Fine structure of cultured glioblasts before and after stimulation by a Glia Maturation Factor

Printed in Sweden Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form resewed ISSN 0014-4827

Experimental

FINE

STRUCTURE

AFTER

Cell Research 106 (1977) 357-372

OF CULTURED

STIMULATION

GLIOBLASTS

BY A GLIA

BEFORE

MATURATION

AND

FACTOR

R. LIM, S. S. TROY and D. E. TURRIFF Departments Research

of Surgety Institute,

(Neurosurgery) University

und

of Chicago,

Biochemistry Chicago,

and IL 60637,

the Bruin USA

SUMMARY The effect of Glia Maturation Factor on glioblasts in a monolayer culture is studied at the ultrastructural level. The most important finding consists of a change from cells with a predominance of sheath microfilaments (SO A) and desmosome junctions to cells with a predominance of 100 A-filaments (gliotilaments) and the puncta adhaerentia type of junctions. The restructuring of the cellular tine components, taken together with our earlier studies on chemistry and cytodynamics. is consistent with the matutation of glial cells in vitro. Although the microtubules do not alter in number, they change from a random orientiation to a parallel alignment with respect to the long axis of the cell processes. Glioblasts in aggregate cultures undergo morphological maturation without the help of the exogenous maturation factor. Glycogen granules characteristic of glia are also observed in these cells. The results suggest that the maturation factor is synthesized by the glial cells during the course of development and that cytodifferentiation is triggered by a local accumulation of the factor above a threshold level.

In 1972 our laboratory [12, 131 observed that one type of embryonic rat brain cell having an epithelial morphology in a monolayer culture can be stimulated by a factor to differentiate into multipolar interconnected astrocyte-like cells. Subsequent studies [14, 151 showed that the factor is a protein with a molecular weight of about 350000. The chemical evidence that the cells are glial in nature came from the high levels of two glia-specific proteins: the S- 100 [ 171 and the GFA or Glial Fibrillary Acidic protein [6] [Lim et al., unpublished]. Cinematographic studies [ 161 showed that the morphologically differentiated cells exhibit dynamic properties characteristic of glia, such as the tugging of processes and pulsation of cell bodies. The coordinated

undulation executed by these cells [ 161 is in great contrast to the random walk characteristic of fibroblasts. The purpose of the present work is to demonstrate electron microscopic changes in cellular fine structures and to relate these changes to alterations in cell shape. MATERIALS

AND METHODS

Cell culture Brain cells were dissociated from IFday SpragueDawley rat fetuses and seeded in Falcon plastic culture flasks as previously described [ 151. After carrying the culture into the second passage, the epithelial glioblasts, which formed a homogeneous cell carpet free of neuroblast contamination, were exposed to an F,,, medium containing the partially purified Glia Maturation Factor [ 151. The medium was supplemented with 5 % fetal calf serum, penicillin (50 U/ml) and streptomycin (100 pg/ml). The cells were grown at 3PC in an

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atmosphere of 5% (v/v) CO, in air with saturated humidity. Feeding was carried out every other day with the above medium. Cells exposed to the factor for a week were fixed for the current study. For comparison the epithelial glioblasts never exposed to the factor were also studied.

Scanning electron microscopy The cells on the plastic flasks were rinsed briefly with Tris-saline (0.15 M NaCl and 0.02 M Tris-HCI. OH 7.4) and fixed at room temperature for 1 h with 2’6 gluta: raldehyde, 1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (modification of Kamovsky [9]). The cells were post-fixed with 1% osmium tetroxide in the same buffer for 2 h and subsequently rinsed with the buffer. An area of the flask, less than 1 cm* in size and previously marked under the guidance of an inverted phase-contrast microscope, was cut out with a razor blade. The ceils were dehydrated by stepwise (IO min each) treatment with 50,75 and 95% ethanol, followed by three treatments with absolute ethanol. Finally, the cells were subjected to critical point drying [2] with liquid CO,. The dried cells were coated first with carbon and subsequently with gold-palladium (60.40). Scanning was conducted with a Hitachi HFS-2 scanning electron microscope using a field emission tip.

