Journal of

Developmental
Biology
Review

Advances in Cardiac Development and Regeneration Using
Zebrafish as a Model System for High-Throughput Research
Nicholas Francoeur † and Rwik Sen *,†Active Motif, Incorporated, 1914 Palomar Oaks Way, Suite 150, Carlsbad, CA 92008, USA;nfrancoeur@activemotif.com* Correspondence: rsen@activemotif.com† These authors contributed equally to this work.

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Citation: Francoeur, N.; Sen, R.
Advances in Cardiac Development
and Regeneration Using Zebrafish asa Model System for High-Throughput

Abstract: Heart disease is the leading cause of death in the United States and worldwide. Understanding the molecular mechanisms of cardiac development and regeneration will improve
diagnostic and therapeutic interventions against heart disease. In this direction, zebrafish is an
excellent model because several processes of zebrafish heart development are largely conservedin humans, and zebrafish has several advantages as a model organism. Zebrafish transcriptomic
profiles undergo alterations during different stages of cardiac development and regeneration which
are revealed by RNA-sequencing. ChIP-sequencing has detected genome-wide occupancy of histone post-translational modifications that epigenetically regulate gene expression and identified
a locus with enhancer-like characteristics. ATAC-sequencing has identified active enhancers in
cardiac progenitor cells during early developmental stages which overlap with occupancy of histone
modifications of active transcription as determined by ChIP-sequencing. CRISPR-mediated editingof the zebrafish genome shows how chromatin modifiers and DNA-binding proteins regulate heartdevelopment, in association with crucial signaling pathways. Hence, more studies in this direction
are essential to improve human health because they answer fundamental questions on cardiac development and regeneration, their differences, and why zebrafish hearts regenerate upon injury, unlike
humans. This review focuses on some of the latest studies using state-of-the-art technology enabledby the elegant yet simple zebrafish.

Research. J. Dev. Biol. 2021, 9, 40.
https://doi.org/10.3390/jdb9040040

Keywords: cardiac development; regeneration; epigenetics; transgenic reporter; transcriptome;
epigenetic; ChIP-seq; ATAC-seq; CRISPR; high-throughput

Academic Editors: Lisa Maves andSimon J. Conway
Received: 1 September 2021
Accepted: 22 September 2021
Published: 25 September 2021

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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Zebrafish is an excellent model system for research on developmental biology, andthis model has been used in highly impactful studies that have defined the field. The
earliest record of zebrafish development research dates back to 1948, when Battle andHiasoka investigated the effect of a carcinogen on zebrafish development to understandits impact on differentiation and organization during embryogenesis [1]. In one of theearliest studies on developmental genetics using zebrafish, Streisinger et al. presented
the production of homozygous diploid zebrafish clones, which laid the foundation forgenerating clonal lines and genetic manipulations in developmental research [2]. The study
focused on treatments of zebrafish eggs by late and early pressure as well as heat shock,and on treatment of zebrafish sperms by irradiation. In 1995, a study by Kimmel et al. [3]focused on developmental staging and cell lineage mapping in zebrafish, providing thefirst detailed documentation of the zebrafish development from zygote period at 0 hours
to protruding mouth at 72 hours post fertilization (hpf) [3]. Several other seminal studies
focused on genetics and mutations in zebrafish [4–6] with a focus on the development ofspecific organs such as the brain [7], heart [8], digestive tract [9], et cetera.
Over the years, there has been a widespread increase in the use of zebrafish as apopular model for research on developmental biology. This popularity can largely be

