(A–F) Expression levels of natriuretic peptide system genes in developing wild-type zebrafish embryos. (A) Total RNA was extracted from whole TL/Wt zebrafish embryos captured at 72 hpf and normalized to 100 ng/µL. Target-specific cDNA synthesis performed followed by multiplex RT-qPCR examining the simultaneous expression of components of the zebrafish natriuretic peptide system (nppc4, nppcl, nppc2, cnp3, nppa, bnp, npra, npr1a, nprb, nprc, corin, furina and furinb). Products were separated using capillary gel electrophoresis and expression quantified using the GenomeLab GeXP analysis system. (B–F) Temporal expression of zebrafish natriuretic peptide pathway genes during development. Total RNA was extracted from whole TL/Wt zebrafish embryos captured at 24, 48, 72, 96 or 120 hpf and normalized to 100 ng/µL. Target-specific cDNA synthesis performed followed by multiplex RT-qPCR examining the simultaneous expression of components of the zebrafish natriuretic peptide system (B: nppa, bnp; C: nppc2, cnp3, nppc4; D: nppcl; E: npr1a, nprb, nprc; F: corin, furina, furinb). Products were separated using capillary gel electrophoresis and expression quantified using the GenomeLab GeXP analysis system. Data shown are normalized to the housekeeping gene (elfα) and presented as the mean ± SEM from 6 to 8 independent RNA extractions (except for furin b, where n = 4); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, significantly different from 24 hpf.
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