Reiji Hirakow

Scanning electron microscopic study on the development of primitive blood vessels in chick embryos at the early somite-stage

SummaryPrimary vasculogenesis in chick embryos at the early somite stage 11–14 somites) was investigated mainly by scanning electron microscopy (SEM), with special reference to the development of primitive blood vessels such as the arteria et vena vitellina (AV, VV), aorta dorsalis (AD) and vena cardinalis (VC). After glutaraldehyde fixation, the endoderm or ectoderm was removed from the embryos to expose either the ventral (AV, VV, AD) or the dorsal (VC), vascular system. The mode of vascular formation was found to be identical in all these blood vessels, arising first in loco as isolated solid masses or cords composed of so-called angioblasts. The angioblasts at this developmental phase could be distinguished from underlying mesenchymal cells, exhibiting a relatively flat surface. The VV was recognized first on both sides of the anterior intestinal portal at the 4-somite stage, whereas the forming AD was identified on the ventral surface of the paired forming AD was identified on the ventral surface of the paired somites at the 6-somite stage, appearing almost simultaneously from the cranial to caudal somite regions. After the 8-somite stage, the AV was formed by transformation of one of the caudal plexuses spreading to the area vasculosa. In the 9-somite stage, the angioblastic cords of the VC appeared on the dorsal side of the mesoderm in the same manner as for other ventral vessels. This finding differs from the statement of a previous author that the VC is formed by longitudinal anastomosis of intersegmental diverticula of the AD.

Quantitative studies on the ultrastructural differentiation and growth of mammalian cardiac muscle cells

The postnatal development of myocardial cells in the bilateral atria and ventricles of rats at days 1, 10, 20, 30 and 60 was studied by means of an ultrastructural quantitative method. No clear differences between the right and left myocardium were found throughout the developmental period or in the adult stage. The myocardial cells increased in thickness more rapidly up to day 20 than during the subsequent period. Myofibrils occupied about 50-60% of the cytoplasm of almost all kinds of cardiac muscle cells except those of the newborn atrium. The fraction of mitochondria increased after day 10, whereas that of glycogen conversely decreased. Although the presence of specific granules characterized the atrial myocardial cells even in newborns (day 1), no differences in other cellular components or fiber thickness between the atrium and the ventricle were seen at this stage. The distinction between atrial and ventricular muscle cells, in terms of fiber thickness, mitochondrial and glycogen content, began to appear at day 10 and became increasingly prominent with age until virtually all muscle fibers reached an almost mature state at day 20.

An ultrastructual topographical study on myofibrillogenesis in the heart of the chick embryo during pulsation onset period

SummaryUltrathin sections of the chick embryonic heart at the 8-, 9- and 10-somite stage were cut serially at an interval of 20 μm and mounted for transmission electron microscopic examination on a copper grid with a sufficiently large hole to survey the entire section area. The grid was supported by a formvar film. Thick filaments were first found to assemble into well-defined bundles in several cells composing the caudal region of the newly formed heart just before onset of the pulsation at the 8-somite stage. Then, at the 9-somite stage when pulsation commences, the cells possessing nascent myofibril(s) increase in number, slightly more in the right side of ventricular region. At the 10-somite stage, the rhythmical contraction is established and striated myofibrils become distinctly discernible. Right side dominance is more conspicuous at this stage than previously. Then, myofibrillogenesis gradually progresses toward the cranial or bulbar region.

TEM-studies on development and canalization of the dorsal aorta in the chick embryo

SummaryTransmission electron microscopic investigations were carried out on the early development of the dorsal aorta in chick embryos during the period ranging from the 6-somite stage to the 16-somite stage. Special attention was paid to the formation of the vascular lumen. The initial angioblasts tended to aggregate into a cluster with a narrow intercellular space. Only a few punctate thickenings of the plasma membrane were found at junctional sites of the angioblasts. The vascular cavity appeared to be produced by expansion of the intercellular space and possibly also by enclosure of the extracellular space with attenuated vasoformative cells. The wall of the early vessels so formed was frequently observed to be open to the extravascular space through clefts originating probably from incomplete closure of the early endothelium. No specific ultrastructural features characterizing the angioblasts or early endothelial cells were recognized.

Freeze-fracture studies of the sinoatrial and atrioventricular nodes of the caprine heart, with special reference to the nexus

SummaryThe caprine sinoatrial node (SAN) and atrioventricular node (AVN) were studied by freeze-fracture techniques, and their nexus or gap junction structure were compared with that of ordinary atrial and ventricular muscle cells. The general features of the nexus in both the SAN and AVN were essentially identical. Approximately two-thirds of the nexuses observed in the nodal cells consisted of typical macular arrangements of nexal particles, and the remaining third, of atypical configurations of either circular arrangements or linear arrays of particles in continuity with the macular nexuses. Such atypical nexuses were never observed in the ordinary adult myocardial cells. Quantitative analysis revealed that all of the nexuses in the nodal cells measured, were less than 0.1 μm2, whereas the majority of the nexuses in ordinary myocardial cells (64% in the atrium and 76% in the ventricle) were larger than 0.1 μm2. No significant differences in diameter and center-to-center distance of nexal particle were found between the nodal cells and ordinary myocardial cells.

Cytoskeletal filaments in embryonic chick myocardial cells as revealed by the quick-freeze deep-etch method combined with immunocytochemistry

SummaryThe three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12–14 nm filaments formed a loose network surrounding nascent myofibrils. These 12–14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8–11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3–5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)-treated samples, subsarcolemmal 8–11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.

Development of the Vertebrate Heart and the Extracellular Matrix

Abstract Three major topics in vertebrate cardiogenesis were surveyed with special attention given to the cellular migration and the extracellular matrix (ECM).

Postnatal differentiation of ventricular myocardial cells of the opossum (Didelphis virginiana Kerr) and T-tubule formation.

SummaryCardiac muscle cells in the right ventricle of the postnatal opposum were studied ultrastructurally, with particular attention to the developmental stage of T-tubule formation. Animals ranging from 5.5cm (day 29 of postnatal life) to 41 cm (young adult) in body length were used. Typical T-tubules were first recognized in a few myocardial cells of the 7.5cm (day 43) opossum. T-tubules increased in number as cardiac muscle cells continued to differentiate until 22cm in body length (about 105 days after birth). At this stage of development most of the T-tubules were completely established. The general mode of differentiation and development of cardiac muscle cells appeared essentially the same as in other mammals.

Effect of growth factors on the differentiation of chick precardiac mesoderm in vitro

We have found that precardiac mesoderm extirpated from chicken blastoderm at stage 5 fails to differentiate into beating tissue when cultured in Eagles minimum essential medium (MEM), while it can pulsate provided either the endoderm is present or serum is added to the MEM. To identify the factor(s) which influence early myocardial differentiation, we examined the effect of insulin-like growth factor 1 (IGF-1), activin A and basic fibroblast growth factor (bFGF). All these growth factors showed a stimulating effect on myocardial differentiation and it is conceivable that these factors exhibit the same effect in vivo.