Image-Based Single-Molecule Analysis of Notch-Dependent Transcription in Its Natural Context.
Image-Based Single-Molecule Analysis of Notch-Dependent Transcription in Its Natural Context.
复制标题
自然背景下缺口依赖性转录的基于图像的单分子分析。
DOI：
10.1007/978-1-0716-2201-8_11
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Kimble,Judith
中科院分区：
文献类型：
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作者：
Lee,ChangHwan;Lynch,Tina;Crittenden,SarahL;Kimble,Judith
Notch信号对动物的发育和动态平衡至关重要。Noch触发其目标基因的转录，根据上下文产生不同的结果。对Notch依赖的转录进行高分辨率和空间精确的评估对于了解Notch在体内如何在其自然环境中正常工作以及Notch缺陷如何导致发病机制至关重要。在这里，我们提出了生物学和计算方法来评估依赖Notch的转录激活在干细胞的生态位内，重点是线虫的生殖系干细胞。具体地说，我们描述了使用单分子RNA荧光原位杂交(SmFISH)显示固定性腺中的单个RNA，使用MS2系统对完整生物体中转录爆发的实时成像，以及定制的MATLAB代码，实现了新的图像处理算法来捕获依赖Notch的转录激活的时空模式。这些方法可以对体内转录激活及其在整个组织中的动力学进行强大的分析。我们的方法基本上可以适用于任何组织或细胞类型的任何转录本。
Notch signaling is crucial to animal development and homeostasis. Notch triggers the transcription of its target genes, which produce diverse outcomes depending on context. The high resolution and spatially precise assessment of Notch-dependent transcription is essential for understanding how Notch operates normally in its native context in vivo and how Notch defects lead to pathogenesis. Here we present biological and computational methods to assess Notch-dependent transcriptional activation in stem cells within their niche, focusing on germline stem cells in the nematodeCaenorhabditis elegans. Specifically, we describe visualization of single RNAs in fixed gonads using single-molecule RNA fluorescence in situ hybridization (smFISH), live imaging of transcriptional bursting in the intact organism using the MS2 system, and custom-made MATLAB codes, implementing new image processing algorithms to capture the spatiotemporal patterns of Notch-dependent transcriptional activation. These methods allow a powerful analysis of in vivo transcriptional activation and its dynamics in a whole tissue. Our methods can be adapted to essentially any tissue or cell type for any transcript.