Development 112, 581-590 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

581

Rodent CNS neuroblasts exhibit both perpendicular and parallel contact

guidance on the aligned parallel neurite bundle

ISAO NAGATA1 and NORIO NAKATSUJI2

'Department of Genetics, Tokyo Metropolitan Institute for Neurosciences, 2-6 Musashidai, Fuchu, Tokyo 183, Japan2Division of Developmental Biology, Meiji Institute of Health Science, 540 Naruda, Odawara 250, Japan

Summary

Mouse cerebellar granule cells showed two types ofmigration behavior in microexplant cultures. They firstmigrated along their neurites, showing the typicalcontact guidance, and then oriented themselves at rightangles to the parallel neurites, thus exhibiting the'perpendicular contact guidance' (Nakatsuji, N. andNagata, I. 1989 Development, 106, 441-447).

To study whether other neurons have the capacity toshow similar 'perpendicular contact guidance', wecultured dissociated neuroblasts from various parts ofCNS or PNS on parallel neurite bundles. The PNSneuroblasts always extended their processes parallel to

the neurite bundle. In contrast, almost all kinds of CNSneuroblasts tested oriented their processes both perpen-dicular and parallel to the neurite bundles that were allfree of glia. Time-lapse video recording revealed thatneuroblasts migrated in both directions. Thus, CNSneuroblasts possess the capacity to migrate and extendtheir processes at right angles to the substratum ofheterotypic neurite bundles, which may play an import-ant role in histogenesis of the CNS during development.

Key words: contact guidance, perpendicular orientation,migration, CNS neuroblasts, neuron-neuron interaction.

Introduction

In the neonatal cerebellum, granule cells migratevertically from the external granular layer to theinternal granular layer by passing through their parallelaxon bundles (Fujita, 1967; Altman, 1972). Thismigration is believed to be guided by Bergmann glialcells (Sidman and Rakic, 1973). However, our recentstudy using microexplant cultures showed that thegranule cells have an inherent tendency to orient theirprocesses and migrate perpendicular to the alignedparallel neurite bundle (Nakatsuji and Nagata, 1989;Nagata and Nakatsuji, 1990). In another cerebellarculture study, Hekmat etal. (1989) showed that GABA-uptaking inhibitory neurons orientate perpendicular tothe parallel neurites. Perpendicular orientation andmigration by granule cells may have a role in theirvertical migration in situ.

To determine whether perpendicular contact guid-ance behavior is specific to cerebellar cells or is alsoshown by other neurons, we examined dissociatedneuroblasts from various regions of the central nervoussystem (CNS) or peripheral nervous system (PNS) byseeding them on the parallel neurite bundle that hadbeen formed by CNS or PNS microexplant cultures.Since orientation of the processes and migrationpatterns of granule cells were closely correlated witheach other in vitro (Nakatsuji and Nagata, 1989), theorientation of neuroblasts was examined systematically

by specific immunocytochemical staining after fixation.In addition, the migration pattern was analysed bytime-lapse video recording in some cases. Resultsshowed that almost all kinds of CNS neuroblasts testedextend their processes and/or migrate in both perpen-dicular and parallel directions, but not randomly, to theheterotypic parallel neurites. We also show thatneurons from the PNS always extend their processesparallel to the CNS or PNS neurite bundles. Prelimi-nary data have been reported previously (Nagata,1989).

Materials and methods

Preparation of single CNS or PNS cellsOlfactory bulbs, cerebral cortices, septa, striata, hypotha-lami, hippocampi, tecta, cerebella, medulla oblongata, spinalcords or adrenal glands were removed from embryonic,prenatal or neonatal mice (BALB/c) or rats (Wistar), atappropriate ages as described in Table 1. In some exper-iments, thick slices of the olfactory bulbs were prepared andpart of the inner cores of the slices, which were rich in granulecells, were isolated under a dissection microscope. Neuro-blasts were dissociated by the following method, except forcerebellar neurons, which were prepared according to themethod described by Nagata et al. (1986). After removing themeninges and pia mater from each tissue, they were treatedwith a trypsin solution (0.1-0.5 % in PBS, GIBCO) for 10 minat room temperature, washed with Tyrode's solution, and

