Abstract
Purpose :
Muller glia (MG) are promising targets for retinal regeneration, due to their ability to reprogram into progenitor-like cells (MGPCs) and differentiate into retinal neurons in lower vertebrates. Chicks have a limited capacity for regeneration: retinal damage induces MGPCs, but very few go on to regenerate neurons. Inhibitor of Differentiation (Id) transcription factors show unique patterns of expression in chick Muller glia and MGPCs. We hypothesize that a) Ids suppress Muller glia cell cycle exit and differentiation, and b) Ids can be targeted to enhance the number of MG progeny that become neuron-like.
Methods :
Young Leghorn (n=16, P7) chicks were intraocularly injected with 73 ug NMDA, and 2 ug EdU 0-2 DPI (days post injury). A small-molecule pan-Id antagonist (AGX-51, 10 ug) was applied to the right eye, while the left (control) eye received the drug vehicle. In 7 chicks, AGX-51 was applied 1 day prior to NMDA; retinas from these chicks were harvested 3 DPI and immunolabeled for Sox2 & EdU to quantify proliferating MGPCs. In 9 chicks, AGX-51 was applied 3-5 DPI; retinas were harvested 11 DPI and labeled for neuronal markers & EdU to quantify regenerated neuron-like cells. Undamaged retinas from (n=6, P7) chicks were also collected 4 hours after AGX-51 treatment to assess early changes in gene expression and protein phosphorylation. Statistical analyses were conducted using two-tailed paired t-tests.
Results :
Application of the Id inhibitor prior to damage decreased the number of MGPCs (17.71±5.22) compared to control retinas (46±10.57, p<0.001). Significantly, application of the Id inhibitor after MGPC induction increased the abundance of EdU+HuD+ amacrine-like cells (3.9±1.8) over those found in control retinas (0.9±0.7, p<0.02). In addition, we found that Id inhibition influenced levels of cFos, p21/CDKN1A, glutamine synthetase, and Sox2 in Muller glia.
Conclusions :
Our findings indicate that Id activity is necessary for MG to dedifferentiate and proliferate, but that Ids also suppress the differentiation of MGPCs into neurons. Id inhibition appears to drive both MG quiescence and suppress neuronal differentiation by upregulating immediate early genes and cell-cycle inhibitors. Targeting Ids is a promising strategy for enhancing the formation of proliferating neurogenic MGPCs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.