Neuron migration within the radial glial fiber system of the developing murine cerebrum: an electron microscopic autoradiographic analysis

Developmental Brain Research, 52 (1990) 39-56 Elsevier

39

BRESD 51020

Neuron migration within the radial glial fiber system of the developing murine cerebrum: an electron microscopic autoradiographic analysis Jean-Franqois Gadisseux 1, Hazim J. Kadhim 1, Philippe van den Bosch de Aguilar 2, V e r n e S. Caviness 3 a n d Philippe E v r a r d 1 1Developmental Neurology Unit, University of Louvain Medical School, Brussels (Belgium), 2Laboratoire de Biologie Cellulaire, Universitd de Louvain, Louvain-la-Neuve (Belgium)and 3Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 (U.S.A.) (Accepted 29 August 1989)

Key words: Neuronoglial relationship; Neuronal migration; Glycogen; Thymidine autoradiography; Mouse brain

The present analysis provides direct evidence in the mouse that in the course of neocortical histogenesis, contact between migrating neurons and the surfaces of radial glial fibers is both invariant and relatively selective. The analysis characterizes in detail the migratory behavior of the individual migrating cell with respect to the overall radial glial fiber system as this system varies systematically in its structure with ascent through the strata of the cerebral wall. A quantitative study of the relationships between the radial glial fibers confidently identified by their glycogen content and the migrating neurons marked autoradiographicallyby injection of [3H]thymidine was also performed at the ultrastructural level on tangential sections at different pallial levels in El6 and El7 embryos. The overall set of observations lend support to the hypothesis that radial glial fibers act specifically as guides to neuronal migration and illustrate the nature of the cell-to-cell interaction which serves this cellular process critical to neocortical histogenesis. INTRODUCTION N e u r o n migration is the critical cellular process which initiates histogenesis of neocortex. Migration involves a series of complex cell interactions and transformations 38. Postmitotic cells must first a d o p t a characteristic conform a t i o n prior to m o v e m e n t . T h e cells are then guided in their ascent b y contact with the surface of a specialized cell of the astroglial lineage, the radial glial cells 36. The ascending fibers of the radial glial cells form a dense system which spans the full width of the cerebral wall. T h e structure of the system varies systematically with ascent through the architectonic strata of the cerebral wall, and t h e r e are dynamic changes in the system at all levels as histogenesis p r o c e e d s t7,18,2°,32. Certain changes in the fiber system a p p e a r to be incidental to growth of the cerebral wall. O t h e r s a p p e a r regulated by mechanisms specific to histogenesis of the cerebral cortex. In particular, gaiting mechanisms at the interface of subcortical and cortical structures as well as within cortical s u b l a m i n a e a p p e a r to regulate the density of fibers which contribute to the transcortical span of the fiber system zl. The present electron microscopic study tracks the centrifugal m o v e m e n t s of migrating neurons through the radial glial fiber system of the developing murine cerebral

wall. The analysis is u n d e r t a k e n in the dorsal pallial wall, a cortical sector that includes the SI, or 'barrel field' representation. It is c o n d u c t e d in the interval e m b r y o n i c days 16-17 when migrations are p r o c e e d i n g maximally 8 and the principal transformations of the radial glial system are realized 17,2°. Migrating neurons are unambiguously identified a u t o r a d i o g r a p h i c a l l y in terms of their p o s t - D N A synthesis age as well as by cytologic criteria. Glial fibers are confidently identified by histological techniques which preserve glycogen 17,27. The t a n d e m application of these m e t h o d s allows characterization of critical transformations which occur in the patterns of interaction of migrating cells and the glial fiber system. MATERIALS AND METHODS Pregnant NMRI female mice were injected intraperitoneally with 10/zCi/g body weight of [3H]thymidine (spec. act. 50-60 Ci/mM) on gestational days 14 (El4), 14.5 (E14.5) and 15 (El5). Males had been placed in cages with the females for a maximum of 6 h. Coitus, marking the day of onset of gestation (E0 of gestation), was ascertained by the presence of a vaginal plug. For each gestational age of thymidine injection, embryos were removed under anesthesia from one set of pregnant females. For another set of animals representing the 3 ages of thymidine injection, pregnancy was allowed to continue to term. Procedures for embryonic animals. The embryos were taken at variable survival times after injection so as to provide animals from

