Fig. 7

Erc1b Rab-binding domain is required for cranial nerve branching, not for NMJ organization or muscle attachment. A, Schematic of WT zebrafish Erc1b and truncated Erc1b^Δ960 constructs, R960 of the Rab-binding domain was replaced with a stop codon. B, Experimental design of mRNA overexpression and imaging of live transgenic or stained larvae for phenotypic analysis. Representative images of live, C, NIC and WT mRNA injected, and D, NIC and erc1b^Δ960 mRNA injected WT (top) and erc1b^−/−/Tg(isl1:GFP) larvae (bottom). Arrows indicate WT nerve midline crossing; open yellow arrowheads indicate ectopic branching in NIC erc1b^−/− larvae. E, Representative images of NMJ (SV2, magenta; αBTX, green) stained NIC, WT mRNA, erc1b^Δ960 mRNA injected WT (top), and erc1b^−/− larvae (bottom). Arrows indicate NMJ clusters, yellow arrowheads indicate small, dispersed NMJs in NIC erc1b^−/− larva. F, Quantification of nerve branching index and G, NMJ count of NIC, WT erc1b mRNA injected, and erc1b^Δ960 mRNA injected larvae. Symbols indicate individual animals, lines indicate mean with SEM. Two-way ANOVA with Sidak's multiple comparison was used for statistical significance, *P < .05, ***P < .001, ****P < .0001. H, Representative images of phalloidin-stained muscles in NIC and erc1b^Δ960 mRNA injected larvae (WT top, erc1b^−/− bottom). Disorganized mhj (yellow arrowhead) in NIC erc1b^−/− is rescued by erc1b^Δ960 mRNA injection (yellow arrow). I, Quantification of percent of larvae displaying ectopic muscle fibers. Sample sizes (n) for each genotype and injection denoted in the table. All images are maximum intensity projections, ventral views with anterior on top. Scale bar = 25 μm