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Over the past several decades, the search for the unifying paradigm between the form and function of the early vertebrate embryo heart has focused on genetic patterns as the blueprints for early heart formation, enhanced by phylogenetic and morphologic observations. More recently, however, there has been a resurgence of interest in epigenetic factors such as intracardiac flow patterns and fluid forces as significant factors in early embryo cardiogenesis and vascular formation. The availability of new techniques such as confocal microscopy, phase contrast magnetic resonant imaging, digital particle velocimetry, and high-frequency ultrasonographic imaging, used for in vivo observation of embryonic flow dynamics, have provided new insights into the early embryo hemodynamics. The existing evidence no longer supports the accepted mode of heart’s peristaltic blood propulsion and has called for a radical re-evaluation of the traditionally accepted model of circulation.

Heart disease is the leading cause of death in the world. Heart tissue engineering holds a great promise for future heart disease therapy by building personalized heart tissues. Here we create heart constructs by repopulating decellularized mouse hearts with human induced pluripotent stem cell-derived multipotential cardiovascular progenitor cells. We show that the seeded multipotential cardiovascular progenitor cells migrate, proliferate and differentiate in situ into cardiomyocytes, smooth muscle cells and endothelial cells to reconstruct the decellularized hearts. After 20 days of perfusion, the engineered heart tissues exhibit spontaneous contractions, generate mechanical force and are responsive to drugs. In addition, we observe that heart extracellular matrix promoted cardiomyocyte proliferation, differentiation and myofilament formation from the repopulated human multipotential cardiovascular progenitor cells. Our novel strategy to engineer personalized heart constructs could benefit the study of early heart formation or may find application in preclinical testing.

The mammalian heart is the first functional organ, the first indicator of life. Its normal formation and function are essential for fetal life. Defects in heart formation lead to congenital heart defects, underscoring the finesse with which the heart is assembled. Understanding the reg-ulatory networks controlling heart development have led to significant insights into its lineage origins and morphogenesis and illuminated important aspects of mammalian embryology, while providing insights into human congenital heart disease. The mammalian heart has very little regenerative potential, and thus, any damage to the heart is life threatening and perma-nent. Knowledge of the developing heart is important for effective strategies of cardiac regen-eration, providing new hope for future treatments for heart disease. Although we still have an incomplete picture of the mechanisms controlling development of the mammalian heart, our current knowledge has important implications for embryology and better understanding of human heart disease. © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.

The delineation of the mechanisms that regulate cardiac gene expression is central to our understanding of cardiac growth and development. Much progress has been made toward the identification of factors involved in tissue-restricted gene expression, especially in skeletal muscle cells. However, the mechanisms regulating the expression of cardiac-specific genes remain less well understood. Certain homeodomain proteins have been implicated in commitment to the cardiac phenotype. Among the best characterized are the murine proteins Csx, Nkx-2.5, and Nkx-2.6, related to the protein tinman, which is essential for heart formation in Drosophila . The expression of these genes precedes that of cardiac-specific genes and is therefore believed to play a critical role in the development of the heart. The GATA proteins are a family of zinc finger proteins that are also expressed early in cardiac development and may act separately from, or in concert with, the homeodomain proteins as crucial regulators of heart development. The myosin heavy and light chain genes, the actin genes, the troponin genes, and the atrial natriuretic factor and muscle creatine kinase genes have served as excellent paradigms for the study of cardiac gene expression. Although differences in cis -acting elements and their behavior in binding assays have been observed between different genes, there exist similarities that are noteworthy. In this review, we will discuss the factors involved in the regulation of cardiac-specific gene expression in an attempt to provide a better understanding of the process of cardiogenesis.

Heart development in both vertebrates and Drosophila is initiated by bilaterally symmetrical primordia that may be of equivalent embryological origin: the anterior lateral plate mesoderm in vertebrates and the dorsal-most mesoderm in arthropods. These mesodermal progenitors then merge into a heart tube at the ventral midline (vertebrates) or the dorsal midline (Drosophila). These observations suggest that there may be similarities between vertebrate and invertebrate heart development. The homeobox gene, tinman, is required for heart as well as visceral mesoderm formation in Drosophila, and at least one of several vertebrate genes with similarities in protein sequence and cardiac expression to tinman is crucial for heart development in vertebrates. Inductive signals are also required for Drosophila heart development: The secreted gene product of wingless (wg) is critical for heart development during a time period distinct from its function in segmentation and neurogenesis. Here, we show that wg is epistatic to hedgehog (hh), another secreted segmentation gene product, in its requirement for heart formation. We also provide evidence show that downstream of wg in the signal transduction cascade, dishevelled (dsh, a pioneer protein) and armadillo (arm, beta-catenin homolog) are mediating the cardiogenic Wg signal. In particular, overexpression of dsh can restore heart formation in the absence of wg function. We discuss the possibility that Wg signaling is part of a combinatorial mechanism to specify the cardiac mesoderm.

Endoderm within the heart forming regions of vertebrate embryos has pronounced effects on myocardial cell development. Previous studies have suggested that these effects are mediated by soluble growth factors, in particular fibroblast growth factor 2 (FGF-2) and activin-A. Since both insulin and insulin-like growth factors (IGFs) are present in developing avian embryos at the time of heart formation, we have investigated the potential role of these molecules in promoting development of premyocardial cells in quail. Culture of precardiac mesoderm explants from stage 5 quail embryos in medium containing insulin, IGF-I, or IGF-II increased proliferation of premyocardial cells, with maximal stimulation observed at approximately 25 nM for each ligand. A direct comparison of the proliferative response of precardiac mesoderm to endoderm, fetal calf serum, insulin, IGF-I, IGF-II, activin-A, and FGF-2 showed that FGF-2 and activin-A increased proliferation of premyocardial cells approximately 2-fold, while insulin, IGF-I, and IGF-II stimulated proliferation approximately 3-fold. Insulin and IGF-I enhanced the rate of myocyte differentiation, similar to previously reported effects of endoderm. In contrast, exposure of precardiac mesoderm explants to transforming growth factor beta (TGFβ) reduced proliferation of premyocardial cells and moderated the proliferative effects of IGF-I. TGFβ did not block the differentiation of stage 5 premyocardial cells. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNAs encoding insulin, IGF-II, insulin receptor, and IGF-I receptor were present in both precardiac mesoderm and endoderm, as well as in the forming heart at stage 8. Since premyocardial cells can survive and differentiate in a defined medium lacking these factors, precardiac mesoderm may produce IGF-II and insulin at levels that are sufficient to stimulate myocyte development. Taken together, these results suggest that insulin and/or IGF-II may promote cardiac development in vivo by both autocrine and paracrine mechanisms. Cardiogenesis may therefore be promoted by the combined action of several classes of growth factors. © 1996 Wiley-Liss, Inc.