Correspondence: Jean-Franqois Gadisseux, Laboratoire de Neurologie du D6veloppement, Avenue Hippocrate 10, UCL 10/1303, B-1200 Brussels, Belgium. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

40 each age of thymidine injection at a final age of El6 and El7. The hysterotomies were perfomed uniformly on the mornings of El6 and El7. The embryos were fixed by transcardial perfusion with z~ fixative containing 2.5% glutaraldehyde and 1% paraformaldehyde with 0.01 M CaCI2 and H20 2 in 0.1 M cacodylate buffer. The brains were immediately dissected and cut coronally into small blocks and then fixed overnight by immersion at 4 °C in the same solution. The blocks were then rinsed in buffer and postfixed for 45-90 rain at room temperature in 1% OsO4, 0.05 M KaFeCno.3H20 in 0.1 M cacodylate buffer 12. After careful rinsing with buffer, blocks were gradually dehydrated and embedded in Epon 812. Two micrometer full thickness sections (ventricular zone-pial surface), were cut coronally from blocks taken from the dorsolateral cerebral wall. These sections were stained with Toluidine blue in order to establish that the plane of section was favorable for optic and ultrastructural autoradiographic study. Adjacent sections were cut and mounted on glass slides, dipped in Ilford K2 photonuclear emulsion diluted 2:1 with distilled water and stored in the dark for 6 weeks at 4 °C. Subsequently, they were developed and stained in 2% Toluidine blue. The distribution of nuclei labeled with [3H]thymidine was plotted on drawings by lucida drawing tube. Ultra-thin sections were cut with a Reichert ultramicrotome. The blocks were first cut coronally, reoriented, and then cut tangentially. One set of serial sections was mounted on Formvar-coated grids and stained with uranyl acetate and lead citrate (U-Pb), or with periodic acid-thiocarbohydrazide-silver proteinate 31'4a(PA-TCH-SP). A second set of ultra-thin sections was mounted on glass slides which had been previously covered with a celloidin film. These were carbonized, dipped in Ilford L4 photo-nuclear emulsion and stored in the dark for 3-4 months at 4 °C. After a 4-min development in the solution described by Boyenval and Fischer3 at 18 °C, the sections were placed on copper and nickel grids and stained with U-Pb or PA-TCH-SP. Quantitative determinations relating to neuron-radial glial interrelationships were obtained from 3 different tissue samples taken at different pallial depths from the cerebrum of El6 and El7 embryos. Each determination was based on a sampling of the radial fiber relationships of 100 neurons. In addition the radial glial relationships of selected migrating neurons at El6, identified by cytologic criteria, were reconstructed from a series of 200 consecutive thin coronal sections which spanned the entire cerebral wall. Neuronal and radial fiber profiles were graphically reconstructed from photomicrographs at a magnification of 4,800× following the method of Hinds and Hinds25. Procedures for postnatal animals. Animals which had been injected with [3H]thymidine at El4, E14.5 and El5 and allowed to continue to term were sacrificed at postnatal day 20 (P20). The animals were perfused transcardially under ether anesthesia with 10% formaldehyde solution adjusted at pH 7.35. The brains were dissected and processed routinely in paraffin-embedded blocks for autoradiographic histology. Serial 10/2m thick sections were cut in the coronal plane, mounted on glass slides, dipped in Ilford K2 photonuclear emulsion, and stored in the dark for 4 weeks at 4 °C. The autoradiographs were developed and stained with Cresyl violet. The distribution of the labelled nuclei was plotted using a camera lucida drawing.

