Effect of cytochalasin B on the surface of neuroepithelial cells of the chick embryo

Neuroepithelial cells of young chick embryos, exposed in vitro for 1.5 and 13hr to cytochalasin B (5μg/ml) and examined by scanning electron microscopy, show changes in their surface morphology. The cell surface loses its cytoplasmic projections, its ruffle appearance and acquires a great number of cavities. These effects of the drug are not reversible after 4hr incubation on control media. Our results show that cytochalasin B does affect the cell surface morphology in our specimens but they give no indication as to manner in which the drug inhibits interkinetic nuclear migration.     

Effect of cytochalasin B on interkinetic nuclear migration in the chick embryo

Interkinetic nuclear migration was totally inhibited in the neural cells of early chicken embryos cultured for 3 hr on media containing 5 μg/ml cytochalasin B. After this treatment the arrangement of apically situated bands of cytoplasmic microfilaments was disturbed although the filaments themselves were not entirely disrupted.     

Quantitative ultrastructural changes of the endodermal cells in the early chick embryo analysed by stereological methods

The ultrastructure of endoderm cells of the area pellucida has been analysed in the chick embryo by stereological methods. These cells show a specific subcellular evolution which can be correlated with several aspects of morphogenetic behaviour. The cell form coefficient (CFc) changes notably from stage 5 (0.683) to stage 8 (0.446) accompanying the transformation of this layer into a squamous epithelium. An increase of the nuclear surface density is observed and is discussed in relation to the control of nucleocytoplasmic interchange. The mitochondrial volume and surface densities remain constant (3.12% of cellular volume and 0.727 mitochondria/mu(3) respectively). The endodermal cells possess higher levels of vitelline reserves (lipid bodies, 6.97% and yolk droplets, 8.90%) than other cellular types of the chick embryo. This fact is discussed with respect to the role of the endoderm in the phagocytosis of yolk. The RER length density shows an increase that could be related to some specific changes of the extracellular matrix during this period, but this fact remains to be demonstrated in relation to changes of Golgi membranes.     

Effects of dissociating agents on the fine structure of embryonic chick thyroid cells

Heads of the boid snakes Python sebae and Python molurus were dissected and the arthrology, myology and dentition studied. Living specimens of these species were observed and their feeding behavior analyzed by means of high- and regular-speed motion pictures. Camera speeds of up to 400 frames per second permitted examination of the jaw movements during the striking and seizing of prey. Motion picture studies conducted at regular speeds provided information on cranial movements during the swallowing of prey. The morphology of the head was correlated with observed movements in an attempt to analyze the functional and adaptive implications of the jaw apparatus. The cranial apparatus was discussed in terms of a linkage or kinematic chain whose constrainment and degrees of freedom were examined and compared with the jaw linkage of lizards. It was concluded that the very rigidly constrained mechanism in lizards is in remarkably sharp contrast to the very loose apparatus in snakes. Motions of various cranial bones were analyzed with particular attention given the mechanical factors involved. In full protraction the maxillae and palatines are lifted and rotated outward about a longitudinal axis. These movements are important in orienting the teeth with respect to the prey and are related to seizing and swallowing.     

Synthesis of serum proteins by cultures of chick embryo yolk sac endodermal cells

Endodermal cells were isolated from yolk sacs of 3-day chick embryos and cultured for 6 days in Eagle's minimal essential media plus 10% fetal calf serum. During this period cells rapidly lost their ability to synthesize DNA as judged by [3H]thymidine incorporation into DNA. In spite of this loss of DNA synthesis serum protein synthesis and secretion remained at a constant 45% of total protein synthesis and secretion. This was determined by immunoprecipitation of culture media using antibodies directed against embryonic chick serum proteins. Media were also analyzed for the synthesis and secretion of specific serum proteins using polyacrylamide gel electrophoresis. The relative synthesis and secretion of the individual serum proteins followed that previously observed in ovo with the exception of alpha-globulin-a which became undetectable. When culture media were supplemented with ovalbumin or insulin the relative synthesis and secretion of certin specific serum proteins were altered. However, analysis of these same media samples showed that the total amounts of serum protein synthesis and secretion were unaffected.     

