Abstract
Purpose :
Both microglia and Müller glia have been shown to phagocytose dying cells in the vertebrate retina, but the phagocytic activities and respective loads of these two cell types appear to differ depending on context. In the zebrafish retina, microglia appear to act as the primary phagocytes during development (Blume et al., 2020, Dev Dyn) while both cell types may phagocytose dying cells in contexts of disease or injury. Müller glia (MG) act as the source of regenerated retinal neurons in zebrafish, and there is a possible link between MG phagocytic activity and the regenerative response. In addition, microglia appear to influence the outcome of retinal regeneration though their functions in this regard are not well understood. We hypothesized that in the absence of microglia, Müller glia take on the primary phagocytic role to clear apoptotic cells during retinal development and that increased phagocytic load may result in changes in MG-expressed genes known to be associated with damage and regenerative responses.
Methods :
Developing retinas from microglia deficient mutant zebrafish and wildtype (wt) siblings were stained and imaged by confocal microscopy to visualize apoptotic cells (TUNEL) and MG (GS). Total numbers of TUNEL+ cells and those phagocytosed by MG were quantified in whole retinas (n=11-13). To analyze MG entry into the cell cycle, retinal cryosections were stained for PCNA and GS and the total number of PCNA+MG were quantified in images from microglia-deficient and wt siblings (n=4-6). Expression of gfap and ascl1a were determined by qPCR (1 pooled sample from 5-10 embryos per genotype).
Results :
We found increased accumulation of TUNEL+ cells in microglia deficient retinas compared to wt (p<0.0001), and increased numbers but not proportions of TUNEL+ cells engulfed by MG (p<0.0001). There was a modest increase in numbers of PCNA+MG in microglia deficient retinas (n=4-6, p=0.008). Preliminary data indicated a modest increase in gfap (~1.8 fold), but not ascl1a in microglia deficient retinas compared to wt.
Conclusions :
In the absence of microglia, Müller glia increase phagocytic load to clear dying cells from the developing retina. However, it appears they are less efficient than microglia at clearance of engulfed cells. Increased phagocytic activity may induce Müller glia proliferation and increase expression of gfap, however phagocytosis alone does not induce a robust regenerative response.
This is a 2021 ARVO Annual Meeting abstract.