Retinoic acid signaling is essential for formation of the heart tube in Xenopus

Retinoic acid is clearly important for the development of the heart. In this paper, we provide evidence that retinoic acid is essential for multiple aspects of cardiogenesis in Xenopus by examining embryos that have been exposed to retinoic acid receptor antagonists. Early in cardiogenesis, retinoic acid alters the expression of key genes in the lateral plate mesoderm including Nkx2.5 and HAND1, indicating that early patterning of the lateral plate mesoderm is, in part, controlled by retinoic acid. We found that, in Xenopus, the transition of the heart from a sheet of cells to a tube required retinoic acid signaling. The requirement for retinoic acid signaling was determined to take place during a narrow window of time between embryonic stages 14 and 18, well before heart tube closure. At the highest doses used, the lateral fields of myocardium fail to fuse, intermediate doses lead to a fusion of the two sides but failure to form a tube, and embryos exposed to lower concentrations of antagonist form a heart tube that failed to complete all the landmark changes that characterize looping. The myocardial phenotypes observed when exposed to the retinoic acid antagonist resemble the myocardium from earlier stages of cardiogenesis, although precocious expression of cardiac differentiation markers was not seen. The morphology of individual cells within the myocardium appeared immature, closely resembling the shape and size of cells at earlier stages of development. However, the failures in morphogenesis are not merely a slowing of development because, even when allowed to develop through stage 40, the heart tubes did not close when embryos were exposed to high levels of antagonist. Indeed, some aspects of left-right asymmetry also remained even in hearts that never formed a tube. These results demonstrate that components of the retinoic acid signaling pathway are necessary for the progression of cardiac morphogenesis in Xenopus.     

Myocardial cell relationships during morphogenesis in normal and cardiac lethal mutant axolotls, Ambystoma mexicanum

Sarcomere formation has been shown to be deficient in the myocardium of axolotl embryos homozygous for the recessive cardiac lethal gene c. We examined the developing hearts of normal and cardiac mutant embryos from tailbud stage 33 to posthatching stage 43 by scanning electron microscopy in order to determine whether that deficiency has any effect on heart morphogenesis. Specifically, we investigated the relationships of myocardial cells during the formation of the heart tube (stage 33), the initiation of dextral looping (stages 34-36), and the subsequent flexure of the elongating heart (stages 38-43). In addition, we compared the morphogenetic events in the axolotl to the published accounts of comparable stages in the chick embryo. In the axolotl (stage 33), changes in cell shape and orientation accompany the closure of the myocardial trough to form the tubular heart. The ventral mesocardium persists longer in the axolotl embryo than in the chick and appears to contribute to the asymmetry of dextral looping (stages 34-36) in two ways. First, as a persisting structure it places constraints on the simple elongation of the heart tube and the ability of the heart to bend. Second, after it is resorbed, the ventral myocardial cells that contributed to it are identifiable by their orientation, which is orthogonal to adjacent cells: a potential source of shearing effects. Cardiac lethal mutant embryos behave identically during these events, indicating that functional sarcomeres are not necessary to these processes. The absence of dynamic apical myocardial membrane changes, characteristic of the chick embryo (Hamburger and Hamilton stages 9-11), suggests that sudden hydration of the cardiac jelly is less likely to be a major factor in axolotl cardiac morphogenesis. Subsequent flexure (stages 38-43) of the axolotl heart is the same in normal and cardiac lethal mutant embryos as the myocardial tube lengthens within the confines of a pericardial cavity of fixed length. However, the cardiac mutant begins to exhibit abnormalities at this time. The lack of trabeculation (normally beginning at stage 37) in the mutant ventricle is evident at the same time as an increase in myocardial surface area, manifest in extra bends of the heart tube at stage 39. Nonbeating mutant hearts (stage 41) have an abnormally large diameter in the atrioventricular region, possibly the result of the accumulation of ascites fluid. In addition, mutant myocardial cells have a larger apical surface area compared to normals.     

