J O U R N A L OF ULTRASTRUCTURE RESEARCH
60, 169-180 (1977)
Intercellular Junctions and Other Membrane Specializations in Developing Spinal Ganglia: A Freeze-Fracture Study 1 E. PANNESE,* L. LUCIANO,t S. IURATO,T+ AND E. R E A L E t *Second Institute of Human Anatomy, University of Milan, Italy, tLaboratory of Electron Microscopy, School of Medicine, Hannover, Federal Republic of Germany, and $Department ofB ioacoustics, University of Bari, Italy Received December 6, 1976 Intercellular junctions and other membrane specializations of neuroblasts and satellite cells were studied in spinal ganglia of chick embryos at the 4th, 10th, and 16th incubation days using the freeze-fracture technique. At the 4th day neuroblasts are closely arranged and connected by gap junctions. At the 10th day, instead, satellite cells appear interposed between neuroblasts so the gap junctions previously joining the latter are no longer evident. These temporary junctions probably play a role in cell differentiation and intercellular adhesion. Together with adhering junctions, gap junctions might maintain the cell organization of the ganglionic rudiment, which at the 4th day still lacks connective tissue. At the 10th day adjacent satellite cells display on their split plasma membrane small gap junctions, short, usually isolated strands recalling those composing zonulae occludentes, and orthogonal particle assemblies. These two latter specializations are still evident at the 16th day. The possible significance of these specializations is briefly discussed.
Evidence accumulated during the last few years suggests that the different types of intercellular junction play relevant (10, 17, 44, 60, 61, 68) and to some extent separate (4, 6, 57) roles in the developmental processes. Until now intercellular junctions in spinal ganglia rudiments were studied only in thin sections (40-42), in which, however, it was not always possible to identify with certainty all the different junctional types. In order to obtain more precise information on the various types of junction which can be found in developing spinal ganglia, the freeze-fracture technique, which allows an unequivocal identification of junctional types, was employed. By using this technique numerous gap junctions were found between adjacent neuroblasts at an early developmental stage, while only some gap junctions were
observed between satellite cells in a later developmental stage. Moreover, other specializations of the plasma membrane of satellite cells were found.
Reprint requests should be sent to Prof. Ennio Pannese, UniversitA degli Studi di Milano, Secondo Istituto di Anatomia Umana Normale, Via Mangiagalli, 14-20133 Milano, Italy.
MATERIALS AND METHODS Chick embryos at the 4th (25 specimens), 10th (10 specimens), and 16th (3 specimens) days of incubation were used. The spine was quickly removed from the embryos placed in the fixative and was cut into segments using a razor blade; then, the single spinal ganglia were isolated from these segments maintained in the fixative. The fixative solution contained 2% formaldehyde obtained from paraformaldehyde (Fluka AG, Buchs, St. Gallen), 2% glutaraldehyde (ultrastructure grade, Polaron Ltd., Watford), 0.1 M sodium cacodylate buffer (pH 7.2) and 25 mg% CaC]2. After fixation (total time, 2 hr), the isolated ganglia were soaked in 30% glycerol in saline for 1 hr, collected in small groups on specimen holders, frozen in liquid Freon 22 (monochlorodifluoromethane) at -150°C, and finally fractured at about -120°C and shadowed with platinum-carbon in a Balzers BA 360 M unit (Balzers AG, Liechtenstein). Some spinal ganglia rudiments at the 4th incubation day were also fractured and replicated without prior fixation. The replicas were cleaned in hypochlorite bleach and, if necessary, chromic acid, and then were repeatedly washed in distilled water.
169 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
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PANNESE E T
The p l a t i n u m - c a r b o n replicas were mounted on Formvar-carbon m e m b r a n e s (15) and examined in a Siemens Elmiskop 101 electron microscope. RESULTS
In replicas of freeze-cleaved developing spinal ganglia both fractured regions of the cells and surface views of their split plasma membranes were revealed. In the fractured regions the nucleus and cytoplasmic organelles were seen, thus allowing an identification of the replicated cell WPe, The only surface views of plasma membranes taken into consideration in this study were those belonging to identifled cells. As in other tissues (7), the plasma membrane of the cells in the developing spinal ganglia splits, giving rise to two complementary membrane faces, the inner or protoplasmic (A or P face) of which showed more randomly scattered particles than the outer or extracellular (B or E face). At the 4th incubation day the rudiment of the spinal ganglion exhibits an epitheiialilike structure. It is built of closely arranged cells (undifferentiated cells, primitive neuroblasts, and a few intermediate neu_roblasts), and lacks connective tissue and blood vessels. In this stage adjacent neuroblasts appeared connected by numerous gap junctions (Figs. 1-7). In the replicas these appeared as assemblies of regularly arranged particles about 8 nm in diameter on the P face of the split plasma membrane, and as arrays of tiny pits on the complementary E face (Fig. 5). While most gap junctions exhibited a compact packing of particles, some showed particle-free regions (Fig. 10). The latter junctions were morphologically similar to those described in other
AL.
