Abstract
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 Jaggedl 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 Notchl 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 Jaggedl 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 Jagged1 expression during
dHSC development. With the line I showed that pre-HSCs express both Jagged1 and
Notch1 and that Jagged1⁺Notch1⁺ cell surface marker phenotype can enrich the pre-HSC population 1 in 8.
To further investigate the implication of Jagged1 in dHSC development the gene was
conditionally deleted in the HSC lineage using a CD41-Cre. Our result indicated that
Jagged1 is not required for HSC development in a cell autonomous manner.
Furthermore, I carried out experiments with a conventional Jagged1 knock -out
mouse line. It has previously been shown that Jagged1 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 Jagged1⁻/⁻ 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.
