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Fig. 5 Whole-mount in situ hybridization analysis of chrna4 RNA expression in 24-96 hours postfertilization (hpf) zebrafish embryos. The purple stain represents chrna4 neuronal nicotinic acetylcholine receptor (nAChR) subunit mRNA in all panels, and orange labeling denotes either krox20 RNA in rhombomeres 3 and 5 or dlx2 RNA in telencephalon, diencephalon, and the pharyngeal arches. All images are lateral views with the anterior to the left unless otherwise noted. Arrows point to specific brain regions and arrowheads point to spinal neurons. A: At 24 hpf, chrna4 expression was localized to a limited area of forebrain, a subset of cells along the yolk sac consistent with neural crest cells, and in a subset of hindbrain neurons with expression beginning in rhombomere 4. B: At 24 hpf, magnification of A, expression in forebrain, hindbrain neurons, and in dlx2-expressing areas likely to be neural crest. C: At 24 hpf, dorsal view whole embryo, possible neural crest expression at the level of, but outside of the rhombomeres, with expression also evident in spinal neurons. D: At 24hpf, chrna4 likely localization in neural crest cells in the dlx2-expressing domain of the mandibular (1), hyoid (2), and brachial (3) pharyngeal arches. E: At 24 hpf, higher magnification of D, chrna4 expression was evident in the dlx2 expression domain of the hyoid (2) pharyngeal arch and brachial (3) arch. F: At 48 hpf, dorsal view, localization consistent with the nucleus of the medial longitudinal fascicle and reticulospinal neurons of the hindbrain. G: At 48 hpf, chrna4 expression was not present in the telencephalon or hypothalamus as evident by using dlx2 expression to localize these structures. There was midbrain and hindbrain expression consistent with the localization pattern of the nucleus of the medial longitudinal fascicle and reticulospinal neurons. H: At 72 hpf, dorsal view and I: At 72 hpf, chrna4 expression was extensive in midbrain and limited in hindbrain. J: At 96 hpf, dorsal view and K: At 96 hpf, chrna4 expression was extensive in midbrain and limited in hindbrain similar to the pattern that seen in 72 hpf embryos (I). dien, diencephalon; fb, forebrain; hb, hindbrain; hn, hindbrain neurons; mb, midbrain; mhb, midbrain hindbrain boundary; nc, neural crest; otic, otic vesicle; pharyngeal arches: (1) mandibular, (2) hyoid, and (3) brachial arch; nmlf, nucleus of the medial longitudinal fascicle; rs, reticulospinal neurons; rhombomeres 3 (r3) and 5 (r5), telencephalon (te).