Haimbaugh Alex, Meyer Danielle, Akemann Camille, Gurdziel Katherine, Baker Tracie R
Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI, United States.
Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, United States.
Front Toxicol. 2022 May 9;4:821116. doi: 10.3389/ftox.2022.821116. eCollection 2022.
In this report, we compare the outcomes and limitations of two methods of transcriptomic inquiry on adult zebrafish testes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during sexual differentiation: conventional or bulk RNA-seq (bulk-seq) and single cell RNA sequencing (scRNA-seq) data. scRNA-seq has emerged as a valuable tool for uncovering cell type-specific transcriptome dynamics which exist in heterogeneous tissue. Our lab previously showed the toxicological value of the scRNA-seq pipeline to characterize the sequelae of TCDD exposure in testes, demonstrating that loss of spermatids and spermatozoa, but not other cell types, contributed to the pathology of infertility in adult male zebrafish exposed during sexual differentiation. To investigate the potential for technical artifacts in scRNA-seq such as cell dissociation effects and reduced transcriptome coverage, we compared bulk-sequenced and scRNA-seq-paired samples from control and TCDD-exposed samples to understand what is gained and lost in scRNA-seq vs bulk-seq, both transcriptomically and toxicologically. We hypothesized that the testes may be sensitive to tissue disruption as they contain multiple cell types under constant division and/or maturation, and that TCDD exposure may mediate the extent of sensitivity. Thus, we sought to understand the extent to which this dissociation impacts the toxicological value of data returned from scRNA-seq. We confirm that the required dissociation of individual cells from intact tissue has a significant impact on gene expression, affecting gene pathways with the potential to confound toxicogenomics studies on exposures if findings are not well-controlled and well-situated in context. Additionally, a common scRNA-seq method using cDNA amplified from the 3' end of mRNA under-detects low-expressing transcripts including transcription factors. We confirm this, and show TCDD-related genes may be overlooked by scRNA-seq, however, this under-detection effect is not mediated by TCDD exposure. Even so, scRNA-seq generally extracted toxicologically relevant information better than the bulk-seq method in the present study. This report aims to inform future experimental design for transcriptomic investigation in the growing field of toxicogenomics by demonstrating the differential information extracted from sequencing cells-despite being from the same tissue and exposure scheme-is influenced by the specific protocol used, with implications for the interpretation of exposure-induced risk.
在本报告中，我们比较了两种转录组学探究方法在性分化期间暴露于2,3,7,8 - 四氯二苯并 - 对 - 二恶英（TCDD）的成年斑马鱼睾丸中的结果和局限性：传统的或批量RNA测序（批量测序）以及单细胞RNA测序（scRNA测序）数据。scRNA测序已成为一种有价值的工具，用于揭示存在于异质组织中的细胞类型特异性转录组动态。我们实验室之前展示了scRNA测序流程在表征睾丸中TCDD暴露后遗症方面的毒理学价值，证明精子细胞和精子的损失而非其他细胞类型导致了性分化期间暴露的成年雄性斑马鱼不育的病理状况。为了研究scRNA测序中的技术假象（如细胞解离效应和转录组覆盖度降低）的可能性，我们比较了来自对照和TCDD暴露样本的批量测序和scRNA测序配对样本，以了解在转录组学和毒理学方面，scRNA测序相对于批量测序的得失。我们假设睾丸可能对组织破坏敏感，因为它们包含多种处于持续分裂和/或成熟状态的细胞类型，并且TCDD暴露可能介导敏感程度。因此，我们试图了解这种解离在多大程度上影响从scRNA测序返回的数据的毒理学价值。我们证实，从完整组织中分离单个细胞所需的解离对基因表达有重大影响，会影响基因通路，如果研究结果没有得到很好的控制和背景定位，就有可能混淆关于暴露的毒理基因组学研究。此外，一种常用的scRNA测序方法，即使用从mRNA 3'端扩增的cDNA，会漏检包括转录因子在内的低表达转录本。我们证实了这一点，并表明scRNA测序可能会忽略与TCDD相关的基因，然而，这种漏检效应不是由TCDD暴露介导的。即便如此，在本研究中，scRNA测序通常比批量测序方法能更好地提取毒理学相关信息。本报告旨在通过展示从测序细胞中提取的差异信息（尽管来自相同组织和暴露方案）受所用特定方案的影响，为毒理基因组学这一不断发展的领域中的转录组学研究的未来实验设计提供参考，这对解释暴露诱导的风险具有重要意义。
