Overlapping Cardiac Programs in Heart Development and Regeneration

Gaining cellular and molecular insights into heart development and regeneration will likely provide new therapeutic targets and opportunities for cardiac regenerative medicine,one of the most urgent clinical needs for heart failure.Here we present a review on zebrafish heart development and regeneration,with a particular focus on early cardiac progenitor development and their contribution to building embryonic heart,as well as cellular and molecular programs in adult zebrafish heart regeneration.We attempt to emphasize that the signaling pathways shaping cardiac progenitors in heart development may also be redeployed during the progress of adult heart regeneration.A brief perspective highlights several important and promising research areas in this exciting field.     

Haploinsufficiency of TAB2 Causes Congenital Heart Defects in Humans

Congenital heart defects (CHDs) are the most common major developmental anomalies and the most frequent cause for perinatal mortality, but their etiology remains often obscure. We identified a locus for CHDs on 6q24-q25. Genotype-phenotype correlations in 12 patients carrying a chromosomal deletion on 6q delineated a critical 850 kb region on 6q25.1 harboring five genes. Bioinformatics prioritization of candidate genes in this locus for a role in CHDs identified the TGF-β-activated kinase 1/MAP3K7 binding protein 2 gene (TAB2) as the top-ranking candidate gene. A role for this candidate gene in cardiac development was further supported by its conserved expression in the developing human and zebrafish heart. Moreover, a critical, dosage-sensitive role during development was demonstrated by the cardiac defects observed upon titrated knockdown of tab2 expression in zebrafish embryos. To definitively confirm the role of this candidate gene in CHDs, we performed mutation analysis of TAB2 in 402 patients with a CHD, which revealed two evolutionarily conserved missense mutations. Finally, a balanced translocation was identified, cosegregating with familial CHD. Mapping of the breakpoints demonstrated that this translocation disrupts TAB2. Taken together, these data clearly demonstrate a role for TAB2 in human cardiac development.     

The atypical Rho GTPase,RhoU, regulates cell-adhesion molecules during cardiac morphogenesis

The vertebrate heart undergoes early complex morphologic events in order to develop key cardiac structures that regulate its overall function (Fahed et al., 2013). Although many genetic factors that participate in patterning the heart have been elucidated (Tu and Chi, 2012), the cellular events that drive cardiac morphogenesis have been less clear. From a chemical genetic screen to identify cellular pathways that control cardiac morphogenesis in zebrafish, we observed that inhibition of the Rho signaling pathways resulted in failure to form the atrioventricular canal and loop the linear heart tube. To identify specific Rho proteins that may regulate this process, we analyzed cardiac expression profiling data and discovered that RhoU was expressed at the atrioventricular canal during the time when it forms. Loss of RhoU function recapitulated the atrioventricular canal and cardiac looping defects observed in the ROCK inhibitor treated zebrafish. Similar to its family member RhoV/Chp (Tay et al., 2010), we discovered that RhoU regulates the cell junctions between cardiomyocytes through the Arhgef7b/Pak kinase pathway in order to guide atrioventricular canal development and cardiac looping. Inhibition of this pathway resulted in similar underlying cardiac defects and conversely, overexpression of a PAK kinase was able to rescue the loss of RhoU cardiac defect. Finally, we found that Wnt signaling, which has been implicated in atrioventricular canal development (Verhoeven et al., 2011), may regulate the expression of RhoU at the atrioventricular canal. Overall, these findings reveal a cardiac developmental pathway involving RhoU/Arhgef7b/Pak signaling, which helps coordinate cell junction formation between atrioventricular cardiomyocytes to promote cell adhesiveness and cell shapes during cardiac morphogenesis. Failure to properly form these cell adhesions during cardiac development may lead to structural heart defects and mechanistically account for the cellular events that occur in certain human congenital heart diseases.     

Expression of sumoylation deficient Nkx2.5 mutant in Nkx2.5 haploinsufficient mice leads to congenital heart defects

Nkx2.5 is a cardiac specific homeobox gene critical for normal heart development. We previously identified Nkx2.5 as a target of sumoylation, a posttranslational modification implicated in a variety of cellular activities. Sumoylation enhanced Nkx2.5 activity via covalent attachment to the lysine residue 51, the primary SUMO acceptor site. However, how sumoylation regulates the activity of Nkx2.5 in vivo remains unknown. We generated transgenic mice overexpressing sumoylation deficient mutant K51R (conversion of lysine 51 to arginine) specifically in mouse hearts under the control of cardiac α-myosin heavy chain (α-MHC) promoter (K51R-Tg). Expression of the Nkx2.5 mutant transgene in the wild type murine hearts did not result in any overt cardiac phenotype. However, in the presence of Nkx2.5 haploinsufficiency, cardiomyocyte-specific expression of the Nkx2.5 K51R mutant led to congenital heart diseases (CHDs), accompanied with decreased cardiomyocyte proliferation. Also, a number of human CHDs-associated Nkx2.5 mutants exhibited aberrant sumoylation. Our work demonstrates that altered sumoylation status may underlie the development of human CHDs associated with Nkx2.5 mutants.     

