Figure 2-figure supplement 1-source data 1.

Figure 2-figure supplement 1-source data 1.

(A–D) Confocal images of Mauthner circuit neurons and stereotypical electrical synaptic contacts in 5-day-post-fertilization, zf206Et zebrafish larvae from gjd2a^-/-; gjd1a^dis3/dis3 (Cx35.5/Cx34.1, respectively) mutant (A,B) and gjd1a/Cx34.1^dis3/dis3 mutant larvae (C,D). Animals are stained with anti-GFP (green), anti-zebrafish-Cx35.5 (cyan), anti-zebrafish-Cx34.1 (yellow), and anti-human-ZO1 (magenta). The gjd1a/Cx34.1^dis3/dis3 mutant was isolated from an ENU-based forward genetic screen for mutants affecting Mauthner electrical synapse formation (Miller et al., 2017). The effects observed from this mutation alone (C,D) are similar to the TALEN-induced frameshift mutation in gjd1a used in Figure 2. The TALEN-induced frameshift mutation is used throughout this paper, except here in this figure supplement, in the gjd2a^-/-; gjd1a^dis3/dis3 mutant images in Figure 3—figure supplement 2, and in the gjd2a^-/-; gjd1a^dis3/dis3 mutant quantitation presented in Figure 2 (M,N). Scale bar = 2 µm in all images. (A,C) Images of the stereotypical of CE contact sites on the Mauthner lateral dendrite. Images are maximum-intensity projections of ~5 µm and neighboring panels show individual channels. (B,D) Images of the sites of contact of Mauthner/CoLo processes in the spinal cord. Images are individual Z-sections and neighboring panels show individual channels. (E,F) Quantification of Cx35.5 (cyan), Cx34.1 (yellow), and ZO1 (magenta) fluorescence intensities at CE (E) and M/CoLo (F) synapses for the noted genotypes. The height of the bar represents the mean of the sampled data normalized to the wt average, and circles represent the normalized value of each individual animal (CE synapses: wt n = 5, gjd1a^dis3/dis3n = 5; M/CoLo synapses: wt n = 4, gjd1a^dis3/dis3n = 4). Error bars are ± SEM. For each comparison, wt and mutant values are significantly different (Welch's t-test, p<0.01). Associated experimental statistics can be found in Figure 2—figure supplement 1—source data 1. (G–L) Confocal images of Mauthner CEs synapses in 5-day-post-fertilization, zf206Et zebrafish larvae from gjd2b/Cx35.1^-/- (G–I) and gjd1b/Cx34.7^-/- (J–L) mutant animals. Images are maximum-intensity projections of G ~ 1.44 µm, H ~ 1.80 µm, I ~ 2.52 µm, J ~ 4.86 µm, K ~ 1.98 µm, L ~ 1.80. In each panel, animals are stained individually with the indicated antibody. Scale bar = 2 µm. (M) Confocal images of Mauthner CEs at 5-day-post-fertilization, zf206Et zebrafish larvae from a gjd2a/Cx35.5^-/- mutant. Animal was stained with anti-GFP (green), anti-zebrafish-Cx35.5 (red), and anti-Cx35/6 (cyan). Images are maximum-intensity projections of ~3.80 µm. Scale bar = 2 µm. gjd2a/Cx35.5^-/- mutants showed residual staining with the Cx35/6 antibody, but Cx35.5 staining at the synapses was not detected.