Transmission electron microscopy The cells on the flask were rinsed with Tris-saline, fixed with glutaraldehyde-paraformaldehyde. postfixed with osmium tetroxide, and dehydrated in ethanol as described above, without cutting the plastic flask. After the absolute alcohol step. the cells were treated successively with ethanol-Epon mixtures (2 : I, 1: I and I : 2) for a period of 30 min each and were left in 100% Epon overnight at room temperature. (Propylene oxide was omitted because it interacts with the plastic wall.) After replacement with a freshly prepared-embedding Epon, the flasks were placed in a 60°C oven for 48 h to complete the polymerization. A piece of the polymerized &on together with the adhering culture flask was punched out with a motordriven saw-tooth bore. The area to be processed was nreviouslv marked under visualization with a phase contrast microscope. Upon trimming the block to the size of I-2 mm in diameter, the oiece of adhering flask wall was separated with pointed pliers; this exposed the cell layer on the Epon block. The pictures presented in this paper are derived from sections, 60-((0 nm thick, made parallel to the flask surface. The ultrathin sections were stained with uranyl acetate and lead citrate. Electron microaraohs were made with a Hitachi HU-IIC electron microscope. For phosphotungstic acid (PTA) staining, the glutaraldehyde-paraformaldehyde fixed cells were dehydrated up to the 95 % alcohol step (post-fixation with osmium tetroxide was omitted). The cells were then stained for 3 h with 2% phosphotungstic acid in absolute ethanol [5]. Followina this. dehvdration was comoleted with absolute ethanol. Epon penetration and embedding were as described above. Sections stained with PTA were not further stained with uranyl acetate or lead citrate. Erp

Cell

Res 106 (1977)

RESULTS Fine structure ofepithelial gliohlasts

A confluent monolayer of epithelial glioblasts never exposed to the maturation factor appears under phase-contrast microscopy as a thin carpet of translucent cells (fig. 1A). Each cell is maximally stretched out, anchoring to and smoothly blending with the surrounding cells. The cell border, when discernible, consists of a relatively pale line decorated with microvilli which appear as black dots. The cells are mononucleated, with each nucleus usually containing more than one nucleolus. In some cells one can see parallel tibrils running through the entire course of a cell, showing no definite orientation with respect to the nucleus or to adjacent cells. The appearAbbreviations wifhin illustrations. b, border; ch, chromatin; dj, desmosome-like junction; er, endoplasmic reticulum;f, 100 A-filament;jb, fibril;fg, fat globule; fp, filopodia; G. Golgi apparatus;gg, glycogen granule; /pp.lamellipodia; ly, lysosome; mf, microtilament; mif, mitochondria; ml, microtubule; mv, microvilli; n, nucleus; ne, nuclear envelope; nl, nucleolus; np. nuclear pore; p, process; pa, puncta adhaerentia; pm, plasma membrane; pt, pit; pv, pinocytotic vesicle; rb, ribosome; rJ tonofilament; v, vesicles; wb, web-like spreading. Fig. 1. Epithelial glioblasts under (A) phase contrast; (B-D) scanning electron microsconv. Parallel oblique lines in (A) a&manufacturer’s markings on the plastic flask surface; (B) vertical view; (C) 45” view of the lower portion of(B); (D) detail of rectangular area in (B). (A) x280; (B) x 1000; (C) x9600; (0) x4WO. Fig. 2. Transmisson electron micrographs of epithelial glioblasts stained with the usual osmium tetroxide method. (A) Nuclear and perinuclear regions, x 12500; (E) region further away from the nucleus, x 12 500. Arrowheads indicate densities in sheath microtilaments (mf). Peripheral region (C) x 100000; (0) x50000. Fig. 3. Transmission electron micrographs of epithelial glioblasts stained with phosphotungstic acid to emphasize the fibrillar structures. (A) Three bundles of sheath microtilaments (mf) (X12500). Arrowheads indicate dense areas. fnser: detail of marked area at x50000. (B) Sheath microfilaments more dispersed than in (A), x37500; (C) a field predominantly of 100 A filaments m, x50000; (0) mixture of microtubules (mr) and 100 A filaments. x50000. Note that with this stain the tibrillar structures appear coarser than with osmium tetroxide, while most other organellcs are either partially or totally destroyed.