Shaping the zebrafish heart: From left-right axis specification to epithelial tissue morphogenesis

Although vertebrates appear bilaterally symmetric on the outside, various internal organs, including the heart, are asymmetric with respect to their position and/or their orientation based on the left/right (L/R) axis. The L/R axis is determined during embryo development. Determination of the L/R axis is fundamentally different from the determination of the anterior-posterior or the dorsal-ventral axis. In all vertebrates a ciliated organ has been described that induces a left-sided gene expression program, which includes Nodal expression in the left lateral plate mesoderm. To have a better understanding of organ laterality it is important to understand how L/R patterning induces cellular responses during organogenesis. In this review, we discuss the current understanding of the mechanisms of L/R patterning during zebrafish development and focus on how this affects cardiac morphogenesis. Several recent studies have provided unprecedented insights into the intimate link between L/R signaling and the cellular responses that drive morphogenesis of this organ.     

Zebrafish heart failure models: opportunities and challenges

Heart failure is a complex pathophysiological syndrome of pumping functional failure that results from injury, infection or toxin-induced damage on the myocardium, as well as genetic influence. Gene mutations associated with cardiomyopathies can lead to various pathologies of heart failure. In recent years, zebrafish, Danio rerio, has emerged as an excellent model to study human cardiovascular diseases such as congenital heart defects, cardiomyopathy, and preclinical development of drugs targeting these diseases. In this review, we will first summarize zebrafish genetic models of heart failure arose from cardiomyopathy, which is caused by mutations in sarcomere, calcium or mitochondrial-associated genes. Moreover, we outline zebrafish heart failure models triggered by chemical compounds. Elucidation of these models will improve the understanding of the mechanism of pathogenesis and provide potential targets for novel therapies.

     

“Casting” light on the role of glycosylation during embryonic development: Insights from zebrafish

Zebrafish (Danio rerio) remains a versatile model organism for the investigation of early development and organogenesis, and has emerged as a valuable platform for drug discovery and toxicity evaluation [1–6]. Harnessing the genetic power and experimental accessibility of this system, three decades of research have identified key genes and pathways that control the development of multiple organ systems and tissues, including the heart, kidney, and craniofacial cartilage, as well as the hematopoietic, vascular, and central and peripheral nervous systems [7–31]. In addition to their application in large mutagenic screens, zebrafish has been used to model a variety of diseases such as diabetes, polycystic kidney disease, muscular dystrophy and cancer [32–36]. As this work continues to intersect with cellular pathways and processes such as lipid metabolism, glycosylation and vesicle trafficking, investigators are often faced with the challenge of determining the degree to which these pathways are functionally conserved in zebrafish. While they share a high degree of genetic homology with mouse and human, the manner in which cellular pathways are regulated in zebrafish during early development, and the differences in the organ physiology, warrant consideration before functional studies can be effectively interpreted and compared with other vertebrate systems. This point is particularly relevant for glycosylation since an understanding of the glycan diversity and the mechanisms that control glycan biosynthesis during zebrafish embryogenesis (as in many organisms) is still developing. Nonetheless, a growing number of studies in zebrafish have begun to cast light on the functional roles of specific classes of glycans during organ and tissue development. While many of the initial efforts involved characterizing identified mutants in a number of glycosylation pathways, the use of reverse genetic approaches to directly model glycosylation-related disorders is now increasingly popular. In this review, the glycomics of zebrafish and the developmental expression of their glycans will be briefly summarized along with recent chemical biology approaches to visualize certain classes of glycans within developing embryos. Work regarding the role of protein-bound glycans and glycosaminoglycans (GAG) in zebrafish development and organogenesis will also be highlighted. Lastly, future opportunities and challenges in the expanding field of zebrafish glycobiology are discussed.     

Atrial natriuretic factor in the developing heart: a signpost for cardiac morphogenesis

The developing heart forms during the early stages of embryogenesis, and misregulated heart development results in congenital heart defects (CHDs). To understand the molecular basis of CHDs, a deep understanding of the morphological and genetic basis of heart development is necessary. Atrial Natriuretic Factor (ANF) is an important and extremely sensitive marker for specific regions of the developing heart, as well as for disturbances in the patterning of the heart. This review summarizes the dynamic expression of ANF in the developing heart and its usefulness in understanding the early molecular defects underlying CHDs.     

