ULTRASTRUCTURE OF CELLS OF THE NEURAL CREST A. A. Sosunov, P. P. Kruglyakov, G. V. Belyanina, and V. N. Shvalev

UDC 611.89-018.82-086

The majority of the ganglia of the vegetative nervous system are derivatives of the neural crest (NC) (ganglionic layer), a temporary formation consisting of cells which have migrated from the dorsal divisions of the closing neural tube [i, 7, 15, 17]. In addition to the neural ganglia, the melanocytes, the connective tissue formations of the heart, and several tissues of the head [13, 15], are also classed as derivatives of the NC. The properties of the NC of the truncal division have been studied in greatest detail [15, 16, 24, 26, 34, 35]; cells migrate from this division along the ventromedial (mainly cells of neuronal determination) and the dorsolateral (the future melanocytes) pathways by means of homo- and heterotransplantation. It has been proven that the direction of migration and the subsequent differentiation of cells into sensory (spinal ganglia) and cholinor adrenergic motor neurons are determined primarily by local factors of the microenvironment, and are not strictly programmed genetically. A large number of investigations have been devoted to the mechanisms of the emergence of cells from the neural tube and to the subsequent goal-directedness of their migration to the sites of definitive location [2, 4, 21, 25]. Fundamental significance has in the process been ascribed to the extracellular matrix which is rich in glycosaminoglycans, collagens, and fibronectin [6, 14, 29]. The preponderant portion of the investigations into the NC, including the electron microscopic investigations, has been carried out on chick embryos. Ruthenium red has only been used in several studies, and only for the study of the extracellular matrix [ii, 23]. The purpose of the present study is the investigation of the ultrastructure of the NC cells of the truncal division up to the point of their exit from the neuroepithelial layer and in the process of migration. Material and Methods. The study was carried out on nine daylold white rat embryos. The first day after mating was considered the zero day of the pregnancy. After extraction from the cavity of the uterus, the embryos were fixed whole by submersion in 3% glutaraldehyde in a 0.i M cacodylate buffer (pH 7.3). Prefixation, dehydration, and embedding in Eponaraldite were carried out by the traditional methods. In order to identify the components of the extracellular matrix, and to analyze the glycocalyx of the NC cells, the embryos were fixed with ruthenium red [19]. Before the preparation of the ultrathin sections, semithin sections were made up which were stained with toluidine blue for orientation. The ultrathin sections were obtained on an Ultracut Om-U3 ultramicrotone, were contrasted with uranyl acetate and lead citrate, and were examined in an EMV-100B electron microscope. Investigation Results and Discussion. The neural tube in the nine-day-old white rat embryos is found in the stage of formation, the closing neural groove is located in the caudal portion of the truncal division; the neural tube is already closed more cranially, and the ectoderm is separated from it by a space (Fig. i, a, b). The dorsal portion of the closing or already closed neural tube is represented by an accumulation of loosely disposed cells, the majority of which do not reach its cavity with their processes (see Fig. i, b, d). The cells are separated by large electron-transparent interstices (see Fig. i, d); many are exiting from the neural epithelial layer, and by that very process disrupting the even contours of the dorsal division of the neural tube (see Fig. i, c). Laboratory of Electron Microscopy, Biology Faculty, N. P. Ogarev Mordovian University, Saransk. Laboratory of Neurohistology and Histochemistry, All-Union Cardiological Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Arkhiv Anatomii, Gistologii i Embriologii, Vol. 94, No. 5, pp. 5-11, May, 1988. Original article submitted July 9, 1987. 0097-0549/90/1901-0057512.50