RESULTS N e u r o n s of the neocortex are generated in a pseudostratified epithelium, the ventricular zone (VZ). Postmitotic n e u r o n s must migrate to the upper margin of the developing cortical plate (CP), at its interface with the overlying molecular layer (ML). In their ascent the migrating n e u r o n s cross a succession of strata (Fig. 1) which includes in turn, the subventricular (SVZ) and

intermediate (lZ) zones at subcorticai levcis and then the cortical strata [subplate (SP) and cortica~ ptate fCP)]". The subcortical strata are densely interlaced with axonal bundles, and a tangentially coursing axonal stratum, the external sagittal stratum (ESS), is sharpl 3 delineated m the outer I Z at its interface with the SP (Fig, 2 ) This fiber stratum is composed largely of ascending thalamocortical axons ~t. Within the IZ, the neuronal populations are

to some extent

' c o m p a r t m e n t a l i z e d ' into tissue

volumes separated from each other by 'septa' formed of axon fascicles (Figs. 2 and 3). A

large c o m p l e m e n t of the

neuronal population

migrating upward from the V Z at E 1 6 - E t 7 have undergone their terminal divisions on E I 4 - E 1 5 (Figs. 1 and 4). Migrating n e u r o n s marked by injections of [31-I]thymidine, staggered at E l 4 , E14.5 and E l 5 . are distributed at complementary though overlapping levels of the l Z and developing cortex at these embryonic ages. At E l 6 (Fig. 4B), it is the E14.5 cohort which is most a b u n d a n t l y represented in the migrating neuronal population crossing the I Z but the E l 4 cohort at intracortical levels. At E l 7 (Fig. 4A) it is principally the E l 5 cohort which is still within the I Z while migrating n e u r o n s of the E14.5 cohort spread through I Z and cortical levels. Cells of the E l 4 cohort have largely completed their migrations to come to rest at the interface of the CP with the ML (Fig. 4A). If 8 h is assumed to represent the S p h a s e - G 1 phase interval of the mitotic cell population of the ventricular zone at El726'2s, the postmitotic cell to initiate its migration immediately upon entering G1 phase and the average length of the migratory excursion from the ventricular zone to the molecular layer in the dorsal pallial cortical sector be accepted as 550/~m, the overall rate of cell migration at E l 7 may be estimated to be 10.5 # m per hour for cells at the vanguard of the E14.5 cohort. The apparent rate of ascent across the intermediate zone is relatively slower at 10/,m per hour; that across the 150 # m wide cortical plate relatively more rapid at 12.5/~m per hour. Eventually the n e u r o n s of these 3 cohorts come to occupy complementary, overlapping positions within the adult cortex (Fig. 4C). The earliest formed of these, the E14 cohort, comes to occupy preferentially layers V-IV. The E14.5 and E l 5 cohorts, following the classic 'insideout' pattern 8'23, occupy preferentially layers I V - I I I and I I I - I I , respectively.

Migrating and postmigrating cells: cytology and distribution Migrating cell. Cells marked autoradiographicaUy by incorporation of [3H]thymidine injected in the interval E I 4 - E 1 5 and located within the IZ. SP and lower levels

41 o f t h e C P in t h e i n t e r v a l E 1 6 - E 1 7 m a y be c o n f i d e n t l y

and bipolar. E a c h has a b r o a d l y t a p e r e d ' l e a d i n g ' process

i d e n t i f i e d as m i g r a t i n g cells (Figs. 5 a n d 6). U n i f o r m l y ,

which ascends radially f r o m t h e s u p e r i o r p o l e . A s o m e -

t h e s e cells w e r e o b s e r v e d to be small, radially e l o n g a t e d

w h a t t h i n n e r a n d short ' t r a i l i n g ' p r o c e s s d e s c e n d s f r o m

CP

$1 a

Fig. 1. Cerebral wall of E17 embryo exposed to [3H]thymidine on E14.5. Toluidine blue-stained autoradiograms of coronal 2-/~m plastic sections. A: labeled cells, assumed to be migrating neurons of the E14.5 cohort, are distributed throughout the IZ, the SP and the CP. They are most numerous in the IZ where they are found in clusters located between interlacing dense axonal bundles. In the CP and the SP the labeled neurons are disposed more homogenously. B,C: upper and lower half of the developing cortex, respectively, at higher magnification. The majority of autoradiographically labeled neurons are relatively small with radially aligned, eliptical nuclei which contain abundant chromatin. Groups of 2-3 of these (*) may appear to be radially entrained. Exceptionally, labeled cells are observed at the outer margin of the CP which have begun to acquire a more differentiated appearance with larger, more circular nuclei staining less densely for chromatin (arrows). Unlabeled relatively differentiated neurons are abundant at subjacent levels of the cortex. Fascicles of radial fibers stained with Toluidine blue are readily recognized at the level of the SP (arrowheads). Bars = 20 pm in A-C.

