Department of Biological Sciences, University at Albany, State University of New York, Albany, NY, USA.
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
Methods Mol Biol. 2022;2472:131-149. doi: 10.1007/978-1-0716-2201-8_11.
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 nematode Caenorhabditis 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.
Notch 信号通路对动物的发育和稳态至关重要。Notch 触发其靶基因的转录，这些靶基因根据上下文产生不同的结果。高分辨率和空间精确评估 Notch 依赖性转录对于理解 Notch 在体内天然环境中正常运作的方式以及 Notch 缺陷如何导致发病机制至关重要。在这里，我们介绍了在其生态位内评估干细胞中 Notch 依赖性转录激活的生物学和计算方法，重点介绍了线虫秀丽隐杆线虫中的生殖干细胞。具体来说，我们描述了使用单分子 RNA 荧光原位杂交 (smFISH) 固定性腺中单 RNA 的可视化，使用 MS2 系统在完整生物体中进行转录爆发的活体成像，以及定制的 MATLAB 代码，实现了新的图像处理算法来捕获 Notch 依赖性转录激活的时空模式。这些方法允许对整个组织中的体内转录激活及其动力学进行强大分析。我们的方法可以适应任何组织或细胞类型的任何转录本。