9 1990 Plenum Publishing Corporation

57

Fig. i. The ultrastructure of the cells of the neural crest (NC) up to the point of their exit from the neuroepithelial layer of the neural tube (NT). a) Cross half-thin section of a rat embryo; b) dorsal portion of the closing neural tube; c) exit from the neuroepithelial layer of the NT cells; d) NT cells; e) fragment of NT cell, mitochondria (M) with single cristae; f) processes of NT cells, directed into the free intercellular space; g) absence of basal membrane on the plasmolemma of NT cells; the extraceliular matrix is represented by single granules. ED, ectoderm; a, b) fixation with ruthenium red. Magn.: a) 320; b) 5500; c) 7500; d) 9000; e) 19,000; f) 14,000; g) 15,000. The NC may be regarded as the cells emerging from the dorsal divisions of the neural groove or of the neural tube, which, while not forming accumulations isolated from the neural epithelium, immediately begin to migrate. The forms of the NC cells are diverse; radially extended cells are not infrequently encountered. Their nuclei are large with large nucleoli. A substantial portion of the cytoplasm is filled with loosely arranged ribosomes. The canal-

58

Fig. 2. The plasmolemma of the growth cone (GC) of the perikaryon of a cell of the neural crest does not react with ruthenium red. Magn. 24,000. iculi of the granular endoplasmic reticulum are few in number. The mitochondria have individual cristae of irregular form (see Fig. i, e). The Golgi complex is small; it is found more often in elongated cells medial to the nucleus. Lipid inclusions and large osmiophilic masses are found not infrequently in the cells. Microtubules are located throughout the entire cytoplasm of the cells. Bundles of parallelly oriented microtubules are typically found in the cell processes. Growth cones filled with characteristic fine polymorphous vacuoles are found in the bodies and processes of the latter (Fig. 2). The NC cells have numerous processes; those lying in the depths have, as a rule, long, thin, radially-oriented processes and short transverse processes. Superficially lying cells usually form curling thin outgrowths directed into the free intercellular space (see Fig. i, f). The NC cells form only simple connections with one another. By contrast with them, the cells of the neural epithelium of the neural tube are connected with its cavity, and form specialized intercellular contacts of the fascia adherens type, the so-called terminal bars, in the ventricular divisions. Some elongated cells in the region of the NC are connected both with the external space, and with the cavity of the neural tube, where the terminal bars are formed. Mitotically dividing cells are often encountered in both the neural tube and the NC. In the dorsal portion of the neural tube or of the neural groove, where the NC cells settle, the basal membrane is not observed (see Fig. i, f, g). Short single fragments of osmiophilic material are observed on the plasmolemma only of individual cells. The basal membrane is absent not only in NC cells which are exiting from the neural tube, but also somewhat more ventrally, even o n t h o s e cells which are connected with the cavity of the neural tube and which form terminal bars in the ventricular divisions. The migrating NC cells are not distinguished by ultrastructure from the surrounding mesenchymal cells [32] or the cells of the somites. Solitary cells or groups of two, or more rarely three cells are found near the NC and between the neural tube and the somites (the ventromedial migration pathway); some of these can be considered to be migrating NC cells (Fig. 3, c). With standard fixation the extracellular matrix at the site of the emergence of the cells from the neural tube and along the ventromedial pathway is represented by single, weakly osmiophilic granules (see Fig. i, g), by fibrils, and by small structures which are reminiscent of transverse sections of thin cell processes, up to 1 ~m in size, the so-called interstitial bodies [18]. With fixation using ruthenium red, a dense network of fibrils, osmiophilic granules, 0.1-0.2 ~m in diameter, and interstitial bodies are found at these sites (see Fig. 3). The osmiophilic granules are usually found at the sites of contact of the fibrils with one another, and can form small aggregates. Two types can be discriminated among the fibrils: thin short fibrils, with a diameter up to i0 nm, and thick long fibrils with an average diameter of 20 rim.

