CRISPR/Cas9-induced zebrafish mutants for ebf3a

Mutations in the transcription factor EBF3 result in a neurodevelopmental disorder, and studies in animal models indicate that it has a critical role in neuronal differentiation. The molecular pathways and neuron types disrupted by its loss, however, have not been thoroughly investigated. Nor have the outcomes of these changes on behavior and brain activity. Here, we generated and characterized a zebrafish ebf3a loss-of-function mutant. We discovered morphological and neural phenotypes, including an overall smaller brain size, particularly in the hypothalamus, cerebellum, and hindbrain. Brain function was also compromised, with activity strongly increased in the cerebellum and abnormal behavior at baseline and in response to visual and acoustic stimuli. RNA-sequencing of developing larvae revealed significant downregulation of genes that mark olfactory sensory neurons, the lateral line, and cerebellar Purkinje neurons. Corroborating the RNA-sequencing, staining revealed fewer lateral line neuromasts and reduced Parvalbumin signal in the cerebellum. This study sets the stage for determining which downstream pathways underlie the emergence of the observed phenotypes and establishes multiple strong phenotypes that could form the basis of a drug screen. Overall design: Total RNA was extracted from the anterior half of the body from 10-12 5 dpf larvae per sample at using the Rneasy Mini Kit (Qiagen). RNA for the 2 dpf RNA-seq samples were collected in the same way, but with 15-20 embryos per pool. The 5 dpf RNA-seq was collected also with 10-12 larvae. The posterior portion of the cuts was used for genotyping. The RNA was isolated from the anterior portion as described and submitted to sequencing by GENEWIZ at Azenta Life Sciences.