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Scaled physical models can be useful in analyzing stage-specific hemodynamics in the embryonic human heart to address correlations between early physical stressors and myocardial wall responses. We generated models of the cardiac blood space from reconstructions of four digitized human embryo images from Carnegie Collection at the Armed Forces Institute of Pathology. From physical scale models manufactured by stereolithography, compliant sleeves can be created for flow dynamics studies. This novel use of Carnegie collection images and graphical modeling software provides tools for broadening our understanding of normal and aberrant heart formation.

A1 adenosine receptors (A1ARs) are expressed in the embryonic heart and influence cardiac function at early developmental stages. To test if A1ARs also affect cardiac structural development, we examined the effects of A1AR activation on heart formation. When pregnant mice were treated with the A1AR agonist, N6-cyclopentyladenosine (CPA), fetuses showed marked reduction of ventricular size and manifested features of heart failure. Suggesting that these findings represent direct effects on embryos, CPA treatment of cultured whole murine embryos resulted in cardiac hypoplasia. When rates of cell division and cell death in the heart were examined, we found decreases in cardiac cell proliferation following A1AR activation. In contrast, no changes in numbers or distribution of apoptotic cells were seen. Collectively, these data show that A1AR activation inhibits cardiac cell proliferation and can lead to cardiac hypoplasia. These data identify adenosine as a potential regulator of cardiac cell division during early development. © 2001 Wiley-Liss, Inc.

Forward-genetic analyses in Drosophila and Caenorhabditis elegans have given us unprecedented insights into many developmental mechanisms. To study the formation of organs that contain cell types and structures not present in invertebrates, a vertebrate model system amenable to forward genetics would be very useful. Recent work shows that a newly initiated genetic approach in zebrafish is already making significant contributions to understanding the development of the vertebrate heart, an organ that contains several vertebrate-specific features. These and other studies point to the utility of the zebrafish system for studying a wide range of vertebrate-specific processes.

Heart formation in vertebrates is believed to occur in a segmental fashion, with discreet populations of cardiac progenitors giving rise to different chambers of the heart. However, the mechanisms involved in specification of different chamber lineages are unclear. The basic helix-loop-helix transcription factor dHAND is expressed in cardiac precursors throughout the cardiac crescent and the linear heart tube, before becoming restricted to the right ventricular chamber at the onset of looping morphogenesis. dHAND is also expressed in the branchial arch neural crest, which contributes to craniofacial structures and the aortic arch arteries. Using a series of dHAND-lacZ reporter genes in transgenic mice, we show that cardiac and neural crest expression of dHAND are controlled by separate upstream enhancers and we describe a composite cardiac-specific enhancer that directs lacZ expression in a pattern that mimics that of the endogenous dHAND gene throughout heart development. Deletion analysis reduced this enhancer to a 1.5 kb region and identified subregions responsible for expression in the right ventricle and cardiac outflow tract. Comparison of mouse regulatory elements required for right ventricular expression to the human dHAND upstream sequence revealed two conserved consensus sites for binding of GATA transcription factors. Mutation of these sites abolished transgene expression in the right ventricle, identifying dHAND as a direct transcriptional target of GATA factors during right ventricle development. Since GATA factors are not chamber-restricted, these findings suggest the existence of positive and/or negative coregulators that cooperate with GATA factors to control right ventricular-specific gene expression in the developing heart.

Over the last 10 years, significant progress has been made in the understanding of molecular and genetic determinants of heart formation. An ever growing number of genes have been identified that are required for cardiogenesis, as evidenced by severe abnormalities in cardiac development produced by inactivation in the mouse or inhibition of gene function in other model organisms.1 In general, scientists have identified these genes because of their expression in early cardiac tissues or because of the severe phenotype produced by mutation or inactivation. Gross abnormalities of cardiac development lead to embryonic demise either during midgestation or in the peripartum period, and the underlying severe defects in cardiac structure or function have been relatively easy to determine and describe.1 2 3 Similar defects in the human versions of these genes may account for embryonic lethal forms of human congenital heart disease. It has been hypothesized that hypomorphic mutations or reduction in gene dosage may result in less severe forms of congenital heart disease, such as those seen in surviving newborns and adults. Alternatively, clinically relevant cardiovascular developmental defects affecting infants and adults generally may result from mutations in genes entirely unrelated to those critical during early stages of cardiac specification and heart morphogenesis, making these factors less attractive for intensive study as disease-causing genes. Data to support the former hypothesis have recently come to light. Perhaps the most intriguing example comes from the study of a mammalian homologue of a gene first described in the fruit fly, Drosophila melanogaster . This organism has an open circulation in which hemolymph is distributed with the assistance of a contracting dorsal aorta. Although structurally dissimilar from the …

CREB-binding protein (CBP) is a transcriptional co-activator which is required by many transcription factors. Rubinstein–Taybi syndrome (RTS), which is an autosomal dominant syndrome characterized by abnormal pattern formation, is associated with mutations in the human CBP gene. Various abnormalities occur at high frequency in the skeletal system of heterozygous Cbp-deficient mice, but some features of RTS such as cardiac anomalies do not, suggesting that some symptoms of RTS are caused by a dominant-negative mechanism. Here we report the characterization of homozygous Cbp-deficient mice. Homozygous mutants died around E10.5–E12.5, apparently as a result of massive hemorrhage caused by defective blood vessel formation in the central nervous system, and exhibited apparent developmental retardation as well as delays in both primitive and definitive hematopoiesis. Cbp-deficient embryos exhibited defective neural tube closure which was similar to those observed in twist-deficient embryos. However, a decrease in the level of twist expression was not observed in Cbp-deficient embryos. Anomalous heart formation, a feature of RTS patients and mice mutated in the CBP-related molecule, p300, was not observed in Cbp-deficient embryos. Since both Cbp and p300 are ubiquitously expressed in embryonic tissues including the developing heart, these results suggest that cardiac anomalies observed in RTS patients may be caused by a dominant negative effect of mutant CBP.