582 /. Nagata and N. Nakatsuji

Table 1. Orientation of the neuroblast processes on the parallel neurite bundle

Substratum

Fraction (%)§

Neuroblastor neuron Animalt Stage Size* Parallel

Inter-mediate

Perpen-dicular

Cerebellum

Hippocampus

Septum

DRG

PL/laminin

Olfactory bulbCerebral cortexCerebellumSpinal cord

Olfactory bulbCerebral cortex

Striatum

SeptumHypothalamus

HippocampusTectumMedulla olbongataSpinal cord

DRGSuperior cervicalGanglionAdrenal gland

Olfactory bulb

Olfactory bulb

Olfactory bulbMedulla oblongata

Olfactory bulb

rrmm

rr

r

mm

rmrm

rm

r

r

r

rr

r

P0-P3E18-P0P3-P5E13-E15

E16-E17E12-E14

E13-E15

E15-E17E14-E16

E16-E18E18-E19E12-E14E12-E14

P0-P1P0-P3

E16-E18

P0-P3

P0-P3

P0-P3E12-E14

P0-P3

SmallSmallSmallSmall

LargeLarge

Large

LargeLarge

LargeLargeLargeLarge

Small

Small

SmallLarge

Small

14341815

1531

•2715

+ 10iy1017

•2015212927

9693

92

27

19

4694

35

Numberof

cellll

Numberof

process^

118813131421191613121610191810

36

6

22

18

115

33

t r , rat; m, mouse.t'small' means small neuroblasts having diameter 7-12/an. 'large' means large neuroblasts having diameter of 15-22 fan.§ Angles measured as shown in Fig. 1 were categorized into three fractions: 'parallel' (0g#

Contact guidance of CNS neurons 583

cortices, hippocampi or septa were dissected out of E(embryonic day) 16-17 mice, cut into small pieces, and placedon the substratum coated with poly-L-lysine and laminin(PL/laminin substratum) (Nagata and Nakatsuji, 1990).Dorsal root ganglia from E18 to E19 rats were cultured on thePL/laminin substratum. These explants were cultured in amodified serum-free defined medium (Fischer, 1982), fromwhich thyroxine (T4) was omitted and sodium pyruvate(1 mM) was added. For explant cultures of dorsal root ganglia,0.5;

584 /. Nagata and N. Nakatsuji

\

Fig. 3. Culture of olfactory bulb neuroblasts immunostainedfor GAB A (A) or MAP2 protein (B,C). The neuroblastswere dissociated from postnatal day-2 mouse olfactory bulband cultured on the PL/laminin substratum (A) for 5 daysor the mouse cerebellar neurite bundle for 3 days (B) or 7days (C). Double-headed arrows indicate the direction ofparallel neurite bundles. Bars=50,um.

B

/ ! • / .

Contact guidance of CNS neurons 585

processes and the neurite bundle were measured as shown inFig. 1.

Time-lapse video recordingMethods for time-lapse video recording and analysis of cellmovement were described by Nakatsuji and Nagata (1989).

Results

PNS neurons on neurite bundles of PNS or CNSmicroexplantsSensory neurons dissociated from rat cervical or dorsalroot ganglia and sympathetic neurons from superiorcervical ganglia, extended bipolar or multipolar longprocesses and made frequent branchings in manydirections on the PL/laminin substratum. In contrast,on parallel neurite bundles of cerebellar or other CNSmicroexplants, they showed a strong tendency toextend their processes along the neurite bundles.Fig. 2A shows large round sensory neurons extendingtheir processes. These processes expressed a160x10 Mr neurofilament protein immunoreactivityand appeared to fasciculate with each other. Thedistribution of angles between the processes and theparallel neurites showed two peaks at 0 and 180 degrees(Fig. 6A). Such strict parallel guidance was alsoobserved when dissociated sensory neurons werecultured on the neurites of dorsal root ganglia.

Adrenal medullar neurons, which were dissociatedfrom adrenal glands, extended several processesradially around the cell body on the PL/lamininsubstratum. On the parallel neurite bundles, thesecatecholaminergic neurons, which expressed tyrosinehydroxylase immunoreactivity, oriented their shortprocesses parallel to the cerebellar granule cell neurites(Fig. 2B, Table 1).

