# Generation and characterization of a knockout mouse of an enhancer of EBF3

## TL;DR

This study creates and analyzes a mouse model with a deletion in a regulatory region linked to neurodevelopmental disorders, showing reduced gene expression and sex-specific behavioral differences.

## Contribution

The study introduces a novel mouse model with a deletion in the Rr169617 enhancer and demonstrates its impact on Ebf3 expression and behavior.

## Key findings

- Deletion of the Rr169617 enhancer leads to reduced Ebf3 expression in mice.
- Rr169617−/− mice show sex-specific differences in mobility and body composition.
- Dysregulated genes in knockout mice include those related to histones and brain development.

## Abstract

Genomic studies of neurodevelopmental disorders (NDDs) have identified several relevant genomic variants. EBF3 is a gene with an excess of protein-coding de novo variants and underlies Hypotonia, Ataxia, and Delayed Development Syndrome. We previously identified noncoding de novo variants in an enhancer of EBF3 and further found enrichment of deletions of this enhancer in NDDs. In this study, we generated a novel mouse line that deletes the highly conserved, orthologous mouse region within the Rr169617 regulatory region, and characterized the molecular and phenotypic aspects of this mouse model. We found a deviation from Mendelian expectation (P=0.02) with significant depletion of the deletion allele (P=5.8×10−4). Rr169617+/− mice had a reduction of Ebf3 expression by 10% and Rr169617−/− mice had a reduction by 20%. Differential expression analyses in E12.5 forebrain, midbrain, and hindbrain in Rr169617+/+ versus Rr169617−/− mice identified dysregulated genes including histone and brain development related genes. A priori phenotyping analysis (open field, hole board and light/dark transition) identified sex-specific differences in mobility only for Rr169617−/− mice across multiple behavioral assays with Rr169617−/− males less mobile than Rr169617−/− females. Furthermore, both sexes when homozygous for the enhancer deletion displayed body composition differences when compared to wildtype mice. Overall, we show that deletion within Rr169617 reduces expression of Ebf3 and results in phenotypic outcomes consistent with potential sex specific behavioral differences.

Summary: This study focuses on the generation and initial characterization of a knockout mouse for a region of noncoding, regulatory DNA that was previously implicated in autism.