Multitype zinc-finger proteins of the Friend of GATA/U-shaped (Ush) class function as transcriptional regulators of gene expression through their modulation of GATA factor activity. To better understand intrinsic properties of these proteins, we investigated the expression and function of the ush gene during Drosophila embryogenesis. ush is dynamically expressed in the embryo, including several cell types present within the mesoderm. The gene is active in the cardiogenic mesoderm, and a loss of function results in an overproduction of both cardial and pericardial cells, indicating a requirement for the gene in the formation of these distinct cardiac cell types. Conversely, ectopic expression of ush results in a decrease in the number of cardioblasts in the heart and the inhibition of a cardial cell enhancer normally regulated by the synergistic activity of the Pannier and Tinman cardiogenic factors. These findings suggest that, similar to its known function in thoracic bristle patterning, Ush functions in the control of heart cell specification through its modulation of Pannier transcriptional activity. ush is also required for mesodermal cell migration early in embryogenesis, where it shows a genetic interaction with the Heartless fibroblast growth factor receptor gene. Taken together, these results demonstrate a critical role for the Ush transcriptional regulator in several diverse processes of mesoderm differentiation and heart formation.

During chick embryogenesis, cells destined to form cardiac myocytes are located within the primitive streak at stage 3 in the same relative anterior-posterior distribution as in the prelooped heart. The most rostral cells contribute to the extreme anterior pole of the heart, the bulbus cordis, and the most caudal to the extreme posterior end, the sinoatrial region. After gastrulation, these cells commit to the myocyte lineage and, retaining their relative positions, migrate to the anterior lateral plate. From stages 5 to 10 they diversify into atrial and ventricular myocytes, with the former located posteriorly and the latter, anteriorly. To determine the effect of a change in the rostro-caudal position of these cells on their diversification, anterior lateral plate mesoderm and the underlying endoderm were cut and rotated 180 degrees along the longitudinal axis, at stages 4-8. The subsequent diversification of these precursor cells into atrial and ventricular myocytes was examined using lineage-specific markers. Our results showed that altering location along the longitudinal axis through stage 6 changed the normal fate of a precursor cell. The orientation of the overlying ectoderm did not alter normal morphogenesis or determination of fate.

Publisher Summary Recently, some of the genetic mechanisms of heart specification have been elucidated in Drosophila. This chapter summarizes the functions and interactions of genes that subdivide the early mesoderm and orchestrate the specification and initial differentiation of the Drosophila heart. In Drosophila, the formation of the mesoderm in ventral regions of blastoderm embryos is largely controlled autonomously, i.e., by mechanisms that act within the nuclei or cells that acquire mesodermal fates. Its mesoderm formation is initiated by a nuclear gradient of the maternally provided, NKkB- related morphogen Dorsal. Peak levels of nuclear Dorsal protein are present along the ventral midline of blastoderm embryos and are required to activate two zygotic genes, twist and snail, which are essential for mesoderm formation. Candidates for target genes of twist at blastoderm stage include folded gastrulation, which codes fora secreted molecule controlling aspects of mesoderm invagination; heartless, which encodes a fibroblast growth factor receptor homolog that is involved in mesoderm migration; the homeobox gene tinman that is crucial for mesoderm patterning and heart formation; mef2, which encodes a MADS domain transcription factor that functions in later aspects of muscle and heart differentiation; and zfh-1, a zinc finger- and homeobox containing gene, which is required for the differentiation of a subset of heart cells.

Publisher Summary This chapter presents the current understanding of the timing and mechanism that establish the myocyte and endocardial cell lineage and their derivatives in the avian heart. Myocardial contractions begin during this double-walled stage of heart formation. Initial pulsations are generated at the right myocardium and spread posteriorly to anteriorly over the whole myocardium. The two-chambered heart further undergoes a series of morphogenetic steps: looping, septation, trabeculation, and thickening of the ventricular walls, and the cranial shift of atrial chambers. Associated with these morphogenetic events, fate diversification takes place within both the myocyte and endocardial endothelial lineages. Diversification in the myocyte lineage generates three distinct cardiac cell types, the atrial myocytes, the ventricular myocytes, and the cells of the cardiac conduction system that coordinates the rhythmic heartbeat. A subpopulation of the endocardial cell lineage further differentiates into valves of the heart. These fate diversification events of cardiogenic mesoderm-derived lineages are regulated by local cell- to-cell communications of neighboring endoderm, ectoderm, migratory neural crest, and coronary vascular cells derived from outside of the heart field. Development and integration of these subcardiac components are sequentially programmed during heart formation in higher vertebrates.

Publisher Summary Discovery of the basic helix-loop-helix (bHLH) proteins, dHAND and eHAND, provides a point of entry into the molecular pathways, which govern both mesodermal and neural crest development of the heart. Moreover, the HAND genes provide a transcriptional basis for a segmental model of cardiac development in which chamber specific regulatory programs operate relatively independently of one another. Finally, through gene knockout experiments of dHAND and other cardiac transcription factors, a variety of critical factors in a molecular pathway controlling individual steps of heart formation is assimilated. Although significant insights have been gained regarding the biology of the HAND transcription factors, many questions remain such as through what mechanism do dHAND and eHAND regulate right and left ventricle development? What are the target genes which lie downstream of these transcription factors? What factors lie upstream of dI-IAND and eHAND and control the chamber-specific expression patterns observed? It is hoped that further understanding of these and other factors will provide answers to the previous and other exciting questions in the future.

