The tjp1b/ZO1b gene is alternatively initiated.

A. Cartoon schematic illustrating the electrical synapses of interest in the Mauthner cell circuit. The image represents a dorsal view with anterior on top. Boxed regions in light grey indicate the Mauthner (M) and CoLo cell bodies, as labeled. Boxed regions in dark grey indicate the stereotypical synaptic contacts used for analysis in this study. Presynaptic auditory afferents contact the postsynaptic Mauthner cell lateral dendrite in the hindbrain forming Club Ending (CE) synapses (denoted by pink circles). In the spinal cord, the presynaptic Mauthner axons form en passant electrical synapses with the postsynaptic CoLo interneurons (M/CoLo synapses, denoted by pink circles) in each spinal cord hemisegment (2 of 30 repeating spinal segments are shown). A cartoon enlargement of an electrical synapse in the Mauthner cell circuit appears to the left. Molecularly asymmetric Connexin hemichannels (Cx35.5 [cyan], Cx34.1 [yellow]) directly couple neurons by forming gap junction (GJ) channels. The formation and function of electrical synapses are regulated by ZO1b scaffolds in the electrical synapse density (ESD, magenta). B. Schematic diagram of the tjp1b/ZO1b gene locus on chromosome 25: 32,031,536–32,271,394 (GRCz11 Ensembl) showing the exon structure located on the forward strand. Horizontal black bar represents the DNA strand and vertical black bars represent individual exons. The region containing alternatively initiated exons for tjp1b/ZO1b-Alpha, -Beta, and -Gamma isoforms is expanded between the dashed lines (Chromosome 25: 32,031,536–32,208,713). Unique initiation exons are represented by white boxes (Exons 1 Alpha, 1 Beta, and 1 Gamma), additional exons are represented by grey boxes, and the first shared exon is represented by a black box (Exon 5). Lengths of exons and introns are indicated. The isoform null mutations generated by CRISPR/Cas9 for each single and double mutant line (b# designates alleles) are indicated below each Exon 1 (white box). Symbols indicate deletion (Δ) or insertion (∇) of the indicated number of base pairs. The location of the in-frame V5 epitope tag for each isoform is indicated in magenta above each Exon 1. C. Schematic diagram representing the alternatively initiated tjp1b/ZO1b isoform mRNAs tjp1b/ZO1b-Alpha (ENSDART00000173656.2; 7618 bp), -Beta (ENSDART00000155992.3; 7626 bp) and -Gamma (ENSDART00000112588.5; 7408 bp). Unique 5’UTRs and initiation exons are represented by white boxes, additionally spliced exons are represented by grey boxes and exons shared by all three isoforms are represented by black boxes (Exons 5–31). Approximate location of the ATG start codon and the shared TGA stop codon are indicated in each transcript. D. Schematic diagram of the ZO1b isoform proteins representing ZO1b-Alpha (2005 aa), -Beta (1853 aa), and -Gamma (1689 aa). The unique N-terminal amino acid sequences are indicated in the dashed box. Note that ZO1b-Alpha and ZO1b-Beta both contain amino acid sequence derived from exons 3 and 4. Domains are depicted as white shapes; CARD, PDZ, SH3, GUK and ZU5 are protein-protein interaction modules. White bars represent regions derived from unique initiation exons, grey bars represent regions derived from additionally spliced exons and black bars represent regions derived from shared exons.

Alternatively initiated isoforms tjp1b/ZO1b-Beta and tjp1b/ZO1b-Gamma are expressed in the Mauthner cell circuit and localize to electrical synapses

