THE SIGNIFICANCE OF THE CAUDAL EPIDERMIS IN ASCIDIAN METAMORPHOSIS

Volume: 124Start Page: 241End Page: 25

Osmium-Fixed and Epon-Embedded Whole Mounts of Delicate Specimens

Osmium-fixed and Epon-embedded whole mounts of delicate specimens. An osmium fixative and an epoxy mountant were used to prepare delicate organisms and tissues as whole mounts for light microscopy. Fine structural details are well preserved by the technique, and common artifacts of whole mount preparation are largely eliminated. The final specimens are suitable as bright field objects or as phase/quasi-phase objects.ye

STYLET FORMATION IN NEMERTEANS

Volume: 162Start Page: 387End Page: 40

Fine Structure and Differentiation of Ascidian Muscle II. MORPHOMETRICS AND DIFFERENTIATION OF THE CAUDAL MUSCLE CELLS OF DlSTAPLlA OCClDENTALlS TADPOLES

The locomotor function of the caudal muscle cells of ascidian larvae is identical with that of lower vertebrate somatic striated (skeletal) muscle fibers, but other features, including the presence of transverse myomuscular junctions, an active Golgi apparatus, a single nucleus, and partial innervation, are characteristic of vertebrate myocardial cells. Seven stages in the development of the compound ascidian Distaplia occidentalis were selected for an ultrastructural study of caudal myogenesis. A timetable of development and differentiation was obtained from cultures of isolated embryos in vitro. The myoblasts of the neurulating embryo are yolky, undifferentiated cells. They are arranged in two bands between the epidermis and the notochord in the caudal rudiment and are actively engaged in mitosis. Myoblasts of the caudate embryo continue to divide and rearrange themselves into longitudinal rows so that each cell simultaneously adjoins the epidermis and the notochord. The formation of secretory granules by the Golgi apparatus coincides with the onset of proteid-yolk degradation and the accumulation of glycogen in the ground cytoplasm. Randomly oriented networks of thick and thin myofilaments appear in the peripheral sarcoplasm of the muscle cells of the comma embryo. Bridges interconnect the thick and thin myofilaments (actomyosin bridges) and the thick myofilaments (H-bridges), but no banding patterns are evident. The sarcoplasmic reticulum (SR), derived from evaginations of the nuclear envelope, forms intimate associations (peripheral couplings) with the sarcolemma. Precursory Z-lines are interposed between the networks of myofilaments in the uesicutate embryo, and the nascent myofibrils become predominantly oriented parallel to the long axis of the muscle cell. Muscle cells of the papittate embryo contain a single row of cortical myofibrils. Myofibrils, already spanning the length of the cell, grow only in diameter by the apposition of myofilaments. The formation of transverse myomuscular junctions begins at this stage, but the differentiating junctions are frequently oriented obliquely rather than orthogonally to the primary axes of the myofibrils. With the appearance of H-bands and M-lines, a single perforated sheet of sarcoplasmic reticulum is found centered on the Z-line and embracing the I-band. The sheet of SR establishes peripheral couplings with the sarcolemma. In the prehatching tadpole, a second collar of SR, centered on the M-line and extending laterally to the boundaries with the A-bands, is formed. A single perforated sheet surrounds the myofibril but is discontinuous at the side of the myofibril most distant from the sarcolemma. To produce the intricate architecture of the fully differentiated collar in the swimming tadpole (J. Morph., 138: 349, 1972), the free ends of the sheet must elevate from the surface of the myofibril, recurve, and extend peripherally toward the sarcolemma to establish peripheral couplings. Morphological changes in the nucleus, nucleolus, mitochondria, and Golgi bodies are described, as well as changes in the ground cytoplasmic content of yolk, glycogen, and ribosomes. The volume of the differentiating cells, calculated from the mean cellular dimensions, and analyses of cellular shape are presented, along with schematic diagrams of cells in each stage of caudal myogenesis. In an attempt to quantify the differences observed ultrastructurally, calculations of the cytoplasmic volume occupied by the mqjor classes of organelles are included. Comparison is made with published accounts on differentiating vertebrate somatic striated and cardiac muscles.yesDepartment of Zoology, University of Washington, Seattle, Washzngton 981 9

Ultrastructure and Differentiation of Ascidian Muscle I. Caudal Musculature of the Larva of Diplosoma Macdonaldi

The larval caudal musculature of the compound ascidian Diplosoma macdonaldi consists of two longitudinal bands of somatic striated muscle. Approximately 800 mononucleate cells, lying in rows between the epidermis and the notochord, constitute each muscle band. Unlike the caudal muscle cells of most other ascidian larvae, the myofibrils and apposed sarcoplasmic reticulum occupy both the cortical and the medullary sarcoplasm. The cross-striated myofibrils converge near the tapered ends of the caudal muscle cell and integrate into a field of myofilaments. The field originates and terminates at intermediate junctions at the transverse cellular boundaries. Close junctions and longitudinal and transverse segments of nonjunctional sarcolemmata flank the intermediate junctions, creating a transverse myomuscular (TMM) complex which superficially resembles the intercalated disk of the vertebrate heart. A perforated sheet of sarcoplasmic reticulum (SR) invests each myofibril. The sheet of SR spans between sarcomeres and is locally undifferentiated in relation to the cross-striations. Two to four saccular cisternae of SR near each sarcomeric Z-line establish interior (dyadic) couplings with an axial analogue of the vertebrate transverse tubular system. The axial tubules are invaginations of the sarcolemma within and adjacent to the intermediate junctions of the TMM complex. The caudal muscle cells of larval ascidians and the somatic striated muscle fibers of lower vertebrates bear similar relationships to the skeletal organs and share similar locomotor functions. At the cellular level, however, the larval ascidian caudal musculature more closely resembles the vertebrate myocardium.ye

