Creation of hematopoietic stem/progenitor cells (HSPCs) early in development is a highly dynamic process whereby flat hemogenic endothelial cells (ECs) change cell fate and emerge as rounded HSPCs with self-renewing capacity and multi-lineage potential. Unfortunately, the molecular mechanisms controlling this endothelial-to-hematopoietic transition (EHT) are still not well defined. This is underscored by the inability to mass-produce long-term reconstituting HSPCs for blood-regenerative therapies. Identification of new regulators of HSPC formation has been challenging in mammalian models likely due to the inability to visualize EHT dynamics in utero. Recently, our lab has taken advantage of the transparency and external development of zebrafish embryos to identify microRNA-223 as a novel inhibitor of HSPC formation. Zebrafish embryos lacking miR-223 activity had an increased number of emerging HSPCs, resulting in mature HSPC expansion from the onset and to later stages of hematopoiesis. How miR-223 controls HSPC production from the endothelium at the cellular and molecular levels is currently unknown. miR-223 belongs to the microRNA (miRNA) class of small noncoding RNAs, which provide precision to signaling pathways by post-transcriptionally repressing gene expression of complementary mRNA targets. Comparative transcriptome profiling of zebrafish miR-223 mutant and wildtype endothelial cells revealed N- glycosylation enzymes as candidate miR-223 molecular targets, and embryos treated with N-glycosylation inhibitors phenocopy the HSPC expansion in miR-223 mutants. Thus, the goal of this proposal is to directly test the hypothesis that miR-223 fine tunes N-glycosylation levels to control HSPC formation from the endothelium. The proposed study will develop novel transgenic and mutant approaches in zebrafish to dynamically visualize the cellular events controlled by miR-223 and to validate the N-glycosylation pathway as the direct target of miR-223 during EHT. Altogether, findings from this study will contribute a novel mechanism of HSPC formation, in which a miRNA dependent N-glycome regulates EC to HSPC cell fate transitions.
The proposed study will develop technologies to facilitate the in vivo study of microRNA function in the development of hematopoietic stem/progenitor cells (HSPCs) from arterial endothelial cells. The resulting data will provide greater mechanistic insight into how HSPCs are initially created. This is of paramount importance, given the therapeutic potential of reconstituting HSPCs for the treatment of a multitude of blood disorders including leukemia.