59

Fig. 3. Cells and extracellular matrix (EM) in the ventromedial migration pathway, a) Ultrastructure of the EM; b) the interconnection of EM components with cells; c) segment of the ventromedial migration pathway. IB, interstitial body; CI, cell; NT, neural tube. Fixation with ruthenium red. Magn.: a) 22,000; b) 25,000; c) 9000. The fibrils and the granules may be connected with the basal membrane of the neural tube, with NC cells, and with cells located on the migration pathways. Such a connection between the components of the extracellular matrix and the cells is observed only in those cases in which the plasmolemma of the cells reacts with ruthenium red, i.e., is covered by a layer of osmiophilic material (see Fig. 3, b). The basal membrane of the neural tube produces an intense reaction with ruthenium red (see Fig. 3, c). The plasmolemmas of the NC cells bind ruthenium red to various degrees, which in many respects depends on the concentration of the dye near the cells. However, visually, the ruthenium-binding capacity of the components of the extracellular matrix is greater than that of the plasmolemmas of the NC cells. The cells found along the ventromedial migration pathway react with ruthenium red nonidentically: the plasmolemma of some is covered by a thick layer of osmiophilic material; the reaction product is practically absent on the plasmolemma of other cells (Fig. 4, a, b). Ruthenium red penetrates into some cells which do not have evident injuries, and causes a diffuse staining of the cytoplasmic matrix, of the membrane structures, the ribosomes, and the nuclear material. The dye does not penetrate into mitochondria, or the canaliculi of the endoplasmic reticulum (see Fig. 4). The diffuse staining of the cells takes place usually

60

Fig. 4. The reaction of cells found on the migration pathways with ruthenium red. a) Reaction product is absent on the plasmolemma of cells filled with ruthenium red; b) the plasmolemma of cells filled with ruthenium red reacts with the latter. Magn.: a) 18,000; b) 17,000. at sites of high concentration of ruthenium red, and is observed in the NC, in the neural tube, in individual cells on the migration pathways, and in the ectoderm. The plasmolemmas of cells filled with the dye do not always give a reaction with ruthenium red (see Fig. 4, a). The plasmolemma of the growth cones of NC cells practically does not react with ruthenium red (see Fig. 2). The main portion of the ruthenium red is found on the external aspect of the ectoderm, and in the extracellular surroundings adjacent to it (Fig. i, a, b). Since the process of the closure of the neural tube unfolds gradually, and since in the time periods studied the caudal divisions of the neural tube of the truncal division are not closed, the ruthenium red penetrates into its cavity as well. The dye diffuses along the cavity into the cranial divisions, where the neural tube is already closed. The reaction product with ruthenium red is identified in the cavity of the closed neural tube in the form of osmiophilic granules of various sizes and shapes. Such osmiophilic masses are found only in the dorsal divisions of the cavity, and are not found in its ventral portion. Thus the NC in the rat embryos must be considered to be the cells of the dorsal portion of the closing or already closed neural tube which have connections with its lumen, and which have lost specialized contacts (terminal bars) with the neighboring cells in the ventricular divisions. Among the causes of the emergence of the NC cells from the neural tube, attention must be paid mainly: to the presence of the extracellular matrix (the substrate of migration), which is necessary for the transiting of the cells; to the absence of the basal membrane of the neural tube, which is a mechanical impediment to the beginning of cell movement; to the formation of cell processes and their adhesion to the extracellular matrix; and to the loss or the weakening of intercellular connections [i0, 22]. In addition, it is assumed that the

61

restructuring of the cytoskeleton, of the microtubules facilitate the emergence of the NC cells [12].

