Nucleoside phosphatase (ATPase) activity in myocardial cells of rat embryos. An electron-microscopic study.

Timed pregnancies were obtained in Sprague-Dawley rats, and cardiac tissues from embryos of days 10, 11, 12, 13, 14 and from newborn rats were used for the cytochemical localization of ATPase activity utilizing a lead phosphate precipitation procedure. Following incubation with ATP as the substrate, granular deposits of reaction product are discernible on the cell membranes of the embryonic myocardium. There is a noticeable decrease in the intensity of reaction product as visualized in the electron micrographs from the 10th day of gestation to the 14th day. No granular reaction product is recognizable in myofibrils, mitochondria or other organelles in the cytoplasm. It appears that there is a selective deposition of the reaction product on the cell membranes or structures derived from it. The intense ATPase activity seen on 10th and 11th days seems to be correlated with the initial appearance of myofilaments and fibrils in the myocardial cells.     

ELECTRON MICROSCOPIC STUDIES ON THE INDIRECT FLIGHT MUSCLES OFDROSOPHILAMELANOGASTER : II. Differentiation of Myofibrils

The differentiation of the indirect flight muscles was studied in the various pupal stages of Drosophila. Fibrillar material originates in the young basophilic myoblasts in the form of short myofilamants distributed irregularly near the cell membranes. The filaments later become grouped into bundles (fibrils). Certain "Z bodies" appear to be important during this process. The "Z bodies" may possibly be centriolar derivatives and are the precursors of the Z bands. The first formed fibrils (having about 30 thick myofilaments) are already divided into sarcomeres by Z bands. These sarcomeres, however, seem to be shorter than those of the adult fibrils.The H band differentiates in fibrils having about 40 thick myofilaments; the fibrils constrict in the middle of each sarcomere during this process. The individual myofibrils increase from about 0.3 µ to 1.5 µ in diameter during development, apparently by addition of new filaments on the periphery of the fibrils. The ribosomes seem to be the only cytoplasmic inclusions which are closely associated with these growing myofibrils. Disintegration of the plasma membranes limiting individual myoblasts was commonly seen during development of flight muscles, supporting the view that the multinuclear condition of the fibers of these muscles is due to fusion of myoblasts.     

The fine structure of differentiating muscle in the salamander tail

Summary Thin methacrylate sections of developing tails of Amblystoma opacum larvae were examined in the electron microscope and a series of stages in the differentiation of the myotome musculature was reconstructed from electron micrographs and earlier light microscopic studies of living muscle. The earliest muscle cell precursor that can be clearly identified is a round or oval cell with abundant cytoplasm containing scattered myofilaments and free ribonucleoprotein granules, but little endoplasmic reticulum. These cells sometimes form a syncytium and they may also be fused with adjacent formed muscle fibers by lateral processes. Nuclei are large and nucleoli are prominent. This cell, called a myoblast here, is distinctly different in its appearance from the adjacent mesenchymal cells which have abundant granular endoplasmic reticulum. The earliest myofilaments are of both the thick and thin varieties and are distributed in a disorganized fashion in the cytoplasm. These filaments are similar to the actin and myosin filaments described by Huxley and they are present in the cytoplasm at an earlier stage of differentiation than heretofore suspected from light microscopy studies. The first myofibrils are a heterogeneous combination of thick and thin filaments and dense Z bands and are not homogeneous as so many light microscopists have contended. As development progresses, cross striations become more orderly and definitive sarcomeres are formed. Thereafter, new myofilaments and Z bands seem to be added to the lateral surfaces and distal ends of existing myofibrils.Free ribonucleoprotein granules are a prominent part of the myoblast cytoplasm and are found in close association with the differentiating myofilaments in all stages of development. In early muscle fibers and some of the formed fibers, similar granules are often concentrated in the I bands. A theory of myofilament differentiation based on current concepts of the role of ribonucleoprotein in protein synthesis is presented in the discussion. Stages in myofibril formation and possible relationships of the filaments in developing muscle cells to other types of cytoplasmic filaments are also discussed.Supported by grant C-5196 from the United States Public Health Service.     

