Investigating novel players in inflammatory cell migration to sites of tissue damage in vivo

Abstract

The use of model organisms is vital for the improvement of our understanding of dynamic immunological processes. Previous work in the developing Drosophila embryo has demonstrated that epithelial wounding by laser ablation induces the rapid production of hydrogen peroxide (H2O2). This early damage signal activates the tyrosine kinase Src42a within the embryonic hemocytes – the innate immune cells of the organism. Src42a subsequently phosphorylates the tissue damage receptor Draper on its intracellular immunoreceptor tyrosine activation motif (ITAM) domain. This recruits a second kinase – Shark – and results in the activation of hemocytes and their subsequent migration to the damage site.
As kinase activation is key to the tissue damage response of these inflammatory cells, we reasoned that other hemocyte-specific proteins may also be phosphorylated following H2O2 signalling. Therefore, a phosphoproteomics screen was conducted in order to uncover novel H2O2/Src42a regulated phosphoproteins. Using this approach, we discovered a hemocyte wound recruitment defect in embryos lacking the PTP type phosphatase Pez. This was shown to be cell-autonomous; and time-lapse imaging revealed a lack of directionality within Pez mutant hemocytes in the presence of an epithelial wound. We also uncovered dynamic localisation of Pez in vivo and it’s colocalization with Draper during the engulfment of apoptotic debris. Finally, we demonstrated that the loss of Draper and Src42a leads to the mislocalisation of Pez.
To investigate whether this signalling pathway is evolutionarily conserved, tail fin clipping of 3 dpf Danio rerio larvae was utilised. Excitingly, Draper orthologue (MEGF10) morphants showed a reduction in both neutrophil and macrophage numbers recruited to the wound margin. This observation was further confirmed by the generation transient CRISPant larvae. Finally, we demonstrated that the Pez orthologue – named PTPN21 – also plays a role in inflammation in the zebrafish following gene disruption by CRISPR.

Date of Award	23 Jan 2020
Original language	English
Awarding Institution	The University of Bristol
Supervisor	Will J Wood (Supervisor)