Distinct Roles for E2f1 and E2f3a in Mediating Division, Death and Differentiation Defects in Rb-Deficient Mouse Retina

Abstract

Purpose:: Rb inhibits E2fs to block proliferation and death, but is thought to potentiate E2f-independent tissue-specific factors to promote differentiation of some tissues such as muscles and bones. In retina it is unclear whether Rb loss perturbs maturation directly or indirectly by disrupting cell cycle exit and/or survival. To resolve this issue it is critical to study differentiation of RbKO cells in the absence of ectopic proliferation and death. The purpose of this study was to investigate the differentiation defects independent of cell cycle or cell death, in RbKO mouse retina, and their underlying mechanisms.

Methods:: We use Cre-Loxp methods to conditionally delete Rb or E2f3 gene in mouse retina. Rb conditional KO mice were also crossed with E2f1, E2f2 or E2f3a germline KO mice to determine whether defects observed in Rb-deficient mice could be rescued by inactivating individual E2fs. Retinal cell proliferation, cell death and differentiation were monitored by Brdu/PH3 labeling, TUNEL labeling and cell type marker immunofluorescence. ERG was used to measure the retinal function.

Results:: E2f1 deletion suppresses division and death in the Rb-deficient retina and, unexpectedly, major cell types, including rod photoreceptors, differentiate and function normally. However, in the rescued Rb/E2f1-double null retina, we uncovered a differentiation defect in cholinergic amacrine cells that, remarkably, reflects a cell-cycle-independent effect of E2f3. E2f3b is thought to mediate Rb function in quiescent cells, but in the first work to reveal E2f isoform function in vivo we show that E2f3a inhibition is critical for SAC differentiation.

Conclusions:: Rb does promote neurogenesis directly, but rather than potentiating tissue-specific factors, it facilitates both cell cycle exit and differentiation independently through distinct E2fs.

Keywords: retina • development • retinoblastoma