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The heart is the first organ to form during embryogenesis, and heart formation is essential for subsequent embryonic development. Since the identification of a cardiac-restricted transcription factor Csx/Nkx-2.5 in the early 1990s, extensive studies on cardiac development have been done in various species ranging from flies to humans. Molecular dissection of regulatory pathways that control multiple steps of cardiogenesis will not only advance our understanding of cardiac development and congenital heart diseases, but will also provide an important clue to novel therapeutic strategies for heart diseases.

X-ray photoemission spectroscopy (XPS) and time-of-flight (TOF) secondary-ion mass spectroscopy (SIMS) have been applied to investigate potsherd samples from Pantanal, Brazil. One of the potsherds presented burnt bone as an additive, which was characterized by XPS as carbonate hydroxyapatite. For shell-tempered ceramics the phase present in shells after firing was identified by X-ray diffraction. TOF-SIMS was used to study the distribution of quartz as temper in one of the sherds. XPS was also applied to the characterization of the finishing external layer of the ceramic vessels. In this case, based on the Fe 2p spectra of the sherd's interior and outermost layers, it was possible to prove that their difference in coloration is due to black heart formation. Hierarchical clustering analysis and principal component analysis of the XPS elemental data enabled the potsherds to be classified with respect to their clay composition.

The vertebrate heart forms initially as a linear tube derived from a primary heart field in the lateral mesoderm. Recent studies in mouse and chick have demonstrated that the outflow tract and right ventricle originate from a separate source of mesoderm that is anterior to the primary heart field. The discovery of this anterior, or secondary, heart field has led to a greater understanding of the morphogenetic events involved in heart formation;however, many of the underlying molecular events controlling these processes remain to be determined. The MADS domain transcription factor MEF2C is required for proper formation of the cardiac outflow tract and right ventricle, suggesting a key role in anterior heart field development. Therefore, as a first step toward identifying the transcriptional pathways upstream of MEF2C, we introduced a lacZ reporter gene into a bacterial artificial chromosome (BAC) encompassing the murine Mef2clocus and used this recombinant to generate transgenic mice. This BAC transgene was sufficient to recapitulate endogenous Mef2c expression,and comparative sequence analyses revealed multiple regions of significant conservation in the noncoding regions of the BAC. We show that one of these conserved noncoding regions represents a transcriptional enhancer that is sufficient to direct expression of lacZ exclusively to the anterior heart field throughout embryonic development. This conserved enhancer contains two consensus GATA binding sites that are efficiently bound by the zinc finger transcription factor GATA4 and are completely required for enhancer function in vivo. This enhancer also contains two perfect consensus sites for the LIM-homeodomain protein ISL1. We show that these elements are specifically bound by ISL1 and are essential for enhancer function in transgenic embryos. Thus, these findings establish Mef2c as the first direct transcriptional target of ISL1 in the anterior heart field and support a model in which GATA factors and ISL1 serve as the earliest transcriptional regulators controlling outflow tract and right ventricle development.

The cardiac neural crest cells migrate from the rostral dorsal neural folds and populate the branchial arches, which contribute directly to the cardiac-outflow structures. Although neural crest cell specification is associated with a number of morphogenic factors, little is understood about the mechanisms by which transcription factors actually implement the transcriptional programs that dictate cell migration and later the differentiation into the proper cell types within the great vessels and the heart. It is clear from genetic evidence that members of the paired box family and basic helix-loop-helix (bHLH) transcription factors from the twist family of proteins are expressed in and play an important function in cardiac neural crest specification and differentiation. Interestingly, both paired box and bHLH factors can function as dimers and, in the case of twist family bHLH factors, partner choice can clearly dictate a change in transcriptional program. The focus of this review is to consider what role the protein-protein interactions of these transcription factors may play in determining cardiac neural crest specification and differentiation, and how genetic alteration of transcription factor stoichiometry within the cell may reflect more than a simple null event.

The earliest step in heart formation in vertebrates occurs during gastrulation, when cardiac tissue is specified. Dorsoanterior endoderm is thought to provide a signal that induces adjacent mesodermal cells to adopt a cardiac fate. However, the nature of this signalling and the precise role of endoderm are unknown because of the close proximity and interdependence of mesoderm and endoderm during gastrulation. To better define the molecular events that underlie cardiac induction, we have sought to develop a simple means of inducing cardiac tissue. We show that the transcription factor GATA4,which has been implicated in regulating cardiac gene expression, is sufficient to induce cardiac differentiation in Xenopus embryonic ectoderm(animal pole) explants, frequently resulting in beating tissue. Lineage labelling experiments demonstrate that GATA4 can trigger cardiac differentiation not only in cells in which it is present, but also in neighbouring cells. Surprisingly, cardiac differentiation can occur without any stable differentiation of anterior endoderm and is in fact enhanced under conditions in which endoderm formation is inhibited. Remarkably, cardiac tissue is formed even when GATA4 activity is delayed until long after explants have commenced differentiation into epidermal tissue. These findings provide a simple assay system for cardiac induction that may allow elucidation of pathways leading to cardiac differentiation. Better knowledge of the pathways governing this process may help develop procedures for efficient generation of cardiomyocytes from pluripotent stem cells.

Congenital heart defects (CHDs) are the most common developmental anomaly and are the leading non-infectious cause of mortality in newborns. Only one causative gene, NKX2-5, has been identified through genetic linkage analysis of pedigrees with non-syndromic CHDs. Here, we show that isolated cardiac septal defects in a large pedigree were linked to chromosome 8p22-23. A heterozygous G296S missense mutation of GATA4, a transcription factor essential for heart formation, was found in all available affected family members but not in any control individuals. This mutation resulted in diminished DNA-binding affinity and transcriptional activity of Gata4. Furthermore, the Gata4 mutation abrogated a physical interaction between Gata4 and TBX5, a T-box protein responsible for a subset of syndromic cardiac septal defects. Conversely, interaction of Gata4 and TBX5 was disrupted by specific human TBX5 missense mutations that cause similar cardiac septal defects. In a second family, we identified a frame-shift mutation of GATA4 (E359del) that was transcriptionally inactive and segregated with cardiac septal defects. These results implicate GATA4 as a genetic cause of human cardiac septal defects, perhaps through its interaction with TBX5.

