Image-Based Single-Molecule Analysis of Notch-Dependent Transcription in Its Natural Context.

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.