Cell Tiss. Res. 163, 313-325 (1975) - 9 by Springer-Verlag 1975
Neuronal Migration during the Early Development of the Cerebral Cortex A Scanning Electron Microscopic Study K. Meller and W. Tetzlaff Institut fiir Anatomie, Arbeitsgruppe Experimentelle Cytologie, Ruhr-Universit~it Bochum
Summary. Fixed cerebral vesicles of mouse foetuses were fractured and examined with the scanning electron microscope. This method provides a study of the three dimensional developmental features of the pseudostratified columnar epithelium up to the formation of the early cortex plate. Matrix cells are a cell population of homogeneous shape, however, mitotic cells are easily identified by their spherical form. The external surface of the brain is formed by the closely packed end feet of these cells covered by a basal membrane. The formation of the cortical plate is the result of a continuous cell migration in columnar arrangement towards the pia. Glioependymal cells extend along the whole brain wall and most likely provide guidance for the migrating cell cords. The formation of the so-called migratory zone is a consequence of the growth of the basal and the horizontal prolongations of emigrating cells. The significance of the cell to cell contacts for the neuronal migration processes is discussed.
Key words: Neurogenesis - Cerebral cortex - Scanning electron microscopy.
Introduction The development of the cerebral cortex occurs within a succession of inseparable events: cell proliferation, cell migration and cell differentiation. These neurogenetic steps of the cortex have been studied by classical and newly developed techniques of neuroembryology. With the use of histoautoradiography, Golgi methods and transmission electron microscopy, a successful contribution to the study of the origin and proliferation of cerebral cells, their mode of emigration and the patterns of their organization has been made. The results of these studies (see reviews by Langman, 1968; Angevine, 1970; Sidman, 1970; Sidman and Rakic, 1973; Sidman, 1974) provide new and basic facts of neurogenesis, although the correlation between electron microscopical results and those provided by S e n d offprint requests to : Prof. Dr. K. Meller, Institut ftir Anatomie, Ruhr-Universit~it Bochum, 4630 Bochum, Universitatsstr. 150, Federal Republic of Germany.
Supported by a grant (Me 276/6) of the Deutsche Forschungsgemeinschaft.
314
K. Meller and W. Tetzlaff
the Golgi technique is not always without discrepancies. Stensaas and Stensaas (1968) demonstrated with the aid of serial section-analysis that the study of the three dimensional shapes and organization of cerebral elements provides additional information. However, this is a very time-consuming method. Hansson (1970) showed the advantages of the scanning electron microscopic technique for the study of the synaptic bodies of the retina. The aim of the present paper is to identify with the aid of the scanning electron microscope the early morphological events occurring in the developing cerebral hemispheres. In this study the scanning observations have been used to describe (1) the three dimensional morphology of the germinal cells, glioblasts and young neurons, (2) the morphology of the migrating cells, (3) the development of the brain external surface, and (4) the formation of the early cerebral cortex plate.
Materials and Methods Brains of mouse foetuses between the 10th and 15th day of gestation were dissected and fixed in buffered glutaraldehyde for 10 to 12 hours, rinsed in buffer and postfixed in o s m i u m tetroxide for 5 hours. After fixation the desired parts of telencephalon were dissected out and fractured. After this relatively long period of fixation, the firmness of the samples is such that they can easily be fractured. Dehydration was carried out in a graded series of methanols. The samples were then transferred to Freon 11 and finally to Freon 13 in a critical point drying apparatus. The specimens were shadowed with gold and examined in a JEOL R-35 scanning electron microscope at 25 kV.
