Consequences of Plk4 overexpression in mouse pancreatic organoids


Type
Thesis
Change log
Authors
Ridings Figueroa, Rebeca 
Abstract

Overexpression of the polo-like kinase 4 (Plk4), the master regulator of centrosome duplication, leads to centrosome amplification, a common feature of cancer, including pancreatic cancer. Previous studies have shown that overexpression of Plk4 in mice carrying a doxycycline-inducible Plk4 transgene (Plk4OE) causes centrosome amplification, a reduction in primary cilia number, and increased proliferation of pancreatic endocrine α-cells and β-cells. This project aimed to investigate the consequences of Plk4 overexpression on pancreas cell biology using mouse pancreatic ductal organoids. In accord with previous observations, immunofluorescence experiments confirmed that Plk4 overexpression leads to centrosome amplification and reduction in primary cilia number. I also investigated the consequences of long-term Plk4 overexpression on the differentiation status of pancreatic organoids. Immunofluorescence and RT-qPCR analyses revealed ductal-to-endocrine transdifferentiation, with insulin secretion and expression of β-cell markers in Plk4OE +dox organoids. I then used whole-transcriptome sequencing to investigate the consequences of Plk4 overexpression on gene expression. Results revealed differential gene expression between control and +dox Plk4OE organoids, with enrichment for genes with centrosome, cilia, cell cycle, cell death, cancer and endocrine function. Finally, I optimised and tested a protocol for the in vitro differentiation of pancreatic organoids, with induced insulin production in pancreatic organoids, irrespective of Plk4 overexpression.

In summary, the findings of this thesis confirm the reported effects of Plk4 overexpression on centrosomes and primary cilia. They also reveal a previously unknown effect of Plk4 overexpression on the identity of pancreatic ductal cells. However, whether this effect is a direct consequence of Plk4 overexpression itself or a secondary consequence of some Plk4-mediated event, such as the downregulation of primary cilia formation, remains unclear. Finally, I present an in vitro differentiation protocol capable of driving ductal organoids into insulin-producing organoids without the need for genetic modification.

Description
Date
2020-12-18
Advisors
Segal, Marisa
Glover, David
Keywords
pancreas, organoids, centrosome, insulin
Qualification
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Sponsorship
Cancer Research UK (S_3518)
Cancer Research UK studentship