Heart formation requires complex interactions among cells from multiple embryonic origins. Recent studies have begun to reveal the genetic pathways that control cardiac morphogenesis. Many of the genes within these pathways are conserved across vast phylogenetic distances, which has allowed cardiac development to be dissected in organisms ranging from flies to mammals. Studies of cardiac development have also revealed the molecular defects underlying several congenital cardiac malformations in humans and may ultimately provide opportunities for genetic testing and intervention.

The emergence of animal form and function depends on cell migrations in the embryo. Some migrations are accomplished by cells individually, and the mechanism of movement is predictable by contemporary models of cell adhesion and cytoskeletal function. However, other migrations occur that involve layers or sheets of cells connected by junctions, and the mechanism of migration is obscure. An example is the precardiac mesoderm, an epithelium that migrates anteriorly and ventrally in the early amniote embryo to the position of heart formation. It moves upon and is influenced by the adjacent endoderm, which has produced an extracellular matrix. The matrix contains the cell adhesion and cytoskeleton-activating glycoprotein fibronectin. Some immunolocalization studies have reported that fibronectin is arrayed in an anterior-to-posterior gradient, and it has been suggested that directional migration results from a haptotactic response of each cell to the gradient, a model derived from and supported by experiments with individual cells in culture. However, we have produced evidence from immunostaining that suggests fibronectin is arrayed as a localized anterior patch rather than a gradient. We propose an alternative model for precardiac epithelial migration in which only the anterior cells attach effectively to fibronectin. Thus adhered, their cytoskeletal contractile activity generates force which propagates throughout the layer of connected cells, and efficiently pulls them in the proper direction, following the bending and extending movements of the foregut, notochord and other structures of the head. Theoretical implications of the two models are discussed.

Much of our current understanding of early vertebrate cardiovascular development has come from study of avian and amphibian embryos (for a review of classical work on early specification of the heart, see Ref. [l]). The physical accessibility of these embryos and their developing cardiovascular tissues has made it possible to begin to examine the inductive interactions leading to heart formation. However, these organisms are relatively intractable to genetic analysis. Genetic screens have been instrumental in providing the tools to dissect complex developmental processes in invertebrates such as Drosophila melanogaster and C. elegans [2,3], but vertebrates more amenable to genetic analysis, notably the mouse, have embryos that develop inside the mother and are relatively inaccessible to observation and experimental manipulation, precluding screening for mutations affecting cardiovascular development. The zebrafish, Danio rerio, is a small tropical freshwater cyprinid with an approximately 3-month generation time. Adult fish measure approximately 1.5 inches in length, allowing large numbers of fish to be maintained economically in a relatively small space (Fig. 1A). Females produce several hundred eggs at frequent intervals, and the eggs are externally fertilized, so embryos are available for observation and experimental manipulation at all stages. In addition, the embryos are optically transparent until relatively late in embryogenesis, making it possible to observe development of internal organs directly and non-invasively (Fig. lC>. We have begun to explore whether the zebrafish might serve as a tractable model system for a combined embryologic and genetic analysis of cardiovascular development. In this review we describe particulars of the developing cardiovascular system in zebrafish and experimental and genetic methods that have been or could be applied to its study, and we illustrate the similarity of early cardiovascular development in zebrafish to that of mammals.

In the United States, Sevin(™) brand insecticide is one of the most commonly used insecticides. The active ingredient in Sevin(™), carbaryl (1-napthyl-N-methylcarbamate), is a known acetylcholinesterase (AChE) inhibitor that prevents the breakdown of acetylcholine to acetate and choline at the synapse. While carbaryl successfully causes the death of insects by paralysis, it has also been shown to have negative effects on the development of several nontarget species. To study the effects of carbaryl on nontarget species, zebrafish (Danio rerio) were used, as they are a good model for both toxicology and development studies. Our study suggests that carbaryl induces changes in morphology, specifically in embryo size and shape. Additionally, carbaryl causes defects in heart formation that is characterized by a decrease in heart rate and a developmental delay/defect in cardiac looping. A significant decrease in the number of spinal cord neurons present was also observed. Further investigation showed that there was an increase in cell death in carbaryl-treated embryos. The results indicate that carbaryl may have a greater environmental impact than initially intended. Our study, which was conducted solely by undergraduates at a liberal arts college, indicates that carbaryl may be detrimental to the development of nontarget species.

An experimental system was devised to study the mecha-nisms by which cells become committed to the cardiac myocyte lineage during avian development. Chick tissues from outside the fate map of the heart (in the posterior primitive streak {PPS} of a Hamburger & Hamilton stage 4 embryo) were combined with potential inducing tissues from quail embryos and cultured in vitro. Species-specific RT-PCR was employed to detect the appearance of the cardiac muscle markers chick Nkx-2.5 (cNkx-2.5), cardiac troponin C and ventricular myosin heavy chain in the chick responder tissues. Using this procedure, we found that stage 4-5 anterior lateral (AL) endoderm and anterior central (AC) mesendoderm, but not AL mesoderm or posterior lateral mesendoderm, induced cells of the PPS to differentiate as cardiac myocytes. Induction of cardiogen-esis was accompanied by a marked decrease in the expression of ρ-globin, implying that PPS cells were being induced by anterior endoderm to become cardiac myocytes instead of blood-forming tissue. These results suggest that anterior endoderm contains signaling molecules that can induce cardiac myocyte specification of early primitive streak cells. One of the cardiac muscle markers induced by anterior endoderm, cNkx-2.5, is here described for the first time. cNkx-2.5 is a chick homeobox-containing gene that shares extensive sequence similarity with the Drosophila gene tinman, which is required for Drosophila heart formation. The mesodermal component of cNkx-2.5 expression from stage 5 onward, as determined by in situ hybridization, is strikingly in accord with the fate map of the avian heart. By the time the myocardium and endocardium form distinct layers, cNkx-2.5 is found only in the myocardium. cNkx-2.5 thus appears to be the earliest described marker of avian mesoderm fated to give rise to cardiac muscle.

By utilizing myosin immunostaining, we were able to identify early rat myocardium as a thin epithelial sheet and realized that its cohesive movement toward the midline leads to the straight heart tube formation. Localization study of fibronectin mRNA and protein was, therefore, carried out to investigate its tissue origin and possible roles in facilitating mesoderm migration and heart formation. Fibronectin mRNAs were first detected throughout the mesoderm during the early primitive streak stage, suggesting that the mesoderm is the source of fibronectin. By pre-head fold (pre-somite) and head fold (early somite) stages, the mesoderm became largely down-regulated for fibronectin mRNAs, while it was also at these stages when myosin-positive myocardium formed itself into the epithelium and was subsequently folding toward the midline. Thus, there appears to be little fibronectin synthesis during and directly relevant to early heart tube formation. Later, during the early straight heart tube stage (5 somite and older), endocardium became highly positive for fibronectin mRNAs, suggesting that the endocardium is the major source of fibronectin for the cardiac jelly. Based on the results, we present a map for the early mammalian heart in which the heart is a single crescentic band lying in front of the prechordal plate. We also suggest a process for heart tube formation based on the cohesive movement of the myocardial epithelium. During heart tube formation, fibronectin protein had been deposited previously by the mesoderm and was found uniformly in the ECM and not newly produced by any adjacent tissue. The data contradict the endodermal guidance of heart migration by fibronectin gradient and suggest, instead, a permissive role for the fibronectin substrate. © 1995 wiley-Liss, Inc.

