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Abstract
ROS and SFK signaling is required for development of hematopoietic stem cells in zebrafish
Bradley Williams and Erich Damm
Background
Hematopoiesis is the process in which the myriad of mature blood cell types, including erythrocytes and leukocytes, develop from hematopoietic stem cells (HSCs) throughout the life of an organism. In vertebrates, HSCs originate from endothelial cells lining the floor of the dorsal aorta. This process is referred to as the endothelial to hematopoietic transition and is likely controlled in part by molecular signals from neighboring cells. Investigation into the molecular signaling mechanisms controlling the development of HSCs is critical to the development of potential treatments to blood disorders, such as training patient-derived induced pluripotent stem cells (iPSCs) to differentiate into replacement HSCs for transplantation into leukemia patients or patients receiving gene therapy for genetic hematologic disorders. Reactive oxygen species (ROS), such as superoxides and peroxides, have been shown to act as cellular signaling molecules. Previous studies have shown a requirement for ROS signaling in the early development of HSCs, although the complete mechanism of ROS signaling involvement has not yet been determined. Additionally, it has been suggested that Src family kinases (SFKs), a family of key cell signaling regulators, can be regulated by ROS signaling, although a role for SFKs in developmental hematopoiesis has not been demonstrated.
Objectives
The aim of this study was to determine if ROS and SFK signaling are required for the earliest stages of HSC development in the zebrafish embryo.
Methods
To evaluate the roles of ROS signaling and SFK activity in HSC development, pharmacological inhibition or anti-sense morpholino oligonucleotides were used to knockdown ROS and SFK activity. Wild type zebrafish (Danio rerio) embryos were treated with either MCI-186, a ROS scavenging compound, or PP2, a pharmacological inhibitor of SFKs. Whole-mount in-situ hybridization (WISH) for key HSC and endothelial cell marker genes was performed to examine the status of HSC and endothelial development under these conditions. To examine the expression of the SFK yes related kinase (yrk) in the zebrafish embryo, WISH was performed. Transgenic zebrafish embryos exhibiting fluorescent vascular tissue (Tg(kdrl:mCherry)) were stained with a phospho-Src antibody to highlight SFK activity in the embryonic vasculature. Transgenic embryos were also injected with splice-blocking morpholino oligonucleotides targeting yrk mRNA transcripts to knock down Yrk protein expression.
Results and Conclusions
We show that inhibition of ROS signaling results in a loss of HSC development and a reduction in phosphorylated Src kinases in the dorsal aorta of zebrafish embryos during the stage when HSCs develop. Importantly, we confirmed that the SFK yrk is expressed in the embryonic vasculature during the stage when HSCs initially develop. We further show that embryos treated with the SFK inhibitor PP2 or injected with morpholinos targeting yrk, exhibit defective HSC development, implying that both ROS and SFKs are required for HSC development. Our results suggest a model where ROS signaling is required for phosphorylation of the SFK yrk, which in turn plays a yet to be determined signaling role in the development of HSCs.
Publication Date
2023
Subject Major(s)
Developmental Biology
Keywords
Developmental Biology, Biology, Hematology, Hematopoiesis, Reactive Oxygen Species, Src family kinase, SFK
Disciplines
Cell Biology | Developmental Biology
Current Academic Year
Senior
Faculty Advisor/Mentor
Erich Damm
Rights
© The Author(s)