Electron microscopic study of the prenatal development of the thoracic aorta in the rat

Prenatal development of the thoracic aorta of the rat during the period ranging from gestational days 12 to 21 was examined by transmission electron microscopic and morphometric studies. The process of wall formation occurred in four major phases. At phase I (gestational day 12), the dorsal aorta consists of an endothelium and loosely surrounding mesenchymal cells. Collagen fibrils and fine filamentous materials are sparsely present in the intercellular space. At phase II (days 13 to 16), the mesenchymal cells begin to differentiate to myoblasts, which have small clusters of myofilaments with dense bodies, rough endoplasmic reticulum, and a discontinuous basal lamina. The differentiating cells form a few compact cell layers around the endothelium. Elastic fibers first occur sparsely in juxtacellular spaces at days 13-14. The thickness of the aorta increases rapidly from 1-3 layers of cells at day 13 to 5-8 layers at day 17, leading to a maximum of 5-9 cell layers at day 20. The differentiation of myoblasts and elastogenesis are initiated in the inner layers, and later progress toward the outer layer of the aortic wall. At phase III (days 17 to 19), the myoblasts continue to develop into typical smooth muscle cells, and elastic fibers rapidly increase in both size and number. At phase IV (day 20 and later), smooth muscle cells have well-developed myofilaments in the cell periphery, and rough endoplasmic reticulum and other organelles tend to accumulate in the apical portion of the cytoplasm. Elastic laminae appear in a few inner layers of the aortic wall.     

Fine structure of the gular gland of the free-tailed bat Tadarida brasiliensis

The gular gland of the bat Tadarida brasiliensis is a specialized sebaceous gland located in the skin of the suprasternal region of adult males. It consists of an aggregation of simple branched tubulo-acinar gland units, the number of which varies seasonally. Each acinus is composed of densely packed sebaceous cells at various stages of differentiation. Acinar basal cells and cells of the epithelium of the ducts can differentiate into sebaceous cells. Two main changes appear in the cytoplasm concurrent with the sebaceous transformation: the differentiation of cytoplasmic organelles and the deposition of lipid material. The appearance of a different type of mitochondrion and the development of large numbers of ribosomes and polyribosomes can be recognized in the cytoplasm at an early stage of differentiation. Concomitant with the deposition of significant numbers of lipid droplets, the cells develop abundant agranular endoplasmic reticulum occurring mainly as scattered tubular cisternae. These at times form whorls surrounding lipid droplets. At later stages, the cisternae of the agranular endoplasmic reticulum often occur in crystalline arrays between secretory oil droplets. The roles of the different cytoplasmic organelles, especially in relation to the production of sebum, are discussed.     

Synthesis of ppGpp by mouse embryonic ribosomes

The unusual nucleotide guanosine tetraphosphate (ppGpp) is synthesized in vitro by ribosomes isolated from mouse embryos, but is not formed by ribosomes isolated from adults. Analysis of specific organs shows a developmental change in detectable ppGpp-forming ability. Eleven day embryonic liver ribosomes synthesize ppGpp, but this ability is essentially lost by 14 day embryonic liver and adult liver. Eleven day embryonic brain ribosomes also synthesize ppGpp at a level comparable to that observed using E. coli ribosomes. The synthesis of ppGpp appears to be associated with the early stages of differentiation, when cell proliferation is rapid and specialized protein synthesis is low or absent. The potential role of ppGpp as an effector molecule or regulator in the eucaryotes is discussed.     

CYTOLOGICAL AND ULTRASTRUCTURAL STUDIES ON MUSCLE DIFFERENTIATION IN THE ASCIDIAN,PEROPHORA ORIENTALIS*,**

Muscle cell differentiation in the tail of the ascidian, Perophora orientalis, from early tail-bud embryos to swimming larvae, were studied cytologically and ultrastructurally. Myogenic cells did not form multinucleated myotubes, but remained as mononucleated cells. Nucleolar component increased prior to a marked increase in cytoplasmic RNA. Cytoplasmic RNA appeared first around nucleus and later concentrated in the peripheral cytoplasm. The fine filaments measuring 20–30 Å in their thin parts and 30–45 Å in their thick parts in diameter appeared initially, forming loose networks, in the peripheral cytoplasm where ribosome clusters had been concentrated. These filaments were tightly attached by particles of various size and density. These filaments tended to be arranged in parallel as they increased in their size. They seemed to be precursors of both actin and myosin filaments of formed myofibrils. Z band precursors were found as dense patches in association with loosely arranged myofilaments and consisted of particulate and filamentous materials. The myofibrils seemed to grow further by organizing free filaments into bundles and further by aligning bundles of myofilaments at both ends.     