and the microfilaments,

can also

The components of the extracellular matrix (fibronectin, hyaluronic acid, some other glycosaminoglycans) are the substrate which governs the movement of the cells, including, possibly, their emergence form the neural tube as well [14, 23, 28, 29, 33]. Fibronectin and glycosaminoglycans are identified electron microscopically in the form of thin fibrils, osmiophilic granules, and interstitial bodies [20]. According to the data of immunohistochemical and electron microscopic investigations [22, 25], and of the present study, a large amount of fibronectin and glycosaminoglycans is found near the NC and along the migration pathways. The disappearance of the basal membrane of the neural tube before the emergence of cells from it has been firmly established [2, 23, 32], and has also been demonstrated in the present study. Before the formation of the NC in chick embryos the fusion of its basal membrane and the membrane of the epidermal ectoderm takes place in the lateral portions of t h e dorsal divisions of the closing neural tube [22]. In the dorsomedial segments of the neural tube, the basal membrane is lacking. The escape of the NC cells in fact takes place precisely here. Before the very beginning of migration, the fused basal membranes of the ectoderm and the neural tube, as it were, become disengaged and, thus, the mechanical impediment to the migration of the cells is eliminated. However, the basal membrane is not the only and main impediment to the beginning of migration. As experiments with the culturing of the NC have shown, there is a delay in the beginning of migration also in culture conditions in which an impediment in the form of fused membranes is lacking [5, 36]. The data obtained in the present investigation to the effect that basal membrane disappears not only in NC cells, but in more ventrally located cells of the neural epithelium of the neural tube as well, also attest to the fact that the absence of the basal membrane is not the principal cause of the exiting of cells from the neural tube. The causes of the disappearance or of the partial lysis of the basal membrane of the neural tube are unknown. Two series of causes are possible a priori: internal causes, depending on the NC cells themselves, and external causes, asssociated with the influence of factors of the extracellular matrix. The presence of processes directed to the outside of the neural tube can be considered a characteristic feature of the NC cells. As is assumed, the processes may increase the adhesion of the cells to the extracellular matrix (with fibronectin, in the first place), and may facilitate their change in position [21, 24, 32]. The absence of desmosome-like (terminal bars) contacts in the ventricular divisions, as well as the absence of specialized connections with one another, is characteristic for NC cells [2, 22]. In addition to the ultrastructures, it is possible that the adhesive properties of the NC cells which are determined by the cellular adhesion molecules of neurons (CAM) [8] may possibly change as well. However, according to immunohistochemistry data, prior to exiting from the neural tube NC cells react to the CAM [31]. This indicates that if the CAM, their quantity or qualitative characteristics in fact change before the beginning of migration, they do so insignificantly. A reaction to the CAM is not observed during the migration of the cells. The ultrastructure of the nucleus and organelles of the NC cells does not differ from that of the cells of the neuroepithelium of the neural tube, and it coincides with the ultrastructure of the NC cells of chick embryos, as previously described [2, 32]. Ruthenium red does not penetrate across the intact ectoderm. The presence of substantial electron-dense masses on the external surface of the ectoderm and in the space surrounding it attests to this. Ruthenium red penetrates into the tissues of the embryo across areas of disruptions in the ectoderm (traumatization of various degrees is practically unavoidable when the material is taken, due to the small size of the embryos and the delicacy of their integuments), and possibly through individual damaged cells, which become filled with the dye, of the ectoderm as well. The results of the present investigation coincide with the data of other authors [23] who have studied the components of the extracellular matrix, also using ruthenium red. The absence of the reaction with ruthenium red at the plasmolemma of the NC cells into which the dye has penetrated, as well as the low ruthenium red-binding capacity of the plas-

