Single-cell RNA-seq analysis of immune cells isolated by FACS from zebrafish embryos after acute muscle injury using a novel irg1 knock-in reporter

The objective of this experiment was to analyze the single-cell transcriptomes of macrophages for molecular profiling of subtypes that emerge during acute muscle injury in zebrafish. This transcriptomic data was used in conjunction with high-resolution live-cell microscopy, in which individual macrophages were tracked to examine both cellular and population-level dynamics. Comparisons were made between baseline (stage-matched, uninjured controls) and distinct phases of injury: the initial inflammatory response at 24 hours post-amputation (hpa) and the resolution phase beginning at 48 hpa of two genotypes, control/wild-type and chronic inflammation mutant (nlrc3l deletion). Single-cell sequencing revealed significant differences in immune cell transcriptome post injury between control and chronic inflammation nlrc3l mutants, and that most irg1 positive cells were macrophages with a fraction of them being altered neutrophils also expressing irg1 after injury particularly in the mutants. Overall design: Transgenic zebrafish carrying the novel GFP knock-in reporter irg1-KI:GFP were used to isolate macrophages at distinct phases of acute muscle injury response. Specifically, samples were collected at 24 hours post-amputation (hpa), representing the initial inflammatory phase, and at 48 hpa, marking the onset of tissue repair following debris clearance. Uninjured ("uncut") animals served as baseline controls. Responses were compared between wild-type (or control sibling) zebrafish and mutants with a deleterious nlrc3l mutation that causes chronic macrophage activation. To induce acute muscle injury, large tail amputations were performed on 2 days post-fertilization (dpf) zebrafish embryos. This procedure causes significant muscle damage, which fully regenerates within four days, forming a new tail tip. Activated macrophages upregulate irg1, displaying strong GFP fluorescence, while steady-state macrophages express minimal irg1, resulting in faint GFP signal. All irg1-KI:GFP+ cells expressing any detectable level of GFP were isolated using fluorescence-activated cell sorting (FACS) on a BD FACSMelody across four conditions: control uncut, control 24 hpa, control 48 hpa, and nlrc3l mutant 24 hpa. Two biological replicates were collected per condition, each consisting of pooled cells from 90 embryos, resulting in a total of eight samples. To maintain cell viability and quality, embryos were dissociated and sorted in batches of 30, with three such batches pooled to constitute a single sample. Sample collection was interleaved to evenly distribute conditions across the processing timeline. Embryo dissociation was performed in a buffer containing ROCK inhibitor, protease, DNase with gentle mechanical grinding, and a holding solution composed of HBSS (without phenol red), 1% BSA, and ROCK inhibitor. Cells were kept on ice until 10x genomics downstream processing and library preparation.