embryonic tissues by Decker and Friend (9). No structural differences were noted between the gap junctions of fixed and unfixed ganglionic rudiments (compare Figs. 9 and 11). Frequently, the fracture plane could be seen to step from one adjacent membrane to the other, thus exposing portions of the P face from one membrane and portions of the E face from the other. If a gap junction was located along one of these steps, only particles on the P face and pits on the complementary E face were recognizable at the level of the junctional area (Figs. 5 and 12). While the adjacent plasma membranes usually appeared separated by a large distance, at the level of the particle and pit assemblies the intercellular space was obliterated (Figs. 5 and 12). This observation indicated the gap junctional hature of the particle and pit assemblies. Gap junctions were the only type of intercellular junction found in the replicas of the developing ganglia at the 4th incubation day. At the l Oth incubation day, undifferentiated cells, neuroblasts, pseudounipolar nerve cells, and satellite cells are recognizable in the rudiment of the spinal ganglion. Satellite cells appear already interposed between the cells of the neuronal line, so that the latter are no longer closely arranged. Moreover, connective tissue and blood vessels are evident in the ganglionic rudiment. In the replicas, gap junctions were not seen on the plasma membrane of neuroblasts, while generally small and only very occasionally larger gap junctions were seldom observed between adjacent
FIas. 1-3. Spinal ganglion of a chick embryo at the 4th incubation day. In these and other figures illustrations are positive images, so regions of p l a t i n u m deposition appear dark while absence of p l a t i n u m appears light. Prints are mounted with the source of p l a t i n u m shadowing below, except as indicated. FIG. 1. The cleavage plane runs through some neuroblasts (Ne), thus revealing cytoplasmic components, and within their plasma membranes. Large extensions of protoplasmic (P) and extracellular (E) faces of these are exposed, x 21 000. FIGs. 2 and 3. Higher magnifications of the areas outlined in Fig. 1. Both areas belong to the P face of the split plasma m e m b r a n e of a neuroblast and show gap junctions. Fig. 2, × 60 000; Fig. 3, x 90 000.
JUNCTIONS IN DEVELOPING SPINAL GANGLIA
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satellite cells (Figs. 20 and 21). the 10th incubation day on the split In the replicas of the split plasma mere- plasma membrane of satellite cells, were brahe of the satellite cells two further spe- still evident in this stage, while gap junccializations were evident. The first one tions were not seen. consisted of short strands, which appeared DISCUSSION as ridges about 15 nm thick on the P face (Figs. 13-15, 18, and 20) and as thinner 1. Junctions between Neuroblasts grooves on the E face (Figs. 19 and 21). In the chick embryo spinal ganglia studThese strands were isolated or grouped un- ied in thin sections at an early developder various angles (Figs. 13-15). Some mental stage (4th incubation day) the adstrands appeared in close relationship jacent neuroblasts were found joined by with small assemblies of only a few parti- two types of junction: adhering junctions cles (Fig. 18) or pits (Fig. 19) lacking a and close membrane appositions (40). geometrical arrangement, while other These latter were then termed fasciae ocstrands were in close relationship with cludentes. In the replicas studied in the larger assemblies of polygonally ordered present research, the adhering junctions particles (Fig. 20) or pits (Fig. 21) possibly between neuroblasts were not identified, belonging to gap junctions. Particles ar- while the close membrane appositions ranged in short rows were sometimes evi- were found to be actually gap junctions. dent (Fig. 16). la. Role of the junctions between neuroThe second specialization observed in blasts. The thin section studies (40) also the plasma membrane of satellite cells ap- showed that in the chick embryo spinal peared on the P face of the split membrane ganglion the plasma membranes of the as assemblies of particles closely packed in neuroblasts are firmly attached to one anan orthogonal array (Fig. 22). The center- other both at the level of adhering juncto-center spacing of these particles mea- tions and close membrane appositions. As sured about 7 nm. On the E face, arrays of the present study has revealed that the smaller pits were detected (Fig. 22, inset). close membrane appositions are actually At the 16th incubation day each nerve gap junctions, it can be concluded that in cell body is completely enveloped by a sat- the chick embryo spinal ganglion both adellite cell sheath, which is in turn sepa- hering and gap junctions contribute to inrated from the sheaths belonging to the tercellular adhesion. other nerve cell bodies by connective tisThe mechanical role played by these sue. The organization of the ganglion, junctions seems to be particularly signifitherefore, already shows the same general cant in the ganglionic early developmental characteristics as those seen at the end of stage, during which the ganglionic rudibody growth. ment lacks connective tissue. During this The strands and rows of linearly ar- stage the junctions might have much to do ranged particles (Fig. 17), as well as the with maintaining the cell organization of orthogonal assemblies of particles found at the ganglionic rudiment. Flas. 4-7. Spinal ganglion of a chick embryo at the 4th incubation day. Fla. 4. Cleaving occurs through two neuroblasts (Ne) and within t h e i r adjoining plasma membranes. nu, Nucleus of a neuroblast; n, processes of neuroblasts, x 8000. FIGS. 5-7. Higher magnifications of the areas outlined in Fig. 4 showing gap junctions between the two neuroblasts. In Fig. 5 the adjacent plasma m e m b r a n e s appear separated by a large distance, but at the level of the gap junction (arrow) the intercellular space disappears; particles are identifiable on the protoplasmic half (P face) of the left plasma m e m b r a n e , pits on the extracellular h a l f (E face) of the r i g h t plasma membrane. Gap junctions can be seen at arrows on the P face in Fig. 6 and on the E face in Fig. 7. Fig. 5, x 60 000; Figs. 6 and 7, x 84 000.