In the Mexican axolotl (salamander), Ambystoma mexicanum, a recessive cardiac lethal mutation causes an incomplete differentiation of the myocardium. Mutant hearts lack organized sarcomeric myofibrils and do not contract throughout their lengths. We have previously shown that RNA purified from normal anterior endoderm or from juvenile heart tissue is able to rescue mutant embryonic hearts in an in vitro organ culture system. Under these conditions as many as 55% of formerly quiescent mutant hearts initiate regular contractions within 48 hours. After earlier reports that transforming growth factor-beta 1 and, to a lesser extent, platelet-derived growth factor-BB could substitute for anterior endoderm as a promoter of cardiac mesodermal differentiation in normal axolotl embryos, we decided to examine the effect of growth factors in the cardiac mutant axolotl system. In one type of experiment, stage 35 mutant hearts were incubated in activin A, transforming growth factors-beta 1 or beta 2, platelet-derived growth factor, or epidermal growth factor, but no rescue of mutant hearts was achieved. Considering the possibility that growth factors would only be effective at earlier stages of development, we tested transforming growth factors-beta 1 and beta 5, and activin A on normal and mutant precardiac mesoderm explanted in the absence of endoderm at neurula stage 14. We found that, although these growth factors stimulated heart tube formation in both normal and mutant mesoderm explants, only normal explants contained contractile myocardial tissue. We hypothesize that transforming growth factor-beta superfamily peptides initiate a cascade of responses in mesoderm that result in both changes in cell shape (the basis for heart morphogenesis) and terminal myocardial cytodifferentiation. The cardiac lethal mutation appears to be deficient only in the latter process.

Coronary vessel development is a highly coordinated process during heart formation. Abnormal development and dysfunction of the coronary network are contributory factors in the majority of heart disease. Understanding the molecular mechanisms that regulate coronary vessel formation is crucial for preventing and treating the disease. We report a zebrafish gene-trap vinculin b (vclb) mutant that displays abnormal coronary vessel development among multiple cardiac defects. The mutant shows overproliferation of epicardium-derived cells and disorganization of coronary vessels, and they eventually die off at juvenile stages. Mechanistically, Vclb deficiency results in the release of another cytoskeletal protein, paxillin, from the Vclb complex and the upregulation of ERK and FAK phosphorylation in epicardium and endocardium, causing disorganization of endothelial cells and pericytes during coronary vessel development. By contrast, cardiac muscle development is relatively normal, probably owing to redundancy with Vcla, a vinculin paralog that is expressed in the myocardium but not epicardium. Together, our results reveal a previously unappreciated function of vinculin in epicardium and endocardium and reinforce the notion that well-balanced FAK activity is essential for coronary vessel development.

The ability of undifferentiated cardiogenic mesoderm to generate diversified myogenic phenotypes was assayed in a minimal culture system. During cardiogenesis in vivo, the anterior and posterior segments of the avian heart have distinct patterns of contractile protein gene expression when they first differentiate. To assess the potential of undifferentiated cardiogenic tissue to diversify into distinct anterior and posterior lineages prior to heart formation, cardiogenic mesoderm and endoderm were removed together from the embryo at Hamburger and Hamilton stages 4-8. Explants from each of these stages differentiated in defined medium as indicated by the expression of muscle-specific genes. However, the ability to express the atrial-specific myosin heavy chain (AMHC1) mRNA was confined to posterior cardiac progenitors. Diversification was not dependent on anterior endoderm, suggesting that inductive interactions between the mesoderm and endoderm are not necessary to maintain diversified cardiac lineages after stage 4. The diversified potential of explanted cardiogenic tissue was altered with retinoic acid treatment, resulting in the activation of AMHC1 gene expression in the anterior progenitors. Anterior cardiogenic cells removed from the embryo at stage 8, when the heart begins to differentiate in vivo, are not susceptible to the alteration of diversified phenotype by retinoic acid treatment. Therefore, the potential to form distinct cardiomyogenic cell lineages is present in the anterior lateral plate mesoderm soon after gastrulation and the maturation of these lineages in a positionally dependent manner is maintained in a simple defined culture system in vitro.

Mesoderm induction has been studied in many systems and some of the factors involved have been identified. Although the heart is mesodermal in origin, the molecular basis of heart development is essentially unknown. The Drosophila heart is a simple tubular structure similar to the early heart tube in vertebrates. The homeobox gene, tinman, has been shown to be crucial for heart formation in Drosophila. Several genes with considerable sequence similarities to tinman are expressed in cardiac primordial tissue of vertebrates and are likely to be required for heart development of higher organisms as well. In addition to transcriptional control factors, heart development might also depend on inductive signals. Here, we demonstrate that the gene wingless (wg), which is known to specify segmental polarity and neuroblast identity in Drosophila, has a novel role in mesoderm development: wg function is specifically required for heart development. A temperature-sensitive mutation of wg was used to inactivate wg function during precise developmental time periods. Elimination of wg function for a short time period after gastrulation results in the selective loss of heart precursors, without significantly affecting the formation of the body wall or visceral muscles, although some pattern defects are observed. This developmental requirement of wg for cardiac organogenesis is distinct from its function in segmentation and neurogenesis. We conclude that wg signaling is a crucial component of heart formation.

Pericardial cavity formation and epithelialization of the cardiac precursor cell population constitute a critical developmental period that precedes stable cardiac cell commitment and differentiation. These events delineate the myocardial and endocardial precursor population in the embryo. Restriction of Na/K-ATPase (the sodium pump) expression to the pre-cardiomyocyte lateral membranes coincides with these events. Na/K-ATPase has been implicated developmentally in cavitation and in maintaining membrane potential. Experiments were undertaken to determine if the effects of perturbing sodium pump activity will affect pericardial cavity formation and, in turn, whether heart formation and/or cardiac cell commitment will be affected. We incubated whole chick embryos in vitro between stages 5 to 8 in the presence of the highly specific Na/K-ATPase inhibitor ouabain. Exposure of whole embryos to 10 μM ouabain (10−5 M) demonstrated that heart development and precardiomyocyte differentiation are inhibited principally between stage 5 through stage 7. In each stage the degree of inhibition follows a rostrocaudal gradient as development proceeds along the anterior to posterior axis. After stage 8 ouabain no longer affects heart development or cardiomyogenesis. The inhibition is concentration- and developmental stage-dependent. The inhibition is reversible by elevating the outside potassium ion concentration [Ko] in the culture medium or by transferring the embryos into normal medium minus ouabain even after 20 hr of ouabain exposure. The results also suggest that the regulation of the formation of the three-dimensional organ is independent from regulation of myogenesis. ©1995 Wiley-Liss, Inc.

