Article Figures & Data
Figures
- Figure 1.
The vestibulo-ocular reflex is defective in mutant asteroid larvae. Representative eye-movement traces are shown in response to 45° movements in the yaw axis at an angular velocity of 45°/s. A, Relative eye movements in a given video frame of a sibling larva (red dots) coordinating with the angular position of the platform (blue triangles). B, Irregular and reduced eye movements in an mutant asteroid larva. Movements were 0.5 mm in response to the rotation of the platform. The gray dots mark the relative motion of a pigment cell in each animal.
- Figure 2.
Positional cloning and identification of the asteroid gene. A, Critical interval of the asteroid gene, including polymorphic markers and the number of meiotic recombinants at each end. B, cDNA spanning exon 2 of vglut3 amplified from wild-type, asteroid sibling and mutant, and vglut3 splice donor site (GT) morpholino-injected larvae. C, Exon/intron structure of vglut3, and position of the genomic lesion (asterisk), a GT>GC donor splice site mutation (blue arrows indicate the morpholino target sites in the transcript). D, Reading frames of the wild-type and mutant transcripts. E, ATG and splice site (GT) morpholinos phenocopied the asteroid/vglut3 mutant. The percentage of larvae with auditory/vestibular defects is as follows: uninjected, 0 ± 0; ATG MO, 62 ± 13; ATG control MO, 0 ± 0; GT MO, 91 ± 12; GT control MO, 0 ± 0 (p ≪ 0.001, Student's t test). Error bars denote SD. Number of total larvae observed in parentheses.
- Figure 3.
A–D, vglut expression in the developing zebrafish. mRNA (blue) was detected at 72 hpf by whole-mount in situ hybridization with vglut1 (A), vglut2a (B), vglut2b and vglut2c (C), and vglut3 (D) antisense riboprobes. A–F, Lateral views of the larval head region. Asterisks indicate labeled neuromasts. The arrows indicate the otic capsule in each specimen. E, F, Cryostat thick sections of the inner-ear hair cells in vglut3- and vglut1-labeled larvae, respectively. Plus signs mark hair-cell bodies. Scale bars: A–D, 100 μm; E, F, 10 μm.
- Figure 4.
Vglut3 protein is absent in asteroid/vglut3 mutants. A–F, Immunolabeling of Vglut3 protein in thick sections of wild-type (wt) and mutant asteroid/vglut3 neuromast hair cells at 120 hpf. A–C, Thick section of a wild-type neuromast. D–F, Thick section of a mutant asteroid/vglut3 neuromast. G–M, Immunolabeling of macular hair cells in the inner ear at 120 hpf. G–I, Thick section of a wild-type macula. K–M, Thick section of a mutant asteroid/vglut3 macula. A, D, H, L, Vglut3 labeling (green). B, E, G, K, Acetylated tubulin staining to outline cell bodies (red). C, F, I, M, Overlays of tubulin and Vglut3 labeling. Scale bars: 3 μm.
- Figure 5.
Ribbon bodies are normal in mutant asteroid/vglut3 larvae. A–F, Immunolabeling of Ribeye b in wild-type (A–C) and mutant (D–F) hair cells. Ribeye b stain is shown in red (B, E), whereas acetylated tubulin is shown in cyan blue to indicate the hair-cell bodies (C–F). G–I, Ribeye b (red) immunofluorescence colocalizes with Vglut3 labeling (green). Images are of a single confocal section (0.5 μm) of a neuromast. Scale bars: 3 μm.
- Figure 6.
Normal hair-cell receptor potentials and absent action currents in mutant asteroid/vglut3 larvae. A, Microphonic potentials from day 5 asteroid/vglut3 wild-type sibling and mutant larvae. Top traces are the average voltage response (200 presentations) to a 20 Hz water-jet stimulus (bottom traces) from a sibling and mutant larva, respectively. B, Extracellular currents recorded from the posterior lateral line ganglion during 20 Hz stimulation of a lateral line neuromast. Top, Fifty consecutive sweeps containing 163 action currents phase locked to one direction of deflection. Inset, Signal average of all 163 events. Middle, Fifty consecutive traces from the sibling wild-type larva in A (note that the 88 action currents are phase locked to the opposite direction of deflection compared with the top trace). Bottom, Fifty consecutive current records from the asteroid/vglut3 mutant in A.
- Figure 7.
asteroid/vglut3 mutants have a reduced number of ribbon-associated synaptic vesicles. A, Schematic of the hair cell afferent synapse. Only vesicles with a diameter of 30–50 nm and proximity to the ribbon were counted (green); outliers (red) were ignored. B, A representative wild-type ribbon; asterisks indicate synaptic vesicles included in analysis. C, A representative section through a mutant ribbon. Scale bars: 200 nm.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Figure 1
- supplemental material - Supplemental Figure 2
- supplemental material - Supplemental Figure 3