Publisher Summary To understand the molecular mechanisms that govern sequentially programmed development and integration of these subcardiac components during heart formation in higher vertebrates, it is essential to define their lineage relationships and migration patterns within the embryo. Molecular and cell biological approaches have uncovered the potentiality or plasticity of embryonic cells in their lineage commitment and terminal differentiation,whereas genetics has been powerful in identifying genes or gene networks involved in these processes. Clone-based cell lineage data, together with fate mapping on a group of cells, define the timing and site at which embryos really induce the cardiac lineages and their differentiation and patterning. Except for neural crest derivatives, the lineage relationships of all cell types in the heart have been identified. It is found that each cardiac cell type is established by lineage diversification of embryonic cells, which arise from one of three distinct origins: cardiogenic mesoderm, neural crest, or proepicardium.

Publisher Summary This chapter examines the embryological studies of heart formation. It is found that the initial steps in establishing cardiac precursors occur during gastrulation, when regions of anterior mesoderm receive signals from the organizer and from adjacent anterior endoderm. It seems likely that the former defines the anterodorsal character of the heart mesoderm, whereas the latter imparts cardiac specificity. Molecular candidates for the organizer signal(s) include members of the fibroblast growth factor and transforming growth factor-β families, along with several other proteins expressed in the early gastrula embryo that have mesoderm-inducing or patterning activity. It is also found that signals from anterior endoderm play an important role in facilitating heart morphogenesis. The distinction between this and the prior role of the endoderm in cardiac specification has sometimes become blurred. Finally, embryo manipulations have demonstrated that the heart field is initially patterned in a dynamic manner. Both cardiac differentiation and morphogenesis are restricted to the anteroventral region of the field within the embryo, even after alteration of the field size. The patterning that underlies this regulative behavior originates at least in part from outside of the heart field since explants of the tissue are freed from such constraints. Inhibition from neural tissue plays some part in this patterning and a role for similar signals within the mesoderm has also been proposed.

Publisher Summary This chapter reviews the current state of knowledge concerning the tissue interactions that regulate heart determination in vertebrates. The method throughout is comparative, with a given phenomenon examined in several species in order to extract fundamental principles as well as to understand how the cardiac developmental program can be modified in different organisms. It is found that avian and amphibian species have a two-step model of vertebrate cardiac induction. Signals from anterior endoderm induce a cardiac field in the overlying mesoderm. Only a subdomain of this field actually becomes heart in vivo. The restriction of the heart field is affected both by BMP signals from the surrounding ventral/lateral tissues that promote cardiogenesis and by signals from dorsal/medial structures, including neural tissues that inhibit heart formation. The end result is specification of heart tissue in the anterior ventral mesoderm. It is also hoped that discussing cardiac development in several vertebrate species will encourage workers on one species to try to integrate their results with the findings of those who work in other experimental systems.

Mutations in the gene encoding the homeobox transcription factor NKX2-5 were found to cause nonsyndromic, human congenital heart disease. A dominant disease locus associated with cardiac malformations and atrioventricular conduction abnormalities was mapped to chromosome 5q35, where NKX2-5 , a Drosophila tinman homolog, is located. Three different NKX2-5 mutations were identified. Two are predicted to impair binding of NKX2-5 to target DNA, resulting in haploinsufficiency, and a third potentially augments target-DNA binding. These data indicate that NKX2-5 is important for regulation of septation during cardiac morphogenesis and for maturation and maintenance of atrioventricular node function throughout life.

In vertebrates, heart formation which integrates different structures and cell types is a complex process that involves a network of genes regulated by transcription factors. Proper spatiotemporal expression of these factors ensure the highly needed tight control of each step in organogenesis. A mistake at any step from cell-commitment to valve formation will have a major impact on heart morphogenesis and function leading to congenital heart disease (CHD). Cardiac abnormalities occur with an incidence of one per 100 live births and represent 25% of all congenital malformations. As an alternative approach to linkage-analysis of familial cases of CHD, we started screening familial and sporadic cases of CHDs in a highly consanguineous population for mutations in genes encoding cardiac-enriched transcription factors. The evolutionarily conserved role of these proteins in cardiac development suggested a role in CHD. In this study, we report a mutation in the gene encoding GATA4, one of the earliest markers of heart development. This mutation was found in two out of 26 patients with Tetralogy of Fallot (TOF), and in none of the 94 patients with different phenotypes included in the study, nor in 223 healthy individuals. The heterozygous mutation results in an amino acid substitution in the first zinc finger of GATA4 that reduced its transcriptional activation of downstream target genes, without affecting GATA4 ability to bind DNA, nor its interaction with ZFPM2. © 2006 Wiley-Liss, Inc.

The limited number of informative phenotypes and similarity among species suggest that there are at least a few elements of heart formation that are “modular” and amenable to dissection as distinct genetic and biochemical pathways. We propose that these were added to a primitive chordate heart (Figure 2Figure 2), a notion similar to that for other modular evolutionary additions (Raff 1996xRaff, R.A. See all ReferencesRaff 1996). Of course, we recognize that it may be overly reductionist to claim that the units we see by standard histology are those for which genetic pathways exist and that those pathways have been added, in a modular way, to a primitive single-chambered peristaltic pump of a presumptive ancestor.The next steps in the evaluation of this hypothesis include identification of the other elements of the modular pathways that regulate morphogenesis and determination of relationships among pathways that define individual cell fate and higher-order form. As mutants that affect other organ systems are characterized, it will also be of interest to determine whether the type of modular logic that appears to underlie cardiac evolution and development reflects a general strategy for organogenesis.‡To whom correspondence should be addressed.

