Zebrafish Neuregulin 1 and the expression of its isoforms.

(A) Gene tree from Clustal-Omega multiple alignment comparison. (B) Schematic of Nrg1 domains encoded by Exons 1–14. (C) Schematic of nrg1 gene structure. Exons are drawn to scale; introns are not to scale. Alternative splicing produces three primary isoforms, nrg1-I, nrg1-IIa-c, and nrg1-III. (D-E) Relative expression of nrg1 isoforms in (D) 3 dpf embryos and (E) dissected hearts from 3 dpf embryos normalized to efl1a. (F) In situ hybridization of anti-sense riboprobe targeting nrg1. Heart is outlined in red. Student’s T-test compared to matched control. Error bars are SEM. N≥3 biological replicates. *p≤0.05–0.01, **p≤0.01–0.001, ***p<0.001.

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.

nrg1 mutants require ErbB2 tyrosine kinase activity to form trabeculae.

(A-F) Representative confocal optical mid-chamber slice of the ventricle at 3–4 dpf in larvae carrying Tg(myl7:dsRed) cardiomyocyte reporters. Boxes include high-resolution image of the outer curvature. Larvae were examined at (A-B) 3 dpf or (C-F) 4 dpf after treatment with (C-D) 1% DMSO or (E-F) 3.75 μM PD168393 from 2–4 dpf. Larvae were genotyped after imaging. Red arrows point to representative trabeculae. N ≥ 4 larvae for each condition and genotype. Relative expression levels of (G) EGF family receptor genes or (H) EGF family receptor ligand genes from isolated hearts of nrg1^WT/WT and nrg1^nc28/nc28 larvae at 3 dpf. N = 3–5 biological replicates with 30–60 hearts pooled per condition normalized to efl1a. N = 1 biological replicates with 30–60 hearts pooled for erbb2 normalized to efl1a. Student’s T-test mutant compared to wild type. Error bars are SEM. N≥3 biological replicates. *p≤0.05–0.01.

nrg1^nc28 survive to adulthood without overt cardiac abnormalities.

(A-C) Representative gross morphology of age-matched (A) nrg1^WT/WT, (B) nrg1^WT/nc28 or (C) nrg1^nc28/nc28 clutch mates, Standard Length (SL) 25–30. Scale bar is 10mm. (D) Weekly survival of fish from sibling nrg1^WT/nc28 and nrg1^nc28/nc28. (E-F) Representative cross section of the heart in H&E stained section of formaldehyde-fixed, paraffin embedded (E) nrg1^WT/WT and (F) nrg1^nc28/nc28adult fish. BA = Bulbous Arteriosus, V = Ventricle, A = Atrium. N = 3 fish per genotype. Scale bar 100 μm.