Ontogeny of lower jaw movement in wildtype zebrafish larvae. (A)Tg(isl1:GFP) larvae from 3, 5, and 7 dpf were mounted in a lateral position, under a brightfield microscope, in agarose with the head free to move, and the opening of the mouth due to lower jaw movement (gape; white triangle and double arrows) was recorded and analyzed using custom software. (B,C,E) Pooled data from 3 experiments (number of larvae in parenthesis). (B) Gape frequency, increased sharply from 3 to 7 dpf, plateauing by 9 dpf. (C) Gape magnitude, a measure of the amount of jaw movement, increased sharply between 3 and 5 dpf, and plateaued thereafter. (D) There were two patterns of gape events – Distributed, with jaw movements distributed uniformly throughout the observation period, and Clustered, with tightly clustered jaw movements with intervals of inactivity. (E) The distributed gape pattern was predominant at every age examined, and there was no significant difference between 5, 7, and 9 dpf. Statistical analysis was carried out with Chi-square test and One-way analysis of variance (ANOVA) with post hoc Tukey’s HSD (honest significance difference). NS, not significant, *p < 0.05, **p < 0.01.

Developmental changes in branchiomotor axon branching and synaptic structures at the jaw neuromuscular junctions. Ventral views with anterior to the left of the jaw musculature. (A,D,G,J) Axon (magenta) outgrowth on jaw muscles (green) in 3, 5, 7, and 9 dpf Tg (α-actin:GFP); Tg (zCREST1:mRFP) larvae. 3D rendering of muscles and axons were overlaid to determine axon position relative to the muscles. Fine axon branches (white arrowheads) across the jaw muscles increased as larvae aged. (B,E,H,K) SV2a antibody (magenta) labeled presynaptic structures on the jaw muscles (green) in 3, 5, 7, and 9 dpf Tg(isl1:GFP); Tg(α-actin:GFP) larvae. 3D rendering was used to visualize the presynaptic regions in contact with the muscles (arrowheads), and calculate their volumes. (M) Presynaptic volumes increased greatly from 3 to 7 dpf larvae, especially for ima/imp and ih muscles. There was a significant increase from 5 to 7 dpf in the ima/imp, ih, and hh muscles. Both ih and hh muscles had a significant decrease in presynaptic volume from 7 to 9 dpf larvae. (C,F,I,L) Ventral view with anterior to the left. Acetylcholine receptor (AChR) clusters (arrowheads) were labeled with alpha-bungarotoxin (αBTX) (magenta) on the jaw muscles (green) in live 3, 5, 7, and 9 dpf Tg(α-actin:GFP) larvae. (N) Although AChR cluster volumes on all muscles tend to increase from 3 to 7 dpf, these changes were not significant. Data pooled from 3 to 5 experiments (number of larvae in parenthesis). Statistical analysis was carried out with Chi-square test and One-way analysis of variance (ANOVA) with post hoc Tukey’s HSD (honest significance difference). NS, not significant; *p < 0.05, **p < 0.01. ima/imp, intermandibularis anterior/intermandibularis posterior; ih, interhyal; hh, hyohal; sh, sternohyoideus.

olt mutants have reduced jaw movement. (A,B) Dorsal views with anterior to the left of the hindbrain of 48 hpf Tg(zCREST1:mRFP) in the olt background. (A) Wildtype (WT) sibling with facial branchiomotor (FBM) neurons (arrowheads) migrating into rhombomere 6 (r6). (B) In the olt mutant, FBM neurons (arrowheads) fail to migrate out of r4. (C) Gape frequencies in 5, 7, and 9 dpf olt mutant larvae were significantly reduced compared to wildtype siblings. Notably, the plateauing of gape frequencies after 7 dpf occurred normally in olt mutants. (D) Gape magnitude was significantly reduced in olt mutants compared to wildtype siblings at 5 and 9 dpf, but not at 7 dpf. (E) Gape events in representative 7 dpf olt mutant and wildtype sibling larvae showing reduced gape frequency in the mutant. (F) Gape event patterns were similarly proportioned between wildtype and olt mutant larvae at 5 dpf, with the distributed pattern being the predominant one. In 7 and 9 dpf wildtype larvae, the distributed pattern was almost exclusively seen. Statistical analysis was performed with a two-tailed student t-test (C,D) or Chi-square test (F). NS, not significant, *p < 0.02, **p < 0.001. Data pooled from 9 experiments (number of larvae in parenthesis).

