INVESTIGATING THE ROLE OF EZH1 IN DEVELOPMENTAL HEMATOPOIESIS

Abstract

Understanding how hematopoietic stem cells (HSCs) are produced and maintained is essential to the goal of generating patient-specific HSCs capable of multi-potent long-term function. HSCs are specified in select arterial niches during embryonic development, however the pathways regulating their emergence remain incompletely defined. Recently, an in vitro loss-of-function screen in human hematopoietic progenitors identified the Polycomb group protein, Enhancer of Zeste 1 (EZH1), as a regulator of definitive hematopoiesis. To evaluate whether this observation was conserved in vivo, functional knockdown techniques were employed in zebrafish. Initial analyses using in situ hybridization indicate that morpholino-mediated ezh1 deficiency promotes expression of the conserved HSC markers runx1/c-myb in the ventral wall of the dorsal aorta (VDA) at 36 hours post fertilization; these studies were confirmed and quantified by qPCR and FACS, indicating enhanced HSPC number. Strikingly, ezh1 knockdown increased hemogenic endothelium at the expense of arterial endothelium without disrupting initial arterial/venous specification, suggesting ezh1 expression is spatiotemporally restricted within the endothelium. As Notch signaling has been implicated in both arterial specification and HSC emergence, we examined the potential role of Notch signaling in ezh1 knockdown-mediated HSPC expansion and observed differential regulation of Notch ligands and receptors throughout the endothelial-to-hematopoietic transition compared to wild-type siblings. Notably, the effect on Notch signaling was specific to ezh1 knockdown, as ezh2 knockdown shows a distinct pattern and temporal impact. This strong conservation of function will serve as a platform to identify chemical or genetic factors that regulate ezh1 expression and function to unlock multi-lineage HSC fate.