Development of electrical rhythmic activity in early embryonic cultured chick double-heart monitored optically with a voltage-sensitive dye

Double-hearted embryos were produced by whole-embryo culture of chick embryos which were microsurgically cut through the tissue of the anterior intestinal portal at the 1- to 6-somite developmental stage, at the time when the cardiac primordia have not yet fused in the bulboventricular region. The cultured embryos were removed from an incubator usually at the 7- to 10-somite stages of development, and then spontaneous electrical action potentials and/or contractions were optically recorded simultaneously from both the right and left half-hearts, using a 10 X 10- element photodiode matrix array together with a voltage-sensitive merocyanine-rhodanine dye (NK 2761). At the 7- to 8-somite stages, spontaneous action potentials were detected from bilateral prebeating half-hearts or sometimes from one half-heart. In each half-heart, the first spontaneous beating was often observed in the half-heart of the 9 somite embryos. In the beating half-hearts regular activity was always observed, while in the prebeating half-hearts at the 7- to 8-somite stages, both the regular and irregular rhythms of action potentials were detected, and the incidence of occurrence of regular activity significantly outnumbered that of the irregular rhythm. The heart rate in the left half-heart was faster than that in the right half-heart in the great majority of the prebeating and beating double-hearted embryos.     

Optical indications of electrical activity and excitation-contraction coupling in the early embryonic heart

Complete understanding of the ontogenesis and early development of electrical activity and its related contraction has been hampered by our inability to apply conventional electrophysiological techniques to the early embryonic heart. Direct intracellular measurement of electrical events in the early embryonic heart is impossible because the cells are too small and frail to be impaled with microelectrodes. Optical signals from voltage-sensitive dyes have provided a new and powerful tool for monitoring changes in membrane potential in a wide variety of living preparations. With this technique it is possible to make optical recordings from cells which are inaccessible to microelectrodes. An additional advantage of the optical method for recording membrane potential activity is that electrical activity can be monitored simultaneously from many sites in a preparation. Thus, applying a multiple-site optical recording method with a 100- or 144-element photodiode array and voltage-sensitive dyes, we have been able to monitor for the first time spontaneous electrical activity in pre-fused cardiac primordia in early chick embryos at the 6- and early 7-somite stages of development; we have been able to determine that the time of initiation of the heartbeat is the middle period of the 9-somite stage. In the rat embryonic heart, the onset of spontaneous electrical activity and contraction occurs at the 3-somite stage. This article describes ionic properties of the spontaneous action potential and genesis of excitation-contraction coupling in the early embryonic chick and rat hearts. In addition, an improved view of the ontogenetic sequence of spontaneous electrical activity and its implications for excitation-contraction coupling in the early embryonic heart are proposed and discussed.     

Effects of calcium on electrical propagation in early embryonic precontractile heart as revealed by multiple-site optical recording of action potentials

The effects of Ca2+ on electrical propagation in early embryonic precontractile chick hearts were studied optically using a voltage-sensitive merocyanine-rhodanine dye. Spontaneous optical signals, corresponding to action potentials, were recorded simultaneously from 25 separate regions of the eight-to-nine-somite embryonic primitive heart, using a square photodiode array. Electrical propagation was assessed by analyzing the timing of the signals obtained from different regions. Electrical propagation in the heart was suppressed by either lowering or raising extracellular Ca2+. Similar effects were produced by a Ca2+ ionophore (A23187). We have also found that electrical propagation across the primordial fusion line at the midline of the heart was enhanced by increasing, and depressed by lowering, external Ca2+. One possible interpretation is that intercellular communication in the embryonic precontractile heart is regulated by the level of the intracellular Ca2+ concentration, and it is suggested that intercellular communication across the primordial fusion line strongly depends on external Ca2+.     

Optical Recording of the Pacemaking Activity of a Congenital Double-heart in an Early Chick Embryo

A congenital double-hearted chick embryo was found among 16,171 embryos, which was at the 11 somite stage of development. The pacemaking activity of its double heart was monitored simultaneously from 9 different regions by optical methods. The right and left half hearts were tubular, and in both, spontaneous rhythmical action potentials and beating were detected, and differences were detected in their rhythms. Action potentials were also monitored in a malformed embryonic heart formed by partial fusion of the primordia. The results are discussed in relation to genesis of intrinsic pacemaking activity in cardiac primordia and to a spatial gradient of rhythmicity in the early stages of cardiogenesis.     

