Generation and analysis of zebrafish with mutations that remove conserved, developmentally regulated microexons.

(A) Pipeline of the screen. Mutant lines with alternatively spliced microexons removed were generated with CRISPR/Cas9, crossed together, and sibling larvae were assessed for changes to brain morphology, brain activity, and behavioral profiling. Created with BioRender.com. (B) The amino acid sequence identity of 95 zebrafish microexons compared to mouse. (C) The amino acid sequence identity of 95 zebrafish microexons compared to mouse and divided by the sequence identity of the entire protein. (D) Gene Ontology analysis of biological processes associated with the 95 mouse microexons that are conserved in zebrafish. The analysis was completed using the PANTHER classification system. (E) Quantification of reverse transcription PCR (RT-PCR) for microexon-containing regions over zebrafish development. These data were clustered using the default seaborn clustermap settings (method = ‘average’).

Larval behavioral phenotypes of zebrafish with microexons removed.

(A) Summary of behavioral pipeline. (B) Baseline behavioral phenotypes for microexon mutants. The labels “1” and “2” indicate biological replicates. The data shown is for homozygous mutant larvae compared to wild-type siblings. The size of the bubble represents the percent of significant measurements in the summarized category, and the color represents the mean of the strictly standardized mean difference (SSMD) of the significant assays in that category. (C) Stimulus-driven behavioral phenotypes for microexon mutants. The labels “1” and “2” indicate biological replicates. The bubble size and color are calculated the same as in panel B. (D) Examples of behavioral phenotypes. The black boxes in panels B and C correspond to the selected plots. Wild-type siblings (black) are compared to the homozygous (red), and the plots show mean ± s.e.m.. All N are in Table S2. Kruskal-Wallis ANOVA p-values for the selected plots are as follows: dop1a center preference during the first night (day0night_boutcenterfraction_3600) = 0.00006/0.0008; eif4g3b p-values are not calculated for response plots (shown is all dark flashes in block 1), p-values for the latency for the first 10 dark flashes in block 1 (day6dpfdf1a_responselatency) = 0.026/0.0008,; ppp6r3 frequency of response to strong acoustic stimuli with a sound frequency of 1000 hertz that precede the habituation block (day5dpfhab1pre_responsefrequency_1_a1f1000d5p) = 0.002/0.016; ptprd-1 pixels moved in each bout for the duration of the experiment (combo_boutcumulativemovement_3600) = 0.001/0.00002; rapgef2 number of bouts for the duration of the experiment calculated using the delta pixel data in each frame (dpix_numberofbouts_3600) = 0.002/0.001.

Whole-brain activity and morphology phenotypes of zebrafish with microexons removed.

(A) Summary of pErk comparisons between homozygous mutants and wild-type control siblings, where magenta represents decreased activity and green represents increased. The signal in each region was summed and divided by the region size. The N for all experiments is available in Table S2. (B) Summary of structure comparisons between homozygous mutants and wild-type control siblings, where magenta represents decreased size and green represents increased. (C) Location of major regions in the zebrafish brain based on masks from the Z-Brain atlas (Randlett et al., 2015). (D) Brain imaging for microexon mutants with repeatable brain activity phenotypes. The brain images represent the significant signal difference between homozygous and wild-type control siblings. They are shown as sum-of-slices projections (Z- and X-axes) with the white outline representing the zebrafish brain. (E) Structural phenotype of the ppp6r3 mutant, with replicates shown side-by-side. The magenta indicates decreased size. (F) Brain imaging for two microexon mutants with brain activity phenotypes that are similar for both the heterozygous and homozygous mutants.