Fig.

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Fig. 4. Transmission electron micrographs of epithelial glioblasts showing cell junctions. (A), (II). stained whh

the usual osmium tetroxide method; (C) stained with phosphorungsk acid. x25 OW.

ante is that of a group of contact-inhibited cells with a tendency to spread out thinly over the substratum. Scanning electron microscopy confirms the above observation, while revealing more details about the microvilli (fig. l&D). When viewed from an angle, these microvilli appear as finger-like projections extending above the cell surface. They have a fairly uniform diameter of about 0.2 pm and a variable length ranging from a mere elevation to 1 pm. Although more concentrated at the border, they are also present in the central area of some cells. Small pits are occasionally seen between the microvilli. Apart from these features, the surface texture is relatively smooch and the terrain is flat throughout the whole monolayer. Blebs (or knobs) having a diameter of 0.5-2.0 pm that are characteristic of dividing cells [23] are not observed.

Transmission electron microscopy (figs 2, 3) reveals relatively few organelles in the cytoplasm and they usually cluster in the perinuclear region. The mitochondria, when observed, are invariably of the tilamentous type, a fact probably attributable to the thinness of the cell layer. Other regions of the cytoplasm are dominated by the fibrillar structures, the most abundant of which are the microfilaments (about 50 8, in diameter) of the “sheath” variety [26]. These microfilaments occur in bundles of variable width and are apparently very long, as they always run off the plane of the thin sections. These long bundles probably correspond to the fibrils seen under phase con&. 5. Moraholonicallv differentiated elioblasts unde% scanning ele&on microscopy. (A) 6eneral view (X 100); (B) detail of (4 I, X 1000: KY detail of (A b x600; (D) detail of cell surface, x’5boo: (E) cell pi01 cesses showing intercellular connections, X2260.

Glioblast

Fig.

5.

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Fig. 6. Transmis ,.I?Exp Cell Res 106 (1977)

rerenuareu guoomsr, showing regions. X25000.

Gliohlast trast. All other organelles, such as endoplasmic reticulum, ribosomes and mitochondria, are completely excluded from the bundles. In the sheath microfilaments one can observe scattered areas of densities like those characteristic of smooth muscle contractile systems. Also present, but to a much lesser degree, are the filaments with a diameter of 100 A. They are found more often toward the periphery of the cells and are totally random in arrangement. The microtubules (200 A in diameter) are very few in number and are scattered throughout the cytoplasm. They show no regular orientation with respect to themselves or to the other types of fibrillar structures. Two features characterize the cell junction (fig. 4). (I) The junction is exclusively of the desmosome type; (2) this desmosome-like junction is coextensive with intercellular contact. Since most of the glioblasts in a confluent culture are surrounded by neighboring cells, one often encounters cells that are completely surrounded by a series of desmosomes arranged in tandem. The tonofilaments (100 A in diameter) characteristic of desmosomes are best demonstrated in our system with phosphotungstic acid stain. A clear space, around 300 A in width, exists between the two plasma membranes. Fine structure of morphologically differentiated glioblasts Scanning electron microscopy (fig. 5) confirms our earlier observation (using phase contrast) on the multipolar appearance of the differentiated cells after exposure to the maturation factor. Numerous processes and filopodia ramify from the cell bodies, which now show depth and volume. The processes either terminates with a web-like spread on the flask surface or become

