Mutations in the microexon splicing regulator srrm4 have minor phenotypic effects on zebrafish neural development.

Abstract

Achieving a diversity of neuronal cell types and circuits during brain development requires alternative splicing of developmentally regulated mRNA transcripts. Microexons are a type of alternatively spliced exon that are 3-27 nucleotides in length and are predominantly expressed in neuronal tissues. A key regulator of microexon splicing is the RNA-binding protein Serine/arginine repetitive matrix 4 (Srrm4). Srrm4 is a highly conserved, vertebrate splicing factor that is part of an ancient family of splicing proteins. To better understand the function of Srrm4 during brain development, we examined neural expression of zebrafish srrm4 from days 1-5 of development using fluorescence in situ hybridization. We found that srrm4 has a dynamically changing expression pattern, with expression in diverse cell types and stages during development. We then used CRISPR-based mutagenesis to generate zebrafish srrm4 mutants. Unlike previously described morphant phenotypes, srrm4 mutants did not show overt morphological defects. Whole brain morphometric analysis revealed a reduction in optic tectum neuropil in G0 crispants that, unexpectedly, was also not replicated in stable mutants. Sequencing of wild-type and mutant transcriptomes revealed only minor changes in splicing and did not support a hypothesis of transcriptional adaptation, suggesting that another, as yet, unidentified mechanism of compensation is occurring. srrm4 thus appears to have a limited role in zebrafish neural development.