Heart induction in Xenopus has been thought to be dependent primarily on the interaction of the heart primordia with the Spemann organizer. We demonstrate, however, that signals derived from the deep dorsoanterior endoderm during early gastrulation are also essential for heart formation. The presence of deep endoderm dramatically enhances heart formation in explants of heart primordia, both in the presence and absence of organizer. Likewise, extirpation of the entire endoderm can decrease the frequency of heart formation in embryos that retain organizer activity. Finally, we show that the combined presence of both endoderm and organizer is necessary and sufficient to induce heart in ventral mesoderm explants that would not otherwise form heart tissue. Xenopus heart induction, therefore, may be a multistep process requiring separate dorsalization and cardiogenic signalling events. This is the first demonstration of a heart-inducing role for the endoderm in Xenopus, indicating that the mechanism of heart formation may be similar in most vertebrates.

The discovery of the vertebrate hox gene clusters and their structural and functional relationship to the Drosophila HOM-C cluster of homeotic genes revealed amazing similarities between the developmental mechanisms by which a major body axis is formed in vertebrates and those of many invertebrates, possibly encompassing all multicellular organisms. Recent data suggest that heart development in Drosophila also resembles vertebrate heart development in several fundamental aspects despite the drastic morphologic differences between them. The discovery of the homeobox gene tinman, which is expressed in the embryonic heart of Drosophila and is required for heart formation, made it possible to compare the determining factors of heart development between Drosophila and vertebrates. tinman has mouse, frog, and fish relatives with considerable sequence similarity, and one of these genes is also specifically expressed in the developing heart. It appears that embryologic orgins and morphogenic movements of heart progenitors, as well as gene expression patterns and possibly their functions, are similar in Drosophila and in vertebrates. If it is true that heart development is conserved between Drosophila and vertebrates, then Drosophila, once again, could serve as a model system for vertebrate development.

There has been considerable recent interest in the roles of vesicle transport proteins in ciliary biology and regulation of signal transduction. We have previously discovered RAB23 mutations in Carpenter syndrome, in which patients present with craniofacial and cardiac malformations, postnatal obesity and tumour formation, and Rab23 regulates trafficking of hedgehog signalling components between cilia and the cytoplasm. In the current study we investigated whether Rab23 may also regulate other signalling pathways. Wnt signalling pathways regulate a variety of developmental processes, yet the mechanisms of Wnt signal transduction remain unclear. We show that zebrafish deficient for Rab23 or its GTPase-activating protein, Evi5l, exhibit abnormal heart formation, which we attribute to a requirement for rab23 in the differentiation of cardiac progenitor cells. We delineate a Wnt11 signalling pathway that is implicated in cardiomyocyte differentiation, which acts via phospholipase C, inositol triphosphate receptor, and protein kinase C, to regulate activator protein 1 (AP-1) transcriptional activity downstream of c-Jun N-terminal kinase. We also show that Rab23 interacts functionally with several components of this pathway to regulate transcription by AP-1. Collectively, these findings identify Rab23 as a novel positive-regulator of non-canonical Wnt11/AP-1 signalling, which may be relevant to cardiovascular disease and stem cell biology. We also present evidence that Rab23 is dispensible for hedgehog signalling in zebrafish, and we propose that this reflects previously reported evolutionary differences in the requirement for cilia in hedgehog signal transduction. Collectively, our findings suggest that Rab23 may integrate multiple signalling pathways, possibly playing both cilia-dependent and –independent roles.

Epithelial-mesenchymal transition (EMT) is a crucial, evolutionarily conserved process that occurs during development and is essential for shaping embryos. Also implicated in cancer, this morphological transition is executed through multiple mechanisms in different contexts, and studies suggest that the molecular programs governing EMT, albeit still enigmatic, are embedded within developmental programs that regulate specification and differentiation. As we review here, knowledge garnered from studies of EMT during gastrulation, neural crest delamination and heart formation have furthered our understanding of tumor progression and metastasis.

International audience; The Drosophila heart has emerged as a powerful model system for cardiovascular research. This simple organ, composed of only 104 cardiomyocytes and associated pericardiac cells, has been the focus of numerous candidate gene approaches in the last 2 decades, which have unraveled a number of transcription factors and signaling pathways involved in the regulation of early cardiac development. Importantly, these regulators seem to have largely conserved functions in mammals. Recent studies also demonstrated the usefulness of the fly circulatory system to investigate molecular mechanisms involved in the control of the establishment and maintenance of the cardiac function. In this review, we have focused on how new technological and conceptual advances in the field of functional genomics have impacted research on the cardiovascular system in Drosophila. Genome-scale genetic screens were conducted taking advantage of recently developed ribonucleic acid interference transgenic lines and molecularly defined genetic deficiencies, which have provided new insights into the genetics of both the developmental control of heart formation and cardiac function. In addition, a comprehensive picture of the transcriptional network controlling heart formation is emerging, thanks to newly developed genomic approaches which allow global and unbiased identification of the underlying components of gene regulatory circuits.

Early cardiac development involves the formation of a heart tube, looping of the tube and formation of chambers. These processes are highly similar among all vertebrates, which suggest the existence of evolutionary conservation of the building plan of the heart. From the jawless lampreys to man, T-box transcription factors like Tbx5 and Tbx20 are fundamental for heart formation, whereas Tbx2 and Tbx3 repress chamber formation on the sinu-atrial and atrioventricular borders. Also, electrocardiograms from different vertebrates are alike, even though the fish heart only has two chambers whereas the mammalian heart has four chambers divided by septa and in addition has much higher heart rates. We conclude that most features of the high-performance hearts of mammals and birds can be traced back to less developed traits in the hearts of ectothermic vertebrates. This article is part of a Special Issue entitled: Cardiomyocyte biology: Cardiac pathways of differentiation, metabolism and contraction.

SummaryMef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart.

Congenital heart defects (CHD) are the most common birth defects, occurring in about 0.7% of all newborn infants. There are multiple lines of evidence that genetic components are involved in developing CHD pathogenesis. An important aspect in understanding disease mechanisms is that in addition to contributions from a single disease-causing gene (usually seen in many familial cases of CHD), a multitude of other genetically interacting loci can also influence the severity or progression of the disease, often diagnosed as idiopathic CHD. It is likely that such genetic interactions underlie a large proportion of cases of idiopathic CHD, where a direct link to known cardiogenic genes yet to be identified. Recent advances in stem cell research and in the growing field of systems biology provide a tremendous amount of new data leading to new hypotheses and to new heart disease gene candidates that may also have potential roles during heart formation and establishment of cardiac function. Usually, these hypotheses are tested in cell-based assays and eventually in the mouse model, however both systems have their own particular set of limitations. In this article we review recent advancements in using Drosophila melanogaster as a model organism to study basic mechanisms of heart development, cardiac function and disease.