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blended into a bundle with processes from other cells. As in the epithelial glioblasts, many microvilli protrude from an otherwise smooth cell surface. The absence of blebs or knobs, as in the flat cells, probably denotes the absence of cell division, as the specimen was taken from a culture seven days after exposure to the maturation factor, a time when cell division is inhibited [16]. The flask surface appears perfectly clean, free of fibrous deposits or any recognizable textural change. Transmission electron microscopy (figs 6-8) reveals many distinctive features inside the cells. The perinuclear region contains many prominent Golgi apparatuses, which are rarely seen in the epithelial g!ioblasts. Likewise, fat globules, never observed in the flat cells, appear in the perinuclear region as well as the proximal part of the processes, ranging in size from 0. I to 0.8 pm in diameter. The cells are rich in mitochondria which now exist in oblong or oval forms and which are distributed throughout the entire cell. The cytoplasm is studded with numerous ribosomes which are either free or attached to the weli developed endoplasmic reticulum. The abundance of organelles and their well-differentiated features indicate the healthy state of the cells. Perhaps the most striking feature is the abundance of the 100 A filaments, which are identical with gliofilaments in mature glial cells in vivo, especially the fibrous astrocytes. Those present in the cell body (fig. 6) assume a wavy appearance: those located in the periphery (fig. 8) look more rigid, probably because of the stretching of the processes. In either place they are arranged in parallel arrays and are directed along the long axis of the processes. It is not unusual to see processes that are completely tilled with the 100 A filaments. ExpCd Xesim

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Unlike the 100 A filaments, the sheath microfilaments which are characteristic of epithelial glioblasts have almost completely disappeared, leaving only a trace in the processes. In the current work we used cells at the 7-day point. Cells taken from the 2-day point, when they are actively engaged in tugging, contain relatively more microfilaments (not shown), but even here the number is considerably less than that in the flattened cells. To our surprise, there appears to be no change in the number of microtubules as a result of morphological differentiation. This observation was confirmed by a chemical assay based on the binding of labeled colchicine [Turriff & Lim, unpublished]. The only change lies in the orientation of the microtubules: they are now parallel to the 100 A filaments with which they are interspersed. Microvilli are seen in the cell body and along the processes. They appear to be simple protrusions from the plasma membrane, devoid of any recognizable structures inside. The process terminals (fig. 8) are rich in lamellipodia (ruffles) and pinocytotic vesicles (a few of which are also scattered in the proximal portion as well as in the perinuclear region). Mitochondria and lysosomal particles are abundant. The tilopodia, which often shoot out from the terminals, are devoid of internal structures, as are the microvilli. In the surface membrane, the desmosome-like junctions characterizing the epithelial glioblasts have disappeared, being replaced by many punctate junctions containing an intercellular space of about 150 A (fig. 9). Electron-dense areas appear on both cytoplasmic sides and occasionally extend into the intercellular space. There are no associated tonofilaments. These juncExp Cell

Res 106 (1977)

tions, designated as “puncta adhaerentia” or adhesive points by Peters et al. [223, are considered a variant of the classical zonulae adhaerentes described by Farquhar & Palade [7]. In the morphologically differentiated glioblasts, adhesive points are commonly observed at the sides of closely apposed processes. Fine structure of reaggregated epithelial glioblasts An experiment was conducted to determine the effect of cell aggregation on the morphological maturation of glioblasts. A homogeneous population of confluent epithelial glioblasts was dissociated by tryp sinization and permitted to reaggregate in the F,, medium supplemented with 5 % fetal calf serum and the usual amounts of antibiotics (see Methods). By rotating at 70 rpm in a gyratory shaker [20] at 37”C, cell groups of about 0.5-1.0 mm in diameter were formed. These reaggregated cells were maintained under the above conditions for 6 weeks, with feeding twice weekly, before being processed for electron microscopic study (fig. 10). The cells share with those morphologically differentiated glioblasts in surface cultures the features characteristic of mature glial cells, such as the abundance of the 100 8, filaments (fig. 10) and the adhesive points (not shown). Glycogen granules, the chemical nature of which was confirmed by its susceptibility to amylase, are abundant in the cytoplasm (fig. 10). The presence of glycogen granules is strongly in favor of the glial nature of the 7. Transmission electron micrograph of a morphologically differentiated ghoblast, showing the proximal region 6f a cell process. (A) Montage, x833j; (B) detail of (A) showing a bundle of microfilaments Cm&, x25000; (C) an &ea comparable to (B) but from a different section stained with phosphotungstic acid, x25000. Arrowheads indicate densities in microtilaments. (0) Detail of (A) showing microtubules (mr) and 100 A filaments m, X2S Ooo. Fig.

