Characterization of zebrafish larval inflammatory macrophages

Abstract

Zebrafish have emerged as a powerful model system to study leukocyte recruitment and inflammation. Here we characterize the morphology and function of inflammatory macrophages in zebrafish larvae. These macrophages can be distinguished from neutrophils by immunolabeling of L-Plastin without MPO co-expression and by an elongated morphology. Live imaging of transgenic zMPO:GFP larvae demonstrate that GFP(lo) macrophages migrate to wounds by extension of thin pseudopods and carry out phagocytosis of tissue debris, and FACS analysis of leukocyte markers indicates expression of CSF1R in these macrophages. These findings identify distinct functional and morphological characteristics of inflammatory macrophages in zebrafish larvae.

Figure 1. Confocal imaging of immunolabeled zebrafish larval leukocytes

(A–L) Wild-type larvae at 3 dpf were sequentially immunolabeled using a rabbit antibody to MPO and a FITC-conjugated anti-rabbit Fab fragment (A,D,G,J), followed by a Rhodamine Red-conjugated rabbit antibody to L-Plastin (B,E,H,K); overlapping signals are yellow in overlay images (C,F,I,L). Arrowheads mark LP+/MPO+ neutrophils, arrows mark LP+/MPO− macrophages. (A–C) Caudal Hematopoietic Tissue (CHT). (D–L) Leukocytes responding to wounds (wedges outlined in yellow) in the tailfin, at 2 hours post-wound (hpw). Note how LP+/MPO− macrophages have accumulated along the edge of a wound (E–F) and infiltrated a small fragment of wounded tissue (marked by an * in G–I). (J–L) Higher magnification views of cells within boxed areas in G–I, note the elongated morphology of LP+/MPO− macrophages and how the granular MPO label does not overlap with the LP signal within co-expressing neutrophils. Scale bars = 25 µm.

Figure 2. L-Plastin-labeled leukocytes in the epidermis of zebrafish larvae

Wild-type larva at 8 dpf immunolabeled with antibodies to L-Plastin (A) and the epithelial cell marker p63 (B); the CHT is denoted by brackets. (A–C) Overlay images of LP and p63 in the xy plane (A–B; anterior is to the left, dorsal side points up) and the yz plane (C; dorsal side points up) of the same embryo; in (D) a second larva labeled as in A–C is shown in the yz plane. Note the localization of LP+ cells within the p63-labeled epithelium. For A–B arrowheads mark neutrophils and arrows mark macrophages identified by morphology. Scale bar = 25 µm.

Figure 3. Hematopoeitic gene expression in zMPO:GFP larvae

(A) Still image (GFP overlaid onto DIC) from a time-lapse movie of zMPO:GFP (3 dpf) leukocytes responding to a wound (*) in the ventral tailfin; GFP^lo leukocytes and GFP^hi neutrophils are marked. (B) Flow cytometry of zMPO:GFP larvae at 3 dpf; GFP-Lo and GFP-Hi fractions are marked with boxes; cells from wild-type larvae were used to set the lowest level of GFP expression. (C) RT-PCR of hematopoietic markers (MPO, CSF1R and L-Plastin; EF-1α is used as a loading control) from GFP-Lo and -Hi fractions marked in B; cells sorted from either fraction and stained by Wright-Giemsa (i,iii–vi) or MPO activity (ii, MPO-A) are also shown. Hematopoietic precursor (hp); polymorphonuclear neutrophil (pmn); macrophage (mac). (D–F) Immunolabeling of zMPO:GFP (3 dpf) larva, 2h following a wound (*) in the caudal tailfin; (D) GFP antibody, (E) L-Plastin, (F) overlay. Note elongated LP+ cells that also express a low level of GFP (arrows); arrowheads mark GFP^hi neutrophils, which also express LP. Scale bar = 10 µm.

Figure 4. Migratory characteristics of GFP^lo macrophages in transgenic zMPO:GFP larvae

Sequential images from time-lapse movies of GFP^lo macrophages; note that brightness and contrast have been increased to view cellular details. (A; from Movie S1) A GFP^lo macrophage (arrow) extends two pseudopods, then “follows” the pseudopod nearest to a wound (out of frame, oriented by an *) by moving the cell body into this pseudopod; 1 min separates each frame. (B; from Movie S3) A GFP^lo macrophage (arrow) extends a pseudopod toward a wound (out of frame, oriented by an *) and maintains the pseudopod in this position for several minutes (3 min separates each frame) before retracting it and migrating in the opposite direction; a GFP^hi neutrophil is marked with arrowheads. Scale bars = 25 µm.

Figure 5. Phagocytosis of Cell Debris by GFP^lo macrophages

A melanocyte over the yolk sac is ruptured by laser wounding (circle denotes targeted area, t=0 is pre-wound), resulting in the recruitment of GFP^lo macrophages (arrows), which phagocytose pigment from the ruptured cell; a GFP^hi neutrophil (arrowheads) also responds. Images are from Movie S5. Scale bar = 20 µm.