42

Fig. 2. Intermediate zone of an E l 6 embryo exposed to [3H]thymidine on EI4.5. Toluidine blue-stained autoradiogram of coronal 2-urn plastic section. Across the ESS at the outer IZ, numerous labeled neurons are aligned in tangential arrays so as to produce a "ripple pattern' along the axon bundles (*). Groups of small, radially elongated and bipolar migrating neurons (arrowheads) are distributed along fascicles of radial fibers. VZ, ventricular zone; SP, subplate. Bar = 20 1~m.

the inferior pole of the cell. The cytologic characteristics of autoradiographically marked migrating cells are essentially those cited in classic electron microscopic descriptions 5,34-37. The autoradiographically defined El4 and E14.5 neuronal cohorts constitute at least 60% of the total cell population within the IZ (Fig. 2) and an even higher proportion of the cell population of the SP on El6 and El7, respectively. On these days migrating cells of these cohorts are a much smaller fraction, no more than 10-15%, of the total cell population of the lower CP. Because the autoradiographic method underestimates the number of neurons in a cohort, these fractions are only approximate and certainly conservative. Postmigratory cells. The neurons migrating upward from the IZ through the SP and lower CP are intermin-

gled at these latter levels with postmigratory neurons. The cytologic features of the neurons identified autoradiographically as migrating cells are substantially different from those of cells presumed to be postmigratory (Figs. 5 and 6). The latter, uniformly unlabeled in autoradiographs, appear to be more differentiated than migrating cells in that they are larger with a more globular somatic and nuclear contour. The more electronlucent cytoplasm of postmigratory cells is tess densely packed with organelles and the nuclear chromatin is less dense than in migrating forms. Particularly in the outer half o f CP, the somata of postmigratory pyriform cells are aligned in columnar fashion with ascending apical dendrites and axons concentrated in the interstices between the columns 7. A columnar pattern of grouping is less apparent in the lower

Fig. 3. Intermediate zone of the cerebral wall of an E17 embryo. Tangential plane of section. A: light micrograph of autoradiogram of a 2-/~m plastic section; [3H]thymidine injected at E14.5. Neurons are clustered in intervals between the interlacing dense axonal bundles (ax). A large proportion of the nuclei are autoradiographically labeled. B-C: electron mierographs of neuronal and glial cells. The circular to elliptical profiles of transected radial glial fibers (*) contain glycogen granules. The fibers are relatively electron lucent and contain few membranous organelles or cytoskeletal elements. C is a higher magnification of the area outlined in B. Glycogen particles are readily visible within the radial glial fibers. Bars = 20/~m in A; 2/~m in B-C.

43

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Radial glial fiber system: cytology and organization Glycogen granules, selectively and intensely stained by postfixation with reduced o s m i u m 6 J 2 followed by impregnation with U-Pb or PA-TCH-SP 44, are readily recognized at the electron microscopic level of resolution. The stained particles are uniformly distributed throughout the full extent of the radial processes of these cells (Figs. 3, 5-7, 9-12) with the consequence that the glycogen stain provides a sensitive means of high resolution analysis of the cytology and organization of the glial fiber system within the embryonic cerebral wall 4'

and rare microtubules

5 . (~ ;ffld

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12)

20,21,32

The tangential plane of the cerebral wall considered, 'unit' of fibers (single fibers or fascicles of 2-8 fibers) are distributed through the tissue separated at intervals of 8-10 Mm. This pattern of fiber unit is relatively uniform at E16-E17, whether at subcortical or cortical levels of the cerebrum (Fig. 7). At subcortical levels the majority of fiber units are multi-fiber fascicles (Figs. 3 and 7). At