FIGS. 8-12. Spinal ganglia of chick embryos at the 4th incubation day. The replicas show the variable extension of the gap junctions between neuroblasts. In the junction of Fig. 10 some pit-free regions can be seen. The junction of Fig. 11 was observed in a replica of an unfixed specimen. In Fig. 12 the adjacent plasma membranes appear separated by a large distance, but at the level of the gap junction (between two asterisks) only the components of the junction are visible; other gap junctions can also be seen on the E face. Figs. 8, 9, and 10, x 80 000; Fig. 11, × 60 000; Fig. 12, × 110 000. 174
;iGs. 13 a n d 14. S p i n a l g a n g l i o n of a cl~ick e m b r y o a t t h e 10th i n c u b a t i o n day. : '~G. 13. N e u r o b l a s t s u r r o u n d e d by f l a t t e n e d s a t e l l i t e cells (SC). O n t h e r i g h t of t h e n e u r o b ~,,~ n u c l e u s (1~: : a l a r g e Golgi c o m p l e x is evident; m i t o c h o n d r i a a n d c i s t e r n a e of t h e e n d o p l ~ s m i c r e t i c u l u ~ ,'~:~ also be s ~ : n, P r o c e s s e s of n e u r o b l a s t s , x 12 00C. ]':fG. 14. H i g h e r m a g n i f i c a t i o n of t h e ar~;a o u t l i n e d in Fig. 13. D i s c o n t i n u o u s r i d g e s a n d a s :~L~i a g g r e ga:! of p a r t i c l e s (arrow) a r e located on t h e P face of t h e p l a s m a m e m b r a n e of a s a t e l l i t e cell. : ~,~ 000. 175
JUNCTIONS IN DEVELOPING SPINAL GANGLIA
A large body of evidence indicates that gap junctions permit the direct intercellular exchange of ions both in the adult and embryo (4, 6, 10, 17, 40-42, 44, 57, 60, 61, 68). As there is some evidence that small cations can affect differentiation as well as influence other cell processes (3, 26, 28, 32), it can ~be held that in the ganglionic rudiment also the intercellular passage of ions through the gap junctions can influence some cell processes, among them cell differentiation. Numerous experiments have shown that cells which are ionically coupled can exchange with one another small molecules [e.g., fluorescein (17, 31, 62), procion yellow (24, 43), labeled sucrose (5), and perhaps even microperoxidase (55)]. The results of Gilula et al. (18) strongly suggest that the passage of ions and certain metabolically important molecules between cultured fibroblasts occurs simultaneously, presumably through gap junctions (see also 4, 33, 38, 62, 66). It is possible that the gap junctions here described are also the anatomic pathway for the transfer of molecules between ganglionic cells. It should be borne in mind, however, that the gap junctions between embryonic cells appear to be less permeable to molecules than the gap junctions found in adult tissues (for a review, see 4). Therefore, the gap junctions between embryonic cells are probably permeable only to smaller mole-
177
cules. As experimental evidence is lacking, the relevance that such a passage of small molecules between neighboring embryonic cells might have to the processes of the ganglionic development is still unclear.
lb. Temporary appearance of the junctions between neuroblasts. After the interposing of the satellite cells between the neuroblasts, junctions previously joining the latter can no longer be observed either in the sectioned material (40) or in the freeze-fractured specimens. These junctions are therefore temporary structures. The number of known cases of temporary junctions increases with time. As can be seen in Table I, in which some examples of these junctions are listed, temporary junctions were observed in actively growing or renewing tissues both in embryos and adults.
2. Plasma Membrane Specializations of Satellite Cells In thin sections of chick embryo spinal ganglia at the 10th and 16th incubation days adjacent satellite cells appeared linked by means of adhering and gap junctions (42). In the replicas studied in the present research adhering junctions escaped observation, while the presence of gap junctions was confirmed at the 10th incubation day. With the freeze-fracture technique strands, which in the sectioned
FIGS. 15-22. Spinal ganglia of chick embryos a t the 10th (Figs. 15, 16, and 18-22) and 16th (Fig. 17) incubation days. Plasma m e m b r a n e specializations of satellite cells. FIG. 15 Arrows point to the tiny cytoplasmic sheaths of two satellite cells surrounding a neuroblast. Cleaving largely exposes the P face of the plasma m e m b r a n e of the innermost satellite cell. Some ridges (crossed arrow) are located on a m a m m i l a t e cell projection, x 21 000. FIGS. 16 and 17. Particles a r r a n g e d in short rows (arrows) and small clusters (crossed arrows in Fig. 17). One cluster is in close relationship with a ridge. Fig. 16, x 44 000; Fig. 17, x 50 000. FIGS. 18 and 19. A ridge in close relationship with a small cluster of particles on the P face (arrow in Fig. 18) and a groove in close relationship with some pits (arrow in Fig. 19) on the E face. Both, x 80 000. FIGS. 20 and 21. A ridge in close relationship with particles (arrow in Fig. 20) and a groove in close relationship with pits (Fig. 21). The polygonal packing of particles a n d pits suggests the gap junctional n a t u r e of these assemblies. Fig. 20, x 75 000; Fig. 21, x 110 000. FIG. 22. Arrows point to assemblies of particles closely packed in a n orthogonal a r r a y on the P face. Inset: highly ordered pits (between arrows) left by a n orthogonal particle array on the E face. x 87 000; inset, x 120 000. Both the figure and inset are mounted with t h e source of p l a t i n u m shadowing from right to left.
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Types of junction
PANNESE E T A L . TABLE I EXAMPLESOF TEMPORARYJUNCTIONS Sites
Author(s)
Adhering junctions Adhering junctions Adhering junctions Adhering junctions Adhering junctions Adhering junctions Adhering junctions Adhering unctions Desmosomes Desmosomes Desmosomes Desmosomes Gap junctions Gap junctions Gap junctions Gap junctions Gap junctions Gap junctions
Developing renal glomerulus (man) Developing spinal ganglion (fowl) Fetal liver (mouse) Maturing cortex (rat) Developing retina (man) Developing spinal cord (Xenopus) Seminiferous tubules (rat) Tooth germ (mouse) Tooth germ (cat) Developing retina (rat) Fetal liver (mouse) Wool follicle (sheep) Blastoderm (Fundulus) Developing retina (Xenopus) Wool follicle (sheep) Developing muscle (rat) Developing retina (man) Developing spinal ganglion (fowl)
Aoki Pannese Rifkind et al. Privat Fisher and Linberg Hayes and Roberts Kaya and Harrison Slavkin and Bringas Pannese Weidman and Kuwabara Rifkind et al. Orwin et al. Lentz and Trinkaus Dixon and Cronly-Dillon Orwin et al. Rash and Staehelin Fisher and Linberg Pannese et al.