The zinc finger transcription factor GATA4 has been implicated in heart development based on its early expression in precardiogenic splanchnic mesoderm and its ability to activate the expression of a number of cardiac-specific genes. To determine the role of GATA4 in embryogenesis, we generated mice homozygous for a GATA4 null allele. Homozygous GATA4 null mice arrested in development between E7.0 and E9.5 because of severe developmental abnormalities. Mutant embryos most notably lacked a primitive heart tube and foregut and developed partially outside the yolk sac. In the mutants, the two bilaterally symmetric promyocardial primordia failed to migrate ventrally but instead remained lateral and generated two independent heart tubes that contained differentiated cardiomyocytes. We show that these deformities resulted from a general loss in lateral to ventral folding throughout the embryo. GATA4 is most highly expressed within the precardiogenic splanchnic mesoderm at the posterior lip of the anterior intestinal portal, corresponding to the region of the embryo that undergoes ventral fusion. We propose that GATA4 is required for the migration or folding morphogenesis of the precardiogenic splanchnic mesodermal cells at the level of the AIP.

Little is known about the molecular mechanisms that govern heart specification in vertebrates. Here we demonstrate that bone morphogenetic protein (BMP) signaling plays a central role in the induction of cardiac myogenesis in the chick embryo. At the time when chick precardiac cells become committed to the cardiac muscle lineage, they are in contact with tissues expressing BMP-2, BMP-4, and BMP-7. Application of BMP-2-soaked beads in vivo elicits ectopic expression of the cardiac transcription factors CNkx-2.5 and GATA-4. Furthermore, administration of soluble BMP-2 or BMP-4 to explant cultures induces full cardiac differentiation in stage 5 to 7 anterior medial mesoderm, a tissue that is normally not cardiogenic. The competence to undergo cardiogenesis in response to BMPs is restricted to mesoderm located in the anterior regions of gastrula- to neurula-stage embryos. The secreted protein noggin, which binds to BMPs and antagonizes BMP activity, completely inhibits differentiation of the precardiac mesoderm, indicating that BMP activity is required for myocardial differentiation in this tissue. Together, these data imply that a cardiogenic field exists in the anterior mesoderm and that localized expression of BMPs selects which cells within this field enter the cardiac myocyte lineage.

We have investigated the early development expressional of the heat shock protein genes (hsps) and HSP synthesis and their role during neuroectoderm induction, differentiation and early CNS formation. The expression and kinetics of 90, 73/71, 47 and 27 HSPs on neuroectoderm differentiation was compared under normal and stressed conditions. The role of HSPs on neuroectoderm cell fate including thermotolerance and apoptosis using a whole in vitro embryo culture system was studied. Hsp expression appears closely linked in early mammalian development to critical differentiation and proliferation stages in early brain and heart formation. The hsps are developmentally activated around blastula stage and HSPs are constitutively expressed at high levels during neural tube closure and are heat shock responsive. Using both Northern analysis, confocal microscopy and whole mount in situ hybridisation we have identified the mRNA hsp transcripts and HSPs during organogenesis. HSPs were detected during neuroectoderm cell induction and differentiation with the hsp mRNA being tightly regulated during the cell cycle of neuroectoderm especially at early fore-, mid-, hindbrain and heart formation. The ‘chaperone’ functions of the HSPs are well known, recently during gastrulation the HSP47 and 27 have been shown to specifically bind and fold to nascent collagen and actin molecules respectively. This role is essential for the formation of the basement membrane, extra cellular matrix and neural crest migration during neural plate development. HSP function was observed by using anti-sense strategy, short ‘5 anti-sense cDNA’ hsp oligonucle otides inhibited hsp expression during gastrulation in the whole embryo cultures. The developmental activation of the heat shock element (HSE) is essential to our understanding of the HSPs role in neuronal cell fate. Using specific polyclonal antibodies to HSF1 and 2 (Dr Nakai, Kyoto University) the expression of heat shock factors (HSFs) during neuroectoderm differentiation was examined. Using Western analysis, confocal microscopy and flow cytometry HSF1 and 2 were identified and studied under both normal and heat shocked conditions. During gastrulation higher levels of HSF1 and 2 were identified in the neuroectoderm layer especially in regions of the fore-, mid- and hindbrain. The heat shock response and activation of the HSPs 90, 70, 47 and 27 families have been correlated with HSF1 and 2. The HSF1 appears to be present in all early embryonic cells but appears not to bind to the HSE untill early head fold stage at gastrulation when the presence of HSF2 is observed. During neuroectoderm differentiation the activation of HSF1 and 2 appears to correlate with high constitutive expression of many of the hsps specificially hsp90, 73, 71, 47 and 27 being tightly regulated by the cell cycle at neurulation.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is a member of the orphan nuclear receptor superfamily. The spatiotemporal expression pattern of COUP-TFII suggests that it participates in mesenchymal-epithelial interactions required for organogenesis. The null mutants of COUP-TFII die around embryonic (E) day E10 due to defects in angiogenesis and heart formation. To further understand the biologic functions of COUP-TFII during development, LacZ knockin mice and floxed COUP-TFII mice were generated. 5-bomo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining of COUP-TFII-LacZ knockin mice shows high COUP-TFII expression in the endothelium of the vein but not in the artery. In contrast, COUP-TFII is highly expressed in the smooth muscle layer surrounding the artery. This differential expression pattern suggests that COUP-TFII may play a role in establishing arterial-venous identity. To further elucidate the functional role of COUP-TFII in the formation of endothelium of the vein, COUP-TFII was specifically knocked out in the endothelium using Tie-2 Cre mice. In mutant mice lacking COUP-TFII in the endothelium, the vein acquires arterial characteristics and ectopic expression of Notch signal pathway. In addition, NP-1, an upstream regulator of Notch signaling, is also up-regulated. Finally, ectopic formation of hematopieitic cell clusters (HCC) is observed in the mutant veins. Our results are consistent with the notion that cell fate determination of the vein is under genetic control rather than deriving from a default pathway as proposed previously. In addition, COUP-TFII is the first transcription factor identified that specifically marks the endothelium of the vein. Understanding the role of COUP-TFII during the vasculature development will provide insights into the molecular mechanism for the establishment of arterial-venous identity.

