The Effect of Cytochalasin B on the Neuroepithelial Cells of the Mouse Embryo WILLIAM WEBSTER I AND J A N LANGMAN Department ofdnatomy, University of Virginia, Charlottesuille, Virginia 22901
ABSTRACT Eleven-day mouse embryos were cultured in a medium containing cytochalasin B (10 pg/ml) to examine the effect of the drug on the developing CNS. The embryos were exposed to the drug for two hours. After culturing, the embryos were prepared for light and electron microscopy and the neuroepithelium of the cerebral vesicles was examined. The following abnormalities were noted in the cytochalasin B-treated embryos: (1) mitotic figures were situated in the middle of the neuroepithelial layer instead of on the lumen, indicating that premitotic nuclear migration had been prevented; (2) binucleate cells were found in the neuroepithelial layer, indicating that cytochalasin B does not interfere with mitosis but prevents cytokinesis; (3) the microfilaments usually seen in the apex of the neuroepithelial cells were disrupted and formed an amorphous mass of filamentous material; (4) some neuroepithelial cells were free in the lumen, whereas others protruded into the ventricle but appeared to remain attached to the internal limiting border by their junctional complexes; and (5) in some regions the neuroepithelial cells had broken away from their end-feet a t the basal lamina. These morphological changes were associated with a change in the internal limiting boundary from concave to convex and vacuolation of the external processes of the cells. The relationship between cytochalasin B, microfilaments and these morphological changes is discussed. Cell proliferation in the early neural tube occurs in a pseudostratified epithelium adjacent t o the ventricles. This neuroepithelial layer consists of radially oriented bipolar cells whose internal (apical) and external (basal) processes span the entire thickness of the brain wall, a t least during the early part of development (Hinds and Ruffett, '71). Autoradiographic experiments have shown that the nuclei of the neuroepithelial cells undergo a to-and-fro migration (interkinetic nuclear migration) during the generation cycle @idman e t al., '59; Sauer and Walker, '59; Fujita, '62; Langman et al., '66). The nuclei in the outer half of the neuroepithelial layer are mostly in S-phase. After completion of DNA synthesis each nucleus moves towards the luminal surface within its cytoplasm (premitotic nuclear migration), while the cell withdraws toward and rounds up a t the lumen, where it divides (Hinds and Ruffett, '71). At early stages of CNS development all neuroepithelial cells undergoing mitosis are located at the ventricular lumen; AM. J. ANAT. (1978) 152: 209-222.
at later stages of development, however, another replicating population is seen external to the neuroepithelial zone in the subependymal layer. After division is completed the daughter cells extend again and re-form external processes. Then, either the nuclei move (postmitotic nuclear migration) to the outer neuroepithelial zone to enter the S-phase or the cell migrates beyond the neuroepithelial layer into the mantle layer. In this layer the cell then differentiates into a neuron. It is still unclear whether the primary site of action of cytochalasin B is on microfilaments or on the cell membrane (Schroeder, '69; Wessells et al., '71; Carter, '72; Sanger and Holtzer, '72; Kletzien and Perdue, '73; Luchtel et al., '76). Several authors, however, suggest that the drug inhibits a number of biological processes thought to be dependent on the contractile abilities of the actin-like miAccepted November 14, '17. ' Present address: Dr. W. Webster, Department of Anatomy, University of Sydney, Sydney 2006, New South Wales, Australia.
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crofilaments (Wessells et al., '71; Spooner et al., '73). Furthermore, inhibition of contractile activity seems to be associated with morphological changes in the microfilamentous network within the cell (Schroeder, '69; Bernfield and Wessells, '70; Karfunkel, '72; Spooner and Wessells, '72; Auersperg, '72; Cloney, '72; Luchtel et al., '76). The suggestion that microfilaments might be the contractile organelles involved in interkinetic migration (Hinds and Ruffett, '71). and the possibility that cytochalasin B may act on this site, led us to investigate the effect of the drug on the neuroepithelium of the mouse embryo. In addition, an attempt was made to equate cytochalasin B-induced changes in histo- and morphogenesis with known microfilament distribution and activity. METHODS
The technique of culturing whole embryos as described by Kochhar ('751, was used to investigate the effect of cytochalasin B on the developing mouse embryo. ICR-strain mice were used throughout the experiment. Following overnight mating, the females were checked for vaginal plugs, and the morning a plug was observed was designated as the beginning of day one of pregnancy. On day 11 the mice were killed and the uterine horns removed and placed in sterile Tyrode's solution. The embryos were removed from the uterus, taking care not to damage Reichert's membrane, the yolk sac and the chorioallantoic placenta. Reichert's membrane was then carefully removed, leaving the visceral yolk sac intact. Most of the chorioallantoic placenta was trimmed away, leaving a small part attached by the allantoic vessels. The embryos were then placed in 10-ml culture vials containing 2 ml of the culture medium, which consisted of 50%Waymouth's solution (Flow Laboratories) and 50%fetal calf serum (Flow Laboratories), to which had been added 12.5 pg/ml streptomycin and 7.5 pgiml penicillin G. Subsequently the vials were gassed with a mixture of 5% carbon dioxide and 95% oxygen, capped tightly and placed on a rotator (36 RPM) a t 37°C. In the control cultures DMSO was added to the medium to give a final concentration of 1% DMSO. The experimental cultures contained 10 pg/ml of cytochalasin B in 1%DMSO. The embryos survived for about two hours in the experimental medium; longer culture resulted in cessation of the heart beat and cell death.
