Figure 1: gpr56 expression and mutant generation in zebrafish.

(a) Representative image of gpr56 expression assessed by RT–PCR from fertilization through larval development (three technical replicates). From left to right: Mat (maternal expression), 8 h (8 h post-fertilization), 1 d (days post-fertilization) to 5 d, RT (-) control and H₂0 control. (b,c) Whole-mount in situ hybridization (WISH) of zebrafish larvae at (b) 1 and (c) 2 dpf shows robust gpr56 expression within the central nervous system (black arrows in b) during larval development (lateral views shown, anterior to the left, dorsal is up, two technical replicates performed). (d) Cross-section through the spinal cord (white dashed line in c depicts approximate location) of gpr56 WISH embryo at 2 dpf shows gpr56 expression in the spinal cord midline (white arrow) and in bands consistent with neural precursors (black arrows). (e) Diagram of the zebrafish gpr56 gene (top) and protein (bottom) structures. TALENs used to generate gpr56 zebrafish mutants targeted between the 8th and 9th exons (text in red). Gpr56 contains a signal sequence (ss), GPCR Autoproteolysis-Inducing Domain (GAIN), GPCR Proteolytic Site (GPS) and the canonical 7-Transmembrane Domain (7TM). (f) Recovered mutant alleles of gpr56—stl13 representing a 6-bp deletion and stl14 representing a 26 bp deletion. (g) Amino-acid sequence alignment of the GPS motif from representative species showing perfect conservation of the Trp residue that is deleted in the gpr56^stl13/stl13 allele (highlighted yellow). Mutation of the second, highly conserved Trp residue within the GPS causes BFPP (black arrowhead).