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3501Development 124, 000-000 (1997)Printed in Great Britain © The Company of Biologists Limited 1997DEV8429

Role of GGF/neuregulin signaling in interactions between migrating neurons

and radial glia in the developing cerebral cortex

E. S. Anton1,*, M. A. Marchionni2, K-F. Lee3 and P. Rakic1,* 1Section of Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06510-8001, USA2Cambridge NeuroScience Inc., 1 Kendall square, Bldg. 700, Cambridge, MA 02139, USA3The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 N. Torrey Pines Rd, La Jolla, CA 92037, USA

*Authors for correspondence (e-mail: [email protected] OR [email protected])

During neuronal migration to the developing cerebralcortex, neurons regulate radial glial cell function and radialglial cells, in turn, support neuronal cell migration anddifferentiation. To study how migrating neurons and radialglial cells influence each others’ function in the developingcerebral cortex, we examined the role of glial growth factor(a soluble form of neuregulin), in neuron-radial glial inter-actions. Here, we show that GGF is expressed by migratingcortical neurons and promotes their migration along radialglial fibers. Concurrently, GGF also promotes the main-tenance and elongation of radial glial cells, which areessential for guiding neuronal migration to the cortex. Inthe absence of GGF signaling via erbB2 receptors, radial

glial development is abnormal. Furthermore, GGF’s regu-lation of radial glial development is mediated in part bybrain lipid-binding protein (BLBP), a neuronally induced,radial glial molecule, previously shown to be essential forthe establishment and maintenance of radial glial fibersystem. The ability of GGF to influence both neuronalmigration and radial glial development in a mutuallydependent manner suggests that it functions as a mediatorof interactions between migrating neurons and radial glialcells in the developing cerebral cortex.

Key words: GGF, migration, glia, neuregulin, cerebral cortex, neuron

SUMMARY

INTRODUCTION

In the developing telencephalon, neurons migrate from theirsite of origin in the ventricular zone to their final position inthe cortical plate, along elongated processes of radial glial cellsthat span the cerebral wall from the ventricular surface to thepial surface (Rakic, 1971, 1972, 1978). Over the course ofembryonic development, the cerebral wall expands several foldand, consequently, the length of radial glial fibers, the distanceneurons have to migrate and the complexity of the environmentthey traverse, increases as well. During this period of neuronalmigration, radial glial cells do not divide (Schmechel andRakic, 1979a), but they do enter the mitotic cycle afterneuronal production ends (Schmechel and Rakic, 1979b). Thisinterdependent pattern of neuronal and glial development in theembryonic cerebral cortex suggests that a reciprocal signalingrelationship exists between migrating cortical neurons andtheir radial glial guides. Defects in neuron-glial interactionsduring the development of cerebral cortex results in abnormalplacement and connectivity of neurons and aberrant patterns ofglial development, which are thought to be the basis for manybrain disorders (Rakic, 1988a; Volpe, 1995).

Interactions between immature cortical neurons and radialglia have been studied in vitro and the results of numerousinvestigations indicate that neurons play a determinant role inregulating and maintaining the function of radial glial cells asneuronal migratory guides (Feng et al., 1994; Feng and Heintz,

1995; Gasser and Hatten, 1990; Hatten, 1985, 1987; Hunter andHatten, 1995; Mason et al., 1988; Sotello et al., 1994). Radialglial cells in turn form specialized cell attachments withmigrating neurons that are thought to be crucial in initiating andmaintaining neuronal cell migration (Anton et al., 1996;Cameron and Rakic, 1994; Edmondson and Hatten, 1987;Edmondson et al., 1988; Fischell and Hatten, 1991; Gao et al.,1991; Gregory et al., 1988; Grumet, 1992; Hatten et al., 1988;Rakic, 1972; Rakic et al., 1994; Zheng et al., 1996). Radial glialcells may also regulate the activity of neuronal ion channels orneurotransmitter receptors involved in cell migration (Komuroand Rakic, 1992, 1993; Parpura et al., 1994). Changes in radialglial cell surface properties are also thought to signal migratingneurons to cease migration and begin their differentiation at theappropriate location in the developing cortical plate (Anton etal., 1996). The molecular signals underlying this reciprocal rela-tionship between migrating cortical neurons and their radialglial guides, where each supports the maintenance of the dif-ferentiated phenotype of the other, remain unknown.

To begin examining the molecular signals exchangedbetween migrating neurons and radial glia, we have focusedon the neuregulins (NRGs) and their receptors. The NRGs area diverse group of membrane-attached and secreted peptidegrowth factors, which are of central importance to nervoussystem development and function (reviewed in Carrway andBurden, 1995; Lemke, 1996). The various forms of NRG aregenerated by alternative splicing of a single gene, and up to

3502 E. S. Anton and others

fifteen variants (Marchionni et al., 1993; Wen et al., 1994),including glial growth factors (GGFs; Marchionni et al.,1993), heregulins (Holmes et al., 1992), neu differentiationfactor (NDF; Wen et al., 1992) and acetylcholine receptor-inducing factor (ARIA; Falls et al., 1993), have been charac-terized. Glial growth factors (GGFs), the first isoforms ofNRG shown to participate in neuron-glia interactions, werediscovered, purified and cloned as Schwann cell mitogens(Brockes, 1978; Lemke and Brockes, 1984; Goodearl et al.,1993; Marchionni et al., 1993). Neuregulins elicit theircellular effects through receptor tyrosine kinases erbB2, erbB3and erbB4 of the epidermal growth factor receptor family(Carraway and Burden, 1995).