CNS neuroblasts on the CNS neuritesVarious kinds of dissociated neuroblasts from CNSextended cell processes in random directions on the PLor PL/laminin substratum (Figs 3A, 6B). In contrast,they extended their processes either perpendicular orparallel to the neurite bundles. Fig. 3B shows smallneuroblasts from a postnatal olfactory bulb cultured for3 days on the cerebellar neurite bundle. Most cellsextended short processes perpendicular to the parallelneurites. Their processes became longer after severaldays of culture (Fig. 3C). Fig. 6C shows a distributionhistogram of the angle between cell processes andneurite bundles. It has one prominently large peak at 90degrees and two smaller peaks at 0 and 180 degrees.These neuroblasts also showed perpendicular orien-tation to the parallel neurite bundles derived from thehippocampus (Fig. 6D) or septum microexplants(Table 1).

The immature cerebellar granule cells extended theirbipolar process first along the cerebellar or otherparallel neurites of CNS, thus exhibiting typical contactguidance (not shown), and then changed their orien-tation by extending perpendicular short thick processes(Fig. 4). Such change of behavior of the cerebellar

Fig. 4. Cerebellar neuroblasts cultured on the cerebellarparallel neurite bundle, immunostained for MAP2 protein.The neuroblasts were dissociated from postnatal day-4mouse cerebellum and cultured on the mouse cerebellarneurite bundle for 5 days. A double-headed arrowindicates the direction of the neurite bundle. Bar=50/^m.

neuroblasts was similar to that described previously(Nagata and Nakatsuji, 1990). The distribution of theangle of the latter processes showed a large peak at 90degrees and smaller peaks at 0 and 180 degrees(Fig. 6E).

Large neuroblasts isolated from many regions of thebrain such as olfactory bulb, cerebral cortex, septum,striatum, hypothalamus, hippocampus, tectum,medulla oblongata or spinal cord at earlier develop-mental stages (embryonic 12-18 day) extended bothlong and fine axon-like processes and short thickdendrite-like ones on the cerebellar neurite bundles.Most of the long fine processes, which did not expressMAP2 protein, oriented perpendicular or parallel tothe parallel neurite bundle. A small number of the longprocesses took oblique orientations. Fig. 5A-C showslarge neuroblasts from the septum, cerebral cortex ormedulla oblongata, extending their axon-like longprocesses (arrowheads) on the cerebellar neurite

586 /. Nagata and N. Nakatsuji

Fig. 5. Septal (A), cerebral cortical (B), or medulla oblogata (C) neuroblasts cultured on the mouse cerebellar neuritebundle, immunostained for MAP2 protein. The neuroblasts were dissociated from E15 mouse septum, E14 rattelencephalic vesicle, or E14 rat medulla oblongata, and cultured for 5 days. Double-headed arrows indicate the directionof parallel neurite bundles. Arrowheads and arrows indicate axon-like long processes and dendrite-like short processes,respectively. Bars=50,um.

bundle. The distribution of the angles of theseprocesses showed three distinct peaks; 90, 0 and 180degrees (Fig. 6F,G). Dendrite-like short processes ofthese cells, which expressed MAP2 protein, alsoshowed similar orientation (arrows in Fig. 5A-C, starsin Table 1).

CNS neuroblasts on PNS neuritesExplants of the dorsal root ganglion produced roughlyparallel neurite bundles on the PL/laminin substratum,but typical distances between neurites (5-20/xm) werewider than that (1-3 ^m) of the cerebellar granule cellneurite bundles. CNS neuroblasts showed low affinityto the PNS neurites (as described by Kapfhammer andRaper, 1987), and growth of the neuritic process wasslow. We managed to make some of the CNSneuroblasts grow on the neurites of sensory ganglia withthe aid of the conditioned medium described in theMaterials and methods section. Such neuroblasts fromthe early postnatal rat olfactory bulb showed both theperpendicular and parallel orientation, but moreshowed the parallel orientation than the perpendicularone (Fig. 6H, Table 1). Long processes of largeneuroblasts from hippocampus or medulla oblongatagrew preferentially along the parallel neurites of

sensory ganglia (Table 1). They did not show perpen-dicular orientation.