A novel cDNA, pCMF1, which is expressed exclusively and transiently in the myogenic cells of the differentiating chicken heart was isolated and characterized. The fulllength cDNA of pCMF1 has one open reading frame encoding 1538 predicted amino acids. While computer analysis predicts the presence of specific structural motifs, the overall sequence of pCMF1 is unique. The pattern of pCMF1 gene expression during heart formation was determined by whole-mount in situ hybridization. pCMF1 is transiently expressed within the myogenic cells of the primitive heart tube from stages 9 to 18 and is not detected in the heart or any other tissue thereafter. A replicationdeficient retrovirus was used to mediate pCMF1 antisense expression in cardiogenic mesoderm. These analyses determined that the presence of pCMF1 antisense sequences disrupted myosin heavy chain expression during cardiac mesoderm differentiation. pCMF1 antisense had no effect on myosin heavy chain expression in differentiated cardiac myocytes. These data suggest a potential function for pCMF1 during cardiac myogenesis.

The murine homeo box gene Nkx2-5 is expressed in precardiac mesoderm and in the myocardium of embryonic and fetal hearts. Targeted interruption of Nkx2-5 resulted in abnormal heart morphogenesis, growth retardation and embryonic lethality at approximately 9-10 days postcoitum (p.c.). Heart tube formation occurred normally in mutant embryos, but looping morphogenesis, a critical determinant of heart form, was not initiated at the linear heart tube stage (8.25-8.5 days p.c.). Commitment to the cardiac muscle lineage, expression of most myofilament genes and myofibrillogenesis were not compromised. However, the myosin light-chain 2V gene (MLC2V) was not expressed in mutant hearts nor in mutant ES cell-derived cardiocytes. MLC2V expression normally occurs only in ventricular cells and is the earliest known molecular marker of ventricular differentiation. The regional expression in mutant hearts of two other ventricular markers, myosin heavy-chain beta and cyclin D2, indicated that not all ventricle-specific gene expression is dependent on Nkx2-5. The data demonstrate that Nkx2-5 is essential for normal heart morphogenesis, myogenesis, and function. Furthermore, this gene is a component of a genetic pathway required for myogenic specialization of the ventricles.

A murine cardiac-specific homeodomain gene named csx (Komuro, I., and Izumo. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8145-8149) and nkx-2.5 (Lints, T. J., Parsons, L. M., Hartley, L., Lyons, I., and Harvey, R. P. (1993) Development 119, 419-431) was identified as a potential vertebrate homologue of Drosophila tinman, a mesoderm determination factor required for insect heart formation (Bodmer, R. (1993) Development 118, 719-729). Bacterial expression of the nkx-2.5 homeodomain allowed us to identify downstream DNA targets from a library of randomly generated oligonucleotides. High affinity nkx-2.5 DNA binding sites, 5'-TNNAGTG-3', represented novel binding sequences, whereas intermediate and weaker affinity sites, 5'-C(A/T)TTAATTN-3', contained the typical 5'-TAAT-3' core required by most homeodomain factors for DNA binding. We also observed that nkx-2.5 served as a modest transcription activator in transfection assays done in 10T1/2 fibroblasts with multimerized binding sites linked to a luciferase reporter gene. Functional dissection of nkx-2.5 revealed a COOH-terminal inhibitory domain composed mainly of clusters of alanines and prolines, which appeared to mask a potent activation domain composed of hydrophobic and highly charged amino acids.

To elucidate the mechanisms of early heart morphogenesis in Xenopus laevis, we examined the effect of endoderm on heart morphogenesis in the early Xenopus neurula. Explants of anterior ventral (presumptive heart) mesoderm from early neurula were cultured alone or in combination with endoderm dissected from various regions. Heart formation was scored by an original heart index based on morphology. These explant studies revealed that anterior ventral endoderm plays a critical role in heart morphogenesis. Furthermore, we found that it was possible to confer this heart-forming ability on posterior ventral endoderm by the injection of poly(A)+ RNA from stage 13 anterior endoderm. These results imply that the heart formative factor(s) is localized in the anterior endoderm of the early neurula and that at least part of this activity is encoded by mRNA(s).

In order to understand how the heart is formed in the embryo, it is essential to understand the cellular movements and interactions that are needed for heart formation. This is the domain of classical embryology studies. It is also important to understand the molecules that mediate these interactions and direct cellular differentiation. This is the domain of molecular biology. In isolation, neither of these fields of study will provide a complete picture of how a heart is formed. Recently, a combination of these approaches has greatly increased the general understanding of embryonic heart development. We wish to present an overview of heart embryology and also to show how current molecular results are enhancing our understanding of heart formation.

The spatially and temporally coordinated interaction between migratory cardiac precursors, endothelial cells and myogenic precursors leads to the formation of the highly differentiated cell lineages in the heart. In a new study, conditional mouse mutants are used to show that Nf1 signaling in cardiac endothelium is essential for proper heart formation.