Subdivision of the cardiac Nkx2.5 expression domain into myogenic and nonmyogenic compartments

Nkx2.5 is expressed in the cardiogenic mesoderm of avian, mouse, and amphibian embryos. To understand how various cardiac fates within this domain are apportioned, we fate mapped the mesodermal XNkx2.5 domain of neural tube stage Xenopus embryos. The lateral portions of the XNkx2.5 expression domain in the neural tube stage embryo (stage 22) form the dorsal mesocardium and roof of the pericardial cavity while the intervening ventral region closes to form the myocardial tube. XNkx2.5 expression is maintained throughout the period of heart tube morphogenesis and differentiation of myocardial, mesocardial, and pericardial tissues. A series of microsurgical experiments showed that myocardial differentiation in the lateral portion of the field is suppressed during normal development by signals from the prospective myocardium and by tissues located more dorsally in the embryo, in particular the neural tube. These signals combine to block myogenesis downstream of XNkx2.5 and at or above the level of contractile protein gene expression. We propose that the entire XNkx2.5/heart field is transiently specified as cardiomyogenic. Suppression of this program redirects lateral cells to adopt dorsal mesocardial and dorsal pericardial fates and subdivides the field into distinct myogenic and nonmyogenic compartments.     

Early myocardial function affects endocardial cushion development in zebrafish

Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih^−/−) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk^−/−) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk^−/− embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfk^s11 variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.     

A mutation in zebrafish hmgcr1b reveals a role for isoprenoids in vertebrate heart-tube formation

In vertebrates, the morphogenetic assembly of the primitive heart tube requires the medial migration and midline fusion of the bilateral myocardial epithelia. Several mutations that result in abnormal heart-tube formation have been studied; however, an understanding of the underlying molecular and cellular mechanisms of the migration and fusion of these epithelial sheets is far from complete. In a forward genetic screen to identify genes regulating early zebrafish heart development, we identified a mutation in the 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 1b (hmgcr1b) gene that affects myocardial migration to the midline and subsequent heart-tube morphogenesis. The mutant phenotype can be rescued with injections of mevalonate, the direct product of HMGCR activity. Furthermore, treatment of embryos with pharmacological inhibitors of isoprenoid synthesis, which occurs downstream of mevalonate production, resulted in defective heart-tube formation. Interestingly, in hmgcr1b mutant embryos and embryos treated with HMGCR inhibitors, both RasCT20-eGFP and RhoaCT32-eGFP fusion proteins were mislocalized away from the plasma membrane in embryonic myocardial cells. We conclude that protein prenylation, acting downstream of Hmgcr1b and possibly through Ras and, or, Rho signaling, is required for the morphogenesis of the myocardial sheets for formation of the primitive heart tube.     

N-cadherin is crucial for heart formation in the chick embryo

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.     

Effect of Vascular Cadherin Knockdown on Zebrafish Vasculature during Development

Background

Vascular endothelial cadherin (VE-cad) is essential for endothelial barrier integrity and vascular sprouting. However, the role of this important protein in cardiovascular development is only recently becoming apparent.

Methodology/Principal Findings

To characterize the role of VE-cadherin in cardiovascular development, we analyzed cardiovascular development in a zebrafish VE-cad knockdown model. Embryos deficient in VE-cad show profoundly impaired cardiac development despite having apparently normal peripheral vasculature. Initial formation of the heart proceeds normally in knockdown embryos, but subsequent looping morphogenesis is impaired. Consistent with these results, VE-cad knockdown embryos demonstrate impaired cardiac function and early circulatory arrest. Histologic examination of knockdown embryos shows persistent, abnormal separation of the endocardial and myocardial layers. Using transmission electron microscopy, we demonstrate that endocardial junctions form poorly in VE-cad knockdown embryos, with resulting leak across the endothelial layer and reduction in the density of the cardiac jelly.

Conclusions

Our results demonstrate a significant role for VE-cadherin in cardiac development independent of its effects on the formation of the peripheral vasculature.     

The anterior heart-forming field: voyage to the arterial pole of the heart

Studies of vertebrate heart development have identified key genes and signalling molecules involved in the formation of a myocardial tube from paired heart-forming fields in splanchnic mesoderm. The posterior region of the paired heart-forming fields subsequently contributes myocardial precursor cells to the inflow region or venous pole of the heart. Recently, a population of myocardial precursor cells in chick and mouse embryos has been identified in pharyngeal mesoderm anterior to the early heart tube. This anterior heart-forming field gives rise to myocardium of the outflow region or arterial pole of the heart. The amniote heart is therefore derived from two myocardial precursor cell populations, which appear to be regulated by distinct genetic programmes. Discovery of the anterior heart-forming field has important implications for the interpretation of cardiac defects in mouse mutants and for the study of human congenital heart disease.     

