Abstract
ABSTRACTZebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing individual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available.This article has an associated First Person interview with the first author of the paper.
Highlights
Neutrophils and macrophages, despite both being phagocytes, have many lineage-specific functions and contribute differentially to physiological and pathological processes
As the injected Gal4 driver lines carried the UAS:NTR-mCherry transgene (Davison et al, 2007), this transgene configuration resulted in lines with mCherry-expressing phagocytes in which, by virtue of the breeding strategy, a red heart indicated Cas9 expression in mCherry neutrophils, and a green eye indicated Cas9 expression in mCherry macrophages (Fig. 1C,D)
Stable transgenic F1 zebrafish embryos expressing the red heart and green eye fluorophore markers were confirmed as carrying the linked Cas9 transgene by polymerase chain reaction (PCR) genotyping (Fig. S1)
Summary
Neutrophils and macrophages, despite both being phagocytes, have many lineage-specific functions and contribute differentially to physiological and pathological processes. Conditional lineagespecific neutrophil or macrophage ablation by metronidazole/ nitroreductase systems reduces numbers of the individual phagocyte type, but by acutely invoking apoptosis, which itself may not be physiologically neutral (Ellett et al, 2018; Okuda et al, 2015; Prajsnar et al, 2012). Transcriptional factor manipulations, such as irf knockdown, can reduce numbers of macrophages, but at the same time increase the abundance of neutrophils, so macrophage requirement is not tested in the absence of any effect on neutrophils (Li et al, 2011; Rhodes et al, 2005). Lineage-specific requirements can be assigned to genes with lineage-specific expression, but this dichotomy only rarely applies and is reliant on the independent marker(s) used to assign leukocyte lineage identity
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call