2026-09-21T23:19:44Zhttps://api.repository.cam.ac.uk/server/oai/requestoai:www.repository.cam.ac.uk:1810/2749092025-12-19T23:09:57Zcom_1810_221811com_1810_256062col_1810_221812
The subnuclear localisation of Notch responsive genes
10.17863/CAM.22060
Jones, Matthew Leslie
0000000236851718
Bray, Sarah
000000021642599X
Title: The subnuclear localisation of Notch responsive genes.
Candidate Name: Matthew Jones
Notch signalling is a highly conserved cell-cell communication pathway with critical
roles in metazoan development and mutations in Notch pathway components are
implicated in many types of cancer. Notch is an excellent and well-studied model of
biological signalling and gene regulation, with a single intracellular messenger, one
receptor and two ligands in Drosophila. However, despite the limited number of
chemical players involved, a striking number of different outcomes arise. Molecular
studies have shown that Notch activates different targets in different cell types and it is
well known that Notch is important for maintaining a stem cell fate in some situations
and driving differentiation in others. Thus some of the factors affecting the regulation of
Notch target genes are yet to be discovered.
Previous studies in various organisms have found that the location of a gene within the
nucleus is important for its regulation and genome reorganisation can occur following
gene activation or during development. Therefore this project aimed to label individual
Notch responsive loci and determine their subnuclear localisation. In order to tag loci of
interest a CRISPR/Cas9 genome-editing method was established that enabled the
insertion of locus tags at Notch targets, namely the well-characterized Enhancer of split
locus and also dpn and Hey, two transcription factors involved in neural cell fate
decisions.
The ParB/Int system is a recently developed locus tagging system and is not well
characterised in Drosophila. It has a number of advantages over the traditional
LacO/LacI-GFP locus tagging system as it does not rely on binding site repeats for
signal amplification and can label two loci simultaneously in different colours. This
thesis characterised the ParB/Int system in the Drosophila salivary gland and larval L3
neuroblast. Using 3D image segmentation hundreds of nuclei were reconstructed and a
volume based normalisation method was applied to determine the subnuclear
localisation of several Notch targets with and without genetic manipulations of the
Notch pathway.
University of Cambridge
2018-05-19
Thesis
Doctoral
Doctor of Philosophy (PhD)
en
Wellcome Trust
https://www.repository.cam.ac.uk/handle/1810/274909
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https://creativecommons.org/licenses/by-nc/4.0/
locus tagging
gene position
genome organisation
nuclear organisation
nuclear architecture
gene regulation
Notch signalling
CRISPR/Cas9
genome-editing
Drosophila
polytene chromosomes
salivary gland
neuroblast
L3 larva
ParB-INT
ParB/parS
gene dynamics
genome reorganisation
fiji
imageJ
image processing
image analysis
3D segmentation
eroded volume distance maps