In the control medium, embryos survived for as long as 36 hours. Twenty-three experimental embryos and 20 control embryos, all viable after two hours' culture, were used for the histological studies. Embryos were fixed for electron microscopic studies in modified Karnovsky fixative ('65) consisting of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer. After two hours' fixation the embryos were washed in buffer and post-fixed in 1%osmium tetroxide in 0.1 M phosphate buffer for one hour. The specimens were dehydrated and then embedded in Araldite. Sections were stained with uranyl acetate and lead citrate, and examined with a Philips EM 301 electron microscope. Light microscopy was performed on l-micrometer-thick sections prepared from the same specimens as used for electron microscopy. The sections were stained with 0.25%azure I1 in 0.5%sodium borate. After 24 hours' fixation in the glutaraldehyde-paraformaldehydefixative the embryos were dehydrated and embedded in 2-butoxyethanolglyco1 methacrylate (Polysciences data sheet 123). The sections were stained with 1%toluidine blue. RESULTS
The wall of the telencephalon of an 11-day mouse embryo, cultured for two hours in 1% DMSO, consisted of a thin, outer layer with a few differentiating neurons, and a thick, inner, neuroepithelial layer (fig. 1).The mitotic figures were located exclusively a t the luminal wall. Electron micrographs showed that the internal (apical) processes of the neuroepithelial cells were joined together by zonular junctional complexes a t the luminal surface (figs. 2, 3). Even cells in mitosis maintained these junctions with adjacent cells. Parts of the junctional complexes, with gapwidths of 20 nm, were associated with bundles of microfilaments 6 nm in diameter, arranged in a purse-string fashion (figs. 3, 18). Also associated with the apical processes were microtubules, often arranged parallel to the long axis of the cells. The external (basal) processes of the neuroepithelial cells extended to the overlying basal lamina, where they formed end-feet, but lacked specialized cell junctions (fig. 4). Similar sections through the wall of the telencephalon of embryos cultured for two hours in cytochalasin B showed a number of differences when compared with the controls. The most striking difference was the location
CYTOCHALASIN B AND NEUROEPITHELIAL CELLS
of the mitotic figures (figs. 5, 15). Many of them were located within the neuroepithelial layer a t varying distances from the lumen. Frequently the long axis of the mitotic spindle was parallel to that of the neuroepithelial cells. The second abnormality seen in the cytochalasin B-treated embryos was the presence of cells with two nuclei (figs. 6-9, 14).Most of these binucleate cells were located close to the ventricular lumen. They are thought to be cells that have completed mitosis but were unable to complete cytokinesis. A third difference from the controls was the presence of cells which seemed to have burst from the neuroepithelium into the ventricles (fig. 17). Some of these cells were binucleate (figs. 6, 121, but others appeared to have only one nucleus (figs. 6, 8, 10). Although some of the cells appeared to be free in the lumen, others were attached by junctional complexes to the neuroepithelium (figs. 10-13). Some cells appeared to be in the process of extrusion (fig. 14). A less frequently seen difference was the separation of the neuroepithelial cells from their end-feet a t the basal lamina (fig. 17). This breakdown of the neuroepithelial layer was usually associated with a large-scale protrusion of cells into the ventricle and a change in shape of the internal limiting layer from concave to convex. This feature may simply represent a pulling away of the extruded neuroepithelial cells. It may, however, have been aided by the increased frequency of vacuoles seen in the external processes of the cytochalasin B-treated neuroepithelial cells (figs. 15, 16), compared to controls (fig. 4). The last difference was the loss of the microfilament bundle from the apical region of the cells. In control embryos this microfilamentous web was easily and frequently seen associated with the junctional complex in the apex of the cells (figs. 3, 18);in the cytochalasin Btreated embryos distinct bundles were absent, being replaced by a rather amorphous mass of filamentous material (figs. 11, 12, 19, 20). DISCUSSION
The abnormal location of mitotic figures in the neuroepithelial layer two hours after culture in cytochalasin B indicates that the drug inhibited premitotic nuclear migration to the lumen of the ventricle. Hinds and Ruffett (’70) reported that the apical processes of neuroepithelial prophase cells of 13-day mouse
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embryos contain many longitudinally oriented microtubules and 6-nm filaments. These microfilaments and/or microtubules may be involved in moving the prophase nucleus from the middle of the neuroepithelium to the lumen. The observation that cytochalasin B inhibited premitotic nuclear migration suggests that the microfilaments play a role in this process. In our work the misplaced mitotic figures were located mainly in the inner half of the neuroepithelial layer but a few were seen in the outer half. In other studies using cytochalasin B, misplaced mitotic figures were also seen throughout the neuroepithelium (Messier and Auclair, ’74). Spooner and Wessells (‘70) reported mitotic figures even a t the basal surface of salivary epithelium after culture in the drug for 18 to 36 hours. It has been suggested that, in addition to the microfilaments, microtubules are also involved in interkinetic nuclear migration. Messier and Auclair (’73) studied nuclear migration in the chick neural tube after exposing the embryos to 2°C for three hours to disrupt the microtubules and then treating them with monoiodoacetamide for 15 minutes to prevent repolymerisation. Although they reported “an abundance of mitotic figures abnormally distributed within the thickness of the neuroepithelium” their single figure of this phenomenon is not clear enough to exclude an accumulation of mitotic figures in the inner zone, as seen in the mouse after colcemid treatment (Webster et al., ’73).Further work by Messier (’761, on the chick embryo with formamide, led him to conclude “that microtubules are part of the active force involved in the displacement of nuclei observed during interkinetic nuclear migration.” Using 0.31 M formamide for six hours he showed not only displaced mitotic figures, but also collapse of the neural tube and the bulging of cells, including mitotic figures beyond the outer edge of the neural tube. In our opinion this may imply the release of cells from the internal limiting border, rather than the inhibition of nuclear migration. Other reports in which substances disrupting microtubules have been used, such as colchicine in the chick neural tube (Woodard and Estes, ’44; Watterson et al., ’56; Watterson, ’65; Karfunkel, ’721, colcemid in the mouse neuroepithelium (Webster et al., ’731, or colcemid in the lens placode of the chick (Pearce and Zwaan, ’70) have failed to indicate any in-
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hibition of premitotic nuclear migration. In these experiments the accumulation of mitotic figures a t the lumen was due to the presence of cells blocked in metaphase. Pearce and Zwaan ('70) even demonstrated the almost total absence of microtubules in the cells of the lens placode after colcemid treatment, but still premitotic nuclear migration took place. Clearly the role of microtubules in premitotic migration remains unsettled. Concanavalin A also prevents interkinetic nuclear migration in the chick neural tube. This was demonstrated by both the presence of displaced mitotic figures and the failure of tritiated thymidine-labeled cells to undergo their normal interkinetic migration as demonstrated by autoradiography (Lee, '76). Concanavalin A is a plant protein whose biological activities depend on its binding to specific cell-surface carbohydrate residues (Goldstein et al., '73). It has been shown to cause agglutination of embryonic cells (Moscona, '71) and transformed cells (Inbar and Sachs, '691, but not of normal adult cells. Moran ('74a) demonstrated that concanavalin A (25 pg/ml) prevented migration of differentiating neural crest cells in vitro, but did not prevent neurulation in the amphibian Ambystoma maculatum (Moran, '74b). In addition it appears that the drug does not affect mitosis or cytokinesis, all indicating that its action is not on microtubules or microfilaments. Moscona ('71) has suggested that embryonic cells possess special cell surface sites, which contain carbohydrate residues. These provide the embryonic cells with their specialized cell movements. When the cells reach an "adult" state these cell surface sites are masked, cell movement ceases and the cells are no longer affected by concanavalin A. If this hypothesis is true it suggests that interkinetic migration of embryonic neuroepithelial cells is dependent not only on nuclear movement but possibly also on accompanying cell-membrane changes. The presence of binucleate cells in the cytochalasin B-treated embryos is in agreement with the well known effect of the drug in preventing cytokinesis, but not mitosis (Carter, '67). All the binucleate cells seen in this study were still a t the lumen, but the short exposure-time precludes us from concluding that post-mitotic migration was prevented. Bernfield and Wessells ('70) reported that multinucleate cells in cytochalasin B-treated lung epithelium did not migrate to the basal
