Genetic Basis For Hematopoietic Stem Cell Generation In The Mammalian Embryo

The generation of hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. Many questions remain unanswered regarding the molecular program in hemogenic endothelium that promotes the budding of hematopoietic cell clusters containing HSCs. Previously, we described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13-14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia (MDS)/leukemia. In contrast to Gata2 knockout mice, which die around E10.5 because of anemia, the prolonged embryonic development of +9.5 site knockout mice provides a unique model system to investigate the potential roles for GATA-2 in HSC production, migration and function, and more specifically, the requirement for the +9.5 element to regulate Gata2 expression during these processes. Using an ex vivo system involving culturing intact AGM, or AGM dissociated into single cells and then reaggregated into an organoid, we demonstrated that the +9.5 deletion reduced Gata2 expression in uncultured AGM (1.4 fold, p<0.05), cultured intact AGM (4 fold, p<0.001) and cultured AGM reaggregates (3.4 fold, p<0.001). The importance of the +9.5 element for Gata2 expression in the AGM suggested that it might control the function of hemogenic endothelium and/or the HSC progeny. The homozygous +9.5 mutation resulted in a complete loss of progenitors and long-term repopulating HSCs in the AGM, as determined by quantitative colony assays and competitive transplantation assays, respectively. To determine whether the ablation of HSC repopulating activity in the +9.5^-/- mutant AGM reflects a +9.5 element requirement for HSC genesis from hemogenic endothelium, we used a whole-mount three-dimensional embryo immunostaining assay to visualize HSC genesis in +9.5^+/+ and +9.5^-/- AGMs. Imaging of E10.5 embryos revealed CD31+c-Kit+ hematopoietic clusters in +9.5^+/+ dorsal aorta, while clusters were absent from the +9.5^-/- embryos. The absence of hematopoietic clusters in the +9.5^-/- dorsal aorta, and the ablation of HSC repopulating activity, demonstrated that the +9.5 element is required for hemogenic endothelium to generate HSCs in the AGM. In principle, the +9.5-dependent genetic network should reveal clues regarding the molecular mechanisms underlying the defective HSC generation in +9.5^-/- AGMs. We conducted RNA-seq to define +9.5^+/+ and +9.5^-/- AGM explant transcriptomes, and this genomic analysis indicated that the +9.5 element instigates a stem cell-regulatory genetic network consisting of genes encoding established regulators of hemogenic endothelium and HSCs, and genes not implicated previously in hematopoiesis. We investigated whether the +9.5 element contributes to the transcriptome of AGM endothelium. Quantitative RT-PCR analysis revealed a similar impact of +9.5 deletion on representative genes in the fraction enriched in endothelial cells (CD31⁺c-Kit^-) from the AGM. These studies establish a new model whereby a composite cis-regulatory element induces Gata2 expression and instigates a complex genetic network in the AGM, which controls the transition of hemogenic endothelium to HSCs in the AGM. Studies are ongoing to establish the genetic network in hemogenic endothelium that mediates the development of the adult hematopoietic system and the applicability of the respective mechanisms to distinct biological and pathological contexts.