The epicardium is a mesothelial cell layer which contributes to the coronary vessels and myocardium and acts as an important source of trophic signals to maintain continued growth and differentiation of the developing heart. The precise lineage potential of the embryonic epicardium has come under recent scrutiny with notable questions around its capacity to give rise to derivative vascular endothelial cells and cardiomyocytes. The importance of the epicardium is not restricted to heart formation. Recent studies in the adult heart have highlighted a paracrine role in modulating injury and have begun to realize its potential as a source of progenitor cells (EPDCs) which may be reactivated toward facilitating neovascularization and myocardial repair after ischemic injury. Thus, the adult epicardium has an embryological origin and emerges as a prime exemplar of the paradigm of activated resident stem cell therapy in regenerative medicine, whereupon a major goal is to restore embryonic plasticity to otherwise dormant adult progenitors and facilitate organ repair. In this review, we will explore current thinking on the origins of the epicardium, its role as a signaling center, lineage heterogeneity, and controversy around epicardial potential within the developing heart. We will extrapolate to the adult injury setting, drawing on key studies in zebrafish and mouse which establish the basis for the adult epicardium as a target for cardiovascular regeneration. Finally, we will consider translation of this potential to the human lineage alongside the prospects for discovery of target cell-based therapeutics.

In Drosophila and Caenorhabditis, signal transduction pathways initiated by the activation of receptor-protein tyrosine kinases can mediate developmental fate decisions. In order to examine whether similar mechanisms are employed during mammalian embryogenesis, we undertook a search for novel protein kinases expressed during heart development in the mouse. The primitive mouse heart is formed between 7.75 and 8.5 days post cotium (dpc) and consists of myocardial ad endocardial cells. A reverse transcriptase polymerase chain reaction-based approach was used to amplify protein kinase specific products from cDNAs obtained from 8.5 dpc heart tissue. Twenty independent PCR products corresponding to either protein serine/threonine or tyrosine kinases were identified. In this report, we describe the characterization of two of the genes corresponding to the novel PCR products (designated Hek2 and msk). Hek2 encodes the mouse ortholog of human HEK2, a recently identified member of the eph receptor-protein tyrosine kinase gene family. Prior to and at the time of heart formation (7.5–8.0 dpc), Hek2 is expressed in the cranial (rostral) region of the embryo from which a subpopulation of cells will give rise to the rudimentary heart. Between 8.0 and 9.5 dpc, Hek2 mRNA expression is observed in myocardial cells, head mesenchyme and paraxial mesoderm. Hek2 transcripts are not detected in endocardial cells. After 9.5 dpc, Hek2 expression is downregulated. msk (for myocardial SNF1-like kinase) encodes a putative protein serine/threonine kinase most similar to the yeast gene SNF1. msk mRNA expression is restricted to myocardial cells and their progenitors in the 7.75–8.5 dpc developing heart. Subsequently, msk mRNA expression is rapidly downregulated. the patterns of Hek2 and msk expression suggest that these protein kinases may function during development of the primitive heart.

Summary The Kenyan maize varieties, Hybrid 511, Hybrid 622, Pwani hybrid, Katumani Composite B, and local populations were evaluated along with the varieties of Multiple Insect-Resistant Tropical (MIRT) pool and experimental varieties from The Maize and Wheat Improvement Centre (CIMMYT), Mexico for resistance to the stem borer, Chilo partellus (Swinhoe). The artificial infestation by first-generation C. partellus on maize during the early whorl stage revealed that the leaf feeding damage, the dead heart formation, and the stem damage on the Kenyan hybrids and populations were significantly greater than on the MIRT varieties. The experimental varieties of CIMMYT were also susceptible to C. partellus. When the maize was infested at anthesis, the Kenyan varieties displayed a moderate level of resistance in terms of stem tunnelling and grain damage by C. partellus. The varieties of Multiple Insect Resistance Tropical pool can be effectively used by the breeders to enhance the resistance of agronomically good, but susceptible, varieties of Kenya.

The complex, partially redundant gene regulatory architecture underlying vertebrate heart formation has been difficult to characterize. Here, we dissect the primary cardiac gene regulatory network in the invertebrate chordate, Ciona intestinalis. The Ciona heart progenitor lineage is first specified by Fibroblast Growth Factor/Map Kinase (FGF/MapK) activation of the transcription factor Ets1/2 (Ets). Through microarray analysis of sorted heart progenitor cells, we identified the complete set of primary genes upregulated by FGF/Ets shortly after heart progenitor emergence. Combinatorial sequence analysis of these co-regulated genes generated a hypothetical regulatory code consisting of Ets binding sites associated with a specific co-motif, ATTA. Through extensive reporter analysis, we confirmed the functional importance of the ATTA co-motif in primary heart progenitor gene regulation. We then used the Ets/ATTA combination motif to successfully predict a number of additional heart progenitor gene regulatory elements, including an intronic element driving expression of the core conserved cardiac transcription factor, GATAa. This work significantly advances our understanding of the Ciona heart gene network. Furthermore, this work has begun to elucidate the precise regulatory architecture underlying the conserved, primary role of FGF/Ets in chordate heart lineage specification.

FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.

Cardiac malformations contribute greatly to cardiovascular disease in the young, constituting a major portion of clinically significant birth defects. Congenital heart disease (CHD) is a common congenital cardiac birth defect, affecting nearly 1% of all live births. Although significant advances have been made in understanding mechanisms controlling heart formation, the causes of most CHD in humans remain undefined in the vast majority of cases. Of the several genes identified for CHD, CRELD1 is an important cell adhesion molecule crucial in cardiac development, which is known to cause atrioventricular septal defect in Down syndrome and also in sporadic forms of atrioventricular septal defect. With informed consent, 100 clinically diagnosed CHD patients and 50 healthy controls in Mysore, South India, were recruited for single-nucleotide polymorphism (SNP) genotyping. MassARRAY analysis of five prominent SNPs of CRELD1 was performed. The analysis revealed the occurrence of the SNP c.985 CT of CRELD1 in two of CHD patients and not in controls. This SNP shows a change from arginine to cysteine in the second calcium-binding epidermal growth factor (EGF) domain, leading to change in the β-sheet in the secondary structure. Therefore, the SNP c.985 CT of CRELD1 is involved in causing CHD in patients of Mysore, South India.

