AUTHOR=Farre Ashley A. , Sun Chi , Starostik Margaret R. , Hunter Samuel S. , English Milton A. , Duncan Audrey , Santhanam Abirami , Shihabeddin Eyad , O’Brien John , Swaroop Anand , Stenkamp Deborah L. 

TITLE=Long wavelength-sensing cones of zebrafish retina exhibit multiple layers of transcriptional heterogeneity

JOURNAL=Frontiers in Cellular Neuroscience

VOLUME=17

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2023.1214084

DOI=10.3389/fncel.2023.1214084

ISSN=1662-5102

ABSTRACT=<sec><title>Introduction</title><p>Understanding how photoreceptor genes are regulated is important for investigating retinal development and disease. While much is known about gene regulation in cones, the mechanism by which tandemly-replicated opsins, such as human long wavelength-sensitive and middle wavelength-sensitive opsins, are differentially regulated remains elusive. In this study, we aimed to further our understanding of transcriptional heterogeneity in cones that express tandemly-replicated opsins and the regulation of such differential expression using zebrafish, which express the tandemly-replicated opsins <italic>lws1</italic> and <italic>lws2</italic>.</p></sec><sec><title>Methods</title><p>We performed bulk and single cell RNA-Seq of LWS1 and LWS2 cones, evaluated expression patterns of selected genes of interest using multiplex fluorescence <italic>in situ</italic> hybridization, and used exogenous thyroid hormone (TH) treatments to test selected genes for potential control by thyroid hormone: a potent, endogenous regulator of <italic>lws1</italic> and <italic>lws2</italic> expression.</p></sec><sec><title>Results</title><p>Our studies indicate that additional transcriptional differences beyond opsin expression exist between LWS1 and LWS2 cones. Bulk RNA-Seq results showed 95 transcripts enriched in LWS1 cones and 186 transcripts enriched in LWS2 cones (FC &gt; 2, FDR &lt; 0.05). <italic>In situ</italic> hybridization results also reveal underlying heterogeneity within the <italic>lws1</italic>- and <italic>lws2</italic>-expressing populations. This heterogeneity is evident in cones of mature zebrafish, and further heterogeneity is revealed in transcriptional responses to TH treatments.</p></sec><sec><title>Discussion</title><p>We found some evidence of coordinate regulation of <italic>lws</italic> opsins and other genes by exogenous TH in LWS1 vs. LWS2 cones, as well as evidence of gene regulation not mediated by TH. The transcriptional differences between LWS1 and LWS2 cones are likely controlled by multiple signals, including TH.</p></sec>