Cardiac development in zebrafish: coordination of form and function

Organogenesis is a dynamic process involving multiple phases of pattern formation and morphogenesis. For example, heart formation involves the specification and differentiation of cardiac precursors, the integration of precursors into a tube, and the remodeling of the embryonic tube to create a fully functional organ. Recently, the zebrafish has emerged as a powerful model organism for the analysis of cardiac development. In particular, zebrafish mutations have revealed specific genetic requirements for cardiac fate determination, migration, fusion, tube assembly, looping, and remodeling. These processes ensure proper cardiac function; likewise, cardiac function may influence aspects of cardiac morphogenesis.     

The zebrafish as a model for studying neuroblastoma

Neuroblastoma is a tumor arising in the peripheral sympathetic nervous system and is the most common cancer in childhood. Since most of the cellular and molecular mechanisms underlying neuroblastoma onset and progression remain unknown, the generation of new in vivo models might be appropriate to better dissect the peripheral sympathetic nervous system development in both physiological and disease states. This review is focused on the use of zebrafish as a suitable and innovative model to study neuroblastoma development. Here, we briefly summarize the current knowledge about zebrafish peripheral sympathetic nervous system formation, focusing on key genes and cellular pathways that play a crucial role in the differentiation of sympathetic neurons during embryonic development. In addition, we include examples of how genetic changes known to be associated with aggressive neuroblastoma can mimic this malignancy in zebrafish. Thus, we note the value of the zebrafish model in the field of neuroblastoma research, showing how it can improve our current knowledge about genes and biological pathways that contribute to malignant transformation and progression during embryonic life.     

Using zebrafish to study podocyte genesis during kidney development and regeneration

During development, vertebrates form a progression of up to three different kidneys that are comprised of functional units termed nephrons. Nephron composition is highly conserved across species, and an increasing appreciation of the similarities between zebrafish and mammalian nephron cell types has positioned the zebrafish as a relevant genetic system for nephrogenesis studies. A key component of the nephron blood filter is a specialized epithelial cell known as the podocyte. Podocyte research is of the utmost importance as a vast majority of renal diseases initiate with the dysfunction or loss of podocytes, resulting in a condition known as proteinuria that causes nephron degeneration and eventually leads to kidney failure. Understanding how podocytes develop during organogenesis may elucidate new ways to promote nephron health by stimulating podocyte replacement in kidney disease patients. In this review, we discuss how the zebrafish model can be used to study kidney development, and how zebrafish research has provided new insights into podocyte lineage specification and differentiation. Further, we discuss the recent discovery of podocyte regeneration in adult zebrafish, and explore how continued basic research using zebrafish can provide important knowledge about podocyte genesis in embryonic and adult environments. genesis 52:771–792, 2014. © 2014 Wiley Periodicals, Inc.     

Functional modulation of cardiac form through regionally confined cell shape changes

Developing organs acquire a specific three-dimensional form that ensures their normal function. Cardiac function, for example, depends upon properly shaped chambers that emerge from a primitive heart tube. The cellular mechanisms that control chamber shape are not yet understood. Here, we demonstrate that chamber morphology develops via changes in cell morphology, and we determine key regulatory influences on this process. Focusing on the development of the ventricular chamber in zebrafish, we show that cardiomyocyte cell shape changes underlie the formation of characteristic chamber curvatures. In particular, cardiomyocyte elongation occurs within a confined area that forms the ventricular outer curvature. Because cardiac contractility and blood flow begin before chambers emerge, cardiac function has the potential to influence chamber curvature formation. Employing zebrafish mutants with functional deficiencies, we find that blood flow and contractility independently regulate cell shape changes in the emerging ventricle. Reduction of circulation limits the extent of cardiomyocyte elongation; in contrast, disruption of sarcomere formation releases limitations on cardiomyocyte dimensions. Thus, the acquisition of normal cardiomyocyte morphology requires a balance between extrinsic and intrinsic physical forces. Together, these data establish regionally confined cell shape change as a cellular mechanism for chamber emergence and as a link in the relationship between form and function during organ morphogenesis.     