Results
Sagittal and frontal fractures of the cerebral vesicles have been examined and compared. Since the samples include the whole hemispheres, the further localization of a desirable zone of the pallium is made with facility. The fracture of the cerebral vesicles from a 10-day-old mouse foetus (E 10) shows two distinguishable parts, a parieto-occipital and a frontal region. The first part is still a neuroepithelium, while on the contrary the fro~al region is a more thickened pseudostratified epithelium (Fig. 1, A). All cells of the neuroepithelium reach from the inner (ventricular) to the outer (pial) surfaces of the brain. These ventricular cells possess an irregular cylindrical shape depending on the variable position of the cell nuclei. The ventricular surface shows many microvilli; cilia cannot be observed in this stage (Fig. 1, B). The lateral borders of the cells are attached with small prolongations to the surface of adjacent cells. These contacts probably represent
Fig. 1 A - D. Sagittal fracture of a cerebral hemisphere of a 10-day-old mouse foetus. (A) Low magnification of the fronto-parietal region of the cerebral vesicle. The arrows indicate mitotic cells near the ventricular surface. • 800. (B) View of the ependymal surface. Note the microvilli and the absence of cilia, x 3 960. (C, D) Portion o f the outermost parts of the prolongations of the neuroepithelial cells that are forming the external surface of the brain. (a) Frontal region, (b) parietal region. Note the channels situated beneath the pia (*). • 8250
Fig. 1 A - D
Fig. 2
Neuronal Migration during the Early Development of the Cerebral Cortex
317
the characteristic desmosomal junctions between the epithelial cells. Beneath the pial surface the basal and lateral portions of the cells possess fine cell prolongations which interdigitate with those of adjacent cells. As a result of this, a complicated extracellular channel system is formed under the pial surface (Fig. 1, C, D). All mitotic cells have a spheric or ovoid shape. Their apical and basal poles occasionally possess numerous microvilli. The second r e g i o n - the frontal r e g i o n - shows an advanced stage of development and contains tightly packed cells. Three cell types can be recognized (Fig. 2) : I. Elongated cells, the glioependymal cells (radial fibres). Their processes span the whole extent of the brain wall. Their nuclei are found in variable positions frequently near the ventricular zone. II. The second cell type is also bipolar but only in contact with the ventricular surface. The free distal processes show lamellipodia and ruffled membranes. III. The third cell type is only in contact with the external pial surface. These cells are ovoid to cylindrical in shape with flattened or elongated processes. The cells of the first and third types form with their distal prolongations the external surface of the cerebral vesicle. The absence of clearly visible boundaries in many of the views obtained from the pial side suggests that the basal lamina stays closely applied to the underside of the cells. Two facts characterize the morphology of the cerebral vesicles of the 11-dayold mouse foetus (E 11): (1) the cerebral vesicle wall increases in thickness as a consequence of an active cell proliferation; (2) there is the formation of a characteristic marginal zone as a cell-sparse layer composed of the outermost cytoplasmic parts of the ventricular cells. Fig. 3 shows a sagittal fracture of the hemisphere wall. The ventricular and subventricular cells are closely packed. However, radial fibres frequently separate groups of cell cords radially oriented from the ventricular to the external surface of the hemispheres. The blunt dissection occasioned by the fracture showes cell groups of glioependymal and migrating cells in clear columnar arrangements. In this stage, cell columns are formed by 20-30 cells lengthwise framed between or grouped around the radial fibres. Near to the ventricle these cells are monopolar; they take a bipolar shape near the periphery. The marginal zone is occupied by numerous interdigitating prolongations which are preferably oriented parallel to the pial surface. A great number of growth cones is found in this layer. The structure of these prolongations is comparable to the features observed in tissue cultures as flattened prolongations with dentate membranes showing the static image of ameboid movement (Fig. 3, B, C). Fig. 4 shows the typical cord arrangement of the matrix and migrating cells of a 12-day-old cerebral vesicle. These cell cords are closely packed. The outermost localized cells show typical membrane boundaries that could be recognized as ameboid features. Glioependymal cells (radial fibres) are also well recognizable. The cells beneath the pia become oriented more horizontally. These features Fig. 2. Sagittal fracture of a cerebral hemisphere of a 10-day-old mouse foetus. Arrows point to the undulating membranes of the different cell types (see text). Blood vessel (F). x 5 440
Fig. 3 A - - D. Sagittal fracture of a cerebral hemisphere of a 1 l-day-old mouse foetus. (A) Low magnification of the brain wall. Observe the numerous cell prolongations in the marginal zone. • 800. (B) Micrograph showing the columnar arrangement of migrating cells (see text). • 4200. (C, D) Morphology of the free end of cell prolongations in the marginal zone. Note the ameboid features of growth cones, x 8000
Fig. 4. Cerebral hemisphere of a 12-day-old mouse foetus, x 3 300
Fig. 5 A - D. Cerebral hemisphere of a 12.5-day-old mouse foetus. (A D) The a r r o w s point to the numerous buds found in many cell types as precursors for the formation of new ramifications, x 6000
Neuronal Migration during the Early Development o f the Cerebral Cortex
32t
Fig. 6. Cerebral hemisphere of a 13.5-day-old mouse foetus. V Z Ventricular zone; I Z intermediate zone (migratory zone); CP cortical plate, x 1200
are identical in the next stages of development. The active migration process that leads to the formation of the cortex plate is clearly demonstrable through the changes of the cell surfaces. These become more irregular and show fine buds on the cell soma and on the cell processes. Because of the rapid proliferation of cell processes found in the next stages, it can be assumed that the buds represent their origin (Fig. 5). In the 13-day-old embryo the uppermost region of the cerebral vesicle is occupied by new migratory cells (Fig. 6). These cells, localized at the end of the cell cords, are cylindrically shaped and send an apical prolongation - the apical d e n d r i t e - t o the pial surface. The growth of basal and laterally oriented prolongations in the middle region of the brain wall provides the formation of the so-called migratory zone. Therefore, all the examined zones of the frontoparietal brain show that during the formation of the cortex plate the emigrating cells from the germinal zone do not lose the cell to cell contacts with the other elements of a given cell cord. The columnar arrangement of the cell somata
322
K. Meller and W. Tetzlaff Fig. 7. Cerebral hemisphere of a 14.5-day-old mouse foetus. For abbreviations consult Fig. 6. x 1200
b e c o m e s o b s c u r e d b y p r o g r e s s i v e g r o w t h o f cellular p r o l o n g a t i o n s (axons a n d b a s a l d e n d r i t e s ) in the following stages o f c o r t e x f o r m a t i o n (Fig. 7).
