Whole mount in situ hybridization of rln3 on embryos at indicated stages. Lateral and dorsal view of the zebrafish brain at 48 hpf (A, B), 72 hpf (C, D), and 96 hpf (H, I). E,F: Transverse sections indicated by the black lines in D. G: A magnification of a transverse section centred at the level of NI. Lateral view showing the expression of rln3 gene in the zebrafish brain at 30 and 40 hpf (J, K). CeP, cerebellar plate; hy, hypothalamus; MO, medulla oblongata; OC, otic capsule; opT, optic tectum; PAG, periaqueductal gray; R, raphe; ReV, rombencephalic ventricle; T, tegmentum.

Whole mount in situ hybridizations with marker genes for specific neural territories. A: penkl expression in the brain of embryos at 96 hpf. B: Double in situ hybridization with penkl (orange) and rln3 (blue). C-H: Double in situ hybridization on embryos at 72 hpf. rln3 (blue), tphR (orange, C, D), crh (orange, E, F), crh-bp (orange, G, H). I: Comparison of single in situ hybridizations for crh1 and rln3. J, K: Transverse section of hybridized embryos with crh1 antisense riboprobe, indicated by the black line in I and magnification as indicated in J. L: Magnification of a transverse section of hybridized embryo with rln3 antisense riboprobe centred at level of NI as indicated by the black line in I. A, B, D, F, H, I: Dorsal view of the brainstem region. C, E, G: Lateral view of the brainstem region. Blue arrowhead indicates rln3-expressing neurons in the NI. Orange arrowhead indicates: tphR-expressing neurons in the dorsal raphe (C, D); crh-expressing neurons in the locus coeruleus (E, F); crh-bp-expressing neurons in the superior raphe (G, H). Blue/orange arrowhead indicates colocalization of rln3 and penkl transcripts in the periaqueductal gray. CeP, cerebellar plate; OC, otic capsule; PAG, periaqueductal gray; R, raphe.

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