Comparison of HuC and DeltaB mRNA expression in the neural plate at the 3-somite stage. HuC (A) is expressed in isolated cells in a pattern very similar to that of DeltaB (B), which is expressed in neuronal precursor cells. Dorsal views, anterior to the left. ps, primary sensory neuron; pin, primary intermediate neuron; pmn, primary motor neuron.

Determination of transcription start site of HuC gene by primer extension. A [γ-³²P]ATP-labeled antisense oligonucleotide primer corresponding to the 24 bp (+169 to +192) of exon-1 of HuC was hybridized with total RNA isolated from 24 hpf zebrafish embryos (Z) or tRNA from yeast (Y) and subjected to primer extension. The extended DNA samples were size-fractionated on a denaturated 6% polyacrylamide gel containing 8 M urea. For the size ladder, a dideoxy DNA sequencing reaction for the zebrafish HuC genomic DNA with the same antisense oligonucleotide primer was performed and then simultaneously electrophoresed with the primer-extended samples on the same gel. An extended cDNA band from zebrafish RNA is indicated by the arrow and the corresponding nucleotide G is marked by an asterisk.

Neuronal specificity of HuC promoter (ΔEco) in living zebrafish embryo. Neuronal specificity of transiently expressed GFP driven by the HuC promoter construct (ΔEco) in 48 hpf zebrafish embryos. (A) GFP fluorescence generated by superimposing a bright-field image on a fluorescence image. (B) Fluorescence image of GFP detected in the nervous system including the telencephalic cluster, retinal ganglion cells, medial longitudinal fasciculus, and dorsal longitudinal fasciculus. (C) GFP was expressed in the trigeminal ganglion neuron and Rohon-Beard neurons (arrow). (D, E) GFP expression in the peripheral process of Rohon-Beard axons (arrow) and dorsal longitudinal fasciculus of spinal cord. dlf, dorsal longitudinal fasciculus; ey, eye; mlf, medial longitudinal fasciculus; rb, Rohon-Beard neurons; rg, retinal ganglion; tc, telencephalic cluster; tg, trigeminal ganglion. Dorsal to the top and anterior to the left.

In vivo functional analysis of HuC promoter constructs in living zebrafish. Promoter deletion constructs for ΔHind (A), ΔBst (B), ΔSac (C, D) were microinjected to the one-cell (A, B, C) or four-cell embryos (D). Fluorescence images of GFP in 48 hpf living zebrafish embryos were captured by the laser confocal microscopy. GFP expression in the neuronal cells and muscle cells are indicated by the arrowheads and arrows, respectively.

GFP fluorescence of neuron in living transgenic zebrafish. The neuronal cells of transgenic zebrafish lines permanently expressing the HuC-GFP minigene (10.7 kb) were analyzed by the fluorescence microscopy. Lateral views of GFP fluorescence detected in the neurons of 24 hpf heterozygotic (A) and homozygotic (B) transgenic embryos. (C) Cranial ganglia are highlighted by asterisks. Ventral motor roots in the boxed area are indicated by arrows. (D) Lateral view of spinal cord of 60 hpf transgenic zebrafish embryo. The Rohon-Beard cells (rb), commissural neurons (co), and primary motorneurons (mo) are visualized by the GFP fluorescence in living transgenic zebrafish embryo. (A, B, C) GFP fluorescence detected by stereomicroscope; (D) Pseudo-color image of GFP fluorescence analyzed by laser confocal microscopy.

Temporal and spatial expression pattern of HuC-GFP minigene in the homozygotic transgenic zebrafish embryos. Expression patterns of GFP (A) and HuC (B) mRNA transcripts detected by whole mount in situ hybridization using synthetic antisense RNAs. Dorsal view of 11 hpf embryos for GFP (A) and HuC (B). (C) Lateral view, expression of acetylated α-tubulin detected by whole mount immunostaining (Sigma mouse Monoclonal, clone 6-11B-1). Expression of GFP labeled by anti-GFP polyclonal antibody in 24 hpf embryos (D–G). (D) Lateral view; expressions of GFP were detected in the telencephalic cluster (tc), anterior commissure (ac), epiphysial cluster (ec), posterior commissure (pc), tract of posterior commissure (tpc), postoptic commissure (poc), and tract of the postoptic commissure (tpoc). (E) Anterior view, expression of GFP in the olfactory placodes. (F) Dorsal view, expression of GFP in the nucleus of the medial longitudinal fasciculus (nMLF) and medial longitudinal fasciculus (MLF). (G) Lateral view of hindbrain; expression of GFP in the trigeminal ganglion (tg) and rhombomeres (v) in the hindbrain.

Characterization of living mib mutant transgenic embryos by GFP fluorescence. The neurogenic phenotype in 2-day-old HuC-GFP^+/-/mib^-/- zebrafish embryo seen by GFP fluorescence with a Leica MZFLIII fluorescence stereomicroscope (right), compared with a heterozygotic wild-type HuC-GFP^+/- transgenic embryo (left).