Neuronal Migrationduringthe EarlyDevelopmentof the CerebralCortex
323
Discussion
In the past ten years a considerable number of publications on the development of the brain using the Golgi method and electron microscopic techniques has supplied numerous morphological data of its cell structure and cell organization (Berry and Rogers, 1965; Stensaas, 1967a, b; Meller et al., 1966, 1968, 1969; Meller and Glees, 1965, 1969; Morest, 1968; Hinds and Ruffett, 1971). However, a three dimensional model of the cell organization and the transitional movements of cell is indispensable for understanding the form changes during the morphogenetic processes. The morphological gap between transmission electron microscopic and Golgi studies can be filled by scanning electron microscopy as demonstrated by the present preliminary observations. The fracture procedure allows the investigation of no natural free surfaces. However, the interpretation of the samples in advanced development stages is more difficult than in the early ones. Histoautoradiographic (Fujita, 1962, 1963) and electron microscopic studies (Meller et al., 1966; Wechsler, 1966a, b) have demonstrated that the matrix cells are a homogeneous population. Therefore, it is not surprising that the scanning electron microscopy of these cells does not reveal any significant differences. The apical-lateral cell contacts are more numerous than shown by transmission electron microscopy. However, the desmosomaljunctions as adhesion sites between the cells are not discernible by scanning electron microscopy. These contacts show variations in number and form at the different stages. For this reason, Potter et al. (1966) suggested that this type of intercellular communication functions as a tool for coordinating embryonic development.
The Process o f Cell Migration
The elongated cells whose processes span the whole extent of the brain wall are of exceptional importance. Rakic (1972) and Sidman (1974) claimed that the immature glial processes provide guidelines for cell emigration through the complex texture of closely packed cell processes and cell bodies that compose the developing cerebral wall. This idea in agreement with the suggestion of one of us (Meller and Glees, 1965) that the orientation of migrating of retinal cells is coordinated by the Miiller cells. The present results supply two new data of main significance: (1) that the migrating cells become radial towards the pia and move to the periphery without breaking their adhesions with other cells. The result is a cellular cord down to the cortical plate, delimited by oriented radial fibres in the same direction. (2) The observation that the further formation of the migratory zone, as a consequence of the development of basal and lateral processes of the migrating cells, is not an obstacle for emigration. The uninterrupted cell to cell contacts provide the direction of migration for the rest of the migrating cells. In other words, the cell to cell guidance is present during the formation of the migratory zone. The cells of the ventricular zone migrating first are leading the emigration of those cells building the upper layer of the cortical plate. This assumption is not in contradiction to the histoautoradiographic results of Sidman et al. (1959)
324
K. Meller and W. Tetzlaff
w h o p o s t u l a t e d t h a t the m o r e m a t u r e a p p e a r i n g cells g e n e r a l l y lie d e e p e r t h a n t h e less m a t u r e o n e s a n d the c o l u m n a r o r g a n i z a t i o n o f t h e cell s o m a t a b e c o m e s o b s c u r e d b y t h e d e v e l o p m e n t o f cell p r o c e s s e s o r i e n t e d in m a n y d i r e c t i o n s . T h e a b o v e d e s c r i b e d m o r p h o l o g i c a l o b s e r v a t i o n s suggest t h e e x i s t e n c e o f a c o n t r o l m e c h a n i s m f o r cell m i g r a t i o n . T h e c o l u m n a r a r r a n g e m e n t o f cells d u r i n g t h e d e v e l o p m e n t o f t h e b r a i n c o u l d be t h e m o r p h o l o g i c a l basis o f the a s s u m p t i o n t h a t t h e cells o f e a c h c o r d d e r i v e f r o m a single p r e c u r s o r cell ( N o r d l a n d e r a n d E d w a r d s , 1969a, b ; 1970). A c l o n a l o r i g i n o f d i f f e r e n t n e r v e cell t y p e s s u p p o s e s t h e e x i s t e n c e o f a n e u r o n a l cell l i n e a g e t h a t p r o v i d e s t h e n e u r o n a l s p e c i f i c i t y a n d the l o c a l i z a t i o n o f e a c h n e r v e cell at t h e r i g h t t i m e a n d r i g h t p l a c e in the b r a i n ( H u n t , 1975).