17,27

The ascending processes of the system are circular to slightly eliptical in outline and of 1-2 Mm average diameter. In addition to the stained glycogen granules, the profiles usually contain small clear vesicles, abundant

A

cisternae

Within the IZ and the S P a few glial fibers are larger a n d contain mostly abundant small clear vesicles as observed in growth cones (Fig. 3). A few glial fibers terminate also on capillaries. The pattern of organization of the fiber system in the developing murine cerebrum, as inferred from the present electron micrographs, accords entirely with that of more comprehensive light microscopic analyses where the fibers have been stained with the selective and sensitive monoclonal antibody, RC2 TM

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45

Fig. 5. Relationship of radial glial fibers to migrating neurons of the lower CP, marked by [3H]thymidine injection at El4, in an El6 embryo. Coronal plane of section. A: neurons confidently identified as migrating neurons autoradiographically and by cytologic criteria (N1, N2) are closely apposed to glial fibers (RGF 1-4) grouped in small fascicles. B,C: contacts between apposed neuron-glial fiber surfaces are illustrated at higher magnification. Glycogen particles (arrows) within the radial fibers are readily visible at the higher magnification. Bars = 1/~m in A-C.

46 El6, with ascent through the ESS into the SP and upward through the CP, there is sharp reduction in the proportion and fiber composition of fascicular units and an apparent corresponding reduction in the overall tissue concentration of fibers (Fig. 7). This trend is progressive such that by E l 6 and later, fascicles crossing the SP are reduced to 2-5 fibers and only single fibers ascend through the upper CP (Figs. 6, 7 and 10).

Interrelationship of neurons and the glial fiber system Migrating neurons in relation to radial glial fibers. The

Fig. 6. Relationship of radial glial fibers to migrating neurons of the CP, marked by [3H]thymidine injection at E14.5, in an E17 embryo. Coronal plane of section. Two labeled cells (N1, N2), with cytologic features of migrating neurons, sectioned parallel to their long axis, are in contact along most of their length with the same single glial fiber (RGF) readily identified by its glycogen content (arrows). Bar = l~m.

consistent generalization to obtain from the uttrastructural analysis is that neurons affirmed to be migrating cells by both autoradiographic and cytological criteria, are closely applied to the surface of one or more radial glial fibers (Fig. 8). In addition the fibers appear to ascend only through tissue zones where neurons are migrating. Through the 1Z (Fig. 3) where glial fiber density is greatest, the number of fibers contacted by a migrating cell is great. Within the dense crowd of fascicles of the IZ, neurons were observed which contacted fibers of one fascicle with its soma but the fibers of another fascicle with its leading process. This p h e n o m e n o n is consistent with neuron-glial interrelationships reconstructed in the earlier study of Rakic et al. 37. Furthermore the fibers span only tissue compartments between axonal fascicles where migrating cells are located and do not cross the septa of axon fascicles between these compartments where migrating neurons are not present (Fig. 3). At the upper level of the IZ, where migrating cells are apposed to glial fibers which do interdigitate with the axon bundles in the ESS, there is a tendency for substantial numbers of migrating neurons to be aligned in a tangential array so as to produce a 'ripple pattern' along the lower margin of axon bundles (Figs. 1A and 2). Through the SP and CP where the density of fibers is much less, the number of fibers in contact with a given migrating cell decreases substantially. At E 1 6 - E I 7 . within the SP (Fig. 9) migrating neurons, usually grouped in small clusters, contacted one R G F or multiple fibers of a fascicle of 2-5 fibers. Within the CP at E l 6 (Figs. 5 and 10A) and the lower CP at E l 7 (Fig. 10B), migrating cells are also unequivocally identified to be apposed or within their respective glial fascicle. In the upper half of the cortical plate at E l 7 (Figs. 6 and 11), invaded only with single glial fibers, migrating cells contacted one or two gliai fibers. Frequently the same glial fiber is contacted with two adjacent migrating neurons. Ninety to 95% of migrating neurons contacted glial fibers through the SP and lower CP at E16-17 (Fig. 8). Only in the outermost level of the CP were autoradiographically labeled neurons encountered which had no

