The Wingless Signaling Pathway Is Directly Involved inDrosophilaHeart Development

The heart is the first organ to form and function during development. In the pregastrula chick embryo, cells contributing to the heart are found in the postero-lateral epiblast. During the pregastrula stages, interaction between the posterior epiblast and hypoblast is required for the anterior lateral plate mesoderm (ALM) to form, from which the heart will later develop. This tissue interaction is replaced by an Activin-like signal in culture. During gastrulation, the ALM is committed to the heart lineage by endoderm-secreted BMP and subsequently differentiates into cardiomyocyte. The right and left precardiac mesoderms migrate toward the ventral midline to form the beating primitive heart tube. Then, the heart tube generates a right-side bend, and the d-loop and presumptive heart segments begin to appear segmentally: outflow tract (OT), right ventricle, left ventricle, atrioventricular (AV) canal, atrium and sinus venosus. T-box transcription factors are involved in the formation of the heart segments: Tbx5 identifies the left ventricle and Tbx20 the right ventricle. After the formation of the heart segments, endothelial cells in the OT and AV regions transform into mesenchyme and generate valvuloseptal endocardial cushion tissue. This phenomenon is called endocardial EMT (epithelial-mesenchymal transformation) and is regulated mainly by BMP and TGFbeta. Finally, heart septa that have developed in the OT, ventricle, AV canal and atrium come into alignment and fuse, resulting in the completion of the four-chambered heart. Altered development seen in the cardiogenetic process is involved in the pathogenesis of congenital heart defects. Therefore, understanding the molecular nature regulating the 'nodal point' during heart development is important in order to understand the etiology of congenital heart defects, as well as normal heart development.

Wg signaling in Drosophila heart development as a pioneering model

Nkx2.5, a homeodomain-containing transcription factor, is known to be necessary for normal heart development in vertebrates. It is one of the earliest lineage-restricted genes expressed in cardiovascular progenitor cells and knowledge of its expression patterns has important therapeutic implications for damaged cardiomyocytes. Mexican axolotl is a unique system to study heart development for two reasons: the presence of a mutant phenotype lacking organized myofibrils due to sarcomeric tropomyosin deficiency and the ability to induce metamorphosis by administration of exogenous thyroid hormone. In this study, we cloned and sequenced the as yet uncharacterized Nkx2.5 cDNA from normal and cardiac mutant axolotl heart RNA. Comparison of cDNA sequences of Nkx2.5 from normal and mutant axolotl hearts did not show differences suggesting that loss of function mutation in Nkx2.5 is not responsible for the mutant phenotype. However, quantitative studies show higher expression of Nkx2.5 in mutant hearts raising the possibility that increased expression of Nkx2.5 may contribute to the mutant phenotype. We also evaluated quantitative changes in expression of Nkx2.5 in axolotl hearts during embryonic and postembryonic heart development induced by exogenous thyroid hormone. There is an apparent increase in Nkx2.5 transcript levels in metamorphosed hearts.

RNA binding proteins are a large family of proteins involved in the post-transcriptional regulation of target genes. These proteins regulate at the level of RNA through several processes including RNA stability, polyadenylation, and alternative splicing. Recently, the RNA binding motif protein, Rbm24a, has been identified for its roles in both eye and heart development in vertebrates, with knockdown and mutation of rbm24a leading to microphthalmia and cardiomyopathy respectively. We modulated rbm24a activity in zebrafish and found a dose-dependent effect leading from normal eyes to severe microphthalmia. We performed visual assays on moderately affected embryos and found visual defects which worsened with more severe phenotypes. In order to further understand the mechanism of rbm24a function in the eye, we identified a target RNA: the proliferative and pluripotency factor sox2. We demonstrated that sox2 RNA levels were reduced in rbm24a knockdown embryos and we examined proliferation and apoptosis in these knockdown zebrafish, finding evidence for increased apoptosis. In functional assays to determine the interaction between Rbm24a and sox2, we found the microphthalmia phenotype induced by depletion of rbm24a can be suppressed with exogenous sox2 RNA. Moreover, we demonstrated functional restoration of vision in embryos which received both rbm24a knockdown and sox2 RNA when compared to rbm24a knockdown alone. Finally, we characterized the cardiomyopathy phenotype and reduced heart tube size in rbm24a depleted embryos when compared to control embryos. A potential cardiac target of Rbm24a: myh6, an atrium-specific factor needed for proper contractions, was decreased in rbm24a depleted embryos. Taken together, my work displays the importance and multifunctionality of post-transcriptional regulation by RNA binding proteins, specifically rbm24a.

