Notch activation and clone fate in intestinal homeostasis and inflammation

The Notch signalling pathway is a highly conserved pathway that has important and often differing roles and functions depending on the tissue. In the intestinal epithelium Notch signals are important for determining cell fate between the absorptive and secretory lineage and regulating the proliferation of intestinal stem cells (ISCs). As a result, Notch is also an important pathway during disease such as inflammation and previous literature has reported conflicting arguments on the role of Notch during inflammation. So far there is a lack of understanding of the effect Notch signals play on ISC dynamics during homeostasis and a disease setting, with most studies focussed on the small intestinal epithelium rather than the colon. In this study I aim to determine the effect of Notch signals on the cell cycle and ISC clone dynamics in the colonic epithelium. Using double nucleotide labelling and pulse-chase lineage tracing experiments along with immunofluorescence and immunohistochemistry methods with a transgenic Notch active mutant mouse model (N1ICD^ΔIEC), cell and clonal dynamics were determined in the normal and inflamed colon. Nucleotide analogue labelling data found N1ICD^ΔIEC crypts had an increased number of proliferative cells with shorter cell cycle times whilst inhibiting secretory cell differentiation. Subsequent pulse-chase experiments determined N1ICD^ΔIEC clones were negatively biased proximally and neutral, distally. This highlighted a disconnect between cell cycle and clone dynamics. Whilst long-term lineage tracing results show these N1ICD^ΔIEC clones are all but lost from the proximal and distal colonic epithelium owing to their increased rate of proliferation and lack of secretory cells. These same dynamics were then explored in a DSS-induced inflammation model. Here, DSS was found to directly inhibit cell proliferation in N1ICD^ΔIEC crypts proximally and following a post-DSS chase period this phenotype was reversed, with rapid N1ICD^ΔIEC clone expansion. In the distal colon, DSS caused N1ICD^ΔIEC clone expansion through increased cell proliferation. Following a post-DSS chase period there was further cell proliferation increases that did not yield further N1ICD^ΔIEC expansion. These data find N1ICD^ΔIEC crypts drive repair and regeneration of the intestinal epithelium during and after inflammation to maintain homeostasis. Therefore, Notch activation on its own in the normal colon does not confer a competitive advantage and requires a disease setting for clonal expansion. Important differences exist between the proximal and distal colon and this information should be considered before using unspecific Notch targeted drugs as these could potentially have varying outcomes and degrees of success.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Lines, George

Advisor dc:contributor.advisor

• Winton, Douglas

• cancer
• cell cycle
• clone dynamics
• colon
• colorectal cancer
• CRC
• differentiation
• epithelial cells
• IBD
• immunofluorescence
• immunohistochemistry
• Inflammation
• inflammatory bowel disease
• intestinal epithelium
• intestinal stem cells
• ISC
• large intestines
• mathematical modelling
• neutral drift
• Notch
• Notch signalling pathway
• small intestines
• stem cell dynamics
• stem cells

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a97b972a-5db7-4e68-aaaf-5c89a121887d/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.109676

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/370161