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Fig. 7 Id2b interacts with Tcf3b to restrict its inhibition on nrg1 expression. (A) Immunoprecipitation (IP) assays of Flag-id2b and HA-tcf3b co-transfected 293T cells. (B) Quantitative real-time PCR (qRT-PCR) analysis of tcf3a, tcf3b, socs1a, and socs3b 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) Two potential Tcf3b-binding sites, with sequences corresponding to the human TCF3 (left) and mouse Tcf3 (right) binding motifs, were predicted in the 2000 bp DNA sequence upstream of the zebrafish nrg1 transcription start site using JASPAR. (D) Luciferase assay showing the expression of nrg1 in embryos with tcf3b overexpression (tcf3b OE) and morpholino-mediated tcf3b knockdown (tcf3b MO). N=3 biological replicates. (E) qRT-PCR analysis of nrg1 mRNA in 72 hpf id2b+/+ and id2b-/- embryonic hearts injected with control and tcf3b morpholino. Data were normalized to the expression of actb1. N=4 biological replicates, with each sample containing 100–200 embryonic hearts. (F) Schematic model for id2b-mediated regulation of myocardium function. During heart development, blood flow operates through primary cilia, initiating endocardial id2b expression. Subsequently, the interaction between Id2b and Tcf3b restricts the activity of Tcf3b, ensuring proper nrg1 expression, which in turn promotes L-type calcium channel (LTCC) expression (left). However, in the absence of Id2b, Tcf3b inhibits nrg1 expression. The reduced Nrg1 hinders LTCC expression in cardiomyocytes, resulting in decreased extracellular calcium entry and disruption of myocardial function. 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. ns, not significant.