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First published online February 6, 2004
Journal of Experimental Biology 207, 923-935 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00845

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The early ontogeny of neuronal nitric oxide synthase systems in the zebrafish

B. Holmqvist^1,*, B. Ellingsen¹, J. Forsell¹, I. Zhdanova² and P. Alm¹

¹ Department of Pathology, Lund University, Sölvegatan 25, S-221 85 Lund, Sweden
² Department of Anatomy and Neurobiology, Boston University Medical School, 715 Albany Street R-91, Boston, MA 02118-2394, USA

View larger version (28K): [in a new window]   Fig. 1. Schematic representation of the spatial and temporal distribution of nNOS mRNA-expressing cell populations (filled circles) in embryonic zebrafish during representative developmental life stages (brain at 19, 24, 30, 40 and 55 h.p.f. and eye and peripheral organs at 72 h.p.f.). Note the initial expression in the brain restricted to the ventrorostral cell cluster (vrc), the subsequent expression in ventrocaudal cell cluster (vcc), dorsorostral cell cluster (drc) and hindbrain cell clusters (hc), followed by the major increase in expression from around 40 h.p.f. and the presence of different nNOS mRNA-expressing cell populations in all major parts of the brain at 55 h.p.f. (hatching). Around hatching, the first nNOS mRNA expression in the eye and peripheral body organs appears, represented in the image of the 72 h.p.f. larvae.

View larger version (122K): [in a new window]   Fig. 2. nNOS mRNA expression in the brain of embryonic zebrafish at different developmental stages. (A-C) Whole mounts; (D–J) cryosections; (H–J) comparison with AT immunoreactive neurons in corresponding regions at a representative stage (24 h.p.f., cryosections). (A,B) 19 h.p.f.; nNOS mRNA expression in the forebrain, in two bilateral cell populations (arrows) as part of vrc (sagittal view in A and frontal view in B). (C) 23 h.p.f.; the bilateral vrc cell clusters (arrow and arrowhead in whole-mount preparation) are seen in a semi-sagittal view. (D) 24–25 h.p.f.; one nNOS mRNA-expressing vrc cell (blue) is shown adjacent to the initial pigmentation of the retinal epithelium (brown; RPE, arrowheads) from a frontal view. (E,F) 30 h.p.f.; nNOS mRNA-expressing cell bodies in vcc (E; sagittal view) and nNOS mRNA-expressing cell populations in drc (F; sagittal view). (G) 34 h.p.f.; nNOS mRNA-expressing cells in hc (sagittal view). (H–J) AT immunoreactivity at 24 h.p.f. in brain regions corresponding to sites of the nNOS mRNA expression in vrc and vcc (H, sagittal view; I, frontal view), and in hc and medulla (J; sagittal view). Scale bars: in A, 100 µm (A,B); 30 µm (C,F); in D, 20 µm (D,E,G); in H, 50 µm (H–J). V, ventricle.

View larger version (184K): [in a new window]   Fig. 3. Distribution of nNOS mRNA expression (A) and AT immunoreactivity (B) in the zebrafish brain at 55 h.p.f. (parallel cryosections). At this stage the nNOS mRNA-expressing cell populations are present in all major brain areas, coinciding with the differentiation of neuronal structures, mainly represented by AT immunoreactive axons, axon arbors and dense fibre nets at putative termination areas. Scale bar, 100 µm. Pin, pineal organ.

View larger version (158K): [in a new window]   Fig. 4. Cryosections from 55 h.p.f. zebrafish demonstrate the distribution of nNOS mRNA expression (blueish cells in A,C,E,F,H,I) in the brain, and its relationship to the neuronal differentiation pattern represented by AT immunoreactivity in adjacent sections (black in B,D,G). (A,B) Central telencephalon (frontal view). (C,D) Thalamic and preoptic area (frontal view). (E) The pretectum, posterior tuberculum (post. tub.), lateral hypothalamus, the brainstem and secondary matrix on the border to the cerebellum (sagittal view). (F,G) Sagittal view; (H) frontal view. (I) Weakly labelled nNOS mRNA-expressing cells in the retina (arrowhead). Scale bars: in A, 50 µm (A,B); in C, 50 µm (C–E,I); in F, 50 µm (F–H).

View larger version (139K): [in a new window]   Fig. 5. nNOS mRNA expression in skin and peripheral organs of the developing zebrafish. (A) Weak labelling of nNOS mRNA (arrows) in the skin of a 20 h.p.f. embryo, treated as a whole-mount preparation before clearing, compared to the strong labelling in the same region of a cryosection from another embryo (B). In the skin, labelling is preferentially present in epithelial cells of the tail (C) and around the yolk sac (D). (E) Absence of labelling after incubation with the sense probe. (F) The initial nNOS mRNA expression in body organs (at 55 h.p.f.), in cells located in the rostral portion of the forming gut (arrows). (G–I) Strong labelling of expression in transversal sections of nNOS mRNA in cells (arrows) located bilateral to the swim bladder (sb) and gut (gut), in relation to the pro-nephritic duct (asterisks in G and H) and to the nNOS-expressing cells in the medulla (arrowheads in G). Brownish structures are pigments. Scale bars: 50 µm (B,C,G); 5 µm (D); 10 µm (E,F,H,I).

View larger version (187K): [in a new window]   Fig. 6. nNOS mRNA expression in peripheral organs of developing zebrafish (at 72 h.p.f.). (A–D) (A) Sagittal section, low magnification, demonstrating nNOS-expressing cell clusters in the posterior portion of the swim bladder (sb; see transversal section in B), and in the rostral portion of the alimentary system (putative enteric ganglia shown in C,D). (E–G) Sagittal sections demonstrating how the rostral nNOS-expressing cell clusters become a population with evenly distributed cells in the mesenchyme along the alimentary tract and nephritic duct. Brownish structures are pigments. Scale bars: in A, 100 µm (A,G); in B, 10 µm (B–E). sb, swim bladder.

View larger version (10K): [in a new window]   Fig. 7. cGMP levels in zebrafish embryos of different age, 8–55 h.p.f. Values are means ± S.D. of two groups of wild-type Danio rerio (Tubingen and local), N=60 eggs per group.