In the developing vertebrate peripheral nervous system,NRGs restrict neural crest stem cells to glial fate (Shah et al.,1994), and further regulate Schwann cell mitosis, migration,apoptosis (Grinspan et al., 1996; Levi et al., 1995; Marchionniet al., 1993; Mahanthappa et al., 1996; Trachtenberg andThompson, 1996) and peripheral neurite growth (Mahan-thappa et al., 1996). In muscle cells, ion channel abundance(Corfas and Fischbach, 1993) and neurotransmitter receptorexpression (Chu et al., 1995; Falls et al., 1993; Jo et al., 1995)is regulated by NRGs. Early nervous system development wasfound to be markedly affected in NRG, erbB2 and erbB4mutants (Lee et al., 1995; Meyer and Birchmeier, 1995;Gassman et al., 1995). Within the central nervous system,NRGs have been shown to have multiple roles in oligoden-drocyte development (Vartanian et al., 1994; Canoll et al.,1996; Marchionni et al., 1996), promote survival and matura-tion of astrocytes (Pinkas-Kramarski et al., 1994), affect interglial communication (Hofer et al., 1996) and stimulatesurvival and growth of retinal neurons (Bermingham-McDonogh et al., 1996).

Since GGF is produced by the developing CNS neuronsand exerts effects on neural and glial development, we soughtto determine whether GGF plays any role in the interactionsbetween radial glial cells and migrating neurons during thedevelopment of cerebral cortex. In particular, we examinedthe effect of GGF on neuronal migration along radial glialfibers and on the development of radial glial cells (we use theterm GGF to refer to both endogenous NRGs and exogenousGGF2). Our results indicate that GGF, expressed bymigrating neurons, promotes cortical neuronal migration onradial glia and, concurrently, supports the lengthening of theradial glial processes. To further implicate the GGF-erbBsignaling pathway in these events, we have analyzed erbB2-deficient mice and found that radial glial developmentappears to be compromised in the absence of GGF signalingvia erbB2. To understand how GGF might mediate neuron-radial glial interactions, we analyzed the role of GGF in theexpression of a radial glial molecule, brain lipid-bindingprotein (BLBP), previously shown to be essential for theestablishment and maintenance of radial glial fiber systemduring neuronal migration in the developing brain (Feng etal., 1994; Feng and Heintz, 1995). Our results suggest thatGGF is a crucial signal for the expression of brain lipid-binding protein (BLBP) on radial glia. Taken together, thesefindings suggest that GGF exerts an important mediatory rolein neuron-glial interactions during the development of thecerebral cortex.

MATERIALS AND METHODS

Preparation of GGF and antibodies Recombinant glial growth factor 2 (rhGGF2) was produced in a stablytransformed CHO cell line and purified by column chromatographyas described previously (Marchionni et al., 1996). The antibodies toGGF2 used in the experiments were generated at Cambridge Neuro-Science. Antibody CN-16 is a rabbit polyclonal raised by immuniz-ing with the entire rhGGF 2 protein. Antibodies 2861 and 11366 arerabbit antisera raised against the N-terminal region and the conservedEGF-like domain of rhGGF2, respectively.

Antibodies to erbB2/neu (AB4, AB1, Oncogene Sciences; 9G6,C18, Santa Cruz), erbB3 (SC-285, Santa Cruz), erbB4 (SC-283, SantaCruz), I-CAM (Sigma) and actin (A-2066, Sigma; mAb1501,Chemicon) were obtained commercially. Affinity-purified polyclonalantibodies to BLBP and αv integrins were generously gifted by Dr N.Heintz (Rockefeller University) and Dr L. F. Reichardt (UCSF),respectively.

Immunohistochemical proceduresCerebral cortices from rats (embryonic day 14 to postnatal day 7;Sprague-Dawley) were removed, fixed in 4% paraformaldehyde, cutinto 10 µm-thick sections in a cryostat and collected onto gelatin-coated glass slides. Sections were washed and blocked in TBS con-taining 5% goat serum, 3% bovine serum albumin and 0.01% Triton-X for 45 minutes, before incubating in primary antibodies for twelvehours at 4°C. Primary antibody binding was detected by incubationwith appropriate cy-3-, rhodamine- or fluorescein-conjugatedsecondary antibodies (1: 200 dilution; Jackson Immunochemicals).Sections were then washed in TBS, counter stained with 10 µm bis-benzimide and mounted in mowiol (Calbiochem; 10% mowiol, 25%glycerol in 0.1 M Tris with p-phenylenediamine) for observation in aZeiss microscope equipped with catecholamine (excitation, 400-440nm; barrier, LP 470 nm), rhodamine and fluorescein filter sets.Cultures of embryonic rat cortical cells were processed identically.However, in some cases, to avoid permeabilizing cells, the blockingbuffer used was devoid of triton-X. Cryostat sections of embryonicbrains from erbB2 mutants and wild types were made as described inLee et al. (1995).

Cortical imprint assay for neuronal migrationCortical imprints containing intact radial glial cells with migratingneurons attached to them were made as described previously (Antonet al., 1996). Briefly, embryonic day 18 rat brains were removed and200 µm-thick coronal sections of the cortices were prepared. Sectionswere then cultured on 10 µg/cm2 cell-tak (Collaborative Research)-coated 35 mm Petri dishes with glass bottoms (Mat Tek corporation)in minimal volume of MEM/10% horse serum media for 10-24 hours.Cortical sections were then gently pressed against the culture sub-stratum with a wet glass coverslip and the culture dish was floodedwith culture medium. This procedure lifts tissue sections off the sub-stratum while leaving behind a few cell layers thick imprint of cerebralwall containing radial glial cells with migrating neurons attached tothem.