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attributed to the presence of human gene orthologs in zebrafish, as well as an abundanceof samples due to their large brood sizes and more accelerated life cycle than that of
mammals [10,11]. Zebrafish are also amenable to sophisticated microscopy and imaging
procedures. Because zebrafish undergo external fertilization and are transparent during
early developmental stages, zebrafish growth during specific developmental windows canbe monitored in real-time. Zebrafish are likewise advantageous for forward and reverse
genetics studies [12], because it is convenient to employ sophisticated techniques to create
mutations [13] and transgenic reporter lines [10,14,15]. Elegant mutational techniques suchas CRISPR are also easy to employ.
In addition to developmental research, the zebrafish model is extremely popular in
the field of regeneration, as the zebrafish can regrow almost any part of its body. Several
studies have focused on the regenerative potential of zebrafish to address similar questions
in humans, where regeneration is not as widespread. Regeneration in the context ofzebrafish, or teleosts which are fish with fully movable maxilla and premaxilla, was first
documented 235 years ago [16]. Over the last several decades, many studies have provenzebrafish to be an excellent model for studying regeneration, especially in relation to
cardiac biology [10,17–23].
Overall, zebrafish provide an excellent model for studying phenotypes and molecularmechanisms associated with development and disease, pharmacological treatments pertaining to drug discovery and environmental research, etc., and genetic manipulations [24–26].
With increasing focus on genome-wide profiling of transcriptomic and epigenomic landscapes, studies on heart development and regeneration in zebrafish have started employing
high-throughput techniques using next-generation sequencing to address the above questions. The zebrafish model has proven to be highly compatible with the new technology, as
the large quantity of samples required can be obtained from zebrafish in a very short spanof time.
As heart diseases are the primary cause of human mortality in the US according to the
CDC (Center for Disease Control and Prevention), future studies of cardiac development
and regeneration in any species with associated orthologs will be invaluable to publichealth. With continuing development and refinement of high-throughput techniques fortranscriptomic and epigenomic profiling, animal models compatible with these efforts willbe critical in the advancement of basic research. In this review, we explore the specificcontributions of the zebrafish model to cardiac development and regeneration research
involving state-of-the-art technology for high-throughput transcriptomic and epigenomic
profiling.
2. Zebrafish Heart Development
For a better context of recent studies using a high-throughput methodology on zebrafish cardiac development and regeneration, an overview of zebrafish heart development
is provided, but in brief, because several impactful studies and reviews have alreadydescribed the process in detail [27–32]. Development of the heart occurs earliest among
all other functional organs in zebrafish when the cardiac crescent begins to form with
contributions from cardiogenic progenitor cells post-gastrulation [33]. Subsequently, thecardiogenic mesoderm gives rise to the first heart field (FHF) and second heart field(SHF) [33–35]. The FHF contributes to the primary heart tube, left ventricle, and most of
the atria, while SHF contributes to the right ventricle, outflow tract, and atria [33,36,37].
Orchestrated signals from mesoderm-derived FHF and SHF, as well as endoderm, arecrucial for cardiogenesis [33]. Broadly, zebrafish heart development comprises of specification and differentiation of myocardial progenitor cells, migration and fusion of bilateralheart fields, and development of endocardium, myocardial tube, cardiac looping, valves,
conduction system, and epicardium [23]. Studies on gene regulatory networks for thedevelopment of the heart in zebrafish show a complex involvement of multiple genes in
the process [6,38–40].

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Zebrafish have a 2-chambered heart with a single circulation system, whereas humans
have a 4-chambered heart and double circulation system. Despite lacking in those complexities, the zebrafish heart is capable of regeneration upon injury, unlike human hearts.
In zebrafish, blood flows into a single atrium, which is connected to a single ventricle
by an atrio-ventricular valve [41]. The ventricle pumps blood into circulation through aprominent outflow tract called the bulbus arteriosus [10].
The absence of cardiac regeneration in humans has focused the attention of many
researchers towards non-mammalian species [10,42,43]. Several studies have established agreat efficiency among zebrafish in their capacity for cardiac regeneration throughout theirlife which can be harnessed to develop novel insights in investigating similar processes inhumans [44,45]. Despite extensive injuries, zebrafish can regenerate a completely functional
heart [46–48]. To date, zebrafish have demonstrated an extraordinarily robust response
to cardiac damage compared to other vertebrates, as zebrafish can regenerate a complete
heart even after the amputation of up to approximately 20% of the ventricle [11]. Althoughprofuse bleeding occurs upon cardiac resection, a fibrin clot is quickly generated which
stops exsanguination of the injured zebrafish [10,11]. In subsequent weeks, new muscle
is observed to replace the fibrin clot with nearly total ventricular recovery between 1 to
2 months post-injury [10,11,49].
Interestingly, studies have reported that cells which contribute to cardiac repair andregeneration are different from cells that participate in processes of original cardiac development during embryogenesis [21] (Figure 1). Lineage tracing studies on cells have shownthat cells from the myocardium, endocardium, etc., contribute to cardiac regeneration, but
the process and underlying mechanisms are not clearly understood. However, studies haveindicated that several genes regulate both cardiac development and regeneration througha tight orchestration of temporal and spatial regulation. In this direction, high-throughput
studies investigating transcriptomic and epigenomic profiles during cardiac development
4 of processes.
16
and regeneration have the potential to provide novel insights into theseThe
discoveries will in turn improve our understanding of related processes in humans fortherapeutic developments against congenital heart defects and cardiac injuries.

