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.

Benzene is an ubiquitous pollutant and industrial solvent that has been identified as a human leukemogen. Early exposure to environmental carcinogens such as benzene has been postulated to play a role in the etiology of childhood leukemia, however the association remains controversial. Genotoxic agents such as benzene can cause an increase in the frequency of DNA double-strand breaks, which may remain unrepaired or result in the initiation of DNA recombinational repair mechanisms. The first objective was to investigate the induction of DNA double-strand breaks following in utero treatment to 200 mg/kg and 400 mg/kg benzene i.p. using the phosphorylated histone γ-H2A.X as a marker. Using immunoblotting, treatment with benzene did not increase the formation of γ-H2A.X in bone marrow cells of adult C57Bl/6N male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells following in utero exposure to 200 mg/kg or 400 mg/kg benzene throughout gestational days 7 to 15. Secondly, the study investigated the induction of micronuclei following in utero exposure to benzene. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells. In utero exposure to 400 mg/kg benzene throughout gestational days 7 to 15 also caused a statistically significant increase in the percentage of micronucleated cells in maternal bone marrow and post-natal bone marrow cells. Fetal liver cells also demonstrated a statistically significant increase in the percentage of micronucleated cells following 200 mg/kg and 400 mg/kg benzene. The third objective was to investigate the initiation of DNA recombination following in utero exposure to benzene using the pKZ1 mutagenesis mouse model as a surrogate marker for non-homologous end joining activity. Adult pKZ1 mouse tissue yielded no recombination events; however, post-natal bone marrow cells did contain detectable recombination frequencies. iii In utero benzene exposure did cause an increasing trend in recombination events, and upon analysis of only the samples containing detectable levels of recombination, in utero exposure to 400 mg/kg of benzene caused a statistically significant increase in recombination frequency within this group. These results demonstrate that benzene does not increase the formation of γ-H2A.X after acute and in utero exposure, however, the induction of micronuclei following acute and in utero benzene exposure confirmed that benzene is a genotoxic agent causing chromosomal breaks. In utero benzene exposure increased the frequency of DNA recombination in bone marrow from post-natal day 9 pups exhibiting detectable levels of recombination. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood leukemias. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-08-22 11:07:49.162