Imprints were then maintained in the incubator for an additional4-24 hours. Subsequently, imprints were transferred into the chamberof a micro-incubator (37°C, 95% air, 5% CO2; Narushige Instru-ments) attached to the microscope stage. Cortical neurons migratingon radial glial cells were monitored using a Zeiss Axiovert 135microscope (GHS filter block, 460-nm excitation wavelengths)equipped with a Zeiss W63 objective lens. Images were recordedevery 5 to 15 minutes using a Panasonic TQ-3031 optic disk recorderand the Attofluor Ratio Vision program (Atto Instruments, Inc). After60-120 minutes of baseline recording, recombinant human glialgrowth factor 2 (2.5, 10, 25 or 50 ng/ml) or purified anti-GGF anti-bodies (CN16; 1: 50 dilution) were added to the cultures and moni-toring continued for an additional 60-240 minutes. As controls,

3503Role of GGF in cortical development

cultures were monitored unperturbed, or after perfusing in mediadevoid of growth factors or antibodies, or media containingnonimmune rabbit immunoglobulins (100 µg/ml). Changes in therate of cell migration, morphological features of migrating neuronsand glial cell substrata, and the extent of neuronal-glial cell contact,were monitored before and after growth factor or antibody perturba-tion. The extent of cell soma movement was divided by time elapsedbetween observations to obtain the rate of cell migration for eachneuron studied. Statistical differences between experimental groupswas tested by Student’s t-test.

In addition to radial glial cells, which had migrating neuronsattached to them, many isolated radial glial cells were also found inthese cultures. Cultures that contained mostly radial glial cells wereused to evaluate the effect of GGF2 on their development. 24 hoursafter imprinting, culture medium of imprints containing radial glialcells were supplemented with MEM/10% HS medium alone, GGF2(25ng/ml), or GGF2 (25 ng/ml) and anti-GGF antibodies (CN16;1:50 dilution). After 24 hours of incubation, images of radial glialcells in each culture were captured as described earlier and thelength of each radial glial cell was measured. Radial glia wereinitially identified by their morphology and eventually by labelingwith RC2 mAbs.

After image acquisition, cultures were fixed in 4% paraformal-dehyde and processed for anti-GFAP (Datco), anti-Neurofilament(Boehringer-Mannheim), Rat-401 mAb (gift from Dr S. Hockfield,Yale University; Hockfield and McKay, 1985), RC2 mAb, or anti-neuron-specific tubulin (TuJ-1 antibodies; generous gift from Dr A.Frankfurter, University of Virginia) immunohistochemistry. GFAP,Rat-401 and RC2 immunohistochemistry was used to analyze radialglial cells both in culture and in embryonic brain sections. Anti-neuron-specific tubulin or neurofilament labeling was used to confirmthe identity of migrating cells as neurons.

Glial cell cultureTo investigate the effect of GGF2 on BLBP expression, purifiedcerebral glial cells were cultured from E18 rat brains as describedpreviously (Cameron and Rakic, 1994; Feng et al., 1994; Feng andHeintz, 1995). After 2 weeks in culture, GGF2 was added to thecultures at a final concentration 25 ng/ml and cultures were allowedto develop for another 48 hours. As control, MEM/10% HS mediumwithout GGF2 was added to some cultures. Cultures were thenrinsed with ice-cold phosphate-buffered saline (PBS) and harvestedin homogenization buffer (40 mM Tris [pH 7.4], 1 mM MgCl2 sup-plemented with 1 µg/ml each of leupeptin, pepstatin A andaprotinin, 0.4 mM phenylmethysulfonylfluoride and 0.005%DNAase) for immunoblotting analysis with anti-BLBP antibodiesas described by Feng et al. (1994). In some cases, cultures werefixed in 4% paraformaldehyde and stained with Rat-401 mAbs andanti-BLBP antibodies to assess the BLBP expression pattern ofglial cells.

To investigate the role of BLBP in GGF-induced radial glial growth,radial glial cultures were supplemented with GGF2 (25 ng/ml), GGF2(25 ng/ml)+ affinity-purified anti-BLBP antibodies (1: 1000 dilution),or GGF2 (25 ng/ml)+ nonimmune rabbit IgGs (100µg/ml). After 24hours, images of radial glial cells were collected and analyzed asdescribed earlier.

Immunoblotting Whole cell extracts were obtained from embryonic brains or glial cellsand polypeptides were separated by one-dimensional SDS-PAGE asdescribed previously (Canoll et al., 1996). Fractionated proteins wereelectrophoretically transferred to nitrocellulose (0.45 µm, Biorad) andprobed with antibodies to GGF, erbB2, erbB3, erbB4, BLBP, I-CAM,neuron-glial junctional proteins (NJPA1) or αv integrins. Immuno-reactive bands were visualized by chemiluminescence as per manu-facturer’s instructions (Amersham, IL).