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Fig. 8. Transmission electron micrograph of a morphologically differentiated glioblast, showing the distal region of a cell process, ~25000. The nucleus of an Exp Cell

Res 106 (1977)

adjacent cell is included in the field. Inset: a process terminal, X 1.5000.

Fig.

9. Transmission electron micrograph of morphologically differentiated glioblasts, showing puncta ad-

haerentia (pa) between two processes, set: detail of the junctions. X50000.

cells, It is important to note that the reaggregated cells attain morphological maturity whether or not the Glia Maturation Factor is added, unlike the monolayer cultures where differentiation occurs only in the presence of the factor. The significance of this point is discussed below.

not completely possible for us to distinguish those ultrastructural events that lead to the change in cell shape from those that might have resulted from it. Although the sheath microfiiaments exist in a wide variety of cell types, they have been observed in cultured glioblasts [26, 271 and are commonly seen in protoplasmic astrocytes in the brain [22]. Wessells [26] reported that the microfilaments not only contain scattered densities but also bind the heavy meromyosin (HMM). Thus it is likely that the microfilaments observed in our flattened cells are the equivalent of actin. However, since the cells in this study are taken from a confluent and contact inhibited culture, the bundles of microfilaments more likely confer a rigid and stretched configuration to the cells than participate in actual cell motility, as has been suggested [I 13. We have not been able to identify the

DISCUSSION The current study establishes a correlation between the change in cell shape and the restructuring of the fine components of the cell, both in the interior and in the plasma membrane. Some of the striking differences between the epithelial and the morphologically differentiated glioblasts include the predominance of sheath microfilaments and desmosome junctions in the former, and the predominance of 100 8, filaments and punctate adhesions in the latter. However, it is

Exp Cdl

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Fig. 10. Transmission electron micrograph of a reaggregated glioblast culture, showing the 100 kilaments v) and glycogen granules kg). (A) Not treated with amylase; (B) after incubating the thin sections

with a-amylase according to the method of Monneron & Bernhard [18], which eliminated the granules. x37500.

“lattice” variety [26] of microfilaments. Nevertheless, its presence in the flattened cells can be inferred by the pronounced effect of cytochalasin B [143, since only the lattice type is susceptible to disruption by the drug [26,27]. As is commonly observed with other cells [l, 81, epithelial glioblasts exposed to cytochalasin B (5 pg/ml medium) rapidly (within 4 h) turn into an arborization of rugged and short cytoplasmic strands. This is in great contrast to the effect of the maturation factor, which shows a long (12 h) latency and which results in the outgrowth of smooth and long processes resembling those naturally occurring in glial cells. It is worth mentioning that, unlike the

glial cells, neurons do not contain sheath microfilaments. Neurons do contain the lattice microfilaments but they are limited to the axonal growth cone [26]. The large number of 100 A filaments in the morphologically differentiatedglioblasts is of course consistent with fibrous astrocytes. Besides these cells, we also observed the presence of a few cells with the electron microscopic features of oligodendroglia (dark and granulated cytoplasm; few 100 A filaments). It is not clear whether the observed astrocytes and oligodendroglia arise from the same stem cells or are derived separately from precommitted glioblasts. The absence of a quantitative change in microtubules is of interest. Since we have

