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

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Notch Signaling Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2472))

Abstract

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.

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Acknowledgements

CHL was supported by the American Heart Association (18POST34030263). TRL was supported by the National Science Foundation Graduate Research Fellowship Program (Grant No. DGE1256259). JK is grateful to NIH R01 GM134119 for support.

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Authors and Affiliations

  1. Department of Biological Sciences, University at Albany, State University of New York, Albany, NY, USA

    ChangHwan Lee

  2. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA

    Tina Lynch, Sarah L. Crittenden & Judith Kimble

Authors
  1. ChangHwan Lee
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  2. Tina Lynch
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  3. Sarah L. Crittenden
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  4. Judith Kimble
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Corresponding author

Correspondence to ChangHwan Lee .

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Editors and Affiliations

  1. Department of Biology, Georgia Southern University, Statesboro, GA, USA

    Dongyu Jia

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Lee, C., Lynch, T., Crittenden, S.L., Kimble, J. (2022). Image-Based Single-Molecule Analysis of Notch-Dependent Transcription in Its Natural Context. In: Jia, D. (eds) Notch Signaling Research. Methods in Molecular Biology, vol 2472. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2201-8_11

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  • DOI: https://doi.org/10.1007/978-1-0716-2201-8_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2200-1

  • Online ISBN: 978-1-0716-2201-8

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