Figure 1. Overview
differencesofbetween
cardiacdevelopment
and regenerationin zebrafish.inUpperand lower
Figure of
1. Overview
differences
betweencardiac development
and regeneration
zebrafish.
Up- panels
outline development
and regeneration,
FHF and SHF—1stand 2ndheart
fields,
LEN—lepb-linked
per and lower
panels outlinerespectively.
developmentKeys:and regeneration,
respectively.
Keys:
FHF
and SHF—1stand 2nd heartenhancer, :—resulting
in. fields, LEN—lepb-linked enhancer, ➔—resulting in.

3. Zebrafish Supports State-of-the-Art High-Throughput Cardiac Research
3.1. Epigenomic Mapping of Zebrafish Reveals New Insights into Cardiac Development
Zebrafish epigenomic profiling was performed by Yuan et al., leading to the discovery of 5598 open chromatin regions that are conserved between zebrafish and humans,
which indicates a diverse collection of ancient enhancers being established before organ
development [50]. Genome-wide chromatin regions which are accessible or open to transcription were mapped using Assay for Transposase-Accessible Chromatin using se-

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3. Zebrafish Supports State-of-the-Art High-Throughput Cardiac Research
3.1. Epigenomic Mapping of Zebrafish Reveals New Insights into Cardiac Development
Zebrafish epigenomic profiling was performed by Yuan et al., leading to the discoveryof 5598 open chromatin regions that are conserved between zebrafish and humans, whichindicates a diverse collection of ancient enhancers being established before organ development [50]. Genome-wide chromatin regions which are accessible or open to transcription
were mapped using Assay for Transposase-Accessible Chromatin using sequencing (ATACseq) [51,52]. The study enabled Yuan et al. to identify enhancers in cardiac progenitor cells
which become activated or expressed earlier than Nkx2.5, the cardiac progenitor marker
whose mutations cause congenital heart disease in humans [53–55].
In this direction, Yuan et al. [50] engineered a green fluorescent protein (GFP) reporterline of zebrafish which is driven by murine Smarcd3 enhancer (Smarcd3-F6), because
its activity is detected in early gastrulating mouse mesoderm [56–58]. Smarcd3 is also
known as Baf60c in humans, a component of the Notch signaling pathway, which regulates
cardiac looping morphology or left-right asymmetry [59], remodels chromatin to regulate
gene expression for heart development and function [60,61], and determines the fate ofcardiomyocyte cells [62]. Moreover, mutations in human NOTCH1 result in aortic valve
anomalies, such as severe calcification and stenosis, bicuspid aortic valve, ascending
aortic aneurysm, atresia of the mitral valve, hypoplastic left ventricle, double-outlet rightventricle [63], and left ventricular noncompaction [64]. Mutations in the Notch ligand in
humans, JAG1, are associated with Alagille syndrome, which contributes to several heart
disorders [65,66].
In the study by Yuan et al., GPF-positive and GFP-negative cells were obtained from10 hpf (hours post fertilization) zebrafish and subjected to ATAC-seq to detect enhancers
that are active at a very early stage of development [50]. The results showed that the
GFP-positive cells contain 155,879 ATAC-seq peaks which correlate to regions of open oraccessible chromatin. The GFP-negative cells showed 153,777 peaks. Overall, significant
quantitative differences were seen in 5471 peaks, among which 3838 were increased in
GFP-positive cells and 1633 were increased in GFP-negative cells [50]. The ATAC-seq peaks
showed significant overlap with histone modifications of H3K4me3 and H3K27ac, whichmark active chromatin at promoters and enhancers, respectively. Prior ChIP-seq (chromatin immunoprecipitation) on zebrafish at similar developmental stages have validated
H3K4me3 and H3K27ac as open chromatin marks [67].
Yuan et al. analyzed the ATAC-seq results using the Genomic Regions Enrichmentof Annotations Tool (GREAT), which was previously developed to study the functionalsignificance of cis-regulatory regions detected by localized quantitation of genome-wide
DNA binding events [68]. GREAT showed that regions of open chromatin in the GFPpositive cells are enriched for processes related to heart development, including heartmorphogenesis and embryonic heart tube formation. The distance of the regions of open
chromatin from the genes of interest was also detected. In summary, the profiles ofaccessible chromatin that are enriched in GFP-positive and -negative 10 hpf zebrafish
suggest that cardiac progenitor cells are marked by the Smarcd3-F6 enhancer.
3.2. Transcriptomic and Chromatin Occupancy Profiling Zebrafish Reveals New Insights into
Cardiac Regeneration
Enhancers are regions of accessible chromatin that are bound by transcription factors
and histone modifications that activate transcription, e.g., histone H3 lysine (K) 27 acetylation (ac) [69,70]. Kang et al. investigated enhancer regulatory elements involved in tissue regeneration, using techniques including RNA-sequencing and ChIP-sequencing [71].
RNA-sequencing is a technique for quantification of all RNA transcripts and their sequence
analysis [72]. ChIP-seq is a technique to map the genome-wide occupancy of proteins ofinterest and analysis of sequences where the proteins are localized [73].
Kang et al. performed transcriptomic analysis to screen for genes that are induced during regeneration in zebrafish. RNA was extracted from uninjured and injured fin and heart