surface, but they also described the cells as showing signs of ill health and necrosis. The mechanism by which cytochalasin B inhibits cytokinesis has been subject to some controversy because cells apparently vary in their permeability to the drug and, hence, in whether or not cleavage-furrow formation is prevented (Luchtel e t al., '76; Selman et al., '76). Schroeder ('69) proposed that in dividing Arbacia eggs a contractile ring of microfilaments just below the cleavage furrow was normally responsible for pinching the cell in two. In the presence of cytochalasin B the microfilament ring disappeared and cytokinesis was prevented. This theory has now been extended to include mammalian (Hela) cells (Schroeder, '70). Cytochalasin B has also previously been reported to cause binucleate cells in cultured salivary, oviductal and lung epithelia (Bernfield and Wessells, '70). Surprisingly, such cells were not reported in the chick neural tube after drug exposure (Karfunkel, '72; Messier and Auclair, '74). The protrusion and eversion of cells from the luminal side of the neuroepithelium is presumably associated with the loss of the microfilament bands a t the apical ends of the cells. In epithelia exposed to cytochalasin B the bands of microfilaments disappear from the cells, to be replaced by an amorphous mass of filamentous material, perhaps depolymerised microfilaments (Bernfield and Wessells, '70; Karfunkel, '72; Messier and Auclair, '74). The function of the apical microfilaments has been suggested from studies on neurulation (Baker and Schroeder, '67; Burnside, '71; Karfunkel, '72). The presence of the purse-string band of microfilaments is necessary for contraction of the apical ends of the neuroepithelial cells and, hence, for the conversion of the neural plate into neural tube. Since, in the developing cerebral vesicles, all the ventricular surfaces of the neuroepithelium are concave compared to the external surface, i t seems probable that the continued presence of the bands of microfilaments is necessary to maintain the curvature. Hence, if cytochalasin B disrupts this band of filaments, the neuroepithelial cells would tend to bulge into the ventricle, and if the external processes of the cells break under the increased tension, the cells would evert through their rings of junctional complexes. The junctional complexes do not break and prevent the cells from becoming free-floating in the lumen. It is interesting to note that the phenomenon of cells protruding
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gration in the absence of microtubules in the chick embryo. J. Embryol. Exp. Morph., 30: 661-671. 1974 Effect of cytochalasin B on interkinetic nuclear migration in the chick embryo. Devel. Biol., 36: 218-223. Moran, D. 1974a The action of concanavalin A on migrating and differentiating neural crest cells. Exp. Cell Res., 86: 365-373. 1974b The inhibitory effects of concanavalin A LITERATURE CITED on the development of the amphibian embryo. J. Exp. Auersperg, N. 1972 Microfilaments in epithelial morZool., 188: 361-365. phogenesis. J. Cell Biol., 52: 206-211. Moscona, A. A. 1971 Embryonic and neoplastic cell surBaker, P. C., and T. E. Schroeder 1967 Cytoplasmic filafaces: availability of receptors for concanavalin A and ments and morphogenetic movement in the amphibian wheat germ agglutinin. Science, 171: 905-907. neural tube. Devel. Biol., 15: 432-450. Pearce, T. L., and J. Zwaan 1970 A light and electron miBernfield, M. R., and N. K. Wessells 1970 Intra- and excroscopic study of cell behavior and microtubules in emtracellular control of epithelial morphogenesis. Devel. bryonic chicken lens using colcemid. J. Embryol. Exp. Biol. Suppl., 4: 195-249. Morph., 23: 491-507. Burnside, B. 1971 Microtubules and microfilaments in Sanger, J. W., and H. Holtzer 1972 Cytochalasin B: Effects newt neurulation. Devel. Biol., 26: 416-441. on cell morphology, cell adhesion and mucopolysaccharide Carter, S. B. 1967 Effects of cytochalasin on mammalian synthesis. Proc. Nat’l. Acad. Sci., 69: 253-257. cells. Nature, 213: 261-264. Sauer, M. E., and B. E. Walker 1959 Radioautographic 1972 The cytochalasins as research tools in cystudy of interkinetic nuclear migration in the neural tology. Endeavour, 31: 77-82. Exp. Biol. Med., 201: 557-560. tube. Proc. SOC. Schroeder, T. E. 1969 The role of “contractile ring” filaCloney, R. A. 1972 Cytoplasmic filaments and morphoments in dividing Arbacia eggs. Biol. Bull., 137: 413-414. genesis: Effects of cytochalasin B on contractile epider1970 The contractile ring. Fine structure of mal cells. Z. Zellforsch., 132: 167-192. dividing mammalian (HeLa) cells and the effects of cytoFujita, S. 1962 Kinetics of cell proliferation. Exp. Cell chalasin B. Z. Zellforsch., 109: 431-449. Res., 28: 52-60. Selman, G. G., J. Jacob and M. M. Perry 1976 The permeaGoldstein, I. J., C. M. Reichert, A. Misaki and P. A. J. Gorin bility to cytochalasin B of the new unpigmented surface 1973 An “extension” of the carbohydrate binding speciin the first cleavage furrow of the newt’s egg. J. Embryol. ficity of concanavalin A. Biochim. Biophys. Acta, 31 7: Exp. Morph., 36: 321-341. 500-504. Sidman, R. L., I. L. Miale and N. Feder 1959 Cell proliferaHinds, J. W., and T. L. Ruffett 1971 Cell proliferation in the tion and migration in the primitive ependymal zone; a n neural tube: An electron microscopic and Golgi analysis autoradiographic study of histogenesis in the nervous in the mouse cerebral vesicle. Z. Zellforsch, 115: 226-264. system. Exp. Neurol., 1: 322-333. Inbar, M., and L. Sachs 1969 Interaction of the carbohySpooner, B. S., J. F. Ash, J. T. Wrenn, R. B. Frater and N. K. drate-binding protein concanavalin A with normal and Wessells 1973 Heavy meromyosin binding to microfilatransformed cells. Proc. Nat’l. Acad. Sci., 63: 1418-1425. ments involved in cell and morphogenetic movements. Karfunkel, P. 1972 The activity of microtubules and miTissue Cell, 5: 37-46. crofilaments in neurulation in the chick. J. Exp. Zool., Spooner, B. S., and N. K. Wessells 1970 Effects of cyto181: 289-302. chalasin B upon microfilaments involved in morphogeneKarnovsky, M. J. 1965 A formaldehyde-glutaraldehyde sis of salivary epithelium. Proc. Nat’l. Acad. Sci., 66: fixative of high osmolarity for use in electronmicroscopy. 360-364. J. Cell Biol., 27: 137A-138A. 1972 An analysis of salivary gland morpbogeneKletzien, R. F., and J. F. Perdue 1973 The inhibition of sis: Role of cytoplasmic microfilaments and microsugar transport in chick embryo filjroblasts by cytotubules. Devel. Biol., 27: 38-54. chalasin B. Evidence for a membrane-specific effect. J. Watterson, R. L. 1965 Structure and mitotic behavior of Biol., Chem., 248: 711-719. the early neural tube. In: Organogenesis. R. L. DeHaan Kochhar, D. M. 1975 The use of in vitro procedures in and H. Ursprung, eds. Holt, New York, pp. 129-159. teratology. Teratology, 11: 273-288. Watterson, R. L., P. Veneziano and A. Bartha 1956 AbLangman, J., R. Guerrant and B. Freeman 1966 Behavior sence of a true germinal zone in neural tubes of young of neuroepithelial cells during closure of the neural tube chick embryos as demonstrated by t h e colchicine techJ. Comp. Neur., 127: 399-412. nique. Anat. Rec., 124: 379 (Abstract). Lee, H-Y. 1976 Inhibition of neurulation and inter. Webster, W., M. Shimada and J. Langman 1973 Effect of kinetic nuclear migration by concanavalin A in explanted fluorodeoxyuridine, colcemid and bromodeoxyuridine on early chick embryos. Devel. Biol., 48: 392-399. developing neocortex of the mouse. Am. J. Anat., 137: Luchtel, D., J. G . Bluemink and S. W. de Laat 1976 The ef67-86. fect of injected cytochalasin B on filament organization Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. Bradley, M. A. in the cleaving egg of Xenopus laevis. J. Ultrastruct. Res., Luduena, E. L. Taylor, J. T. Wrenn and K. M. Yamada 54: 406-419. 1971 Microfilaments in cellular and developmental processes. Science, 171: 135-143. Messier, P-E. 1976 Effects of formamide on neuroepiWocdard, T. M., and S. B. Estes 1944 Effect of colchicine on thelial cells and on interkinetic nuclear migration in the mitosis in the neural tube of forty-eight hour chick emchick embryo. J. Embryol. Exp. Morph., 35: 197-212. bryos. Anat. Rec., 90: 51-54. Messier, P-E., and C. Auclair 1973 Inhibition of nuclear mi-
into the lumen is opposite to that seen after microtubule disruption, when the cells protrude from the basal surface (Messier and Auclair, ’73). This suggests that the microtubules and microfilaments are in balance to maintain the neuroepithelium.
