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Fig. 3 In situ hybridization analysis of chrna6 RNA expression in 24 hours postfertilization (hpf) and 48 hpf zebrafish embryos. The purple stain represents chrna6 neuronal nicotinic acetylcholine receptor (nAChR) subunit mRNA in all panels, and the orange labeling is either krox20 in rhombomeres 3 and 5, dlx-2 in telencephalon, diencephalon and pharyngeal arches, or tyrosine hydroxylase (TH) in catecholaminergic neurons. Use of these additional probes is indicated in each panel. All images are lateral views with the anterior to the left unless otherwise noted. Arrows point to specific brain regions and arrows with an asterisk denote co-labeling with TH. A: In 24 hpf embryos, the chrna6 transcript was seen in a subset of spinal neurons and a subset of Rohon-Beard sensory neurons in the trunk. B: 24 hpf, chrna6 expression was present in pineal, ventral forebrain, and Rohon-Beard sensory neurons. C: At 24 hpf, dorsal view, chrna6 expression in trigeminal ganglion and Rohon-Beard sensory neurons. D: At 24 hpf, chrna6 expression was seen in rhombomere 1 as well as forebrain, pineal, and spinal neurons. The Krox20 probe (orange) labeled rhombomeres 3 and 5. E: At 24 hpf, chrna6 expression was identified in diencephalon as shown by the colocalization with dlx2, a marker for telencephalon, diencephalon, and hypothalamus. Additionally, chrna6 is labeled in pineal and rhombomere 1. F: At 48 hpf, dorsal view, chrna6 transcript was heavily expressed in the retina with limited expression in midbrain and continued expression in pineal and trigeminal ganglion. G: At 48 hpf, dorsal view, expression in midbrain is not colocalized with dlx2 and is dorsal to the dlx2 expression domain (orange labeling) in telencephalon, diencephalon, and hypothalamus. Additionally, expression in retina and trigeminal ganglion was present. H: At 48 hpf, chrna6 was expressed in tectum, retina, pineal, and trigeminal ganglion. The diencephalic catecholaminergic cluster and locus coeruleus are detected by the TH probe (orange). As heavy retinal expression is evident in H that impedes with visualizing midbrain, I-N are 8-12-micron sagittal sections though the diencephalic regions marked by a line in H and L. I: At 48 hpf, chrna6 expression was clear in the pineal, retinal ganglion cells, trigeminal ganglion, and colocalization with TH in the locus coeruleus was evident. The insert is a magnification of locus coeruleus. J: At 48 hpf, indicates chrna6 expression in the most lateral regions of the diencephalic catecholaminergic cluster colocalizing with TH. Additionally, chrna6 expression in the pineal and retinal ganglion cells was present. K: At 48 hpf, higher magnification of K, colocalization of TH and chrna6 transcript in the lateral most region of the diencephalic catecholaminergic cluster in the midbrain. L: At 48 hpf, a midsagittal section through the diencephalic catecholaminergic cluster indicates chrna6 colocalization with TH. M,N: At 48 hpf, medial sagittal sections, indicate areas of colocalization with TH and areas solely expressing chrna6 RNA. dien, diencephalon; dcc, diencephalic catecholaminergic cluster; fb, forebrain; hypo, hypothalamus; lat, lateral; lc, locus coeruleus; 1, mandibular pharyngeal arch; mb, midbrain; obc, olfactory cluster in olfactory bulbs; pin, pineal; rgc, retinal ganglion cells; r1, rhombomere 1; rb, Rohon-Beard sensory neurons; sn, spinal neurons; tec, tectum; tg, trigeminal ganglion.