Fig. 4: In vivo longitudinal and time-lapse imaging tracks macrophage activation during acute injury response, revealing severe deficiencies in auto-inflammatory nlrc3l mutant.

a Schematic of injury model involving tail amputation at 2 dpf, where the cut is positioned ~ tenth somite down from the end of yolk extension (or the midpoint between yolk extension and tail end). Created in BioRender. Shiau, C. (2026) https://BioRender.com/rxau1o9. b In vivo confocal images from a longitudinal study of wild-type embryos (n = 6) post-injury, representative data from one individual. c Longitudinal analysis plots comparing wild-type (n = 6) and nlrc3l mutants (n = 4) during injury from initiation to resolution. Wild-type individuals are plotted in shades of black, and nlrc3l mutants in shades of red. The dotted box in b highlights the quantified region. Thicker plot lines indicate group averages, with SEM shown by shaded regions. d Quantification of macrophage and neutrophil responses between nlrc3l control siblings (sib, heterozygous and wild-type, n = 4 uncut and 5 cut) and homozygous mutants (mut, n = 4 uncut and 5 cut) at baseline and peak inflammation ( ~ 24 hpa). Each data point represents an individual animal. e Representative static time series from time-lapse confocal imaging of double transgenic zebrafish labeling macrophages and neutrophils at 24 hpa. Images were captured every 2 min using a 40x objective. Three distinct macrophage subtypes (1, 2, 3) are labeled by numbers and arrows, with mutants lacking subtypes 2 (cluster) and 3 (muscle-encasing). For more details, see Supplementary Movies 3 and 5 (timelapse imaging) and Supplementary Movies 4 and 6 (rendered movies with subtype annotations). f Quantification of macrophage cell behavior and morphology along with representative microscopy images of the three subtypes (1: mobile, white; n = 7 sib cells; n = 7 mut cells; 2: cluster, blue; n = 5 sib cells; 3: muscle-encasing, red, n = 8 sib cells)(shown in g), shows differences between nlrc3l mutants and control siblings. See also Supplementary Fig. 12 for supporting data. Statistical significance for all scatter bar plots was determined using a one-way ANOVA followed by multiple comparisons: *, p < 0.05, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. A.U., arbitrary units. Each experiment was repeated at least twice. Data are presented as mean values +/- SD in d and f.