Murine embryonic stem (ES) cells are derived from the inner cell mass of the developing embryonic blastocyst. These cells can self renew which allows them to be propagated indefinitely in the laboratory and they can differentiate into cell types derived from all three germ layers. Manipulation of the mouse genome using gene targeting techniques in conjunction with ES cell technology has provided valuable insights into embryonic development and cell lineage specification. KIT is a trans-membrane receptor tyrosine kinase (RTK) that dimerises upon binding to its ligand, stem cell factor (SCF) resulting in the auto-phosphorylation of intracellular kinase domains. This activity is crucial for the transmission of signals from the cell surface to the nucleus. KIT is expressed on stem and progenitor cells of many lineages and defects in the SCF/KIT signaling pathway causes detrimental effects at both the cellular and physiological level. This project aimed to investigate the role of the SCF/KIT signalling pathway during murine ES cell differentiation and survival. To assess the role of SCF/KIT signalling in ES cell proliferation and survival, we knocked out the c-kit gene in mouse ES cells to produce heterozygous (KitW-lacZ/+) and KIT Null (KitW-lacZ/W-lacZ) cell lines. The self renewal and differentiation profile of these cell lines revealed an auxiliary role for SCF/KIT during ES cell self renewal and an absolute role in survival upon in vitro differentiation. This phenotype of apoptosis upon differentiation was recapitulated in wild type E14 ES cells treated with a KIT neutralising antibody (ACK2). Wild type cells that were treated with the JNK inhibitor, SP600125 had a comparable phenotype to KIT null cells indicating that this could be one of the mediators of KIT signalling that has a protective role in the survival of differentiating ES cells. We hypothesised that blocking classical apoptotic pathways might prevent the death on differentiation observed in KIT null cells. However, neither blocking the pro-apoptotic P38 pathway with the chemical inhibitor PD169316 nor over-expressing the pro-survival protein BCL2 in KIT Null cells could prevent their apoptosis upon differentiation phenotype. This strongly suggests that these pathways are not involved in KIT mediated survival of differentiating ES cells. Although compensatory mechanisms are thought to exist for defective KIT signaling in vivo, an absolute role is assigned to KIT during ES cell differentiation. Further analysis of micro array data comparing gene expression from wild type E14 and KIT Null cell lines may reveal the specific mechanisms of KIT mediated survival during differentiation onset.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:563502 |
| Date | January 2011 |
| Creators | Fraser, Lindsay |
| Contributors | Forrester, Lesley. ; Harrison, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/5564 |
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