In order to fully describe the expression pattern of the transcription factor FoxO1, we have screened the ES cell genetrap repository databases and obtained a clone that contains the ß-geo reporter gene inserted within intron 1 of FoxO1. We then used the ES cell clone to generate a new mouse strain (B6;129P2- Foxo1(Gt(AD0086)Wtsi/JJC)), which expresses ß-geo according to the endogenous FoxO1 pattern, and collected embryo stages from 7.0dpc to 18.5dpc. We show that the expression of FoxO1 is highly dynamic, starting in the neuroepithelium and then extending into the developing vasculature, including all early stages of heart formation. There is a dramatic switch of expression at 11.5dpc in which most vascular expression is abolished and replaced by skeletal muscle expression. In addition FoxO1 is also expressed in several epithelial structures including the olfactory and otic systems, the cornea and at different levels of the gut depending on developmental stage. At later foetal stages, FoxO1 is upregulated again in the same tissues were it is active during early development, including skeletal muscle, vascular system and neuroepithelium., This work was supported by The Institute of Cancer Research, Cancer Research UK Grant C1178/A4520 and the MYORES Network of Excellence, contract 511978, from the European Commission 6th Framework Programme. B.V. held a Studentship from The Institute of Cancer Research.

Publisher Summary This chapter reviews the biology of cardiac stem cells and the growing body of knowledge regarding cell-based therapeutics. Mammalian cardiogenesis involves proper synchrony of function among a diverse population of cells that comprise the heart such as atrial/ventricular cardiomyocytes, smooth muscle cells, endothelial cells, epicardial cells, conduction system cells, valvular components, and connective tissue. The role of cardiac neural crest precursors is ultimately to give rise to the vascular smooth muscle of the aortic arch, ductus arteriosus, and great vessel, as well as to contribute to the cardiac autonomic nervous system. The proepicardial cells give rise to the smooth muscle cells of the coronary vessels and other epicardial cells. Finally, cardiogenic mesodermal precursor(s) are responsible for the formation of the cardiomyocytes and are most likely to be the source of reservoirs of precursor cells post-natally. Currently there are several contemplated mechanisms of actions underlying successful cell-based therapeutics, each with varying degrees of experimental support. These include differentiation, paracrine signaling, fusion, and cell autonomous niche reconstitution. Ex vivo culture of adult and embryonic stem cells under specific conditions such as the hanging drop technique, stimulation by biochemical compounds, or coculture with cardiac myocytes have demonstrated the capacity of stem cells to differentiate into beating cardiomyocytes and vascular lineages.

We have isolated two murine homeobox genes, Nkx-2.5 and Nkx-2.6, that are new members of a sp sub-family of homeobox genes related to Drosophila NK2, NK3 and NK4/msh-2. In this paper, we focus on the Nkx-2.5 gene and its expression pattern during post-implantation development. Nkx-2.5 transcripts are first detected at early headfold stages in myocardiogenic progenitor cells. Expression preceeds the onset of myogenic differentiation, and continues in cardiomyocytes of embryonic, foetal and adult hearts. Transcripts are also detected in future pharyngeal endoderm, the tissue believed to produce the heart inducer. Expression in endoderm is only found laterally, where it is in direct apposition to promyocardium, suggesting an interaction between the two tissues. After foregut closure, Nkx-2.5 expression in endoderm is limited to the pharyngeal floor, dorsal to the developing heart tube. The thyroid primordium, a derivative of the pharyngeal floor, continues to express Nkx-2.5 after transcript levels diminish in the rest of the pharynx. Nkx-2.5 transcripts are also detected in lingual muscle, spleen and stomach. The expression data implicate Nkx-2.5 in commitment to and/or differentiation of the myocardial lineage. The data further demonstrate that cardiogenic progenitors can be distinguished at a molecular level by late gastrulation. Nkx-2.5 expression will therefore be a valuable marker in the analysis of mesoderm development and an early entry point for dissection of the molecular basis of myogenesis in the heart.

Vitamin A insufficiency has profound adverse effects on embryonic development. Major advances in understanding the role of vitamin A in vertebrate heart formation have been made since the discovery that the vitamin A active form, all-trans-retinoic acid, regulates many genes, including developmental genes. Among the experimental models used, the vitamin A-deficient avian embryo has been an important tool to study the function of vitamin A during early heart formation. A cluster of retinoic acid-regulated developmental genes have been identified that participate in building the heart. In the absence of retinoic acid the embryonic heart develops abnormally leading to embryolethality.

Fetal hearts show a remarkable ability to develop under hypoxic conditions. The metabolic flexibility of fetal hearts allows sustained development under low oxygen conditions. In fact, hypoxia is critical for proper myocardial formation. Particularly, hypoxia inducible factor 1 (HIF-1) and vascular endothelial growth factor play central roles in hypoxia-dependent signaling in fetal heart formation, impacting embryonic outflow track remodeling and coronary vessel growth. Although HIF is not the only gene involved in adaptation to hypoxia, its role places it as a central figure in orchestrating events needed for adaptation to hypoxic stress. Although "normal" hypoxia (lower oxygen tension in the fetus as compared with the adult) is essential in heart formation, further abnormal hypoxia in utero adversely affects cardiogenesis. Prenatal hypoxia alters myocardial structure and causes a decline in cardiac performance. Not only are the effects of hypoxia apparent during the perinatal period, but prolonged hypoxia in utero also causes fetal programming of abnormality in the heart's development. The altered expression pattern of cardioprotective genes such as protein kinase c epsilon, heat shock protein 70, and endothelial nitric oxide synthase, likely predispose the developing heart to increased vulnerability to ischemia and reperfusion injury later in life. The events underlying the long-term changes in gene expression are not clear, but likely involve variation in epigenetic regulation.

Publisher Summary This chapter reviews the utility of zebrafish for the study of heart development. It summarizes the experimental approaches that have been particularly useful for analysis of heart formation and outlines the major phases of heart development in zebrafish, emphasizing current understanding of the molecular and cellular regulation of each step. It also reflects on the application of the zebrafish model to the study of human heart disease and highlights the important impact of the combinatorial use of imaging, embryology, and genetics in zebrafish on the understanding of how genes regulate cardiac development. By combining multiple genetic and embryological experimental approaches, investigators have taken considerable strides toward elucidating the mechanisms that drive heart patterning and morphogenesis, including and in addition to the work encapsulated here. Notably, the ability to study development at the cellular level in the zebrafish embryo has been especially useful for linking essential genes to the specific cell behaviors that they regulate. As the number of investigators using zebrafish increases and the sophistication of zebrafish technology advances, the pace of discovery will undoubtedly continue to accelerate. In particular, emerging methods for targeted gene inactivation, enhanced strategies for spatial and temporal manipulation of gene function, and new techniques for automated image analysis are likely to facilitate high-throughput analysis of phenotypes generated by forward, reverse and chemical genetics.