RESULTS

Distribution of GGF and its receptors in thedeveloping cerebral cortexThe temporal, spatial and cellular distribution of GGF andGGF receptors in the developing rat cerebral wall wasexamined during the period of neuronal cell migration usingboth immunohistochemical and immunoblotting procedures.Both procedures utilized specific antibodies to GGF, CN16 and11366, which have been used to localize GGF immunoreac-tivity in the retina (Bermingham-McDonogh et al., 1996). Inimprint cultures of developing cerebral cortex, containingradial glia and migrating neurons, CN16 labeled migratingneurons prominently (Fig. 1A-D), but occasional punctatelabeling of nuclear and peri-nuclear regions of radial glial cellswas also observed. In the developing rat cerebral cortex, GGFimmunoreactivity was present on migrating neurons during theperiod of intense migratory activity between embryonic day 14and postnatal day 1 (Fig. 1F). In general, neurons in the devel-oping cortical plate labeled more intensely with anti-GGF anti-bodies than the migratory neurons that are traversing the inter-mediate zone or the mitotic neural precursors situated in theventricular zone. Of the receptors for GGF, immunoreactivityfor erbB2 is present primarily on radial glia in the developingcerebral wall (Fig. 1E). ErbB2 may also be expressed in devel-oping neuroblasts as well. However, erbB2 immunoreactivityin the cerebral cortex decreased with advancing embryonicdevelopment. ErbB3 immunoreactivity is present predomi-nantly in postmigratory cortical plate neurons and cells in theventricular zone (Fig. 1G). ErbB4 immunoreactivity is presenton all cell types of the developing cerebral wall (Fig. 1H).However, postmigratory cortical plate neurons and cells in theventricular zone labeled more prominently than others. Incultures of cortical neurons and glia, a similar pattern of erbB2,erbB3 and erbB4 immunoreactivity was seen: erbB2 wasexpressed mainly in glia, whereas erbB3 and erbB4 were foundin both neurons and glia (Fig. 2).

Western blot analysis of whole-cell extracts from thecerebral cortices of E14, E16, E18 and P0 rats indicate thatGGFs, erbB2, erbB3 and erbB4 receptors are present through-out the cortical development (Fig. 1I-K). The level of erbB2expression decreases with age, in agreement with the immuno-histochemical observations. A slight decrease in erbB3 levelfrom E18 to P0, and a small increase in erbB4 level from E14to E16 were also noticed.

Taken together, our analysis of the developmental distribu-tion of GGF and its receptors in the cerebral cortex suggest thatneuronal GGF and its receptors are present in appropriatespatial and temporal patterns to participate in neuron-glialinteractions during the process of neuronal migration in thedeveloping cerebral cortex. Therefore, we examined the effectof GGF, a soluble form of neuregulin, on cortical neuronalmigration on radial glia and in radial glial development.

Effect of GGF on neuronal migrationWe used cortical imprint cultures containing migrating neuronsattached to radial glia (Anton et al., 1996) to study the role ofGGF in cortical neuron-radial glial interactions. Following aperiod of baseline observation (1-2 hours) of neuronalmigration, GGF was added to the cultures at a concentrationof 2.5, 10, 25 or 50 ng/ml. Changes in the rate of neuronal


bution of GGF and its receptors in vitro and in vivo. GGF is expressedigrating (white arrow; A,C) on radial glia in cortical imprint cultures.

light images of A and C, respectively. Glial processes (black arrow; B,D)with anti-GGF antibodies. In the developing cerebral wall, neurons in the region labeled more intensely with anti-GGF antibodies than theurons that traverse the intermediate zone and the neural precursors in theone (F). In the inset of F, a confocal microscope image of a cordon ofurons (arrows) from the intermediate zone labeled with anti-GGF shown. Of the receptors for GGF, erbB2 immunoreactivity is present radial glia in the developing cerebral cortex (E). The arrow in E points to fiber that spans the cerebral wall. The brightly labeled structure on thenel is a blood vessel and the cerebral wall in this panel is presented at anbB3 immunoreactivity is present primarily in the postmigratory cortical

s and cells in the ventricular zone. (H) ErbB4 is present all across theerebral wall, but postmigratory cortical plate neurons and cells in theone labeled more prominently than others. Coronal sections from E16-H) telencephalon were used to detect immunoreactivity. Horizontal barscate the different regions of the cerebral wall: top, cortical plate; middle, zone; bottom, ventricular zone. Whole-cell extracts from E14 (1), E16and P0 (4) rat cortices were resolved by SDS-PAGE, transferred toe and probed with antibodies specific to erbB2 (I), erbB3 (J), erbB4 (K). Scale bar, A-D, 15 µm; E, 40 µm; F-H, 50 µm.

migration, neuronal and glial cell morphologies was monitoredas described before (Anton et al., 1996). The average rate ofneuronal migration in this assay is 10.1±0.3 µm/hour (n=354).

At 25 ng/ml, GGF maximally promoted neuronal migrationon radial glia by 58% (Fig. 3). The response was dosedependent. The increase in the rate of neuronal migration inresponse to GGF was often accompanied by changes in themorphology of migrating neurons. Neuronal cell somabecomes more elongated in shape, and leading and trailingprocesses lengthen in response to GGF. Blocking endogenousGGF activity in the migration assays with antibodies to GGF,reduced the rate of neuronal migration by 56% (Fig. 3). Basalneuronal migration on radial glia therefore is dependent uponendogenous GGF. No discernible changes inneuronal attachment to radial glial cells wereobserved in the presence of GGF or antibodies toit. Addition of culture medium devoid of GGF orcontrol antibodies did not induce any changes inthe migratory behavior of cortical neurons. Thus,the rate of neuronal migration on radial glia seemsto be tightly coupled to the level of available GGF.