A. scRNA-seq dataset of tjp1b/ZO1b expression in early development where grey represents low expression and magenta represents the highest level of expression. Cells are derived from whole embryos at 1, 2, and 5 days post fertilization (dpf) and graphed together in UMAP space. Annotated cell types of clusters are indicated and each dot represents a single cell. B. Confocal tile scan of fluorescent RNA in situ in whole 5 dpf wt zebrafish larvae for negative control DapB, tjp1b/ZO1b-Alpha, tjp1b/ZO1b-Beta, and tjp1b/ZO1b-Gamma. Images are maximum intensity projections of ~68–98 μm. Anterior left. Contrast is inverted for clarity. Black arrowhead denotes signal in spinal cord. Black arrow denotes signal in hindbrain. While there is possible expression of all three isoforms in the vacuolar cells of the notochord as notochord expression was detected by scRNA-seq methods (Fig 2A), we note that the fluorescent RNA in situ method often non-specifically detects signal in the notochord (blue arrowhead). Scale bar = 20 μm. C-J. Confocal images of fluorescent RNA in situ (white) for the negative control DapB (C, D), tjp1b/ZO1b-Alpha (E, F), tjp1b/ZO1b-Beta (G, H), and tjp1b/ZO1b-Gamma (I, J) in the Mauthner circuit cell bodies of 5 days post fertilization (dpf) zf206Et zebrafish larvae from wildtype (wt). Animals are co-stained with anti-GFP (green). Scale bars = 2 μm. Images of the Mauthner cell body (C, E, G, I) are maximum intensity projections of ~15–30 μm. Boxed regions are enlarged in C’, E’, G’ and I’ and neighboring panels show individual channels. Images of the CoLo cell body (D, F, H, J) are maximum intensity projections of ~6–12 μm and neighboring panels show individual channels. The green dashed line in the probe channel indicates the outline of the CoLo cell body. In F, the arrow indicates the Alpha signal in unidentified nearby cells. In G-J, the Beta and Gamma signals appear as discrete nuclear granules in the neuronal cell body [30, 31]. K-N. Confocal images of Mauthner circuit neurons and stereotypical electrical synaptic contacts in 5dpf zf206Et zebrafish larvae from heterozygous V5-tjp1b/ZO1b Beta (K, L) and heterozygous V5-tjp1b/ZO1b-Gamma (M, N) animals. Animals are stained with anti-GFP (green), anti-Cx35.5 (cyan), anti-Cx34.1 (yellow), and anti-V5 (magenta). Scale bars = 2 μm. Images of the lateral dendrite in the hindbrain (K, M) are maximum intensity projections of ~5–6 μm. Neighboring panels show individual channels. Images of the sites of contact of M/CoLo processes in the spinal cord (L, N) are maximum-intensity projections of ~3–4 μm. Boxed regions denote stereotyped location of electrical synapses and regions are enlarged in neighboring panels. In L’ and N’, the white dashed circle denotes the location of the M/CoLo site of contact. Neighboring panels show individual channels. Anterior is up in all images.

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

A-J. 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-pan^-/- (C, D), tjp1b/ZO1b-alpha^-/- (E, F), tjp1b/ZO1b-beta^-/- (G, H), and tjp1b/ZO1b-gamma^-/- (I, J) 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, G, I) are maximum intensity projections of ~10–20 μm. In A’, C’, E’, G’, and I’, images are maximum-intensity projections of ~3–6 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, H, J) are maximum-intensity projections of ~6–8 μm. In B’, D’, F’, H’ and J’, 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.

Quantification of ZO1 isoform mutants.

A. 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 = 10; tjp1b/ZO1b-pan^-/- n = 5, tjp1b/ZO1b-alpha^-/- n = 5, tjp1b/ZO1b-beta^-/- n = 10, and tjp1b/ZO1b-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 by ANOVA with Dunnett’s test. For ZO1 (magenta circles), **** indicates p<0.0001 and ** indicates p = 0.0093 by ANOVA with Dunnett’s test. B. Quantification of Cx35.5, Cx34.1, and ZO1 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 = 11, tjp1b/ZO1b-pan^-/- n = 3, tjp1b/ZO1b-alpha^-/- n = 5, tjp1b/ZO1b-beta^-/- n = 5, and tjp1b/ZO1b-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 by ANOVA with Dunnett’s test. For ZO1 (magenta circles), **** indicates p<0.0001, ** indicates p = 0.008, and * indicates p = 0.0364 by ANOVA with Dunnett’s test.

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.

Model of tjp1b/ZO1b isoform contribution to electrical synapse assembly.

Diagram summarizing the contribution of each ZO1b isoform to electrical synapse formation in the Mauthner circuit. Unique first exons of tjp1b/ZO1b-Alpha (black), -Beta (magenta), and -Gamma (blue) are denoted by triangles on the tjp1b-ZO1b locus (grey bar) with relative exon structure denoted (dark grey rectangles). Curved arrows indicate unique transcription initiation sites. ZO1b-Beta and -Gamma both localized to the electrical synapse, yet only the ZO1b-Beta isoform is required for robust Connexin (grey ovals) localization to synaptic contacts.