Abstract
Purpose :
Human vision is largely dependent upon cone function, however death or dysfunction of rods results in secondary loss of cones and blindness. We previously reported genetic models in zebrafish that recapitulate the rod death associated with common forms of inherited retinitis pigmentosa. The purpose of this study was to test the utility of these models to screen small molecules or genetic manipulations to alter the time course of the rod degeneration.
Methods :
The following zebrafish lines were used: The transgenic Tg[Xops:mCFP] line expressing a palmitoylated-CFP reporter gene under control of the Xenopus opsin promoter results in rod death but allows visualization of the surviving rods in live larvae. CRISPR/Cas9-induced 5’ and 3’ alleles of the endogenous zebrafish rhodopsin locus (rho1-1) that recapitulate class II and class I RHO mutations respectively were placed on the Tg[Xops:eGFP] background to visualize rod survival. 4-6 embryos were arrayed into each well of multi-well dishes. Commercially available small molecules libraries in DMSO (Sigma-Aldrich, Enzo Life Sciences) were added using a pin transfer device to embryos at 2 days post fertilization (dpf). Reporter gene expression was monitored at 4-5 dpf. Gene knockdown was accomplished by injection of morpholinos into 1-cell stage embryos. Embryos were treated with PTU to block melanin production, and expression of fluorescent reporter genes was monitored using an inverted fluorescent microscope outfitted with low magnificationn, high NA objective lenses and captured using a digital camera.
Results :
Reporter gene expression in live larvae showed that in the various degeneration models, by 4-5 dpf loss of rods in the central and ventral retina left a rim of fluorescent cells 1 to 3 cell layers in width at the retinal margin. Knockdown of p53 resulted in readily observable increase in the number of fluorescent rods at the margin and in the central retina consistent with rod death by apoptosis. In our pilot screen of targeted libraries containing ~600 compounds, transient alterations in the number of fluorescent rods at the retinal margin were noted with steroid hormone analogues and treatments that affect RA synthesis. However no survival was observed in the central retina.
Conclusions :
The data indicate that these zebrafish models provide valuable tools to systematically screen compounds and test gene manipulations for effects upon inherited rod degeneration.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.