Abstract
Purpose :
Retinal ganglion cells (RGCs) differentiate from multipotent retinal progenitor cells (RPCs); however, the genetic mechanisms are incompletely understood. Atonal homolog 7 (ATOH7) - positive RPCs generate RGCs, yet Atoh7 loss only reduces the RGC population by ~50%, suggesting the existence of an alternative pathway responsible for RGC differentiation. We have previously demonstrated that Tbx3 loss leads to ISLET1/2+ and OPN4+ dorsal RGCs loss. Here, we tested the hypothesis that TBX3 mediates an ATOH7-independent dorsal RGC differentiation pathway
Methods :
Conditional removal of Tbx3 from retinal progenitors was performed (cKO: BAC-Dkk3-Cre+;Tbx3ΔFl/ΔFl). Embryos were collected at the indicated times, cryo-sectioned and analyzed using standard immunohistochemical techniques. Cells were counted in double-blind experiments. Statistical significance was determined using Prism v10.0 software.
Results :
TBX3 expression was detected in differentiating RGCs. Tbx3 loss increased RPC proliferation (Ki67 WT: 1.0±0.017; cKO: 1.40±0.09; p = 0.007), and the number of ATOH7+ RPCs (WT: 0.97±0.05; cKO: 1.16±0.05; p = 0.03) while reducing the number of differentiated (TUBB3/b-tubulin-positive) dorsal RGCs (WT: 1.0±0.05; cKO: 0.74±0.09). Consistent with reduced TUBB3/b-tubulin expression, we detected fewer TBX5+ and RXRΥ+ RGCs. Since Atoh7+ RPCs differentiate into BRN3B+ and ISL1/2+ RGCs, we determined the effect of TBX3 loss on these RGC subpopulations. While the number of BRN3B+ RGCs was unchanged, the ISL1/2 population was reduced in both the E13.5 (WT: 1.0±0.001; cKO: 0.80±0.02; p = 0.01) and P6 dorsal retina (WT: 37.7±2.8 cells/300µm dorsal retina; cKO: 27.7±2.4; p = 0.02).
Conclusions :
We provide evidence that TBX3 is required for the differentiation of a sub-class of dorsal RGCs. These findings support the idea that TBX3 mediates an independent RGC differentiation pathway involving Isl1/2, but not Brn3b RGCs. Future studies will determine the direct targets of TBX3 and the genetic network it controls.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.