Understanding how hematopoietic stem cells (HSCs) are specified from mesodermal precursors is essential to the goal of generating patient-specific HSCs capable of multi-potent long-term function. HSCs are born from hemogenic endothelium in select arterial niches during embryonic development through a transdifferentiation process turned endothelial-to-hematopoietic transistion (EHT). Despite increasing efforts to recapitulate this process in vitro, current differentiation protocols largely fail to produce long-lived multi-lineage progenitors from human induced pluripotent stem cell (iPSC) sources. Recently, an in vitro loss-of-function screen in human hematopoietic progenitors identified the Polycomb group protein, Enhancer of Zeste Homolog 1 (EZH1), as a regulator of definitive hematopoietic commitment, as assayed by acquisition of lymphoid competence. To determine the mechanism by which Ezh1 regulates HSPC fate in vivo we employed functional knockdown and epistasis investigations using the zebrafish model. Morpholino-mediated knockdown of ezh1 promoted expression of the conserved HSC markers runx1 and c-myb in the ventral wall of the dorsal aorta (VDA) at 36 hours post fertilization (hpf), as assessed by whole mount in situ hybridization (WISH); additionally, expression of the lymphoid marker rag1 was found to be enhanced at 120 hpf, as assayed by WISH and fluorescent activated cell sorting (FACS), in line with our in vitro observations. An impact on HSPCs was confirmed and quantified by qPCR for runx1 (**p < 0.01) and FACS using the CD41:GFP reporter line (**p < 0.01), indicating significantly increased HSPC number. Importantly, this enhancement in HSPC production had no effect on gross vascular morphology of the niche as determined by confocal microscopy for flk1. Assessment of arterial versus venous fate indicated that while the latter was unchanged in morphant embryos, expression of the arterial markers, epbrinb2a, dll4, dlc and tbx20, was strongly reduced by WISH and qPCR (**p < 0.01, *p < 0.05, **p < 0.01, and **** p < 0.001, respectively). In contrast, markers of hemogenic commitment, gata2b, and scl/flk1, were significantly increased, suggesting that loss of ezh1 enhanced hematopoietic potential at the expense of maintaining arterial fate. Profiling of single-cell RNA-sequencing data obtained from sorted populations of E10.5 mouse embryos revealed EZH1 to be more highly expressed in cells undergoing the endothelial-to-hematopoietic transition, consistent with a role of EZH1 in regulating arterial verses hematopoietic fate. Gene set enrichment analysis (GSEA) from our prior in vitro studies revealed the Notch pathway to be significantly altered following EZH1 knockdown. As Notch signaling has been implicated in both arterial specification and HSC emergence, we next examined the potential role of Notch signaling in ezh1 knockdown-mediated HSPC expansion. Consistent with a hypothesized interaction, differential regulation of Notch ligands and receptors was observed in ezh1 morphants compared to wild-type siblings; specifically, expression of arterial ligands, dll4 and dlc were decreased, while hematopoietic ligands and receptors, jag1a and notch1a were enhanced. Notably, the effect on Notch signaling was specific to ezh1 knockdown, as ezh2 loss shows a distinct pattern and temporal impact, reducing HSC production rather than enhancing it, consistent with recent reports. The strong conservation of ezh1-mediated regulation of HSC number, and our identification of its mechanistic role at the level of Notch receptor/ligand interactions, position zebrafish as a platform to identify chemical mediators that can be used to regulate ezh1 function during in vitro differentiation to unlock multi-lineage HSC commitment of human iPSC for therapeutic application.
Disclosures
Daley: Epizyme, Inc: Other: Equity & Consulting Fees; 28/7 Therapeutics: Other: Equity & Consulting Fees.
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