Identification of id2b as a blood flow sensitive gene.

(A) Schematic showing the experimental procedures, including treatment, heart collection, and RNA-sequencing of zebrafish embryonic hearts. (B) KEGG enrichment analysis depicting differentially expressed genes encoding transcription factors and transcriptional regulators between control (ctrl) and tricaine-treated embryonic hearts. Red and blue rectangles represent up-regulated and down-regulated gene sets, respectively. |log2fold change|≥0.585, adjusted p-value<0.1. Each replicate contains approximately 1,000 hearts. (C) Heat map exhibiting representative genes from KEGG pathways mentioned in (B). (D) qRT-PCR analysis of id2b mRNA in control and tricaine-treated embryonic hearts. Data were normalized to the expression of actb1. Each sample contains ∼1,000 embryonic hearts. (E) In situ hybridization of id2b in 72 hpf and 96 hpf ctrl, tricaine (1 mg/mL), and blebbistatin (10 μM)-treated embryos. Numbers at the bottom of each panel indicate the ratio of representative staining. (F) In situ hybridization showing reduced id2b expression in tnnt2a morpholino-injected embryos at 72 hpf compared to control. *** p<0.001. Scale bars: 50 μm.

The spatiotemporal expression of id2b.

(A) Schematic of the intron targeting-mediated eGFP knockin at the id2b locus using the CRISPR-Cas9 system. The sgRNA targeting sequence and the protospacer adjacent motif (PAM) sequence are shown in orange and blue, respectively. The donor plasmid comprises left and right arm sequences and a linker-FLAG-P2A-eGFP cassette denoted by black lines with double arrows and green box, respectively. The linker-FLAG-P2A-eGFP cassette was integrated into the id2b locus upon co-injection of the donor plasmid with sgRNA and zCas9 protein, enabling detection by PCR using two pairs of primers (F1, R1 and F2, R2)-the former length yielding a length of about 2.2 kb and the latter about 2.7 kb. (B) Zebrafish id2b expression pattern, as indicated by in situ hybridization of embryos at designated time points, was consistent with the fluorescence localization of id2b:eGFP, revealing expression in the heart, brain, retina, notochord, pronephric duct, and other tissues. (C) Three dimensional (3D) reconstructions (top) and confocal sections (bottom) of id2b:eGFP; Tg(myl7:mCherry) hearts at designated time points. (D) 3D reconstructions (top) and confocal sections (bottom) of id2b:eGFP; Tg(kdrl:mCherry) embryos at specific time points. Magenta: id2b:eGFP; yellow: kdrl:mCherry. (E) Confocal z-stack maximal intensity projection of id2b:eGFP;Tg(kdrl:mCherry) embryos at 96 hpf showing the whole body (lateral view) and the head (top view). (F) Immunofluorescence of adult id2b:eGFP; Tg(kdrl:mCherry) heart section (middle panel). Enlarged views of boxed areas are shown in the left and right panels. Green: eGFP; red: mCherry; blue, DAPI. Scale bars: 500 μm (B, E, left and F); 100 μm (E, right); 50 μm (C and D).

Cardiac contraction promotes endocardial id2b expression through primary cilia.

(A) Representative confocal z-stack (maximal intensity projection) of id2b:eGFP embryos under different conditions: control, tricaine-treated, blebbistatin-treated, and tnnt2a morpholino-injected. (B) Quantification of mean fluorescence intensity (MFI) of id2b:eGFP in the working myocardium (atrium and ventricle, A+V) and atrioventricular canal (AVC) in (A). Data normalized to the mean fluorescence intensity of control hearts. (C) Representative confocal z-stack (maximal intensity projection) of control and ift88 morpholino-injected id2b:eGFP embryos. (D) Normalized MFI of id2b:eGFP in the working myocardium (A+V) and AVC in (C). (E) Whole-mount in situ hybridization showing id2b expression in control, klf2a-/-, and klf2b-/- embryos at 48 hpf and 72 hpf. Numbers at the bottom of each panel indicate the ratio of representative staining. Data are presented as mean ± sem. ** p<0.01, *** p<0.001, **** p<0.0001. Scale bars: 50 μm.

id2b-/- adults exhibit thinner atrioventricular valve leaflets and prominent retrograde blood flow.