Conserved origin of the ventral pancreas in chicken

To determine the origin of the ventral pancreas, a fate map of the ventral pancreas was constructed using DiI crystal or CM-DiI to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. First, the region lateral to the 7- to 9-somite level, which has been reported to contribute to the ventral pancreas, was shown to contribute mainly to the intestine or the dorsal pancreas. At the 10 somite stage (ss), the ventral pre-pancreatic cells reside laterally at the 2-somite level, at the lateral boarder of the somite. At this stage, however, the fate of these cells has not yet segregated and they contribute to the ventral pancreas and to the intestine or bile duct. The ventral pancreas fate segregated at the 17 ss; the cells residing at the somite boarder at the 4-somite level at the 17 ss were revealed to contribute to the ventral pancreas. Interestingly, the dorsal and the ventral pancreatic buds are different in both origin and function. These two pancreatic buds begin to fuse at day 7 (HH 30) of embryonic development. However, whereas the dorsal pancreas gives rise to both Insulin-expressing endocrine and Amylase-expressing exocrine cells, the ventral pancreas gives rise to Amylase-expressing exocrine cells, but not insulin-expressing endocrine cells before day 7 (HH 30) of embryonic development.     

Zebrafish cypher is important for somite formation and heart development

Mammalian CYPHER (Oracle, KIA0613), a member of the PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36), has been associated with cardiac and muscular myopathies. Targeted deletion of Cypher in mice is neonatal lethal possibly caused by myopathies. To further investigate the role of cypher in development, we have cloned the zebrafish orthologue. We present here the gene, domain structure, and expression pattern of zebrafish cypher during development. Cypher was not present as a maternal mRNA and was absent during early development. Cypher mRNA was first detected at the 3-somite stage in adaxial somites, and as somites matured, cypher expression gradually enveloped the whole somite. Later, cypher expression was also found in the heart, in head and jaw musculature, and in the brain. We further identified 13 alternative spliced forms of cypher from zebrafish heart and skeletal muscle tissue, among them a very short form containing the PDZ domain but lacking the ZM (ZASP-like) motif and the LIM domains. Targeted gene knock-down experiments using cypher antisense morpholinos led to severe defects, including truncation of the embryo, deformation of somites, dilatation of the pericardium, and thinning of the ventricular wall. The phenotype could be rescued by a cypher form, which contains the PDZ domain and the ZM motif, but lacks all three LIM domains. These findings indicate that a PDZ domain protein is important for normal somite formation and in normal heart development. Treatment of zebrafish embryos with cyclopamine, which disrupts hedgehog signaling, abolished cypher expression in 9 somite and 15-somite stage embryos. Taken together, our data suggest that cypher may play a role downstream of sonic hedgehog, in a late stage of somite development, when slow muscle fibers differentiate and migrate from the adaxial cells.     

Myogenesis and contraction in the early embryonic heart of the rainbow trout

Summary Myogenesis in the embryonic heart of the rainbow trout, Salmo galrdneri (Rich.), was investigated electron microscopically from the 29th to the 41st somite stage. Thick and thin myofilaments are formed simultaneously as well as precursors of Z-lines, to which the thin filaments are attached. The genesis of filaments takes place in the region around the intracellular yolk droplets. The first myofibrils appear by the 33rd somite stage, probably formed by a mechanism of self-assembly in which the binding sites of actin and myosin participate. A- and I-bands do not develop before the 38th somite stage. The contraction already begins during the 33rd somite stage in the middle of the tubular heart. Gradually, the peristaltic waves spread increasingly to other parts of the heart. In the 41st somite stage the entire heart is contractile and all myocytes contain myofibrils.     

The development of the early atrioventricular conduction system in the embryonic heart

Recent electrophysiological evidence indicates that periodic spontaneous depolarizations occur in the primordial heart of the bird (and presumably mammal) even before the myocardial cells can contract, and these are initiated in the primordial sinoatrial region. As contractions are generated, these then establish a peristaltic wave. From that time on, during ontogenesis, the contractile sequence follows a regular pattern of development. As chambers form they contract sequentially in the direction of blood flow, even though, in the twisted configuration, myocardial continuities suggest the possibility of short-circuiting the electrical conduction pathways from atrium to bulbus. This implies that, even at these early stages, the electrical properties of the myocardium are not isotropic, and that specialized conduction pathways must exist. To the present time, electrophysiological techniques have limited the direct evidence that can be obtained on these delicate electrically specialized pathways. However, microscopical techniques have permitted studies on the morphological development of the tissue and of the cells in the various regions of the myocardium. The present paper traces the development of cell morphology in these regions, including the development of structural nodes and proximal ventricular fibre pathways, and from these observations, the manner in which the electrical conduction pathways are believed to develop is suggested.     