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tissue of zebrafish, and subjected to library preparation, sequencing, and bioinformatics
analysis. One of the genes that was found to be drastically upregulated upon transcriptomic
analysis of regenerating caudal fins and cardiac tissue was the energy homeostasis regulator leptin b (lepb) [71]. Interestingly, the expression of leptin is upregulated in the failinghuman heart, suggesting a cardioprotective role [74]. Leptin has been further implicated inheart failure with preserved ejection fraction and left-ventricular hypertrophy [75,76], riskof coronary heart disease and stroke [77], and obesity-associated impairment of cardiac
function and CVD [78,79].
Kang et al. [71] observed in zebrafish that partial resection-induced local injury of
the cardiac ventricle resulted in lepb expression in the endocardium, which forms theendothelial lining of inner myofibers and participates in regeneration [80,81].
To detect enhancers that regulate cardiac regeneration and reside in the proximity oflepb, ChIP-seq for H3K27ac was performed in uninjured and injured regenerating zebrafish
cardiac tissue. Chromatin from injured and uninjured ventricles was subjected to sonication,
followed by immunoprecipitation with antibodies against H3K27ac, library preparation,
sequencing, and bioinformatics analysis. Chromatin from the regenerating samples showed
an enrichment of H3K27ac in two regions, 7 kb and 3 kb, upstream of lepb start codon,
which was absent in the uninjured samples.
Further analysis revealed the region at 7 kb to be an enhancer because it showedstrong blastemal expression upon fin amputation and strong fluorescence expression in theendocardium upon cardiac injury. Hence, ChIP-seq identified a short loci 7 kb upstreamof lepb start codon as an enhancer of gene expression in adult tissues during regeneration.
Further analysis of the ChIP-seq results revealed a short intergenic element called lepblinked enhancer (LEN) which regulates regeneration-activated transcription from multiple
promoters. Enrichment of H3K27ac during regeneration was also detected at several other
1–2 kb intergenic regions. Fins and hearts of transgenic zebrafish with cmlc2 (marker ofdifferentiated cardiomyocytes) or α-cry (lens marker) promoters also displayed robust
fluorescence expression during regeneration, which indicates that these promoters havea role in the regeneration process. cmlc2 is also known as mlc7 or myl7, and it displayed
endogenous post-transcriptional down-regulation in murine models of Down’s syndrome,
which implicates this gene in human congenital heart defects that are detected in a majority
of Down’s syndrome patients [82,83]. MYL7 is also investigated in studies on cardiac
biology using human pluripotent stem cells [84,85], cardiac regeneration in zebrafish [86],and cardiac failure [87].
3.3. CRISPR-Mediated Disruption of an Epigenetic Factor and Transcriptomic Analysis Reveal
How a Signaling Pathway for Cardiac Development Is Impacted
RNA-sequencing of zebrafish has been extended to understanding the role of epigenetic factors in cardiac development. Epigenetic factors such as KMT2D and KDM6A are
mutated in humans with Kabuki Syndrome, where a varying percentage of patients show
cardiac defects [88]. Cardiac defects seen in Kabuki Syndrome patients include left-sided
obstructions/aortic dilation, septation defects, coarctation of the aorta, etc. [89]. Studies
have shown that disruptions of the zebrafish orthologs, kmt2d and kdm6a, recapitulate
phenotypes of Kabuki Syndrome [88,90]. In this direction, Serrano et al. disrupted kmt2d inzebrafish by the elegant gene editing technology called CRISPR [91]. Next, they performed
RNA-seq on single 1-day post-fertilization (dpf) zebrafish embryos with homozygous
mutations in kmt2d and corresponding wild-types [90]. RNA was extracted, libraries wereprepared and sequenced, followed by bioinformatics analysis. The timeline of 1 dpf waschosen so that alterations in pathways resulting in Kabuki Syndrome in kmt2d mutants canbe detected immediately before the earliest phenotypes appear, because transcriptomic
information at 1 dpf is not yet confounded by any of its own downstream effects.
The results showed differential expression of 276 genes on the mutants. Further analysis revealed that 72.1% of the top 50 genes are implicated in neural and/or cardiovascularsystems, while the remainder are linked to reproductive system, muscle, and pharyngeal
arches development [90]. For example, three genes detected in the analysis, krt18, bgnb,