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Fig. 7. Variation in density and differential patterns of distribution of radial glial fibers of the IZ (A), SP (B), lower CP (C) and upper CP (D) as revealed in the tangential plane of section through the cerebral wall of El6 embryos. The outlines of both neuronal somata and radial fibers are represented in the drawing in A, only the outline of radial fibers are represented in the drawings in B-D. All glycogen-marked radial fibers are represented so that each drawing is a quantitative map. Each map corresponds to an area of 1000/~m2. The set of analyses illustrate a progressive reduction in the number (N) and fiber content of glial fiber fascicles with centrifugal progression through the strata of the cerebral wall. Single fibers occur in isolation within the CP.

demonstrable contact with the surface of at least one radial glial fiber. In the interval E 1 6 - E 1 7 the proportion of such neurons encountered in the upper part of the CP was still no more than 20% and the majority of these were found to be separated from nearby glial fibers by only one or two neuronal processes (Fig. 8). The cytologic interrelationship of migrating cells and radial glial fibers has been considered in closest detail at the levels of the SP and the lower CP at E l 6 embryos (Fig. 12). For this purpose, our analysis is based upon reconstruction of neuron-glial relations from electron micrographs of tissue sectioned serially in coronal plane. The serial reconstructions have been limited to neurons

affirmed to be migrating cells by cytological criteria. The somata and leading process of each migrating cell contacts one fiber or multiple fibers of the same fascicle, More than one migrating neuron in close relation to each other are entrapped into glial fascicle and may contact adjacent portions of the surface of the same glial fiber, Characteristically the fiber or fibers, in contact with the somata of a migrating cell are enclosed within a cytoplasmic invagination or are 'embraced' by cytoplasmic expansions. Contact with the trailing process, in contrast, is limited to close apposition rather than invagination and apposition may be interrupted over short segments. The number of fibers in contact with a given migrating cell

48 decreases substantially with advancing embryonic age and with ascent through the SP (Fig. 12I) and into the CP (Fig. 12J,K) where the density and grouping of fibers is much less.

Postmigratory neurons in relation to radial glial fibers. The postmigratory neurons of the CP and SP, in contrast to the migrating cells, bear no consistent relationship to glial fibers (Fig. 8). In particular, less than 50% of those of the CP and SP were demonstrated to have direct contact with radial glial fibers.

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DISCUSSION All migrating cells, defined in this analysis by autoradiographic and cytologic criteria, were observed to contact one or more radial glial fibers. Fherc is close contact between apposed surfaces of migrating cell and glial fiber which extends the full radial length of the migrating cell. The invariant association with glial fibers is not similarly characteristic of postmigratory cells or even of predifferentiated young neurons at the completion of their migrations at the C P - M L interface. For postmigratory cells contact with glial fibers appeared to be only incidental to proximity. Through the cortical strata, only some 30-50% of these latter two populations have demonstrable contacts with fibers, and the contacts are of only minimal extent on the surfaces of the neurons. Within the IZ, the glial density is great and all neurons, both migratory and post-migratory, are bordered by glial fibers. However, glial fibers in close apposition to migrating cells are associated in the same tissue compartments. Neither glial fibers nor migrating cells ascend independently through the axonal bundles which form the septa between these compartments. This set of observations, consistent with those of other high resolution studies 34 37 lend support to the hypothesis that cell migration is guided specifically by contact with the surfaces of radial glial cells.

u p p e r CP

Varied patterns of neuron-glial fiber interrelationship

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lower CP

upper CP

Fig. 8. Histograms representing the proportion of migrating and non-migrating neurons which directly contact one or more radial glial cells in the separate strata of the cerebral walt of E16 (A) and E17 (B) embryos. The determinations were made from ultramicrographs of autoradiograms from animals injected with [SH]thymidine on E14 (A) and E14.5 (B). Each histogram is based upon an analysis of 100 cells from each of 3 separate animals. Series 1 (open bars) and 2 (hatched bars) correspond to the mean proportion in the 3 samples of labeled and non-labeled neurons, respectively, in direct contact with one or more glial fibers. Series 3 (dotted bars) and 4 (dark bars) correspond respectively, to unlabeled and labeled neurons without demonstrated contact with a gliai fiber.

