Approach of Tol2-mediated microinjection in zebrafish. CNE was inserted into the enhancer activity detecting vector. Together with the tol2 mRNA, plasmid containing the CNE was co-injected into zebrafish embryos at the single-cell stage.

Identification of tissue-specific CNEs around eya1 gene. a CNEs distribution near the zebrafish eya1 locus based on the danRer7 assembly. Genes are shown as blue rectangles and the orientation of arrows indicates the translational initiation sites of genes. The CNEs located near the eya1 gene are indicated by red rectangles. The horizontal line at the bottom indicates the studied range of the DNA sequence range. b Sequence conservation between zebrafish (danRer7 assembly) and humans (hg19 assembly). c CNE16.39 and CNE 16.45 regulate GFP expression mainly in zebrafish ear. Photos were taken with the GFP channel at 24 hpf, 48 hpf, and 72 hpf. Red arrow, location of ear.

Schematic diagram of dissection. Red color represents the region where sequences that are more than 80% similar in the human and zebrafish genome sequence alignment are located. a The case of CNE16.39 dissection. b The case of CNE16.45 dissection.

Expression of each full-length and dissected CNE plasmid in zebrafish embryos at different hpf.

Ear-specific enhancers CNE16.39 and CNE16.45-R mediate GFP-specific expression in zebrafish inner ear crista.