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and rbp7b, are associated with human cardiac disorders. A study on humans and miceindicated Krt18 as a cardioprotective factor induced by stress in failing hearts [92]. It
is regulated by NHLRC2, whose mutations in humans cause severe fibrosis in tissues
including the heart [93,94]. Fibrosis leads to loss and death of cardiomyocytes, leading toheart failure [95]. Loss-of-function mutations in BGN show a severe syndromic form ofthoracic aortic aneurysms and dissections in humans [96]. Rbp7 is expressed in the heart
endothelium and other tissues [97], and likely regulates the antioxidant properties of theendothelium in association with PPARγ [98].
A small subset of genes appeared to be enriched during gene set enrichment analysis(GSEA), where one of the associations of each set was found to be structural extracellularmatrix (ECM) glycoproteins. The observations indicate crucial variations in ECM content
or topography prior to the onset of Kabuki Syndrome phenotypes. Interestingly, ECM is
implicated in cardiac development, function, failure, and diseases [99–102].
The kmt2d mutants also showed an upregulation in her4.4, a specific downstream
target of Notch, a signaling pathway conserved in metazoans that plays crucial roles
in development including that of the zebrafish heart [49,90,103–106]. The observationis consistent with their finding of hyperactivation of Notch signaling in kmt2d mutantsin the same study [90]. The transcriptomic profiles observed in 1 dpf zebrafish with
kmt2d mutation support the mutant phenotypes observed at 3 dpf in this study. Suchhigh-throughput transcriptomic analysis provides an excellent basis for discoveries ondevelopment and disease using zebrafish.
It is interesting to note that in addition to RNA-seq, the above study also employed
CRISPR, adding to the growing popularity of zebrafish as a subject for CRISPR-mediatedgene editing. Another study used CRISPR in a pioneering approach towards precise gene
editing in zebrafish, to reveal cardiac developmental roles of a DNA-binding factor called
pbx3 [107], whose single nucleotide variant is associated with human disease. A cohort ofpatients with a mutation in PBX3 showed a range of phenotypes for congenital heart defects
including outflow tract malformations such as tetralogy of Fallot, atrioventricular septal defect,
persistent truncus arteriosus, bicuspid aortic valve, coarctation of the aorta, and hypoplastic
left heart syndrome [108]. The zebrafish study on pbx3 identified its potential new role asa modifier in congenital heart defects [107]. Severe cardiac phenotypes were also observed
upon CRISPR-mediated mutation of its family member, pbx4, which is known to regulate
myocardial differentiation [109]. Patients with a mutation in PBX4 showed an atrioventricularseptal defect [108]. CRISPR-mediated gene editing of zebrafish extends to developmental
studies beyond the heart, e.g., eye [110], craniofacial [111,112], muscle [113], etc.
3.4. CRISPR-Mediated Disruption of a Chromatin Modifier and Transcriptomic Analysis Reveal
New Insights into Multiple Signaling and Biological Pathways Regulating Cardiac Development
Xiao et al. performed CRISPR-mediated disruption of another histone methyltransferase called smyd4 in zebrafish, which disrupted histone modifications and caused severe
cardiac defects [114]. smyd4 is one of the five members of the Smyd family of genes
which are lysine methyltransferases. Significant homology is maintained among all family
members of Smyd proteins, from fish to humans [115]. Studies have implicated various
members of the Smyd family in heart development and disease in different species. One
member, Smyd1, shows a high upregulation in endogenous expression during human heart
failure [115–118], which underscores the significance of the study by Xiao et al.
However, the roles of Smyd family members in adult heart development are notwell-explored. In terms of genetic conservation for smyd1, it is known that zebrafish havesmyd1a and smyd1b genes which could have resulted from whole-genome duplication inteleosts, mice have Smyd1a, Smyd1b, and Smyd1c, while humans have a single transcriptfrom the SMYD1 gene [115,119]. Other family members such as smyd3 show modulation of
mesodermal commitment during development in zebrafish and mice [120], while smyd5
regulates hematopoiesis during zebrafish embryogenesis [121].
Xiao et al. used a CRISPR mutant of smyd4 in a transgenic zebrafish line wherecardiomyocyte marker cmlc2 is labeled with green fluorescence protein. Transcriptomes of