The purpose of the present study was to determine the relationship between concentration of Zn, Cu and Fe, and the catalase, glutathione peroxidase and superoxide dismutase activities in the heart and liver of newborn rats whose dams were fed a diet supplemented with caffeine. Heart Zn levels of the 22- and 30-day-old rats of the caffeine group showed a decrease, whereas liver Zn levels showed an increase compared to the control. Cu levels in the liver at day 22 in the caffeine group were less than in the control. Cu- and Zn-containing superoxide dismutase activities showed an increase in the hearts of the caffeine group compared to the control. The activity of catalase and glutathione peroxidase showed no difference in the heart and liver between the groups. The present study suggests the possible involvement of superoxide dismutase enzyme in the impairment of heart formation as a result of chronic caffeine intake in the early growing period.

A unique myosin heavy chain cDNA (AMHC1), which is expressed exclusively in the atria of the developing chicken heart, was isolated and used to study the generation of diversified cardiac myocyte cell lineages. The pattern of AMHC1 gene expression during heart formation was determined by whole-mount in situ hybridization. AMHC1 is first activated in the posterior segment of the heart when these myocytes initially differentiate (Hamburger and Hamilton stage 9+). The anterior segment of the heart at this stage does not express AMHC1 although the ventricular myosin heavy chain isoform is strongly expressed beginning at stage 8+. Throughout chicken development, AMHC1 continues to be expressed in the posterior heart tube as it develops into the diversified atria. The early activation of AMHC1 expression in the posterior cardiac myocytes suggests that the heart cells are diversified when they differentiate initially and that the anterior heart progenitors differ from the posterior heart progenitors in their myosin isoform gene expression. The expression domain of AMHC1 can be expanded anteriorly within the heart tube by treating embryos with retinoic acid as the heart primordia fuse. Embryos treated with retinoic acid prior to the initiation of fusion of the heart primordia express AMHC1 throughout the entire heart-forming region and fusion of the heart primordia is inhibited. These data indicate that retinoic acid treatment produces an expansion of the posterior (atrial) domain of the heart and suggests that diversified fates of cardiomyogenic progenitors can be altered.

We have isolated a Xenopus homeodomain sequence, XNkx-2.5, which shows significant similarity to mouse Nkx-2.5 and to the Drosophila tinman gene product. In Drosophila, tinman is required for formation of the heart and visceral mesoderm structures. In situ hybridization studies show that XNkx-2.5 is expressed in the heart region during early Xenopus development and later is also expressed in gut tissue. The observed similarity of sequences and expression patterns suggests that the regulatory mechanisms underlying heart formation may be conserved between distant species.

Publisher Summary Recent advances in the study of endothelial cell origin and assembly were made possible by the production of monoclonal antibodies that labeled angioblasts and endothelium. Another useful property of these monoclonal antibodies is that they label quail angioblasts but not chick cells so that endothelial cell differentiation and migration in quail/chick chimeras can be followed. Descriptive studies of quail embryos using the QH-1 antibody revealed that angioblasts arise at discrete locations and then assemble into cords at the sites of the blood vessel rudiments. These antibodies have also been used at older stages of embryogenesis in parallel with corrosion casting to examine vessel remodeling. Risau and co-workers were the first to distinguish between two different mechanisms of embryonic blood vessel formation: vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells and ungiogenesis, the sprouting of capillaries from preexisting vessels. Blockage and transplantation studies have been used to demonstrate the important role of angioblast migration in both of these mechanisms. These studies are discussed in detail in this chapter. Noden has also found evidence from grafting experiments for a subset of invasive angioblasts in the head that migrate extensively as individual cells initially unlabeled by QH-1 or related antibodies. The formation of dorsal aortae, intersomitic arteries, and endocardium has been followed in mouse embryos using a fluorescent lectin and antisera against von Willebrand factor. Serial sections, reconstructions, and scanning electron microscopy have been used to carefully examine the origin of the endocardium as a single tube in mouse embryos, particularly in relation to the foldings of the embryo that accompany heart formation.

Background: Congenital heart diseases (CHD) are the most common heart developmental anomaly disease. It occurs in approximately 0.4%b5% of all newborns and continues to be a significant cause of death in infancy. Nevertheless, the aetiology of CHD in the majority of cases remains unknown. Heart formation is a complex process regulated by many transcription factors. Although most of CHD cases are sporadic, there are several genes which were found to be associated with CHD, including NKX2.5, TBX5, GATA4, etc.

The homeobox-containing gene tinman (msh-2, Bodmer et al., 1990Development 110, 661-669) is expressed in the mesoderm primordium, and this expression requires the function of the mesoderm determinant twist. Later in development, as the first mesodermal subdivisions are occurring, expression becomes limited to the visceral mesoderm and the heart. Here, I show that the function of tinman is required for visceral muscle and heart development. Embryos that are mutant for the tinman gene lack the appearance of visceral mesoderm and of heart primordia, and the fusion of the anterior and posterior endoderm is impaired. Even though tinman mutant embryos do not have a heart or visceral muscles, many of the somatic body wall muscles appear to develop although abnormally. When the tinman cDNA is ubiquitously expressed in tinman mutant embryos, via a heatshock promoter, formation of heart cells and visceral mesoderm is partially restored. tinman seems to be one of the earliest genes required for heart development and the first gene reported for which a crucial function in the early mesodermal subdivisions has been implicated.

