June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Control of selected transcripts in LWS1 vs LWS2 cones by thyroid hormone in zebrafish
Author Affiliations & Notes
  • Ashley Alice Farre
    University of Idaho, Moscow, Idaho, United States
  • CHI SUN
    Washington University in St Louis, St Louis, Missouri, United States
  • Audrey Duncan
    University of Idaho, Moscow, Idaho, United States
  • Sam Hunter
    University of California Davis, Davis, California, United States
  • Margaret Starostik
    National Eye Institute, Bethesda, Maryland, United States
  • Linn Gieser
    National Eye Institute, Bethesda, Maryland, United States
  • Milton English
    National Eye Institute, Bethesda, Maryland, United States
  • Abirami Santhanam
    The University of Texas Health Science Center at Houston, Houston, Texas, United States
  • Eyad Shihabeddin
    The University of Texas Health Science Center at Houston, Houston, Texas, United States
  • John O'Brien
    The University of Texas Health Science Center at Houston, Houston, Texas, United States
  • Anand Swaroop
    National Eye Institute, Bethesda, Maryland, United States
  • Deborah L Stenkamp
    University of Idaho, Moscow, Idaho, United States
  • Footnotes
    Commercial Relationships   Ashley Farre None; CHI SUN None; Audrey Duncan None; Sam Hunter None; Margaret Starostik None; Linn Gieser None; Milton English None; Abirami Santhanam None; Eyad Shihabeddin None; John O'Brien None; Anand Swaroop None; Deborah Stenkamp None
  • Footnotes
    Support  William Stamps Farish Fund, NEI-IRP ZIAEY000450, NIH R01 EY012146, NIH R01 EY012146-16S, NIH F31 EY031962, NSF REU Site Award #1757826, Summer Undergraduate Research Fellowship (U. Idaho)
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 4278. doi:
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      Ashley Alice Farre, CHI SUN, Audrey Duncan, Sam Hunter, Margaret Starostik, Linn Gieser, Milton English, Abirami Santhanam, Eyad Shihabeddin, John O'Brien, Anand Swaroop, Deborah L Stenkamp; Control of selected transcripts in LWS1 vs LWS2 cones by thyroid hormone in zebrafish. Invest. Ophthalmol. Vis. Sci. 2022;63(7):4278.

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

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Abstract

Purpose : In humans, the long and middle wavelength-sensing cone opsins (LWS/MWS) are encoded in a tandemly replicated array. Zebrafish possess a tandemly replicated long wavelength-sensitive (lws) array; the zebrafish lws genes and human LWS and MWS genes share a common ancestor. The differential regulation of human LWS vs. MWS is viewed as a stochastic mechanism, while there is evidence for trans-regulatory mechanisms in zebrafish. Our lab demonstrated thyroid hormone (TH) promotes lws1 at the expense of lws2 (Mackin et al., 2019, PNAS). The present work aims to identify additional transcriptional differences in the LWS1 vs. LWS2 cone populations, toward elucidating the genetic mechanisms underlying the differential regulation of LWS cone opsin expression.

Methods : We isolated GFP+ (LWS1) cones and RFP+ (LWS2) cones from adult male lws:PAC(H) zebrafish using established FACS methods (Sun et al., 2018, Exp Eye Res). Bulk RNA-Seq was used to identify differentially expressed (DE) transcripts of interest in these two cone populations. A separate, single cell RNA-Seq dataset was used to complement this DE list with transcripts enriched in lws1- or lws2-expressing cones. Larval zebrafish were treated with TH, and qPCR and multiplex in situ hybridization (HCR) of DE transcripts were performed.

Results : Based on a false discovery rate (FDR)<0.05, ~130 transcripts were enriched in LWS1 cones (~1.6 % of LWS1 transcriptome), and ~93 transcripts were enriched in LWS2 cones (1.2% of LWS2 transcriptome), suggesting that these cone types are highly similar yet with a subset of distinct transcripts. Among the DE transcripts were those encoding phototransduction components [gngt2b], nuclear receptors [nr2f2], extracellular proteins [si:busm1-57f23.1], transcription factors [vax2], and regulators of circadian rhythm [cry1ba, aanat2]. qPCR and HCR data show some transcripts are DE in control vs TH-treated larvae, including gngt2b (qPCR: p<0.05, n=6). For some transcripts such as si-busm1, HCR was able to detect expression domain changes in the eye upon TH treatment.

Conclusions : This study identified several transcripts that were DE in LWS1 vs. LWS2 cones, which have been presumed identical aside from opsin expression. Some of those DE transcripts are regulated by TH. This dataset provides foundations to investigate the mechanism by which lws1 and lws2 are differentially regulated, including candidates for functional testing.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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