GGF could exert its effect on neuronal migrationdirectly via its actions on neurons or (and) in-directly through its effects on radial glial cells.Supporting the latter possibility, in the course ofanalyzing neuronal migration in imprint cultures,it was apparent that the length of radial glial cellswas also affected by GGF. In some cortical imprintcultures where the entire length of a radial glial cellwith migrating neurons attached to its processeswas within the field of observation, increases inradial glial length were observed in GGF-treatedcultures (Fig. 4).

Effect of GGF on radial glial developmentSince the maintenance and lengthening of radialglial migratory substrata during neuronalmigration are essential components of the neuronalmigratory process in the cerebral cortex(Schmechel and Rakic, 1979a,b), we next soughtto determine if GGF plays any role in the devel-opment of radial glial cells. We established imprintcultures containing predominantly radial glial cellsfrom the same embryonic age cerebral cortex usedin the migration assays, and fed them withMEM/10% HS medium with or without GGF (25ng/ml). After 24 hours of incubation, images ofevery radial glial cell in the culture dish werecollected and the total length of each cell wasmeasured. Radial glial cells were initially identi-fied by their unique morphology: unipolar orbipolar cells with elongated, pear-shaped cell somaand long, slender processes. Following imageacquisition, cultures were stained with radial glial-cell-specific RC2 antibodies to confirm that all ofthe cells that we identified morphologically asradial glial cells indeed were radial glial cells by asecond criterion. Occasionally, few cells not iden-tified morphologically as radial glia were labeledwith RC2 antibodies and these were then includedin the sample. When compared to the untreated

Fig. 1. Distriby neurons m(B,D) Phase do not label cortical platemigratory neventricular zmigrating neantibodies isprimarily ona radial glialleft of this paangle. (G) Erplate neurondeveloping cventricular z(E) or E18 (Fon F-I demarintermediate(2), E18 (3) nitrocellulosand actin (L)

radial glial cells, GGF increased the radial glial length by 32%(Fig. 5). This increase in the length of radial glial fibers wasnot observed when GGF was neutralized with anti-GGF anti-bodies prior to addition to the culture medium (Fig. 5).

To further analyze the role of GGF in radial glial cell devel-opment, we studied the morphology and distribution of radialglia in mutant embryos lacking erbB2, a functional receptor forGGF. ErbB2 is primarily expressed by radial glia in the devel-oping cerebral cortex (Fig. 1E). In wild-type mice, individualradial glial fibers usually split into several branches (end feet)at the border of marginal zone (prospective layer 1) and theunderlying cortical plate (Fig. 6A,B). In erbB2-deficientembryos, however, radial glial fibers formed abnormal glial


Fig. 2. Distribution of erbB receptors in neurons and glia.Developing cortical neurons and radial glial cells wereimmunostained with polyclonal antibodies to erbB2 (A), erbB3 (B-D) and erbB4 (E,F). (C,F,H) Phase light images of B, E and G,respectively. (A) ErbB2 is expressed primarily by radial glial cells.ErbB3 (B) and erbB4 (E) immunoreactivity was found in bothmigrating neurons (arrows, B,E) and radial glial cells (arrowheads,B,E). (D) erbB3 immunoreactivity in a postmitotic, differentiatingneuron with processes. No immunoreactivity was observed withpreimmune rabbit serum (G,H) in neurons (arrow, H) or radial glialcells (arrowhead, H). Scale bar, A-C, E-H, 10 µm; D, 8 µm.

Fig. 3. GGF promotes cortical neuronal migration on radial glia.(A) A representative image from a cortical imprint assay illustratingcortical neurons (black arrow) migrating on radial glia (white arrow).The characteristic morphology of migrating neurons with theirleading and trailing processes is visible. (B) GGF promoted neuronalmigration in this assay. Maximal effect was observed at 25 ng/ml.Blocking of endogenous GGF with anti-GGF polyclonal antibodies(CN16) retarded neuronal migration. Exposure to medium devoid ofGGF (MEM) or rabbit immunoglobulins (Rbt. IgGs) did not affectneuronal migration. Data shown are the mean ± s.e.m for each group.Asterisks indicate that the effect is significant when compared withthe respective controls at P<0.01. n=33, MEM; 13, GGF-2.5 ng/ml;10, 10 ng/ml; 28, 25 ng/ml; 11, 50 ng/ml; 38, rabbit IgGs (Rbt. IgG);24, anti-GGF. Scale bar, 4 µm.

end feet. The ends of the radial glia in mutants are minimallyarborized (Fig. 6C-E). We did not detect any other clearlydefinable changes in radial glial morphology in the cerebralwall of erbB2 mutant embryos. Due to the lethality of theerbB2 mutants, analysis of the developing cerebral wall beyondE10.5-11 is not possible. Thus the effect of this radial glialabnormality on neuronal migration in erbB2 mutant embryosis unclear at this juncture.

Taken together, these observations suggest that GGF plays avital role in radial glial growth that accompanies neuronalmigration in the developing cerebral wall. In the absence of afunctional erbB2 receptor, radial glial development isabnormal. We therefore sought to identify some of themolecular mediaries of this GGF-modulated radial glial devel-opment.

Effect of GGF on brain lipid-binding protein (BLBP),a radial glial molecule that functions in glialdevelopment Of the various candidate molecules that are thought to play arole in radial glial development, BLBP represents a potentialmolecular mediator of GGF-induced radial glial development.