微电极矩阵研究小鼠胚胎心脏电生理活动

本实验采用一种新方法——微电极矩阵技术从整体水平研究小鼠胚胎离体整体心脏电生理活动。我们用微电极矩阵记录与60个电极相接触的心肌细胞的电活动(细胞外记录),称为场电位(field potentials,FPs),并与全细胞膜片钳记录的动作电位(action potentials,APs)(细胞内记录)进行比较,发现心房、心室处场电位形态类似于负向的细胞动作电位,场电位时程亦与动作电位时程类似。为研究兴奋的传导,我们比较了不同电极处场电位发生时间,发现在房室结构还未形成的胚胎发育第9．5天(E9．5)已经观察到明显的房室传导延迟(A-V delay)[(50．21±9．7)ms],而心室不同部位兴奋几乎是同步的。在发育晚期(E16．5),房室传导延迟为(82．21±10．50)ms。进一步研究基本的神经体液因素对心脏兴奋的调控,表明: 在E9．5,异丙肾上腺素(isoproterenol,Iso)使胚胎兴奋频率加快(34．04±7．31)％,房室传导延迟缩短(20．00±6．44)％,同时场电位时程增宽;相反,卡巴唑(carbachol,CCh)则使兴奋频率降低(42．32±5．36)％,房室传导缩短(26．00±4．81)％, 场电位时程减小。而在E16．5,Iso的作用显著增强,兴奋频率加快(101．54±10．23)％,房室传导延迟缩短(56．62±6．43)％, 而CCh则几乎使所有晚期心脏兴奋完全消失。所以,心脏的传导系统在胚胎发育早期4个腔室还未形成时已经建立,神经体液因子对心脏基本电生理活动的调控是在发育过程中逐渐成熟的。     

Effects of embryotrophic factors on the embryogenesis and organogenesis of mouse embryos in vitro

In order to study embryogenesis and organogenesis in vitro, two cell mouse embryos were cultured with alpha-MEM supplemented 10% FCS and embryotrophic factors (ETFs). The ETFs were separated from the conditioned medium of a SKG-II-SF cell line derived from a human uterine cervical epidermoid carcinoma. IL-1 beta, IL-6, IL-8, EGF, GH, PDGF-AB, basic FGF, VEGF were also detected in the conditioned media of this cell line. Using ETFs and a 10% FCS supplemented culture medium, 23% of the mouse two cell stage embryos developed to the bilaminar disc stage, 13% to the trilaminar germ disc stage, 9% were observed with blood islets in the yolk sac, and the heart beat was noted in 7% (28 embryos) of the embryos. Furthermore, primordial organs, such as the liver, heart, kidney, notochord, retina-like structure, etc. were observed. Usually, structures associated with the primordial streak stage (bilaminar germ disc embryo) developed in vitro using ETFs from two cell stage embryos. These closely resemble structures found at the same stage in the normal embryo in vivo. After the primordial streak stage, the cultured embryos showed no resemblance to the same stage in normal embryos. None of the external appearances of these embryos appeared normal. On the other hand, trilaminar disc stage embryos never developed when using only a 10% FCS supplemented culture system.     

Cardiac neural crest of the mouse embryo: axial level of origin, migratory pathway and cell autonomy of the splotch (Sp2H) mutant effect

A sub-population of the neural crest is known to play a crucial role in development of the cardiac outflow tract. Studies in avians have mapped the complete migratory pathways taken by 'cardiac' neural crest cells en route from the neural tube to the developing heart. A cardiac neural crest lineage is also known to exist in mammals, although detailed information on its axial level of origin and migratory pattern are lacking. We used focal cell labelling and orthotopic grafting, followed by whole embryo culture, to determine the spatio-temporal migratory pattern of cardiac neural crest in mouse embryos. Axial levels between the post-otic hindbrain and somite 4 contributed neural crest cells to the heart, with the neural tube opposite somite 2 being the most prolific source. Emigration of cardiac neural crest from the neural tube began at the 7-somite stage, with cells migrating in pathways dorsolateral to the somite, medial to the somite, and between somites. Subsequently, cardiac neural crest cells migrated through the peri-aortic mesenchyme, lateral to the pharynx, through pharyngeal arches 3, 4 and 6, and into the aortic sac. Colonisation of the outflow tract mesenchyme was detected at the 32-somite stage. Embryos homozygous for the Sp2H mutation show delayed onset of cardiac neural crest emigration, although the pathways of subsequent migration resembled wild type. The number of neural crest cells along the cardiac migratory pathway was significantly reduced in Sp2H/Sp2H embryos. To resolve current controversy over the cell autonomy of the splotch cardiac neural crest defect, we performed reciprocal grafts of premigratory neural crest between wild type and splotch embryos. Sp2H/Sp2H cells migrated normally in the +/+ environment, and +/+ cells migrated normally in the Sp2H/Sp2H environment. In contrast, retarded migration along the cardiac route occurred when either Sp2H/+ or Sp2H/Sp2H neural crest cells were grafted into the Sp2H/Sp2H environment. We conclude that the retardation of cardiac neural crest migration in splotch mutant embryos requires the genetic defect in both neural crest cells and their migratory environment.     