Publisher Summary This chapter discusses the functions of T-box genes in normal and abnormal heart formation. T-box genes expressed in the heart have important functions in the development of most, if not all, cardiac cell lineages and anatomical structures. Studies in a variety of animal systems, including Drosophila, Xenopus, zebrafish, chick, and mouse, as well as human genetic studies, have shown that T-box proteins are critical for cell differentiation, patterning, proliferation and migration in the developing heart. Members of the T-box gene family are dynamically expressed in distinct and overlapping patterns in myocardial, endothelial and epicardial cell lineages, as well as in valvuloseptal, venous, and arterial structures of the heart. Individual T-box family members have distinct transcriptional functions and dosage effects, which supports coordinated regulation of target genes by the balance of multiple T-box proteins where they are coexpressed. T-box proteins function with other cardiac transcription factors, including Nkx2.5 and GATA4, in normal heart development to regulate target gene expression. Likewise, human genetic studies provide evidence that compromised functions of any of the proteins in these regulatory complexes can lead to similar spectra of congenital heart malformations.

The size and morphology of organs are largely determined by a genetic program. However in some cases, an epigenetic mechanism influences the process of organ development. Particularly, epigenetic factors such as hemodynamic stress and blood pressure affect the morphogenesis of cardiac chambers and valves. Here, we report that the epigenetic influences affect the cardiomyocyte production. Taking advantage of longer developmental period of medaka fish, we could examine the later emerging tissue responses to the defect of ventricular beating, which occurred in the hozuki (hoz) mutant that harbors the mutated ventricular myosin heavy chain (vmhc) gene. The mutant showed a remarkable ventricular enlargement, and we showed that this enlargement was due to an excess production of ventricular cardiomyocytes in addition to the lack of concentric chamber growth. By experimental blockade of blood flow, we demonstrated that an elevated cardiac pressure was responsible for the aberrant cardiomyocyte production. From these data, we propose that the epigenetic tissue response to a stressed situation controls the production of cardiomyocytes to attain a fine tuning of heart formation.

Recent studies have shown that the primary heart tube continues to grow by addition of cells from the coelomic wall. This growth occurs concomitantly with embryonic folding and formation of the coelomic cavity, making early heart formation morphologically complex. A scarcity of data on localized growth parameters further hampers the understanding of cardiac growth. Therefore, we investigated local proliferation during early heart formation. Firstly, we determined the cell cycle length of primary myocardium of the early heart tube to be 5.5 days, showing that this myocardium is nonproliferating and implying that initial heart formation occurs solely by addition of cells. In line with this, we show that the heart tube rapidly lengthens at its inflow by differentiation of recently divided precursor cells. To track the origin of these cells, we made quantitative 3D reconstructions of proliferation in the forming heart tube and the mesoderm of its flanking coelomic walls. These reconstructions show a single, albeit bilateral, center of rapid proliferation in the caudomedial pericardial back wall. This center expresses Islet1 . Cell tracing showed that cells from this caudal growth center, besides feeding into the venous pole of the heart, also move cranially via the dorsal pericardial mesoderm and differentiate into myocardium at the arterial pole. Inhibition of caudal proliferation impairs the formation of both the atria and the right ventricle. These data show how a proliferating growth center in the caudal coelomic wall elongates the heart tube at both its venous and arterial pole, providing a morphological mechanism for early heart formation.

The purpose of this study was to expand an existing semi-mechanistic forest model, ANAFORE (ANAlysing Forest Ecosystems), to allow for the prediction of log quality and the accompanying uncertainty as influenced by climate and management. The forest stand is described as consisting of trees of different cohorts, either of the same or of different species (deciduous or coniferous). In addition to photosynthesis, transpiration, total growth and yield, the model simulates the daily evolution in vessel biomass and radius, parenchyma and branch development. From these data early and latewood biomass, wood tissue composition, knot formation and density are calculated. The new version presented here, includes the description of log quality, including red heart formation of beeches. A Bayesian optimisation routine for the species parameters was added to the stand model. From a given range of input parameters (prior), the model calculates an optimised range for the parameters (posterior) based on given output data, as well as an uncertainty on the predicted values. A case study was performed for Slovenian beech forests to illustrate the main model functioning and more in particular the simulation of the wood quality. The results indicate that the ANAFORE model is a useful tool for analyzing wood quality development and forest ecosystem functioning in response to management, climate and stand characteristics. However, the Bayesian optimization showed that the remaining uncertainty on the input parameters for the chosen stand was very large, due to the large number of input parameters in comparison to the limited stand data.

The vitamin A derivative retinoic acid has previously been shown to have teratogenic effects on heart development in mammalian embryos. The craniomedial migration of the precardiac mesoderm during the early stages of heart formation is thought to depend on a gradient of extracellular fibronectin associated with the underlying endoderm. Here, the effects of retinoic acid on migration of the precardiac mesoderm have been investigated in the early chick embryo. When applied to the whole embryo in culture, the retinoid inhibits the craniomedial migration of the precardiac mesoderm resulting in a heart tube that is stunted cranially, while normal or enlarged caudally. Similarly, a local application of retinoic acid to the heart-forming area disrupts the formation of the cardiogenic crescent and the subsequent development of a single mid-line heart tube. This effect is analogous to removing a segment of endoderm and mesoderm across the heart-forming area and results In various degrees of cardia bifida. At higher concentrations of retinoic acid and earlier developmental stages, two completely separate hearts are produced, while at lower concentrations and later stages there are partial bifurcations. The controls, in which the identical operation is carried out except that dimethyl sulphoxide (DMSO) is used instead of the retinoid, are almost all normal. We propose that one of the teratogenic effects of retinoic acid on the heart is to disrupt the interaction between precardiac cells and the extracellular matrix thus inhibiting their directed migration on the endodermal substratum.

The outermost layer of the vertebrate heart originates from migratory mesothelial cells (epicardium) that give rise to coronary vascular smooth muscles and fibroblasts. The role of the epicardium in myocardial morphogenesis and establishment of normal heart function is still largely unknown. Here, we use Drosophila to investigate non-autonomous influences of epicardial-like tissue surrounding the heart tube on the structural and functional integrity of the myocardium. It has previously been shown that during Drosophila heart formation, mesodermal expression of the homeobox transcription factor even-skipped (eve) is required for specification of a subset of non-myocardial progenitors in the precardiac mesoderm. These progenitors may share some similarities with the vertebrate epicardium. To investigate a non-autonomous epicardial-like influence on myocardial physiology, we studied the consequences of reduced mesodermal Eve expression and epi/pericardial cell numbers on the maturation of the myocardial heart tube, its contractility, and acquisition of a normal heart rhythm in the Drosophila model. Targeting the eve repressor ladybird early (lbe) with the minimal eve mesodermal enhancer efficiently eliminates the mesodermal Eve lineages. These flies exhibit defects in heart structure, including a reduction in systolic and diastolic diameter (akin to ‘restrictive cardiomyopathy’). They also exhibit an elevated incidence of arrhythmias and intermittent asystoles, as well as compromised performance under stress. These abnormalities are restored by eve reexpression or by lbe-RNAi co-overexpression. The data suggest that adult heart function in Drosophila is likely to be modulated non-autonomously, possibly by paracrine influences from neighboring cells, such as the epi/pericardium. Thus, Drosophila may serve as a model for finding genetic effectors of epicardial–myocardial interactions relevant to higher organisms.

