Data_Sheet_4_Long wavelength-sensing cones of zebrafish retina exhibit multiple layers of transcriptional heterogeneity.XLSX

IntroductionUnderstanding 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 lws1 and lws2.
MethodsWe performed bulk and single cell RNA-Seq of LWS1 and LWS2 cones, evaluated expression patterns of selected genes of interest using multiplex fluorescence in situ hybridization, and used exogenous thyroid hormone (TH) treatments to test selected genes for potential control by thyroid hormone: a potent, endogenous regulator of lws1 and lws2 expression.
ResultsOur 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 > 2, FDR < 0.05). In situ hybridization results also reveal underlying heterogeneity within the lws1- and lws2-expressing populations. This heterogeneity is evident in cones of mature zebrafish, and further heterogeneity is revealed in transcriptional responses to TH treatments.
DiscussionWe found some evidence of coordinate regulation of lws 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.

引言
解析光感受器基因的调控机制，对于研究视网膜发育与疾病具有重要意义。尽管目前对视锥细胞（cones）中的基因调控已有较多研究，但串联复制视蛋白（tandemly-replicated opsins）——例如人类长波长敏感视蛋白与中波长敏感视蛋白——的差异调控机制仍不明确。本研究以表达串联复制视蛋白lws1与lws2的斑马鱼为模型，旨在深入解析表达串联复制视蛋白的视锥细胞中的转录异质性，以及此类差异表达的调控机制。

材料与方法
我们对LWS1与LWS2型视锥细胞开展了批量RNA测序（bulk RNA-Seq）与单细胞RNA测序（single cell RNA-Seq）；采用多重荧光原位杂交（multiplex fluorescence in situ hybridization）技术，对筛选得到的目标基因的表达模式进行了验证；并通过外源性甲状腺激素（thyroid hormone, TH）处理实验，探究目标基因是否受甲状腺激素调控——甲状腺激素是调控lws1与lws2表达的强效内源性调控因子。

结果
本研究结果显示，LWS1与LWS2型视锥细胞之间，除视蛋白表达差异外，还存在额外的转录组差异。批量RNA测序结果显示，在LWS1型视锥细胞中富集的转录本共有95个，LWS2型视锥细胞中富集的转录本则有186个（折叠变化FC>2，错误发现率FDR<0.05）。荧光原位杂交结果同样显示，表达lws1与lws2的视锥细胞群体内部存在潜在的转录异质性。该异质性在成熟斑马鱼的视锥细胞中已可观测到，而经甲状腺激素处理后的转录应答进一步揭示了更多的异质性特征。

讨论
我们发现，在外源性甲状腺激素处理下，LWS1与LWS2型视锥细胞中的lws视蛋白及其他基因存在协同调控的证据，同时也观测到了不受甲状腺激素介导的基因调控现象。LWS1与LWS2型视锥细胞之间的转录组差异，可能由包括甲状腺激素在内的多种信号通路共同调控。