62

molemma of the cells as compared with the extracellular matrix, make it possible to hypothe= size the weak development of the carbohydrate component of their plasmolemma. It is known that NC cells are not capable of synthesizing fibronectin, and that this glycoprotein is absent on their surface. It is precisely with this that the capacity of NC cells, as of other cells which do not synthesize fibronectin, and even of latex beads, to migrate actively through the extraceliular matrix is associated [3, 9]. The cells which are found along the migration pathways react variably with ruthenium red. It is possible that the cells which actively bind ruthenium red on their surface are mesenchymal cells, whereas the migrating NC cells weakly react weakly with ruthenium red. The absence of the reaction product on the plasmolemma of growth cones is probably associated with the undeveloped state of glycocalyx of the newly formed cytolemma. The specific features of the structure of the plasmolemma of the growth cones of neuronal cells have been noted by many authors [27]. The presence of ruthenium red only in the dorsal divisions of the cavity of the neural tube, and the absence of ruthenium red in its ventral portion, may indicate the heterogeneity and the dorsoventral polarity of the disposition in it of the extracelluiar matrix. Analysis of ruthenium red-fixed material is made significantly more difficult as a result of the weak penetrating capacity of the dye, and, therefore, judgments must be made very carefully about the intensity of its binding reaction. Ruthenium red penetrates, so it is believed, only into damaged cells [30]. At the same time it has been shown in this study that many cells are filled with the dye. It is possible that this is the consequence of traumatization of the cells; however, it is impossible to exclude as well the probability of the existence of differences in the reaction of embryonal and definitive tissues with ruthenium red. LITERATURE CITED i. 2. 3. 4. 5.

6. 7.

8. 9. 10. ii. 12. 13. 14. 15. 16. 17.

A. G. Knorre and L. V. Suvorova, The Development of the Vegetative Nervous System in the Embryogenesis of the Vertebrate Animals and Man [in Russian], Meditsina, Moscow (1984). M. Bancroft and R. Bellairs, "The neural crest of trunk region of the chick embryo studied by SEM and TEM," Zool. [sic], ~, 73-85 (1976). M. Bronner-Franzer, "Distribution of latex beads and retinal pigment epithelial cells along the ventral neural crest pathway," Dev. Biol., 91, 50-63 (1982). M. Bronner-Franzer and A. M. Cohen, "Analysis of the neural crest ventral pathway using injected tracer cells," Dev. Biol., 77, 130-141 (1980). A. Chevallier, "Localization et dure~-des potentialites medulla,surrenaliennes des cretes neurales chez l'embryon de poulet," J. Embryol. Exp. Morphol., 27, 603-614 (1972). M.A. Derby, "Analysis of glycosaminoglycans within the extracellular environment encountered by migrating neural crest cells," Dev. Biol~, 66, 321-336 (1978). J. L. Duband, G. C. Tucker, Th. J. Poole, et al., "How do the migratory and adhesive properties of the neural crest govern ganglia formation in the avian peripheral nervous system?" J. Cell Biochem., 27, 189-203 (1985). G. M. Edelman, "Cell adhesion and the molecular processes of morphogenisis," Annu. Rev. Biochem., 54, 135-169 (1985). C. A. Erickson, K. W. Tosney, and J. A. Weston, "Analysis of migratory behavior of neural crest and fibroblastic cells in embryonic tissues," Dev. Biol., 77, 142-156 (1980). C. A. Erickson and J. A. Weston, "A SEM analysis of neural crest migration in the mouse, '~ J. Embryol. Exp. Morphol., 74, 97-118 (1983). E. D. Hay, "Fine structures of embryonic matrices and their relation to the cell surface in ruthenium red-fixed tissue," Growth, 42, 399-423 (1978). M. Jacobson, Developmental Biology, Plenum Press, New York, London (1978). M. L. Kirby, Th. F. Gale, and D. E. Stewart, "Neural crest cells contribute to normal aorticopulmonary septation," Science, 220, 1059-1061 (1983). B. E. Lacey, "Neural crest cells migration and the extracellular matrix," J. Wash. Acad. Sci., 73, 128-140 (1983). N. M. Le Douarin, The Neural Crest, Cambridge Univ. Press, Cambridge (1982). N.M. Le Douarin, "Cell migration in embryos," Cell, ~, 353-360 (1984). N.M. Le Douarin, J. Smith, andC. S. Le Lievere, "From the neutral crest to the ganglia of the peripheral nervous system," Annu. Rev. Physiol., 43, 653-671 (1981). 63

18.

F. N. Low, "Interstitial bodies in the early chick embryo," Am. J. Anat., 128, 45-56

19.