Acquisition of a cardiac fate by embryonic mesodermal cells is a fundamental step in heart formation. Heart development in frogs and avians requires positive signals from adjacent endoderm, including bone morphogenic proteins, and is antagonized by a second secreted signal, Wnt proteins, from neural tube. By contrast, mechanisms of mesodermal commitment to create heart muscle in mammals are largely unknown. In addition, Wnt-dependent signals can involve either a canonical β-catenin pathway or other, alternative mediators. Here, we tested the involvement of Wnts and β-catenin in mammalian cardiac myogenesis by using a pluripotent mouse cell line (P19CL6) that recapitulates early steps for cardiac specification. In this system, early and late cardiac genes are up-regulated by 1% DMSO, and spontaneous beating occurs. Notably, Wnt3A and Wnt8A were induced days before even the earliest cardiogenic transcription factors. DMSO induced biochemical mediators of Wnt signaling (decreased phosphorylation and increased levels of β-catenin), which were suppressed by Frizzled-8/Fc, a soluble Wnt antagonist. DMSO provoked T cell factor-dependent transcriptional activity; thus, induction of Wnt proteins by DMSO was functionally coupled. Frizzled-8/Fc inhibited the induction of cardiogenic transcription factors, cardiogenic growth factors, and sarcomeric myosin heavy chains. Likewise, differentiation was blocked by constitutively active glycogen synthase kinase 3β, an intracellular inhibitor of the Wnt/β-catenin pathway. Conversely, lithium chloride, which inhibits glycogen synthase kinase 3β, and Wnt3A-conditioned medium up-regulated early cardiac markers and the proportion of differentiated cells. Thus, Wnt/β-catenin signaling is activated at the inception of mammalian cardiac myogenesis and is indispensable for cardiac differentiation, at least in this pluripotent model system.

Initial events involved in the process of heart formation consist of myocardial differentiation as well as development of endothelial and endocardial tissues. As only limited means are allocated to the studying of cardiovascular system development, embryonic stem cells (ESCs) isolated from the inner cell mass (ICM) of developing mice or human blastocysts offer the first step toward the understanding of these complex and intriguing events. ESCs are able to differentiate into a wide range of cell types, including various vascular cells and cardiomyocytes, and their self-renewal capability renders them a unique, homogeneous, and unlimited preliminary population of cells for the investigation of early developmental events of cardiovascular system and lineage commitment. This review summarizes the accumulated knowledge of the cellular and molecular mechanisms involved in the development of the cardiovascular system.

The way in which spatially patterned cellular identities are generated is a central question of organogenesis. In the case of Drosophila heart formation, the cardiac progenitors are specified in precise mesodermal positions, giving rise to multiple cell types in a highly ordered arrangement. Here, we study the mechanisms by which positional information conveyed by signaling pathways and a combinatorial code of activating and repressing transcription factors work together to confine the expression of the homeobox gene even-skipped (eve) to a small region of the dorsal mesoderm. By manipulating both expression patterns and binding sites for transcription factors, we show that a complex combination of regulatory activities converge on a single enhancer of eve to generate precisely targeted gene expression within the cardiac mesoderm. In particular, ladybird early (lbe), a homeobox gene expressed adjacent to eve, restricts the positive actions of factors downstream of wingless, decapentaplegic, and ras to generate the eve pattern. Mutation of a Lbe binding site causes dramatic expansion of expression and abolishes the responsiveness to repression by lbe. Conversely, eliminating eve in the mesoderm expands lbe expression into the normal eve-expressing territory, suggesting that mutual repression between eve and lbe is essential for delineating the spatial patterns of gene expression that specify cell types within the cardiac mesoderm.

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.

Background: Gene targeting studies indicate that desmin is not required for embryonic myogenesis and heart formation but is essential for maintaining the structural and functional integrity of myocytes. Desmin-null mouse hearts developed hypertrophy and dilatation. However, the expression of desmin is very often upregulated rather than downregulated in many cardiac diseases. Altered distribution of desmin protein has been observed in dilated cardiomyopathy, as well as desminopathies to which a number of mutations of desmin and a missense mutation of αB-crystallin (CryAB) have been linked. Methods and Results: Using mouse transgenesis, we have recently demonstrated that modest overexpression of a mutant form of either desmin or CryAB in the heart leads to aberrant aggregation of desmin and CryAB proteins, disruption of the normal desmin network, perturbation of myofibril alignment, and distortion of the nuclear shape. The transgenic hearts develop concentric cardiac hypertrophy and diastolic malfunction at the early stage and ventricular dilatation and congestive heart failure subsequently. Moderate overexpression of either wild-type protein, however, did not cause discernible morphological and functional abnormalities or increased mortality. Conclusions: Thus cardiac overexpression of wild-type desmin is not detrimental whereas aberrant aggregation of desmin and disruption of the desmin network remodel the heart and compromise cardiac function.

Genetic disruption of endothelin (ET) 1, endothelin-converting enzyme (ECE) 1, and endothelin receptor A (ET(A)) in "knockout" or mutant mouse models result in defects in branchial arch derived craniofacial tissues and in cardiac outflow and great vessel structures. Interestingly, certain types of human congenital cardiovascular malformations such as Catch 22 syndrome and type B interruption of the aortic arch strongly resemble defects seen in knockout animal models. To better address the exact involvement of the ET system in heart formation we explored the spatiotemporal pattern of expression of the components of the ET system during critical phases of cardiogenesis in the human embryo (3-6 weeks of development; Carnegie stages 10-17) by in situ hybridization. We detected high ET-1 mRNA expression in endocardial cells lining the heart outflow tract in the region where the future aortic valves will form. No hybridization signal corresponding to pre-pro-ET-3 was observed in the heart. At the same location, the underlying myocytes express ET(A) mRNA. Whereas a functional role of ET in the valve formation can be proposed because of the simultaneous presence of all the components of the endothelin system (ET-1/ECE-1/ET(A)), this seems not to be the case for the formation of the ventricular septum where endocardial cells do not express ET-1, and only a weak ET(A) hybridization signal was detected in the surrounding myocardium. An abnormal hemodynamism indirectly due to valve malformation may be the indirect cause of this septal defect. The results of this study suggest an important role for the ET system in the formation of certain anatomical structures of the developing human heart.

Congenital heart disease in humans results from abnormal morphogenesis of the embryonic cardiovascular system. The characterization of mutations affecting cardiovascular development in animal models ranging from flies to mice has identified many of the key signaling molecules and transcriptional regulators of heart formation. Many of these molecules are also mutated in familial forms of human congenital heart disease. Through the use of animal models combined with analysis of human pedigrees, a molecular framework that controls formation of the vertebrate heart is beginning to emerge.

Formation of the heart is dependent on an intricate cascade of developmental decisions. Analysis of the molecules and mechanisms involved in the specification of cardiac cell fates, differentiation and diversification of cardiac muscle cells, and morphogenesis and patterning of different cardiac cell types has revealed an evolutionarily conserved network of signaling pathways and transcription factors that underlies these processes. The regulatory network that controls the formation of the primitive heart in fruit flies has been elaborated upon to form the complex multichambered heart of mammals. We compare and contrast the mechanisms involved in heart formation in fruit flies and mammals in the context of a network of transcriptional interactions and point to unresolved questions for the future.