Tight junctions Tight junctions Tight junctions Tight junctions Tight junctions
Embryonic mesenchyme (fowl) Small intestine (rat) Neural groove (frog) Developing renal glomerulus (man and rat) Neural tube (fowl)
Revel et al. Staehelin Decker and Friend Humbert et al. Revel and Brown
material are difficult to differentiate from small gap junctions, and orthogonal assemblies of particles were also demonstrated on the split plasma membrane of satellite cells. 2a. Gap junctions. The small gap junctions linking adjacent satellite cells in the ganglionic rudiment at the 10th incubation day could play a role in cell differentiation, coordination of cell activities, intercellular adhesion, and so on. In this respect, the comments made beforehand for the gap junctions between neuroblasts could be repeated here. The lack of gap junctions in replicas of satellite cells at the 16th incubation day is possibly due to both the only very occasional occurrence of these junctions at this developmental stage and the freeze-fracture technique, which generally exposes areas quite small in comparison to the very extended plasma membrane of satellite cells. This interpretation seems to be supported by the observation of gap junc-
Year 1967 1968 1969 1974 1975 1975 1976 1976 1962 1968 1969 1973 1971 1972 1973 1974 1975 Present study 1973 1973 1974 1976 1976
tions in thin sections of satellite cells of adult specimens (42). 2b. Strands. These strands show the same morphological charactersitics as those composing the tight junctions in other tissues [for reviews, see Refs. (33) and (66)]. Since these strands appeared as ridges on the P face of the split plasma membrane and as grooves on the complementary E face of the adjacent plasma membrane, they should be interpreted as cell junctions. These strands could link mechanically, as tiny maculae occludentes, adjacent cells. This hypothesis also seems to be supported by the fact that at times strands were found on mammilate cell projections (see Fig. 15). Similar membrane specializations were described in replicas of freeze-fractured embryonic tissues by Revel et al. (57) and Decker and Friend (9), who interpreted these structures as remnants of the well developed tight junctions (zonulae occludentes) found between the cells of the
JUNCTIONS IN DEVELOPING SPINAL GANGLIA
same tissue in previous stages of development. This hypothesis, however, would be difficult to apply to the strands here described because tight junctions could not be found between undifferentiated cells, from which satellite cells take origin. The strands observed in the replicas of the split plasma membrane of the spinal ganglion satellite cells (which, as is known, arise from the neural crest) instead could represent rudiments of tight junctions. In this connection, it can be recalled that zonulae and maculae occludentes are very numerous in other kinds of cells which probably arise from the neural crest [cells of the arachnoid (13, 35, 50), cells of the perineurium (1, 53, 54), and cells forming the myelin sheath (34, 52,
59)]. At the 10th and 16th incubation days, particles arranged in short rows were found together with the strands in the replicas of the split plasma membrane of the satellite cells. Particles showing a similar arrangement were observed in replicas of freeze-fractured embryonic tissues (9, 57), and were interpreted either as the result of the breaking up of the strands into their constituent subunits, or as a step in the formation of new strands. At present, sufficient data to confirm either one or the other hypotheses is lacking.
2c. Orthogonal assemblies of particles. As these assemblies were not evident in the plasma membrane of undifferentiated cells, from which satellite cells originate, it can be supposed that they are formed either at satellite cell differentiation or after. Similar assemblies were also found in the plasma membrane of other neuroglial cells (8, 11, 12, 19, 29, 35, 51). However, such assemblies a r e not peculiarities of the glial membranes as they were also noted in the plasma membrane of hepatocytes (27), intestinal epithelial cells (64), skeletal muscle fibers (21, 46, 47, 49), light cells of the kidney collecting tubule (23),
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and cerebellar sarcoma cells (67). The orthogonal assemblies of particles observed in the plasma membrane of the satellite cells represent a further example of localization of this specialization, the functional significance of which is still obscare. The excellent technical assistance of Mrs. G. Voss and Mr. H. Heidrich is gratefully acknowledged. This research was partly supported by a grant of the National Research Council (CNR), Italy. REFERENCES 1. AKERT, K., SANDRI, C., WEIBEL, E. R., PEPER, K., AND MOOR, H., Cell Tissue Res. 165, 281 (1976). 2. AOKI, A., Develop. Biol. 15, 156 (1967). 3. BARTH, L. G., AND BARTH, L. J., Develop. Biol. 20, 236 (1969). 4. BENNETT,M. V. L., Fed. Proc. 32, 65 (1973). 5. BENNETT, M. V. L., AND DUNHAM, P. B., Biophys. J. 10, 114a (1970). 6. BENNETT, M. V. L., AND TRINKAUS,J. P., J. Cell Biol. 44, 592 (1970). 7. BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY,M. J., MOOR,H., MOHLETHALER, K., NORTHCOTE,D. H., PACKER,L., SATIR,B., SATIR, P., SPETH, V., STAEHELIN, L. A., STEERE, R. L., AND WEINSTEIN,R. S., Science 190, 54 (1975). 8. BRIGHTMAN, M. W., AND REESE, T. S., in TOWER, D. S. (Ed.), The Nervous System, Vol. 1, The Basic Neurosciences, p. 267. Raven Press, New York, 1975. 9. DECKER, a. S., AND FRIEND, D. S., J. Cell Biol. 62, 32 (1974). 10. DEHAAN, R. L., AND SACHS, H. G., Curr. Top. Dev. Biol. 7, 193 (1972). 11. DERMIETZEL, n., Naturwissenschaften 60, 208 (1973). 12. DERMIETZEL, R., Cell TissueRes. 149, 121 (1974). 13. DERMIETZEL,R., Cell TissueRes. 164,309 (1975). 14. DIXON, J. S., AND CRONLY-DILLON,J. R., J. Embryol. Exp. Morphol. 28, 659 (1972). 15.. DOWELL, W. C. T., Optik (Stuttgart) 21, 47 (1964). 16. FISHER, S. K., AND LINBERG, K. A., J. Ultrastruct. Res. 51, 69 (1975). 17. FURSHPAN,E. J., AND POTTER, D. D., Curr. Top. Dev. Biol., 3, 95 (1968). 18. GILULA,N. B., REEVES, O. R., AND STEINBACI-I, A., Nature (London) 235, 262 (1972). 19. HANNA, R. B., HIRANO, A., AND PAPPAS, G. D., J. Cell Biol. 68, 403 (1976).
