Assessment of the cardiac expression of gatad1 in zebrafish. (A) The relative expression level of gatad1 in different tissues were revealed by real-time RT-PCR using 18s as internal control. Cardiac expression was defined as 1 and used as a reference. The brain has the highest gatad1 expression; (B) Different expression levels of gatad1 in embryonic and adult zebrafish hearts were revealed by RNA sequencing. gatad1 expression in the embryonic heart is higher than that in the adult heart; (C) Cardiac expression of gatad1 transcripts was also revealed by in situ hybridization in 2 dpf zebrafish embryos. RPKM: number of reads per kilobase per million reads.

Subcellular localization of the Gatad1 protein in the embryonic zebrafish heart. After the myl7:gatad1-GFP construct was injected into 1-cell staged embryos, hearts from 2 dpf embryos were dissected for immunostaining and imaging. (A) Gatad1-GFP shows strong expression in nuclei and relatively weak expression in myofibrils, overlapping with Actin as revealed by phalloidin staining; (B) Gatad1-GFP partially overlaps with Z-discs marked by Actinin (arrows); (C) Gatad1-GFP forms alternatively striated patterns with M-line marked by Myomesin (arrowheads). * Nucleus; Scale bar 5 μm.

Generating and phenotyping GATAD1 transgenic fish. (A) Schematic illustration of constructs that were used to generate two transgenic fish lines expressing either human wild type GATAD1 or GATAD1-S102P mutation. The GATAD1 gene is flanked by the myl7 enhancer at its 5′ terminal to drive cardiomyocyte-specific expression, and it has an HA tag and IRES-EGFP at its 3′ terminal to facilitate detection of ectopic gene expression and fish propagation; (B) The transgenic fish expressing mutant GATAD1 started to die at approximately 12 months of age, and all transgenic fish expressing wild type GATAD1 were able to survive to 16 months; (C) Severe cardiac hypertrophy in a Tg(myl7:hGATAD1S102P) fish. Left panel, images of a heart dissected from a Tg(myl7:hGATAD) and a Tg(myl7:hGATAD1S102P) fish at 17 months of age, separately. The heart from this single Tg(myl7:hGATAD1S102P) fish exhibits significantly enlarged ventricle and out flow tract. Right panel, phalloidin staining revealed less dense myofibril and wider myofibril in the heart of this single Tg(myl7:hGATAD1S102P) fish. V, ventricle; O, out flow tract; Scale bar 0.5 mm.