Gliohlast demonstrated with colchicine and vinblastine [ 141 that the integrity of microtubules is essential for morphological differentiation, the current observation underscores the importance of their organization in the shaping of the cells. Alternatively, it is possible that only those microtubules that might be associated with the plasma membrane [lo] and whose presence and changes may not be readily detectable might be involved in the alteration of cell shape. One should also be cognizant of the other effects of colchicine, such as those on membrane transport and general metabolism (see [ 11). Adhesive points are frequently observed among brain cells in vivo, both between neurons and between glia [22]. The appearance of these junctions with morphological differentiation is in line with the concept of glial maturation. In reference to our cinematographic study of cultured glioblasts [16], these junctions probably serve as points of contact at which the cells tug against each other. Materials adhering to the culture flask, either introduced by the medium or extruded by the cells, may affect cell shape through contact guidance. Weiss & Garber [25], for example, reported that the shape of fibroblasts can be framed by the fibrin network in a plasma clot culture. The complete absence of any fibrous material on our flask surface excludes the possibility of contact guidance. Furthermore, the presence of collagen-producing fibroblasts is ruled out, since the extracellular collagen fibers would have been detected under scanning electron microscopy [21, 231. The study of cell morphology and its relationship with chemical differentiation is basic to the understanding of life processes. In cell cultures, the observed histotypic pattern is the net effect of a number of interacting forces. Such forces include the ad-

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hesiveness of the cell surface to the substratum, the affinity among adjacent cells, and the intrinsic cellular structures such as the number, orientation and distribution of the fibrillar elements, the organization of the membrane systems, and the consistency and movement of the cytoplasm and its organelles. The complexity of the interaction, as yet poorly understood, leads to the common notion that morphology is a poor criterion to use in identifying cultured cells. Such unpredictability is by no means an indication of a random and chaotic underlying mechanism. On the contrary, the plasticity of cell shape and the ease of chemical manipulation make tissue culture a useful model for the study of cellular morphogenesis. The choice of glioblasts provides further advantages in that their histotypic pattern changes dramatically during differentiation. The results of the current study, like our earlier studies on chemistry and cytodynamics, strongly indicate the shift of the cells from a lower to a higher state of maturation following stimulation by the protein factor. A possible clue to the biological role of the factor comes from the observation that the protein is required for cytodifferentiation only in a monolayer culture but not in an aggregate culture. A number of reasons (its high molecular weight; its strict localization in solid organs) led us to speculate that the protein might exist on the cell surface membrane [lS]. By further assuming that the receptor for this protein also lies on the cell surface, a mechanism could be proposed to explain why cells can activate each other when they are in physical contact, but not when they are separated. It is likely that in cell aggregates sufficient numbers of the receptors in each cell are stimulated. In a monolayer, however, the minimum cell contact results in the stimulation of only a

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small number of receptors; the addition of the protein factor could then stimulate the remaining receptors, partially reconstituting the conditions enjoyed by the cells in a three-dimensional culture. Such communication through surface macromolecules could complement the already known ionic interactions across the gap junctions (for review, see [24]). Alternatively, the maturation factor could simply be a soluble protein which needs to build up within or around glial cells to a threshold concentration before cytodifferentiation occurs. In aggregate cultures this threshold level is reached, but in monolayers the protein is continuously lost to the culture medium. In either hypothesis, it seems as if a local accumulation of the protein factor is essential for its function. Examples abound that demonstrate the attainment of higher levels of maturation when cells are permitted to grow in an aggregated form. For instance, the induction by hydrocortisone of glutamine synthetase in neuroretinal cells is effective in aggregate cultures but not in surface cultures [19]. In a separate study [3, 41 it was reported that the astrocytoma cell line RGC-6 produces the glia-specific GFA protein only in three-dimensional cultures. Conversely, it is well known that cells dissociated from an organ tend to revert to a lower state of differentiation. Perhaps the study of the protein factor might in the long run bring us to a better understanding of this phenomenon that is universal to all multicellular organisms. We thank Dr L. M. H. Larramendi and Mr Joseph Zientarski for nrovidina facilities and assistance for transmission electron microscopy. We are grateful to the Enrico Fermi Institute of the University of Chicaao for the use of the scanning microscope; and to 6r Paul S. D. Lin and Mr Telmec Peterson for valuable assistance. Dr Arthur Arnold and Dr Robert Richardson participated in the preliminary phase of this work. Drs Troy and Turriff (NIH postdoctoral fellowship NS-05017) are Research Associates in Dr Lim’s labora-

