A: Diagram of the Mauthner cell circuit illustrating the electrical synapses of interest. The image represents a dorsal view with anterior on top. Boxed regions indicate the stereotypical synaptic contacts used for analysis. Presynaptic auditory afferents contact the postsynaptic Mauthner cell lateral dendrite in the hindbrain forming Club Ending (CE) synapses. Presynaptic Mauthner axons form en passant electrical synapses with the postsynaptic CoLo interneurons (M/CoLo synapses) in the spinal cord (2 of 30 segments are depicted). B–E: Confocal images of Mauthner circuit neurons and stereotypical electrical synaptic contacts in 5 dpf zf206Et zebrafish larvae from wildtype ( wt ) ( B, C ) and dis2 ^-/- mutant ( D, E ) animals. Animals are stained with anti-GFP (green) and anti-Cx36 (white). Scale bars = 2 µm. Boxed regions denote stereotyped location of electrical synapses and regions are enlarged in neighboring panels. B, D : Images of the Mauthner cell body and lateral dendrite in the hindbrain. Images are maximum intensity projections of ~10 µm. In B’ and D’, images are maximum-intensity projections of ~5 µm and neighboring panel shows the Cx36 channel. C, E : Images of the sites of contact of Mauthner/CoLo processes in the spinal cord. Images are maximum-intensity projections of ~4 µm. In C’ and E’, the white dashed circle denotes the M/CoLo site of contact and the neighboring panel shows the Cx36 channel. F, G: Quantification of Cx36 fluorescence intensities at CE ( F ) and M/CoLo ( G ) synapses for the noted genotypes. The height of the bar represents the mean of the sampled data normalized to the wt average. Circles represent the normalized value of each individual animal. All CEs (~10) of both Mauthner cells were sampled per animal, and between 12 and 18 M/CoLo synapses were sampled per animal. CE synapses: wt n=6, dis2 ^-/- n=5, p<0.0001; M/CoLo synapses: wt n=7, dis2 ^-/- n=7, p<0.0001. Error bars are ± SEM. H: Genome wide RNA-seq-based mapping data. The average frequency of mutant markers (black marks) is plotted against genomic position. A single region on chromosome 25 was linked to the dis2 mutation (red arrow). I: Mutant reads are shown aligned to the reference genome identifying a T to A transversion (highlighted in red font) creating a premature splice acceptor site in the intron and introducing 11 base pairs of intronic sequence (boxed in black) into the transcript that causes a frameshift. A sample of aligned reads are shown as grey boxes. The coverage (cov.) of aligned reads is depicted as a histogram at each genomic position. J–M: Confocal images of Mauthner circuit neurons and stereotypical electrical synaptic contacts in 5 dpf zf206Et zebrafish larvae from wt ( J, K ) and tjp1b ^dis2/∆16 ( L, M ) animals. Animals are stained with anti-GFP (green), anti-Cx35.5 (cyan), anti-Cx34.1 (yellow), and anti-ZO1 (magenta). Scale bars = 2 µm. Boxed regions denote stereotyped location of electrical synapses and regions are enlarged in neighboring panels. J, L : Images of the Mauthner cell body and lateral dendrite in the hindbrain. Images are maximum intensity projections of ~20 µm. In J’ and L’, images are maximum-intensity projections of ~10 µm and neighboring panels show the individual channels. K, M : Images of the sites of contact of M/CoLo processes in the spinal cord. Images are maximum-intensity projections of ~8 µm. In K’ and M’, images are from a single 0.42 µm Z-plane and the white dashed circle denotes the location of the M/CoLo site of contact. Neighboring panels show individual channels. N, O: Quantification of Cx35.5 (cyan), Cx34.1 (yellow), and ZO1 (magenta) fluorescence intensities at CE ( N ) and M/CoLo ( O ) synapses for the noted genotypes. The height of the bar represents the mean of the sampled data normalized to the wt average. Circles represent the normalized value of each individual animal. CE synapses: wt n=4, tjp1b ^dis2/∆16 n=4, Cx35.5 p=0.003, Cx34.1 p=0.0005, ZO1 p=0.0002; M/CoLo synapses: wt n=4, tjp1b ^dis2/∆16 n=4, Cx35.5 p=0.0121, Cx34.1 p=0.0024, ZO1 p=0.0005. Error bars are ± SEM.