Identification of sensory hair-cell transcripts by thiouracil-tagging in zebrafish.
Identification of sensory hair-cell transcripts by thiouracil-tagging in zebrafish.
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DOI：
10.1186/s12864-015-2072-5
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发表时间：
2015-10-23
期刊：
影响因子：
4.4
通讯作者：
Nicolson T
中科院分区：
文献类型：
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作者：
Erickson T;Nicolson T
感觉毛细胞对机械刺激非常敏感，因此，在解剖或体外操作过程中，容易发生损伤和凋亡。硫尿嘧啶(Thiouracil，TU)标记是一种非侵入性的方法，可以标记完整生物体中特定细胞类型的转录本，从而解决了如何在不需要分选细胞的情况下分析毛细胞中基因表达的挑战。我们将TU标签应用于斑马鱼，以确定在发育中的听觉外侧器官的感觉毛细胞中表达的新的转录本。我们创建了一个转基因斑马鱼品系，该转基因品系在内耳和侧线器官的毛细胞中特异性地表达毛滴虫尿嘧啶磷酸核糖基转移酶(UPRT)酶。将受精后3天(DPF)的UPRT转基因幼虫暴露于2.5 mM 4-硫氧嘧啶(4TU)中15h，标记RNA。在总RNA提取、Poly(A)mRNA浓缩和TU标记RNA纯化后，对输入和TU标记的RNA样本进行了深度测序。对RNA测序数据的分析显示，28个转录本相对于输入样本显著富含(调整p值 <； 0.05)。在25个带有TU标记的哺乳动物同源物转录本中，18个以前没有在斑马鱼毛细胞中表达过。其中17个转录本在3只DPF幼虫中的毛细胞限制性表达经整体mRNAs原位杂交证实。这些基因的毛细胞限制性表达模式提供了对这种受体细胞类型的生物学的洞察，并可能作为有用的标记来研究感觉毛细胞的发育和功能。此外，我们的研究证明了TU标记在研究特定细胞类型的新生转录物方面的作用，这些转录物在整个斑马鱼幼体的背景下相对罕见。本文的在线版本(doi：10.1186/s12864-0152072-5)包含补充材料，可供授权用户使用。
Sensory hair cells are exquisitely sensitive to mechanical stimuli and as such, are prone to damage and apoptosis during dissections or in vitro manipulations. Thiouracil (TU)-tagging is a noninvasive method to label cell type-specific transcripts in an intact organism, thereby meeting the challenge of how to analyze gene expression in hair cells without the need to sort cells. We adapted TU-tagging to zebrafish to identify novel transcripts expressed in the sensory hair cells of the developing acoustico-lateralis organs. We created a transgenic line of zebrafish expressing the T.gondii uracil phospho-ribosyltransferase (UPRT) enzyme specifically in the hair cells of the inner ear and lateral line organ. RNA was labeled by exposing 3 days post-fertilization (dpf) UPRT transgenic larvae to 2.5 mM 4-thiouracil (4TU) for 15 hours. Following total RNA isolation, poly(A) mRNA enrichment, and purification of TU-tagged RNA, deep sequencing was performed on the input and TU-tagged RNA samples. Analysis of the RNA sequencing data revealed the expression of 28 transcripts that were significantly enriched (adjusted p-value < 0.05) in the UPRT TU-tagged RNA relative to the input sample. Of the 25 TU-tagged transcripts with mammalian homologs, the expression of 18 had not been previously demonstrated in zebrafish hair cells. The hair cell-restricted expression for 17 of these transcripts was confirmed by whole mount mRNA in situ hybridization in 3 dpf larvae. The hair cell-restricted pattern of expression of these genes offers insight into the biology of this receptor cell type and may serve as useful markers to study the development and function of sensory hair cells. In addition, our study demonstrates the utility of TU-tagging to study nascent transcripts in specific cell types that are relatively rare in the context of the whole zebrafish larvae. The online version of this article (doi:10.1186/s12864-015-2072-5) contains supplementary material, which is available to authorized users.