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.

In the formed heart, it is convention to distinguish working myocardium (the primary function of which is contraction) from the conduction system (the primary function of which is the generation and conduction of the electrical impulse). The conduction system comprises separate components with distinct functions. The SAN, which contains the leading pacemaker, generates the impulse. The impulse is subsequently conducted, via the atrial myocardium, which in this sense is part of the conduction pathway as well, toward the AVN. With a delay, the impulse is then rapidly transmitted from the AVN via the bundle branches and PPN to ensure a coordinated activation of the ventricular myocardium from apex to base. Classic reports cover the anatomy,1 pathology,1 and histology2 of the adult and developing conduction system. The myocytes of the conduction system share with those of the ordinary working myocardium four basic elements: (1) contraction, (2) autorhythmicity, (3) intercellular conduction, and (4) electromechanical coupling. In the early embryonic heart tube, an ECG, similar to an adult ECG, can be recorded, indicating the presence of sequentially activated chambers.3 Given this observation, it is as confusing to accept the presence of a conduction system because it is functionally present as it is to deny its existence because it is not morphologically recognizable. Rather, it is of paramount importance to appreciate that the arrangement of myocyte populations, with distinct contractile, conductive, and pacemaking properties, establishes the coordinated activation of the heart. Departures from these tenets have led to a confusing and fruitless search for so-called “cardiac specialized tissues” during development. The obvious key question is how this arrangement is being achieved. Early cardiac development starts with the formation of a primary heart tube from the cardiogenic mesoderm (Fig 1⇓); this topic has been reviewed recently.4 The primary heart …

The canonical Wingless signaling pathway is required but not sufficient for inflow tract formation in the Drosophila melanogaster heart

In zebrafish, two paralogous genes, activating molecule in beclin-1 (BECN1)-regulated autophagy ambra1a and ambra1b, both required for the autophagic process and during development, encode the protein AMBRA1, a positive regulator of early steps of autophagosome formation. As transcripts for both genes are expressed during embryogenesis in the heart region, in this work, we investigated the effects of ambra1a and ambra1b knockdown on heart development by means of morpholino oligonucleotides (MOs). Silencing of the two proteins by MOs directed against the ATG translation initiation codon affects cardiac morphogenesis, resulting in a small, string-like heart with pericardial edema, whereas treatment with splice-blocking MOs does not lead to overt cardiac phenotypes, thus revealing the relevance of maternally supplied ambra1 transcripts for heart development. Co-injection of both ATG-MOs determines a more severe cardiac phenotype, with prominent pericardial edema. Whole-mount in situ hybridization (WMISH) for myosin light chain 7 (myl7), as well as ambra1 ATG-MO microinjection in zebrafish transgenic line expressing green fluorescent protein in the heart, revealed defects with the heart jogging process followed by imperfect cardiac looping. Moreover, WMISH of homeodomain transcription factor 2 isoform c (pitx2c) transcripts showed both bilateral and reversed pitx2c expression in morphants. The morphants' cardiac phenotypes were effectively rescued by co-injection of MOs with human AMBRA1 (hAMBRA1) messenger RNA (mRNA), pointing at the conservation of Ambra1 functions during evolution. Co-injections of ambra1 ATG-MOs with a hAMBRA1 mRNA mutated in the protein phosphatase 2a (PP2A) binding sites (hAMBRA1PXP) were not able to rescue the cardiac phenotypes, at the difference from wild-type hAMBRA1 mRNA, and treatment of zebrafish embryos with the specific PP2A inhibitor cantharidin resulted in similar developmental cardiac defects. These results suggest a critical role for AMBRA1 in vertebrate heart development, likely involving the binding site for the PP2A phosphatase.

