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Blood, Vol. 111, Issue 1, 132-141, January 1, 2008
Origins and unconventional behavior of neutrophils in developing zebrafish
Blood Le Guyader et al.
111: 132
Supplemental materials for: Le Guyader et al
Files in this Data Supplement:
- Figure S1. The first Sudan Black stained leukocyte granules appear by 35 hpf (JPG, 250 KB)
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DIC images from embryos in left lateral view. (A) Yolk sac; (B) Just ventral to somite 1; (C-E) In the head mesenchyme near the midbrain-hindbrain boundary (C), near the yolk sac (D), and near the inner ear (E); (F) On the pericardium, just beneath the epidermis; (G-J) in the ventral tail. (A-I) are all at the same scale. (K-P) VE-DIC images of likely myelocytes in the ventral tail, between caudal artery and vein, of a live 35 hpf embryo. Arrowheads point at granules clustered at the indentation of the kidney shaped nucleus. Panels L-P are a time sequence showing the mitosis of such a myelocyte (arrow); time is indicated in the upper left corner. P, pericardial cavity; oe, otic epithelium; y, yolk sac; ca, caudal artery; cv, caudal vein; m, somitic muscle. Scale bars, 10 µm.
- Figure S2. Electron microscopy of neutrophilic granulocytes in the tissues of the zebrafish swimming larva (JPG, 215 KB)
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(A-E) 4 dpf. Zebrafish neutrophils contain two types of granules1-3: round, with homogeneous density (arrowheads), and needle-like, often containing crystalline inclusions (arrows). (A) Neutrophil in the epidermis, with bilobed nucleus; note the actin ridges of the outer periderm (p) forming the body wall. (B) Beneath the periderm (p) and underlying epidermis (ep) is a neutrophil (n), in extended contact with a macrophage (m ) and a melanocyte (m). (C-E) Neutrophils in the cephalic mesenchyme. (F) 5 dpf; neutrophil infiltrated in the intestinal mucosa.
- Figure S3. All SB stained granulocytes express L-plastin (JPG, 145 KB)
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SB staining was followed by immunodetection of L-plastin (red/orange). All panels are left lateral views of 2 dpf (A-F), 3 dpf (G-I) and 5 dpf (J) larvae.
(A) Macrophages in the retina and brain (A) are L-plastin+ and SB-; so are likely macrophages in the mesenchyme, e.g. near the ear (B) and in the trunk horizontal myoseptum (C); all SB+ cells (granulocytes) are also L-plastin+, e.g. in the cephalic mesenchyme (A,E) and nascent caudal hematopoietic tissue (D,F); note that erythrocytes in the caudal artery are negative for either staining. (G) By 3 dpf, the retinal macrophages, which have become primitive microglia4 are still L-plastin+; so are all SB+ granulocytes, shown here in the cephalic mesenchyme (G,I) and on the pericardium (H). Thymocytes in the nascent left thymus are also L-plastin+ (I,J; at these early stages, the only leukocytes in the thymus are lymphoblasts1; at 3 dpf they are still very few, and only the few dorsal-most ones are seen in panel I). R, retina; mb, midbrain; c, cerebellum; hb, hindrain; e, ear; p, pericardium; y, yolk sac; ca, caudal artery; cv, caudal vein; m, muscle; t, thymus; ba, branchial arches; prc, photoreceptor cell layer; bnc, bipolar neuron cell layer; rgc, retinal ganglion neuron cell cell layer; op, outer plexiform layer; ip, inner plexiform layer; l, lens. Bars, 50 µm in A, 10 µm in B-F,J, 20 µm for G-I.
- Figure S4. Granulocyte vs. macrophage progeny of primitive myeloid precursors photolabelled at 20 hpf (JPG, 84.5 KB)
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Following the laser-mediated uncaging of caged fluorescein dextran in 10 primitive myeloid progenitors in the yolk sac per embryo at 20 hpf, the resulting larvae were fixed at 72 hpf and the labelled progeny was counted throughout each larva after combined tyramide-based detection of endogenous peroxidase activity (marking granulocytes) and anti fluorescein immunohistochemistry, as in Figure 5C.
- Figure S5. Injection of sterile PBS into the ear cavity, if not finely tuned, recruits neutrophils as efficiently as injection of bacteria (JPG, 129 KB)
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The experiment done here was the same as in Figure 7E (Sudan Black staining, 5 hrs post injection in the left ear at 3.5 dpf), except that PBS or bacteria were micro-injected in the inner ear with a 2-fold higher pressure (40 psi) and duration (40msec); see Materials and methods.
- Figure S6. Altered distribution of Sudan Black stained granulocytes in a 21-day malformed larva (JPG, 524 KB)
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This malformed wild-type larva (B,D,G-I) is compared to one of its normal siblings (A,C,E,F). (A,B) overall lateral view. (C,D) dorsal view; (E-I) higher-magnification left lateral views, except (G) which shows the right eye. The malformed larva notably displays a depletion of granulocytes from the head kidney (K), and an accumulation of them all around both lenses (G,H), which are free of granulocytes in the normal sibling (E), and over the intestinal bulb (I). K, head kidney; IB, intestinal bulb, SB, swimbladder; r, retina.
- Video 1. Neutrophilic granulocyte wandering in the epidermis at 48 hpf (MOV, 5.93 MB)
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Real-time DIC video-microscopy. The further progression of this neutrophil between the epidermal cells in the next 9 min. is shown in Figure 2A. The flow visible out of focus all over the image is that of blood cells in the yolk sac circulation valley (duct of Cuvier), just beneath this epidermal layer. The dark shadow in the upper right corner is from an out-of-focus melanophore.
- Video 2. Interaction of a neutrophil with two macrophages in the yolk sac circulation valley (MOV, 5.39 MB)
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This DIC video sequence opens on a macrophage (note the two dot-like nucleoli in its nucleus) interacting with four seemingly degenerating erythrocytes. It is initially in real-time. A neutrophil (with typical moving refractile granules and nucleus also showing two dot-like nucleoli), brought by the blood flow, “lands” on the macrophage (see also Figure 2D). After 7 seconds, the movie accelerates from real-time to 8-fold faster. At 13 sec. of movie, an eythrocyte lands on the neutrophil, then is taken again by the blood flow at 19 sec. At 24 sec., a second macrophage has stopped by (top) and come in contact with the neutrophil.
- Video 3. Macrophage and granulocyte in the CHT at 56 hpf (MOV, 2.23 MB)
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DIC video-microscopy of the CHT; lateral view, rostral to the left. The macrophage is to the left, in the lumen of the future definitive caudal vein (cv), attached to the endothelium wall, with phagocytosed material and endocytosed methylene blue. The neutrophil is to the right, in the abluminal mesenchyme. Note its moving granules and indented nucleus. Cv, caudal vein.
REFERENCES
1. Willett CE, Cortes A, Zuasti A, Zapata AG. Early hematopoiesis and developing lymphoid organs in the zebrafish. Dev Dyn. 1999;214:323-336.
2. Lieschke GJ, Oates AC, Crowhurst MO, Ward AC, Layton JE. Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish. Blood. 2001;98:3087-3096.
3. Bennett CM, Kanki JP, Rhodes J, et al. Myelopoiesis in the zebrafish, Danio rerio. Blood. 2001;98:643-651.
4. Herbomel P, Thisse B, Thisse C. Zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a M-CSF receptor-dependent invasive process. Dev Biol. 2001;238:274-288.
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