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of alpha-actinin dots within titin spots at the time of the first myofibril formation

In whole mount preparations of the 9 somite stage chick embryonic hearts that were immunofluorescently double labeled for titin and alpha- actinin, presumptive myofibrils were recognized as rows of several periodically aligned titin spots. Within these titin spots, smaller alpha-actinin dots were observed. These periodical arrangements of titin spots and alpha-actinin dots were not found in the 7 somite stage hearts. In wide myofibrils in the 10 somite stage hearts, the alpha- actinin dots and titin spots simultaneously became 'lines.' To study the ultrastructural features of the titin-positive regions in the 6-9 somite stage hearts, the thoracic portions of the embryos were immunofluorescently labeled for titin and embedded in resin. Ultrathin sections were mounted on electron microscopic grids and examined in immunofluorescence optics. The titin-positive regions thus identified were then examined in the electron microscope. No readily discernable specific ultrastructural features were found in titin-positive regions of the 6 somite stage cardiac primodia. Examination of the sections of the 9 somite stage hearts, on the other hand, revealed the occasional presence of small dense bodies, Z bodies, in the titin-positive regions. These observations strongly suggest that these Z bodies are the ultrastructural counterparts of the alpha-actinin dots seen by immunofluorescence optics and that they are formed nearly at the time of the formation of the first myofibrils. In some of the nascent myofibrils the Z bodies were found to be considerably narrower than the myofibrils, implying that the Z bodies are required not for the assembly of myofibrils per se but for their stabilization. Immunofluorescent labeling for titin and alpha-actinin revealed that the length of the shortest sarcomeres in the first myofibrils is approximately 1.5 micron, approximately the width of the A bands of mature myofibrils. The possibility that the A bands might define the initial length of nascent sarcomeres was indicated.     

Development of the hypochord and dorsal aorta in the zebrafish embryo (Danio rerio)

The hypochord of the zebrafish embryo (Danio rerio) emerges at the 9-somite stage as a single row of cells in the dorsomedial endoderm immediately ventral to the notochord. It is recognizable from the 2(nd) or 3(rd) somite and extends along the trunk to the same extent as the somites. A basal lamina surrounds the hypochord and its cells are slightly larger than the nearby endoderm cells. TEM studies have shown that the hypochord cells contain, in addition to mitochondria, well-developed rough endoplasmic reticula and Golgi networks, indicating synthetic activity. Once formed, the hypochord will stay in close association with the notochord, and this axial complex gradually moves dorsally, separating the hypochord from the endoderm as a one-cell-wide, rod-like structure that is bean-shaped in transverse section. This is the situation in the 15-somite embryo, at the level of the 4-5(th) somites. In the gap between the hypochord and the endoderm, angioblast cells aggregate and start to form the dorsal aorta, which becomes intimately associated with the hypochord. In the 17-somite embryo the aortic rudiment is established just ventral to the hypochord as a tube with a lumen. As development proceeds, the size of the hypochord decreases. In the pec fin embryo the hypochord is still recognizable in the posterior trunk, but has apparently vanished in anterior regions. The temporal correlation between the appearance of the hypochord and the formation of the dorsal aorta, coupled with the intimate relationship between these structures, suggest that the hypochord may play a role in the positioning of the dorsal aorta.     

Modulating L-type calcium current affects discontinuous cardiac action potential conduction.

