Neuron navigator 3 (NAV3) is required for heart development in zebrafish

As a tightly controlled biological process, cardiogenesis requires the specification and migration of a suite of cell types to form a particular three-dimensional configuration of the heart. Many genetic factors are involved in the formation and maturation of the heart, and any genetic mutations may result in severe cardiac failures. The neuron navigator (NAV) family consists of three vertebrate homologs (NAV1, NAV2, and NAV3) of the neural guidance molecule uncoordinated-53 (UNC-53) in Caenorhabditis elegans. Although they are recognized as neural regulators, their expressions are also detected in many organs, including the heart, kidney, and liver. However, the functions of NAVs, regardless of neural guidance, remain largely unexplored. In our study, we found that nav3 gene was expressed in the cardiac region of zebrafish embryos from 24 to 48 h post-fertilization (hpf) by means of in situ hybridization (ISH) assay. A CRISPR/Cas9-based genome editing method was utilized to delete the nav3 gene in zebrafish and loss of function of Nav3 resulted in a severe deficiency in its cardiac morphology and structure. The similar phenotypic defects of the knockout mutants could recur by nav3 morpholino injection and be rescued by nav3 mRNA injection. Dual-color fluorescence imaging of ventricle and atrium markers further confirmed the disruption of the heart development in nav3-deleted mutants. Although the heart rate was not affected by the deletion of nav3, the heartbeat intensity was decreased in the mutants. All these findings indicate that Nav3 was required for cardiogenesis in developing zebrafish embryos.


Background
Cardiogenesis is an indispensable process in vertebrates during embryogenesis, and the heart plays a vital role in the distribution of nutrients and oxygen in the embryos. Any failures during cardiogenesis will lead to cardiac malformations, further resulting in the death of the embryos. It is challenging to study heart development during embryogenesis. Compared with other mammalian models, such as mice, zebrafish offers several unique advantages for studying cardiac development (Gore et al., 2012;Liu and Stainier, 2012). First, the zebrafish embryos develop externally after fertilization, allowing direct non-invasive observation of heart development during cardiogenesis. Second, the optical transparency of zebrafish embryos allows high-resolution optical imaging. Third, zebrafish embryos can survive up to 7 days without a functional cardiovascular system to supply the nutrients and oxygen due to their tiny size, offering the possibility to analyze the phenotype of the cardiac defects. Besides, essential genes responsible for cardiac development are also highly conserved throughout vertebrates. Therefore, zebrafish is a valuable animal model for exploring the mechanisms underlying cardiac development.
In zebrafish, the formation of a mature heart includes a series of cellular processes, including cell migration, proliferation, and differentiation (Buckingham et al., 2005;Keegan et al., 2004). Numerous genes are involved in embryonic cardiogenesis, such as bmp4 (Jiao et al., 2003), nppa (Grassini et al., 2018), vcana (Mittal et al., 2019), amhc, and vmhc (Berdougo et al., 2003;Yelon et al., 1999), as well as transcription factors nkx2.5 (Targoff et al., 2013), hand2 (Schindler et al., 2014), and GATA family members (Haworth et al., 2008;Holtzinger and Evans, 2007;Reiter et al., 1999). Nav3, the vertebrate homology of C. elegans gene uncoordinated-53 (unc-53), is one of the neural guidance genes, which plays a role in shaping the developing nervous system by modulating/guiding neural cell migration (Maes et al., 2002;Stringham et al., 2002). Moreover, NAV3 is identified as a microtubule-binding protein that regulates actin assembly in extensions of lamellipodia and filopodia . Although nav genes are mainly expressed in the brain, their slight expressions are also detected in the heart, kidney, and liver, suggesting potential roles of NAVs in organogenesis (Maes et al., 2002). In the present study, we focused on the effects of Nav3 on heart development in zebrafish. We found that nav3 was temporally expressed in the cardiac region from 24 h post-fertilization (hpf) to 48 hpf through in situ hybridization (ISH) assay, indicating its function in ventricle-atrium differentiation (Bakkers, 2011).
Deletion of nav3 in zebrafish via CRISPR/Cas9 genome editing technique resulted in severe defects in heart development. Fluorescent imaging of ventricle marker (vmhc) and atrium marker (amhc) further confirmed the malformed morphogenesis of the cardiac structure in nav3-null mutants. In addition to structural alterations, a significant decrease in heartbeat intensity (HI) was also detected in nav3 −/− mutants. Moreover, the phenotypic defects caused by loss of function of Nav3 could be rescued by nav3 mRNA injection. Collectively, our current work revealed a novel role of Nav3 in heart development, which might provide potential strategies for heart disease therapy and cardiac regeneration.

