Reply to Edemir: Physiological regulation and single-cell RNA sequencing.
Reply to Edemir: Physiological regulation and single-cell RNA sequencing.
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回复Edemir：生理调节和单细胞RNA测序。
DOI：
10.1073/pnas.1720333115
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发表时间：
2018
影响因子：
11.1
通讯作者：
Knepper,MarkA
中科院分区：
文献类型：
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作者：
Chen,Lihe;Lee,JaeWook;Chou,Chung-Lin;Nair,AnilV;Battistone,MariaA;Păunescu,TeodorG;Merkulova,Maria;Breton,Sylvie;Verlander,JillW;Wall,SusanM;Brown,Dennis;Burg,MauriceB;Knepper,MarkA
Edemir（1）指出了单细胞RNA测序实验数据解释中的一个重要问题。任何给定的细胞都可以存在于各种受细胞外信号和细胞先前历史影响的调节状态中。因此，来自单细胞RNA测序的数据，例如在我们的论文（2）中进行的，仅提供给定细胞类型的部分表征。从事大规模尝试识别体内每种细胞类型转录组的工作人员（3）最好注意到这一限制。然而，这样的细胞图谱项目的有效性是支持的观察，即在后生动物细胞的生理调节通常是通过增量变化的监管状态的许多基因和它们的蛋白质，而不是通过选择性诱导或消融一个或另一个基因的表达。因此，我们预计，在一个给定的细胞类型中表达的基因将相对不受生理因素的影响，虽然相对mRNA水平预计会有所不同。在考虑潜在的使用单细胞RNA测序的生理调节的研究所提出的Edemir，有重要的警告。首先，在研究上皮细胞时，必须记住相邻上皮细胞之间的细胞间相互作用产生影响转录的重要信号，例如来自紧密连接的ZO-1（4），来自粘附连接和桥粒的β-连环蛋白（5），整合素连接的信号分子（6），以及来自紧密连接和粘附连接的Yes相关蛋白（雅普）（7，8）。分离单细胞的过程必然会去除这些信号，从而可能干扰所研究的生理反应。除此之外，组织解离和细胞隔离可能会造成与营养物质损失或氧可用性改变相关的不利条件，这是Potter及其同事最近提出的观点（9）。因此，代谢活跃的细胞类型，如近曲小管细胞和亨利氏袢粗升支的细胞可能会受到不利影响，改变其基因表达谱，而代谢更稳定的细胞，如我们研究中的集合管细胞，可能不太容易受到压力的影响。因此，在完整的上皮细胞中而不是在单个细胞中进行生理调节的研究可能是明智的。我们已经证明（10），基于多年前开发的用于快速解剖有活力的肾小管节段的技术（11），在单个显微解剖的肾小管中进行转录组学分析是可行的。这是我们推荐给Edemir和其他希望研究肾脏上皮细胞生理调节的人的方法。
Tacitly, Edemir (1) points to an important issue in the interpretation of data from single-cell RNA-sequencing experiments. Any given cell can exist in a variety of regulatory states that are affected both by extracellular signals and the prior history of the cell. Consequently, data from single-cell RNA sequencing, such as carried out in our paper (2), provide only a partial characterization of a given cell type. Workers engaged in large-scale attempts to identify transcriptomes of every cell type in the body (3) would do well to take note of this constraint. However, the validity of such Cell Atlas projects is supported by the observation that physiological regulation in metazoan cells is generally achieved through incremental changes in the regulatory state of numerous genes and their proteins and not by selective induction or ablation of expression of one gene or another. Thus, we would expect that the genes expressed in a given cell type will be relatively unaffected by physiological factors, although relative mRNA levels would be expected to vary.In considering the potential use of single-cell RNA sequencing for studies of physiological regulation as proposed by Edemir, there are important caveats. First of all, when studying epithelial cells, it must be remembered that cell–cell interactions between neighboring epithelial cells create important signals that influence transcription, eg, ZO-1 from tight junctions (4), β-catenin from adherens junctions and desmosomes (5), integrin-linked signaling molecules (6), and Yes-associated protein (YAP) from both tight junctions and adherens junctions (7, 8). The process of isolating single cells necessarily removes these signals, potentially interfering with physiological responses under study. Beyond this, tissue dissociation and cell isolation may create adverse conditions related to loss of nutrients or altered oxygen availability, a point made recently by Potter and colleagues (9). Thus, metabolically active cell types like proximal tubule cells and cells from the thick ascending limb of the loop of Henle may be adversely affected, altering their gene expression profiles, while cells that are metabolically more stable like collecting duct cells in our study may be less vulnerable to stress. Therefore, it may be wise to conduct studies of physiological regulation in intact epithelia rather than in single cells. We have shown (10) that it is feasible to carry out transcriptomic profiling in single microdissected renal tubules based on techniques developed many years ago for rapid dissection of viable kidney tubule segments (11). This is the approach that we recommend to Edemir and others wishing to study physiological regulation in epithelial cells of the kidney.