References Angevine, J.B.,Jr.: Critical cellular events in the shaping of neural centers. In: The Neurosciences second study program (F.O. Schmitt, ed.), p. 62-72. New York: The Rockefeller University Press 1970 Berry, M., Rogers, A.W. : The migration of neuroblasts in the developing cerebral cortex. J. Anat. (Lond.) 99, 691-709 (1965) Fujita, S. : Kinetics of cellular proliferation. Exp. Cell Res. 28, 52~0 (1962) Fujita, S. : The matrix cell and cytogenesis in the developing central nervous system. J. comp. Neurol. 120, 37~t2 (1963) Hansson, H.-A.: Scanning electron microscopic studies on the synaptic bodies in the rat retina. Z. Zellforsch. 107, 45-53 (1970) Hinds, J.W., Ruffett, T.L. : Cell proliferation in the neural tube : an electron microscopic and Golgi analysis in the mouse cerebral vesicle. Z. Zellforsch. 115, 226-264 (1971) Hunt, R.K. : The cell cycle, cell lineage, and neuronal specificity. In: Cell cycfe and cell differentiation (H. Rennert and H. Holtzer, eds.), p. 43-62. Berlin-Heidelberg-New York: Springer 1975 Langman, J. : Histogenesis of the central nervous system. In: The structure and function of nervous tissue (G.H. Bourne, ed.), vol. I, p. 33-66. New York: Academic Press 1968 Meller, K., Breipohl, W., Glees, P. : Early cytological differentiation in the cerebral hemisphere of mice. An electron microscope study. Z. Zellforsch. 72, 525-533 (1966) Meller, K., Breipohl, W., Glees, P. : Synaptic organization of the molecular and the outer granular layer in the motor cortex in white mouse during postnatal development. A Golgi and electron microscopical study. Z. Zellforsch. 92, 217-231 (1968) Meller, K., Breipohl, W., Glees, P.: Ontogeny of the mouse motor cortex. The polymorph layer or layer VI. A Golgi and electron microscopical study. Z. Zellforsch. 99, 443458 (1969a) Meller, K., Glees, P. : The differentiation of neuroglia-Miiller-cells in the retina of chick. Z. Zellforsch. 66, 321-332 (1965b) Meller, K., Glees, P. : The development of the mouse cerebellum. A Golgi and electron microscopical study. In: Neurobiology of cerebellar evolution and development (R. Llin/ts, ed.), p. 783-801. Chicago: Amer. Med. Assn. Educ. Res. Fdn. 1969 Morest, D.K.: The growth of synaptic endings in the mammalian brain: A study of the calyces of the trapezoid body. Z. Anat. Entwickl.-Gesch. 127, 201-220 (1968) Nordlander, R.H., Edwards, J.S. : Postembryonic brain development in the monarch butterfly, Danaus plexippus plexippus L. I. Cellular events during brain morphogenesis. Wilhelm Roux' Archiv 162, 197-217 (1969) Nordlander, R.H., Edwards, J.S. : Postembryonic brain development in the monarch butterfly, Danaus plexippusplexippus L. II. The optic lobes. Wilhelm Roux' Archiv 163, 197-220 (1969) Nordlander, R.H., Edwards, J.S. : Postembryonic brain development in the monarch butterfly, Danaus plexippus plexippus L. III. Morphogenesis of centers other than the optic lobes. Wilhelm Roux' Archiv 164, 247~260 (1970)
Neuronal Migration during the Early Development of the Cerebral Cortex
325
Potter, D.D., Furshpan, E.J., Lennox, E.S.: Connections between cells of the developing squid as revealed by electrophysiological methods. Proc. nat. Acad. Sci. (Wash.) 55, 328-336 (1966) Rakic, P.: Mode of cell migration to the superficial layers of fetal monkey neocortex. J. comp. Neurol. 145, 61 84 (1972) Sidman, R.L. : Cell proliferation, migration, and interaction in the developing mammalian central nervous system. In: The neurosciences second study program (F.O. Schmitt, ed.), p. 100-107. New York: The Rockefeller University Press 1970 Sidman, R.L. : Contact interaction among developing mammalian brain cells. In: The cell surface in development (A.A. Moscona, ed.), p. 221~53. New York-London: A. Wiley & Sons 1974 Sidman, R.L., Miale, I.L., Feder, N. : Cell proliferation and migration in the primitive ependymal zone; an autoradiographic study of histogenesis in the nervous system. Exp. Neurol. 1, 322-333 (1959) Sidman, R.L., Rakic, P. : Neuronal migration, with special reference to developing human brain: A review. Brain Res. 62, 1-25 (1973) Stensaas, L.J.: The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits. I. Fifteen millimeter stage, spongioblast morphology. J. comp. Neurol. 129, 59-70 (1967a) Stensaas, L.J.: The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits. II. Twenty millimeter stage, neuroblast morphology. J. comp. Neurol. 129, 71 84 (1967b) Stensaas, L.J., Stensaas, S.S. : An electron microscope study of cells in the matrix and intermediate laminae of the cerebral hemisphere of the 45 mm rabbit embryo. Z. Zellforsch. 91, 341 365 (1968) Wechsler, W. : Elektronenmikroskopischer Beitrag zur Histogenese der weiBen Substanz des Rfickenmarks yon Hfihnerembryonen. Z. Zellforsch. 74, 232-251 (1966a) Wechsler, W. : Elektronenmikroskopischer Beitrag zur Nervenzelldifferenzierung und Histogenese der grauen Substanz des Riickenmarks von Hfihnerembryonen. Z. Zellforsch. 74, 401-422 (1966b)
Received July 14, 1975
Neuronal Migration during the Early Development of the Cerebral Cortex A Scanning Electron Microscopic Study K. Meller and W. Tetzlaff Institut fiir Anatomie, Arbeitsgruppe Experimentelle Cytologie, Ruhr-Universit~it Bochum
Summary. Fixed cerebral vesicles of mouse foetuses were fractured and examined with the scanning electron microscope. This method provides a study of the three dimensional developmental features of the pseudostratified columnar epithelium up to the formation of the early cortex plate. Matrix cells are a cell population of homogeneous shape, however, mitotic cells are easily identified by their spherical form. The external surface of the brain is formed by the closely packed end feet of these cells covered by a basal membrane. The formation of the cortical plate is the result of a continuous cell migration in columnar arrangement towards the pia. Glioependymal cells extend along the whole brain wall and most likely provide guidance for the migrating cell cords. The formation of the so-called migratory zone is a consequence of the growth of the basal and the horizontal prolongations of emigrating cells. The significance of the cell to cell contacts for the neuronal migration processes is discussed.