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Intercellular Junctions and Other Membrane Specializations in Developing Spinal Ganglia: A Freeze-Fracture Study 1 E. PANNESE,* L. LUCIANO,t S. IURATO,T+ AND E. R E A L E t *Second Institute of Human Anatomy, University of Milan, Italy, tLaboratory of Electron Microscopy, School of Medicine, Hannover, Federal Republic of Germany, and $Department ofB ioacoustics, University of Bari, Italy Received December 6, 1976 Intercellular junctions and other membrane specializations of neuroblasts and satellite cells were studied in spinal ganglia of chick embryos at the 4th, 10th, and 16th incubation days using the freeze-fracture technique. At the 4th day neuroblasts are closely arranged and connected by gap junctions. At the 10th day, instead, satellite cells appear interposed between neuroblasts so the gap junctions previously joining the latter are no longer evident. These temporary junctions probably play a role in cell differentiation and intercellular adhesion. Together with adhering junctions, gap junctions might maintain the cell organization of the ganglionic rudiment, which at the 4th day still lacks connective tissue. At the 10th day adjacent satellite cells display on their split plasma membrane small gap junctions, short, usually isolated strands recalling those composing zonulae occludentes, and orthogonal particle assemblies. These two latter specializations are still evident at the 16th day. The possible significance of these specializations is briefly discussed.
Evidence accumulated during the last few years suggests that the different types of intercellular junction play relevant (10, 17, 44, 60, 61, 68) and to some extent separate (4, 6, 57) roles in the developmental processes. Until now intercellular junctions in spinal ganglia rudiments were studied only in thin sections (40-42), in which, however, it was not always possible to identify with certainty all the different junctional types. In order to obtain more precise information on the various types of junction which can be found in developing spinal ganglia, the freeze-fracture technique, which allows an unequivocal identification of junctional types, was employed. By using this technique numerous gap junctions were found between adjacent neuroblasts at an early developmental stage, while only some gap junctions were
observed between satellite cells in a later developmental stage. Moreover, other specializations of the plasma membrane of satellite cells were found.
Reprint requests should be sent to Prof. Ennio Pannese, UniversitA degli Studi di Milano, Secondo Istituto di Anatomia Umana Normale, Via Mangiagalli, 14-20133 Milano, Italy.
MATERIALS AND METHODS Chick embryos at the 4th (25 specimens), 10th (10 specimens), and 16th (3 specimens) days of incubation were used. The spine was quickly removed from the embryos placed in the fixative and was cut into segments using a razor blade; then, the single spinal ganglia were isolated from these segments maintained in the fixative. The fixative solution contained 2% formaldehyde obtained from paraformaldehyde (Fluka AG, Buchs, St. Gallen), 2% glutaraldehyde (ultrastructure grade, Polaron Ltd., Watford), 0.1 M sodium cacodylate buffer (pH 7.2) and 25 mg% CaC]2. After fixation (total time, 2 hr), the isolated ganglia were soaked in 30% glycerol in saline for 1 hr, collected in small groups on specimen holders, frozen in liquid Freon 22 (monochlorodifluoromethane) at -150°C, and finally fractured at about -120°C and shadowed with platinum-carbon in a Balzers BA 360 M unit (Balzers AG, Liechtenstein). Some spinal ganglia rudiments at the 4th incubation day were also fractured and replicated without prior fixation. The replicas were cleaned in hypochlorite bleach and, if necessary, chromic acid, and then were repeatedly washed in distilled water.
169 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
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PANNESE E T
The p l a t i n u m - c a r b o n replicas were mounted on Formvar-carbon m e m b r a n e s (15) and examined in a Siemens Elmiskop 101 electron microscope. RESULTS
In replicas of freeze-cleaved developing spinal ganglia both fractured regions of the cells and surface views of their split plasma membranes were revealed. In the fractured regions the nucleus and cytoplasmic organelles were seen, thus allowing an identification of the replicated cell WPe, The only surface views of plasma membranes taken into consideration in this study were those belonging to identifled cells. As in other tissues (7), the plasma membrane of the cells in the developing spinal ganglia splits, giving rise to two complementary membrane faces, the inner or protoplasmic (A or P face) of which showed more randomly scattered particles than the outer or extracellular (B or E face). At the 4th incubation day the rudiment of the spinal ganglion exhibits an epitheiialilike structure. It is built of closely arranged cells (undifferentiated cells, primitive neuroblasts, and a few intermediate neu_roblasts), and lacks connective tissue and blood vessels. In this stage adjacent neuroblasts appeared connected by numerous gap junctions (Figs. 1-7). In the replicas these appeared as assemblies of regularly arranged particles about 8 nm in diameter on the P face of the split plasma membrane, and as arrays of tiny pits on the complementary E face (Fig. 5). While most gap junctions exhibited a compact packing of particles, some showed particle-free regions (Fig. 10). The latter junctions were morphologically similar to those described in other
AL.
