Hematopoietic stem cells emerge from a specialized subset of endothelial cells in the midgestation mouse aorta. This subset, the so-called hemogenic endothelium (HE), undergoes a morphological and molecular change to a hematopoietic cell type, as part of the endothelial-to- hematopoietic transition (EHT). Previously, lack of specific markers prevented mechanistic studies of HE, as well as studies into its developmental origin. Runx1 is a critical regulator of developmental hematopoiesis and is expressed in all cell intermediates of EHT. Identification of the Runx1 +23 enhancer led to the development of enhancer-reporter tools in order to isolate HE for further analysis. Here, I investigated the cell-type specific activity of another Runx1 enhancer, located 204 kb downstream of the ATG in exon 1. I generated a novel enhancer-reporter mouse line (204GFP) and determined the expression pattern and lineage potential of 204GFP+ cells. It was established that the +204 enhancer marks all HE and part of the HSCs. Hematopoietic progenitor cells, in contrast, were not marked by the 204GFP transgene. Interestingly, the 204GFP reporter also marks part of the Runx1- expressing sub-aortic mesenchyme. To test whether the 204GFP reporter could enrich for HE when combined with a Runx1 +23 enhancer-reporter transgene, I generated and characterized a 23Cherry transgenic mouse line. Expression analysis of aortic endothelial cells marked by both the 204GFP and 23Cherry transgenes using the Fluidigm platform indicated an enrichment of cells with a HE expression signature. This enrichment will facilitate further analysis of the molecular networks active in HE using whole genome expression profiling. The Runx1 enhancer-reporter models are also valuable tools to track the developmental origin of HE, which remains to be established in the mouse embryo. To this end, I mapped the precise spatio-temporal expression pattern of the 23GFP transgene in pre- somitic embryos and established lineage tracing experiments. This provides the basis to revisit fate mapping of the primitive streak to determine the origin(s) of the HE lineage.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:729948 |
| Date | January 2016 |
| Creators | Rode, Christina |
| Contributors | De Bruijn, Marella Theophiel |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ora.ox.ac.uk/objects/uuid:49b91ea8-36a3-4bcd-8842-baa1ee31c7b9 |
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