Retinoic acid signaling in heart development

Retinoic acid (RA) is a vitamin A metabolite that acts as a morphogen and teratogen. Excess or defective RA signaling causes developmental defects including in the heart. The heart develops from the anterior lateral plate mesoderm. Cardiogenesis involves successive steps, including formation of the primitive heart tube, cardiac looping, septation, chamber development, coronary vascularization, and completion of the four‐chambered heart. RA is dispensable for primitive heart tube formation. Before looping, RA is required to define the anterior/posterior boundaries of the heart‐forming mesoderm as well as to form the atrium and sinus venosus. In outflow tract elongation and septation, RA signaling is required to maintain/differentiate cardiogenic progenitors in the second heart field at the posterior pharyngeal arches level. Epicardium‐secreted insulin‐like growth factor, the expression of which is regulated by hepatic mesoderm‐derived erythropoietin under the control of RA, promotes myocardial proliferation of the ventricular wall. Epicardium‐derived RA induces the expression of angiogenic factors in the myocardium to form the coronary vasculature. In cardiogenic events at different stages, properly controlled RA signaling is required to establish the functional heart.     

Recent insights on the role and regulation of retinoic acid signaling during epicardial development

The vitamin A metabolite, retinoic acid, carries out essential and conserved roles in vertebrate heart development. Retinoic acid signals via retinoic acid receptors (RAR)/retinoid X receptors (RXRs) heterodimers to induce the expression of genes that control cell fate specification, proliferation, and differentiation. Alterations in retinoic acid levels are often associated with congenital heart defects. Therefore, embryonic levels of retinoic acid need to be carefully regulated through the activity of enzymes, binding proteins and transporters involved in vitamin A metabolism. Here, we review evidence of the complex mechanisms that control the fetal uptake and synthesis of retinoic acid from vitamin A precursors. Next, we highlight recent evidence of the role of retinoic acid in orchestrating myocardial compact zone growth and coronary vascular development.     

Glycolipid glycosyltransferase activities during early development of Xenopus: effect of retinoic acid

Retinoic acid (RA) plays an important role in differentiation stage in which it also influences glycoconjugate metabolism. Previous work in our laboratory has shown that treatment with RA modifies glycolipid synthesis and distribution in total Xenopus embryos during development. In this study we have investigated the activity of the following anabolic enzymes involved in glycolipid biosynthesis: sialyltransferase-1 (SAT-1), GM3(beta1, 4)-N-acetylgalactosaminyltransferase (GalNAcT-1) and LacCer(beta1, 3)N-acetylglucosaminyltransferase (GlcNAcT-1). These enzymes are located at the branching point of lactosylceramide (Lc(2)) metabolism. Enzyme activities were assayed after treatment with different doses of RA added exogenously to the medium during the first 7 days of Xenopus embryo development. Our results show that RA activates GlcNAcT-1, the enzyme that drives Lc(2)to the glycolipids of the lacto-series, and SAT-1 that inserts Lc(2)in the ganglio-series pathway. These data support our previous analysis of glycolipid pattern in Xenopus embryos after RA treatment (Rizzo et al., 1995;Cell Biol Int19: 895-901) indicating a possible correlation between the distribution of glycolipids and the enzymes involved in their metabolism.     

Pharmacological evidence for the role of RAR in axon guidance and embryonic development of a protostome species

Retinoic acid (RA), the active metabolite of vitamin A, functions through nuclear receptors, one of which is the retinoic acid receptor (RAR). Though the RAR is essential for various aspects of vertebrate development, little is known about the role of RAR in nonchordate invertebrates. Here, we examined the potential role of an invertebrate RAR in mediating chemotropic effects of retinoic acid. The RAR of the protostome Lymnaea stagnalis is present in the growth cones of regenerating cultured motorneurons, and a synthetic RAR agonist (EC23), was able to mimic the effects of retinoic acid in inducing growth cone turning. We also examined the ability of the natural retinoids, all‐trans RA and 9‐cis RA, as well as the synthetic RAR agonists, to disrupt embryonic development in Lymnaea. Developmental defects included delays in embryo hatching, arrested eye, and shell development, as well as more severe abnormalities such as halted development. Developmental defects induced by some (but not all) synthetic RAR agonists were found to mimic those induced by addition of high concentrations of the natural retinoid isomers. These pharmacological data support a possible physiological role for the RAR in axon guidance and embryonic development of an invertebrate protostome species.     

