Hyperinflation of the swim bladder in zebrafish lateral line mutants (lhfpl5b^−/−). (A) Representative examples of over-, regular- and under-inflated swim bladders (arrows) in larval zebrafish at 6 dpf (top: lhfpl5b^−/−; middle and bottom: wild-type). (B) Correlation between buoyancy and swim bladder volume. Line of best fit applied through the combination of mutant and wild-type data (linear model, R²=0.8574). (C) Percentage of larvae that exhibit over-, regular and under-inflation of the swim bladder, as defined by positive, neutral and negative buoyancy. Legend in D applies to both C and D. A χ² test was used to determine significance. (D) Swim bladder volume (mm³) in wild-type and lhfpl5b mutant larvae at 6 dpf. A Welch's t-test was used to determine significance. ***P<0.001, ****P<0.0001. Scale bars: 0.2 mm.

Blocking access to the surface eliminates the hyperinflation phenotype in zebrafish lateral line mutants (lhfpl5b^−/−). (A) Diagram of experimental set-up with a filter blocking access to the surface. (B) Percentage of larvae that exhibit over-, regular- and under-inflation of the swim bladder at 6 dpf. A χ² test was used to determine significance. (C) Swim bladder volume (mm³) of larvae at 6 dpf. A one-way ANOVA was used to determine significance. ****P<0.0001, **P<0.01; n.s., not significant. Complete ANOVA and Tukey statistics are available in Table S1.

Rescue of zebrafish lateral line mutants (lhfpl5b^−/−) with the Tg(myo6b:EGFP-lhfpl5a)vo23 transgene. (A) Percentage of larvae that exhibit swim bladder under-, regular- and over-inflation at 6 dpf. A χ² test was used to determine significance. (B) Swim bladder volume (mm³) of larvae at 6 dpf. A one-way ANOVA was used to determine significance. ****P<0.0001; n.s., not significant. Complete ANOVA and Tukey statistics are available in Table S1.

Head-specific ablations of the zebrafish lateral line recapitulates the lhfpl5b mutant hyperinflation phenotype. (A) Diagram of experimental protocol. (B) Percentage of larvae that exhibit swim bladder under-, regular- and over-inflation at 5 dpf. A χ² test was used to determine significance. (C) Swim bladder volume (mm³) of larvae at 5 dpf. A one-way ANOVA was used to determine significance. *P<0.05, ****P<0.0001; n.s., not significant. Complete ANOVA and Tukey statistics are available in Table S1. Representative images of live larvae following region-specific CuSO₄ treatment are in Fig. S3.

Decreasing interfacial surface tension with mineral oil results in over-filling of the swim bladder. (A) Diagram of experiment with 2 mm layer of oil on surface. (B) Images of swim bladder phenotypes resulting from surface oil and surface air exposure in wild-type larvae at 6 dpf. (C) Swim bladder volume (mm³) at 6 dpf. A one-way ANOVA was used to determine significance. ****P<0.0001; n.s., not significant. Scale bars: 0.1 mm. Complete ANOVA and Tukey statistics are available in Table S1.

Optogenetic activation of lateral line hair cells with Channelrhodopsin-2 (ChR2) in wild-type and lhfpl5b mutant larval zebrafish. (A) Diagram of experiment with blue light stimuli delivered for 25 ms at 1 s intervals. Treatments were started at 4 dpf and swim bladder volume was assessed at 6 dpf. (B) Swim bladder volume (mm³) of larvae at 6 dpf, n=16–21 per condition. One-way ANOVA was used to determine significance. **P<0.001; n.s., not significant. Complete ANOVA and Tukey statistics are available in Table S1.

Quantification of the surfacing behaviors of wild-type and lhfpl5b mutant zebrafish larvae during initial swim bladder inflation. (A) Total number of individual visits taken by larval fish to the air–water interface on 4 dpf for 12 h (09:00–21:00 h). (B) Total amount of time larval fish spent at the surface for all visits combined on 4 dpf. (C) Time to first surface visit after access was allowed. A one-way ANOVA was used to determine significance. ***P<0.001, ****P<0.0001; n.s., not significant. Complete ANOVA and Tukey statistics are available in Table S1.

Summary of the surfacing behavior of wild-type and lateral line-deficient zebrafish larvae. (A) Wild-type larvae use their lateral line hair cells (in green) to mediate interactions with the air–water interface during surfacing. As a result of accurate surface detection, larvae take in an appropriate volume of air for swim bladder (SB) inflation and achieve neutral buoyancy. (B) Larvae with a genetic loss of lateral line function (hair cells in white) misinterpret the air–water interface, leading to increased interactions with the surface. Consequently, lateral line mutants (lhfpl5b^−/−) take in an excess volume of air, resulting in hyperinflation of the swim bladder for approximately half of mutant larvae. Selective chemical ablations of the head neuromasts produced similar results, implicating the anterior lateral line as the primary sensory organ for interactions with the air–water interface during the surfacing behavior.