Background Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis. Methodology/Principal Findings Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential. Conclusions/Significance These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

Background Vertebrate limb development involves a reciprocal feedback loop between limb mesenchyme and the overlying apical ectodermal ridge (AER). Several gene pathways participate in this feedback loop, including Fgf signaling. In the forelimb lateral plate mesenchyme, Tbx5 activates Fgf10 expression, which in turn initiates and maintains the mesenchyme/AER Fgf signaling loop. Recent findings have revealed that Tbx5 transcriptional activity is regulated by dynamic nucleocytoplasmic shuttling and interaction with Pdlim7, a PDZ-LIM protein family member, along actin filaments. This Tbx5 regulation is critical in heart formation, but the coexpression of both proteins in other developing tissues suggests a broader functional role. Results Knock-down of Pdlim7 function leads to decreased pectoral fin cell proliferation resulting in a severely stunted fin phenotype. While early gene induction and patterning in the presumptive fin field appear normal, the pectoral fin precursor cells display compaction and migration defects between 18 and 24 hours post-fertilization (hpf). During fin growth fgf24 is sequentially expressed in the mesenchyme and then in the apical ectodermal ridge (AER). However, in pdlim7 antisense morpholino-treated embryos this switch of expression is prevented and fgf24 remains ectopically active in the mesenchymal cells. Along with the lack of fgf24 in the AER, other critical factors including fgf8 are reduced, suggesting signaling problems to the underlying mesenchyme. As a consequence of perturbed AER function in the absence of Pdlim7, pathway components in the fin mesenchyme are misregulated or absent, indicating a breakdown of the Fgf signaling feedback loop, which is ultimately responsible for the loss of fin outgrowth. Conclusion This work provides the first evidence for the involvement of Pdlim7 in pectoral fin development. Proper fin outgrowth requires fgf24 downregulation in the fin mesenchyme with subsequent activation in the AER, and Pdlim7 appears to regulate this transition, potentially through Tbx5 regulation. By controlling Tbx5 subcellular localization and transcriptional activity and possibly additional yet unknown means, Pdlim7 is required for proper development of the heart and the fins. These new regulatory mechanisms may have important implications how we interpret Tbx5 function in congenital hand/heart syndromes in humans.

We have examined the tissue interactions responsible for the expression of heart-forming potency during gastrulation. By comparing the specification of different regions of the marginal zone, we show that heart-forming potency is expressed only in explants containing both the dorsal lip of the blastopore and deep mesoderm between 30° and 45° lateral to the dorsal midline. Embryos from which both of these 30°-45° dorsolateral regions have been removed undergo heart formation in two thirds of cases, as long as the dorsal lip is left intact. If the dorsal lip is removed along with the 30° – 45° regions, heart formation does not occur. These results indicate that the dorsolateral deep mesoderm must interact with the dorsal lip in order to express heart-forming potency. Transplantation of the dorsal lip into the ventral marginal zone of host embryos results in the formation of a secondary axis; in over half of cases, this secondary axis includes a heart derived from the host mesoderm. These findings suggest that the establishment of heart mesoderm is initiated by a dorsalizing signal from the dorsal lip of the blastopore.

The zinc-finger transcription factor Ovol2 (Movo, Movo2) is a mouse homologue of Drosophila ovo, which is essential for the survival and differentiation of female germ line cells. To elucidate OVOL2 function in mammals, we generated Ovol2-deficient mice by gene targeting. The Ovol2 mutants died at embryonic days 9.5-10.5 (E9.5-E10.5), as a result of defects in extraembryonic and embryonic vascularization, and in heart formation. Although the Ovol2 expression was weak, severe defects were detected in extraembryonic and embryonic vascularization, and in heart formation at E8.5-E9.5. In Ovol2(-/-) placentas, allantoic blood vessel expansion and development of the labyrinthine layer were impaired at E10.5. In an endothelial cell line, siRNAs for Ovol2 reduced the expression of Ovol2 and inhibited the capillary-like network formation on Matrigel in vitro. These results demonstrate that Ovol2 may play a critical role in vascular angiogenesis during early embryogenesis.

Mutations in several transcription factors that regulate cardiac development have recently been described to cause congenital heart disease in humans. A greater understanding of the molecular mechanisms that regulate heart formation may help identify other genes that when mutated will result in human congenital heart disease. FOG-2 is one such gene that encodes a transcriptional corepressor expressed in the developing heart. It is critical for proper cardiac morphogenesis as mice deficient in this factor die during midgestation of cardiac malformations. We have previously shown that FOG-2 physically interacts with GATA4 and attenuates GATA49s ability to activate cardiac specific gene expression. This repression is mediated by a domain of FOG-2 localized to its N-terminus, termed the FOG repression motif. To gain further insights into the molecular mechanism responsible for this repression, we took a biochemical approach to identify factors that interact with FOG repression motif. Using MALDI-TOF mass spectrometry, we identified seven proteins from rat neonatal cardiac nuclear extracts that copurified with a FOG-2-GST fusion protein. All of these proteins have been previously described to be subunits of a nucleosome-remodeling complex called the NuRD complex. To determine which of the NuRD subunits directly interact with the FOG repression motif, we used a series of in vitro binding assays. We found that MTA-1 and MTA-2 specifically bound to the N-terminus of FOG-2 but not to a mutant form of the N-terminus that is unable to mediate repression. In situ hybridization revealed that both MTA-1 and MTA-2 are expressed in the developing heart during mouse embryogenesis. Taken together, these results suggest that FOG-2 mediates transcriptional repression during cardiac development by the recruitment of the NuRD complex to GATA-dependent promoters leading to the remodeling of the local chromatin structure and the attenuation of gene expression. This work was supported by NIH HL071063 and a grant from the Schweppe Foundation.

