Abstract
Purpose :
The transcription factor Tbr2 is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) and is required during development for their viability, however its role in adult ipRGCs is not clear. Additionally, ipRGCs survive better than conventional RGCs (cRGCs) after optic nerve injury for reasons that are not well understood. We tested the following hypotheses: 1) Tbr2 is required for the maintenance of ipRGCs, 2) Tbr2 can induce expression of ipRGC-specific proteins (the photopigment melanopsin) in cRGCs, 3) Tbr2 is necessary for ipRGC survival after optic nerve injury.
Methods :
1) We administered tamoxifen to adult Tbr2CreER/flox;R26tdTomato mice to remove Tbr2 from Tbr2+ RGCs while simultaneously labeling their somas, dendrites, and axons with tdTomato. Controls are littermates lacking the floxed Tbr2 allele (Tbr2CreER/+;R26tdTomato). We analyzed brains (coronal sections) and retinas (flatmounts and sections) >30 days after tamoxifen administration.
2) We injected one eye intravitreally with a virus containing Tbr2 and GFP, and the contralateral control eye with a virus containing GFP alone in wildtype, Tbr2-CKO, Tbr2CreER/flox;R26tdTomato, and Tbr2CreER/+;R26tdTomato mice and analyzed their retinas 2-5 weeks later.
3) We performed the optic nerve crush (ONC) procedure in adult tamoxifen-induced Tbr2CreER/flox;R26tdTomato and control mice and analyzed their retinas 2 weeks later.
Three or more mice were used per experimental condition. Images were taken with an Olympus BX51 microscope. 4-8 fields of view (446.15 μm x 333.33 μm) per retina were imaged for quantification. Cells were manually counted in FIJI. Statistical analyses (Student's t-test or two-way ANOVA) and graph generation were performed using GraphPad’s Prism9 software.
Results :
Loss of Tbr2 in adult Tbr2+ RGCs does not affect their viability but results in loss of melanopsin expression (23.5±10 vs. 95.8±6 cells/mm2, P<0.001). Tbr2 is able to induce melanopsin expression in Tbr2 RGCs (P=0.0156) but not in Tbr2-deficient RGCs (P=0.2651). We find that there is decreased survival of Tbr2-deficient RGCs relative to Tbr2+ RGCs after optic nerve crush (P=0.0153), however, Tbr2-deficient RGC survival is still greater than the survival of cRGCs (P=0.007).
Conclusions :
Our findings demonstrate important roles for Tbr2: its regulation of melanopsin expression and its involvement in RGC survival after ONC.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.