Wheat Streak Mosaic Virus-induced Cytoplasmic Expansion and Membrane Proliferation

The cytoplasmic volume of wheat streak mosaic virus-infected cells was significantly greater than that of cells in healthy control tissue. Ultrastructural examination revealed that mainly cellular membranes and ribosomes filled the expanded cytoplasm. Imperfectly spherical inclusions, containing continuous endoplasmic reticulum membranes at the periphery and a mixture of membranes and ribosomes in the centre, were observed near nuclei at early infection stages. The inclusions became larger as infection progressed. Membranes and ribosomes proliferated also throughout the cell, forming a matrix in which organelles and various cytopathic structures were enclosed. Numerous vesicles were observed in the cytoplasm of other WSMV-infected cells. Multi-layered membrane bodies were found at later infection stages. Virus particles were present in the central space of these myelin-like structures. The presence of apparently intermediate stages in myelin-like structure development in chloroplasts suggest that at least some of the myelin-like structures originated from the chloroplasts.     

FINE STRUCTURE OF SMOOTH MUSCLE CELLS GROWN IN TISSUE CULTURE

The fine structure of smooth muscle cells of the embryo chicken gizzard cultured in monolayer was studied by phase-contrast optics and electron microscopy. The smooth muscle cells were irregular in shape, but tended to be elongate. The nucleus usually contained prominent nucleoli and was large in relation to the cell body. When fixed with glutaraldehyde, three different types of filaments were noted in the cytoplasm: thick (150–250 A in diameter) and thin (30–80 A in diameter) myofilaments, many of which were arranged in small bundles throughout the cytoplasm and which were usually associated with dark bodies; and filaments with a diameter of 80–110 A which were randomly orientated and are not regarded as myofilaments. Some of the aggregated ribosomes were helically arranged. Mitochondria, Golgi apparatus, and dilated rough endoplasmic reticulum were prominent. In contrast to in vivo muscle cells, micropinocytotic vesicles along the cell membrane were rare and dense areas were usually confined to cell membrane infoldings. These cells are compared to in vivo embryonic smooth muscle and adult muscle after treatment with estrogen. Monolayers of cultured smooth muscle will be of particular value in relating ultrastructural features to functional observations on the same cells.     

Zygote shrinkage and subsequent development in some Hibiscus hybrids

Summary Early embryonic development was compared in self-fertilized embryos of the diploid species, Hibiscus costatus, and triploid hybrid embryos, H. costatus-aculeatus and H. costatus-furcellatus, the paternal parent in both hybrids being tetraploid. The self-fertilized zygotes shrank to 50% of the volume of the unfertilized egg. These young embryos showed marked polarity. There was a concentration of cytoplasm in the apical cells and large vacuoles in the basal cells. There was also a polar distribution of organelles within the embryo as a whole which probably reflected initial differentiation. In comparison, hybrid zygotes shrank only about 20% of their original volume but started division at about the same time as selffertilized zygotes. There appeared to be no polarization and little proliferation of the cytoplasm in the hybrids. Large vacuoles remained prominent throughout the hybrid embryos, while organelles were few in the scant cytoplasm and no polarization of these was evident. These highly divergent hybrid embryos had become necrotic and aborted by the time the normal, self-fertilized embryos had reached the late globular stage. This altered developmental sequence of the hybrids suggests that shrinkage and rearrangement of the zygote cytoplasm is essential for normal embryonic differentiation.     

