The Interaction Between the asb5a and asb5b Subtypes Jointly Regulates the L-R Asymmetrical Development of the Heart in Zebrafish

The <i>asb5</i> gene, a member of the Asb protein subfamily characterized by six ankyrin repeat domains, is highly conserved and comprises two subtypes, <i>asb5a</i> and <i>asb5b</i>, in <i>zebrafish</i>. Our previous research has demonstrated that a deficiency of the <i>asb5</i> gene significantly impairs early cardiac contractile function, highlighting its close relationship with heart development. <i>Zebrafish asb5</i> 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 <i>asb5</i> 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 <i>zebrafish asb5</i> morphants. Rescue experiments in the <i>asb5</i>-deficiency group (inactivating both <i>asb5a</i> and <i>asb5b</i>) demonstrated that the injection of either <i>asb5a</i>-mRNA or <i>asb5b</i>-mRNA alone was insufficient to rectify the abnormal L-R asymmetry of the heart. In contrast, the simultaneous injection of both <i>asb5a</i>-mRNA and <i>asb5b</i>-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 <i>asb5</i>-deficiency group. WISH further confirmed that the expression of genes such as <i>fli1a</i>, <i>acta1b</i>, <i>hand2</i>, <i>has2</i>, <i>prrx1a</i>, <i>notch1b</i>, and <i>foxa3</i> were upregulated, while the expression of <i>mei2a</i> and <i>tal1</i> was downregulated. These results indicated that loss of the <i>asb5</i> gene in <i>zebrafish</i> 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 <i>lefty1</i> was downregulated at early stages in the <i>asb5</i>-deficiency group, and the expression of <i>spaw</i> and <i>lefty2</i>, which are specific to the left lateral plate mesoderm (LPM), was disrupted. This study reveals that the two subtypes of the <i>asb5</i> gene in <i>zebrafish</i>, <i>asb5a</i> and <i>asb5b</i>, interact and jointly regulate the establishment of early cardiac L-R asymmetry through the Nodal-<i>spaw</i>-<i>lefty</i> signaling pathway.