Neuroblast motility on neuritesObservations described above showed that CNS neuro-blasts extended their processes in both perpendicularand parallel orientations to the parallel neurite bundles.But such static observations do not give any infor-mation about whether there are two groups ofneuroblasts each of which extends processes only ineither perpendicular or parallel orientation, or whetheran individual neuroblast can extend a process in bothperpendicular and parallel orientations by changingdirections from time to time. To answer this questionand also to analyze cell migration patterns, weexamined movement of neuroblasts isolated from theCNS or dorsal root ganglion on cerebellar granule cellneurites by using a time-lapse video recording system.

Small neuroblasts from the inner part of postnatalolfactory bulb moved actively on the cerebellar neuritebundle by taking on a bipolar shape and extending shortprocesses. They migrated in both perpendicular andparallel paths relative to the neurite bundle by changingdirection from time to time (Fig. 7A). The neuronalprocess in front of moving neuroblasts appeared to

0 R C / C E R E B E . OLF . B . / L A M I N I N O I F B / C E R E B E .

S E P T U M / C E H E B E O L F B. / D R 6

Contact guidance of CNS neurons 587

O L F B. / H I P P O

Fig. 6. Histograms showingdistribution of angles made bythe neuroblast process and theparallel neurite bundle.Neurons from sensory ganglia(DRG) were cultured on themouse cerebellar neuritebundle (A). Neuroblasts frompostnatal 0- and 3-day mouseolfactory bulbs (OLF.B)(B,C,D,H), postnatal 3- to 5-day old mouse cerebellum (E,CEREBE.) or E12-14 ratcerebral cortex (F, CEREB.)or E15-17 mouse septum (G,SEPTUM) were cultured onthe neurite bundle of themouse cerebellar (A,C,E,F,G),hippocampal (HIPPO.) (D) ordorsal root ganglional (H)microexplants, or on thelaminin substratum withoutneurite bundles (B).

determine the direction of movement by contact withthe parallel neurite bundle.

The large neuroblasts from earlier stages of theembryonic cerebral cortex also migrated actively on theneurite bundle until they extended long axon-likeprocess. They underwent both perpendicular andparallel translocation relative to the neurite bundle, bychanging directions, although they moved more fre-quently in the parallel direction than the smallneuroblasts from the olfactory bulb (Fig. 7B). Fig. 8shows one of the cerebral cortical neuroblasts thatchanged its direction of movement from parallel toperpendicular and again to parallel orientation relativeto the neurite bundle.

Sensory neurons from the dorsal root ganglion didnot translocate on the neurite bundle, but extendedlong processes. Their orientation was always parallel tothe neurite bundle, and they never extended theirprocess in a perpendicular direction.

Discussion

Many studies have shown that neurons extend their

processes along the glial cell, neurites of other neuronsor even artificial micro-structures, and migrate alongthese oriented structures in vivo (Sidman and Rakic,1973; Singer etal. 1979; Silver and Sidman, 1980; Hynesetal. 1986) and in vitro (Gundersen, 1987; Hirono etal.1988; Kleinfeld et al. 1988; Hatten, 1990). Contactguidance of the growth cone plays an important role insuch pathfinding and migration (Letourneu, 1975,1979;Dodd and Jessel, 1988). Most of the previous studies invitro, however, used PNS neurons such as sensory orsympathetic neurons. We recently found that cerebellargranule cells have a strong tendency to orient andmigrate perpendicular to the aligned parallel neuritebundle in mouse cerebellar microexplant cultures, andnamed such behavior as 'perpendicular contact guid-ance' (Nakatsuji and Nagata, 1989). In the presentstudy, we showed that almost all kinds of neuroblastsfrom CNS oriented their processes and migrated in bothperpendicular and parallel orientations on the neuritebundles of CNS or PNS in a heterotypic culture system.We also confirmed that neuroblasts from PNS showedconventional contact guidance but no perpendicularorientation. Thus, it is possible that neuroblasts of CNSdiffer from those of PNS in the regulation of cellmigration and pathfinding of the neuritic process.

588 /. Nagata and N. Nakatsuji

Fig. 7. Trajectories of small neuroblasts from the postnatalday-1 rat olfactory bulb (A) or large neuroblasts from theE13 rat cerebral cortex (B) on the cerebellar neuritebundle. They were made by marking the approximatecenter of the cell body at every hour and connecting themwith straight lines. They indicate movement for 20 h duringthe 2nd and 3rd days of culture. Solid circles indicatestarting points. Broken lines indicate orientation of theneurite bundles. Bar=100/zm.

