Fig. 4

Delayed macrophage recruitment in zebrafish compromises neovascularization, CM proliferation and scar resolution.

Adult zebrafish were injected with PBS or clodronate liposomes 1 day before injury. Hearts were collected at 1 dpci (A), 7 dpci (B and C) and 1 mpci (E–G) to examine macrophages by mpeg1:EGFP expression and IB4 staining (A and B), neovascularization by alkaline phosphatase (AP) staining (B and E, a side view as well as an apex view are shown), CM proliferation by PCNA/Mef2 immunostaining (C), and scar resolution by Acid Fuchsin Orange G (AFOG) staining (F). Mef2 and PCNA double positive CMs within 200 μm of the injured area were quantified in (D). Sections with the largest injured area/scar for each 1 mpci heart (delineated by black dotted lines) were stained with AFOG and quantified in G (n > 5, shown in Figure 4—figure supplement 2). Dotted lines delineate the injured area, arrowheads point to vessels, asterisks mark the initial injury site; scale bars, 200 μm. N ≥ 3 for each treatment and time point. Clodronate injections diminished macrophage recruitment at 1 dpci, and macrophage numbers recovered to control levels at 7 dpci (A and B, quantification in Figure 4—figure supplement 1 and Figure 4—figure supplement 1—source data 1). Delayed macrophage recruitment compromised neovascularization (B and E), and CM proliferation (C and D), and resulted in delayed scar resolution (F and G).