Figure 1

trio loss-of-function causes malformation of neural crest derivatives in zebrafish. (A) WISH of trio in zebrafish embryos at 24, 36, 48 and 72 hpf. Bar = 100 μm. At 24 hpf, trio staining was detected in cranial NCC migrating around the eye (blue arrow) and developing ear (red asterisk), and in the NCC on either side of the neural tube (red arrow). Dorsal and lateral views of zebrafish at 36 hpf display prominent staining in the cranial and trunk NCCs (black arrow). Views of embryos at 48 and 72 hpf indicate trio expression around the pharyngeal arch tissues (pa). hpf: hours post-fertilization; WISH: whole-mount in situ hybridization. (B) Western blot showing the trio knockdown efficiency in the trio MO embryos, followed with quantification (n = 3). MO: morpholino. (C) Statistics of deformed, dead, and normal zebrafish embryos after the trio MO injection (n = 200). (D) Bright-field images of con MO embryos and trio MO embryos at 96 hpf. Red dotted line: mandible. Bar = 100 μm. (E) Images of eye iridophore (white arrow) at 96 hpf. Bar = 100 μm. (F) Quantification of eye iridophore amount and distribution at 96 hpf (n = 10). (G) Ventral view of con MO and trio MO embryos by Alcian blue staining at 120 hpf. Bar = 100 μm. Line A served as a baseline for later measurements and was used to normalize line B. Line B represents the distance from line A to the anterior of Meckel's cartilage. (H) Schematic of the ventral view of the pharyngeal arch cartilage structure in zebrafish embryos at 120 hpf. bh: basihyal; cb: ceratobranchial; ch: ceratohyal; hs: hyosymplectic; ih: interhyal; m: Meckel's cartilage; p: palatoquadrate. The quantification of the B/A ratio between con MO and trio MO embryos is shown. (I, J) Ventral (I) and lateral (J) views of 96 hpf Tg(sox10:egfp) con MO and trio MO zebrafish. Red arrowheads indicate mandible. Bar = 500 μm. For (F) and (H), data are represented as mean ± S.E.M. (two-tailed t test **p < 0.01).