Zebrafish nav3 is expressed in the heart during embryogenesis
Zebrafish Nav3 has been previously reported as an ortholog of UNC-53 in C. elegans. Compared with other vertebrate NAV3 in humans, mice, and rats, as well as UNC-53 in C. elegans, zebrafish Nav3 was highly conserved among all these species (Supplemental Fig. S1). NAV3 is a huge protein consisting of more than 2,000 amino acids. It contains several conserved domains, including a putative calponin-homology domain (CH-domain), an LKK actin-binding domain, two Src Homology 3 (SH3) domains, and an ATP/GTP-binding AAA domain (Schmidt et al., 2009) (Fig. 1A). The CH-domain is essential for the interaction with actin filaments, suggesting its role in the formation of lamellipodia and filopodia, and it would further drive cell movements. Phylogenic alignment also revealed the close relationship between zebrafish Nav3 and NAV3 of other species (Fig. 1B).
WISH was performed with zebrafish embryos from 18 hpf to 96 hpf to explore the spatiotemporal expression of zebrafish nav3 during embryogenesis. The nav3 mRNA transcripts could be detected at an early embryonic stage (18 hpf), while its expression was mainly restricted to the brain and somites (Fig. 1C). A slight expression was also presented in cardiac primordium at the same developmental stage (Fig. 1D, E). As nkx2.5 is specifically expressed in cardiac primordium, the ISH experiment with nkx2.5 probe could further confirm the expression of nav3 in the region of cardiac primordium (Fig. 1F). At 24 hpf, nav3 was more specifically expressed in the brain, heart, and somites (Fig. 1G-I). However, the expression of nav3 in somites became weaker, and it was rarely detected from 48 hpf. At 48 hpf, its expression was highly regionalized to the brain and heart (Fig. 1J-L). At 96 hpf, there was almost no expression of nav3 in the heart, and its expression was found to be accumulated in other tissues, such as the gill arch, swim bladder, and intestine (Fig. 1M-O), suggesting that its potential roles were not only restricted to nervous or cardiovascular system.
Nav3 loss-of-function in zebrafish exhibits severe phenotypic defects and low survival rates3e4r
To explore the effects of Nav3 deletion in heart development, a CRISPR/Cas9-based genome editing method was utilized to generate a nav3-deleted mutant (Fig. 2A). A 55-bp deletion at the second exon of nav3 led to the occurrence of a truncated protein (a 79-amino acid sequence versus the full-length sequence with 2,270 amino acids). The homozygous nav3−/− mutants were further obtained through genetic selection. An obvious heart malformation phenotype was observed in nav3-null zebrafish embryos. Compared with the wild-type (WT) counterparts, the nav3−/− mutants displayed severe pericardial edemas not only at the early developmental stage but also in adult zebrafishes, although more than 70% of the nav3−/− mutants died within 24 hpf (Fig. 2B-H). Afterwards, only 8% of the mutants could successfully develop into adults (Fig. 2H).
Heart morphogenesis and function are disrupted in nav3 mutant zebrafish embryos
To further evaluate the changes in cardiac morphology, structure, and function in nav3-null mutants, the fluorescently labeled atrium-specific marker amhc and ventricle-specific marker vmhc were respectively adopted. Transgenic lines Tg(vmhc:mCherry::amhc:EGFP) and Tg(nav3−/−-vmhc:mCherry::amhc:EGFP) were used to compare the heart morphological differences between WT and mutants. Both atrium and ventricle in nav3−/− mutants at 72 hpf exhibited a long tubular-shaped morphology, and the boundary line between atrium and ventricle was not as clear as the WT (Fig. 3A-F). Next, we compared the heart rate as well as the HI between WT and nav3−/− mutants to check whether the heart functions were affected upon Nav3 loss-of-function. We found that the heart rate was not affected by the deletion of nav3. The frequency was within a range of 150 to 200 times per minute in both WT and mutant strains (Fig. 3S). However, the HI in WT and mutant strains was different. Here, we introduced a parameter defined as HI to indicate cardiac capacity. The areas of the GFP-labeled heart were measured in the dilated and contracted states (Fig. 3G-R). The area difference between these two states was regarded as HI. The calculation formula was shown in Methods. The HI was significantly lower in the mutants compared with the WT siblings (Fig. 3T). However, there was no obvious difference in heart rate between WT and mutants (Fig. 3U). All these findings indicated that Nav3 deficiency resulted in heart development defects during zebrafish embryogenesis, including the cardiac structure and functional disruption.
nav3 mRNA injection can rescue the cardiac defects in nav3-deleted embryos
To further confirm whether the abnormal phenotype of the mutant was caused by the deletion of nav3, we injected a morpholino against nav3 mRNA into transgenic line Tg(myl7:mCherry) to delete nav3. Tg(myl7:mCherry) line was under a WT background, and the cardiac marker myl7 (myosin light chain 7) was fluorescently labeled with mCherry fluorescent protein. Upon nav3 expression was down-regulated through morpholino injection, WT zebrafish embryos displayed a similar phenotype to nav3−/− mutants. Moreover, fluorescent imaging of the heart in morpholino-injected embryos mimicked the abnormal cardiac morphology of nav3-null mutants (Fig. 4).
To verify that the phenotype of nav3-morphants was attributed to the loss-of-function of Nav3 rather than non-specific effects, we performed a rescue experiment by injecting nav3 mRNA together with nav3 morpholino into 1-cell-stage zebrafish embryos of Tg(myl7:mCherry). The nav3 mRNA injection partially rescued the phenotypic defects caused by the down-regulation of nav3. Upon nav3 mRNA injection, the tubular-shaped ventricle and atrium structure of the nav3-morphants were almost recovered to a normal state. However, compared with the WT counterparts, the rescued heart was slightly misshaped in the morphology, such as tubular-shaped ventricle. Additionally, the boundary between the ventricle and atrium became observable after nav3 mRNA injection (Fig. 4).