Results
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. 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 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 rates 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 when compared to 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 the "Methods" section. 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 downregulated 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 downregulation 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). Vol.: (0123456789)

Discussion
As a complicated but coordinated biological process, the assembly of an intact heart involves the proliferation, differentiation, and migration of cardiomyocytes to form a functional contractile organ (Buckingham et al., 2005;Keegan et al., 2004;Liu and Stainier, 2012). A cohort of genes participate in this process and play important roles. Nav3, the vertebrate homology of unc-53 in C. elegans, is first discovered as a neural guidance gene that is functional in cell migration and outgrowth of axons. Because cell movement is highly relevant to embryonic organogenesis and NAV3 is also involved in liver development , we hypothesized that NAV3 played a novel role in cardiogenesis. In our present study, we first examined the expression of nav3 in zebrafish during embryogenesis. The specific spatiotemporal expression of nav3 in the zebrafish heart region suggested its potential function in embryonic heart development beyond the nervous system. To confirm our prediction, we generated the nav3-deleted mutants. Loss of function of Nav3 in zebrafish embryos displayed severe heart development defects, such as pericardial edemas and chamber malformations. Moreover, the function of the heart was affected by the deletion of Nav3. For instance, the HI was significantly decreased in mutants. However, the heart rates were similar in both WT and mutant embryos. The functional analysis revealed that loss of Nav3 resulted in an abnormal dilation and contraction in the heart.
To confirm whether the phenotypic defects of the nav3-null mutants were caused by the loss of function of Nav3, a morpholino-mediated gene knockdown experiment and a nav3 mRNA rescue experiment were performed. The pericardial edemas and heart morphogenesis malformations were reproduced in nav3-deleted mutants and could also be partially rescued by co-injection of morpholino and nav3 mRNA. Additionally, the mRNA rescue experiment also recapitulated that the severe cardiac edema was not caused by the off-target effects triggered by sgRNA or morpholino injection.
In our present work, we also noticed that the survival rates of nav3-deleted mutants were significantly lower compared with their WT counterparts, especially from the very early developmental stage (from 24 hpf). Since zebrafish could survive up to 7 days without a functional cardiovascular system, the high mortality upon Nav3 deletion might not result from the cardiac defects and more probably be attributed to its dysfunction in the nervous system or immunity (Carlsson et al., 2013;Karenko et al., 2005).
As mentioned above, Nav3 loss of function could lead to severe defects in zebrafish heart development. However, it remains largely unknown whether some Fig. 4 Nav3 mRNA injection rescues the cardiac defects caused by nav3 deletion. Transgenic zebrafish line Tg(myl7:mCherry) embryos injected with nav3 morpholino was compared with the control. Co-injection of nav3 morpholino and nav3 mRNA partially rescued the defects in cardiac chamber morphogenesis. The ventricle and atrium are outlined with red and blue dotted lines, respectively Fish Physiol Biochem (2022) 48:173-183 179 essential cardiac-related genes are altered in nav3-null mutants. In this work, we examined the expressions of nkx2.5, hand2, vmhc, and amhc in nav3 −/− mutants and WT zebrafish embryos using ISH. We found that the expressions of nkx2.5 and hand2 were slightly decreased after the deletion of nav3 (Supplemental Fig. S1). Although they both play roles in embryonic heart development, each of them also owns a distinct and specific function in regulating this process. Nkx2.5 determines the cardiomyocyte identity and can further maintain the vascular and atrial chamber morphology (Targoff et al., 2013). Hand2 is implicated as a regulator of cardiomyocyte production, which promotes the generation of a proper number of cardiomyocytes during cardiogenesis (Schindler et al., 2014). As key regulators in heart development, the lower expressions of nkx2.5 and hand2 in nav3 −/− mutants supported its deficiency in cardiac development. This finding was consistent with the previous view that nkx2.5 maintains cardiac chamber identity, and hand2 regulates cardiac differentiation and morphogenesis (Schindler et al., 2014;Targoff et al., 2013). Although the structure and morphology of the atrium and ventricle were severely affected by the loss of nav3, the expressions of both their markers remained unchanged (Supplemental Fig. S1). As the expressions of vmhc and amhc are regulated by upstream transcription factors (Bakkers, 2011;Zhang and Xu, 2009), we assumed that the heart morphogenesis defects might result from these factors, like nkx2.5 or hand2.
Although nav3 is initially discovered as a neural guidance gene in the nervous system, the NAV family members have also been widely accepted as regulators in cell migration among several species (Cohen-Dvashi et al., 2015;Klein et al., 2011;Maes et al., 2002;Schmidt et al., 2009;Stringham et al., 2002). Moreover, it has been reported to act as a positive modulator in regulating the actin assembly in the extension of filopodia and lamellipodia during zebrafish liver development . As a complicated but orchestrated morphogenetic process, heart formation involves specification, differentiation, and migration of cardiac progenitor cells. Therefore, NAV3 might also play a role in heart development by facilitating the migration of cardiac progenitor cells or cardiomyocytes. Besides, crosstalk between NAV3 and other essential factors, such as nkx2.5, might be involved in these crucial processes during cardiac development. Further efforts are needed to decipher the mechanism of NAV3-regulated cardiogenesis. Taken together, NAV3 was a potential regulator in cardiac development during embryogenesis.