Growth of the developing mouse heart: an interactive qualitative and quantitative 3D atlas

Analysis of experiments aimed at understanding the genetic mechanisms of differentiation and growth of the heart, calls for detailed insights into cardiac growth and proliferation rate of myocytes and their precursors. Such insights in mouse heart development are currently lacking. We quantitatively assessed the 3D patterns of proliferation in the forming mouse heart and in the adjacent splanchnic mesoderm, from the onset of heart formation till the developed heart at late gestation. These results are presented in an interactive portable document format (Suppl. PDF) to facilitate communication and understanding. We show that the mouse splanchnic mesoderm is highly proliferative, and that the proliferation rate drops upon recruitment of cells into the cardiac lineage. Concomitantly, the proliferation rate locally increases at the sites of chamber formation, generating a regionalized proliferation pattern. Quantitative analysis shows a gradual decrease in proliferation rate of the ventricular walls with progression of development, and a base-to-top decline in proliferation rate in the trabecules. Our data offers clear insights into the growth and morphogenesis of the mouse heart and shows that in early development the phases of tube formation and chamber formation overlap. The resulting interactive quantitative 3D atlas of cardiac growth and morphogenesis provides a resource for interpretation of mechanistic studies.     

Heart and soul/PRKCi and nagie oko/Mpp5 regulate myocardial coherence and remodeling during cardiac morphogenesis

Organ morphogenesis requires cellular shape changes and tissue rearrangements that occur in a precisely timed manner. Here, we show that zebrafish heart and soul (Has)/protein kinase C iota (PRKCi) is required tissue-autonomously within the myocardium for normal heart morphogenesis and that this function depends on its catalytic activity. In addition, we demonstrate that nagie oko (Nok) is the functional homolog of mammalian protein associated with Lin-seven 1 (Pals1)/MAGUK p55 subfamily member 5 (Mpp5), and we dissect its earlier and later functions during myocardial morphogenesis. Has/PRKCi and Nok/Mpp5 are required early for the polarized epithelial organization and coherence of myocardial cells during heart cone formation. Zygotic nok/mpp5 mutants have later myocardial defects, including an incomplete heart tube elongation corresponding with a failure of myocardial cells to correctly expand in size. Furthermore, we show that nok/mpp5 acts within myocardial cells during heart tube elongation. Together, these results demonstrate that cardiac morphogenesis depends on the polarized organization and coherence of the myocardium, and that the expansion of myocardial cell size contributes to the transformation of the heart cone into an elongated tube.     

BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium

The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium.     

Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish.

The embryonic vertebrate heart is divided into two major chambers, an anterior ventricle and a posterior atrium. Although the fundamental differences between ventricular and atrial tissues are well documented, it is not known when and how cardiac anterior-posterior (A-P) patterning occurs. The expression patterns of two zebrafish cardiac myosin genes, cardiac myosin light chain 2 (cmlc2) and ventricular myosin heavy chain (vmhc), allow us to distinguish two populations of myocardial precursors at an early stage, well before the heart tube forms. These myocardial subpopulations, which may represent the ventricular and atrial precursors, are organized in a medial-lateral pattern within the precardiac mesoderm. Our examinations of cmlc2 and vmhc expression throughout the process of heart tube assembly indicate the important role of an intermediate structure, the cardiac cone, in the conversion of this early medial-lateral pattern into the A-P pattern of the heart tube. To gain insight into the genetic regulation of heart tube assembly and patterning, we examine cmlc2 and vmhc expression in several zebrafish mutants. Analyses of mutations that cause cardia bifida demonstrate that the achievement of a proper cardiac A-P pattern does not depend upon cardiac fusion. On the other hand, cardiac fusion does not ensure the proper A-P orientation of the ventricle and atrium, as demonstrated by the heart and soul mutation, which blocks cardiac cone morphogenesis. Finally, the pandora mutation interferes with the establishment of the early medial-lateral myocardial pattern. Altogether, these data suggest new models for the mechanisms that regulate the formation of a patterned heart tube and provide an important framework for future analyses of zebrafish mutants with defects in this process.