The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development

We have used confocal microangiography to examine and describe the vascular anatomy of the developing zebrafish, Danio rerio. This method and the profound optical clarity of zebrafish embryos make it possible to view the entire developing vasculature with unprecedented resolution. A staged series of three-dimensional images of the vascular system were collected beginning shortly after the onset of circulation at 1 day postfertilization through early- to midlarval stages at approximately 7 days postfertilization. Blood vessels in every region of the animal were imaged at each stage, and detailed "wiring patterns" were derived describing the interconnections between every major vessel. We present an overview of these data here in this paper and in an accompanying Web site "The interactive atlas of zebrafish vascular anatomy" online at (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We find a highly dynamic but also highly stereotypic pattern of vascular connections, with different sets of primitive embryonic vessels severing connections and rewiring in new configurations according to a reproducible plan. We also find that despite variation in the details of the vascular anatomy, the basic vascular plan of the developing zebrafish shows strong similarity to that of other vertebrates. This atlas will provide an invaluable foundation for future genetic and experimental studies of vascular development in the zebrafish.     

Epicardium‐derived cells (EPDCs) in development,cardiac disease and repair of ischemia

The proepicardial-derived epicardium covers the myocardium and after a process of epithelial–mesenchymal transition (EMT) forms epicardium-derived cells (EPDCs). These cells migrate into the myocardium and show an essential role in the induction of the ventricular compact myocardium and the differentiation of the Purkinje fibres. EPDCs are furthermore the source of the interstitial fibroblast, the coronary smooth muscle cell and the adventitial fibroblast. The possible differentiation into cardiomyocytes, endothelial cells and the recently described telocyte and other cells in the cardiac stem cell niche needs further investigation. Surgically or genetically disturbed epicardial and EPDC differentiation leads to a spectrum of abnormalities varying from thin undifferentiated myocardium, which can be embryonic lethal, to a diminished coronary vascular bed with even absent main coronary arteries. The embryonic potential of EPDCs has been translated to both structural and functional congenital malformations and adult cardiac disease, like development of Ebstein’s malformation, arrhythmia and cardiomyopathies. Furthermore, the use of adult EPDCs as a stem cell source has been explored, showing in an animal model of myocardial ischemia the recapitulation of the embryonic program with improved function, angiogenesis and less adverse remodeling. Combining EPDCs and adult cardiomyocyte progenitor cells synergistically improved these results. The contribution of injected EPDCs was instructive rather than constructive. The finding of reactivation of the endogenous epicardium in ischemia with re-expression of developmental genes and renewed EMT marks the onset of a novel therapeutic focus.     

When an epithelium ceases to exist – an ultrastructural study on the fate of the embryonic coelom in Epiperipatus biolleyi (Onychophora, Peripatidae)

It is an accepted fact that fusion between the coelomic cavities and the primary body cavity occurs during development in the Arthropoda. However, such a fusion is much disputed in the Onychophora. In order to clarify this subject, the fate of embryonic coelomic cavities has been studied in an onychophoran. Ultrastructural investigations in this paper provide evidence that embryonic coelomic cavities fuse with spaces of the primary body cavity in Epiperipatus biolleyi. During embryogenesis, the somatic and splanchnic portions of the mesoderm separate and the former coelomic linings are transformed into mesenchymatic tissue. The resulting body cavity therefore represents a mixture of primary and secondary (coelomic) body cavities, i.e. the ‘mixocoel’. The nephridial anlage is already present, when the ‘mixocoel’ is formed, although there is no trace of a sacculus yet. The lumen of the nephridial anlage, thus, communicates with the newly formed ‘mixocoel’. Accordingly, the lumen of the nephridial sacculus cannot be regarded as a kind of ‘persisting coelomic cavity’ in E. biolleyi. Our findings support the hypothesis that the ‘mixocoel’ was already present in the common stem species of the Onychophora and Euarthropoda.     