embryonic tissues by Decker and Friend (9). No structural differences were noted between the gap junctions of fixed and unfixed ganglionic rudiments (compare Figs. 9 and 11). Frequently, the fracture plane could be seen to step from one adjacent membrane to the other, thus exposing portions of the P face from one membrane and portions of the E face from the other. If a gap junction was located along one of these steps, only particles on the P face and pits on the complementary E face were recognizable at the level of the junctional area (Figs. 5 and 12). While the adjacent plasma membranes usually appeared separated by a large distance, at the level of the particle and pit assemblies the intercellular space was obliterated (Figs. 5 and 12). This observation indicated the gap junctional hature of the particle and pit assemblies. Gap junctions were the only type of intercellular junction found in the replicas of the developing ganglia at the 4th incubation day. At the l Oth incubation day, undifferentiated cells, neuroblasts, pseudounipolar nerve cells, and satellite cells are recognizable in the rudiment of the spinal ganglion. Satellite cells appear already interposed between the cells of the neuronal line, so that the latter are no longer closely arranged. Moreover, connective tissue and blood vessels are evident in the ganglionic rudiment. In the replicas, gap junctions were not seen on the plasma membrane of neuroblasts, while generally small and only very occasionally larger gap junctions were seldom observed between adjacent
FIas. 1-3. Spinal ganglion of a chick embryo at the 4th incubation day. In these and other figures illustrations are positive images, so regions of p l a t i n u m deposition appear dark while absence of p l a t i n u m appears light. Prints are mounted with the source of p l a t i n u m shadowing below, except as indicated. FIG. 1. The cleavage plane runs through some neuroblasts (Ne), thus revealing cytoplasmic components, and within their plasma membranes. Large extensions of protoplasmic (P) and extracellular (E) faces of these are exposed, x 21 000. FIGs. 2 and 3. Higher magnifications of the areas outlined in Fig. 1. Both areas belong to the P face of the split plasma m e m b r a n e of a neuroblast and show gap junctions. Fig. 2, × 60 000; Fig. 3, x 90 000.
JUNCTIONS IN DEVELOPING SPINAL GANGLIA
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satellite cells (Figs. 20 and 21). the 10th incubation day on the split In the replicas of the split plasma mere- plasma membrane of satellite cells, were brahe of the satellite cells two further spe- still evident in this stage, while gap junccializations were evident. The first one tions were not seen. consisted of short strands, which appeared DISCUSSION as ridges about 15 nm thick on the P face (Figs. 13-15, 18, and 20) and as thinner 1. Junctions between Neuroblasts grooves on the E face (Figs. 19 and 21). In the chick embryo spinal ganglia studThese strands were isolated or grouped un- ied in thin sections at an early developder various angles (Figs. 13-15). Some mental stage (4th incubation day) the adstrands appeared in close relationship jacent neuroblasts were found joined by with small assemblies of only a few parti- two types of junction: adhering junctions cles (Fig. 18) or pits (Fig. 19) lacking a and close membrane appositions (40). geometrical arrangement, while other These latter were then termed fasciae ocstrands were in close relationship with cludentes. In the replicas studied in the larger assemblies of polygonally ordered present research, the adhering junctions particles (Fig. 20) or pits (Fig. 21) possibly between neuroblasts were not identified, belonging to gap junctions. Particles ar- while the close membrane appositions ranged in short rows were sometimes evi- were found to be actually gap junctions. dent (Fig. 16). la. Role of the junctions between neuroThe second specialization observed in blasts. The thin section studies (40) also the plasma membrane of satellite cells ap- showed that in the chick embryo spinal peared on the P face of the split membrane ganglion the plasma membranes of the as assemblies of particles closely packed in neuroblasts are firmly attached to one anan orthogonal array (Fig. 22). The center- other both at the level of adhering juncto-center spacing of these particles mea- tions and close membrane appositions. As sured about 7 nm. On the E face, arrays of the present study has revealed that the smaller pits were detected (Fig. 22, inset). close membrane appositions are actually At the 16th incubation day each nerve gap junctions, it can be concluded that in cell body is completely enveloped by a sat- the chick embryo spinal ganglion both adellite cell sheath, which is in turn sepa- hering and gap junctions contribute to inrated from the sheaths belonging to the tercellular adhesion. other nerve cell bodies by connective tisThe mechanical role played by these sue. The organization of the ganglion, junctions seems to be particularly signifitherefore, already shows the same general cant in the ganglionic early developmental characteristics as those seen at the end of stage, during which the ganglionic rudibody growth. ment lacks connective tissue. During this The strands and rows of linearly ar- stage the junctions might have much to do ranged particles (Fig. 17), as well as the with maintaining the cell organization of orthogonal assemblies of particles found at the ganglionic rudiment. Flas. 4-7. Spinal ganglion of a chick embryo at the 4th incubation day. Fla. 4. Cleaving occurs through two neuroblasts (Ne) and within t h e i r adjoining plasma membranes. nu, Nucleus of a neuroblast; n, processes of neuroblasts, x 8000. FIGS. 5-7. Higher magnifications of the areas outlined in Fig. 4 showing gap junctions between the two neuroblasts. In Fig. 5 the adjacent plasma m e m b r a n e s appear separated by a large distance, but at the level of the gap junction (arrow) the intercellular space disappears; particles are identifiable on the protoplasmic half (P face) of the left plasma m e m b r a n e , pits on the extracellular h a l f (E face) of the r i g h t plasma membrane. Gap junctions can be seen at arrows on the P face in Fig. 6 and on the E face in Fig. 7. Fig. 5, x 60 000; Figs. 6 and 7, x 84 000.