Introduction: Neural crest (NC) cells are involved in cardiac development, and NC defects account for over 10% of all congenital heart disease. The migratory and morphogenic patterns of these NC cells are not well understood because conventional optical microscopes struggle to acquire images of live embryos with sufficient temporal and spatial resolution without causing major photo-damage. To address these issues, the lab has designed and built a light-sheet fluorescence microscope (LSFM) that is capable of acquiring dual-channel signals with high spatial and temporal resolution. Hypothesis: We hypothesize that the LSFM system will capture heart tube development at a cellular level and show the time point and location at which the NC cells integrate with the heart tube. Methods and Results: The transgenic line that is manipulated for this study is the Tg(neural crest:nfsB-mCherry, cardiac: eGFP) in which the heart tube fluoresces green while NC-derived cells fluoresce red. 10 zebrafish were imaged using the 3-D time-lapse LSFM setup from 15 hours post fertilization (hpf) to 36 hpf. The imaging data was qualitative and consistent across fish. Fig. 1 (a) shows mCherry NC-derived cells (red) integrating with eGFP cardiomyocytes (green) and areas of colocalization (yellow). Fig 1 (b) and (c) show the time course of the NC cell migration into the heart tube. By 26-27 hpf, the NC cells contacted the dorsal surface of the heart tube. By the 29 hpf, the NC-derived cells integrated into the heart tube and expressed nuclear eGFP, which is visualized by the yellow fluorescing portions of the image. Conclusions: We conclude that the LSFM was successful in acquiring distinct signals from the heart tube cells and the NC-derived cells. The temporal and spatial resolution was sufficient to observe NC migration at the single cell level and map out exactly where along the heart tubule the NC-cells entered.

Convergent extension movements occur ubiquitously in animal development. This special type of cell movement is controlled by the Wnt/planar cell polarity (PCP) pathway. Here we show that Xenopus paraxial protocadherin (XPAPC) functionally interacts with the Wnt/PCP pathway in the control of convergence and extension (CE) movements in Xenopus laevis. XPAPC functions as a signalling molecule that coordinates cell polarity of the involuting mesoderm in mediolateral orientation and thus selectively promotes convergence in CE movements. XPAPC signals through the small GTPases Rho A and Rac 1 and c-jun N-terminal kinase (JNK). Loss of XPAPC function blocks Rho A-mediated JNK activation. Despite common downstream components, XPAPC and Wnt/PCP signalling are not redundant, and the activity of both, XPAPC and PCP signalling, is required to coordinate CE movements.

Homeotic Genes Autonomously Specify the Anteroposterior Subdivision of the Drosophila Dorsal Vessel into Aorta and Heart

In the developing vertebrate heart, cells that contribute to vascular smooth muscle, cardiac fibroblasts, and coronary endothelial cells are derived from the proepicardium and associated cells originally located at the venous pole of the primitive heart tube. Recently, there has been much interest in the regulatory mechanisms that control epicardium-derived cell (EPDC) lineage development and mobilization with adult cardiac injury. However, less is known of the embryonic origins and inductive mechanisms that control the initial induction of the proepicardium. The study by Schlueter and Brand1 in the current issue of Circulation Research reports fate mapping of proepicardial progenitors to the somatic mesoderm and demonstrates a critical role for the basic helix-loop-helix transcription factor Twist1 in the earliest stages of proepicardial development. Article, see p 1128 The heart is the first organ to function in the developing vertebrate embryo, and beating of the primitive heart tube is apparent by embryonic day (E)8 in mouse and by 40 hours of development in chicken embryos.2 As the primitive heart tube loops, lateral plate mesoderm cells on the right side of the embryo at the cardiac venous pole begin to form the proepicardium.3 Schlueter and Brand1 demonstrate through fate mapping studies in avian embryos that at least some cells of the proepicardium are derived from the somatic layer of lateral plate mesoderm. The somatic compartment (somatopleure) of the lateral plate mesoderm is adjacent to the ectoderm and forms the body wall.4 The new data provide initial evidence that cells of the proepicardium also are derived from somatic mesodermal layer. The proepicardium shares many characteristics with other mesothelia of the major coelomic organs that contribute progenitors of fibroblasts and smooth muscle to the lung, gut, kidney, etc.4 The embryonic origins of coelomic mesothelia are not …