The pattern of arrangement of glial fibers changes with ascent through the successive strata of the cerebral wall, and there are corresponding changes in the interrelationship of migrating cells with the fiber system. Fiber density is maximum across the I Z where fibers are essentially all grouped in fascicles. The fascicles virtually saturate the interstices between radial streams of ascending cells with the consequence that migrating cells are in intimate contact with multiple fascicles and multiple fibers forming the external contour of each of these fascicles. The pattern changes abruptly at the level of the ESS in transition to the SP as'2°. There is a step down in the density of fiber fascicles and a progressive replacement of fascicles by single fibers with ascent through the SP and CP. By E17, only single fibers span the upper CP. There is a correlated change in the relation of migrating cells to the fiber system at this level of the cerebral wall. Specifically, the neurons are observed to associate with a single fascicle or only a single fiber. Where the association is with a fascicle, the migrating neuron is observed to penetrate among the fibers which constitute the fascicle. Where the association is with a single fiber, and particularly at the level of the CP, the cell makes contact with a single fiber only.

49

Fig. 9. A migrating neuron within the SP of an El7 embryo marked by [3H]thymidine injection on E14.5. Tangential plane of section. The neuron has direct contact with a fascicle of 3 glycogen-marked radial glial fibers (RGF1, RGF2, RGF3) on one side but a single radial fiber (RGF4) on the opposite side. RGF4 may have been separated from other fibers of the fascicle by the passage of the migrating neuron. Bar = 1/~m.

Not infrequently within the cortical plate the paired fiber and migrating cell are observed to be completely enclosed within a dendritic bundle. Pattern changes at the ESS-SP zone of transition The transition from the fasciculated to the single fiber pattern of arrangement of glial fibers in ascent through the lower cortical strata appears to be the result of two distinct cellular mechanisms: a surging extension of fibers through the cortical strata from the I Z occurring concurrently at E16-E17 with a decomposition of fascicles into single fibers 17'2°. The observed penetration of migrating cells among the fibers of glial fascicles suggests the possibility that this is the mechanism by which the fascicles are broken apart. That could also explain the partial glial defasciculation in reeler neocortex 17'19 where late generated neurons do not complete their migration 24" 35. Fascicle decomposition is initiated at the ESS-SP zone

of transition but not, apparently, in the I Z below the ESS. The decomposition phenomenon implies a drop in the strength of adhesivity of physical-chemical interglial fiber bonds. Putative molecular mechanisms which might underly such physical-chemical changes include, on the one hand, variations in the strffcture or density of specific glial homophilic cell adhesion molecules 14'29'4°'41 and the elaboration/activation of serine proteases by the migrating cell itself33 on the other. Correlated phenomena at the ESS-SP zone of transition Multiple phenomena reflect the transitional character of the ESS-cortical interface. Changes in the patterns of arrangement of the glial fibers and the interrelationship of migrating cells to the fibers are cited in the foregoing paragraphs. Another analysis of the radial fiber system of the developing cerebral wall zl, a quantitative immunohistochemical analysis, provides evidence that large

50

Fig. 10. Relationship of radial glial fibers to migrating neurons of the upper CP at E l 6 (A) and of the lower CP at El7 (B) after l~H]thymidine injection at E l 4 and E14.5 respectively. Tangential plane of section. Radial fibers ascend singly or gather in small fascicles of 2~-3 fibers. Labeled, presumably migrating neurons contact multiple glial fibers (RGF). One of these fibers (A) is in contact with two adjacent migraling neurons. Bars = 1 p m in A and B.

51

Fig. 11. Relationship of radial glial fibers (RGF 1-3) to migrating neurons (N1, N2) of the upper CP, marked by [3H]thymidine injection at E14.5, in an E17 embryo. Tangential plane of section. A: autoradiographically labeled migrating neurons (N1, N2) contact one or two glial fibers (RGF 1,2). The same glial fiber (RGF 1) may contact two adjacent migrating neurons. Glial fibers (RGF) ascend only singly. B,C: higher magnification of RGF1 and RGF2 of Fig. A, marked by the presence of glycogen particles (arrows), which have intimate contacts with adjacent migrating neuron. Bars = 1/zm in A-C.