FIGS. 8-12. Spinal ganglia of chick embryos at the 4th incubation day. The replicas show the variable extension of the gap junctions between neuroblasts. In the junction of Fig. 10 some pit-free regions can be seen. The junction of Fig. 11 was observed in a replica of an unfixed specimen. In Fig. 12 the adjacent plasma membranes appear separated by a large distance, but at the level of the gap junction (between two asterisks) only the components of the junction are visible; other gap junctions can also be seen on the E face. Figs. 8, 9, and 10, x 80 000; Fig. 11, × 60 000; Fig. 12, × 110 000. 174
;iGs. 13 a n d 14. S p i n a l g a n g l i o n of a cl~ick e m b r y o a t t h e 10th i n c u b a t i o n day. : '~G. 13. N e u r o b l a s t s u r r o u n d e d by f l a t t e n e d s a t e l l i t e cells (SC). O n t h e r i g h t of t h e n e u r o b ~,,~ n u c l e u s (1~: : a l a r g e Golgi c o m p l e x is evident; m i t o c h o n d r i a a n d c i s t e r n a e of t h e e n d o p l ~ s m i c r e t i c u l u ~ ,'~:~ also be s ~ : n, P r o c e s s e s of n e u r o b l a s t s , x 12 00C. ]':fG. 14. H i g h e r m a g n i f i c a t i o n of t h e ar~;a o u t l i n e d in Fig. 13. D i s c o n t i n u o u s r i d g e s a n d a s :~L~i a g g r e ga:! of p a r t i c l e s (arrow) a r e located on t h e P face of t h e p l a s m a m e m b r a n e of a s a t e l l i t e cell. : ~,~ 000. 175
JUNCTIONS IN DEVELOPING SPINAL GANGLIA
A large body of evidence indicates that gap junctions permit the direct intercellular exchange of ions both in the adult and embryo (4, 6, 10, 17, 40-42, 44, 57, 60, 61, 68). As there is some evidence that small cations can affect differentiation as well as influence other cell processes (3, 26, 28, 32), it can ~be held that in the ganglionic rudiment also the intercellular passage of ions through the gap junctions can influence some cell processes, among them cell differentiation. Numerous experiments have shown that cells which are ionically coupled can exchange with one another small molecules [e.g., fluorescein (17, 31, 62), procion yellow (24, 43), labeled sucrose (5), and perhaps even microperoxidase (55)]. The results of Gilula et al. (18) strongly suggest that the passage of ions and certain metabolically important molecules between cultured fibroblasts occurs simultaneously, presumably through gap junctions (see also 4, 33, 38, 62, 66). It is possible that the gap junctions here described are also the anatomic pathway for the transfer of molecules between ganglionic cells. It should be borne in mind, however, that the gap junctions between embryonic cells appear to be less permeable to molecules than the gap junctions found in adult tissues (for a review, see 4). Therefore, the gap junctions between embryonic cells are probably permeable only to smaller mole-
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cules. As experimental evidence is lacking, the relevance that such a passage of small molecules between neighboring embryonic cells might have to the processes of the ganglionic development is still unclear.
lb. Temporary appearance of the junctions between neuroblasts. After the interposing of the satellite cells between the neuroblasts, junctions previously joining the latter can no longer be observed either in the sectioned material (40) or in the freeze-fractured specimens. These junctions are therefore temporary structures. The number of known cases of temporary junctions increases with time. As can be seen in Table I, in which some examples of these junctions are listed, temporary junctions were observed in actively growing or renewing tissues both in embryos and adults.
2. Plasma Membrane Specializations of Satellite Cells In thin sections of chick embryo spinal ganglia at the 10th and 16th incubation days adjacent satellite cells appeared linked by means of adhering and gap junctions (42). In the replicas studied in the present research adhering junctions escaped observation, while the presence of gap junctions was confirmed at the 10th incubation day. With the freeze-fracture technique strands, which in the sectioned
FIGS. 15-22. Spinal ganglia of chick embryos a t the 10th (Figs. 15, 16, and 18-22) and 16th (Fig. 17) incubation days. Plasma m e m b r a n e specializations of satellite cells. FIG. 15 Arrows point to the tiny cytoplasmic sheaths of two satellite cells surrounding a neuroblast. Cleaving largely exposes the P face of the plasma m e m b r a n e of the innermost satellite cell. Some ridges (crossed arrow) are located on a m a m m i l a t e cell projection, x 21 000. FIGS. 16 and 17. Particles a r r a n g e d in short rows (arrows) and small clusters (crossed arrows in Fig. 17). One cluster is in close relationship with a ridge. Fig. 16, x 44 000; Fig. 17, x 50 000. FIGS. 18 and 19. A ridge in close relationship with a small cluster of particles on the P face (arrow in Fig. 18) and a groove in close relationship with some pits (arrow in Fig. 19) on the E face. Both, x 80 000. FIGS. 20 and 21. A ridge in close relationship with particles (arrow in Fig. 20) and a groove in close relationship with pits (Fig. 21). The polygonal packing of particles a n d pits suggests the gap junctional n a t u r e of these assemblies. Fig. 20, x 75 000; Fig. 21, x 110 000. FIG. 22. Arrows point to assemblies of particles closely packed in a n orthogonal a r r a y on the P face. Inset: highly ordered pits (between arrows) left by a n orthogonal particle array on the E face. x 87 000; inset, x 120 000. Both the figure and inset are mounted with t h e source of p l a t i n u m shadowing from right to left.
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Types of junction
PANNESE E T A L . TABLE I EXAMPLESOF TEMPORARYJUNCTIONS Sites
Author(s)
Adhering junctions Adhering junctions Adhering junctions Adhering junctions Adhering junctions Adhering junctions Adhering junctions Adhering unctions Desmosomes Desmosomes Desmosomes Desmosomes Gap junctions Gap junctions Gap junctions Gap junctions Gap junctions Gap junctions
Developing renal glomerulus (man) Developing spinal ganglion (fowl) Fetal liver (mouse) Maturing cortex (rat) Developing retina (man) Developing spinal cord (Xenopus) Seminiferous tubules (rat) Tooth germ (mouse) Tooth germ (cat) Developing retina (rat) Fetal liver (mouse) Wool follicle (sheep) Blastoderm (Fundulus) Developing retina (Xenopus) Wool follicle (sheep) Developing muscle (rat) Developing retina (man) Developing spinal ganglion (fowl)
Aoki Pannese Rifkind et al. Privat Fisher and Linberg Hayes and Roberts Kaya and Harrison Slavkin and Bringas Pannese Weidman and Kuwabara Rifkind et al. Orwin et al. Lentz and Trinkaus Dixon and Cronly-Dillon Orwin et al. Rash and Staehelin Fisher and Linberg Pannese et al.