Heart formation in vertebrates and fruit flies requires signaling by bone morphogenetic proteins (BMPs) to cardiogenic mesodermal precursor cells. The vertebrate homeobox gene Nkx2–5 and its Drosophila ortholog, tinman, are the earliest known markers for the cardiac lineage. Transcriptional activation of tinman expression in the cardiac lineage is dependent on a mesoderm-specific enhancer that binds Smad proteins, which activate transcription in response to BMP signaling, and Tinman, which maintains its own expression through an autoregulatory loop. Here, we show that an evolutionarily conserved, cardiac-specific enhancer of the mouse Nkx2–5 gene contains multiple Smad binding sites, as well as a binding site for Nkx2–5. A single Smad site is required for enhancer activity at early and late stages of heart development in vivo, whereas the Nkx2–5 site is not required for enhancer activity. These findings demonstrate that Nkx2–5, like tinman, is a direct target for transcriptional activation by Smad proteins; however, the independence of this Nkx2–5 enhancer of Nkx2–5 binding suggests a fundamental difference in the transcriptional circuitry for activation of Nkx2–5 and tinman expression during cardiogenesis in vertebrates and fruit flies.

In the forming vertebrate heart, bone morphogenetic protein signaling induces expression of the early cardiac regulatory gene nkx-2.5. A similar regulatory interaction has been defined in Drosophila embryos where Dpp signaling mediated by the Smad homologues Mad and Medea directly regulates early cardiac expression of tinman. A conserved cluster of Smad consensus binding sequences was identified in early cardiac regulatory sequences of the mouse nkx-2.5 gene. The importance of the nkx-2.5 Smad consensus region in early cardiac gene expression was examined in transgenic mice and in cultured mouse embryos. In transgenic mice, deletion of the Smad consensus region delays induction of embryonic DeltaSmadnkx-2.5/lacZ gene expression during early heart formation. Induction of DeltaSmadnkx-2.5/lacZ expression is also delayed in the outflow tract myocardium and visceral mesoderm. Targeted mutation of the three Smad consensus sequences inhibited nkx-2.5/lacZ expression in the cardiac crescent, demonstrating a specific requirement for the Smad consensus sites in early cardiac gene induction. Cultured DeltaSmadnkx-2.5/lacZ transgenic mouse embryos also exhibit delayed induction of transgene expression. In the four-chambered heart, deletion of the Smad consensus region resulted in expanded DeltaSmadnkx-2.5/lacZ transgene expression. Thus, the nkx-2.5 Smad consensus region can have positive or negative regulatory function, depending on the developmental context and cellular environment.

Receptor tyrosine kinases (RTKs) participate in numerous developmental decisions. Ror RTKs are a family of orphan receptors that are related to muscle specific kinase (MuSK) and Trk neurotrophin receptors. MuSK assembles acetylcholine receptors at the neuromuscular junction [1, 2], and Trk receptors function in the developing nervous system (reviewed in [3-5]). Rors have been identified in nematodes, insects and mammals. Recent studies have begun to shed light on Ror function during development. In most species, Rors are expressed in many tissue types during development. Analyses of mutants that are defective in the single nematode Ror demonstrate a role in cell migration and in orienting cell polarity. Mice lacking one of the two Ror gene products display defects in bone and heart formation. Similarly, two different human bone development disorders, dominant brachydactyly B and recessive Robinow syndrome, result from mutations in one of the human Ror genes.

This issue of microcirculation focusses on the special topic of "microvessels of the heart" and contains five state-of-the-art reviews and one expert article that reflect current efforts to address the major gaps in our understanding of these key microvessels. In the adult heart, most attention until recently (especially among the clinical cardiology community) has been given to the main coronary arteries, which are the culprit vessels in patients with coronary artery disease, including its most serious manifestation, acute MI. However, due to major advances in efficiently reopening the acutely blocked coronary arteries, MI is no longer the killer disease it once was. In contrast, there are few treatment options for patients who develop microvascular obstruction during acute MI. Indeed, we have a very poor understanding of this disease, or even how heart vessels are initially formed in development. This is surprising in light of the essential nature of the cardiac microvessels for efficient cardiac function throughout life. The articles in this issue are from six keynote speakers at the 66th annual meeting of the BMS at Newcastle University and review our understanding of these key vessels from initial development to their role in adult heart disease.

Studies on population dynamics of yellow stem borer on different rice varieties revealed that the dead heart formation was observed from the 2nd week of August and peak deadheart formation was observed during 4th week of September in PS-15 (30.3%) followed by PR-113 (26.7%) and was minimum in HKR-4 (14.6%). The white ear head formation started from the 1st week of September and attained its peak during 2nd week of November with maximum white ears in NDR-359 (44.9%) followed by PR-113 (44.3%) and minimum in variety PS-5 (27%). White earhead symptom and weather parameters, showed that varieties, PR-113, HKR-47 and PS-15 exhibited significant negative correlation with maximum temperature. Significant negative correlation values -0.962, -0.927, -0.903, -0.931, -0.940 and -0.931 were recorded in varieties, PR-113, HKR-47, PS-4, NDR-359, Sarju-52 and PS-21, respectively with minimum temperature. Varieties PR-113, NDR-359, and Sarju-52 showed significant negative correlation with minimum relative humidity.

Planar cell polarity (PCP) signaling is an evolutionarily conserved mechanism that coordinates polarized cell behavior to regulate tissue morphogenesis during vertebrate gastrulation, neurulation and organogenesis. In Xenopus and zebrafish, PCP signaling is activated by non-canonical Wnts such as Wnt11, and detailed understanding of Wnt11 expression has provided important clues on when, where and how PCP may be activated to regulate tissue morphogenesis. To explore the role of Wnt11 in mammalian development, we established a Wnt11 expression and lineage map with high spatial and temporal resolution by creating and analyzing a tamoxifen-inducible Wnt11‐CreER BAC (bacterial artificial chromosome) transgenic mouse line. Our short- and long-term lineage tracing experiments indicated that Wnt11‐CreER could faithfully recapitulate endogenous Wnt11 expression, and revealed for the first time that cells transiently expressing Wnt11 at early gastrulation were fated to become specifically the progenitors of the entire endoderm. During mid-gastrulation, Wnt11‐CreER expressing cells also contribute extensively to the endothelium in both embryonic and extraembryonic compartments, and the endocardium in all chambers of the developing heart. In contrast, Wnt11‐CreER expression in the myocardium starts from late-gastrulation, and occurs in three transient, sequential waves: first in the precursors of the left ventricular (LV) myocardium from E7.0 to 8.0; subsequently in the right ventricular (RV) myocardium from E8.0 to 9.0; and finally in the superior wall of the outflow tract (OFT) myocardium from E8.5 to 10.5. These results provide formal genetic proof that the majority of the endocardium and myocardium diverge by mid-gastrulation in the mouse, and suggest a tight spatial and temporal control of Wnt11 expression in the myocardial lineage to coordinate with myocardial differentiation in the first and second heart field progenitors to form the LV, RV and OFT. The insights gained from this study will also guide future investigations to decipher the role of non-canonical Wnt/PCP signaling in endoderm development, vasculogenesis and heart formation.

