AbstractIntestinal homeostasis is governed by fate choices of stem cells residing in the intestinal crypt base. This must involve niche-specific co-ordination of cell division to guarantee that epithelial cells divide at the right time and place. These mechanisms operate to ensure precise control of the numbers of stem and differentiated cells. Little is known about how proliferative fate decisions are regulated in intestinal crypts. Both the placement of daughter cells within a particular niche, and their decision to enter and progress through the cell cycle, contribute. This thesis investigates the spatiotemporal control of cell division in intestinal crypts to understand the relationship between cell-cycle specific fate choices and intestinal homeostasis.
Firstly, I describe a novel mode of asymmetric cell division within intestinal crypts. Using high resolution microscopy of intestinal organoids, I show that a subset of mitoses produce daughters that become displaced from one another after cytokinesis. This post-mitotic separation or the ‘positional asymmetry’ of daughter cells occurs in all cycling epithelial cells. These divisions may facilitate divergent fate of daughter cells and provides a general mechanism for stochastic niche exit. Post-mitotic separation is facilitated by interkinetic nuclear migration and selective tethering to the basement membrane during mitosis. Importantly, these mechanisms are altered in tissue carrying mutations in Adenomatous polyposis coli (Apc), highlighting its importance for normal tissue homeostasis.
Secondly, I aimed to understand the dynamics of cell-cycle commitment in intestinal crypt compartments by investigating the DNA Replication Licensing System. The licensing system is a master regulator of proliferative fate in all cells in adult tissue. At its core is the regulated loading of the Mcm2-7 protein complex onto origins of replication exactly once per cell cycle. Engagement of the licensing system directly indicates commitment to proliferative cell fate. A technique to visualise licensing in intestinal crypts was developed. This revealed distinct proliferation zones in intestinal crypts. Mcm licensing was most prevalent in the lower transit-amplifying compartment, the zone enriched for early TA progenitors. Licensing is inhibited in terminally differentiated cells, and not detected in the transit-amplifying cells most proximal to the differentiated zone. Strikingly, the majority of ‘active’ intestinal stem cells were found in an unlicensed state. These data suggest that licensing decisions are delayed or inhibited until late G1 phase in intestinal stem cells and explains their longer cell-cycle. We postulate that this may provide a time window for niche cues to act, either stimulating cell-cycle entry or allowing retention in a ‘shallow’ G0 state.
High resolution imaging of cell-cycle phases throughout the epithelium revealed remarkable cell-cycle co-ordination. This manifested in uninterrupted ‘ribbons’ of cells in similar cell-cycle states. This was due to lineage specific cell cycle co-ordination where adjacent daughter cells progress through the cell cycle at the same rate. These field effects are the result of co-ordinated cell-cycle progression between daughter cells. These observations were validated using living organoids expressing fluorescent ubiquitination-based cell cycle indicators (FUCCI). These ribbons were occasionally interrupted by cells in other cell cycle phases suggesting the separation of sisters by daughters from another lineage. This suggests that cell-cycle coordination can facilitate post-mitotic separation, and influence stochastic niche exit.
|Date of Award||2016|
|Sponsors||Medical Research Council|
|Supervisor||Inke Nathke (Supervisor) & Julian Blow (Supervisor)|
- Intestinal stem cell
- DNA replication