Fine Structure and Differentiation of Ascidian Muscle I. DIFFERENTIATED CAUDAL MUSCULATURE OF DlSTAPLlA OCClDENTALlS TADPOLES

The structure of the caudal muscle in the tadpole larva of the compound ascidian Distaplia occidentalis has been investigated with light and electron microscopy. The two muscle bands are composed of about 1500 flattened cells arranged in longitudinal rows between the epidermis and the notochord. The muscle cells are mononucleate and contain numerous mitochondria, a small Golgi apparatus, lysosomes, proteid-yolk inclusions, and large amounts of glycogen. The myofibrils and sarcoplasmic reticulum are confined to the peripheral sarcoplasm. Myofibrils are discrete along most of their length but branch qear the tapered ends of the muscle cell, producing a Felderstmktur. The myofibrils originate and terminate at specialized intercellular junctional complexes. These myomuscular junctions are normal to the primary axes of the myofibrils and resemble the intercalated disks of vertebrate cardiac muscle. The myofibrils insert at the myomuscular junction near the level of a Z-line. Thin filaments (presumably actin) extend from the terminal Z-line and make contact with the sarcolemma. These thin filaments frequently appear to be continuous with filaments in the extracellular junctional space, but other evidence suggests that the extracellular filaments are not myofilaments. A T-system is absent, but numerous peripheral couplings between the sarcolemma and cisternae of the sarcoplasmic reticulum (SR) are present on all cell surfaces. Cisternae coupled to the sarcolemma are continuous with transverse components of SR which encircle the myofibrils at each I-band and H-band. The transverse component over the I-band consists of anastomosing tubules applied as a single layer to the surface of the myofibril. The transverse component over the H-band is also composed of anastomosing tubules, but the myofibrils are invested by a double or triple layer. Two or three tubules of sarcoplasmic reticulum interconnect consecutive transverse components. Each muscle band is surrounded by a thin external lamina. The external lamina does not parallel the irregular cell contours nor does it penetrate the extracellular space between cells. In contracted muscle, the sarcolemmata at the epidermal and notochordal boundaries indent to the level of each Z-line, and peripheral couplings are located at the base of the indentations. The external lamina and basal lamina of the epidermis are displaced toward the indentations. The location, function, and neuromuscular junctions of larval ascidian caudal muscle are similar to vertebrate somatic striated muscle. Other attributes, including the mononucleate condition, transverse myomuscular junctions, prolific gap junctions, active Golgi apparatus, and incomplete nervous innervation are characteristic of vertebrate cardiac muscle cells.yesDepartment of Zoology, University of Washington, Seattle, Washington 9819

Tetracycline Labeling Studies of Calcification in Nemertean Worms

Calcification of the stylet apparatus in nemertean worms was investigated by fluorescence microscopy following incubation of living specimens in sea water solutions of tetracycline. the synthesis of nail-shaped stylets that contain calcium phosphate, and the composition of the granular basis that anchors the central stylet tot he worm's pro-boscis, wer examined in five species of nemerteans belonging to the order Hoplonemertea. After a two-week treatment with either tetracycline-HCI or chlortetracycline, the basis appeared intensely fluorescent in all specimens. Such observations, coupled with results from electron microprobe analyses, indicate that the basis is calcified. None of the developing stylets, however, exhibited fluorescence after incubation in tetracycline. Hypotheses acounting for the lack of tetracycline labeling by stylets are discussed.ye

Laser irradiation of centrosomes in newt eosinophils: evidence of a centriole role in motility

ABSTRACT Newt eosinophils are motile granulated leukocytes that uniquely display a highly visible centrosomal area. Electron microscope and tubulin antibody fluorescence confirms the presence of centrioles, pericentriolar material, and radiating microtubules within this visible area. Actin antibodies intensely stain the advancing cell edges and tail but only weakly stain pseudopods being withdrawn into the cell. Randomly activated eosinophils follow a roughly consistant direction with an average rate of 22.5 gm/min. The position of the centrosome is always located between the trailing cell nucleus and advancing cell edge. If the cell extends more than one pseudopod, the one closest to or containing the centrosome is always the one in which motility continues. Laser irradiation of the visible centrosomal area resulted in rapid cell rounding. After several minutes following irradiation, most cells flattened and movement continued. However, postirradiation motility was uncoordinated and directionless, and the rate decreased to an average of 14.5 Am/min. Electron microscopy and tubulin immunofluorescence indicated that an initial disorganization of microtubules resulted from the laser microirradiations. After several minutes, organized microtubules reappeared, but the centrioles appeared increasingly damaged. The irregularities in motility due to irradiation are probably related to the damage