Previous studies have shown that BLBP, a ~15×103 Mr brain-specific member of a family of hydrophobic ligand binding-proteins, is required for the establishment and maintenance ofthe radial glial fiber system in the developing brain (Feng etal., 1994; Kurtz et al., 1994). The expression of BLBP in radialglia throughout the developing CNS is strictly correlated withmigration of neurons upon these cells. Furthermore, theinduction and maintenance of radial glial BLBP duringneuronal migration in the developing CNS requires either thepresence of neurons or a soluble neuronally derived factor(Feng et al., 1994; Feng and Heintz, 1995). Since migratingcortical neurons release GGF and GGF influences radial glialdevelopment and neuronal migration on glial cell surfaces, wetested whether GGF plays any role in the induction and main-tenance of BLBP expression in radial glia.

Purified glial cells from embryonic cerebral cortex werecultured without neurons for 2 weeks. Glial cultures were thenfed either with MEM/10% HS medium or MEM/10% HSmedium supplemented with GGF and the cultures wereanalyzed 48 hours later. Cells were either harvested forimmunoblotting with anti-BLBP antibodies or fixed in 4%paraformaldehyde for immunohistochemical analysis withanti-radial glial and anti-BLBP antibodies. In earlier studies,Feng et al. (1994, 1995) have demonstrated that, in the absence


Fig. 5. GGF promotes radial glial development. GGF was added toradial glial cultures at 25 ng/ml and changes in morphology wereevaluated after 24 hours. GGF promoted radial glial lengthening.(A) A radial glial cell cultured in MEM culture medium withoutGGF and (B) a radial glial cell maintained in the presence of GGF.GGF addition lead to a 31% increase in glial length, which was notobserved when GGF was neutralized with antibodies prior toaddition to the cultures (C). Data shown are the mean± s.e.m for eachgroup. Asterisks indicate that the effect is significant when comparedwith the controls at P<0.01. n=96, MEM; 65, GGF; 81, GGF+ anti-

of neurons, BLBP expression is drastically down-regulated;addition of differentiating neurons back to the purified glialcultures restores glial BLBP expression. In our experiments,exposure to GGF induced BLBP expression in glial cells thatwere maintained in the absence of neurons (Fig. 7). WhenGGF-treated and untreated glial cells were double labeled withanti-BLBP and radial glial lineage-specific Rat-401 antibodies,only GGF-treated cells were found to express BLBP (Fig. 7).In western blot analysis of glial cells treated with and withoutGGF, significant levels of BLBP were detected only in GGF-treated glial cells (Fig. 8). These results suggest that soluble,neuronally derived GGF might function to induce and maintainBLBP expression in radial glial cells during neuronalmigration.

BLBP is thought to play a role in radial glial process growth,since radial glial cells fail to extend processes in the presenceof anti-BLBP antibodies (Feng et al., 1994). We found that theGGF-mediated growth of radial glial processes was blocked inthe presence of anti-BLBP antibodies (Fig. 9). Radial glial cellsexposed to both GGF and anti-BLBP antibodies displayedstunted processes, in contrast to the untreated cells or cellstreated with GGF or GGF plus nonimmune rabbit IgGs. Thus,BLBP appears to be an essential component of GGF-inducedradial glial cell elongation that accompanies neuronalmigration on radial glia.

Taken together, these studies suggest that GGF, expressed bymigrating neurons, promotes the maintenance and develop-ment of radial glial guides, which are necessary for neuronal

Fig. 4. GGF promotes neuronal migration and radial glial growthconcurrently. Cortical neurons migrating on a radial glial processwere monitored before (A, 60 minutes; B, 30 minutes; C, 0 minutes)and after (D, 60 minutes) the addition of GGF (25 ng/ml). GGFexposure led to increased rate of neuronal migration (see neuronmarked by white arrow) and enhanced radial glial process (blackarrow) elongation. Scale bar, 12 µm.

GGF. Scale bar, 4 µm.

migration. The stage-specific radial glial molecule BLBP is akey intermediary in this neuron-glia interaction.

Effect of GGF on glial expression of cell adhesionmolecules and integrin receptors To analyse GGF’s effects on glial molecules known to directlymodulate neuronal migration, we tested GGF’s effect on glialexpression of neuron-glial junctional proteins, I-CAM and αvintegrin receptors as described above for BLBP (Anton et al.,1996; O’Shea et al., 1990; Chong et al., 1987, Bacus et al.,1993). GGF did not affect the expression of these molecules,indicating that these three glial molecules are unlikely to bepart of GGF-induced promotion of neuronal migration on glialprocesses.

DISCUSSION

Although there is considerable evidence to suggest that themigrating neurons and their radial glial guides influence eachothers’ growth and differentiation during the development ofcentral nervous system (reviewed in Rakic et al., 1994), themolecular mediaries of these interactions are not fully under-stood. Here, we demonstrate that GGF, expressed by develop-ing cortical neurons, enhances the migration of corticalneurons on radial glia and, concurrently, promotes the main-tenance and development of radial glial cells. The maintenanceand growth of radial glial scaffold is essential for neuronalmigration in the developing cerebral cortex. Further support for


Fig. 6. Abnormal radial glial development in the absence of GGFsignaling in erbB2 knock out mice. Radial glial cells in telencephalonfrom erbB2 mutant embryos and wild-type mice were labeled with Rat-401 mAbs. Images of the radial glial end feet near the pial surface wereobtained with a Biorad confocal microscope. (A,B) Wild-type embryos;(C-E) erbB2 null embryos. Arrows in A,B point to the elaboratelybranched, tufted radial glial end feet in wild-type embryos. In theabsence of the erbB2 receptor for GGF, radial glial cells did not formthe elaborately arborized end feet (arrows in C-E). Scale bar, 7 µm.