PLATE 1 EXPLANATION OF FIGURES
1
Photomicrograph of one-micrometer plastic section of the wall of the telencephalon from a n 11-day mouse control embryo. A thin mantle layer (ml) and a thick neuroepithelial layer (ne) can be distinguished. Mitotic figures are located adjacent to the ventricular lumen (vl). X 600.
2 Electron micrograph of the luminal side of the neuroepithelium from a control embryo. Adjacent internal (apical) processes are joined by junctional complexes (jc). Similar dark-staining junctions are seen between the metaphase figure (m) and internal processes of adjacent cells. X 3,400.
3 Section through the internal processes of neuroepithelial cells from a control embryo. Note the junctional complexes (jc) and the bundles of microfilaments (mf) traversing the apical parts of the cells. 4
X
25,000.
Electron micrograph of the outer part of the cerebral vesicle from a control embryo. The cytoplasm of the external (basal) processes of the neuroepithelial cells expand to form broad end-feet (ef) a t the basal lamina (bl). Adjacent end-feet are not connected to each other by cell junctions. Differentiating neurons (n) in the mantle layer are also visible. x 1,900.
5 Photomicrograph of a 1-pm section of the wall of the cerebral vesicle from a n 11-day mouse embryo cultured for two hours in cytochalasin B. Mitotic figures (mit) are seen in the middle of the neuroepithelial layer. Some of the neuroepithelial cells have everted into the lumen (arrow). X 600.
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CYTOCHALASIN B AND NEUROEPITHELIAL CELLS William Webster and Jan Langman
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
6 Electron micrograph of the neuroepithelium of a cerebral vesicle from a cytochalasin B-treated embryo. Among the normal-looking neuroepithelial cells are two binucleate cells (*). Free cells with one or two nuclei can also be seen in the lumen of the ventricle. X
3,400.
7 Low-power electron micrograph of the neuroepithelium from a cytochalasin B-treated embryo. A short distance from the lumen is a cell in telophase (t,).I t shows no cytoplasmic constriction and will presumably become binucleate. A second cell in telophase (t,) is a t the lumen. This cell is dividing in a plane perpendicular to the neuroepithelial cells and shows evidence of a cleavage furrow. X 3,400. 8 Low-power electron micrograph of the neuroepithelium from a cytochalasin B-treated embryo. Note the presence of two displaced mitotic figures. The internal limiting boundary marked by junctional complexes (jc) is unclear, a s many cells have protruded into the lumen of the ventricle (vl). Some of these cells appear to be connected to the neuroepithelium by junctional complexes. Note the binucleate cell (*). x 3,400.
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CYTOCHALASIN B AND NEUROEPITHELIAL CELLS Willlam Webster and J a n Langman
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
9 Low-power electron micrograph of t h e neuroepithelial layer from a cytochalasin B-treated embryo. Note the displaced anaphase-figure and the adjacent binucleate cell. X 3,400. 10 Electron micrograph of a protruded neuroepithelial cell, with a single nucleus, still attached to the neuroepithelial layer. X 3,400.A higher magnification of the cell junction seen in the rectangle is shown in figure 11. 11 Higher magnification of the junctional complex between a n extruded cell (ec) and the neuroepithelium. Although the junctional complex appears normal there are no obvious microfilaments. The filaments seem to be replaced by a rather ill-defined amorphous mass. X 19,000.
12 Electron micrograph of a binucleate cell (*I extruded into the ventricular lumen (vl). The junctional complex shown in the rectangle is seen a t higher magnification in figure 13. X 3,400.
13 Higher magnification of junctional complex between the extruded binucleate cell (ec) and the neuroepithelium. The junctional complex appears normal but clearly defined microfilaments are absent. x 25,000. 14 Micrograph of the neuroepithelial layer of a cytochalasin B-treated embryo. Two binucleate cells are present. Both are still attached to adjacent cells by junctional complexes. Another cell is seen in the process of extrusion. The cell appears to have been ejected through its ring of junctional complexes. x 1.900.
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CYTOCHALASIN B AND NEUROEPITHELIAL CELLS William Webster and J a n Langman
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
15 Light micrograph showing displaced mitotic figures throughout the neuroepithelium from a cytochalasin B-treated embryo. X 800. 16 Electron micrograph showing the outer part of cerebral vesicle from a n experi-
mental embryo. Note the vacuoles in the external (basal) processes of the neuroepithelial cells and in the differentiating neurons of the mantle layer. X 3,400. Compare with control, figure 4.
17 Light micrograph of the cerebral vesicle from a n experimental embryo. The neuroepithelial and mantle layer appear to have pulled away from t h e external limiting layer (ell). The end-feet and the basal lamina remain in position and are intact. The normal concave appearance of the internal limiting boundary is lost and many cells have been extruded into the lumen of the ventricle (vl). X 600. 18 High-power electron micrograph of the internal (apical) processes of the neuroepithelium from a control embryo. Note presence of junctional complexes between adjacent cells and the microfilamentous band (mf) extending across the internal processes. X 25,000.
