Figure 1. tmc1/2a/2b triple-mutant larvae have auditory/vestibular defects. A, Truncation mutant alleles are shown for tmc1 (frame shift), tmc2a (frame shift), and tmc2b (point mutation) genes. A diagram of each gene is included. Vertical bars represent exons. Horizontal lines indicate introns. 1 cm = 3 kb. Dotted lines indicate the target locus for each gene. Mutant sequences are aligned to corresponding WTs. Dashes represent deleted bases. Spaces represent inserted bases. For tmc1, the 20 bp Cas9 target sequence (bold) and adjacent PAM sequence (underlined) are indicated. For tmc2a, the TALEN-deleted sequence is indicated (bold). For tmc2b, a G > T point mutation yields a TGA stop codon (bold). All tmc1/2a/2b double- and triple-mutant larvae include the tmc1Ex3, +1bp(1) allele; however, the tmc1Ex3, +1bp(2) allele also appears in tmc1/2a mutants used for microphonics assays. The tmc1 single mutants are homozygous for the tmc1Ex3, −5bp allele. B, The AEBR is absent in tmc1/2a/2b triple-mutant larvae (n = 9) at 5 dpf, suggesting complete deafness (WT = 70 ± 20.5%; mutant = 0 ± 0%; t = 10.26; df = 8.0). C, *tmc1/2a/2b triple-mutant larvae show characteristics of vestibular dysfunction. They rest on their sides or backs in a Petri dish without regard for gravitational direction, and they fail to inflate their swim bladders. A WT sibling is included for comparison. D, VIEM in tmc1/2a/2b triple mutants at 5 dpf is lacking, further demonstrating the absence of vestibular function (WT = 1.0 ± 0.22 AU; mutant = 0.1 ± 0.057 AU; t = 7.88; df = 3.26).