GGF’s role in radial glial development is evident in ouranalysis of erbB2-deficient mice, which display abnormalitiesin the radial glial scaffold. GGF was found to promote the

Fig. 7. Induction of BLBP expression in radial glial cells by GGF. Purifiedneuronal contact for 14 days prior to the addition of GGF or plain MEM/1labeled with Rat-401 monoclonal antibodies (A,C,E,G,I,K) and polyclonacells of radial glial lineage (i.e. radial glial cells and reactive astrocytes). Inexpress BLBP. In the absence of GGF, neither type of glial cells express B

development of radial glial cells via BLBP, whose presence iscorrelated with and is necessary for neuronal migration alongradial glial fibers. Blocking the activity of BLBP also appearsto prevent the actions of GGF on radial glial cells. The main-tenance of radial glial cell phenotype and the expression ofBLBP on radial glial cells depend on neuronal signals (Fenget al., 1995). GGF, expressed by migrating neurons appears tobe a crucial signal in this process.

A role for GGF in the modulation of neuronalmigrationThe actions of GGF during neuronal migration in the devel-oping cerebral cortex may occur via multiple modes ofsignaling. Since cortical neurons express both GGF and GGFreceptors and are capable of binding and responding to GGF,autocrine signaling is a likely possibility for some aspects ofcortical neuron development. However, paracrine signalingfrom neurons can explain several of the observed effects onradial glial cells, including the induction of BLBP and growthand development of glial processes. The lengthening of radialglial fiber migratory substrata may result in response toincreased rate of neuronal migration. Alternatively, neuronsmay increase their rate of migration in response to GGF-induced lengthening of radial glial fibers. Both possibilitiessuggest that GGF is a crucial link in the reciprocal interactionsbetween migrating neurons and radial glia.

GGF, in addition to its effect on radial glial growth thataccompanies neuronal migration, may also modulate a plethoraof radial glial molecules that could impact neuronal migrationmore directly. Evidence from other cell types suggests thatGGF can regulate the expression of neurotrophic factors, celladhesion molecules, neurotransmitter receptors, ion channels,extracellular matrix molecules and their receptors (Pinkas-

glial cells from E18 cerebral cortex were maintained in culture without0% HS cell culture medium. After 48 hours cells were fixed and doublel anti-BLBP antibodies (B,D,F,H,J,L). Rat-401 mAbs specifically label the presence of GGF, radial glial cells (A-D) and astrocytes (E,F)LBP (G-L). Scale bar, A-D, G-J, 10 µm; E,F,K,L, 20 µm.


42

18.6

GGF

7.7+ −

§ Actin

§ BLBP

Fig. 8. Induction of BLBP expression in radial glial cells by GGF.GGF-treated and untreated cells were harvested and samples (25 µgof protein) were subjected to immunoblot analysis with anti-BLBPantibodies. BLBP expression was induced in glial cells treated withGGF. Identical lanes of immunoblots were also probed with anti-actin antibodies. No significant change in the level of actin wasobserved in response to GGF exposure, thus supporting thespecificity of GGF effect on BLBP expression.

Cha

nge

in R

adia

l Glia

l Len

gth

(fol

d ba

sal)

0

1

1.4

GG

F+A

nti-BLB

P

GG

F+R

bt.IgG

ME

M

GG

F

Fig. 9. Inhibition of GGF-induced radial glial development by anti-BLBP antibodies. Radial glial cells were cultured in MEM media, orin MEM media supplemented with GGF, GGF plus anti-BLBPantibodies, or GGF plus nonimmune rabbit IgGs. GGF significantlypromoted the lengthening of radial glial cells. However, when thefunction of BLBP was blocked in the presence of anti-BLBPantibodies, radial glial cells fail to lengthen in response to GGF. Datashown are the mean ± s.e.m for each group. n=58, MEM; 25, GGF;33, GGF+ anti-BLBP; 32, GGF+ Rbt. IgGs.

Kramarski et al., 1994; Falls et al., 1993; Corfas and Fischbach,1993; Mahanthappa et al., 1996; Verdi et al., 1996). Regula-tion of such molecules in radial glia by neuron-derived GGFmay allow it to directly modulate the cell surface properties ofradial glia and, thus, neuronal migration on them. Alterna-tively, neurotrophic factors secreted by radial glia in responseto GGF might exert a general trophic effect on cortical neuronsleading to altered patterns of neuronal migration. We testedGGF’s effects on three radial glial molecules – neuron-glialjunctional proteins, I-CAM and αv integrin receptors – that areknown to directly influence neuronal migration on radial glialcells (Anton et al., 1996; O’Shea et al., 1990; Chong et al.,1987; Bacus et al., 1993). GGF did not directly affect theexpression of these molecules, indicating that it probably actsthrough a different set of glial cell surface molecules or solublesignals to mediate its effect on neuronal migration along glialprocesses. Additional studies, with function-blocking probes toerbB receptors, and co-cultures of neurons and radial gliaobtained from the different neuregulin and erbB mutants willbe necessary to characterize how the different modes of GGFsignaling impact different aspects of the cortical development.

