Decoding the Notch signal: investigating dynamics of transcription directed by Notch responsive enhancers

Information from developmental signalling pathways must be accurately decoded to generate transcriptional outcomes. Notch is an evolutionarily conserved signalling pathway, which, despite its apparent simplicity, regulates very different processes across tissues and developmental contexts. After activation of the pathway via interaction of the Notch receptor with its ligands on adjacent cells, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. In order to determine how enhancers respond to Notch activity in real time, I have used the MS2-MCP system to visualize nascent transcripts in Drosophila embryos. To do so, I used two well-characterized Notch-responsive enhancers that drive expression in a stripe of mesectoderm (MSE) cells and analyzed their transcription profile over time at the single cell level. Strikingly, all MSE cells initiated transcription within a few minutes of one another, and once active, each nucleus produced a sustained profile of transcription. By manipulating NICD levels and altering key motifs within the enhancers, I uncover two key principles. First, the ability of NICD to confer synchronized and sustained activity in MSE requires that the enhancers be primed by tissue-specific transcription factors Twist and Dorsal. In their absence, MSE enhancers confer stochastic and bursty transcription profiles, demonstrating that different response profiles can be generated from a single enhancer according to which other factors are present. Second, changing Notch levels modulate the transcription burst size but not the inter-burst periods, in contrast to most current examples of enhancer activation. Next, I performed a small knockdown screen of maternal factors that could be required to promote sustained transcription in response to Notch. I found that reduced activity of some of these factors decreased the mean levels of transcription in different ways. Unexpectedly, knockdown of transcriptional repressors reduced the levels of transcription, although it is unclear if they affect Notch dependent transcription directly or indirectly. Lastly, I investigated the relationship between morphogenetic processes occurring at this stage of embryogenesis and Notch dependent transcription. I found cellularization acts as a limiting factor in the onset of transcription, and that Notch-Delta signalling likely occurs at the lateral membranes before cellularization finishes. Notch responsive transcription presents a clear transition in levels at the time of gastrulation, which correlates with the start of mesoderm invagination in genetic perturbation experiments. I hypothesize that gastrulation influences transcription in a Notch sensitive but not specific manner, through an unknown mechanism that likely occurs downstream of pathway activation.

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Falo Sanjuan, Julia

Advisor dc:contributor.advisor

• Bray, Sarah

• Notch signalling
• transcription
• live imaging
• genetics
• Drosophila
• Developmental Biology
• enhancers

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/abb19966-3c58-4380-abed-b8418221afe0/download
• https://creativecommons.org/licenses/by-nc/4.0/

Language dc:language

eng

Author Identifier

0000-0002-3563-4789

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/307130