Background The vertebrate heart makes its first sign of appearance at the gastrulation. Gene regulatory network controlling heart development is still not completely understood. Wnt signaling plays a pivotal role in orchestrating different aspects of early and late embryonic development including heart formation. Wnt ligands through canonical and non-canonical pathways have been shown to be essential for cardiac specification and morphogenesis. For example, Wnt3A and Wnt8 found to be capable of inhibiting endogenous heart induction, antagonizing Dkk-1 and Crescent and sufficient to induce heart formation in non-cardiogenic ventral marginal zone mesoderm [1]. Wnt receptors, Frizzleds, are seven-transmembrane proteins to which Wnts bind to their extracellular part, the cysteine-rich domain (CRD) [2]. Little is still known about the mechanism of Wnt-Frizzled receptors signaling during cardiogenesis. The Wnt receptor, Frizzled-7 (fzd7), has been shown by us and others to play important roles in different embryonic developmental contexts including neural crest induction [3] and convergent extension movements during gastrulation [4]. Xfz7 expression pattern during cardiogenesis in comparison to other early heart markers has not been yet investigated. In the current study, we have analyzed Xfzd7 expression in relation to that of XNkx2.5, XTroponin1-C, XGata-4, -5 and -6 and show its co-localization with these heart markers. We are aiming by this analysis to provide some clues about gene expression profile during cardiogenesis through which we might be able to shed some light on the cause of abnormal cardiac development that are Wnt-related. Materials and methods Xenopus laevis embryos were staged according to the normal time table of Nieuwkoop and Faber [5]. Riboprobes of Xfzd7, XNkx2.5, XTroponin-1C, Gata-4, -5 and -6 were synthesized. Single and double in situ hybridisation was carried out according to the methods described by Harland [6] and Knecht, et al. [7]. Embryos were frozen sectioned and imaged using Leica microscopes with Axiovision imaging software.

The Snail family member snail encodes a zinc finger-containing transcriptional factor that is involved in heart formation. Yet, little is known about how Snail regulates heart development. Here, we identified that one of the duplicated snail genes, snai1b, was expressed in the heart region of zebrafish embryos. Depletion of Snai1b function dramatically reduced expression of α5 integrin, disrupted Fibronectin layer in the heart region, especially at the midline, and prevented migration of cardiac precursors, resulting in defects in cardiac morphology and function in zebrafish embryos. Injection of α5β1 protein rescued the Fibronectin layer and then the myocardial precursor migration in snai1b knockdown embryos. The results provide the molecular mechanism how Snail controls the morphogenesis of heart during embryonic development.

Over the past decade, rapid advances in imaging technologies have enabled state-of-the-art image-based analysis of cardiac function at a variety of scales and from multiple aspects, such as cardiac electrophysiology, cardiac biomechanics, and cardiovascular system. This special issue aims to showcase developments in cardiac image computing over the past five years, with a special emphasis given to novel computing techniques used to analyze cardiac anatomy and functions for better understanding of cardiac diseases. The final issue includes 8 high quality manuscripts that addressed some very interesting and timely research questions faced by researchers worldwide. The topics range from the improvement of real-time imaging acquisition, through the design of novel image analysis techniques and image-guided surgical planning tools, to cardiovascular risk assessment and detection. Studying the evolution of heart formation has become an active research area [1]. Embryonic heart morphogenesis (EHM) is a complex and dynamic process where the heart transforms from a single tube into a four-chambered pump. Conventional imaging techniques fail to capture this process due to limited penetration depth. H. Mao et al. utilized confocal microscopy imaging with tissue optical immersion clearing technique to image the heart at different stages of development for an EHM study of quail. This area of study not only showed promising results in obtaining snapshots of hearts during growth, but also laid the foundation to investigate data-driven modeling of heart growth, which in turn could deepen our understanding of the cause, progression, treatment, and prevention of diseases related to cardiac growth. Improving the speed of image reconstruction directly enhances the abundance of the data. In the work of N. Cai et al., a new method was developed to improve compressive sensing (CS) based reconstruction for dynamic cardiac MRI leveraging the theory of structured sparse representation. Their experimental results demonstrated that the proposed method improved the reconstruction quality of dynamic cardiac cine MRI over the state-of-the-art CS method [2]. Whilst numerous researches have been dedicated to imaging cardiac structure and mechanical function, noninvasive imaging of cardiac electrical activities remains to be a challenging task which entails the use of sophisticated mathematical models and optimization techniques [3, 4]. A. Rahimi et al. presented the superiority of an Lp-norm model over its L1 and L2 counterparts in imaging cardiac current sources with various spatial extents and distributions. Through computer-simulated and real-data experiments, they further demonstrated the feasibility of the proposed method in imaging the complex structure of excitation wavefront, as well as current sources distributed along the postinfarction scar border—the region that plays a critical role in the triggering and sustaining of lethal arrhythmias. Extracting anatomical and physiological information of the heart from cardiac images requires robust yet simple segmentation techniques that suit different application purposes and different image qualities [5]. The work of J. Bayer et al. attempted to fit continuous parametric surfaces to scattered geometric data points forming frontiers delimiting physiologic structures in segmented images, and they successfully verified their methods by reconstructing a geometric model of a mouse's ventricles from a CT scan. This type of segmentation technique provides useful means of visualizing anatomical structures from low-quality images. C. Zhong et al. also investigated model-based image segmentation technique to reconstruct coronary artery (CA) segments that correspond to myocardial segments typically shown in conventional echocardiographic sectional images. By incorporating the existing Chinese visible human (CVH) data and 5 sets of clinical CT data, they established 3D CA models and studied their application in localization of CA segments. The fully reconstructed 3D model allowed virtual dissection to simulate convectional sections of transthoracic echocardiography. A preliminary study involving 170 patients was also undertaken to assess their proposed technique. With coronary artery disease (CAD) becoming one of the most frequent causes of heart failure, there continues to be an urgent need to design prompt and accurate clinical diagnosis for CAD [6]. For example, noncalcified plaques (NCPs) on coronary artery trees are associated with the presence of lipid-core plaques that are prone to rupture. Thus, it is important to detect and monitor the development of NCPs. Y. Li et al. proposed a mathematical morphological approach to quantitatively analyze the noncalcified plaques on a three-dimensional coronary artery wall model (3D-CAWM). This work combined Voxel-Map analysis techniques, plaque locating, and anatomical location related labeling, which was demonstrated to achieve more detailed and comprehensive coronary tree wall visualization. Image-guided surgery has now become one of the most active research topics in cardiac imaging computing [7]. Y. Wu et al. reconstructed a three-dimensional digitized visible model of human thoracic structures to provide morphological data for imaging diagnosis and thoracic-cardiovascular surgery. This research provided an efficient learning platform for medical students or junior surgeons to better interpret human thoracic anatomy and practice virtual thoracic and cardiovascular surgery. This special issue concludes with a clinical study that examined the relationship between blood pressure variation (BPV) and carotid intima-media thickness (IMT) for 60 subjects (aged between 33 and 79) in Shenzhen. H. Xiong et al. found that both the daytime systolic BPV and 24-hour systolic BPV evaluated by three indices are positively associated with IMT. They conclude that daytime systolic BPV is the best variable to represent the increasing of carotid IMT. In addition, after adjusting by age, sex, smoking, hypertension, and mean blood pressure (BP) and peak-to-peak values, 24 h diastolic BPV evaluated with standard deviation also presents the favorable performance. Overall, the 8 manuscripts included in this special issue provide a sample of the current state in image-based computational cardiology, including the classic topics such as image segmentation and image reconstruction, the relatively young areas such as growth modeling and electrophysiological imaging, and the use of computational techniques in image-based understanding, diagnosis, and surgery planning of cardiac diseases. The continuing effort in all these expanding and interconnected areas will likely improve our understanding of the mechanism, treatment, and prevention of cardiac diseases.