The effect of GGF on radial glial cell development In the absence of GGF signaling via a key functional receptor(erbB2), radial glial development is compromised. During thedevelopment of cerebral cortex, radial glial fibers becomeelongated and their end feet, which begin as single processes,branch extensively at the interface between marginal zone andcortical plate as development proceeds. Changes in the cellsurface properties of radial glial end feet region are alsothought to modulate the terminal phase of neuronal migrationin the cerebral cortex (Anton et al., 1996; Rakic, 1972; Rakicand Caviness, 1995; Schmechel and Rakic, 1979b). In theerbB2 mutant embryos, the characteristic radial glial end feetarborization is minimal or absent. Similar defects in radial glialprocesses were also observed in reeler mutant mice whereneuronal migration along radial glial cells is grossly abnormaland cells fail to stop at the interface between cortical plate andmarginal zone (Caviness and Rakic, 1978; Caviness et al.,1989; Derer, 1985; Hunter and Hatten, 1996; Rakic andCaviness, 1995). This aberrant radial glial development in theabsence of proper GGF signaling is likely to affect cortical

neuronal proliferation and migration, since earlier studies havedemonstrated that radial glial cells in culture normally inhibitneuronal proliferation and promote neuronal differentiation(Gao et al., 1991; Gasser and Hatten, 1990; Hatten et al., 1984,1988). Further studies with neuronal and radial glial cellsobtained from late-surviving erbB2 mutant and wild-typeembryos will strengthen the support for this hypothesis.

After the completion of neuronal migration to the cerebralcortex, radial glial cells transform into astrocytes or ependymalcells (reviewed in Cameron and Rakic, 1991). Previous studieson peripheral and central glia of various lineages suggest thatGGF can influence cell-fate decisions or otherwise impact pro-gression of cells along the lineage (Shah et al., 1994; Dong etal., 1995; Canoll et al., 1996). The relevant example in the CNSis the oligodendrocyte lineage, where the differentiation ofprogenitors appears to be slowed or inhibited by high concen-trations of GGF (Canoll et al., 1996). Similarly, high levels ofGGF expression might prevent the premature onset of radialglial differentiation and delay it until neuronal migration iscompleted, thus ensuring the continued presence of radial glialguides for directing migration. Towards the end of neuronalmigration, down-regulation of neuronal GGF expressioncoupled with the down-regulation of BLBP and erbB-2receptors in radial glia, may then trigger the transformation ofradial glial cells into astrocytes.

Regulation of GGF activity in the developing CNS Despite the continued expression of GGF in neurons, cells inboth oligodendrocyte and radial glial lineages ultimately doadvance to a fully differentiated state. Thus, the influence ofGGF in this respect is diminished as development proceeds.


These changes could reflect either a reduced level or change inthe isoform of GGF expression, altered responsiveness of thecells to GGF, or the influence of other epigenetic cues takingprecedence. Studies on Schwann cells and their progenitors inthe PNS have shown that GGF promotes different cellulareffects in culture, depending on the concentration of the factorpresent in the medium (Dong et al., 1995; Mahanthappa et al.,1996). Moreover, GGFs exist in a variety of soluble andmembrane-associated forms (Marchionni et al., 1993; seeLemke, 1996 for review). Of the two main functional domainsof GGF, the Ig-like domain binds to ECM molecules such asheparin sulfate proteoglycans (Loeb and Fischbach, 1995,Sudhalter et al., 1996) and the EGF-like domain binds to cellsurface receptor tyrosine kinases. Binding of GGF to heparin-like molecules has been shown to modulate GGF’s signaling(Sudhalter et al., 1996). The relative abundance of GGFs andtheir receptors also changes during different embryonic stagesand across the different subregions of the developing cerebralwall (Canoll et al., 1996; Chen et al., 1994; Marchionni et al.,1993; Meyer and Birchmeier, 1994; Orr-Urtreger et al., 1993).Hence, it is possible that GGF in the CNS can elicit differentcellular effects depending on concentration and the structuralform(s) present. Recently, bone morphogenetic proteins(BMPs) have been shown to influence the fate of astroglial cellsin the cortex at advanced developmental stages (Gross et al.,1996). Conceivably, BMPs could predominate over GGF atlater stages of radial glial cell differentiation.

Implications for the cerebral cortical developmentIn summary, present study suggests that GGF modulatesneuron-glial interactions during the period of neuronalmigration to the developing cerebral cortex. Neuronallyderived GGF appears to be an essential signal for the main-tenance and function of radial glial scaffold until the comple-tion of neuronal migration to the cortex. GGF may be part ofa developmentally regulated cascade of epigenetic signals thatregulate radial glial function and differentiation and, thus, thephenotypic differentiation of neurons interacting with theseglial cells. As evident here and in other studies of mousemutants of GGF and GGF receptors (Meyer and Birchmeier,1995; Gassmann et al., 1995; Lee et al., 1995), disturbance ofGGF signaling results in highly aberrant neural phenotypes.Further analysis of how the activity of this molecule isregulated during neuronal migration on radial glia and uncov-ering its down-stream signaling pathways will be crucial toelucidate, not only the mechanisms of neuronal migration andthe accompanying development of radial glial scaffolding, butalso the resultant laminar patterning of cerebral cortex.

This research was supported by NSF grant 9006752 and NIH grantNS02253 to PR, NIH grant HD 34534 to KFL, and a PHS award toEA.

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(Accepted 9 July 1997)

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