(A) Two sgRNAs, represented by short vertical lines, were designed to create id2b-/- mutants. Co-injection of the two sgRNAs with zCas9 protein induces a 157 bp truncation in the exon 1 of id2b, which can be detected by genotyping primers marked with arrows. This genetic modification leads to the formation of a premature stop codon and the subsequent loss of the HLH domain. (B) No discernible morphological differences were observed between id2b+/+ and id2b-/- larvae at both 72 hpf and 96 hpf. (C) Kaplan-Meier survival curve analysis and logrank test of id2b+/+ (n=50) and id2b-/- (n=46). Wpf: weeks post-fertilization. (D) Pericardial edema and an enlarged atrium are evident in a subset of id2b-/- adults. V, ventricle; A, atrium. (E) id2b-/- adults developed thinner atrioventricular valve leaflets (denoted by arrowheads) compared to id2b+/+. Enlarged views of boxed areas are shown in the right panels. (F, G) Echocardiograms of adult id2b+/+ (F) and id2b-/- (G) hearts. Unidirectional blood flow was observed in the id2b+/+ heart, while retrograde blood flow was evident in the id2b-/- heart. (H) Ratio of retrograde flow area over inflow area shows a significant increase in retrograde flow in id2b-/- (n=13) compared to id2b+/+ (n=10). Data are presented as mean ± sem. ** p<0.01. Scale bars: 200 μm (E); 500 μm (B and D, bottom); 2 mm (D, top).

Reduced cardiac contractile function and compromised calcium handling in id2b-/- mutants.

(A) Time-lapse imaging (from T1 to T8) illustrates the cardiac contraction-relaxation cycle of 120 hpf id2b+/+ and id2b-/- hearts carrying myl7:mCherry. (B and C) id2b-/- larvae display a significant decrease in heart rate and fractional area change compared to id2b+/+. (D) Echocardiograms of adult id2b+/+ and id2b-/- hearts. (E and F) id2b-/- fish exhibit reduced cardiac contractile function with preserved heart rate compared to id2b+/+. (G) Time-lapse imaging illustrates the calcium dynamics of 120 hpf id2b+/+ and id2b-/- hearts carrying actb2:GCaMP6s. (H) Ratio of maximal fluorescence intensity (F) over basal fluorescence intensity (F0) of GCaMP6s signal. (I) qRT-PCR analysis of cacnα1c mRNA in id2b+/+ and id2b-/- hearts at 120 hpf (N=three replicates, with each sample containing 500-1,000 embryonic hearts) and adult stage (N=five replicates). Data were normalized to the expression of actb1. (J) The action potential of ventricular cardiomyocyte in adult id2b+/+ and id2b-/- hearts. (K) Statistical data showed a notable difference between id2b+/+ and id2b-/- accordingly. Data are presented as mean ± sem. *** p<0.001, **** p<0.0001, ns, not significant. Scale bar: 50 μm.

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) qRT-PCR analysis of nrg1 mRNA in 120 hpf id2b+/+ and id2b-/- embryonic hearts. Data were normalized to the expression of actb1. N=four replicates, with each sample containing 500-1,000 embryonic hearts. (C) id2b+/+ and id2b-/- larvae were injected with nrg1 mRNA at the 1-cell stage, followed by qRT-PCR analysis of cacnα1c mRNA at 72 hpf. Data were normalized to the expression of actb1. N=three replicates, with each samples containing 100-200 embryonic hearts. (D) The heart rate of 72 hpf id2b+/+ and id2b-/- larvae injected with nrg1 mRNA at 1-cell stage. (E) Heart rate in 120 hpf larvae treated with AG1478 and DMSO. Data are presented as mean ± sem. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. ns, not significant.

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) qRT-PCR analysis of tcf3a, tcf3b, socs1a, and socs3b mRNA in 120 hpf id2b+/+ and id2b-/- embryonic hearts. Data were normalized to the expression of actb1. N=three replicates, with each samples containing 500-1,000 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 2,000bp 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=three 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=three replicates, with each samples 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 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 ± sem. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. ns, not significant.