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Ashley Alice Farre, CHI SUN, Sam Hunter, Margaret Starostik, Linn Gieser, Milton English, Abirami Santhanam, Eyad Shihabeddin, John O'Brien, Anand Swaroop, Deborah L Stenkamp; Transcriptional distinctions between LWS cone subtypes are regulated by thyroid hormone in zebrafish. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1687.
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© ARVO (1962-2015); The Authors (2016-present)
In humans, the long and middle wavelength-sensing cone opsins (LWS/MWS) are encoded in a tandemly replicated array. Zebrafish possess an orthologous tandemly replicated long wavelength-sensitive (lws) array. 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.
We isolated GFP+ (LWS1) cones and RFP+ (LWS2) cones from dissociated retinas of adult male lws:PAC(H) zebrafish, using established FACS methods (Sun et al., 2018, Exp Eye Res). Bulk RNA-Seq was performed to identify differentially expressed (DE) transcripts of interest in these two cone populations. A separate, single cell RNA-Seq dataset obtained from adult retinas was used to complement this DE list with transcripts enriched in identified lws1- or lws2-expressing cones. Larval zebrafish were treated with TH, and qPCR and multiplex in situ hybridization of DE transcripts were performed.
Based on a false discovery rate (FDR)<0.05, ~130 transcripts were enriched in LWS1 cones (~1.6 [AF1] % 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 [SD(2] [AF3] were phototransduction components [gngt2b], transcriptional regulators [hmgb1b, sox4a, meis2a], and synaptic/cell adhesion molecules [syt2, sypb, adrm1], and regulators of circadian rhythm [cry1ba, aanat2]. Preliminary qPCR and in situ data show some transcripts may also be DE in control vs TH-treated larvae, including gngt2b (qPCR: p<0.05, n=6).
This study identified several transcripts that were DE in LWS1 vs. LWS2 cones, which have been presumed identical aside from opsin expression. The DE transcripts include some involved in cone function. Some of those DE transcripts appear to be 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 is a 2021 ARVO Annual Meeting abstract.
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