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zebrafish hearts were analyzed to study how smyd4 affects cardiac development. Zebrafish
hearts were excised at 72 hpf based on the expression of green fluorescence, and RNA-seq
was performed.
Differential expression of 3856 genes was observed in the mutant hearts, with upregulation in 2648 genes and downregulation in 1208 genes. Upregulation was seen in 10
genes associated with cardiac muscle contraction, as well as 36 and 15 genes associated with
important cardiac signaling pathways such as canonical Wnt and Hedgehog, respectively.
Downregulation was seen in 22, 10, and 3 genes which are respectively associated with cardiac
muscle contraction, Wnt, and Hedgehog signaling pathways [114]. It is important to notethat cardiac development and disease are associated with both Hedgehog [122–124] and Wnt
signaling [125–127], with the reactivation of fetal non-canonical WNT gene program in humanright ventricular failure [128].
Further analysis was performed to detect if the alterations in the transcriptome werelinked to specific biological pathways. Analysis of KEGG (Kyoto Encyclopedia of Genes
and Genomes) pathways indicated an enrichment of upregulated genes in the protein
processing pathway of the endoplasmic reticulum in the Biological Processes domain [114].
Enrichment of downregulated genes was observed in the carbon metabolism and glycolysis/gluconeogenesis pathways. Enrichment of 975 genes in cellular metabolic processes
was detected upon gene ontology analysis of upregulated genes.
Analysis of downregulated genes revealed enrichment of 185 genes in organonitrogencompound metabolic processes, 49 genes in ATP metabolic processes, and 55 genes inglycosyl compound metabolic processes. Overall, the results indicate that smyd4 regulates epigenetic processes, which, as detected by the above analysis, likely results froman orchestration of the pathways involved in zebrafish cardiac development [114]. Aninteresting observation highlighted in this study is that the transcriptomic analysis of wildtype versus smyd4-mutant hearts showed differential expression of more than 3000 genes,
but less than 100 genes among them have known roles in cardiac muscle contraction andcardiac signaling pathways, such as canonical Wnt and Hedgehog signaling. Instead,the analysis showed that several pathways of cellular metabolism were overwhelminglyenriched [114]. The observation steers our attention towards investigating links between
metabolism and cardiac development in zebrafish, which are currently not well understood. The study further mentions that smyd4 likely regulated cellular metabolism as oneof its unique and specific biological functions [114]. Recent studies on the zebrafish heart
indicate that metabolism regulates physiological phenomenon, including cell proliferationand differentiation, which crucially impact development and regeneration [129–133], andthat antioxidants can be potential therapies against cardiac arrhythmia [134].
3.5. Lineage Tracing and Transcriptomic Analyses of Transgenic Reporter Zebrafish Reveals theRoles of a Distinct Subpopulation of Cells in Cardiac Regeneration
As mentioned earlier, the zebrafish model is also advantageous for research on cardiac
regeneration using high-throughput methods involving next-generation sequencing. A
study on cardiac regeneration by Sande-Melón et al. employed genetic fate mapping,transgenic reporter zebrafish lines, and RNA-seq to discover the role of a subset of cardiomyocytes in heart regeneration [135]. Sande-Melón et al. analyzed transgenic zebrafish
and revealed that cardiomyocytes derived from sox10 have distinct transcriptomic profiles
compared to other cardiomyocytes. sox10 is a transcription factor that crucially regulates
the maturation of neural crest cells (NCCs) which are a population of precursors withstem cell-like properties originating in vertebrate embryos [136–138]. In humans, SOX10
mutation impacts autonomic control of the heart dynamics [139], which indicates its role inheart innervation [140].
The study used hearts from adult transgenic zebrafish sox10:CreERT2 ;vmhcl:loxPtagBFP-loxP-mCherry-NTR, which were subjected to cryoinjury or no injury. The rationale
behind this transgenic line is explained below. Cre (causes recombination) is an enzymethat is classified as a recombinase or Type I topoisomerase and is a product of the bacteriophage P1 gene [141]. Cre causes site-specific recombination between loxP (locus of

