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Title: Jagged1 and Notch1 involvement in haematopoietic stem cell development
Author: Murphy, Fiona L.
ISNI:       0000 0004 7658 8832
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2014
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Previous studies have identified the Notch signalling pathway as an important regulator of haematopoietic development. However its role in definitive haematopoietic stem cell (dHSC) development is still unclear mainly due to the fact that Notch mutants die around mid- gestation before the emergence of the first dHSC. Here I investigated the role of the Notch signalling pathway in dHSC development focusing on the ligand Jagged1 and the receptor Notchl. I carried out a detailed characterisation of the expression pattern of Notchl and Jaggedl in the aorta- gonad- mesonephros (AGM) region, where dHSCs first emerge in the embryo, by immunocytochemistry and immunohistochemistry. I then determined, by sorting cells from the AGM region based on their level of Notchl, that Notch1 was highly expressed in endothelial cells, precursors of dHSCs (called pre-HSC) and dHSCs, and its expression then decreases in haematopoietic progenitors. I also generated a Jagged1 dtTomato knock-in reporter mouse using a combination of recombineering and traditional cloning to produce a targeting vector, followed by targeting a B16 ES cell line, and producing a mouse line from a correctly targeted ES cell clonal line. This mouse line allowed me to visualise Jaggedl expression during dHSC development. With the line I showed that pre-HSCs express both Jaggedl and Notchl and that Jaggedl+Notchl+ cell surface marker phenotype can enrich the pre - HSC population 1 in 8. To further investigate the implication of Jaggedl in dHSC development the gene was conditionally deleted in the HSC lineage using a CD41 -Cre. Our result indicated that Jaggedl is not required for HSC development in a cell autonomous manner. Furthermore, I carried out experiments with a conventional Jaggedl knock -out mouse line. It has previously been shown that Jaggedl null embryos die around E10.5 and contain fewer intra-haematopoietic progenitors. I used an explant culture system to culture E10.5 AGM regions from Jaggedl-/- embryos past the point of embryo lethality and in culture HSCs were produced. This result indicates that Jagged1-/- embryos contain pre -HSCs that can mature efficiently into dHSC in vitro.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available