July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Thyroid hormone regulates the tandemly-quadruplicated rh2 cone opsin gene array in zebrafish
Author Affiliations & Notes
  • Ashley Farre
    University of Idaho, Moscow, Idaho, United States
  • Robert Mackin
    University of Idaho, Moscow, Idaho, United States
  • Deborah L Stenkamp
    University of Idaho, Moscow, Idaho, United States
  • Footnotes
    Commercial Relationships   Ashley Farre, None; Robert Mackin, None; Deborah Stenkamp, None
  • Footnotes
    Support  NIH R01 EY012146, NIH R01 EY012146S, NSF REU Site award 1460696
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 6039. doi:https://doi.org/
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      Ashley Farre, Robert Mackin, Deborah L Stenkamp; Thyroid hormone regulates the tandemly-quadruplicated rh2 cone opsin gene array in zebrafish. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6039. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The mechanism by which tandemly replicated opsins are differentially regulated is thought to be stochastic in humans (l/m array); however, there is evidence for trans-regulatory mechanisms for the orthologous lws array in zebrafish. Retinoic acid (Mitchell et al., 2015, PLOS Genet 11(8):e1005483) and thyroid hormone (TH) (unpublished data) promote lws1 at the expense of lws2. This work aims to determine the effect of TH on expression from the rh2 cone opsin gene array, toward elucidating the mechanism by which tandemly replicated opsins are differentially regulated.

Methods : Wildtype and reporter transgenics rh2-2:GFP and rh2-3:GFP;rh2-4:RFP were treated with 100nM triiodothyronine (T3) or DMSO (control) for 2-4 days post-fertilization (dpf) for qPCR (n=7) and in situ hybridization (wildtype), and for confocal imaging and cone quantification (transgenics) (n=3). In thyroid ablation studies, there were four treatment groups (n=4-9). Tg(tg:nVenus-2a-nfnB)wp.rt8 transgenics treated with 1) DMSO (control) or 2) 10mM metronidazole, 2-3dpf, which ablates the thyroid gland (LOF). 3) LOF transgenics treated with 386nM thyroxine (T4), 26-31dpf (rescue). 4) Transgenic controls treated with T4, 26-31dpf (GOF). Opsin expression was measured by qPCR at 31dpf.

Results : In embryos, T3 downregulated native rh2-1 (qPCR p=8.1E-6) and upregulated rh2-2 (p=2.0E-7). However, preliminary results from whole eyes of rh2-2:GFP embryos did not show increased numbers of rh2-2+ cones (p>0.05). Preliminary results from rh2-3:GFP;rh2-4:RFP embryos showed T3 increased numbers of rh2-3+ cones (p<0.01) but had no effect on rh2-4 (no RFP+ cones in either condition). In juveniles, T4 GOF downregulated rh2-1 (p=8.7E-25) and rh2-2 (p=0.0002), upregulated rh2-3 (p=0.001), and had no effect on rh2-4 (p=0.75). Thyroid ablation downregulated rh2-2 (p=1.9E-8), rh2-3 (p=6.7E-19), and rh2-4 (p=7.0E-5) and did not affect rh2-1 (p=0.82). In the rescue experiment, T4 rescued rh2-3 (p=3.12E-16) and rh2-4 (p=0.001), but not rh2-2, compared to LOF.

Conclusions : TH differentially affects the expression of the rh2-type cone opsins in a complex manner, which may be dependent upon life history stage. High TH tends to favor the expression of the distal genes in the array, which encode opsins sensitive to higher wavelengths, while low TH tends to favor the expression of the proximal genes in the array, which encode opsins sensitive to lower wavelengths.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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