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Robert Mackin, Diana Mitchell, Deborah L Stenkamp; Lws opsin promoter analysis and phenotype plasticity of LWS cones in zebrafish retina in response to thyroid hormone.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3106. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Human trichromatic color vision requires that the tandemly-duplicated LWS/MWS (long- and medium-wavelength sensitive) cone opsin genes are differentially expressed in subsets of cones. The trans-acting nuclear signaling molecule thyroid hormone (TH) is endogenously involved in differential regulation of the orthologous, tandemly-duplicated lws1/lws2 cone opsin genes in zebrafish embryos (Mackin et al., 2017 IOVS, vol. 58, 124). Here we identify response elements on the lws locus important for the response to TH, and use gain- and loss-of-function (GOF; LOF) approaches to determine the plasticity of LWS cone phenotype in post-embryonic zebrafish.
Response element studies used the transgenic lines lws1up2.6kb:GFP (2.6kb upstream of lws exon 1, driving GFP), and LAR-lws2up1.8kb:GFP (lws-activating region or LAR, and the lws intergenic region, driving GFP). Both lines were treated with 100nM triiodothyronine (T3) or DMSO control, 2-4 days post-fertilization (dpf). Analysis was by confocal microscopy and quantification of GFP+ cones. For cone plasticity studies, there were four treatment groups. 1. Tg(tg:nVenus-2a-nfnB)wp.rt8 transgenics treated with 10mM metronidazole, 2-3dpf, which ablates the thyroid gland (LOF). 2. DMSO-treated transgenic controls. 3. LOF transgenics treated with 386nM thyroxine (T4), 26-31dpf (“rescue”). 4. Transgenic controls treated with T4, 26-31dpf (GOF). Lws1 vs. lws2 expression was measured by qPCR at 31dpf.
In control lws1up2.6kb:GFP embryos, lws1 (GFP) was not detected, but was detected in T3 treated transgenics. In LAR-lws2up1.8kb:GFP embryos, numbers of lws2 (GFP)+ cells were not decreased when treated with T3 compared to control transgenics (p=.621). Preliminary results of plasticity studies suggested that thyroid LOF resulted in increased lws2 and reduced lws1 expression compared to controls. T4 rescue reduced lws2 expression compared to LOF, but lws1 expression was not rescued. T4 GOF increased lws1 and decreased lws2 compared to controls.
Promoter-reporter analyses indicated that the 2.6kb region upstream of lws1 contains response elements sufficient for lws1 upregulation, and necessary for lws2 downregulation, in response to T3. Rescue studies suggested some plasticity in differential expression of lws1 vs. lws2, implying that expression of these opsins can be manipulated post-embryonically.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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