Conclusions
The nav3 gene can be expressed in a variety of tissues or organs although it is initially identified as a neural guiding factor. Gene knockout of nav3 in zebrafish leads to severe development malformation of the heart. Moreover, the defective heart development can be rescued by nav3 mRNA injection. All these findings indicate that Nav3 was required for cardiogenesis in the development of zebrafish embryos.

Zebrafish husbandry and strains
The zebrafish (TU line) in this study was obtained from China Zebrafish Resource Center and maintained in Jiangsu Key Laboratory of Neuroregeneration of Nantong University. The transgenic zebrafish lines Tg(vmhc:mCherry::amhc:EGFP) and Tg(myl7:mCherry) were kindly provided by Dr. R. Zhang and Dr. T. Zhong,respectively. Tg(mef2a:EGFP) transgenic line was constructed by our lab previously (Lv et al., 2017).

Whole-mount in situ hybridization (WISH)
WISH with antisense RNA probes was performed as previously described . The probe for the detection of NAV3 (NM_001045143.2) was cloned from cDNA fragments. The nav3 cDNA fragment was amplified with a forward primer: 5′-TCA CCT TCT GAC TCC ACC AG-3′ and a reverse primer: 5′-GTT CGA TGT TAC GCG CTC AC-3′. After hybridization, images 1 3 Vol.: (0123456789) of the embryos were acquired with an Olympus stereomicroscope MVX10 equipped with an Olympus DP71 camera.

Generation of nav3-null mutants
A CRISPR/Cas9-mediated approach was used to generate nav3-deleted mutants. The target site of CRISPR/Cas9 designed to identify the sequence in the second exon of nav3 was 5′-CAT ACC CGA TGG AGT TCT GCTGG -3′ (underlined was the PAM sequence). The template sequence of sgRNA for in vitro transcription was 5′-TAA TAC GAC TCA CTA TAG CAT ACC CGA TGG AGT TCT GCG TTT TAG AGC TAG AAA TAG CAA GTT AAA ATA AGG CTA GTC CGT TAT CAA CTT GAA AAA GTG GCA CCG AGT CGG TGC TTT TTT T-3′. For the sgRNA synthesis, a forward primer (5′-TAA TAC GAC TCA CTA TAG CAT ACC CGA TGG AGT TCT GCG TTT TAG AGC TAG AAA TAGC-3′) containing a T7 promoter region and a nav3 gene-targeting region and a universal reverse primer (5′-AAA AAA AGC ACC GAC TCG GTG CCA C-3′) were used in the PCR amplification with pT7 plasmid as the template to obtain the sgDNA, which was then transcribed into sgRNA in vitro using the T7 mMessage mMachine kit (Ambion). Micro-injection was performed with 1-cell stage zebrafish embryos, and each embryo was co-injected with 100 pg sgRNA and 200 pg Cas9 mRNA. G0 generations were examined by PCR, followed by Sanger sequencing. A mutant with a 55-bp deletion at the second exon was identified, leading to the occurrence of a stop codon downstream of the Cas9 cutting site. The heterozygous nav3 ± mutants were incrossed to obtain the homozygous F2 progenies.

