Luoqing Gao

The asb5 gene, a member of the Asb protein subfamily characterized by six ankyrin repeat domains, is highly conserved and comprises two subtypes, asb5a and asb5b, in zebrafish. Our previous research has demonstrated that a deficiency of the asb5 gene significantly impairs early cardiac contractile function, highlighting its close relationship with heart development. Zebrafish asb5 expression was disrupted by both morpholino (MO) antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. A high-throughput RNA-Seq analysis was used to analyze the possible molecular regulatory mechanism of asb5 gene deletion leading to left–right (L-R) asymmetry defects in the heart. Whole-mount in situ hybridization (WISH) was conducted to evaluate gene expression patterns of Nodal signaling components and the positions of heart organs. Heart looping was defective in zebrafish asb5 morphants. Rescue experiments in the asb5-deficiency group (inactivating both asb5a and asb5b) demonstrated that the injection of either asb5a-mRNA or asb5b-mRNA alone was insufficient to rectify the abnormal L-R asymmetry of the heart. In contrast, the simultaneous injection of both asb5a-mRNA and asb5b-mRNA successfully rescued the morphological phenotype. A high-throughput RNA-Seq analysis of embryos at 48 h post fertilization (hpf) revealed that numerous genes associated with L-R asymmetry exhibited expression imbalances in the asb5-deficiency group. WISH further confirmed that the expression of genes such as fli1a, acta1b, hand2, has2, prrx1a, notch1b, and foxa3 were upregulated, while the expression of mei2a and tal1 was downregulated. These results indicated that loss of the asb5 gene in zebrafish led to the disordered development of L-R asymmetry in the heart, resulting in an imbalance in the expression of genes associated with the regulation of L-R asymmetry. Subsequently, we examined the expression patterns of classical Nodal signaling pathway-related genes using WISH. The results showed that the midline barrier factor gene lefty1 was downregulated at early stages in the asb5-deficiency group, and the expression of spaw and lefty2, which are specific to the left lateral plate mesoderm (LPM), was disrupted. This study reveals that the two subtypes of the asb5 gene in zebrafish, asb5a and asb5b, interact and jointly regulate the establishment of early cardiac L-R asymmetry through the Nodal-spaw-lefty signaling pathway.

Ankyrin repeat and suppression-of-cytokine-signaling box (Asb) proteins, a subset of ubiquitin ligase E3, include Asb5 with six ankyrin-repeat domains. Zebrafish harbor two asb5 gene isoforms, asb5a and asb5b. Currently, the effects of asb5 gene inactivation on zebrafish embryonic development and heart function are unknown. Using CRISPR/Cas9, we generated asb5a-knockout zebrafish, revealing no abnormal phenotypes at 48 h post-fertilization (hpf). In situ hybridization showed similar asb5a and asb5b expression patterns, indicating the functional redundancy of these isoforms. Morpholino interference was used to target asb5b in wild-type and asb5a-knockout zebrafish. Knocking down asb5b in the wild-type had no phenotypic impact, but simultaneous asb5b knockdown in asb5a-knockout homozygotes led to severe pericardial cavity enlargement and atrial dilation. RNA-seq and cluster analyses identified significantly enriched cardiac muscle contraction genes in the double-knockout at 48 hpf. Moreover, semi-automatic heartbeat analysis demonstrated significant changes in various heart function indicators. STRING database/Cytoscape analyses confirmed that 11 cardiac-contraction-related hub genes exhibited disrupted expression, with three modules containing these genes potentially regulating cardiac contractile function through calcium ion channels. This study reveals functional redundancy in asb5a and asb5b, with simultaneous knockout significantly impacting zebrafish early heart development and contraction, providing key insights into asb5’s mechanism.