19,20 High-power electron micrographs of similiar region of internal processes as seen in figure 18 but from cytochalasin B-treated embryos. Junctional complexes appear normal but there are no microfilament bands. Instead, there are amorphous masses of filamentous material. X 19,000,
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CYTOCHALASIN B AND NEUROEPITHELIAL CELLS William Webster and Jan Langman
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ABSTRACT Eleven-day mouse embryos were cultured in a medium containing cytochalasin B (10 pg/ml) to examine the effect of the drug on the developing CNS. The embryos were exposed to the drug for two hours. After culturing, the embryos were prepared for light and electron microscopy and the neuroepithelium of the cerebral vesicles was examined. The following abnormalities were noted in the cytochalasin B-treated embryos: (1) mitotic figures were situated in the middle of the neuroepithelial layer instead of on the lumen, indicating that premitotic nuclear migration had been prevented; (2) binucleate cells were found in the neuroepithelial layer, indicating that cytochalasin B does not interfere with mitosis but prevents cytokinesis; (3) the microfilaments usually seen in the apex of the neuroepithelial cells were disrupted and formed an amorphous mass of filamentous material; (4) some neuroepithelial cells were free in the lumen, whereas others protruded into the ventricle but appeared to remain attached to the internal limiting border by their junctional complexes; and (5) in some regions the neuroepithelial cells had broken away from their end-feet a t the basal lamina. These morphological changes were associated with a change in the internal limiting boundary from concave to convex and vacuolation of the external processes of the cells. The relationship between cytochalasin B, microfilaments and these morphological changes is discussed. Cell proliferation in the early neural tube occurs in a pseudostratified epithelium adjacent t o the ventricles. This neuroepithelial layer consists of radially oriented bipolar cells whose internal (apical) and external (basal) processes span the entire thickness of the brain wall, a t least during the early part of development (Hinds and Ruffett, '71). Autoradiographic experiments have shown that the nuclei of the neuroepithelial cells undergo a to-and-fro migration (interkinetic nuclear migration) during the generation cycle @idman e t al., '59; Sauer and Walker, '59; Fujita, '62; Langman et al., '66). The nuclei in the outer half of the neuroepithelial layer are mostly in S-phase. After completion of DNA synthesis each nucleus moves towards the luminal surface within its cytoplasm (premitotic nuclear migration), while the cell withdraws toward and rounds up a t the lumen, where it divides (Hinds and Ruffett, '71). At early stages of CNS development all neuroepithelial cells undergoing mitosis are located at the ventricular lumen; AM. J. ANAT. (1978) 152: 209-222.
at later stages of development, however, another replicating population is seen external to the neuroepithelial zone in the subependymal layer. After division is completed the daughter cells extend again and re-form external processes. Then, either the nuclei move (postmitotic nuclear migration) to the outer neuroepithelial zone to enter the S-phase or the cell migrates beyond the neuroepithelial layer into the mantle layer. In this layer the cell then differentiates into a neuron. It is still unclear whether the primary site of action of cytochalasin B is on microfilaments or on the cell membrane (Schroeder, '69; Wessells et al., '71; Carter, '72; Sanger and Holtzer, '72; Kletzien and Perdue, '73; Luchtel et al., '76). Several authors, however, suggest that the drug inhibits a number of biological processes thought to be dependent on the contractile abilities of the actin-like miAccepted November 14, '17. ' Present address: Dr. W. Webster, Department of Anatomy, University of Sydney, Sydney 2006, New South Wales, Australia.
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crofilaments (Wessells et al., '71; Spooner et al., '73). Furthermore, inhibition of contractile activity seems to be associated with morphological changes in the microfilamentous network within the cell (Schroeder, '69; Bernfield and Wessells, '70; Karfunkel, '72; Spooner and Wessells, '72; Auersperg, '72; Cloney, '72; Luchtel et al., '76). The suggestion that microfilaments might be the contractile organelles involved in interkinetic migration (Hinds and Ruffett, '71). and the possibility that cytochalasin B may act on this site, led us to investigate the effect of the drug on the neuroepithelium of the mouse embryo. In addition, an attempt was made to equate cytochalasin B-induced changes in histo- and morphogenesis with known microfilament distribution and activity. METHODS
The technique of culturing whole embryos as described by Kochhar ('751, was used to investigate the effect of cytochalasin B on the developing mouse embryo. ICR-strain mice were used throughout the experiment. Following overnight mating, the females were checked for vaginal plugs, and the morning a plug was observed was designated as the beginning of day one of pregnancy. On day 11 the mice were killed and the uterine horns removed and placed in sterile Tyrode's solution. The embryos were removed from the uterus, taking care not to damage Reichert's membrane, the yolk sac and the chorioallantoic placenta. Reichert's membrane was then carefully removed, leaving the visceral yolk sac intact. Most of the chorioallantoic placenta was trimmed away, leaving a small part attached by the allantoic vessels. The embryos were then placed in 10-ml culture vials containing 2 ml of the culture medium, which consisted of 50%Waymouth's solution (Flow Laboratories) and 50%fetal calf serum (Flow Laboratories), to which had been added 12.5 pg/ml streptomycin and 7.5 pgiml penicillin G. Subsequently the vials were gassed with a mixture of 5% carbon dioxide and 95% oxygen, capped tightly and placed on a rotator (36 RPM) a t 37°C. In the control cultures DMSO was added to the medium to give a final concentration of 1% DMSO. The experimental cultures contained 10 pg/ml of cytochalasin B in 1%DMSO. The embryos survived for about two hours in the experimental medium; longer culture resulted in cessation of the heart beat and cell death.
