Fig 2

Fig 2 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.