Loss of Mgat5a-mediated N-glycosylation stimulates regeneration in zebrafish.

We are using genetics to identify genes specifically involved in hearing regeneration. In a large-scale genetic screening, we identified mgat5a, a gene in the N-glycosylation biosynthesis pathway whose activity negatively impacts hair cell regeneration. We used a combination of mutant analysis in zebrafish and a hair cell regeneration assay to phenotype the loss of Mgat5a activity in zebrafish. We used pharmacological inhibition of N-glycosylation by swansonine. We also used over-expression analysis by mRNA injections to demonstrate how changes in N-glycosylation can alter cell signaling. We found that mgat5a was expressed in multiple tissues during zebrafish embryo development, particularly enriched in neural tissues including the brain, retina, and lateral line neuromasts. An mgat5a insertional mutation and a CRISPR/Cas9-generated truncation mutation both caused an enhancement of hair cell regeneration which could be phenocopied by pharmacological inhibition with swansonine. In addition to hair cell regeneration, inhibition of the N-glycosylation pathway also enhanced the regeneration of lateral line axon and caudal fins. Further analysis showed that N-glycosylation altered the responsiveness of TGF-beta signaling. The findings from this study provide experimental evidence for the involvement of N-glycosylation in tissue regeneration and cell signaling.