Defective jaw movements greatly reduce food intake in olt mutants. (A) A semi-quantitative food intake assay for zebrafish larvae. Lateral views of 7 dpf larvae fed yellow–green fluorescent microspheres coated with fish food for 3 h. The fluorescent contents (arrowheads) in their guts were visualized under GFP epifluorescence, and ranged from no food with a feeding score (FS) of 0, less than 25% of the gut FS = 1, 50% of the gut (FS = 2), to a full gut (FS = 3). e, eye; s, swim bladder. (B) Distribution of feeding scores showing that a population of olt mutants ate significantly less than wildtype siblings. Pooled data from 3 experiments. Chi-square test used for testing significance (**p < 0.01). (C,D) Cartilage morphology at 7 dpf showing that the various elements develop and pattern normally in olt mutants. (E,F) Pooled data from 4 experiments. The pharyngeal arch (numbered) was measured for (E) length (white line in D) and (F) angle (yellow arc in D) in wildtype siblings and olt mutants. There was no difference in cartilage elements’ lengths or the angles subtended by the elements between olt mutants and wildtype siblings. (G,H) Swimming parameters in 6 dpf larvae. (G) distanced moved and (H) moving duration were analyzed and compared between olt mutant and wildtype sibling larvae with the DanioVision and EthoVision locomotion tracking software (Noldus). There was no significant difference in swimming distance or duration between olt mutants and wildtype siblings. Pooled data from 2 experiments. Number of larvae in parenthesis. The two-tailed student t-test was performed to test for significance (E–H).

Axon guidance and outgrowth are unaffected in olt mutant larvae. (A,B) Lateral view with anterior to the left of the hindbrains of 5 dpf Tg(zCREST1:mRFP) larvae. The morphologies of trigeminal (V) and FBM (VII) axons were analyzed and number of branches were calculated with Leica Application Suite X (LAS X) software. e, eye. (C) The total lengths of trigeminal motor axons and FBM axons and their branches were similar between olt mutants and wildtype siblings. (D) Trigeminal motor and FBM axon branching numbers showed no significant differences between wildtype siblings and olt mutants. Statistical analysis was performed with a two-tailed student t-test (C,D). NS, not significant. Pooled data from 4 experiments (number of larvae in parenthesis). (E,F) Ventral view with anterior to the left of the jaw musculature in 5 dpf Tg(zCREST1:mRFP); Tg(α-actin:GFP) larvae showing motor axons (magenta) and jaw and gill muscles (green). (E) Thick motor axon fascicles were seen on the ima/imp, ih, and hh muscles in wildtype siblings. This phenotype was defined as Normal Fasciculation (NF). (F) In olt mutants, these axon fascicles appeared thinner and were frequently defasciculated (arrowheads), especially on the ih muscle. This phenotype was defined as Defasciculation (DF). (G) There was a preponderance of the defasciculated axon (DF) phenotype in olt mutants, and the proportion of normal fasciculation (NF) to defasciculated (DF) axons was significantly different between wildtype siblings and olt mutants. Statistical analysis was performed using Fisher’s two-tailed exact test. Data pooled from 4 experiments (number of larvae in parenthesis). **p < 0.0001. ima/imp, intermandibularis anterior/intermandibularis posterior; ih, interhyal; hh, hyohal. Scale bar (A,E), 100 μm.

Neuromuscular junctions on jaw muscles in olt mutants are unaffected. (A–D) Ventral view with anterior to the left of the jaw musculature in 7 dpf Tg(isl1:GFP);Tg(α-actin:GFP) larvae. (A,B) SV2a antibody staining (magenta) of presynaptic structures on the jaw muscles (green). (C,D) Postsynaptic Acetylcholine receptor (AChR) clusters were labeled with alpha-bungarotoxin (αBTX) (magenta) on the jaw muscles (green) in live larvae. Volumes of the presynaptic structures and postsynaptic clusters were calculated from 3D renderings by using the muscle as a mask. There was no significant difference in the volumes of SV2a-stained structures (E) or αBTX-labeled structures (F) on any muscle groups between wildtype siblings and olt mutants. Data pooled from 3 experiments (number of larvae in parenthesis). The two-tailed student t-test was performed to test for significance. NS, not significant. ima/imp, intermandibularis anterior/intermandibularis posterior; ih, interhyal; hh, hyohal; am, adductor mandibularis; sh, sternohyoideus. Scale bar (A,C), 100 μm.

Facial branchiomotor neurons are less active in olt mutants. (A,B) Dorsal views, with anterior to the left, of the hindbrains of 7 dpf Tg(zCREST1:GCaMP6s);TgzCREST1:mRFP larvae showing GCaMP fluorescence intensities. (A) In a wildtype larva, fluorescence can be seen in the FBM neurons (arrowheads) in r6, as well as the trigeminal motor neurons (nV) in r2. (B) In an olt mutant, fluorescence is evident in the nV neurons in r2 and in the FBM neurons (arrowheads) that have failed to migrate out of r4. (C) Overlay of GCaMP6s ΔF/F traces for a representative wildtype sibling (blue) and an olt mutant (orange) showing fewer Ca²⁺ events (asterisks) in the mutant. Amplitudes of the Ca²⁺ events were not affected. (D) The frequency of Ca²⁺ events in FBM neurons was significantly lower in olt mutants compared to wildtype siblings. The Ca²⁺ event frequency for trigeminal motor (nV) neurons was not affected in mutants. Data pooled from 6 experiments (number of larvae in parenthesis). The two-tailed student t-test was performed to test for significance, *p < 0.01.