Despite recent treatment advances, an increase in cardiovascular diseases (CVD) mortality is expected for the next years. Mediator (MED) complex plays key roles in eukaryotic gene transcription. Currently, while numerous studies have correlated MED alterations with several diseases, like cancer or neurological disorders, fewer studies have investigated MED role in CVD initiation and progression. The first finding of MED involvement in these pathologies was the correlation of missense mutations in MED13L gene with transposition of the great arteries. Nowadays, also MED13 and MED15 have been associated with human congenital heart diseases and others could be added, like MED12 that is involved in early mouse development and heart formation. Interestingly, a missense mutation in MED30 gene causes a progressive cardiomyopathy in homozygous mice suggesting a potential role for this subunit also in human CVDs. Moreover, several subunits like MED1, MED13, MED14, MED15, MED23, MED25 and CDK8 exert important roles in glucose and lipid metabolism. Although these evidences derive from in vitro and animal model studies, they indicate that their deregulation may have a significant role in human CVD-related metabolic disorders. Finally, alternative transcripts of MED12, MED19 and MED30 are differently expressed in circulating endothelial progenitor cells thus suggesting they can play a role in the field of regenerative medicine. Overall, further functional studies exploring MED role in human CVD are warranted. The results could allow identifying novel biomarkers to use in combination with imaging techniques for early diagnosis; otherwise, they could be useful to develop targets for novel therapeutic approaches. © 2014 Elsevier B.V.

Development of the mature heart is a complex process that turns a field of mesodermal progenitors into a four-chambered organ with divided circulation. When this process goes awry, cardiac anomalies arise, and abnormalities of the heart present at birth, or congenital heart disease (CHD), have been described for centuries. CHD is highly prevalent, representing the most frequent birth defect worldwide. Tremendous strides have been made in understanding the developmental biology of cardiac morphogenesis and the human genetics of CHD. These fields of research are beginning to merge to provide an understanding of the molecular mechanisms underlying CHD. In this chapter, we present the major developmental and molecular pathways of heart formation and how they are related to human CHDs that arise when these pathways are perturbed. While CHD is generally considered conceptually as a single disease, it is much more accurate to consider CHD as a compilation of numerous distinct specific diseases, each with their own developmental and molecular etiology.

The beetle Tribolium castaneum has increasingly become a powerful model for comparative research on insect development. One recent resource is a collection of piggyBac transposon-based enhancer trap lines. Here, we provide a detailed analysis of three selected lines and demonstrate their value for investigations in the second half of embryogenesis, which has thus far lagged behind research on early stages. Two lines, G12424 and KT650, show enhanced green fluorescent protein (EGFP) expression throughout the extraembryonic serosal tissue and in a few discrete embryonic domains. Intriguingly, both lines show for the first time a degree of regionalization within the mature serosa. However, their expression profiles illuminate distinct aspects of serosal biology: G12424 tracks the tissue’s rapid maturation while KT650 expression likely reflects ongoing physiological processes. The third line, G04609, is stably expressed in mesodermal domains, including segmental muscles and the heart. Genomic mapping followed by in situ hybridization for genes near to the G04609 insertion site suggests that the transposon has trapped enhancer information for the Tribolium orthologue of midline (Tc-mid). Altogether, our analyses provide the first live imaging, long-term characterizations of enhancer traps from this collection. We show that EGFP expression is readily detected, including in heterozygote crosses that permit the simultaneous visualization of multiple tissue types. The tissue specificity provides live, endogenous marker gene expression at key developmental stages that are inaccessible for whole mount staining. Furthermore, the nonlocalized EGFP in these lines illuminates both the nucleus and cytoplasm, providing cellular resolution for morphogenesis research on processes such as dorsal closure and heart formation. In future work, identification of regulatory regions driving these enhancer traps will deepen our understanding of late developmental control, including in the extraembryonic domain, which is a hallmark of insect development but which is not yet well understood.

Background: One of the earliest steps in heart formation is the generation of two chambers, as cardiogenic cells deployed in the epithelial sheet of mesoderm converge to form the nascent heart tube. What guides this transformation to organotypic form is not known. Results: We have identified a small molecule, concentramide, and a genetic mutation called heart-and-soul ( has ) that disrupt heart patterning. Both cause the ventricle to form within the atrium. Here, we show that the has gene encodes PKCλ. The effect of the has mutation is to disrupt epithelial cell-cell interactions in a broad range of tissues. Concentramide does not disrupt epithelial interactions, but rather shifts the converging heart field rostrally. What is shared between the concentramide and has effects is a reversal of the order of fusion of the anterior and posterior ends of the heart field. Conclusions: The polarity of cardiac tube assembly is a critical determinant of chamber orientation and is controlled by at least two distinct molecular pathways. Combined chemical/genetic dissection can identify nodal points in development, of especial importance in understanding the complex patterning events of organogenesis.

Although several cardiac-specific transcription factors have been shown to play vital roles in various steps during the heart formation, the precise mechanism of the early stage of cardiogenesis has yet to be elucidated. By differential display technique, we tried to identify molecules that are expressed earlier than cardiac transcription factors such as CSX/NKX2-5 and GATA-4 and are involved in cardiomyocyte differentiation using the P19CL6 cell line, which efficiently differentiates into cardiomyocytes when treated with dimethyl sulfoxide. We isolated a novel gene designated Midori. Its deduced amino acid sequence contained an ATP/GTP-binding site, Ig-like domain, and Kringle-like domain. Northern blot analysis revealed that expression of Midori was restricted to the fetal and adult heart and adult skeletal muscle in mice. In whole mount in situ hybridization, Midori was expressed in cardiac crescent and developing heart but not in somites. The MIDORI protein was localized in the nucleus and overexpression of Midori induced expression of endogenous Midori itself, suggesting that MIDORI may act as a transcriptional regulator. Permanent P19CL6 cell lines overexpressing Midori more efficiently differentiated into cardiomyocytes than did parental cells, whereas those overexpressing the antisense Midori less efficiently differentiated. These results suggest that Midori may promote the differentiation of P19CL6 into cardiomyocytes.

