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

Background: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.Methods: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.Results: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.Conclusions:The findings from this study provide experimental evidence for the involvement of N-glycosylation in tissue regeneration and cell signaling.