In the control medium, embryos survived for as long as 36 hours. Twenty-three experimental embryos and 20 control embryos, all viable after two hours' culture, were used for the histological studies. Embryos were fixed for electron microscopic studies in modified Karnovsky fixative ('65) consisting of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer. After two hours' fixation the embryos were washed in buffer and post-fixed in 1%osmium tetroxide in 0.1 M phosphate buffer for one hour. The specimens were dehydrated and then embedded in Araldite. Sections were stained with uranyl acetate and lead citrate, and examined with a Philips EM 301 electron microscope. Light microscopy was performed on l-micrometer-thick sections prepared from the same specimens as used for electron microscopy. The sections were stained with 0.25%azure I1 in 0.5%sodium borate. After 24 hours' fixation in the glutaraldehyde-paraformaldehydefixative the embryos were dehydrated and embedded in 2-butoxyethanolglyco1 methacrylate (Polysciences data sheet 123). The sections were stained with 1%toluidine blue. RESULTS
The wall of the telencephalon of an 11-day mouse embryo, cultured for two hours in 1% DMSO, consisted of a thin, outer layer with a few differentiating neurons, and a thick, inner, neuroepithelial layer (fig. 1).The mitotic figures were located exclusively a t the luminal wall. Electron micrographs showed that the internal (apical) processes of the neuroepithelial cells were joined together by zonular junctional complexes a t the luminal surface (figs. 2, 3). Even cells in mitosis maintained these junctions with adjacent cells. Parts of the junctional complexes, with gapwidths of 20 nm, were associated with bundles of microfilaments 6 nm in diameter, arranged in a purse-string fashion (figs. 3, 18). Also associated with the apical processes were microtubules, often arranged parallel to the long axis of the cells. The external (basal) processes of the neuroepithelial cells extended to the overlying basal lamina, where they formed end-feet, but lacked specialized cell junctions (fig. 4). Similar sections through the wall of the telencephalon of embryos cultured for two hours in cytochalasin B showed a number of differences when compared with the controls. The most striking difference was the location
CYTOCHALASIN B AND NEUROEPITHELIAL CELLS
of the mitotic figures (figs. 5, 15). Many of them were located within the neuroepithelial layer a t varying distances from the lumen. Frequently the long axis of the mitotic spindle was parallel to that of the neuroepithelial cells. The second abnormality seen in the cytochalasin B-treated embryos was the presence of cells with two nuclei (figs. 6-9, 14).Most of these binucleate cells were located close to the ventricular lumen. They are thought to be cells that have completed mitosis but were unable to complete cytokinesis. A third difference from the controls was the presence of cells which seemed to have burst from the neuroepithelium into the ventricles (fig. 17). Some of these cells were binucleate (figs. 6, 121, but others appeared to have only one nucleus (figs. 6, 8, 10). Although some of the cells appeared to be free in the lumen, others were attached by junctional complexes to the neuroepithelium (figs. 10-13). Some cells appeared to be in the process of extrusion (fig. 14). A less frequently seen difference was the separation of the neuroepithelial cells from their end-feet a t the basal lamina (fig. 17). This breakdown of the neuroepithelial layer was usually associated with a large-scale protrusion of cells into the ventricle and a change in shape of the internal limiting layer from concave to convex. This feature may simply represent a pulling away of the extruded neuroepithelial cells. It may, however, have been aided by the increased frequency of vacuoles seen in the external processes of the cytochalasin B-treated neuroepithelial cells (figs. 15, 16), compared to controls (fig. 4). The last difference was the loss of the microfilament bundle from the apical region of the cells. In control embryos this microfilamentous web was easily and frequently seen associated with the junctional complex in the apex of the cells (figs. 3, 18);in the cytochalasin Btreated embryos distinct bundles were absent, being replaced by a rather amorphous mass of filamentous material (figs. 11, 12, 19, 20). DISCUSSION
The abnormal location of mitotic figures in the neuroepithelial layer two hours after culture in cytochalasin B indicates that the drug inhibited premitotic nuclear migration to the lumen of the ventricle. Hinds and Ruffett (’70) reported that the apical processes of neuroepithelial prophase cells of 13-day mouse
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embryos contain many longitudinally oriented microtubules and 6-nm filaments. These microfilaments and/or microtubules may be involved in moving the prophase nucleus from the middle of the neuroepithelium to the lumen. The observation that cytochalasin B inhibited premitotic nuclear migration suggests that the microfilaments play a role in this process. In our work the misplaced mitotic figures were located mainly in the inner half of the neuroepithelial layer but a few were seen in the outer half. In other studies using cytochalasin B, misplaced mitotic figures were also seen throughout the neuroepithelium (Messier and Auclair, ’74). Spooner and Wessells (‘70) reported mitotic figures even a t the basal surface of salivary epithelium after culture in the drug for 18 to 36 hours. It has been suggested that, in addition to the microfilaments, microtubules are also involved in interkinetic nuclear migration. Messier and Auclair (’73) studied nuclear migration in the chick neural tube after exposing the embryos to 2°C for three hours to disrupt the microtubules and then treating them with monoiodoacetamide for 15 minutes to prevent repolymerisation. Although they reported “an abundance of mitotic figures abnormally distributed within the thickness of the neuroepithelium” their single figure of this phenomenon is not clear enough to exclude an accumulation of mitotic figures in the inner zone, as seen in the mouse after colcemid treatment (Webster et al., ’73).Further work by Messier (’761, on the chick embryo with formamide, led him to conclude “that microtubules are part of the active force involved in the displacement of nuclei observed during interkinetic nuclear migration.” Using 0.31 M formamide for six hours he showed not only displaced mitotic figures, but also collapse of the neural tube and the bulging of cells, including mitotic figures beyond the outer edge of the neural tube. In our opinion this may imply the release of cells from the internal limiting border, rather than the inhibition of nuclear migration. Other reports in which substances disrupting microtubules have been used, such as colchicine in the chick neural tube (Woodard and Estes, ’44; Watterson et al., ’56; Watterson, ’65; Karfunkel, ’721, colcemid in the mouse neuroepithelium (Webster et al., ’731, or colcemid in the lens placode of the chick (Pearce and Zwaan, ’70) have failed to indicate any in-
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hibition of premitotic nuclear migration. In these experiments the accumulation of mitotic figures a t the lumen was due to the presence of cells blocked in metaphase. Pearce and Zwaan ('70) even demonstrated the almost total absence of microtubules in the cells of the lens placode after colcemid treatment, but still premitotic nuclear migration took place. Clearly the role of microtubules in premitotic migration remains unsettled. Concanavalin A also prevents interkinetic nuclear migration in the chick neural tube. This was demonstrated by both the presence of displaced mitotic figures and the failure of tritiated thymidine-labeled cells to undergo their normal interkinetic migration as demonstrated by autoradiography (Lee, '76). Concanavalin A is a plant protein whose biological activities depend on its binding to specific cell-surface carbohydrate residues (Goldstein et al., '73). It has been shown to cause agglutination of embryonic cells (Moscona, '71) and transformed cells (Inbar and Sachs, '691, but not of normal adult cells. Moran ('74a) demonstrated that concanavalin A (25 pg/ml) prevented migration of differentiating neural crest cells in vitro, but did not prevent neurulation in the amphibian Ambystoma maculatum (Moran, '74b). In addition it appears that the drug does not affect mitosis or cytokinesis, all indicating that its action is not on microtubules or microfilaments. Moscona ('71) has suggested that embryonic cells possess special cell surface sites, which contain carbohydrate residues. These provide the embryonic cells with their specialized cell movements. When the cells reach an "adult" state these cell surface sites are masked, cell movement ceases and the cells are no longer affected by concanavalin A. If this hypothesis is true it suggests that interkinetic migration of embryonic neuroepithelial cells is dependent not only on nuclear movement but possibly also on accompanying cell-membrane changes. The presence of binucleate cells in the cytochalasin B-treated embryos is in agreement with the well known effect of the drug in preventing cytokinesis, but not mitosis (Carter, '67). All the binucleate cells seen in this study were still a t the lumen, but the short exposure-time precludes us from concluding that post-mitotic migration was prevented. Bernfield and Wessells ('70) reported that multinucleate cells in cytochalasin B-treated lung epithelium did not migrate to the basal