Tight junctions Tight junctions Tight junctions Tight junctions Tight junctions
Embryonic mesenchyme (fowl) Small intestine (rat) Neural groove (frog) Developing renal glomerulus (man and rat) Neural tube (fowl)
Revel et al. Staehelin Decker and Friend Humbert et al. Revel and Brown
material are difficult to differentiate from small gap junctions, and orthogonal assemblies of particles were also demonstrated on the split plasma membrane of satellite cells. 2a. Gap junctions. The small gap junctions linking adjacent satellite cells in the ganglionic rudiment at the 10th incubation day could play a role in cell differentiation, coordination of cell activities, intercellular adhesion, and so on. In this respect, the comments made beforehand for the gap junctions between neuroblasts could be repeated here. The lack of gap junctions in replicas of satellite cells at the 16th incubation day is possibly due to both the only very occasional occurrence of these junctions at this developmental stage and the freeze-fracture technique, which generally exposes areas quite small in comparison to the very extended plasma membrane of satellite cells. This interpretation seems to be supported by the observation of gap junc-
Year 1967 1968 1969 1974 1975 1975 1976 1976 1962 1968 1969 1973 1971 1972 1973 1974 1975 Present study 1973 1973 1974 1976 1976
tions in thin sections of satellite cells of adult specimens (42). 2b. Strands. These strands show the same morphological charactersitics as those composing the tight junctions in other tissues [for reviews, see Refs. (33) and (66)]. Since these strands appeared as ridges on the P face of the split plasma membrane and as grooves on the complementary E face of the adjacent plasma membrane, they should be interpreted as cell junctions. These strands could link mechanically, as tiny maculae occludentes, adjacent cells. This hypothesis also seems to be supported by the fact that at times strands were found on mammilate cell projections (see Fig. 15). Similar membrane specializations were described in replicas of freeze-fractured embryonic tissues by Revel et al. (57) and Decker and Friend (9), who interpreted these structures as remnants of the well developed tight junctions (zonulae occludentes) found between the cells of the
JUNCTIONS IN DEVELOPING SPINAL GANGLIA
same tissue in previous stages of development. This hypothesis, however, would be difficult to apply to the strands here described because tight junctions could not be found between undifferentiated cells, from which satellite cells take origin. The strands observed in the replicas of the split plasma membrane of the spinal ganglion satellite cells (which, as is known, arise from the neural crest) instead could represent rudiments of tight junctions. In this connection, it can be recalled that zonulae and maculae occludentes are very numerous in other kinds of cells which probably arise from the neural crest [cells of the arachnoid (13, 35, 50), cells of the perineurium (1, 53, 54), and cells forming the myelin sheath (34, 52,
59)]. At the 10th and 16th incubation days, particles arranged in short rows were found together with the strands in the replicas of the split plasma membrane of the satellite cells. Particles showing a similar arrangement were observed in replicas of freeze-fractured embryonic tissues (9, 57), and were interpreted either as the result of the breaking up of the strands into their constituent subunits, or as a step in the formation of new strands. At present, sufficient data to confirm either one or the other hypotheses is lacking.
2c. Orthogonal assemblies of particles. As these assemblies were not evident in the plasma membrane of undifferentiated cells, from which satellite cells originate, it can be supposed that they are formed either at satellite cell differentiation or after. Similar assemblies were also found in the plasma membrane of other neuroglial cells (8, 11, 12, 19, 29, 35, 51). However, such assemblies a r e not peculiarities of the glial membranes as they were also noted in the plasma membrane of hepatocytes (27), intestinal epithelial cells (64), skeletal muscle fibers (21, 46, 47, 49), light cells of the kidney collecting tubule (23),
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and cerebellar sarcoma cells (67). The orthogonal assemblies of particles observed in the plasma membrane of the satellite cells represent a further example of localization of this specialization, the functional significance of which is still obscare. The excellent technical assistance of Mrs. G. Voss and Mr. H. Heidrich is gratefully acknowledged. This research was partly supported by a grant of the National Research Council (CNR), Italy. REFERENCES 1. AKERT, K., SANDRI, C., WEIBEL, E. R., PEPER, K., AND MOOR, H., Cell Tissue Res. 165, 281 (1976). 2. AOKI, A., Develop. Biol. 15, 156 (1967). 3. BARTH, L. G., AND BARTH, L. J., Develop. Biol. 20, 236 (1969). 4. BENNETT,M. V. L., Fed. Proc. 32, 65 (1973). 5. BENNETT, M. V. L., AND DUNHAM, P. B., Biophys. J. 10, 114a (1970). 6. BENNETT, M. V. L., AND TRINKAUS,J. P., J. Cell Biol. 44, 592 (1970). 7. BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY,M. J., MOOR,H., MOHLETHALER, K., NORTHCOTE,D. H., PACKER,L., SATIR,B., SATIR, P., SPETH, V., STAEHELIN, L. A., STEERE, R. L., AND WEINSTEIN,R. S., Science 190, 54 (1975). 8. BRIGHTMAN, M. W., AND REESE, T. S., in TOWER, D. S. (Ed.), The Nervous System, Vol. 1, The Basic Neurosciences, p. 267. Raven Press, New York, 1975. 9. DECKER, a. S., AND FRIEND, D. S., J. Cell Biol. 62, 32 (1974). 10. DEHAAN, R. L., AND SACHS, H. G., Curr. Top. Dev. Biol. 7, 193 (1972). 11. DERMIETZEL, n., Naturwissenschaften 60, 208 (1973). 12. DERMIETZEL, R., Cell TissueRes. 149, 121 (1974). 13. DERMIETZEL,R., Cell TissueRes. 164,309 (1975). 14. DIXON, J. S., AND CRONLY-DILLON,J. R., J. Embryol. Exp. Morphol. 28, 659 (1972). 15.. DOWELL, W. C. T., Optik (Stuttgart) 21, 47 (1964). 16. FISHER, S. K., AND LINBERG, K. A., J. Ultrastruct. Res. 51, 69 (1975). 17. FURSHPAN,E. J., AND POTTER, D. D., Curr. Top. Dev. Biol., 3, 95 (1968). 18. GILULA,N. B., REEVES, O. R., AND STEINBACI-I, A., Nature (London) 235, 262 (1972). 19. HANNA, R. B., HIRANO, A., AND PAPPAS, G. D., J. Cell Biol. 68, 403 (1976).
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