(A) (i–iii) Live images of 4 dpf otic vesicles, lateral view. (i) Wild-type sibling, (ii) adgrg6tb233c, (iii) adgrg6fr24 showing the swollen, unfused projections in the homozygous mutant otic vesicles in ii and iii compared with the fused pillars in the wild-type ear (marked with dotted lines). (iv–vi) ISH with vcanb at 4 dpf. (iv) Wild-type sibling, (v) adgrg6tb233c, (vi) adgrg6fr24 mutant ears showing overexpression of vcanb in the unfused projections. Stronger staining is seen in the stronger allele, fr24. (vii–xi) ISH with mbp at 4 dpf, (vii-ix) dorsal views, (x–xii) lateral views. (vii, x) wild-type sibling, (viii, xi) adgrg6tb233c, (ix, xii) adgrg6fr24 showing complex staining patterns in the PNS (black arrows and arrowheads) and CNS (white arrowheads). mbp staining around the PLLg is absent in both tb233c and fr24 alleles (black arrowheads); staining in the posterior lateral line nerve is variable in tb233c mutants and absent in fr24 mutants (black arrows). (B) Schematic diagram showing the structure of the Adgrg6 receptor and the positions of the predicted amino acid changes for the two adgrg6 mutant alleles used in this study. (C) Light-sheet microscope images using a transgenic line expressing GFP in the otic epithelium, showing a dorsal view of the ear (anterior to the top). (i–iii) Wild-type sibling showing anterior and posterior pillars formed from fused projections (iii). Note that images are flipped horizontally from the originals for ease of comparison (see Video 1; t0 on the stills corresponds to ~100 mins into the video). (iv–vi) Still images from a time-lapse video of adgrg6fr24 mutant with unfused projections that roll around each other (see Video 2). Abbreviations: AC, lumen of anterior semicircular canal; ap, anterior projection; CTF, carboxy-terminal fragment; CUB, Complement C1r/C1s, Uegf, BMP1 domain; ECD, extracellular domain; GAIN, GPCR auto-proteolysis domain; GPS, GPCR proteolytic site; HBD, hormone binding domain; ICD, intracellular domain; NTF, amino-terminal fragment; PC, lumen of posterior semicircular canal; pp, posterior projection; PTX, Pentraxin domain; vp, ventral projection; 7TM, 7-transmembrane domain. Scale bars: 50 μm in Ai, for Aii–vi; 100 μm in Avii, for Aviii–xii; 20 μm in Ci, for Cii–vi.