surface, but they also described the cells as showing signs of ill health and necrosis. The mechanism by which cytochalasin B inhibits cytokinesis has been subject to some controversy because cells apparently vary in their permeability to the drug and, hence, in whether or not cleavage-furrow formation is prevented (Luchtel e t al., '76; Selman et al., '76). Schroeder ('69) proposed that in dividing Arbacia eggs a contractile ring of microfilaments just below the cleavage furrow was normally responsible for pinching the cell in two. In the presence of cytochalasin B the microfilament ring disappeared and cytokinesis was prevented. This theory has now been extended to include mammalian (Hela) cells (Schroeder, '70). Cytochalasin B has also previously been reported to cause binucleate cells in cultured salivary, oviductal and lung epithelia (Bernfield and Wessells, '70). Surprisingly, such cells were not reported in the chick neural tube after drug exposure (Karfunkel, '72; Messier and Auclair, '74). The protrusion and eversion of cells from the luminal side of the neuroepithelium is presumably associated with the loss of the microfilament bands a t the apical ends of the cells. In epithelia exposed to cytochalasin B the bands of microfilaments disappear from the cells, to be replaced by an amorphous mass of filamentous material, perhaps depolymerised microfilaments (Bernfield and Wessells, '70; Karfunkel, '72; Messier and Auclair, '74). The function of the apical microfilaments has been suggested from studies on neurulation (Baker and Schroeder, '67; Burnside, '71; Karfunkel, '72). The presence of the purse-string band of microfilaments is necessary for contraction of the apical ends of the neuroepithelial cells and, hence, for the conversion of the neural plate into neural tube. Since, in the developing cerebral vesicles, all the ventricular surfaces of the neuroepithelium are concave compared to the external surface, i t seems probable that the continued presence of the bands of microfilaments is necessary to maintain the curvature. Hence, if cytochalasin B disrupts this band of filaments, the neuroepithelial cells would tend to bulge into the ventricle, and if the external processes of the cells break under the increased tension, the cells would evert through their rings of junctional complexes. The junctional complexes do not break and prevent the cells from becoming free-floating in the lumen. It is interesting to note that the phenomenon of cells protruding
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gration in the absence of microtubules in the chick embryo. J. Embryol. Exp. Morph., 30: 661-671. 1974 Effect of cytochalasin B on interkinetic nuclear migration in the chick embryo. Devel. Biol., 36: 218-223. Moran, D. 1974a The action of concanavalin A on migrating and differentiating neural crest cells. Exp. Cell Res., 86: 365-373. 1974b The inhibitory effects of concanavalin A LITERATURE CITED on the development of the amphibian embryo. J. Exp. Auersperg, N. 1972 Microfilaments in epithelial morZool., 188: 361-365. phogenesis. J. Cell Biol., 52: 206-211. Moscona, A. A. 1971 Embryonic and neoplastic cell surBaker, P. C., and T. E. Schroeder 1967 Cytoplasmic filafaces: availability of receptors for concanavalin A and ments and morphogenetic movement in the amphibian wheat germ agglutinin. Science, 171: 905-907. neural tube. Devel. Biol., 15: 432-450. Pearce, T. L., and J. Zwaan 1970 A light and electron miBernfield, M. R., and N. K. Wessells 1970 Intra- and excroscopic study of cell behavior and microtubules in emtracellular control of epithelial morphogenesis. Devel. bryonic chicken lens using colcemid. J. Embryol. Exp. Biol. Suppl., 4: 195-249. Morph., 23: 491-507. Burnside, B. 1971 Microtubules and microfilaments in Sanger, J. W., and H. Holtzer 1972 Cytochalasin B: Effects newt neurulation. Devel. Biol., 26: 416-441. on cell morphology, cell adhesion and mucopolysaccharide Carter, S. B. 1967 Effects of cytochalasin on mammalian synthesis. Proc. Nat’l. Acad. Sci., 69: 253-257. cells. Nature, 213: 261-264. Sauer, M. E., and B. E. Walker 1959 Radioautographic 1972 The cytochalasins as research tools in cystudy of interkinetic nuclear migration in the neural tology. Endeavour, 31: 77-82. Exp. Biol. Med., 201: 557-560. tube. Proc. SOC. Schroeder, T. E. 1969 The role of “contractile ring” filaCloney, R. A. 1972 Cytoplasmic filaments and morphoments in dividing Arbacia eggs. Biol. Bull., 137: 413-414. genesis: Effects of cytochalasin B on contractile epider1970 The contractile ring. Fine structure of mal cells. Z. Zellforsch., 132: 167-192. dividing mammalian (HeLa) cells and the effects of cytoFujita, S. 1962 Kinetics of cell proliferation. Exp. Cell chalasin B. Z. Zellforsch., 109: 431-449. Res., 28: 52-60. Selman, G. G., J. Jacob and M. M. Perry 1976 The permeaGoldstein, I. J., C. M. Reichert, A. Misaki and P. A. J. Gorin bility to cytochalasin B of the new unpigmented surface 1973 An “extension” of the carbohydrate binding speciin the first cleavage furrow of the newt’s egg. J. Embryol. ficity of concanavalin A. Biochim. Biophys. Acta, 31 7: Exp. Morph., 36: 321-341. 500-504. Sidman, R. L., I. L. Miale and N. Feder 1959 Cell proliferaHinds, J. W., and T. L. Ruffett 1971 Cell proliferation in the tion and migration in the primitive ependymal zone; a n neural tube: An electron microscopic and Golgi analysis autoradiographic study of histogenesis in the nervous in the mouse cerebral vesicle. Z. Zellforsch, 115: 226-264. system. Exp. Neurol., 1: 322-333. Inbar, M., and L. Sachs 1969 Interaction of the carbohySpooner, B. S., J. F. Ash, J. T. Wrenn, R. B. Frater and N. K. drate-binding protein concanavalin A with normal and Wessells 1973 Heavy meromyosin binding to microfilatransformed cells. Proc. Nat’l. Acad. Sci., 63: 1418-1425. ments involved in cell and morphogenetic movements. Karfunkel, P. 1972 The activity of microtubules and miTissue Cell, 5: 37-46. crofilaments in neurulation in the chick. J. Exp. Zool., Spooner, B. S., and N. K. Wessells 1970 Effects of cyto181: 289-302. chalasin B upon microfilaments involved in morphogeneKarnovsky, M. J. 1965 A formaldehyde-glutaraldehyde sis of salivary epithelium. Proc. Nat’l. Acad. Sci., 66: fixative of high osmolarity for use in electronmicroscopy. 360-364. J. Cell Biol., 27: 137A-138A. 1972 An analysis of salivary gland morpbogeneKletzien, R. F., and J. F. Perdue 1973 The inhibition of sis: Role of cytoplasmic microfilaments and microsugar transport in chick embryo filjroblasts by cytotubules. Devel. Biol., 27: 38-54. chalasin B. Evidence for a membrane-specific effect. J. Watterson, R. L. 1965 Structure and mitotic behavior of Biol., Chem., 248: 711-719. the early neural tube. In: Organogenesis. R. L. DeHaan Kochhar, D. M. 1975 The use of in vitro procedures in and H. Ursprung, eds. Holt, New York, pp. 129-159. teratology. Teratology, 11: 273-288. Watterson, R. L., P. Veneziano and A. Bartha 1956 AbLangman, J., R. Guerrant and B. Freeman 1966 Behavior sence of a true germinal zone in neural tubes of young of neuroepithelial cells during closure of the neural tube chick embryos as demonstrated by t h e colchicine techJ. Comp. Neur., 127: 399-412. nique. Anat. Rec., 124: 379 (Abstract). Lee, H-Y. 1976 Inhibition of neurulation and inter. Webster, W., M. Shimada and J. Langman 1973 Effect of kinetic nuclear migration by concanavalin A in explanted fluorodeoxyuridine, colcemid and bromodeoxyuridine on early chick embryos. Devel. Biol., 48: 392-399. developing neocortex of the mouse. Am. J. Anat., 137: Luchtel, D., J. G . Bluemink and S. W. de Laat 1976 The ef67-86. fect of injected cytochalasin B on filament organization Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. Bradley, M. A. in the cleaving egg of Xenopus laevis. J. Ultrastruct. Res., Luduena, E. L. Taylor, J. T. Wrenn and K. M. Yamada 54: 406-419. 1971 Microfilaments in cellular and developmental processes. Science, 171: 135-143. Messier, P-E. 1976 Effects of formamide on neuroepiWocdard, T. M., and S. B. Estes 1944 Effect of colchicine on thelial cells and on interkinetic nuclear migration in the mitosis in the neural tube of forty-eight hour chick emchick embryo. J. Embryol. Exp. Morph., 35: 197-212. bryos. Anat. Rec., 90: 51-54. Messier, P-E., and C. Auclair 1973 Inhibition of nuclear mi-
into the lumen is opposite to that seen after microtubule disruption, when the cells protrude from the basal surface (Messier and Auclair, ’73). This suggests that the microtubules and microfilaments are in balance to maintain the neuroepithelium.
