mRNA expression analysis of zebrafish unc5 family genes, unc5a, unc5b, unc5c, unc5da and unc5db. Amplification of GAPDH served as a control to calibrate the amount of the cDNA templates used in PCR reaction.

Expression analysis of unc5a, unc5b unc5c, unc5da and unc5db at bud stage and 20 hpf by whole mount in situ hybridization. All embryos are in the lateral view and rostral to the left. The probes used for in situ hybridization are listed in the upper right corner of each panel. Developmental stages are indicated in the lower right corners. The arrow heads represent the head region, tail notochord and hindbrain as indicated. Abbreviations: hr, head region; hb, hind brain; n, notochord. Scale bar (under E2): A1–E1, 200 μm; A2–E2 250 μm.

Expression analysis of unc5a, unc5b, unc5c, unc5da and unc5db in zebrafish at 24, 36 and 48 hpf by whole mount in situ hybridization. The probes used for in situ hybridization are listed in the upper right corner of each panel. Developmental stages are indicated in the lower right corners. A1–A3, unc5a gene expression in zebrafish. Arrow heads mark signals in the trunk region. B1–B3, unc5b gene expression in zebrafish. Arrow heads mark signals in the otic vesicle and roof plate. C1–C3, unc5c gene expression in zebrafish. D1–D3, unc5da gene expression in zebrafish. E1–E3, unc5db gene expression in zebrafish. Abbreviations: ov, otic vesicle; rp, roof plate. Scale bar (under E3): 200 μm.

Expression analysis of unc5a, unc5b, unc5c, unc5da and unc5db in the zebrafish brain region at 24, 36 and 48 hpf by whole mount in situ hybridization. The probes used for in situ hybridization are listed in the upper right corner of each panel. Developmental stages are indicated in the lower right corners. A–C1, unc5a gene expression in zebrafish brain. Dashed line marks the telencephalon-diencephalon border. D: dorsal; A: anterior ventral. D–F1, unc5b gene expression in zebrafish brain. G–I1, unc5c gene expression in zebrafish brain. J–L1, unc5da gene expression in zebrafish brain. M–O1, unc5db gene expression in zebrafish brain. A–O are lateral view with anterior to the left; A1–O1 are frontal view with dorsal to the top. Abbreviations: ce, cerebellum; di, diencephalon; dt, dorsal thalamus; e, eye; ep, epiphysis; h, hindbrain; m, mouth; rf, roof plate; te, telencephalon; th, thalamus; vt, ventral thalamus. Scale bar (under O1): A–O, 100 μm; A1–O1, 75 μm.

Expression pattern of unc5a-unc5da and unc5a-unc5db in zebrafish forebrain examined by fluorescent in situ hybridization (FISH). The probes used for in situ hybridization are listed in the lower left corner of each panel. The embryos are in the frontal view, dorsal to the top. The embryo was counterstained with DAPI to show the cell nuclei (C, C1, F, F1). The dorsal telencephalon regions in C and F are marked with a white square, and enlarged views are shown in A1–C1 and D1–F1. Projections of the image stacks are shown in A-C and D–F; images of single confocal planes are shown in A1–C1 and D1–F1. The white arrows in B, C, E, and F indicate the unspecific staining for epidermis. Scale bar (under F): A–C, D–F, 40 μm; A1–C1, D1–F1, 16 μm.