Figure 2.

Figure 2.

(A) Schematic of the screening assay protocol. Homozygous adult adgrg6^tb233c mutant fish were paired to raise large numbers of adgrg6^tb233c mutant embryos. Embryos were grown until 60 hpf, when the lateral, anterior and posterior epithelial projections in the inner ear are evident. Three embryos were aliquoted into each well of a mesh-bottomed multiwell plate in E3 medium. The mesh-bottomed plate was then transferred to the drug plate containing control compounds as shown in the plate layout and library compounds at 25 µM in 250 µL of E3 embryo medium. Plates were incubated at 28°C until 90 hpf. The mesh-bottomed plate and embryos were then transferred to 4% PFA for fixation (4°C, overnight) and then processed for ISH to vcanb. Micrographs show a selection of typical results. Treatment with 100 µM IBMX (positive control, top) results in loss (rescue) of otic vcanb expression (white arrowhead). Strong otic vcanb expression (black arrowhead) is evident in embryos where the compound had no effect (non-hit) and in negative control wells (not shown). Note the spot of stain in each embryo, marking expression in the otic vesicle. Three examples are shown of wells where compounds were scored as a hit; one of these (Hit 2) was IBMX, represented in the Spectrum collection. (B). Pipeline of the compound screening strategy and chemoinformatics analysis. The left hand side describes the flow of experimental work and the right hand side describes the complementary chemoinformatics processes. For details, see the text.