We have used pairs of cardiac cells (i.e., one real guinea pig ventricular cell and a real-time simulation of a numerical model of a guinea pig ventricular cell) to evaluate the effects on action potential conduction of a variable coupling conductance in combination with agents that either increase or decrease the magnitude of the L-type calcium current. For the cell pairs studied, we applied a direct repetitive stimulation to the real cell, making it the "leader" cell of the cell pair. We have demonstrated that significant delays in action potential conduction for a cell pair can occur either with a decreased value of coupling conductance or with an asymmetry in size such that the follower cell is larger than the leader cell. In both conditions we have shown that isoproterenol, applied to the real cell at very low concentrations, can reversibly decrease the critical coupling conductance (below which action potential conduction fails) for a cell pair with fixed cell sizes, or, for a fixed value of coupling conductance, increase the maximum allowable asymmetry in cell size for successful conduction. For either of these effects, we were able to show that treatment of the real cell with BayK 8644, which more specifically increases the magnitude of the L-type calcium current, was able to mimic the actions of isoproterenol. Treatment of the leader cell of the cell pair (the real cell) with nifedipine, which selectively lowers the magnitude of the L-type calcium current, had effects opposite those of isoproterenol or BayK 8644. The actions of nifedipine, isoproterenol, and BayK 8644 are all limited to conditions in which the conduction delay is on the order of 5 ms or more, whether this delay is caused by limited coupling conductance or by asymmetry in size of the cells. This limitation is consistent with the time course of the L-type calcium current and suggests that the effects of calcium channel blockers or beta-adrenergic blocking drugs, in addition to being selective for regions of the heart that depend on the L-type calcium current for the upstroke of the action potential, would also be somewhat selective for regions of the heart that have discontinuous conduction, either normally or because of some pathological condition.     

Influence of copper on the early post-implantation mouse embryo: An in vivo and in vitro study

Summary Female mice were injected intravenously with copper sulphate on either the 7th day (early egg cylinder stage of development), the 8th day (late egg cylinder stage), or the 9th day (early somite stage of development), and examined on the 10th day of gestation. Injection on the 7th day was found to be embryo-lethal; when females were injected on the 8th day, the majority of the surviving embryos exhibited anomalies of the neural tube and/or the heart, while injection on the 9th day resulted in a very low incidence of anomalies. The most common malformations seen on the 10th day involved failure of closure of the neural tube in the head region of the embryo, and various types of anomalies of cardiac rotation and shape. When additional females injected on the 8th day were examined on the 12th day, a high proportion of the fetuses examined had developed exencephaly.A further group of embryos from untreated females were explanted on the 9th day and cultured in vitro in various concentrations of copper sulphate. The lowest levels tested had little obvious effect on neural tube closure. Intermediate doses resulted in, retarded and anomalous embryonic development, while the highest levels employed resulted in neural tube and cardiac anomalies similar to those produced in vivo.The results demonstrate both the direct toxic effect of copper on embryonic development and that the stage of embryonic development at the time of exposure determines both the nature and the extent of the effect.     

Cyclic nucleotides and calcium: their role in the control of cell communication in the heart

The effects of theophylline and di-butyryl cyclic adenosine 3',5'-monophosphate (db-cAMP) on the electrical coupling of heart cells were investigated in rat trabeculae. Theophylline (4 X 10(-4) M) and db-cAMP (5 X 10(-5) M) increased both the space constant and conduction velocity. The time constant of the membrane was not changed by either drug. Measurements of the time constant of the foot of the action potential and conduction velocity were used to calculate the intracellular longitudinal resistance. Both theophylline and db-cAMP were found to enhance cell-to-cell communication in the heart by decreasing the intracellular longitudinal resistance.     

Adrenergic regulation of conduction velocity in cultures of immature cardiomyocytes

During cardiac maturation, increased exposure of the heart to circulating catecholamines correlates with increased conduction velocity and growth of the heart. We used an in vitro approach to study the underlying mechanisms of adrenergic stimulation induced changes in conduction velocity. By combining functional measurements and molecular techniques, we were able to demonstrate that the increased conduction velocity after β-adrenergic stimulation is probably not caused by changes in intercellular coupling. Instead, RT-PCR experiments and action potential measurements have shown an increased excitability that may well explain the observed increase in conduction velocity. Apart from being relevant to cardiac maturation, our findings are relevant in the context of stem cells and cardiac repair. Preconditioning of stem cell derived cardiomyocytes may help to enhance electrical maturation of de novo generated cardiomyocytes and consequently reduce their proarrhythmogenic potential. (Neth Heart J 2008;16:106-9.)     

体外培养小白鼠胚胎从胚泡期到体节期的初步研究

初步报道小白鼠3.5天受孕卵体外连续培养10天,从胚泡期到达体节期。重点介绍各阶段在显微镜下所见的特征。培养到第7—8天出现心跳,心跳一般持续2—3天。