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Fig. 6 Nrg1 serves as a pivotal mitogen mediating the function of id2b. (A) Identification of genes (fgf8a, nkx2.5, myh6, nrg1, and nkx2.7) associated with four distinct heart development processes: cardiac muscle tissue development, cardiomyocyte differentiation, heart morphogenesis, and cardiac chamber development. The heatmap illustrates scaled-normalized expression values for the mentioned genes. (B) Quantitative real-time PCR (qRT-PCR) analysis of nrg1 mRNA in 120 hr post-fertilization (hpf) id2b+/+ and id2b-/- embryonic hearts. Data were normalized to the expression of actb1. N=4 biological replicates, with each sample containing 500–1000 embryonic hearts. (C) Heart rate in 120 hpf larvae treated with AG1478 (n=20) and DMSO (n=20). (D) id2b+/+ and id2b-/- larvae were injected with nrg1 mRNA at the one-cell stage, followed by qRT-PCR analysis of cacnα1c mRNA at 72 hpf. Data were normalized to the expression of actb1. N=3 biological replicates, with each sample containing 100–200 embryonic hearts. (E) The heart rate of 72 hpf id2b+/+ and id2b-/- larvae injected with nrg1 mRNA at one-cell stage. n=(16, 12, 14, 14). Data are presented as mean ± s.e.m. p-values were calculated by unpaired two-tailed Student’s t-tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.