A DNA nonbinding mutant of the NK2 class homeoprotein Nkx2.5 dominantly inhibits cardiogenesis in Xenopus embryos, causing a small heart to develop or blocking heart formation entirely. Recently, ten heterozygous CSX/NKX2.5 homeoprotein mutations were identified in patients with congenital atrioventricular (AV) conduction defects. All four missense mutations identified in the human homeodomain led to markedly reduced DNA binding. To examine the effect of a DNA binding-impaired mutant of mouse Csx/Nkx2.5 in the embryonic heart, we generated transgenic mice expressing one such allele, I183P, under the beta-myosin heavy chain promoter. Unexpectedly, transgenic mice were born apparently normal, but the accumulation of Csx/Nkx2.5(I183P) mutant protein in the embryo, neonate, and adult myocardium resulted in progressive and profound cardiac conduction defects and heart failure. P-R prolongation observed at 2 weeks of age rapidly progressed into complete AV block as early as 4 weeks of age. Expression of connexins 40 and 43 was dramatically decreased in the transgenic heart, which may contribute to the conduction defects in the transgenic mice. This transgenic mouse model may be useful in the study of the pathogenesis of cardiac dysfunction associated with CSX/NKX2.5 mutations in humans.

During Drosophila embryogenesis, the homeobox gene tinman is expressed in the dorsal mesoderm where it functions in the specification of precursor cells of the heart, visceral, and dorsal body wall muscles. The GATA factor gene pannier is similarly expressed in the dorsal-most part of the mesoderm where it is required for the formation of the cardial cell lineage. Despite these overlapping expression and functional properties, potential genetic and molecular interactions between the two genes remain largely unexplored. Here, we show that pannier is a direct transcriptional target of Tinman in the heart-forming region. The resulting coexpression of the two factors allows them to function combinatorially in the regulation of cardiac gene expression, and a physical interaction of the proteins has been demonstrated in cultured cells. We also define functional domains of Tinman and Pannier that are required for their synergistic activation of the D-mef2 differentiation gene in vivo. Together, these results provide important insights into the genetic mechanisms controlling heart formation in the Drosophila model system.

GATA-family transcription factors are critical to the development of diverse tissues. In particular, GATA-4 has been implicated in formation of the vertebrate heart. As the mouse Gata-4knock-out is early embryonic lethal because of a defect in ventral morphogenesis, the in vivo function of this factor in heart development remains unresolved. To search for a requirement for Gata4 in heart development, we created mice harboring a single amino acid replacement in GATA-4 that impairs its physical interaction with its presumptive cardiac cofactor FOG-2. Gata4ki/ki mice die just after embryonic day (E) 12.5 exhibiting features in common with Fog2−/− embryos as well as additional semilunar cardiac valve defects and a double-outlet right ventricle. These findings establish an intrinsic requirement for GATA-4 in heart development. We also infer that GATA-4 function is dependent on interaction with FOG-2 and, very likely, an additional FOG protein for distinct aspects of heart formation.

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.

Supraventricular tachycardias are the most prevalent group of arrhythmias observed in the fetus and infant and their incidence increases through early childhood. The molecular pathogenesis of embryonic cardiac dysfunction is poorly understood, due in part to the absence of imaging techniques that provide functional information at the cellular and molecular levels in the developing mammalian heart, particularly during early heart formation. The combination of protein engineering, genetic specification, and high-resolution optical imaging enables new insights into cardiac function and dysfunction during cardiac development. Here we describe the use of GCaMP2, a genetically encoded Ca(2+) indicator (GECI), to determine the processes of cardiac electrical activation during cardiac organogenesis. Transgenic specification of GCaMP2 in mice allows sufficient expression for Ca(2+) imaging as early as embryonic day (e.d.) 9.5, just after the heart begins to function at e.d. 8.5. Crosses with knockout lines in which lethality occurs due to cardiac dysfunction will enable precise determination of the conduction or excitation-contraction coupling phenotypes and thereby improve the understanding of the genetic basis of heart development and the consequence of gene mutations. Moreover, lineage-specific targeting of these sensors of cell signaling provides a new window on the molecular specification of the heart conduction system. We describe mouse lines and imaging methods used to examine conduction in the pre-septated heart (e.d. 10.5), which occurs through dramatically slowed atrioventricular (AV) canal conduction, producing a delay between atrial and ventricular activation prior to the development of the AV node. Genetic constructs including single and bi-allelic minimal promoter systems, and single allele BAC transgenes, enable general or lineage-specific targeting of GCaMP2. High-resolution imaging of embryonic heart conduction provides a new window on one of the most complex events in the mammalian body plan.

In these studies, we have taken advantage of a transient transgenic strategy in Xenopus embryos to demonstrate that BMP signaling is required in vivo for heart formation in vertebrates. Ectopic expression of dominant negative Type I (tALK3) or Type II (tBMPRII) BMP receptors in developing Xenopus embryos results in reduction or absence of heart formation. Additionally, blocking BMP signaling in this manner downregulates expression of XNkx2-5, a homeobox gene required for cardiac specification, prior to differentiation. Notably, however, initial expression of XNkx2-5 is not affected. Mutant phenotypes can be rescued by co-injection of mutant with wild-type receptors or co-injection of mutant receptors with XSmad1, a downstream mediator of BMP signaling. Whole-mount in situ analyses indicate that ALK3 and XSmad1 are coexpressed in cardiogenic regions. Together, our results demonstrate that BMP signaling is required for maintenance of XNkx2-5 expression and heart formation and suggest that ALK3, BMPRII, and XSmad1 may mediate this signaling.

The T-box gene tbx5 is expressed in the developing heart, forelimb, eye, and liver in vertebrate embryos during critical stages of morphogenesis and patterning. In humans, mutations in the TBX5 gene have been associated with Holt–Oram syndrome, which is characterized by developmental anomalies in the heart and forelimbs. In chicken and mouse embryos, tbx5 expression is initiated at the earliest stages of heart formation throughout the heart primordia and is colocalized with other cardiac transcription factors such as nkx-2.5 and GATA4. As the heart differentiates, tbx5 expression is restricted to the posterior sinoatrial segments of the heart, consistent with the timing of atrial chamber determination. The correlation between tbx5 expression and atrial lineage determination was examined in retinoic acid (RA)-treated chicken embryos. tbx5 expression is maintained throughout the hearts of RA-treated embryos under conditions that also expand atrial-specific gene expression. The downstream effects of persistent tbx5 expression in the ventricles were examined directly in transgenic mice. Embryos that express tbx5 driven by a β-myosin heavy chain promoter throughout the primitive heart tube were generated. Loss of ventricular-specific gene expression and retardation of ventricular chamber morphogenesis were observed in these embryos. These studies provide direct evidence for an essential role for tbx5 in early heart morphogenesis and chamber-specific gene expression.

The twin-reversed arterial perfusion (TRAP) sequence, or acardia, is the most severe complication in monozygotic twinning. Although more than 400 cases with TRAP sequence were reported since 1533, thorough investigations of the brain in those cases with a rudimentary head remained infrequent. We report a TRAP sequence with microcephaly and a severely rudimentary brain anlage. Neuropathologic examination clearly demonstrated two types of change: (1) developmental arrest of brain at the prosencephalic stage (holoprosencephaly), and (2) hypoxic damage to the holospheric brain mantle with cystic change (hydranencephaly). With reference to previous studies in experimental animals showing that lack of oxygen during early embryogenesis can induce severe disruptions of head-brain and heart formation, it is concluded that oxygen deficiency due to TRAP may be responsible not only for the encephaloclastic changes in the acardius anceps, but for the developmental arrest of the brain cases as well. This would make it unnecessary to postulate additional primary causes such as asymmetric zygote cleavage (Schwalbe, '07) for the maldevelopment.