Retinoic acid signaling in regulation of meiosis during embryonic development in mice

In the embryonic gonads of mice, the genetic and epigenetic regulatory programs for germ cell sex specification and meiosis induction or suppression are intertwined. The quest for garnering comprehensive understanding of these programs has led to the emergence of retinoic acid (RA) as an important extrinsic factor, which regulates initiation of meiosis in female fetal germ cells that have attained a permissive epigenetic ground state. In contrast, germ cells in fetal testis are protected from the exposure to RA due to the activity of CYP26B1, an RA metabolizing enzyme, which is highly expressed in fetal testis. In this review, we provide an overview of the molecular mechanisms operating in fetal gonads of mice, which enable regulation of meiosis via RA signaling.     

Chimeric analysis of retinoic acid receptor function during cardiac looping

Retinoids (vitamin A and its derivatives) play essential roles during vertebrate development. Vitamin A deprivation leads to severe congenital malformations affecting many tissues, including diverse neural crest cell populations and the heart. The vitamin A signal is transduced by the retinoic acid receptors (RARalpha, RARbeta, and RARgamma). However, these receptors exhibit considerable functional redundancy, as judged by the mild phenotype of RAR single null mutants relative to the defects evoked by loss of multiple RARs. To circumvent this redundancy, the endogenous RARgamma2 allele was replaced with a ligand-binding RARgamma mutant (RARgammaE(305)) by gene targeting in mouse embryonic stem (ES) cells. Chimeric embryos derived from hemizygous RARgammaE(305) ES cells displayed several defects similar to those observed in certain RAR double null mutants, including hypoplasia or absence of the caudal pharyngeal arches and myocardial deficiencies. The latter defects were not due to abnormal cardiac specification as affected hearts still expressed chamber-specific markers in an appropriate manner. Chimeras also displayed cardiac looping anomalies, which were associated with a reduction of Pitx2. This work suggests a role for RAR signaling in late looping morphogenesis and illustrates the utility of using a dominant-negative gene substitution approach to circumvent the functional redundancy inherent to the RAR family.     

Bioassay development: The implications of cardiac myocyte motility in vitro

Summary Cardiac myocytes cultured over microfabricated extracellular recording devices can be used to assay bioactive compounds. However, electrophysiological signals recorded from these devices vary in amplitude with time. Theoretically, changes in signal amplitude arise from myocytes being moved over recording sites by cocultured fibroblasts. To test this, neonatal rat cardiac myocytes were cultured at high densities and low densities on fibronectin-coated glass. After 36.5 h, myocytes were identified by their rhythmic contractions and then time-lapse-recorded for 3.5 h. Length, width, and angle of orientation was then determined every 30 min for five cells in low density and five cells in high-density culture. Low-density cells had mean lengths of 65.3 μm and widths of 35.1 μm, whereas cells in high-density culture had greater mean lengths of 74.2 μm and lower mean widths of 24.3 μm. Length, width, and angle of orientation of cells in low- and high-density culture changed by 4.1%, 11.8%, and 2.7 degrees, and 6.4%, 10%, and 4.6 degrees, respectively, every half hour. We found no evidence of myocyte-fibroblast interactions influencing cell position or shape in low density, but in high density, we found evidence that fibroblast-myocyte interactions could transiently influence cell shape. We conclude that fibroblast-independent changes in cell shape are largely responsible for the changes in signal amplitude recorded from cardiac myocytes cultured on microfabricated extracellular recording devices. However, there is some evidence that myocyte-fibroblast interactions may augment this process in high-density culture. The implications of these findings for bioassay development are discussed.