The fine localization of acid phosphatase activity in the unvacuolated notochordal cells of the early chick embryo

Summary The electron microscopical localization of acid phosphatase activity was investigated in ultra-thin and semi-thin sections of unvacuolated notochordal cells of chick embryos from stages 9 to 14 (as defined by Hamburger & Hamilton). At stage 9, many notochordal cells show a lightly positive reaction for acid phosphatase activity. Thereafter, the acid phosphatase-positive cells of the notochord increase in number and, at stage 14, the reaction products for the enzyme are distributed throughout almost all the cisternae of the nuclear envelope and a well-differentiated endoplasmic reticulum, the parallel cisternal and reticular parts of the Golgi complex, and various lysosomes in nearly all notochordal cells. In the cisternae of the nuclear envelope and endoplasmic reticulum, the acid phosphatase reaction products are in a fine granular form. In the outermost layer of the cisternal parts of the Golgi complex, faint lead deposits similar to those in the endoplasmic reticulum are found, but in other cisternal and reticular regions which may correspond to the GERL, considerable amounts of reaction products are present. Knob-like projections are also seen protruding from the reticular parts of the Golgi complex. These results suggest that, at least up to stage 14, the notochordal cells are actively synthesizing acid phosphatase which is directly transported from the endoplasmic reticulum to the Golgi complex. The enzyme may be accumulated by the Golgi complex from which primary lysosomes are formed. Furthermore, the pattern of the ultrastructural localization of acid phosphatase activity in embryonic notochordal cells of the chick differs from that of adult cells of other animals.     

Concanavalin A-binding by cells of the early chick embryo

The surfaces of cells from the early embryo of the chick were examined using electron microscope techniques for the visualization of concanavalin A-binding sites. Horseradish peroxidase and Ferritin labelled concanavalin A were used to determine the distribution of the binding sites. All surfaces of the epiblast and hypoblast layers which were accessible to concanavalin A showed the presence of binding sites in stage 1 embryos. The ventral surface of the epiblast showed a high lectin affinity which may reflect the development of a basal lamina on this surface. The individual hypoblast cells at this stage showed a non-uniform distribution of binding sites, having a greater affinity on the dorsal surface than the ventral. By the time of primitive streak formation (stage 4-5) the dorsal surface of the epiblast displayed increased binding sites, while the frequency of sites on the ventral surface of the endoblast was reduced. The latter may reflect a change from one cell population to another, which occurs in the lower layer of the embryo at this time. No consistent correlation could be drawn between changes in motility of cells actually invaginating through the primitive streak and changes in affinity for concanavalin A. An overall increase in affinity of the dorsal surface of the epiblast was revealed by Ferritin and may reflect the changes in surface structure occurring in readiness for the morphogenetic migrations of gastrulation.     

Effects of cytochalasin B on the aggregation, electrophoretic mobility and surface morphology of chick neural retina cells.

Over a concentration range of o-5-10 mug/cm-3, cytochalasin B caused a biphasic change in the electrophoretic mobility of disaggregated neural retina cells. An initial rise in anodal mobility at low concentrations of the drug was transformed into a reduction in the mobility below that of the control at a concentration of 10 mug/cm-3. The effect of cytochalasin B was found to be reversible by washing treated cells in cytochalasin B-free media. This was investigated at a concentration of cytochalasin at which the greatest difference existed between the mobilities of the control and experimental cell suspensions. Reaggregation of cell dispersions failed to show any significant difference in the rate of aggregation between cytochalasin B-treated cells and the control. Scanning electron microscopy of cells fixed while in suspension also showed little significant change in the surface morphology upon application of cytochalasin B. In high concentrations of the drug cells appeared somewhat smoother in outline, but no correlation was found between changes in surface morphology and the variations in cell electrophoretic mobility. It is concluded that the observed changes in electrophoretic mobility may be attributed to a binding of cytochalasin B to the cell membrane. This lends support to the hypothesis that the primary site of action of cytochalasin B may be the plasma membrane.     

Acetylcholinesterase activity in neural crest cells of the early chick embryo

The appearance and distribution of AChE activity in the neural crest cells of the chick embryo were histochemically investigated. Prior to closure of the neural tube, neural crests were not demonstrated and most of the cells constituting the neural plate and the more lateral ectoderm were AChE-negative. With the closure of the neural tube, the neural crests assumed the form of a cell mass in its mid-dorsal portion and AChE activity was demonstrated in some elements of both tube and crests. The neural crest cells beginning to migrate ventrally or laterally were AChE-positive, and some showed intense enzymatic activity. Electron microscopically, the neural crest cells and the cells migrating from the neural crest displayed AChE activity in the cisternae of the nuclear envelope and in a few r-ER profiles, but were morphologically undifferentiated. As assessed by 3H-thymidine autoradiography, these cells possessed the potential to proliferate. These findings indicate that with the formation of the neural tube and neural crest, cells constituting these structures begin to differentiate with respect to AChE activity and that the enzyme appears in the neural crest cells before the onset of neuronal differentiation.     

