Fig 5

ZO1b-Beta is sufficient for robust Connexin localization to electrical synapses.

A-H. Confocal images of Mauthner circuit neurons and stereotypical electrical synaptic contacts in 5 days post fertilization (dpf) zf206Et zebrafish larvae from wt (A, B), tjp1b/ZO1b-alpha-gamma^-/- double mutant (C, D), tjp1b/ZO1b-alpha-beta^-/- double mutant (E, F), and tjp1b/ZO1b-beta-gamma^-/- double mutant (G, H) 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. Images of the Mauthner cell body and lateral dendrite in the hindbrain (A, C, E and G) are maximum intensity projections of ~10–25 μm. In A’, C’, E’ and G’, images are maximum-intensity projections of ~3–7 μm and neighboring panels show the individual channels. Images of the sites of contact of M/CoLo processes in the spinal cord (B, D, F and H) are maximum-intensity projections of ~3–7 μm. In B’, D’, F’ and H’, 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. Anterior up. I. Quantification of Cx35.5, Cx34.1, and ZO1 fluorescence intensities at CE 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. Mean is shown ± SEM. wt n = 15, tjp1b/ZO1b-alpha-gamma^-/- n = 5, tjp1b/ZO1b-alpha-beta^-/- n = 5, and tjp1b/ZO1b-beta-gamma^-/- n = 5. For Cx35.5 (cyan circles), **** indicates p<0.0001 by ANOVA with Dunnett’s test. For Cx34.1 (yellow circles), **** indicates p<0.0001 and * indicates p = 0.0478 by ANOVA with Dunnett’s test. For ZO1 (magenta circles), **** indicates p<0.0001 and * indicates p = 0.0197 by ANOVA with Dunnett’s test. J. Quantification of Cx34.1 fluorescence intensities at M/CoLo 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. Mean is shown ± SEM. wt n = 15, tjp1b/ZO1b-alpha-gamma^-/- n = 5, tjp1b/ZO1b-alpha-beta^-/- n = 5, and tjp1b/ZO1b-beta-gamma^-/- n = 5. For Cx35.5 (cyan circles), ** indicates p = 0.0031 and **** indicates p<0.0001 by ANOVA with Dunnett’s test. For Cx34.1 (yellow circles), ** indicates 0.0013 and **** indicates p<0.0001 by ANOVA with Dunnett’s test. For ZO1 (magenta circles), tjp1b/ZO1b-alpha-beta^-/- ** indicates p = 0.0029 and tjp1b/ZO1b-beta-gamma^-/- ** indicates p = 0.0017 by ANOVA with Dunnett’s test.