Jan 2015 "The program of endothelial-to-hematopoietic transition (EHT) and HSC generation" January 27, 2016 Doug Vernimmen hosted Professor Elaine Dzierzak, University of Edinburgh, for a seminar at The Roslin Institute. ABSTRACT Our knowledge and an understanding of the genetic program that directs HSC generation during embryonic development is key to devising strategies for patient-specific HSC production and understanding HSC dysfunction. HSCs are generated through a natural transdifferentiation process occurring in specialized embryonic vascular cells, known as hemogenic endothelial cells. These cells undergo endothelial-to-hematopoietic transition or EHT as shown in several vertebrate embryo models. As the first site to initiate generate HSCs with robust adult repopulating and self-renewing activity, the aorta-gonad-mesonephros (AGM) region has been our major research focus. Using mouse transgenic reporters and knockout models we have been able to characterize and isolate AGM HSCs, as well as HSCs and other progenitors from hematopoietic tissues where they are detected (yolk sac, placenta and the liver). Most recently we have characterized HSCs that are generated in the embryonic. Live imaging has provided visual evidence of EHT in the AGM of Ly6A-GFP transgenic embryos, where GFP expression begins in a few aortic endothelial cells. Subsequently, expression is found in cells bulging from the endothelium, and hematopoietic cells in the proximal and distal parts of the clusters. In our current studies exploring the genetic program leading to HSC formation, this mouse model has facilitated the quantitation and high enrichment of midgestation aortic hemogenic endothelial cells undergoing EHT. We identified the whole transcriptome of small numbers of enriched aortic HSCs, hemogenic endothelial cells and endothelial cells by a highly sensitive RNAseq method and found 530 differentially expressed genes. Amongst these are signalling pathway, transcription factor and receptor genes, many are relevant to endothelial and hematopoietic cells. Processes (gene ontology terms) involved include organ morphogenesis, development, regulation of cell migration, cell cycle progress, regulation of histone methylation, immune system and hematopoiesis. The datasets also revealed that the heptad complex of hematopoietic transcription factors are upregulated during EHT, with Gata2 being a pivotal factor. The consistent but small quantitative level upregulation of some of these factors suggests that even 2-fold differences are likely to be important for precise regulation of EHT and HSC generation. Interestingly, we found that the G-protein receptor GPR56, is one of the most highly upregulated genes and is required for hematopoietic cluster formation during EHT. Current work on the processes and the regulators involved in EHT and HSC development will be presented.
