AbstractThe Wnt/β-catenin signalling plays a crucial role in many developmental and homeostatic processes. It has been shown to participate in the maintenance of self-renewal of a wide range of mammalian stem cells including mouse embryonic stem cells (mESCs). Stem cells reside in micro-environments called the “niche”, where they receive signals determining their cell fate. Stem cells can divide asymmetrically; within a cell, proteins are segregated unequally and the division machinery including the spindle is often oriented, so that the cells divide in relation to this particular signal.
To mimic the niche environment and to induce an oriented asymmetric cell division (ACD) in vitro, I used a localised Wnt signal (Wnt3a-bead) applied to single mESC. Wnt3a-bead induce ACD and is characterised by the recruitment of Wnt/β-catenin pathway components, including β-catenin and the tumour suppressor Adenomatous Polyposis Coli (APC). These two proteins are at the junction of the Wnt pathway and thus important to understand the mechanisms of Wnt-mediated ACD.
In chapter 3, we generated null mutants for different components of the Wnt/β-catenin pathway in wild-type (WT) mESCs (with a CRISPR/Cas9 based approach) to investigate their capacity to induce ACD. We knocked-out the co-receptors Lrp5 and Lrp6 independently or together (Lrp5/6 double KO). Using the same method, we generated a null mutant for Dvl2 (a downstream component of the Wnt/β-catenin pathway). In addition to these cell lines, a conditional KO for β-catenin (βf/-) inducible with 4-hydroxy-tamoxifen (4OHT) is also available in the laboratory. I characterised the different cell lines (WT, Lrp5KO, Lrp6KO, Lrp5/6dKO, Dvl2KO and βKO) by quantifying pluripotency markers (Nanog and Rex1), the levels of β-catenin and APC, and their proliferation rate.
In chapter 4, using Wnt3a-beads, I studied the capacity of the different null mutants to induce ACD. Using the βf/- cell line, I showed that one functional allele of β-catenin is sufficient to polarise APC towards the Wnt-proximal cell. When both alleles of β-catenin are not functional, I showed that APC asymmetric distribution is lost. Similar results have been found in Lrp6KO where APC asymmetric distribution is also lost.
The results shown in chapter 4 are based on fixed samples and immunofluorescence, which implies limitations at the level of spatial-temporal resolution. To investigate the subcellular localisation and the dynamics of endogenous APC and β-catenin, I generated targeting vectors to knock-in the two genes by gene targeting. In chapter 5, I first optimised a protocol of gene targeting where I knocked-in a fluorescent protein (Venus) at the C-terminal end of Nanog locus, in WT mESCs (R1, JM8, W4), using a published targeting vector (kindly gifted by Professor Anastassiadis). After successful results of gene targeting, I generated targeting vectors to knock-in β-catenin and APC with a fluorescent protein at their C-terminal end. Subsequently, the targeting vectors were electroporated into mESCs. I am the first to successfully knock-in APC gene with a fluorescent protein (mVenus) in mESCs.
In chapter 6, APC-mVenus cell line was validated by PCR amplification, and I performed functional validations. Preliminary observations were acquired by confocal microscopy, where I described the subcellular localisation of endogenous APC proteins during Wnt3a-mediated ACD.
|Date of Award||1 Jun 2020|
|Supervisor||Shukry Habib (Supervisor) & Karen Liu (Supervisor)|