Fig. 2

Zebrafish nrg1-I/II mutants.

(A) CRISPR/Cas9 gene targeting and validation of nrg1^nc28 and nrg1^nc29 alleles showing target site and mutations. (B) Sanger sequence of nrg1^WT, nrg1^nc28 and nrg1^nc29 alleles spanning target site in Exon 3. (C) Gel electrophoresis and densitometry of nrg1 amplified from 10ng cDNA derived from nrg1^WT or nrg1^nc28/nc28 embryos at 5 dpf. Student’s T-test compared to matched control. Error bars are SEM. N≥3 biological replicates. *p≤0.05–0.01. (D) Representative Mitotracker stain for neuromasts. Heterozygous adult fish carrying nrg1^WT/nc28, nrg1^WT/nc26, or nrg1^WT/z26 alleles were inbred, and resulting offspring were evaluated for supernumerary neuromasts (red arrows). (E-F) Frequency distribution of the number of neuromasts per embryo. Blue bar marks range of neuromasts found in wild type larvae; red bars mark supernumerary neuromasts. Similar results were obtained with nrg1^nc29 lines (data not shown). N = 15–20 embryos imaged per pairing; N = 2 biological replicates.