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8 of 15crossing (x) over, P1) sites in the genome [141]. Tamoxifen induces Cre/loxP recombination at embryonic stages in the sox10:ERT2 -Cre zebrafish line as previously reported [142].
Hence, it is an efficient transgenic model to label NCCs using tamoxifen-induced Cre/loxP
recombination, which helps to identify many established derivatives of NCCs in juvenile
and adult zebrafish [142]. The recombination is induced upon treatment with an active
metabolite of tamoxifen (4-OHT) that binds to estrogen receptors.
Other components of the transgene, such as vmhcl stands for ventricular myosin heavy
chain-like; while NTR denotes nitroreductase; and vmhcl:loxP-tagBFP-loxP-mCherry-NTR(vmBRN) is known for specific tracing of vmhcl-expressing ventricular cardiomyocytesthat emit red fluorescence after recombination [143]. It is interesting to note that zebrafish
vmhcl is also known as myh7l, whose human ortholog is unknown, but human MHY7 isassociated with the heart. Mutations in MYH7 are implicated in atrial fibrillation, which iscommon among hypertrophic cardiomyopathy patients [144], and MYH7B encodes a longnon-coding RNA that significantly regulates the biology of cardiomyocytes [145].
In the transgenic zebrafish line used by Sande-Melón et al., the recombination inducedby 4-OHT causes sox10-derived cardiomyocytes to express mCherry, or red fluorescence,
while other cardiomyocytes express blue fluorescence. 4-OHT treatment for recombination
was performed at different time points before cryoinjury, followed by harvesting the
cardiac tissue at 7 days post-injury for transcriptomic comparison between injured hearts
and uninjured hearts used as a control. Ventricles were obtained from uninjured andinjured hearts which were subdivided into several pools followed by fluorescence-activatedcell (FAC) sorting of 20 cardiomyocytes from each pool. RNA libraries were prepared,
sequenced, and bioinformatics analysis was performed.
Cardiomyocytes derived from sox10 or expressing red fluorescence in the uninjured
ventricles showed upregulation of 101 genes and downregulation of 129 genes compared to
the remaining cardiomyocytes [135]. Uninjured hearts showed metabolic contrasts between
transcriptional profiles of sox10-derived cardiomyocytes and the remaining population
upon gene enrichment analysis. Pathways diagnosed with differences include oxidative
phosphorylation and nucleic acid metabolism [135].
In the injured hearts, sox10-derived cardiomyocytes showed higher transcriptional activity, with an upregulation of 415 genes compared to 30 genes in other cardiomyocytes. The
injured hearts also showed a significant upregulation of sox10 mRNA itself in sox10-derived
cardiomyocytes. The observation verifies that cells expressing sox10 can be endogenously
traced using the sox10:ERT2 line.
sox10-derived cardiomyocytes showed an upregulation of T-box transcription factors,
tbx20 and tbx5a, which are expressed in cardiomyocytes participating in cardiac regeneration [143] and actively regulate the regeneration of injured cardiac tissue [146,147]. In
humans, TBX20 mutations are associated with a spectrum of congenital heart defects including abnormalities in valvulogenesis, chamber septation, and growth [148]. Mutations
in TBX5 cause Holt–Oram syndrome, which includes cardiac defects in the conduction
system, atrial and ventricular septation, and tetralogy of Fallot [149].
sox10-derived cardiomyocytes of injured hearts display a profile that favors regeneration when subjected to gene enrichment analysis, since they show inhibition of Gene
Ontology (GO) biological processes associated with negative regulation of the cell cycle [135]. On the other hand, the cells show enrichment in pathways associated with thedevelopment of myocardium and cardiac cells, including cardiomyocyte differentiationand cardiac muscle contraction. The distinct transcriptomic profile of sox10-derived cardiomyocytes reported in the study confirms their crucial roles in regenerating the injured
myocardium [135].
4. Conclusions
It is readily apparent that high-throughput techniques for genetic profiling are crucial to understanding heart development and regeneration (Figure 2). Considering thenumerous orthologous genes related to cardiac function between zebrafish and humans,