PLATE 1 EXPLANATION OF FIGURES
1
Photomicrograph of one-micrometer plastic section of the wall of the telencephalon from a n 11-day mouse control embryo. A thin mantle layer (ml) and a thick neuroepithelial layer (ne) can be distinguished. Mitotic figures are located adjacent to the ventricular lumen (vl). X 600.
2 Electron micrograph of the luminal side of the neuroepithelium from a control embryo. Adjacent internal (apical) processes are joined by junctional complexes (jc). Similar dark-staining junctions are seen between the metaphase figure (m) and internal processes of adjacent cells. X 3,400.
3 Section through the internal processes of neuroepithelial cells from a control embryo. Note the junctional complexes (jc) and the bundles of microfilaments (mf) traversing the apical parts of the cells. 4
X
25,000.
Electron micrograph of the outer part of the cerebral vesicle from a control embryo. The cytoplasm of the external (basal) processes of the neuroepithelial cells expand to form broad end-feet (ef) a t the basal lamina (bl). Adjacent end-feet are not connected to each other by cell junctions. Differentiating neurons (n) in the mantle layer are also visible. x 1,900.
5 Photomicrograph of a 1-pm section of the wall of the cerebral vesicle from a n 11-day mouse embryo cultured for two hours in cytochalasin B. Mitotic figures (mit) are seen in the middle of the neuroepithelial layer. Some of the neuroepithelial cells have everted into the lumen (arrow). X 600.
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CYTOCHALASIN B AND NEUROEPITHELIAL CELLS William Webster and Jan Langman
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
6 Electron micrograph of the neuroepithelium of a cerebral vesicle from a cytochalasin B-treated embryo. Among the normal-looking neuroepithelial cells are two binucleate cells (*). Free cells with one or two nuclei can also be seen in the lumen of the ventricle. X
3,400.
7 Low-power electron micrograph of the neuroepithelium from a cytochalasin B-treated embryo. A short distance from the lumen is a cell in telophase (t,).I t shows no cytoplasmic constriction and will presumably become binucleate. A second cell in telophase (t,) is a t the lumen. This cell is dividing in a plane perpendicular to the neuroepithelial cells and shows evidence of a cleavage furrow. X 3,400. 8 Low-power electron micrograph of the neuroepithelium from a cytochalasin B-treated embryo. Note the presence of two displaced mitotic figures. The internal limiting boundary marked by junctional complexes (jc) is unclear, a s many cells have protruded into the lumen of the ventricle (vl). Some of these cells appear to be connected to the neuroepithelium by junctional complexes. Note the binucleate cell (*). x 3,400.
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CYTOCHALASIN B AND NEUROEPITHELIAL CELLS Willlam Webster and J a n Langman
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
9 Low-power electron micrograph of t h e neuroepithelial layer from a cytochalasin B-treated embryo. Note the displaced anaphase-figure and the adjacent binucleate cell. X 3,400. 10 Electron micrograph of a protruded neuroepithelial cell, with a single nucleus, still attached to the neuroepithelial layer. X 3,400.A higher magnification of the cell junction seen in the rectangle is shown in figure 11. 11 Higher magnification of the junctional complex between a n extruded cell (ec) and the neuroepithelium. Although the junctional complex appears normal there are no obvious microfilaments. The filaments seem to be replaced by a rather ill-defined amorphous mass. X 19,000.
12 Electron micrograph of a binucleate cell (*I extruded into the ventricular lumen (vl). The junctional complex shown in the rectangle is seen a t higher magnification in figure 13. X 3,400.
13 Higher magnification of junctional complex between the extruded binucleate cell (ec) and the neuroepithelium. The junctional complex appears normal but clearly defined microfilaments are absent. x 25,000. 14 Micrograph of the neuroepithelial layer of a cytochalasin B-treated embryo. Two binucleate cells are present. Both are still attached to adjacent cells by junctional complexes. Another cell is seen in the process of extrusion. The cell appears to have been ejected through its ring of junctional complexes. x 1.900.
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PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
15 Light micrograph showing displaced mitotic figures throughout the neuroepithelium from a cytochalasin B-treated embryo. X 800. 16 Electron micrograph showing the outer part of cerebral vesicle from a n experi-
mental embryo. Note the vacuoles in the external (basal) processes of the neuroepithelial cells and in the differentiating neurons of the mantle layer. X 3,400. Compare with control, figure 4.
17 Light micrograph of the cerebral vesicle from a n experimental embryo. The neuroepithelial and mantle layer appear to have pulled away from t h e external limiting layer (ell). The end-feet and the basal lamina remain in position and are intact. The normal concave appearance of the internal limiting boundary is lost and many cells have been extruded into the lumen of the ventricle (vl). X 600. 18 High-power electron micrograph of the internal (apical) processes of the neuroepithelium from a control embryo. Note presence of junctional complexes between adjacent cells and the microfilamentous band (mf) extending across the internal processes. X 25,000.
19,20 High-power electron micrographs of similiar region of internal processes as seen in figure 18 but from cytochalasin B-treated embryos. Junctional complexes appear normal but there are no microfilament bands. Instead, there are amorphous masses of filamentous material. X 19,000,
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PLATE 4
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