It has been generally assumed that the initial rudiment of the heart ventricle is divided by the longitudinal interventricular septum into the right and left ventricles. This paper presents evidence for the hypothesis that the right and the left ventricles are produced during normal development from different sequentially located segments of the cardiac tube. These segments yielding rudiments of the right and left ventricles could be detected even during early embryogenesis. This hypothesis requires a new explanation for the process of the formation of two separate outlets from the heart ventricles.

BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart.

We have used serial histological sections to document heart formation in Xenopus laevis, from the formation of a linear heart tube to the appearance of morphologically distinct atrial and ventricular chambers. 3D reconstruction techniques have been used to derive accurate models from digital images, revealing the morphological changes that accompany heart differentiation. To demonstrate the utility of this approach in analysing cardiac gene expression, we have reexamined the distribution of Hand1 transcripts in the linear and looped heart tube. Our results demonstrate that prior to looping, an initial asymmetric, left-sided pattern is replaced by more symmetrical localisation of transcripts to the ventral portion of the myocardium. After the onset of looping, Hand1 expression is restricted to the ventral ventricular myocardium and extends along the entire length of the single ventricle.

The Late Coniacian, shallow-marine Bad Heart Formation of the Western Canada foreland basin is very unusual in that it contains economically significant ooidal ironstone. Deposition of shallow-water and iron-rich facies appears to have been localized over the crest and flanks of a subtle intrabasinal arch, in part interpreted as a forebulge and partly attributed to reactivation of the long-lived Peace River Arch. The formation comprises two upward-shoaling allomembers, typically 5–10 m thick, that are bounded by regionally mappable ravinement surfaces. The lower unit, allomember 1, grades up from laminated mudstone to bioturbated silty sandstone, which is abruptly overlain by bioturbated ooidal silty sandstone grading into an almost clastic-free ooidal ironstone up to 7 m thick. Ooidal ironstone was concentrated into NW- to SE-trending ridges, kilometres wide and tens of kilometres long. Ironstone formation appears to have been promoted by: (a) drowning of the arch, which progressively curtailed sediment supply; and (b) enhanced reworking over the shallowly submerged arch and over a fault-bounded block that underwent episodic vertical movement of 10–20 m during Bad Heart deposition. Allomember 2 also shoals upwards from mudstone to bioturbated and laminated silty sandstone but lacks ooids, apparently reflecting a rejuvenated supply of detrital sediment from the arch. The marine ravinement surface above allomember 2 is a Skolithos firmground, above which is developed a regional blanket of ooidal sediment. In the east, ooids are dispersed in a bioturbated silty sandstone with abundant evidence of repeated reworking and early siderite and phosphate cements. Westwards, this facies grades, over about 40 km, into almost clastic-free ooidal ironstone about 5 m thick; the lateral facies change may reflect progressive clastic starvation distal to a low-relief source area. The two allomembers are interpreted to reflect eustatic oscillations of about 10 m, superimposed on episodic tectonic warping and block-faulting events. The development of ooidal ironstone immediately above initial marine flooding surfaces indicates a close relationship to marine transgression, reflecting sediment-starved conditions. Ironstone does not appear to be related to either sequence boundaries or maximum flooding surfaces. The Bad Heart Formation is blanketed by marine mudstone deposited in response to major flexural subsidence and rejuvenation of clastic sources in the Cordillera to the SW.

Heart formation in Drosophila is dependent on the homeobox gene tinman. The homeobox gene Nkx2-5 is closely related to tinman and is the earliest known marker for cardiogenesis in vertebrate embryos. Recent studies of cis-regulatory elements required for Nkx2-5 expression in the developing mouse heart have revealed an extraordinary array of independent cardiac enhancers, and associated negative regulatory elements, that direct transcription in distinct regions of the embryonic heart. These studies demonstrate the modularity in cardiac transcription, in which different regulatory elements respond to distinct sets of transcription factors to control gene expression in different compartments of the developing heart. We consider the potential mechanisms underlying such transcriptional complexity, its possible significance for cardiac function, and the implications for evolution of the multichambered heart.

In Drosophila, the homeobox gene tinman is required for specification of dorsal vessel and a number of mesodermal subtypes. Six tinman homologues have now been found in diverse vertebrate species: Nkx2-3, 2-5, 2-6, 2-7, 2-8 and 2-9. Of these, Nkx2-5 appears to be the mostly highly conserved among species, in terms of both primary protein sequence and mRNA expression pattern. Of the others, some have been found as yet only in a single species. Although expression patterns of vertebrate tinman homologues indicate that they may play a role in the specification of several mesodermal or endodermal tissues, to date most attention have been focussed on their role in cardiac development. Results of these studies indicate that, as for Drosophila tinman, vertebrate tinman homologues may be required for heart formation, but may not be sufficient. Studies in Drosophila are defining other pathways which are required in concert with tinman for dorsal vessel formation. Circumstantial evidence suggests that similar pathways may be operative in vertebrate heart formation. This review summarizes recent advances in our understanding of vertebrate tinman homologues and interacting genetic pathways.

Tinman is a Drosophila homeodomain protein that is required for formation of both visceral and cardiac mesoderm, including formation of the dorsal vessel, a heart-like organ. Although several vertebrate tinman homologues have been characterized, their requirement in earliest stages of heart formation has been an open question, perhaps complicated by potential functional redundancy of tinman homologues. We have utilized a novel approach to investigate functional redundancy within a gene family, by coinjecting DNA encoding dominantly acting repressor derivatives specific for each family member into developing Xenopus embryos. Our results provide the first evidence that vertebrate tinman homologues are required for earliest stages of heart formation, and that they are required in a functionally redundant manner. Coinjection of dominant repressor constructs for both XNkx2-3 and XNkx2-5 is synergistic, resulting in a much higher frequency of mutant phenotypes than that obtained with injection of either dominant repressor construct alone. Rescue of mutant phenotypes can be effected by coinjection of either wild-type tinman homologue. The most extreme mutant phenotype is a complete absence of expression of XNkx2-5 in cardiogenic mesoderm, an absence of markers of differentiated myocardium, and absence of morphologically distinguishable heart on the EnNkxHD-injected side of the embryo. This phenotype represents the most severe cardiac phenotype of any vertebrate mutant yet described, and underscores the importance of the tinman family for heart development. These results provide the first in vivo evidence that XNkx2-3 and XNkx2-5 are required as transcriptional activators for the earliest stages of heart formation. Furthermore, our results suggest an intriguing mechanism by which functional redundancy operates within a gene family during development. Our experiments have been performed utilizing a recently developed transgenic strategy, and attest to the efficacy of this strategy for enabling transgene expression in limited cell populations within the developing Xenopus embryo.