J. H. Luft, "Ruthenium red and violet. II. Fine structural localization in animal tissues," Anat. Rec., 171, 369-416 (1971). B. W. Mayer, E. D. Hay, and R. O. Hynes, "Immunocytochemica! localization of fibronectin in embryonic chick trunk and area vasculosa," Dev. Biol., 82, 267-286 (1981). D. F. Newgreen, "Adhesion to extracellular materials by neural crest cells at the stage of initial migration," Cell Tiss. Res., 227, 297-317 (1982). D. F. Newgreen and I. L. Gibbins, "Factors controlling the time of onset of the migration of neural crest cells in the fowl embryo," Cell Tiss. Res., 224, 145-160 (1982). D. F. Newgreen, I. L. Gibbins, J. Santer, et al., "Ultrastructurai and tissue-culture studies on the roll of fibronectin collagen and glycosaminoglycans in the migration of neural cells in the fowl embryo," Cell Tiss. Res., 221, 521-549 (1982). D. F. Newgreen, M. Ritterman, and E. A. Peters, "Morphology and behavior of neural crest cells of chick embryo in vitro," Cell Tiss. Res., 203, 115-140 (1979). D. F. Newgreen and J. P. Thiery, "Fibronectin in early avian embryos: synthesis and distribution along the migration pathways of neural crest cells," Cell Tiss. Res., 211,

(1970). 20. 21. 22. 23.

24. 25.

269-291 (1980). 26.

27.

28.

29. 30~ 31. 32. 33.

34. 35. 36.

64

D. N. Noden, "Interactions directing the migration and cytodifferentiation of avian neural crest cells," in: Receptors and Recognition, B4, Specificity of Embryonic Interactions, ed. D. R. Garrod, Chapman and Hall, London (1978), pp. 3-5. K. H. Pfenninger, "Molecular biology of the nerve growth cone: a perspective," in: Gene Expression and Cell-Cell Interactions in the Developing Nervous System, New York, London (1984), pp. 1-14. R. A. Rovasio, A. Delouvee, K. M. Yamada, et al., "Neural crest cell migration: requirements for exogenous fibronectin and high cell density," J. Cell Biol., 96, 462473 (1983). J. R. Sanes, "Roles of extracellular matrix in neural development," Annu. Rev. Physiol. 45, 581-600 (1983). E. Tani and T. Ametani, "Substructure of microtubules in brain nerve cells as revealed by ruthenium red," J. Cell Biol., 46, 159-165 (1970). J. P. Thiery, J. L. Duband, U. Rutfshauser, and G. M. Edelman, "Cell adhesion molecules in early chicken embryogenesis," Proc. Natl. Acad. Sci. USA, 79, 6737-6741 (1982). K. W. Tosney, "The early migration of neural crest cells in the trunk region of the avian embryo: an electron microscopic study," Dev. Biol., 62, 317-333 (1978). F. Tuckett and G. M. Morris-Kay, "The distribution of fibronectin, laminin and entactin in the neuruiating rat embryo studied by indirect immunofluorescence," J. Embryol. Exp. Morphol., 94, 95-112 (1986). J. A. Weston, "The migration and differentiation of neural crest cells," Adv. Morphogeno, 8, 41-114 (1970). Jo A. Weston, "Motile and social behavior of neural crest cells," in: Cell Behavior, Cambridge Univ. Press, London (1982), pp. 429-470. J. A. Weston and S. L. Butler, "Temporal factors affecting localization of neural crest cells in the chicken embryo," Dev. Biol., 14, 246-266 (1966).

Ultrastructure of cells of the neural crest.

ULTRASTRUCTURE OF CELLS OF THE NEURAL CREST A. A. Sosunov, P. P. Kruglyakov, G. V. Belyanina, and V. N. Shvalev UDC 611.89-018.82-086 The majority o...
2MB Sizes 0 Downloads 0 Views