Diverse classes of molecules regulate cell migration, one of the most fundamental processes in development and disease. In the vascular system, smooth muscle cell migration mediated by the PDGF-B pathway (1) is required for both normal blood vessel formation during development and pathogenesis of vascular diseases. In this paracrine signaling pathway, endothelial cells secrete PDGF-B, which acts on the PDGF-β receptor expressed on the surface of smooth muscle cell precursors and initiates the migration of the latter cells toward endothelial cells during angiogenesis. In this issue of the JCI, Liu et al. implicate another receptor and another class of mediator — bioactive glycolipids — in smooth muscle cell migration in the mouse (2). Remarkably, this same class of molecule was recently shown to mediate the migration of cardiac muscle progenitor cells during the initial formation of the heart tube in zebrafish development (3). Together, these two studies establish the presence of a common pathway, mediated by receptors for sphingosine-1-phosphate (S1P), underlying both smooth muscle and cardiac muscle migration during vascular and cardiac development (Figure ​(Figure11). Figure 1 New roles of lipid receptors in cardiac and vascular development. Receptors for S1P are critical for the migration of cardiac muscle precursors and smooth muscle cells during heart tube formation and blood vessel formation, respectively. See the text ... In the present study, Liu et al. (2) address the putative biological functions of one of the S1P receptors, Edg-1, by generating knockout mice for its gene. Edg-1 belongs to a family of G protein–coupled receptors for S1P or lysophosphatidic acid (4, 5). Edg-1 is a specific receptor for S1P and interacts directly with the Gi pathway. Targeted mutation of Edg-1 resulted in embryonic lethality (2), but the most intriguing feature of the Edg-1 knockout phenotype is the apparent lack of normal layers of smooth muscles surrounding endothelial cells of vessels (2). Interestingly, smooth muscle cells are clustered to the ventral side of the vessels and fail to surround the endothelial lumen of the vessels in a uniform fashion. Smooth muscle cell precursor cells have been thought to be recruited from any direction in the vessel’s vicinity (1). However, this polarized clustering of smooth muscle cells in Edg1 mutant embryos suggests an alternative model in which smooth muscle cells are recruited in a unidirectional manner (6, 7) and only later spread to surround the endothelium. This unidirectional recruitment is not understood, but it might be explained by an asymmetrically distributed chemoattractant gradient or by the presence of a critical adhesive factor on the ventral side of the developing vessel. In PDGF-B–deficient mice, in contrast, pericytes (contractile cells for capillaries equivalent to smooth muscle cells for larger vessels) are not unevenly distributed but are actually absent from vessels (8). This difference suggests the following model for the smooth muscle formation in developing vessels (Figure ​(Figure2):2): PDGF-B, secreted by endothelial cells undergoing angiogenesis, mediates the recruitment of smooth muscle precursors toward the immediate vicinity of the endothelial cells, where they accumulate in an asymmetric fashion. Once the smooth muscle precursors are immediately adjacent to endothelial cells, they are induced to differentiate to mature smooth muscle cells via signaling pathways including TGF-β, endoglin, and SMADs (1, 9–11). Following the formation of a complete vascular lumen by endothelial cells, S1P mediates the migration of smooth muscle cells either directly or indirectly to surround the endothelial layer. Because smooth muscle cell migration goes awry in many types of vascular disease (12, 13), this model could have profound clinical as well as biological implications. It is conceivable that the same or a related lipid receptor signaling pathway is critically involved in or could be exploited to treat human diseases. Figure 2 Sequential roles of PDGF-B/TGF-β/S1P in smooth muscle infiltration into vessels. During angiogenesis, endothelial cells that are yet to form a mature vascular lumen secrete PDGF-B to recruit smooth muscle precursor cells to their immediate vicinity. ...

See related article, pages 1000–1007 Abnormal development of the arterial pole of the heart underlies a significant fraction of congenital heart defects. Critical steps in arterial pole development are formation of the myocardial outflow tract (or conotruncal region) and its subsequent division into separate left and right ventricular outlets. Division of the cylindrical outflow tract is a complex morphogenetic process driven by cardiac neural crest cell influx and associated with rotation of the myocardial wall and cell death, ensuring alignment of the ascending aorta and pulmonary trunk with the left and right ventricles.1–3 The transient nature of the embryonic outflow tract raises existential but also clinically relevant questions as to the origin of this structure and its fate. In an article in this issue, Rana et al have addressed the latter in the developing chick heart with important inferences for the origin of the right ventricle.4 Using scanning confocal microscopy, Rana et al monitored the rise and fall of the myocardial outflow tract. After a 4-fold increase in length to reach a maximum extension the myocardial outflow tract shortens 5-fold. At the same time a nonmyocardial component appears, giving rise to the ascending aorta and pulmonary trunk. Rana et al focused on the retraction phase by following the fate of clusters of DiI labeled cells in ovo at 2 developmental timepoints and concluded that the proximal outflow tract gives rise to a large part of the right ventricular free wall. Although the concept of ventricularisation of the proximal outflow tract (or conal absorption) to form the right ventricular outlet is not new,5–7 the extent to which myocardium initially in the outflow tract contributes to the trabeculated part of the free ventricular wall …

The roles of GATA-4, -5 and -6 in vertebrate heart development.