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

Microexons show remarkable neuronal switch-like regulation and evolutionary conservation and they can impact protein-protein interactions and other molecular functions. Neuronal microexons are regulated in a tightly coordinated manner by the splicing factors Srrm3 and Srrm4. Depletion of Srrm3/4 in mice causes dramatic neurological phenotypes and their misregulation is associated with human disease. However, which microexon targets are associated with these phenotypes and how individual microexon deletions could impact neurodevelopment, physiology and behavior is unknown. To address this question, we generated zebrafish KO lines for 18 individual microexons and characterized their phenotypes at multiple levels, together with srrm3/4 mutant lines. We found that, while mutation of the regulators, specially srrm3, shows multiple alterations in neuritogenesis, locomotion and social behavior, the impact of deleting of individual microexons was usually mild or non-existing. Nonetheless, for various microexons, we identified neuritogenesis defects (evi5b, vav2, itsn1, src) and social defects (vti1a, kif1b). In addition, microexon deletions led to transcriptomic alterations that are likely compensatory. Overall, our data suggest that the defects caused by srrm3/4 depletion are due to a cumulative effect of multiple small impacts that can individually be well tolerated by zebrafish. Overall design: We deleted individual microexons in zebrafish using CRISPR-Cas9 with a double guide-RNA strategy. Stable mutant lines were generated for each microexon deletion, and rise to the F2 generation. To generate RNA-seq data, we cross two adult heterozygous individuals, and genotyped 5 dpf larva through fin clipping. Larve of WT or DEL (homozygous deletion) genotype were grouped together, RNA extracted and submitted for polyA-selected stranded RNA-seq. A similar approach was taken for the mutant of the regulators. All fish had a HuC:GFP background.