Key words: Neurogenesis - Cerebral cortex - Scanning electron microscopy.
Introduction The development of the cerebral cortex occurs within a succession of inseparable events: cell proliferation, cell migration and cell differentiation. These neurogenetic steps of the cortex have been studied by classical and newly developed techniques of neuroembryology. With the use of histoautoradiography, Golgi methods and transmission electron microscopy, a successful contribution to the study of the origin and proliferation of cerebral cells, their mode of emigration and the patterns of their organization has been made. The results of these studies (see reviews by Langman, 1968; Angevine, 1970; Sidman, 1970; Sidman and Rakic, 1973; Sidman, 1974) provide new and basic facts of neurogenesis, although the correlation between electron microscopical results and those provided by S e n d offprint requests to : Prof. Dr. K. Meller, Institut ftir Anatomie, Ruhr-Universit~it Bochum, 4630 Bochum, Universitatsstr. 150, Federal Republic of Germany.
Supported by a grant (Me 276/6) of the Deutsche Forschungsgemeinschaft.
314
K. Meller and W. Tetzlaff
the Golgi technique is not always without discrepancies. Stensaas and Stensaas (1968) demonstrated with the aid of serial section-analysis that the study of the three dimensional shapes and organization of cerebral elements provides additional information. However, this is a very time-consuming method. Hansson (1970) showed the advantages of the scanning electron microscopic technique for the study of the synaptic bodies of the retina. The aim of the present paper is to identify with the aid of the scanning electron microscope the early morphological events occurring in the developing cerebral hemispheres. In this study the scanning observations have been used to describe (1) the three dimensional morphology of the germinal cells, glioblasts and young neurons, (2) the morphology of the migrating cells, (3) the development of the brain external surface, and (4) the formation of the early cerebral cortex plate.
Materials and Methods Brains of mouse foetuses between the 10th and 15th day of gestation were dissected and fixed in buffered glutaraldehyde for 10 to 12 hours, rinsed in buffer and postfixed in o s m i u m tetroxide for 5 hours. After fixation the desired parts of telencephalon were dissected out and fractured. After this relatively long period of fixation, the firmness of the samples is such that they can easily be fractured. Dehydration was carried out in a graded series of methanols. The samples were then transferred to Freon 11 and finally to Freon 13 in a critical point drying apparatus. The specimens were shadowed with gold and examined in a JEOL R-35 scanning electron microscope at 25 kV.
Results
Sagittal and frontal fractures of the cerebral vesicles have been examined and compared. Since the samples include the whole hemispheres, the further localization of a desirable zone of the pallium is made with facility. The fracture of the cerebral vesicles from a 10-day-old mouse foetus (E 10) shows two distinguishable parts, a parieto-occipital and a frontal region. The first part is still a neuroepithelium, while on the contrary the fro~al region is a more thickened pseudostratified epithelium (Fig. 1, A). All cells of the neuroepithelium reach from the inner (ventricular) to the outer (pial) surfaces of the brain. These ventricular cells possess an irregular cylindrical shape depending on the variable position of the cell nuclei. The ventricular surface shows many microvilli; cilia cannot be observed in this stage (Fig. 1, B). The lateral borders of the cells are attached with small prolongations to the surface of adjacent cells. These contacts probably represent
Fig. 1 A - D. Sagittal fracture of a cerebral hemisphere of a 10-day-old mouse foetus. (A) Low magnification of the fronto-parietal region of the cerebral vesicle. The arrows indicate mitotic cells near the ventricular surface. • 800. (B) View of the ependymal surface. Note the microvilli and the absence of cilia, x 3 960. (C, D) Portion o f the outermost parts of the prolongations of the neuroepithelial cells that are forming the external surface of the brain. (a) Frontal region, (b) parietal region. Note the channels situated beneath the pia (*). • 8250
Fig. 1 A - D
Fig. 2
Neuronal Migration during the Early Development of the Cerebral Cortex
317
