Abstract
Purpose :
The human long wavelength sensitive (LWS) and medium wavelength sensitive (MWS) opsins arose from a tandem duplication event which resulted in opsins with divergent spectral sensitivities. Little is known about regulation of these genes, but studies have been conducted using the zebrafish lws locus. Zebrafish independently underwent tandem duplication of their lws opsin, which resulted in two spectrally divergent opsins, lws1 and lws2. In support of a transregulatory mechanism of lws expression, we have shown that thyroid hormone (TH) regulates these opsins (Mackin et al., 2019). In this study, we seek to uncover the putative cis-regulatory sequence responsible for the TH-mediated regulation of the zebrafish lws locus.
Methods :
We used genomic prediction tools to find a predicted palindromic TH response element (ppTRE) in the 0.6kb region upstream of lws1. We generated two transgenic reporter constructs with the 2.6kb region upstream of lws1 driving expression of GFP and the 1.8kb region upstream of lws2 driving expression of RFP: one with intact ppTRE (control) and one that lacks a ~25bp element containing the ppTRE (ΔppTRE). To assess lws opsin expression in athyroid conditions, we crossed each line with Tg(tg:nVenus-2a-nfsB)wp.rt8. This construct expresses nitroreductase in the cells of the thyroid, which catabolizes metronidazole (MTZ) into a cytotoxic compound to selectively ablate the thyroid. Embryos were treated with MTZ or DMSO (control) from 2-4 days post-fertilization (dpf), then collected at 6dpf. Whole eyes were fixed for confocal imaging of the GFP and RFP reporters, reporter positive cells were counted, and Kruskal-Wallis test was used.
Results :
In embryos with intact ppTRE, lws2 is expressed in greater quantities than lws1 and is spread over most of the retina, with lws1 isolated to the ventral periphery. Increased TH allows for the premature expression of lws1 and decreases lws2 expression. Athyroidy has the opposite effect. In ΔppTRE embryos (n=12), more cones expressed lws2 reporter than lws1 reporter (p=2e-4). The ratio of lws1 and lws2 reporter+ cells was not altered by athyroidy, with lws2 reporter expressed in a greater number of cells than lws1 reporter (n=12; p=9e-6).
Conclusions :
The element containing the ppTRE may be involved in the differential expression of lws opsins in response to TH.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.