Expression profiling of nos2b in zebrafish embryos by reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization (WISH). A: Chromosomal mapping of nos2b. Linkage map showing the localization of nos2b (=unp2188) to LG15 (obtained from http://wwwmap.tuebingen.mpg.de/). The distances between markers on the main map (left) are given in centimorgans (cM) and in centirays (cR) for the expanded region (right). B: Detection of nos2b transcripts by RT-PCR revealed a lower maternal expression in contrast to a higher expression after 16 hours postfertilization (hpf). An internal control using the house-keeping gene, elongation factor 1 alpha (ef1α) is also shown. C-E: nos2b transcripts were first detected at 14 hpf in the prospective ventral diencephalon. Expression of nos2b (arrowheads in C,D,E,F and G) is confined to the anterior part of the forebrain as shown in the frontal view (D), lateral view and the flat mount ventral view (E) of the 14 hpf embryo double stained with nos2b and catL1b. The gray dotted line in E outlined the optic primordium (op). F: By 20 hpf nos2b remained in the ventral floor of the forebrain. G: However, nos2b is excluded from the lim3-positive anterior pituitary. H-J: In addition, nos2b expression domain is ventrad to the nk2.1-positive hypothalamus (H,I) and colocalizes with fgf3 (J). d, diencephalon; e, eye; hg, hatching gland; ht, hypothalamus; pit, pituitary; t, telencephalon.

nos2b is expressed in two distinct primordial by 36 hours postfertilization (hpf). A: By 36 hpf, nos2b expression was found in two domains, one in the hypothalamus posterior to the hypophysis (arrow) and the second domain (arrowhead), at the base of the primordium of the lower jaw ventral of the anterior hindbrain. B: By 48 hpf, nos2b expression was maintained in the hypothalamus posterior to the hypophysis (arrow), but the expression level decreased. At the same time, the expression level in the domain (arrowhead) projecting onto the primordium of the lower jaw increased. C-H: This centrally localized domain of nos2b expression, flanked by tyrosine hydroxylase (TH) -positive arch associated cells (aac, C) and closely associated with the thyroid primordium marker tg (D), is absent in both smu^-/- and noi^-/- mutants (E-H). The arrowheads in E and G point to the localization of the second nos2b domain in noi^-/+ and smu^-/+ siblings respectively that is otherwise missing in the homozygous mutants. I,J: At 72 hpf, this domain of intense expression was localized at position of basihyal cartilage (bh, I,J). J shows the cross section; the section is shown by a straight line on I. The arrowhead in J indicates the nos2b-positive cells in position of the basihyal cartilage, whereas the arrows point to areas of lower level of expression in oral roof ectoderm. On all whole-mounts anterior is to the left. bh, basihyal; lj, lower jaw; in, infundibulum; hyp, hypophysis; tg, tegmentum.

Unillustrated author statements