Effects of cytochalasin B on pancreatic acinar cell structure and secretion

Summary The effects of cytochalasin B (CB) on pancreatic structure and amylase release were studied by use of pancreatic fragments, isolated acini and isolated acinar cells. In pancreatic fragments and isolated acini CB caused the disappearance of microfilaments underlying the apical plasma membrane, loss of apical microvilli and luminal swelling, the last of which was greatly enhanced by addition of protein secretagogues. CB had no effect on basal amylase release but inhibited bethanechol-stimulated amylase in both fragments and acini. Isolated acinar cells, while retaining overall polarity, had lost most of the apical specialization including the microfilament and microvillous complex. Cells were still able to release amylase in response to bethanechol but this release was not affected by CB. The only structural effect of CB on isolated cells was margination of zymogen granules against the plasma membrane. This was, however, not accompanied by increased amylase release. It is concluded that microfilaments are important in maintaining the pancreatic acinar structure. Interference with this structure by CB leads to inhibition of bethanechol-stimulated amylase release. Microfilaments, however, may not play a direct role in secretion.Supported by NIH grant GM-19998 from the United States Public Health Service. — We are indebted to Dr. John Heuser for advice throughout this project and assistance in the preparation of rapidly frozen acini, and to M. Lee for technical assistance     

Organelle distribution in chick neuroepithelial cells: effects of colchicine and cytochalasin B

The intracellular distribution of mitochondria, cytoplasmic inclusions and rough endoplasmic reticulum cisternae of chick neuroepithelial cells was investigated at neurulation stages 6, 8, 10 and 12. These neuroepithelial cells were subdivided into three zones: apical, median and basal and the distribution percentages of distribution of these organelles were obtained. Mitochondrial distribution was related to the energy supply that mitochondria provide for apical microfilament contraction. Cytoplasmic inclusions were distributed preferentially in the apical zone of the neuroepithelial cells during the four stages. Rough endoplasmic reticulum cisternae were homogeneously distributed in the three zones at stages 10 and 12, but at stages 6 and 8 there are more elevated percentages of rough endoplasmic reticulum in the apical zones than in the other zones. Experimental treatments with colchicine and cytochalasin B does not modify the patterns of mitochondria and rough endoplasmic reticulum cisternae but alters the distribution of cytoplasmic inclusions. Finally, there is a correlation in the normal neurulating neuroepithelial cells between the distributions of mitochondria and rough endoplasmic reticulum distribution and between the distributions of mitochondria and cytoplasmic inclusions distribution. This relationship is retained in the treated neuroepithelial cells.     

The effect of cytochalasin B on chondrogenesis in chick limb-bud mesoderm cells grown in vitro

Summary Cytochalasin B (CB) has been shown to have many biological effects on cultured cells. We report that an initial 48-hr treatment of freshly plated chick embryo limb mesoderm cells with CB irreversibly inhibits chondrogenesis. A slight inhibition in the amount of matrix is seen when limb cells are allowed to grow in culture for 24 hr prior to treatment for the second 24 hr of culture. If the cells are allowed to plate-out and grow for 48 hr or longer prior to being treated with CB for 24 hr, the amount of matrix produced is essentially the same as that seen in the controls. However, if the initial 48-hr culture period is followed by a 48- or 72-hr treatment, chondrogenesis is reduced, but not to the same extent as that seen in cultures treated for the first 48 or 72 hr. The irreversible inhibition of chrondrogenesis does not appear to be due to irreversible inhibition of protein synthesis or hexose uptake because, although these are reduced during treatment, they return to control levels within 48 hr following the removal of the drug. We cannot mimic the effect of CB treatment using glucose-deficient medium, thereby eliminating the possiblity that a critical glucose level is necessary to permit chondrogenesis. Multinucleation of limb cells treated with CB is reversed within 4 to 7 days following the removal of the drug. Therefore multinucleation alone is probably not responsible for the CB effect on chondrogenesis. However, other subtle permanent changes may occur during the period of multinucleation which result in the irreversible inhibition of chondrogenesis. This work was supported in part by a grant to R. A. F. from the North Carolina United Way and a grant to C. L. P. from the General Research Support Grant RR-5404 from the National Institutes of Health. A portion of these results were presented at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, 1977.