Regulation of stem cell development by Runx transcription factors

Abstract

The origin of haematopoietic stem cells (HSCs) and the relative roles of the yolk sac and the aorta-gonad-mesonephros (AGM) region in the establishment of definitive haematopoiesis are controversial. Definitive HSCs were thought to originate from the AGM, while contribution from the yolk sac was restricted to the primitive blood. However, recent mammalian studies have demonstrated a contribution of yolk sac progenitors to the formation of definitive erythroid and myeloid lineages. This is supported by the recent discovery in zebrafish of erythromyeloid progenitors (EMPs) that arise within the posterior blood island (PBI), before emergence of HSCs in the AGM. The cell type that gives rise to HSCs is also unknown, with possible contributions from a bipotential haemangioblast, the haemogenic endothelium and the mesenchyme. The Runx transcription factors are important regulators of development in a number of organ systems. Runx1 is essential for the development of definitive HSCs and is transcribed from two promoters, P1 and P2, generating two major Runx1 isoforms. To further understand the role of Runx1 in HSC biology and the roles of the two isoforms during the establishment of definitive haematopoiesis, transgenic runx1 promoter reporter zebrafish were generated. The Tg(runx1P1:EGFP) line displays fluorescence in the PBI, where EMPs develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the AGM that are shown, by lineage tracing, to later populate the pronephros and thymus. Direct tracking of marked cells from the PBI in the runx1P1 transgenic confirms the two definitive blood lineages are distinct. Time-lapse imaging of a compound Tg(runx1P2:EGFP)/ Tg(kdrl:nls-mCherry) embryo expressing the red fluorescent protein mCherry in endothelial cells shows the emergence of HSCs from endothelial cells. This is the first demonstration of the direct generation of definitive HSCs from the haemogenic endothelium in a living embryo. The differential expression and functions of Runx3 isoforms were analysed. Depletion of Runx3P2 delays the entry of primitive erythrocytes into the circulation, and blocks the initiation of definitive haematopoiesis. Runx3P1 is expressed in the gut endoderm of the embryo and may function in regulating the activity of signalling pathways controlling angiogenesis through the Hedgehog and BMP signalling pathways.