Abstract
Mutations in the human growth differentiation factor 6 gene (GDF6) have been shown to contribute to several syndromic diseases, but their role in sensorineural hearing loss is rarely studied. Bademci et al. (2020) reported that homozygous deletions of a GDF6 cis-regulatory element are associated with nonsyndromic profound hearing loss in humans due to cochlear aplasia and that Gdf6 knockout mice demonstrate cochlear aplasia at the fetal stage but cannot survive postnatally. To further understand the biological role of growth differentiation factor 6 in the evolutionary context of hearing development, we employed the larval zebrafish model because of its rapid development, experimental accessibility due to external fertilization, and transparent nature which allows noninvasive imaging studies to be performed. The zebrafish carries two homologs (gdf6a and gdf6b) of the human GDF6. Using phenotypic screening, confocal imaging, and electrophysiological methods, we performed multiple morphological assessments of body plan development, saccular hair cell counts, and functional assessment of hearing in gdf6 mutant zebrafish and siblings at 3 and 7 days postfertilization (dpf). Heterozygous carriers of the gdf6as327 mutation from the Herwig Baier lab were bred to generate the gdf6as327/s327 mutants along with the gdf6WT/WT and gdf6aWT/s327 siblings used in this study. Knockout of gdf6b was obtained using CRISPR-Cas9 gene editing. We found that gdf6b rather than gdf6a mutations significantly reduce the sizes of the otic vesicle and saccular otolith at 3 dpf, but not 7 dpf. In addition, gdf6a/b double mutants have fewer saccular hair cells and higher hearing thresholds than their wild-type siblings at 3 dpf. Thus, our results indicate that gdf6 genes play a role in early zebrafish inner ear development and that gdf6b is likely the primary gdf6 gene involved in sensorineural hearing. In contrast, gdf6a primarily influences the early development of the eye and swim bladder formation.