Date of Award
Doctor of Philosophy
James A. Roth
Rac2 is a small Rho GTPase known to have roles regulating actin cytoskeletal reorganization and gene expression in cells of hematopoietic origin. Loss of Rac2 activity in humans and mice results in neutrophil function deficits and severe bacterial infections. While rac2 transcript had been confirmed in whole adult zebrafish preparations, little else was known about Rac2 in the zebrafish. This work substantially increases what is known about Rac2 in the zebrafish.
It was hypothesized that Rac2 function in zebrafish is similar to that in humans. In order to investigate this, a three-pronged approach was taken: 1) a descriptive study of the expression of Rac2 protein, using immunocytochemistry, and transcript, using qPCR, in the zebrafish neutrophil; 2) in vitro neutrophil function assays and Rac-specific small-molecule inhibition; 3) morpholino oligonucleotide gene knockdown of rac2 in zebrafish embryos followed by exposure to one of two fish pathogens, Aeromonas hydrophila or Edwardsiella ictaluri, and assessment of embryo survival.
The presence of Rac2 protein is described for the first time in the adult zebrafish neutrophil. It was localized to the cytoplasm, as in human neutrophils. rac2 transcript was found in whole embryo preparations by 12 hours post fertilization, earlier than previously described. Only rac2, and not rac1 or rac3, was found in adult zebrafish neutrophils. Inhibition of Rac caused significant decreases in the key neutrophil functions of respiratory burst, phagocytosis, and NETs release. Following exposure to Aeromonas hydrophila, rac2 morpholino-injected embryos exhibited significant increases in mortality as compared to uninjected or control morpholino-injected embryos.
Rac2 clearly plays a role in neutrophil function in the zebrafish. That role is similar to the one that Rac2 plays in human and mouse neutrophils.
Rachel Maureen Tell
Tell, Rachel Maureen, "Rac2 in the zebrafish (Danio rerio) with a focus on neutrophils" (2012). Graduate Theses and Dissertations. 12870.