Introduction: Cardiac malformations occur in some humans who have chromosome abnormalities or malformation syndromes. Genetic control of heart development has been investigated in animal models, and early pregnancy failure is observed in mice when certain genes are altered. We hypothesize that some Growth Disorganized (GD) human embryos have had a failure of cardiac development. We suggest that cardiovascular abnormalities contributed to the pregnancy failure and spontaneous abortion (SAB). Methods: To test this, we reviewed the Embryo Collection of the Perinatal Pathology Service of Magee-Womens Hospital, comparing 35 externally normal embryos, Carnegie Stages 11-16, to 41 externally abnormal embryos, GD 2 and 3, for external evidence of normal heart formation: the presence of a heart bulge. We also evaluated the gross appearance of placenta] villi, if these were stored with the embryo. A difference in frequency of observations between the two groups was sought by χ2 analysis. Results: Each 35 of the normal embryos had a heart bulge, and all 10 of the embryos with stored villi had grossly normal villi. The GD embryos were significantly different. Only 11 of 41 GD embryos (27%) had a heart bulge (X2 =39.3, p

SummaryPoly(A)-attached RNA (mRNA) was isolated from chicken and calf hearts. Both mRNAs were found to induce the development of explanted postnodal pieces (PNP) of stage 4 chick blastoderm into beating tubes and/or tissues. Heart formation is specific and the frequency was found to depend entirely upon the concentration of mRNA used. In contrast, the concentration had no influence on other tissue formation e.g., neural tube, tubules, myoblasts, and/or notochord.The uptake of heart [3H]RNA by PNP cells was analyzed using high-resolution autoradiography. Silver grains were localized more in nucleus than in cytoplasm. The effect of heart mRNA on PNP was sensitive to actinomycin D and bromodeoxyuridine. It appears, therefore, that exogenous mRNA plays an important role in nuclear function. This RNA may affect the nuclei of intact cells in two ways: (1) as a derepressor, and (2) as a primer of new DNA synthesis. These alternatives were tested experimentally using the isozyme, lactate dehydrogenase, as marker....

The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.

An anterior-posterior concentration difference of fibronectin associated with the endoderm in early chick embryos has been implicated in the directional migration of precardiac mesoderm cells. We have examined the effect of increasing concentrations of an antibody to fibronectin (FN) to test the essentiality of FN to precardiac cell migration. For controls embryos were incubated in the presence of antibodies produced against several other extracellular components, such as laminin and anti-collagen types I and IV, as well as against integrin, a cell surface FN receptor. Embryos were also incubated in the presence of a high concentration of exogenous FN, as well as in the presence of an RGD-containing synthetic pentapeptide that is recognized by the FN receptor. After incubation of chick embryos in various concentrations of anti-FN (5 to 80 micrograms/ml), a dose-dependent effect of anti-fibronectin was observed, whereby heart development was arrested at high concentrations of anti-FN. Early developmental stages were more susceptible to lower antibody concentrations than later stages. Incubation in the presence of the RGD-containing synthetic peptide resulted in partial cardiabifida. None of the antibodies serving as controls affected cell migration or early heart development. These results support the hypothesis that FN is a major component in the migratory pathway and plays a role in the directional migration of precardiac cells to the embryonic midline.

The physiological effect of fresh calf heart-RNA was studied on the explanted chick blastoderm at the definitive streak stage. It was found that heart-RNA interferes with normal development of the central nervous system, especially forebrain, and of the body axis, but not with normal development of the heart. To analyse this effect further, the untreated and RNA-treated fragments of the antero-lateral blastoderm were investigated by intrablastodermal transplant and in vitro. Approximately 50% of the treated grafts transplanted intrablastodermally developed into heart, but none of the controls. In vitro formation of the heart-like structure was found in 45% of the heart-RNA-treated series as opposed to 20% of the PC saline controls and none of the liver-RNA series. When the explants of the presumptive forebrain were treated with heart-RNA and cultured in isolation in vitro, 11% developed into brain vesicle compared with 76% of the controls. It appears, therefore, that heart-RNA has somehow collaborated with the macromolecules responsible for heart formation but interfered with those responsible for the development of the central nervous system.

T HE rhythmic contractions of the lateral body walls in grasshopper embryos (Melanoplus diferentialis) were observed by Nelsen (i93i), who suggested that they represented rudiments of heart beats. These contractions have been employed as physiological indicators by Slifer (i934), working on the influence of anisotonic solutions, and by Walker (1935), in his study on carbon dioxide effects. It was, therefore, considered desirable to study the rate of behavior of these movements at different temperatures from the time of their initiation, through the period of heart formation, and up to the time of hatching.

The study of the embryology of Hemimysis Lamornæ , of which the following pages contain an account, was first undertaken with a view of tracing the later development of the mesoderm, and the formation of the heart and segmental excretory organs. The later embryological stages of the Malacostraca have not been investigated in much detail, and usually with poor fixation of the material. In view of the recent work of Cannon (1924 and 1927) on the development of Estheria and Chirocephalus , where the presence of segmental cœlomic cavities in the mesoderm and their relation to the heart formation has been demonstrated, it became highly desirable that some Malacostracan should be examined with similar care. A Mysid, although it is not liberated as a freeswimming nauplius, can be taken as one of the least specialised Malacostraca, possessing a long tubular heart, simple antennal gland and most of the external features of the caridoid facies. For these reasons, and for the fact that abundant material is available, the type selected was Hemimysis Lamornæ . However, it became evident that an examination of the earliest stages was equally necessary, and the development from segmentation stages onwards has been followed. In spite of abundant literature on Malacostracan germ-layer formation, the accounts are incomplete and often inaccurate, owing chiefly to inferior technique. In the Peracarida the origin of the germ layers and the formation and closure of the blastoporal area have not been satisfactorily worked out in any one type. Among the Decapod a the recent work of Sollaud (1923) on the Palæmoninæ is the most complete and careful account so far produced. Very little is known of the development of the Hoplocarida apart from the complicated metamorphosis. The embryology of the Syncarida is unknown. The literature contains much that is contradictory, especially concerning the Peracarida, so that a thorough account of the early development of any one form, and particularly of one of the Mysidacea which occupy a central position in the Peracarida, seems called for. Such a study would be useless unless a new technique was found.