Understanding the role of Deltex in adult tissue homeostasis

Abstract

The evolutionarily conserved Notch pathway is involved in many developmental and stem cell processes and its dysfunction leads to disorders such as cancer. In addition to the canonical pathway, Notch can be activated ligand-independently by the E3 ubiquitin ligase Deltex, which allows for rapid responses to changes in the cellular context. This is particularly important in fast-renewing tissues, such as the gut, and in developmental contexts. This project investigated the requirements and roles of endocytic-dependent Notch signalling and explored Deltex function conservation between flies and vertebrates using the zebrafish model. Two this extent, phenotypes in the deltex null and RING finger deficient mutants were analysed. The two alleles were used in conjunction with mitotic clone induction to show that ubiquitination of the Notch receptor is not required for endocytosis from the plasma membrane, but that its interaction with Deltex is crucial. Disruption of Deltex function altered follicle stem cell proliferation and progeny differentiation in a manner dependent on the specific mutation, age and environmental temperature. Moreover, Deltex is involved in the regenerative response of the adult intestine after dextran sulfate sodium-induced damage. The mutants showed altered lifespan and sensitivity to irritant feeding, as well as disruption of the intestinal cell behaviour during and after damage induction. The alleles often induced contrasting phenotypes in both cellular contexts, suggesting that the RING finger deficient variant may result in overactivation of Notch, rather than act as a dominant negative allele, in vivo. To examine conservation of Deltex function in vertebrates, CRISPR/Cas9 was used to generate null mutations in 3 of the zebrafish deltex genes (dtx2, dtx4a and dtx4b) and a precise RING finger domain deletion of dtx2. All individual mutants were found to be homozygous viable with no gross developmental abnormalities, although dtx2 RING finger deficient homozygous fish exhibit decreased survival at the embryonic, as well as the adult stages. Together with the dtx4a knockout line, these fish displayed ovarian phenotypes such as reduced egg laying frequency and number, while histological analysis revealed a decreased supply of primary oocytes and the presence of tissue masses in the posterior ovary. Additionally, mutants exhibit overproliferation in midgut compartments, which worsens with ageing and disrupts the gross morphology of the intestinal tissues. Altogether, these findings highlight the context-dependent effects of deltex mutations on stem cell behaviour, tissue regeneration, and organismal viability, while providing valuable insights into the evolutionary conservation of Notch regulatory mechanisms.

Date of Award	12 Jul 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Baron (Main Supervisor) & Shane Herbert (Co Supervisor)