Phenotypic impact of individual conserved neuronal microexons and their master regulators in zebrafish

Microexons exhibit striking evolutionary conservation and are subject to precise, switch-like regulation in neurons, orchestrated by the splicing factorsSrrm3andSrrm4. Disruption of these regulators in mice leads to severe neurological phenotypes, and their misregulation is linked to human disease. However, the specific microexons involved in these phenotypes and the effects of individual microexon deletions on neurodevelopment, physiology, and behavior remain poorly understood. To explore this, we generated zebrafish lines with deletions of 18 individual microexons, alongsidesrrm3andsrrm4mutant lines, and conducted comprehensive phenotypic analyses. We discovered that while loss ofsrrm3, alone or together withsrrm4, resulted in significant alterations in neuritogenesis, locomotion, and social behavior, individual microexon deletions typically produced mild or no noticeable effects. Nonetheless, we identified specific microexons associated with defects in neuritogenesis (evi5b,vav2,itsn1,src) and social behavior (vti1a,kif1b). Additionally, microexon deletions triggered coordinated transcriptomic changes in neural pathways, suggesting the presence of molecular compensatory mechanisms. Our findings suggest that the severe phenotypes caused bySrrm3/4depletion arise from the combined effects of multiple subtle disruptions across various cellular pathways, which are individually well-tolerated.