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further applying these techniques to zebrafish as a model of study will undoubtedly leadto the discovery of the equivalent processes in humans. Two zebrafish-based clinical trials
against heart disease are already underway, (NCT04009759) and (NCT02641145), where
the latter has collaborations with the American Heart Association and the National Institutes of Health (NIH). Although significant discoveries have been made in the field of
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cardiac development and regeneration, our understanding of the mechanisms
behindtheirsimilarities and differences is not completely clear.

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Zebrafish
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ATAC-seq,
ChIP-seq, transgenic reporter line generation, gene editing by CRISPR, etc. Keys: ●●●—amino acid
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networks,
considerable
genetics[17], distinct
cell types, and signaling
pathwaysrequire morethorough impact
investi- of epigenetics[17],
distinctcell
types,
andsignaling
pathwaysrequiremore
thoroughinvestigations
gations using high-throughput technologies. For a better understanding of regenerationusing
high-throughput
technologies.
understanding
ofisregeneration
responses
responses
so that human cardiac
injuries For
can abebetteraddressed,
further focus
needed on theregeneration
microenvironment[19,22]andcellularroles[150–153].
These
discoveries
so that human cardiac injuries can be addressed, further focus is needed on the regeneration
may, in turn, illuminate
new andpathwaysfor roles
the early
diagnosisof damageto cardiac
tissuemicroenvironment[19,22]
cellular[150–153].
These
discoveries
may,
in turn, illuas
occursinheartdisease,suchasthroughtheidentificationofrelevant
promoters[50], in heart
minate new pathways for the early diagnosis of damage to cardiac tissue as occurs
enhancerssuch[50,68],transcription
factors [114,135],
as well as
new methods
treatment [50,68],
disease,as andthroughthe identificationof relevant
promoters[50],ofenhancers
to encourage regeneration of cardiac tissue. To address the above questions, advanced
and transcription factors [114,135], as well as new methods of treatment to encouragehigh-throughput genome-wide profiling of transcriptome, epigenome, proteome, metabregeneration of cardiac tissue. To address the above questions, advanced high-throughput
olome, etc. would be extremely beneficial.
genome-wide
profiling of transcriptome, epigenome, proteome, metabolome, etc. would
As evidenced by the studies discussed in this review, zebrafish serve as an excellentbeextremely
beneficial.
modelsystem forthe study of cardiac development, and regeneration due to its multifacAs evidenced
by thestudies
discussed
thisreview,zebrafish serveas an excellent
eted ability
to regenerate
cardiactissue,as well asinthe
species’compatibility
with efficientmodel
systemfor the studyresearchof cardiac
development, and regeneration due to its muland accurate
high-throughput
techniques.

tifaceted ability to regenerate cardiac tissue, as well as the species’ compatibility with
Author Contributions:
Allhigh-throughput
authors contributedresearchequally. All
authors have read and agreed to theefficientand accurate
techniques.
published version of the manuscript.

AuthorAll authors
contributed
Funding:Contributions:This research received
no externalfunding. equally. All authors have read and agreed to the
published version of the manuscript.
Institutional Review Board Statement: Not applicable.

Funding:
This researchreceived
no external funding.
Informed Consent
Statement:
Not applicable.
Institutional
Review
Statement:
Notwhoseapplicable.
Acknowledgments:
We Board
apologize
to all authors
publications could not be cited due to spaceconstraints.

Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

Acknowledgments: We apologize to all authors whose publications could not be cited due to space constraints.

References
1.
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Conflicts of Interest: The authors declare no conflict of interest.

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