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Deborah L Stenkamp, Ashley L Farre, CHI SUN, Margaret Starosik, Linn Gieser, Milton English, Anand Swaroop; Transcriptome analysis of adult zebrafish LWS1 vs LWS2 (long wavelength sensitive) cones. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6045.
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© ARVO (1962-2015); The Authors (2016-present)
In humans, the genes for red and green-sensing cone opsins (LWS/MWS) are arrayed in tandem (L/M array) and share a single locus control region. These genes evolved through tandem duplication and sequence divergence. Independently, a similar evolutionary process occurred in the orthologous long wavelength sensitive (lws) array of zebrafish. The process by which tandemly replicated opsins are differentially regulated is viewed as a stochastic mechanism in humans, while there is evidence for trans-regulatory mechanisms in zebrafish. Our lab demonstrated retinoic acid promotes lws1 at the expense of lws2 in zebrafish (Mitchell et al., 2015, PLOS Genet 11(8):e1005483). Here we determine transcriptional differences in the LWS1 vs. LWS2 cone populations, toward elucidating genetic mechanisms underlying the differential regulation of LWS cone opsin expression, and addressing the assumption that LWS1 and LWS2 cones are identical except for the opsin expressed.
We isolated GFP+ (LWS1) cones and RFP+ (LWS2) cones from dissociated retinas of adult male lws:PAC(H) zebrafish, using FACS methods established in our lab (Sun et al., 2018, Exp Eye Res 177:130-44). RNA was extracted, cDNA libraries were prepared, and then sequenced using Illumina Hiseq 2500.
Based on a cutoff of absolute fold change >2 and false discovery rate <0.05, approximately 90 transcripts were enriched in GFP+ (LWS1) cones (~0.6% of the LWS1 transcriptome), and approximately 180 transcripts were enriched in RFP+ (LWS2) cones (~1.2% of the LWS2 transcriptome), suggesting that these cone types are indeed highly similar. Among the transcripts showing increased expression in the GFP+ (LWS1) cones were those encoding phototransduction components [gngt2a] and transcription factors [thrab, vax1, neurod1]. The RFP+ (LWS2) population was also enriched for phototransduction [gngt2b] and transcription factor [sox4a, stox2a] transcripts.
This study identified several transcripts that were differentially expressed (DE) in zebrafish LWS1 vs. LWS2 cones, which have previously been presumed identical aside from opsin expression. DE transcripts include those involved in cone function and those potentially involved in cone differentiation. This dataset provides foundational information to investigate mechanisms through which lws1 vs. lws2 expression are differentially regulated, including multiple candidates for functional testing.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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