52

Fig. 12. A - D . (For legend, see page 55.)

53

J

i._._.t

G Fig. 12. E - H . (For legend, see page 55.)

-"

H

54

~iiii

J Fig. 12. I-K. (For legend, see page 55.)

i(

55

Fig. 12. Schematic representation of the intimate relationships between migrating neurons (N1-5) and the glial fibers (arrowheads) in the SP and lower part of the CP of an El6 embryo. The neuron and glial elements are reconstructed from micrographs of a series of sections cut in the coronal plane. Much of the radial extent of the neuron N3 of the lower CP is presented at relatively low magnification in A,B,E. Glycogen particles (arrows) are readily seen at higher magnification to mark adjacent radial glial fibers (arrowheads) in corresponding representative fields in C,D,F. The cells and fibers were drawn with the aid of superimposed outlines. The migrating cell (N3) and the ensheathing set of radial glial fibers are presented in separate reconstructions in G and H, respectively. It is 'reassembled' from the perspective of exterior and interior views of the glial fascicle in J. Schematic representation of other migrating neurons in the SP and CP are also represented in I and K respectively. During their migration, the neurons are entrapped into the glial fascicles and realized with their glial guides, an extensive and close relationship. The intrafascicular penetration of the leading process and the somata, contribute to the progressive isolation of the glial fibers. Into the CP (J,K), the grouping and density of glial fibers is much less than into the SP (I) and the number of fibers in contact with a given migrating cell decreases substantially. Bars = 2 pm in A-J.

numbers of radial fibers are 'gated' at the ESS zone of transition. For some 25-40% of fibers extending through

cerebral wall 1'10'14'3°'38'41. The transitional architectural

the I Z , further extension into the cortical strata appears

and, perhaps physical-chemical, e n v i r o n m e n t of the E S S - S P interface 13'43 appear to impose dramatic changes

to be p r e v e n t e d or delayed until E l 6 and later.

upon the migratory behavior of the cell.

O t h e r observations suggest that the transitional properties of this zone affect significantly the facility of cell

The E S S - S P

migration. A m o n g these is the 'ripple' pattern of tangen-

opment

tial arrays of cells, apparently 'gated' briefly along the i n n e r margins of the axon fascicles that form the ESS. With ascent through the ESS, on the other hand, there appears to be an acceleration in the rate of cell migration. Rates estimated here are 10 p m per hour below but 12.5

It is arresting that the E S S - S P strata interface appears to gate the ascent of migrating n e u r o n s in a spectrum of genetically and non-genetically d e t e r m i n e d disorders of neocortical histogenesis 9A6'39. In certain specimens of the h u m a n disorders 9'16'22'39'42 and as a result of the reeler

p m per hour above the ESS. (The error of these calculations is uncertain and, of course, may be substantial because of the uncertain validity of f u n d a m e n t a l assumptions and the considerable potential for error of m e a s u r e m e n t . In relation to similar calculations for n e u r o n a l migration the values are, however, closely in accord with calculated rates of n e u r o n a l migration in developing rhesus m o n k e y cerebral wall 34'39 and of

m u t a t i o n in mice 7'9'24'35, for example, large n u m b e r s of

granule cells along glial cables in microwell cultures15). The principal inference to be made from this set of observations at this time, in the absence of critical experimental analysis, is that a heterogeneous succession of c e l l u l a r - m o l e c u l a r interactions must serve n e u r o n migration as the cell traverses the various strata of the

Acknowledgements. The authors thank Prof. G. Lyon, Dr. A.M. Goffinet, J.P. Misson, and Ph. de Saint Georges for their stimulating discussions. Mrs. A. Gonzalez-Gentille and Mrs. Claes Hermine provided valuable help during this work. This work was supported by the Action Concertre Gouvernementale (Belgium), Foundation Reine Elisabeth, Foundation Madame A. Froelich, EN.R.S., ER.S.M. number 3.454081, Foundation Marie-Christine (LEG.), NATO Grants (J.EG.) and NS 12005 (V.S.C.).

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