Abstract
Purpose :
Dorsal patterning of the retina is established, in part, by the T-box transcription factors, Tbx2 and Tbx5. Both are expressed in the dorsal retina when the optic vesicle develops into the optic cup. Loss or misexpression of either Tbx2 or Tbx5 causes abnormal eye formation in mouse, chick and fish. The role in optic cup formation for a third T-box gene, Tbx3, with a similar expression pattern as Tbx2/Tbx5, has not been determined. The purpose of this study was to find out if Tbx3 was also required for normal optic cup patterning in mouse.
Methods :
We crossed BAC-Dkk3-CREmice to mice with a conditional allele of Tbx3. Tbx3 is detected in the dorsal optic cup at E10.5. In BAC-Dkk3-CREmouse retina, CRE activity is first detected in the central retina at E10.5 and is detected throughout the optic cup by E13.5. We used western blot analysis on P0 retinal tissue to determine Tbx3 loss. We immunostained sections of frozen tissue and retinal flat mounts of postnatal animals using standard techniques. Samples were coded and counted in double-blind experiments. Statistical significance was determined using Prism v6.0 software.
Results :
We observed that Tbx3 was significantly reduced in retinal tissue from conditional knockout mice at P0. We also found the retinas of conditional knockout mice had statistically thinner optic nerves. Counting Islet1/2-positive retinal ganglion cells (RGCs), we found a 33% reduction in the number of dorsal RGCs at P9, which lead to less axon bundles at P26. Previous studies have shown that metabolites produced by RGCs drive blood vessel formation. Consistent with less dorsal ganglion cells in our mutants, we observed a dramatic lag in primary blood vessel formation at P9 (percent surface area of IB4-positive endothelial cells: wildtype, 94±1.7%; mutant, 47±2.3%), which lack superficial blood vessels in the dorsal/temporal region at P26.
Conclusions :
We showed that Tbx3 is required for normal optic cup development. Specifically, removal of Tbx3 causes a loss of retinal ganglion cells and blood vessels in the dorsal retina. Future studies will determine the cellular mechanism underlying retinal ganglion cell loss. We will also determine if Tbx3 affects the formation of retinal blood vessels directly or through a retinal ganglion cell-dependent mechanism.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.