Deletion of a Long-Range Dlx5 Enhancer Disrupts Inner Ear Development in Mice.

Distal enhancers are thought to play important roles in the spatiotemporal regulation of gene expression during embryonic development, but few predicted enhancer elements have been shown to affect transcription of their endogenous genes or to alter phenotypes when disrupted. Here, we demonstrate that a 123.6-kb deletion within the mouse Slc25a13 gene is associated with reduced transcription of Dlx5, a gene located 660 kb away. Mice homozygous for the Slc25a13 deletion mutation [named hyperspin (hspn)] have malformed inner ears and are deaf with balance defects, whereas previously reported Slc25a13 knockout mice showed no phenotypic abnormalities. Inner ears of Slc25a13hspn/hspn mice have malformations similar to those of Dlx5-/- embryos, and Dlx5 expression is severely reduced in the otocyst but not the branchial arches of Slc25a13hspn/hspn embryos, indicating that the Slc25a13hspn deletion affects otic-specific enhancers of Dlx5 In addition, transheterozygous Slc25a13+/hspn Dlx5+/- mice exhibit noncomplementation with inner ear dysmorphologies similar to those of Slc25a13hspn/hspn and Dlx5-/-embryos, verifying a cis-acting effect of the Slc25a13hspn deletion on Dlx5 expression. CRISPR/Cas9-mediated deletions of putative enhancer elements located within the Slc25a13hspn deleted region failed to phenocopy the defects of Slc25a13hspn/hspn mice, suggesting the possibility of multiple enhancers with redundant functions. Our findings in mice suggest that analogous enhancer elements in the human SLC25A13 gene may regulate DLX5 expression and underlie the hearing loss that is associated with split-hand/-foot malformation 1 syndrome. Slc25a13hspn/hspn mice provide a new animal model for studying long-range enhancer effects on Dlx5 expression in the developing inner ear.