the characteristic desmosomal junctions between the epithelial cells. Beneath the pial surface the basal and lateral portions of the cells possess fine cell prolongations which interdigitate with those of adjacent cells. As a result of this, a complicated extracellular channel system is formed under the pial surface (Fig. 1, C, D). All mitotic cells have a spheric or ovoid shape. Their apical and basal poles occasionally possess numerous microvilli. The second r e g i o n - the frontal r e g i o n - shows an advanced stage of development and contains tightly packed cells. Three cell types can be recognized (Fig. 2) : I. Elongated cells, the glioependymal cells (radial fibres). Their processes span the whole extent of the brain wall. Their nuclei are found in variable positions frequently near the ventricular zone. II. The second cell type is also bipolar but only in contact with the ventricular surface. The free distal processes show lamellipodia and ruffled membranes. III. The third cell type is only in contact with the external pial surface. These cells are ovoid to cylindrical in shape with flattened or elongated processes. The cells of the first and third types form with their distal prolongations the external surface of the cerebral vesicle. The absence of clearly visible boundaries in many of the views obtained from the pial side suggests that the basal lamina stays closely applied to the underside of the cells. Two facts characterize the morphology of the cerebral vesicles of the 11-dayold mouse foetus (E 11): (1) the cerebral vesicle wall increases in thickness as a consequence of an active cell proliferation; (2) there is the formation of a characteristic marginal zone as a cell-sparse layer composed of the outermost cytoplasmic parts of the ventricular cells. Fig. 3 shows a sagittal fracture of the hemisphere wall. The ventricular and subventricular cells are closely packed. However, radial fibres frequently separate groups of cell cords radially oriented from the ventricular to the external surface of the hemispheres. The blunt dissection occasioned by the fracture showes cell groups of glioependymal and migrating cells in clear columnar arrangements. In this stage, cell columns are formed by 20-30 cells lengthwise framed between or grouped around the radial fibres. Near to the ventricle these cells are monopolar; they take a bipolar shape near the periphery. The marginal zone is occupied by numerous interdigitating prolongations which are preferably oriented parallel to the pial surface. A great number of growth cones is found in this layer. The structure of these prolongations is comparable to the features observed in tissue cultures as flattened prolongations with dentate membranes showing the static image of ameboid movement (Fig. 3, B, C). Fig. 4 shows the typical cord arrangement of the matrix and migrating cells of a 12-day-old cerebral vesicle. These cell cords are closely packed. The outermost localized cells show typical membrane boundaries that could be recognized as ameboid features. Glioependymal cells (radial fibres) are also well recognizable. The cells beneath the pia become oriented more horizontally. These features Fig. 2. Sagittal fracture of a cerebral hemisphere of a 10-day-old mouse foetus. Arrows point to the undulating membranes of the different cell types (see text). Blood vessel (F). x 5 440
Fig. 3 A - - D. Sagittal fracture of a cerebral hemisphere of a 1 l-day-old mouse foetus. (A) Low magnification of the brain wall. Observe the numerous cell prolongations in the marginal zone. • 800. (B) Micrograph showing the columnar arrangement of migrating cells (see text). • 4200. (C, D) Morphology of the free end of cell prolongations in the marginal zone. Note the ameboid features of growth cones, x 8000
Fig. 4. Cerebral hemisphere of a 12-day-old mouse foetus, x 3 300
Fig. 5 A - D. Cerebral hemisphere of a 12.5-day-old mouse foetus. (A D) The a r r o w s point to the numerous buds found in many cell types as precursors for the formation of new ramifications, x 6000
Neuronal Migration during the Early Development o f the Cerebral Cortex
32t
Fig. 6. Cerebral hemisphere of a 13.5-day-old mouse foetus. V Z Ventricular zone; I Z intermediate zone (migratory zone); CP cortical plate, x 1200
are identical in the next stages of development. The active migration process that leads to the formation of the cortex plate is clearly demonstrable through the changes of the cell surfaces. These become more irregular and show fine buds on the cell soma and on the cell processes. Because of the rapid proliferation of cell processes found in the next stages, it can be assumed that the buds represent their origin (Fig. 5). In the 13-day-old embryo the uppermost region of the cerebral vesicle is occupied by new migratory cells (Fig. 6). These cells, localized at the end of the cell cords, are cylindrically shaped and send an apical prolongation - the apical d e n d r i t e - t o the pial surface. The growth of basal and laterally oriented prolongations in the middle region of the brain wall provides the formation of the so-called migratory zone. Therefore, all the examined zones of the frontoparietal brain show that during the formation of the cortex plate the emigrating cells from the germinal zone do not lose the cell to cell contacts with the other elements of a given cell cord. The columnar arrangement of the cell somata
322
K. Meller and W. Tetzlaff Fig. 7. Cerebral hemisphere of a 14.5-day-old mouse foetus. For abbreviations consult Fig. 6. x 1200
b e c o m e s o b s c u r e d b y p r o g r e s s i v e g r o w t h o f cellular p r o l o n g a t i o n s (axons a n d b a s a l d e n d r i t e s ) in the following stages o f c o r t e x f o r m a t i o n (Fig. 7).
