Knockdown of miR-1 affects neural crest derivatives. (A) qPCR showed that miR-1 MO knocked down miR-1 efficiently (the experiments were repeated three times). Data are expressed as the mean ± SD, from at least three independent experiments, n=15, **P < 0.01. (B, C) Lateral views of live zebrafish embryos showed pigment cells at 3 dpf. Negative control-injected zebrafish exhibited neural crest-derived pigment cells: black melanophores and iridescent iridophores. The distribution and number of melanophores were reduced in miR-1 morphants. The number of iridophores was reduced in the eye and trunk. (D, E) Lateral views of embryos at 4 dpf. miR-1 morphants displayed a shorter body length with a smaller head and edema around the heart. The lower jaw was reduced in size in the miR-1 morphants (arrows) compared with the negative control embryos. (F) Alcian blue staining showed cartilage development at 4 dpf; ventral views of embryos. (G, H) Ratios B: A and C: A, as shown in (F), in the miR-1 MO-injected and negative MO-injected groups. The definition for lines A, B, and C has been described previously. microRNA-1, miR-1; morpholino, MO; dpf, days post fertilization; mc, Meckel's cartilage; pq, palatoquadrate; ch, ceratohyal cartilage; Data are expressed as the mean ± SD, from at least three independent experiments, n=15, **P < 0.01; N.S., not significant.

Expression of selected genes in neural crest induction and specification in miR-1 MO-injected embryos at 11 hpf. The photographs show a dorsal view with the anterior to the top at 11 hpf. Expression of foxd3 (A), msxb (B), pax3 (C), dlx3b (D), and snai1b (E) in embryos is shown. The expression of foxd3, msxb, pax3, dlx3b and snai1b was similar in negative control embryos and miR-1 morphants. microRNA-1, miR-1; morpholino, MO.

miR-1 morphants displayed defective migration of neural crest cells. Live embryo imaging in miR-1 morphants and negative control Tg (sox10: EGFP) embryos was used to analyze the movement of neural crest cells, viewed dorsally. (A-D) Representative images are shown from the time-lapse photography from 6 segment stage (ss) to 20 ss. At 6ss, 10 ss, 16 ss and 20 ss, neural crest cells in miR-1 morphants displayed ectopic EGFP expression (red arrowheads) in the dorsal midline (A, B, C and D). At 24 hpf, expression of snai1b (E) and tfap2a (F) in negative control and miR-1 morphants is shown. At this time, increasing numbers of the migrating lateral line primordium (arrow) expressed tfap2a and snail1b. The expression of neural crest migration markers (snai1b, tfap2a) was reduced (arrows) (E, F). microRNA-1, miR-1; morpholino, MO; ss, segment stage.

miR-1 MO-injected embryos displayed defective neural crest cells differentiation. Embryos were viewed dorsally, anterior to the top, at 24 hpf. Expression of dlx2a (A), dlx3b (B), ngn1 (C), and crestin (D) is shown. Expression of the pharyngeal homing marker gene dlx2a was reduced in the neural crest stream (S1, S2, S3) in miR-1 MO-injected embryos (A). General features of dorso-ventral patterning appeared affected as shown (arrows) by decreased expression of dlx3b (B). ngn1, a marker of neuron differentiation, was downregulated in miR-1 morphants (arrows) (C). The expression of neural crest marker crestin was reduced in the cranial region and the branchial primordia (arrows) (D). microRNA-1, miR-1; morpholino, MO. Taken together, our data suggest that miR-1 loss of function interfered with the development of normal neural crest derivatives through disruption of neural crest cell migration and differentiation.

miR-1 directly targeted sec63. (A) Subcellular localization of potentially targeted proteins differentially expressed in miR-1 morphants and negative control embryos identified by gene ontology annotations. 9 proteins were upregulated and 23 proteins were downregulated in miR-1 morphants. (B) Protein expression of Sec63 in the control and miR-1 MO-injected groups. (C) A candidate protein for upregulation, sec63, was determined by iTRAQ analysis. Validation of increased sec63 expression in miR-1 morphants by qPCR in vivo at 24 hpf. Data are expressed as the mean ± SD, from at least three independent experiments, n=15, **P < 0.01. (D) Expression of sec63 in embryos at 24 hpf. Embryos were viewed laterally, with the anterior to the left. sec63-3ʹUTR binding site of miR-1 was predicted using TargetScan (E, F). (G) Mutant 3ʹUTR of sec63 in dual luciferase reporter plasmids. Overexpression of miR-1 (miR-1-pre) reduced sec63-3ʹUTR luciferase activity in vitro, but not the luciferase activity of mutated sec63-3ʹUTR. Three independent experiments were performed, and each experiment was carried out in duplicate (H).

Reducing sec63 partly restored the neural crest derivative defects in miR-1 morphants. (A) The defects in melanophores and iridophores were reversed in embryos co-injected with both miR-1 MO and sec63 MO. (B) The craniofacial cartilage were corrected in embryos co-injected with miR-1-MO and sec63-MO. Data are expressed as the mean ± SD, from at least three independent experiments, n=15, **P < 0.01. (C) The expression of dlx2a, ngn1 and crestin were partly rescued at 24 hpf in embryos co-injected with both miR-1 MO and sec63 MO (S1, S2, S3, arrows). (D) The number of ectopic neural crest cells was reduced. microRNA-1, miR-1; morpholino, MO.