Morpholino-mediated gene deletion of nav3 in zebrafish
The nav3 gene-specific morpholino (Gene Tools, LLC) was used to block the splicing of nav3 pre-mRNA. The sequence of the morpholino antisense oligomer was 5′-CAG CCC ATG TGC CAT GCT GCT TCT G-3′. In the present study, 2 nL of 0.3 mM morpholino oligo was micro-injected into the embryos at the 1-cell stage.

mRNA rescue experiment
In the rescue experiment, 200 pg nav3 mRNA was co-injected into the embryos with the nav3 antisense morpholino. The cDNA fragment of nav3 was first subcloned into pCS2 + vector. To make the mRNA, the DNA plasmid was linearized with an appropriate restriction enzyme and transcribed in vitro using the SP6 mMessage mMachine kit (Ambion).

Survival rate quantification
Zebrafish embryos of WT and nav3-null mutants were respectively collected after fertilization and divided into 10 tanks (100 embryos in each tank). The counts of surviving fishes per tank were determined at 1, 2, 3, 20, and 60 days post-fertilization (dpf). Afterward, the proportions of fish surviving at each time point were assessed.
The heart rate of the embryo was defined as the beats per minute and counted under a dissecting microscope (Olympus). To examine the HI, the cardiac-labeled transgenic zebrafish lines Tg(mef2a:EGFP) and Tg(nav3 − / − -mef2a:EGFP) were used to visualize the hearts of WT and mutants, respectively. The cross-sectional areas of the chambers in GFP-labeled hearts in the dilated state (A d ) and contracted state (A c ) were respectively measured with ImageJ (Rueden et al., 2017). The HI was calculated by the equation as follows, HI = A d -A c . In our present analysis, five WT and mutant fishes were respectively selected, and each fish was measured for 20 successive heartbeats.

Data analysis
A two-tailed, unpaired Student's t-test was used to determine statistical significance when comparing Fish Physiol Biochem (2022) 48:173-183 independent groups. p < 0.05 was considered statistically significant. All results were presented as mean ± SD.
Author contribution CC and DL designed and conceptualized the project. FL, XG, PQ, and XL performed the experiment. FL and CC analyzed the data. CC wrote the manuscript. All authors read and approved the final manuscript.
Funding The research conducted in this manuscript was funded by the National Natural Science Foundation of China (82000458) to CC and start-up funding for Doctoral Research of Nantong Science and Technology College (NTKY-Dr2017001) to FL. It is also funded by Nantong Science and Technology Program (JC2021088), Open Program of Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province (2019fjsccq08), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (20KJB180008), and the Science Foundation of Nantong City (JC2020023).
Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files.
Code availability Not applicable.

Declarations
Ethics approval All animal-related experiments were carried out following the NIH Guidelines for the care and use of laboratory animals (http:// oacu. od. nih. gov/ regs/ index. htm), and animal protocols were ethically approved by the Administration Committee of Experimental Animals of Nantong University, Jiangsu Province, China (Approval ID: 20180608-Z001). Transgenic zebrafish lines provided by others were approved by the owners with written informed consent.

Competing interests
The authors declare no competing interests.

Disclaimer
The funding sources provided financial support for the experiments described, but had no role in the design of the study or collection, analysis, and interpretation of data or in writing the manuscript.