Neuronal Migrationduringthe EarlyDevelopmentof the CerebralCortex
323
Discussion
In the past ten years a considerable number of publications on the development of the brain using the Golgi method and electron microscopic techniques has supplied numerous morphological data of its cell structure and cell organization (Berry and Rogers, 1965; Stensaas, 1967a, b; Meller et al., 1966, 1968, 1969; Meller and Glees, 1965, 1969; Morest, 1968; Hinds and Ruffett, 1971). However, a three dimensional model of the cell organization and the transitional movements of cell is indispensable for understanding the form changes during the morphogenetic processes. The morphological gap between transmission electron microscopic and Golgi studies can be filled by scanning electron microscopy as demonstrated by the present preliminary observations. The fracture procedure allows the investigation of no natural free surfaces. However, the interpretation of the samples in advanced development stages is more difficult than in the early ones. Histoautoradiographic (Fujita, 1962, 1963) and electron microscopic studies (Meller et al., 1966; Wechsler, 1966a, b) have demonstrated that the matrix cells are a homogeneous population. Therefore, it is not surprising that the scanning electron microscopy of these cells does not reveal any significant differences. The apical-lateral cell contacts are more numerous than shown by transmission electron microscopy. However, the desmosomaljunctions as adhesion sites between the cells are not discernible by scanning electron microscopy. These contacts show variations in number and form at the different stages. For this reason, Potter et al. (1966) suggested that this type of intercellular communication functions as a tool for coordinating embryonic development.
The Process o f Cell Migration
The elongated cells whose processes span the whole extent of the brain wall are of exceptional importance. Rakic (1972) and Sidman (1974) claimed that the immature glial processes provide guidelines for cell emigration through the complex texture of closely packed cell processes and cell bodies that compose the developing cerebral wall. This idea in agreement with the suggestion of one of us (Meller and Glees, 1965) that the orientation of migrating of retinal cells is coordinated by the Miiller cells. The present results supply two new data of main significance: (1) that the migrating cells become radial towards the pia and move to the periphery without breaking their adhesions with other cells. The result is a cellular cord down to the cortical plate, delimited by oriented radial fibres in the same direction. (2) The observation that the further formation of the migratory zone, as a consequence of the development of basal and lateral processes of the migrating cells, is not an obstacle for emigration. The uninterrupted cell to cell contacts provide the direction of migration for the rest of the migrating cells. In other words, the cell to cell guidance is present during the formation of the migratory zone. The cells of the ventricular zone migrating first are leading the emigration of those cells building the upper layer of the cortical plate. This assumption is not in contradiction to the histoautoradiographic results of Sidman et al. (1959)
324
K. Meller and W. Tetzlaff
w h o p o s t u l a t e d t h a t the m o r e m a t u r e a p p e a r i n g cells g e n e r a l l y lie d e e p e r t h a n t h e less m a t u r e o n e s a n d the c o l u m n a r o r g a n i z a t i o n o f t h e cell s o m a t a b e c o m e s o b s c u r e d b y t h e d e v e l o p m e n t o f cell p r o c e s s e s o r i e n t e d in m a n y d i r e c t i o n s . T h e a b o v e d e s c r i b e d m o r p h o l o g i c a l o b s e r v a t i o n s suggest t h e e x i s t e n c e o f a c o n t r o l m e c h a n i s m f o r cell m i g r a t i o n . T h e c o l u m n a r a r r a n g e m e n t o f cells d u r i n g t h e d e v e l o p m e n t o f t h e b r a i n c o u l d be t h e m o r p h o l o g i c a l basis o f the a s s u m p t i o n t h a t t h e cells o f e a c h c o r d d e r i v e f r o m a single p r e c u r s o r cell ( N o r d l a n d e r a n d E d w a r d s , 1969a, b ; 1970). A c l o n a l o r i g i n o f d i f f e r e n t n e r v e cell t y p e s s u p p o s e s t h e e x i s t e n c e o f a n e u r o n a l cell l i n e a g e t h a t p r o v i d e s t h e n e u r o n a l s p e c i f i c i t y a n d the l o c a l i z a t i o n o f e a c h n e r v e cell at t h e r i g h t t i m e a n d r i g h t p l a c e in the b r a i n ( H u n t , 1975).
