The promise of zebrafish: a novel resource to study the myeloid response in vivo

Thesis

Oncogenic transformation, local tissue damage at a site of injury, or invading pathogens all induce signals resulting in the recruitment of macrophages and neutrophils to the inciting stimulus. This study describes the development of an innovative binary approach in a zebrafish animal model that enables genome-wide profiling of specific cell types isolated directly from the in vivo context. The modular system harnesses the power of zebrafish genetics using an in vivo biotinylation system consisting of a zebrafish line ubiquitously expressing a nuclear envelope with an Avi-tag. The Avi-tag is biotinylated by the biotin ligase, BirA, specifically expressed by macrophages and neutrophils, and subsequently the nuclei of these cells types can be isolated by streptavidin affinity purification. The model was used to profile the active transcriptome of neutrophils in two disease pathologies, Mycobacterium marinum infection and the earliest stages of melanoma. For studies of the immune response to M. marinum infection, a granuloma-forming model was optimised, which recapitulates human tuberculosis. Profiling revealed a unique transcriptional signature for neutrophils, characterised by both factors that are host protective and those that may contribute to the pathogenesis of the disease. For studies of melanoma onset, we have generated a novel inducible model for melanocyte oncogenic transformation. The model for melanoma onset was crossed to the binary neutrophil-specific in vivo biotinylation model. Profiling of the neutrophils in response to oncogene-transformed melanocytes revealed a neutrophil transcriptional signature consistent with a pleiotropic role for neutrophils at the onset of melanoma. Not only does the model generated in this study prove to be an excellent resource for studying inflammation in vivo, but the data obtained provides novels insights into the role of neutrophils in mycobacterium infection and the onset of melanoma, that can be extended to human studies and harnessed for immunotherapy.

Authors: Kenyon, A

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford