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 potential regulators of these genes, but studies have been conducted using the zebrafish lws locus. Zebrafish independently underwent a 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, our lab has shown that thyroid hormone (TH) is involved in the regulation of these opsins (Mackin et al., 2019). In this study, we seek to uncover the genetic sequence responsible for the TH-mediated regulation of the zebrafish lws locus.
Methods :
We used genomic prediction tools to find two predicted palindromic TH response elements (ppTREs) in the 0.6kb region upstream of lws1, which we refer to as ppTRE1 and ppTRE2. We generated three 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. The first construct contains intact ppTRE1 and 2 (control), the second lacks a ~25bp element containing ppTRE1 (ΔppTRE1), and the third lacks a ~25bp element containing ppTRE2 (ΔppTRE2). We treated embryos with TH or DMSO (control) from 2-4DPF, then fixed their whole eyes for fluorescent imaging of the GFP and RFP reporters. GFP and RFP positive cells were counted, and Kruskal-Wallis test was used for statistical analysis.
Results :
In wildtype embryos, increased TH allows for the premature expression of lws1 and decreases lws2 expression. F0 studies in ΔppTRE1 and 2 embryos determined that in the absence of ppTRE1, lws1 is prematurely expressed and lws2 is not expressed. In the absence of ppTRE2, lws1 is prematurely expressed in equal quantities as lws2. Reporter expression did not change with increased TH. Recent experiments on F3 ΔppTRE2 embryos (DMSO, n=6; TH, n=7) confirm the observations from F0 embryos. In DMSO treated embryos, lws1 and lws2 were expressed in roughly equal numbers of cones (p= 0.09). This ratio was not altered by TH (p= 0.65).
Conclusions :
The element containing ppTRE2 may be involved in the repression of lws1 during early zebrafish development, and necessary for the differential expression of lws opsins in response to TH.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.