Lrrc10 is required for early heart development and function in zebrafish

Leucine-rich Repeat Containing protein 10 (LRRC10) has recently been identified as a cardiac-specific factor in mice. However, the function of this factor remains to be elucidated. In this study, we investigated the developmental roles of Lrrc10 using zebrafish as an animal model. Knockdown of Lrrc10 in zebrafish embryos (morphants) using morpholinos caused severe cardiac morphogenic defects including a cardiac looping failure accompanied by a large pericardial edema, and embryonic lethality between day 6 and 7 post fertilization. The Lrrc10 morphants exhibited cardiac functional defects as evidenced by a decrease in ejection fraction and cardiac output. Further investigations into the underlying mechanisms of the cardiac defects revealed that the number of cardiomyocyte was reduced in the morphants. Expression of two cardiac genes was deregulated in the morphants including an increase in atrial natriuretic factor, a hallmark for cardiac hypertrophy and failure, and a decrease in cardiac myosin light chain 2, an essential protein for cardiac contractility in zebrafish. Moreover, a reduced fluorescence intensity from NADH in the morphant heart was observed in live zebrafish embryos as compared to control. Taken together, the present study demonstrates that Lrrc10 is necessary for normal cardiac development and cardiac function in zebrafish embryos, which will enhance our understanding of congenital heart defects and heart disease.     

Imaging circuit formation in zebrafish

The study of nervous system development has been greatly facilitated by recent advances in molecular biology and imaging techniques. These approaches are perfectly suited to young transparent zebrafish where they have allowed direct observation of neural circuit assembly in vivo. In this review we will highlight a number of key studies that have applied optical and genetic techniques in zebrafish to address questions relating to axonal and dendritic arbor development,synapse assembly and neural plasticity. These studies have revealed novel cellular phenomena and modes of growth that may reflect general principles governing the assembly of neural circuits.     

Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart. Dev. Genet. 22:288–299, 1998. © 1998 Wiley-Liss, Inc.     

The regenerative capacity of the zebrafish heart is dependent on TGFβ signaling

Mammals respond to a myocardial infarction by irreversible scar formation. By contrast, zebrafish are able to resolve the scar and to regenerate functional cardiac muscle. It is not known how opposing cellular responses of fibrosis and new myocardium formation are spatially and temporally coordinated during heart regeneration in zebrafish. Here, we report that the balance between the reparative and regenerative processes is achieved through Smad3-dependent TGFβ signaling. The type I receptor alk5b (tgfbr1b) is expressed in both fibrotic and cardiac cells of the injured heart. TGFβ ligands are locally induced following cryoinjury and activate the signaling pathway both in the infarct area and in cardiomyocytes in the vicinity of the trauma zone. Inhibition of the relevant type I receptors with the specific chemical inhibitor SB431542 qualitatively altered the infarct tissue and completely abolished heart regeneration. We show that transient scar formation is an essential step to maintain robustness of the damaged ventricular wall prior to cardiomyocyte replacement. Taking advantage of the reversible action of the inhibitor, we dissected the multifunctional role of TGFβ signaling into three crucial processes: collagen-rich scar deposition, Tenascin C-associated tissue remodeling at the infarct-myocardium interface, and cardiomyocyte proliferation. Thus, TGFβ signaling orchestrates the beneficial interplay between scar-based repair and cardiomyocyte-based regeneration to achieve complete heart regeneration.     

Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development

Defects in cardiac valve morphogenesis and septation of the heart chambers constitute some of the most common human congenital abnormalities. Some of these defects originate from errors in atrioventricular (AV) endocardial cushion development. Although this process is being extensively studied in mouse and chick, the zebrafish system presents several advantages over these models, including the ability to carry out forward genetic screens and study vertebrate gene function at the single cell level. In this paper, we analyze the cellular and subcellular architecture of the zebrafish heart during stages of AV cushion and valve development and gain an unprecedented level of resolution into this process. We find that endocardial cells in the AV canal differentiate morphologically before the onset of epithelial to mesenchymal transformation, thereby defining a previously unappreciated step during AV valve formation. We use a combination of novel transgenic lines and fluorescent immunohistochemistry to analyze further the role of various genetic (Notch and Calcineurin signaling) and epigenetic (heart function) pathways in this process. In addition, from a large-scale forward genetic screen we identified 55 mutants, defining 48 different genes, that exhibit defects in discrete stages of AV cushion development. This collection of mutants provides a unique set of tools to further our understanding of the genetic basis of cell behavior and differentiation during AV valve development.     

Normal and abnormal development of the cardiac conduction system; implications for conduction and rhythm disorders in the child and adult

The cardiac conduction system is a specialized network that initiates and closely coordinates the heart beat. Cardiac conduction system development is intricately related to the development and maturation of the embryonic heart towards its four-chambered form, as is indicated by the fact that disturbed development of cardiac structures is often accompanied by a disturbed formation of the CCS. Electrophysiological studies have shown that selected conduction disturbances and cardiac arrhythmias do not take place randomly in the heart but rather at anatomical predilection sites. Knowledge on development of the CCS may facilitate understanding of the etiology of arrhythmogenic events. In this review we will focus on embryonic development of the CCS in relation to clinical arrhythmias, as well as on specific cardiac conduction abnormalities that are observed in patients with congenital heart disease.