Figure 4.

The generation of new macrophages is reduced in MRKO larvae, and macrophages migrating toward the wound exhibit a less inflammatory phenotype. (A) Representative images of macrophage proliferation at 48 hours postfertilization (hpf), 4 hours postamputation (hpa), where the nuclei are stained with DAPI (blue), new cells have incorporated the thymidine nucleoside analogue EdU (red), and macrophages are visualized by immunohistochemistry using a Mfap4 antibody (green). White dotted lines denote the location of amputation. (B) Number of Mfap4+ macrophages localized to the wound site at 4 hpa (n = 7). (C) The percentage of new (EdU+) macrophages localized to the wound site at 4 hpa (n = 7). (D and E) Transcript abundance of macrophage-specific chemokines cxcl11aa (D) and ccl2 (E), determined by qPCR analysis of RNA isolated from larvae at 4 hpa (n = 4). (F) Circularity index of macrophages that had migrated towards the wound at 2 hpa. (G) Transcript abundance of il1b, cxcr3.2, and ccr2 in FACS-sorted macrophages determined by qPCR (n = 3). (H) Number of mpeg+ macrophages that have migrated to the hindbrain 4 hours after CCL2 injection at 48 hpf (n = 6-9). (I) Representative images of WT and MRKO Tg(mpeg:mCherry) at 48 hpf, injected with either PBS or recombinant human CCL2 in the hindbrain ventricle. Bars show mean ± SEM of data pooled from 3 different experiments (B,C,H; each data point representing a single larva) or mean ± SEM of 3 individual experiments. Data were analyzed using a t-test (B, C, G) or a 2-way ANOVA (D, E, H; Holm-Sidak post hoc test). Statistical significance is indicated by *P ≤ .05, **P ≤ .01, ***P ≤ .001.