Cytodifferentiation of the chick pancreas. II. Ultrastructure of the acinar cells

The ultrastructural changes occurring during the differentiation of the pancreatic acinar cell were studied in White Leghorn chick embryos from the onset of pancreatic morphogenesis on day 3 of incubation (day 3) to hatching. Generally, the changes included a loss of some structures, the addition of others and modification of existing structures. Numerous cytoplasmic filaments which were present in the early migrating cells of the pancreatic bud were no longer present on day 5. The nucleoli enlarged temporarily on days 5–6 and then resumed a reduced size. The Golgi apparatus enlarged by day 6 and remained this way throughout the embryonic period. Associated with these changes was the initial appearance of the zymogen granules on day 5. The endoplasmic reticulum was present initially in both the smooth and the rough forms. The rough form and the outer nuclear membrane were both initially studded intermittently with aggregates of ribosomes. Subsequently, there was an increase in the number of attached ribosomes, an increase in the amount of rough reticulum and a decrease in smooth membranes. The ribosomes attached to the membranes appeared to augment the large free ribosome population characteristic of the early cells. Mitochondria did not appear to increase in number but there was an increase in size. The granules varied in kind, number and size with developmental age. The first granules formed (days 3–5) appeared to be miniatures of the mature type. Subsequently, a heterogeneity of granule morphologies was present.     

Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study

The formation of neointimal thickenings in the rat carotid artery after balloon injury was studied by a combination of electron-microscopic and stereological methods. All smooth muscle cells in the normal media had a contractile phenotype, the cytoplasm being dominated by myofilaments. Seven days after endothelial denudation, the smooth muscle cells in the innermost part of the media had assumed a synthetic phenotype by loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. These cells moved through fine openings in the internal elastic lamina and gave rise to a growing neointima by proliferation and secretion of extracellular matrix components. Fourteen days after the operation, the neointima had almost reached its final size, and mitoses were no longer noted. Nevertheless, the cells maintained a synthetic phenotype with prominent secretory organelles, although myofilaments had started to become more abundant again. They were surrounded by an extracellular matrix made up of collagen fibrils and coalescing patches of elastin. Thirty-five days after the operation, an endothelial cell layer had reformed and covered most of the luminal vessel surface. In parallel, the smooth muscle cells in the neointima had returned to a contractile phenotype with a cytoplasm dominated by myofilaments. These findings provide a morphological basis for further analysis of the cellular and molecular interactions involved in the formation of neointimal thickenings after endothelial injury, and for the search for agents interfering with this process.     

Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo

Embryonic chick myocardium (stages 8+ to 12?) was studied by light and electron microscopy. The myocardium, which is initially comprised of radially oriented cells with large intercellular spaces gradually becomes more tightly packed. Intercellular spaces decrease and the cells assume a circumferential orientation. Myocardial cells remain epithelial throughout formation of the functional tubular heart and specialized epithelial junctions (apical junctional complex or terminal bars) undergo modification to form intercalated discs. Embryonic myocardial cells contain large amounts of free ribosomes and particulate glycogen, the latter often associated with portions of granular reticulum. Unlike developing skeletal muscle. The amount of granular reticulum contained in the myocardial cell cytoplasm is large and, along with a hypertrophied Golgi apparatus, suggests that these cells may have a secretory function. These organelles persist during the initial period of fibril formation. Myofibrils apparently form from non filamentous precursor material and not by alignment of sequentially synthesized components.     

Ultrastructural observations on the embryonic development of the integument of Lacerta muralis (Lacertilia,Reptilia)

Integumentary development on the dorsal and ventral aspects of the body of 14, 21, 26, 33, and 40-day incubated embryos of the European Wall Lizard (Lacerta muralis) is described. While the earliest stages of epidermal differentiation resemble those reported for other tetrapods, precocious differentiation of dermal collagen more resembles that of anamniotes than that of birds and mammals. Anchoring complexes comprising cellular components, anchor filaments, and collagen are described, and their possible relationship to the formation of scale anlagen is discussed. The first embryonic epidermal generation differentiates beneath the periderm; most features of its histogenesis resemble those that have been described for the epidermis of adult squamates, but certain previously ignored organelles, including possible earlier β-keratin precursors, are reported. Different strategies regarding in ovo peridermal loss and posthatching shedding behavior are described and discussed in light of presently available data concerning control of cell differentiation in the squamate epidermis.     