The anteroposterior (A-P) patterning of the developing heart underlies atrial and ventricular lineage specification and heart chamber morphogenesis. The posteriorization of cardiomyogenic phenotype with retinoic acid (RA) treatment of primitive streak stage chicken embryos is suggestive of a role for the clustered homeobox (Hox) genes in early heart patterning (Yutzey et al. [1994] Development 120:871-873; [1995] Dev. Biol. 170:531-541). A screen for Hox genes expressed in chick heart primordia and primitive heart led to the isolation of anterior genes of the Hox clusters expressed during cardiogenesis. Specific hoxd-3, hoxa-4, and hoxd-4 transcripts were detected at the early stages of heart formation and full-length cDNA clones were isolated. Expression of hoxd-3 was detected in the heart forming region of embryos prior to heart tube formation. Expression of hoxa-4, hoxd-3, and hoxb-5 was increased in cardiogenic tissue treated with RA in culture conditions that also produced changes in positionally restricted cardiomyogenic phenotypes. Hox genes expressed in cardiac explants exhibited distinct sensitivities to RA and ouabain treatment when compared to genes, such as nkx-2.5, that are involved in cardiac commitment and differentiation. These studies support a role for Hox genes in early heart patterning and suggest that positional information in the cardiogenic region is established by regulatory mechanisms distinct from early heart lineage specification.

In Drosophila induction of the homeobox gene tinman and subsequent heart formation are dependent on dpp signaling from overlying ectoderm. In order to define vertebrate heart-inducing signals we screened for dpp-homologues expressed in HH stage 4 chicken embryos. The majority of transcripts were found to be BMP-2 among several other members of the BMP family. From embryonic HH stage 4 onwards cardiogenic mesoderm appeared to be in close contact to BMP-2 expressing cells which initially were present in lateral mesoderm and subsequently after headfold formation in the pharyngeal endoderm. In order to assess the role of BMP-2 for heart formation, gastrulating chick embryos in New culture were implanted with BMP-2 producing cells. BMP-2 implantation resulted in ectopic cardiac mesoderm specification. BMP-2 was able to induce Nkx2-5 expression ectopically within the anterior head domain, while GATA-4 was also induced more caudally. Cardiogenic induction by BMP-2, however remained incomplete, since neither Nkx2-8 nor the cardiac-restricted structural gene VMHC-1 became ectopically induced. BMP-2 expressing cells implanted adjacent to paraxial mesoderm resulted in impaired somite formation and blocked the expression of marker genes, such as paraxis, Pax-3, and the forkhead gene cFKH-1. These results suggest that BMP-2 is part of the complex of cardiogenic signals and is involved in the patterning of early mesoderm similar to the role of dpp in Drosophila.

During heart formation in all vertebrate species, the linear heart tube undergoes rightward looping followed by the formation of the atrial and ventricular chambers. The direction of cardiac looping is determined in part by the asymmetric expression of members of the transforming growth factor beta family across the left-right axis of the embryo. The basic helix-loop-helix (bHLH) transcription factors dHAND and eHAND are expressed in the heart tube within specific cardiogenic precursors destined to form the right and left ventricular regions, respectively, and loss-of-function experiments have demonstrated the importance of these factors for looping morphogenesis and ventricular development. We propose a model in which the HAND gene products interpret asymmetric positional information in the developing heart and participate in transcriptional programmes that control development of the right and left ventricular compartments of the heart.

Heart formation provides an excellent model for studying the molecular basis of cell determination in vertebrate embryos. By combining molecular assays with the experimental approaches of classic embryology, a model for the cell signalling events that initiate cardiogenesis is emerging. Studies of chick, amphibian, and fish embryos demonstrate the inductive role of dorso-anterior endoderm in specifying the cardiac fate of adjacent mesoderm. A consequence of this signalling is the onset of cardiomyogenesis and several transcription factors--Nkx2-5-related, HAND, GATA and MEF-2 families--contribute to these events.

The tissue-restricted GATA-4 transcription factor and Nkx2-5 homeodomain protein are two early markers of precardiac cells. Both are essential for heart formation, but neither can initiate cardiogenesis. Overexpression of GATA-4 or Nkx2-5 enhances cardiac development in committed precursors, suggesting each interacts with a cardiac cofactor. We tested whether GATA-4 and Nkx2-5 are cofactors for each other by using transcription and binding assays with the cardiac atrial natriuretic factor (ANF) promoter_the only known target for Nkx2-5. Co-expression of GATA-4 and Nkx2-5 resulted in synergistic activation of the ANF promoter in heterologous cells. The synergy involves physical Nkx2-5-GATA-4 interaction, seen in vitro and in vivo, which maps to the C-terminal zinc finger of GATA-4 and a C-terminus extension; similarly, a C-terminally extended homeodomain of Nkx2-5 is required for GATA-4 binding. Structure/function studies suggest that binding of GATA-4 to the C-terminus autorepressive domain of Nkx2-5 may induce a conformational change that unmasks Nkx2-5 activation domains. GATA-6 cannot substitute for GATA-4 for interaction with Nkx2-5. This interaction may impart functional specificity to GATA factors and provide cooperative crosstalk between two pathways critical for early cardiogenesis. Given the co-expression of GATA proteins and NK2 class members in other tissues, the GATA/Nkx partnership may represent a paradigm for transcription factor interaction during organogenesis.

The developing heart primordium strongly expresses N-cadherin. In order to investigate the role of this adhesion molecule in heart morphogenesis, chicken embryos were cultured at stages 5-12, and injected with anti-N-cadherin antibodies that can specifically block the activity of this cadherin. In the injected embryos, the epimyocardial layers, which develop bilaterally from the splanchnic mesoderm, did not fuse to form a single cardiac tube. Moreover, each of the unfused layers became fragmented into epithelioid clusters. At the cellular level, large intercellular gaps were observed in the antibody-treated myocardial layers. These disorganized myocardial layers beat to some extent, suggesting that their differentiation was not blocked; however, their contraction was not coordinated. Morphogenesis of other tissues, not only N-cadherin-negative but also N-cadherin-positive tissues, such as the neural tube and notochord, proceeded normally even in the presence of anti-N-cadherin antibodies. These results suggest that N-cadherin is indispensable for heart formation, but not for morphogenesis of the other tissues, at the developmental stages examined. For the latter processes, expression of other cadherin subtypes presumably compensated for the loss of N-cadherin activity.

The NK-type homeobox gene tinman and the MADS box gene D-mef2 encode transcription factors required for the development and differentiation of the Drosophila heart, and closely related genes regulate cardiogenesis in vertebrates. Genetic analyses indicate that tinman and D-mef2 act at early and late steps, respectively, in the cardiogenic lineage. However, it is unknown whether regulatory interactions exist between these developmental control genes. We show that D-mef2 expression in the developing Drosophila heart requires a novel upstream enhancer containing two Tinman binding sites, both of which are essential for enhancer function in cardiac muscle cells. Transcriptional activity of this cardiac enhancer is dependent on tinman function, and ectopic Tinman expression activates the enhancer outside the cardiac lineage. These results define the only known in vivo target for transcriptional activation by Tinman and demonstrate that D-mef2 lies directly downstream of tinman in the genetic cascade controlling heart formation in Drosophila.