Abstract
Purpose :
Microglia derive from erythromyeloid precursors in the yolk sac and migrate to the retina at embryonic stages. Despite their ability to regulate proliferation, differentiation and survival in other contexts, their role in the developing retina is unknown. Here, we investigate microglia function during embryonic mouse retina development.
Methods :
Using B6.CX3CR1gfp/+ mice of both sexes we assess microglia distribution by immunostaining and cell counts at e12.5, e16.5 and P0. We pharmacologically inhibit microglia activity by administering minocycline (120mg/kg) to pregnant dams. To deplete microglia we conditionally knockout Csf1r in Cx3cr1-expressing microglia by administering 3mg tamoxifen to pregnant dams and analyze Cx3cr1creERT2;Csf1rfl/fl embryos versus control Csf1rfl/fl embryos lacking Cre. We analyze retinal ganglion cell (RGC) density at e14.5, e16.5 and P0 by Brn3 immunostaining. We performed immunostaining for cleaved caspase 3 (CC3) to assess cell death, and BrDU and EdU labeling to assess RGC genesis. We use qRT-PCR to analyzed changes in microglial gene expression after inhibition. To test the role of complement signaling we analyze knockout mice lacking complement receptor 3 (CR3; Itgam).
Results :
The majority of microglia in embryonic retina were directly contacting Brn3+ RGCs. Depletion of microglia results in a 22% increase in RGC density at e14.5 (n=5, p<0.001) that was sustained at birth (n=5, p<0.01). Pharmacological inhibition also results in increased RGCs (n=6, p=0.03), with no significant effect on retinal progenitor proliferation, RGC genesis, apoptosis, or other cell types. Microglia in the embryonic retina were enriched for phagocytic markers and we observe engulfment of non-apoptotic (CC3-) Brn3+ RGCs. By qPCR (n=8) we test microglia cell engulfment pathways and find selective downregulation of complement genes Cd11b (p<0.01), C1q (p<0.01) and C3 (p=0.011) with microglia inhibition, and show that C1q protein marks a subset of RGCs in the embryonic retina. Knockout of complement receptor 3 (CR3; Itgam), which is only expressed by microglia, results in increased RGC density at birth (n=10, p<0.01), similar to that observed after depletion or inhibition of microglia.
Conclusions :
These findings reveal a new mechanism of RGC cell elimination during retina development and suggest a role for complement in mediating the phagocytosis of non-apoptotic newborn RGCs by microglia.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.