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Hematopoietic stem cells (HSCs) are an integral part of modern biomedical research because they are precursors to all blood cell lineages in vertebrates. Further understanding of hematopoietic development will allow for improvements in the success of therapeutic bone marrow transplantation; which is a crucial treatment in management of both malignant and non-malignant blood disorders. Although HSC transplantation is curative for many blood disorders, finding compatible donors is difficult due to a need for immunologic compatibility. Consequently, recent research efforts have been directed towards generating HSCs for the patient, from the patient themselves through the directed differentiation of induced pluripotent stem cells (iPSCs). To achieve this overarching goal, it is imperative to have a thorough understanding of genetics and molecular signals involved in the specification, determination, and differentiation of HSCs during embryonic development so that this process can be replicated using patient derived cells. In all vertebrates, hemogenic endothelium cells, located in the dorsal aorta floor, are the origin of HSCs. Hemogenic endothelial must undergo an endothelial to hematopoietic transition to become HSCs. Oxidative stress, as a result of glycolysis, is required for this transition and to generate proliferating, quiescent, and differentiating HSCs. Deoxyribose phosphate aldolase (Dera) is an enzyme that is involved in controlling the development of stress granules in cells that are under stress. These stress granules are thought to play an important role in mediating the stress response in cells. Dera is expressed in the embryonic vasculature early in zebrafish development – at the time when HSCs first develop. Trunk expression is present in the intermediate cell mass at 18 hours post fertilization (hpf) and in the pronephric ducts, vasculature, and neural crest. Publicly available RNA sequencing data suggests that dera might be somewhat enriched in the hemogenic endothelium. In this study, we knocked down Dera expression in zebrafish embryos by antisense morpholino oligonucleotide and examined expression of the conserved hematopoietic markers: runx1, cmyb and rag1. We found a significant reduction in the expression of these markers, indicating defective HSC development in dera morphant embryos. We further examined the expression of markers of embryonic vasculature development in morphant embryos and found expression to be normal suggesting that defective HSC development was not a result of abnormal vascular patterning. We also conducted drug treatments with cycloheximide and sodium arsenite, at varying concentrations, to determine if embryos are more sensitive to stress in the absence of Dera. In conclusion, normal HSC development requires dera where its role is likely to moderate the cellular stress response to keep it within physiological levels that are required for HSC development.
zebrafish, oxidative stress, developmental niche, hemogenic endothelium, HSCs, Dera, blood cell development, hematopoietic stem cells, embryo, arsenite, cycloheximide, transcription factor, vasculature, knockdown, development, morpholino
Animals | Developmental Biology
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Dr. Erich W Damm
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