apparent primary purpose of holding cargo for microglial clearance. With increased rod cell death, both cell types increase phagocytic load. Expression of phagocytic receptors showed redundancy in PtdSer receptors for MiG and limited receptors for MG.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.