We review the anatomic development of the sturgeon’s (Acipenser naccarii) heart. Attention has been focussed on the main developmental events that take place during the embryonic and early post-hatching periods. The study examines identification of the early heart tube, cardiac loop formation, and the transformation of the tubular heart into a multi-chambered organ in a temporal sequence. Also included are the development of the heart valves and that of the epicardium. Many of these processes have been followed into adulthood to illustrate the maturation of the different structures with age. On the whole, sturgeon heart formation appears to share many developmental mechanisms with other vertebrates. This indicates the conservation of the mechanisms along the phyletic scale. The development of the A. naccarii heart appears to be a very slow process in relation both to other sturgeon species and to other fish classes. This should allow detailed investigation of specific morphologic events. Many of the developmental changes experienced by the heart could well prove useful in establishing the chronology of both embryonic and juvenile specimens.

The pacemaker is composed of specialized cardiomyocytes located within the sinoatrial node (SAN), and is responsible for originating and regulating the heart beat. Recent advances towards understanding the SAN development have been made on the genetic control and gene interaction within this structure. Here we report that the Shox2 homeodomain transcription factor is restrictedly expressed in the sinus venosus region including the SAN and the sinus valves during embryonic heart development. Shox2 null mutation results in embryonic lethality due to cardiovascular defects, including an abnormal low heart beat rate (bradycardia) and severely hypoplastic SAN and sinus valves attributed to a significantly decreased level of cell proliferation. Genetically, the lack of Tbx3 and Hcn4 expression, along with ectopic activation of Nppa, Cx40, and Nkx2-5 in the Shox2−/− SAN region, indicates a failure in SAN differentiation. Furthermore, Shox2 overexpression in Xenopus embryos results in extensive repression of Nkx2-5 in the developing heart, leading to a reduced cardiac field and aberrant heart formation. Reporter gene expression assays provide additional evidence for the repression of Nkx2-5 promoter activity by Shox2. Taken together our results demonstrate that Shox2 plays an essential role in the SAN and pacemaker development by controlling a genetic cascade through the repression of Nkx2-5.

Congenital heart disease is the commonest malformation in humans and contributes greatly to the burden of disease in infancy. Increasingly, developmental origins are also implicated in heart disease in adults. Significant advances have been made over the past decade in elucidating morphogenetic events of heart formation and their underlying molecular cascades, mostly in animal models. Clinical studies are increasingly successful in quantifying and unraveling genetic factors. This review focuses on recent progress made in understanding the genetic underpinnings of normal and abnormal heart formation and highlights the importance of understanding these mechanisms to improve patient management.

The C-terminal binding proteins (CtBPs) are ubiquitous corepressors that recruit histone-modifying enzymes to a variety of sequence specific DNA-binding proteins and other transcriptional regulators. CtBPs appear to play an important role in mediating repression and transforming activities of a variety of hematopoietic transcription factors such as Basic Kruppel-like Factor/Kruppel-like Factor 3 (BKLF/KLF3), Friend of GATA (FOG), Evi-1 and members of the Ikaros family. Mice lacking CtBPs die during embryonic development and exhibit defects in a wide range of developmental processes, including aberrant heart formation and absence of blood vessels in the yolk sac. The ongoing identification of repressed target genes and interacting transcriptional partners will help to unravel the contributions of CtBP proteins to hematopoiesis.

Aims Cyclin-dependent kinase inhibitors (CDKIs) play a critical role in negatively regulating the proliferation of cardiomyocytes, although their role in cardiac differentiation remains largely undetermined. We have shown that the most prominent CDKI in Xenopus, p27Xic1(Xic1), plays a role in neuronal and myotome differentiation beyond its ability to arrest the cell cycle. Thus, we investigated whether it plays a similar role in cardiomyocyte differentiation. Methods and results Xenopus laevis embryos were sectioned, and whole-mount antibody staining and immunofluorescence studies were carried out to determine the total number and percentage of differentiated cardiomyocytes in mitosis. Capped RNA and/or translation-blocking Xic1 morpholino antisense oligonucleotides (Xic1Mo) were microinjected into embryos, and their role on cardiac differentiation was assessed by in situ hybridization and/or PCR. We show that cell-cycling post-gastrulation is not essential for cardiac differentiation in Xenopus embryos, and conversely that some cells can express markers of cardiac differentiation even when still in cycle. A targeted knock-down of Xic1 protein by Xic1Mo microinjection decreases the expression of markers of cardiac differentiation, which can be partially rescued by co-injection of full-length Xic1 RNA, demonstrating that Xic1 is essential for heart formation. Furthermore, using deleted and mutant forms of Xic1, we show that neither its abilities to inhibit the cell cycle nor the great majority of CDK kinase activity are essential for Xic1’s function in cardiomyocyte differentiation, an activity that resides in the N-terminus of the molecule. Conclusion Altogether, our results demonstrate that the CDKI Xic1 is required in Xenopus cardiac differentiation, and that this function is localized at its N-terminus, but it is distinct from its ability to arrest the cell cycle and inhibit overall CDK kinase activity. Hence, these results suggest that CDKIs play an important direct role in driving cardiomyocyte differentiation in addition to cell-cycle regulation.

Heart formation is a complex morphogenetic process, and perturbations in cardiac morphogenesis lead to congenital heart disease. NKX2-5 is a key causative gene associated with cardiac birth defects, presumably because of its essential roles during the early steps of cardiogenesis. Previous studies in model organisms implicate NKX2-5 homologs in numerous processes, including cardiac progenitor specification, progenitor proliferation, and chamber morphogenesis. By inhibiting function of the zebrafish NKX2-5 homologs, nkx2.5 and nkx2.7, we show that nkx genes are essential to establish the original dimensions of the linear heart tube. The nkx-deficient heart tube fails to elongate normally: its ventricular portion is atypically short and wide, and its atrial portion is disorganized and sprawling. This atrial phenotype is associated with a surplus of atrial cardiomyocytes, whereas ventricular cell number is normal at this stage. However, ventricular cell number is decreased in nkx-deficient embryos later in development, when cardiac chambers are emerging. Thus, we conclude that nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number. Our data suggest that morphogenetic errors could originate during early stages of heart tube assembly in patients with NKX2-5 mutations.