References Angevine, J.B.,Jr.: Critical cellular events in the shaping of neural centers. In: The Neurosciences second study program (F.O. Schmitt, ed.), p. 62-72. New York: The Rockefeller University Press 1970 Berry, M., Rogers, A.W. : The migration of neuroblasts in the developing cerebral cortex. J. Anat. (Lond.) 99, 691-709 (1965) Fujita, S. : Kinetics of cellular proliferation. Exp. Cell Res. 28, 52~0 (1962) Fujita, S. : The matrix cell and cytogenesis in the developing central nervous system. J. comp. Neurol. 120, 37~t2 (1963) Hansson, H.-A.: Scanning electron microscopic studies on the synaptic bodies in the rat retina. Z. Zellforsch. 107, 45-53 (1970) Hinds, J.W., Ruffett, T.L. : Cell proliferation in the neural tube : an electron microscopic and Golgi analysis in the mouse cerebral vesicle. Z. Zellforsch. 115, 226-264 (1971) Hunt, R.K. : The cell cycle, cell lineage, and neuronal specificity. In: Cell cycfe and cell differentiation (H. Rennert and H. Holtzer, eds.), p. 43-62. Berlin-Heidelberg-New York: Springer 1975 Langman, J. : Histogenesis of the central nervous system. In: The structure and function of nervous tissue (G.H. Bourne, ed.), vol. I, p. 33-66. New York: Academic Press 1968 Meller, K., Breipohl, W., Glees, P. : Early cytological differentiation in the cerebral hemisphere of mice. An electron microscope study. Z. Zellforsch. 72, 525-533 (1966) Meller, K., Breipohl, W., Glees, P. : Synaptic organization of the molecular and the outer granular layer in the motor cortex in white mouse during postnatal development. A Golgi and electron microscopical study. Z. Zellforsch. 92, 217-231 (1968) Meller, K., Breipohl, W., Glees, P.: Ontogeny of the mouse motor cortex. The polymorph layer or layer VI. A Golgi and electron microscopical study. Z. Zellforsch. 99, 443458 (1969a) Meller, K., Glees, P. : The differentiation of neuroglia-Miiller-cells in the retina of chick. Z. Zellforsch. 66, 321-332 (1965b) Meller, K., Glees, P. : The development of the mouse cerebellum. A Golgi and electron microscopical study. In: Neurobiology of cerebellar evolution and development (R. Llin/ts, ed.), p. 783-801. Chicago: Amer. Med. Assn. Educ. Res. Fdn. 1969 Morest, D.K.: The growth of synaptic endings in the mammalian brain: A study of the calyces of the trapezoid body. Z. Anat. Entwickl.-Gesch. 127, 201-220 (1968) Nordlander, R.H., Edwards, J.S. : Postembryonic brain development in the monarch butterfly, Danaus plexippus plexippus L. I. Cellular events during brain morphogenesis. Wilhelm Roux' Archiv 162, 197-217 (1969) Nordlander, R.H., Edwards, J.S. : Postembryonic brain development in the monarch butterfly, Danaus plexippusplexippus L. II. The optic lobes. Wilhelm Roux' Archiv 163, 197-220 (1969) Nordlander, R.H., Edwards, J.S. : Postembryonic brain development in the monarch butterfly, Danaus plexippus plexippus L. III. Morphogenesis of centers other than the optic lobes. Wilhelm Roux' Archiv 164, 247~260 (1970)
Neuronal Migration during the Early Development of the Cerebral Cortex
325
Potter, D.D., Furshpan, E.J., Lennox, E.S.: Connections between cells of the developing squid as revealed by electrophysiological methods. Proc. nat. Acad. Sci. (Wash.) 55, 328-336 (1966) Rakic, P.: Mode of cell migration to the superficial layers of fetal monkey neocortex. J. comp. Neurol. 145, 61 84 (1972) Sidman, R.L. : Cell proliferation, migration, and interaction in the developing mammalian central nervous system. In: The neurosciences second study program (F.O. Schmitt, ed.), p. 100-107. New York: The Rockefeller University Press 1970 Sidman, R.L. : Contact interaction among developing mammalian brain cells. In: The cell surface in development (A.A. Moscona, ed.), p. 221~53. New York-London: A. Wiley & Sons 1974 Sidman, R.L., Miale, I.L., Feder, N. : Cell proliferation and migration in the primitive ependymal zone; an autoradiographic study of histogenesis in the nervous system. Exp. Neurol. 1, 322-333 (1959) Sidman, R.L., Rakic, P. : Neuronal migration, with special reference to developing human brain: A review. Brain Res. 62, 1-25 (1973) Stensaas, L.J.: The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits. I. Fifteen millimeter stage, spongioblast morphology. J. comp. Neurol. 129, 59-70 (1967a) Stensaas, L.J.: The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits. II. Twenty millimeter stage, neuroblast morphology. J. comp. Neurol. 129, 71 84 (1967b) Stensaas, L.J., Stensaas, S.S. : An electron microscope study of cells in the matrix and intermediate laminae of the cerebral hemisphere of the 45 mm rabbit embryo. Z. Zellforsch. 91, 341 365 (1968) Wechsler, W. : Elektronenmikroskopischer Beitrag zur Histogenese der weiBen Substanz des Rfickenmarks yon Hfihnerembryonen. Z. Zellforsch. 74, 232-251 (1966a) Wechsler, W. : Elektronenmikroskopischer Beitrag zur Nervenzelldifferenzierung und Histogenese der grauen Substanz des Riickenmarks von Hfihnerembryonen. Z. Zellforsch. 74, 401-422 (1966b)
Received July 14, 1975