tory. We thank Dr Jane Ovetton and Dr Aron A. Moscona for the critical reading of this manuscript. This work was supported by USPHS grants nos. NS-09228, CA-14599, CA-19266 and NS-07376.

REFERENCES I. Allison, A C, Locomotion of tissue cells. Ciba Foundation Symposium I4 (new series) (ed M Abercrombie) p. 109. Associated Scientific Publishers, Amsterdam (1973). 2. Anderson, T F, Physical techniques in biological research (ed A W Pollister) vol. 3, p. 178. Academic Press. New York (1956). 3. Bissell, M G, Eng, L F, Herman, M M, Bensch, K G & Miles, L E M, Nature 255 (1975) 633. 4. Bissell, M G, Rubinstein, L J, Bignami, A & Herman, M M, Brain res 82 (1974) 77. 5. Bloom, F E & Aghajanian, G K, J ultrastruct res 22 (1968) 361. 6. Bock, E; Jorgensen, 0 S, Dittmann, L & Eng, L F, J neurochem 25 (1975) 867. 7. Farquhar, M G & Palade, G E, J cell biol 17 (1963) 375. 8. Goldman, R D, Germaine, B & Bushnell, A, Locomotion of tissue cells. Ciba Foundation Symposium I4 (new series) (ed M Abercrombie) p. 83. Associated Scientific Publishers, Amsterdam (1973). Kamovsky, M J, J cell bio127 (1%5) l37a. 1;: Kochhar, 0 S, J cell biol70 (1976) 143a. 11. Lazarides, E, J cell biol70 (i976) 359a. 12. Lim. R. Li. W K P & Mitsunobu. K. Abstr of 2nd arm ‘meeting sot neurosci, Houston,’ Texas (1972) 181. 13. Lim, R, Mitsunobu, K & Li, W K P, Exp cell res 79 (1973) 243. 14. Lim, R & Mitsunobu, K, Science 185 (1974) 63. IS. - Biochim biophys acta 400 (1975) 200. 16. Lim, R, Turriff, D E & Troy, S S, Brain res 113 (1976) 165. 17. Lim, R, Turriff, D E, Troy, S S, Moore, B W & Eng, L F, Science 195 (1977) 195. 18. Monneron. A & Bernhard. W. J microsc 5 (1966) 697. 19. Morris, J E & Moscona, A A, Dev biol 25 (1971) 420. 20. Moscona, A A, Exp cell res 22 (l%l) 455. 21. Overton, J & Collins, J, Dev bio148 (1976) 80. 22. Peters, A, Palay, S L & Webster, H de F, Fine structure of the nervous system. Harper & Row, New York (1970). 23. Porter. K R, Puck. T T, Hsie, A W & Kelley, D, Cell 2 (1974) 145. 24. Sheridan, J D, Cell communication (ed R P Cox) p. 31. John Wiley & Sons, New York (1974). 25. Weiss, P & Garber, B, Proc natl acad sci US 38 (1952) 264. Wessells, N K, Neurosci res prog bull 1 I (1973) 24. f ;: Wessells, N K, Spooner. B S & Ludueha, M A, Locomotion of tissue cells. Ciba Foundation Symposium 14 (new series) (ed M Abercrombie) p. 53. Associated Scientific Publishers, Amsterdam (1973). Received October 13, 1976 Accepted January 4, 1977