Conditions for perpendicular orientation andmigrationThe perpendicular orientation and migration of cellseem to occur only when the following three conditionsare satisfied. (1) Cells should be immature neurons orneuroblasts. Neither GFA-protein-immunoreactiveastrocytes, muscle cells, Schwann cells nor neuro-2aneuroblastoma cells showed any perpendicular orien-tation or migration (unpublished observation). Thesecells always oriented parallel to the neurite bundle.(2) The immature neuron has to be derived from theCNS, but not from the PNS. Neuroblasts from the CNSsuch as olfactory bulb, cerebral cortex, septum,striatum, hypothalamus, hippocampus, tectum, brainstem or spinal cord displayed the perpendicularorientation as well as the parallel orientation. Ganglionor adrenal medullar PNS neurons, which originate fromthe neural crest, did not show the perpendicularorientation. Thus, origin of cells, such as from theneuroepithelium or neural crest, may be important forsuch a difference. (3) Tightly aligned neurite bundles ofthe CNS or PNS are necessary as the substratum.

Neuroblasts showed no perpendicular orientation whenneurites were not aligned, or when there were widedistances between neurites.

Mechanism of perpendicular contact guidanceThe mechanism of the perpendicular orientation andmigration is not known, but there are several lines ofevidence for speculation. This orientation is probablycaused by contact of motile filopodia, extending fromthe growth cone and/or from the shaft of the process, tothe parallel neurite bundles, as described previously(Nakatsuji and Nagata, 1989). Interaction of thefilopodia and neurite bundles could be brought aboutby recognition of either the physical surface structure ormore specific surface molecules such as the celladhesion molecules. The former possibility wouldpredict that artificial surface structures similar to thefine neurite bundles should cause similar perpendicularorientation. The latter possibility would suggest in-volvement of the cadherins, N-CAM or LI. We arecurrently testing these possibilities.

Perpendicular contact guidance and CNS developmentFew investigators have noted that immature neuronsmigrate and/or orient their soma or processes perpen-dicular to non-myelinated aligned neurite bundles.Cerebellum, olfactory bulb, cerebral cortex, hippocam-pus and spinal cord have such cyto-architecturesuggesting perpendicular migration or orientation invivo.

The cerebellum is one such region, in which one canrecognize mutually perpendicular orientation involvingfour kinds of neurons: parallel fibers of the granule cellsare oriented perpendicular to the dendrites of Purkinjecells, axons of the basket cells, stellate cells and granulecells (Palay and Chan-Palay, 1974). In the olfactorybulb, the granule cells migrate parallel to the pialsurface in the subependymal zone and then turnvertically (Kishi, 1987). In the cerebral cortex forma-tion, bipolar neuroblasts seem to rotate by 90 degrees atleast once in the intermediate zone (Stensaas, 1976;Altman and Bayer, 1990) or in the ventricular zone(Walsh and Cepko, 1988) during the migration alongradial fibers.

In our culture system, small neuroblasts such as thosefrom cerebellum or olfactory bulb showed the perpen-dicular orientation at high frequencies. Larger neuronsalso extended long or short processes in the perpendicu-lar orientation, but at various frequencies depending onthe origin of neurons. Thus, the perpendicular contactguidance seems to be a common feature of the CNSneurons, but not of the PNS neurons. It may playimportant roles in histogenesis and network formationof the CNS.

This study was supported, in part, by a grant (02804066)from the Minister of the Department of Science andEducation of the Japanese Government to I.N. We thank DrH. Shimazu for critical reading of the manuscript.

Contact guidance of CNS neurons 589

Fig. 8. Photographic prints made from the time-lapse video recording of the cerebral cortical neuroblasts cultured on thecerebellar neurite bundle shown in Fig. 7B. A neuroblast (star) changed its direction twice during the migration; fromparallel (A-B), then perpendicular (C-D) and again to parallel (E-F) directions. An arrow in C indicates a leadingprocess that grew in advance of the perpendicular movement in its direction. Time is shown in the upper-right corner ofeach print in hours and minutes. Double-headed arrows indicate the direction of the neurite bundle. Bar=50,um.

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