Ultrastructure of the sympathetic paravertebral ganglia in rat embryogenesis. I. Cytogenesis and cell differentiation

The development of sympathetic paravertebral ganglia was studied in rat embryos by electron microscopy. The main attention was paid to the initial stages of ganglion formation. The first aggregations of presumptive ganglionic cells were observed in 12 day-old embryos. Single preganglionic terminals appeared in contact with cell bodies sometime later. The appearance of large granular vesicles in the cytoplasm is the first ultrastructural feature of the beginning of neural differentiation of cells. Small granulated cells observed from the 12th day of gestation and neuroblasts differentiate earlier than glial cells. In the ganglia of late fetuses nerve cells varied in the electron density of the cytoplasm, in the degree of distention of rough endoplasmic reticulum and in vacuolization of mitochondria.     

First ultrastructural differentiation of myoblasts of chicken embryos: appearance of the initial filaments

A study is made of the structure and ultrastructure of myoblasts located in the myotome in chicken embryos, between stages 18 and 19, for electron microscope observation of the occurrence of the first myogenetic filamentous molecules. We suggest a hypothesis for the formation of initial filaments consisting of an initial synthesis of actin globular molecules, carried out in the centre of the myoblast, near the nucleus, with the participation of RNA and the ribosomes. These molecules accumulate peripherically in areas below the plasmatic membrane where they polymerize into actin filamentous molecules which form the initial filaments. These move towards the sharp ends of the myoblast, under the plasmatic membrane, and thence to the interior of the cytoplasm, where they are evenly distributed. This genesis of initial filaments is independent of the influence of the nervous system. We postulate the existence of a single type of myoblast, of fibrillar form, with a dark central area containing the nucleus and the cell organelles, and two sharp, light end zones which contain only the initial filaments in a very light cytoplasmic matrix.     

Perinuclear organelles of the sensory cell of the Grandry cutaneous corpuscle. Ultrastructure and formation

Perinuclear organelles are found in the sensory cell of the Grandry cutaneous corpuscle in the duck. They are ovoid, fusiform or conical. They measure up to 5 µ length and l µ in diameter. They are formed by regular alternation of granular layers (mornolayers of RNA-rich granules which can be interpreted as ribosomes) and fibrous layers (generally formed by two sublayers of parallel fibrils). These fibrils (60-80 Å diameter) are in continuity with the intra-cytoplasmic fibrils which are very abundant in the Grandry cell. The central part of the organelles is devoid of RNA-rich granules.The formation of these organelles begins about one week after hatching, in the cytoplasmic perinuclear area where abundant granular endoplasmic reticulum and very numerous ribosomes and fibrils are present. The function of these perinuclear organelles remains unknown.     

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells.

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.     

Embryogenesis and germination in rye (Secale cereale L.)

Summary When rye embryos imbibe water they rapidly return to a condition of biochemical and structural complexity. Three stages of imbibition can be recognised: Phase I a short period (10 min) of physical wetting; Phase II a longer period (1 h) when little further imbibition occurs, followed by Phase III a continuous phase of active water uptake. The latter coincides with an increase in respiration rate and an increase both in the number of mitochondria and of cristae within them. Changes in fine structure become evident in all organelles in Phase III, after 2 h of imbibition. In the unimbibed embryo endoplasmic reticulum is present only as short crescents associated with electron lucent bodies, but in Phase III the endoplasmic reticulum proliferates to form many surrounding cirlets. After 6 h these circlets become fewer and instead the endoplasmic reticulum is seen in close association with the nuclear membrane. Concurrently incorporation of radioactive uridine and thymidine is first detectible. This suggests that the large increase in protein synthesis occurs on new ribosomes present on the reticulum associated with the nuclear membrane. For the first 6 h protein synthesis must occur either on polysomes within the dense packing of ribosomes or on these circlets of endoplasmic reticulum associated with electron lucent bodies.