Expression of the gpr177 gene in zebrafish embryos. Expression of gpr177 mRNA was analyzed by whole mount in situ hybridization (A–I). A: Early somitogenesis, lateral view. B: 18 hpf, lateral view. C: 24 hpf, lateral view. D: 48 hpf, lateral view of head. E: 48 hpf, dorsal view of head. F: 48 hpf, lateral view of ear. G: 72 hpf, lateral view of head. H: 72 hpf, dorsal view of head. I: 72 hpf, lateral view of ear. All views are anterior to the right. Arrowheads indicate protrusions of semicircular canals. BA, branchial arches; T, tectum; HB, hindbrain, MHB, midbrain-hindbrain boundary; SC, spinal cord; TB, tail bud; VZ, ventricular zone. J: Western blot containing lysates from embryonic zebrafish between 1 and 10 hpf were probed using anti-GPR177 antibodies. Data are representative of four separate experiments.

Knockdown of gpr177 mRNA expression. An antisense gpr177 MO was used to knock down gpr177 mRNA translation. All images are lateral views with anterior to the right. Morphants produced by the ATG-MO are in C, D, G, H, while morphants produced by the UTR-MO are in J, K. A: 24 hpf wild type embryo. B: 24 hpf, head of wild type embryo. C: 24-hpf morphant injected with 4 ng of ATG-MO. D: Head of 24-hpf morphant. E: 48-hpf wild type embryo. F: Head of 48-hpf embryo. G: 48-hpf embryo injected with 4 ng ATG-MO. H: Head of 48-hpf morphant. J: 48-hpf embryo injected with 6 ng of UTR-MO. K: Head of 48-hpf morphant. Arrows indicate otoliths. Arrowhead indicates protrusions of semicircular canals. MHB, midbrain-hindbrain boundary; OV, otic vesicle; CE, cardiac edema. L: Phenotypes of morphants injected with UTR-MO scored at 48 hpf. N = 19–21 at each dose. M: UTR-MO injected embryos (scored in L) were harvested at 48 hpf and GPR177 expression compared to that in wild type embryos. Arrowhead indicates position of zebrafish GPR177 (<43 kDa).

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