Expression of Transforming GrowthFactor-� in Developing Rat Cerebral

Cortex: Effects of PrenatalExposure to Ethanol

MICHAEL W. MILLER

Department of Neuroscience and Physiology, State University of New York-UpstateMedical University, and Research Service, Veterans Affairs Medical Center,

Syracuse, New York 13210

ABSTRACTThe effects of prenatal ethanol exposure on the spatiotemporal expression of transform-

ing growth factor-� (TGF�) and its receptors in developing rat cerebral cortex in vivo wereexamined. Pregnant Long-Evans rats were fed ad libitum with a diet containing ethanol fromgestational day (G) 6 through G21 or were pair fed an isocaloric nonalcoholic liquid diet. Aquantitative immunoblotting study showed that expression of TGF� ligands was differen-tially affected by ethanol; ethanol decreased TGF�1 expression fetally and in the maturecortex and increased TGF�2 at most ages. A complementary immunohistochemical experi-ment generated similar results so far as the timing of ligand expression was concerned. Inboth control and ethanol-treated rats, TGF�1 was expressed by cells in the two neocorticalproliferative zones and neurons in the cortical plate. TGF�2 was expressed principally byradial glia and astrocytes in developing rats. In the adult, both ligands were expressed by gliaand neurons. Ethanol virtually eliminated the TGF�1 expression in the perinatal subven-tricular zone. The TGF�2-positive radial glial labeling was transient and was lost earlier inethanol-treated neonates than in controls. Concomitantly, the appearance of TGF�2-positiveglia occurred earlier in the ethanol-treated rats. The expression of only one receptor (TGF�Ir)was affected by ethanol; it was increased during the pre- and early postnatal periods. TGF�Irwas expressed by glia perinatally and by all cell types in weanlings. As with TGF�2, ethanolexposure promoted the loss of TGF�Ir expression in radial glia and the precocious expressionamong astrocytes. TGF�IIr was expressed primarily by neurons. Thus, TGF� ligands andreceptors are strategically placed both in time and space to regulate cell proliferation andmigration. Ethanol, which affects both of these processes, has marked effects on the TGF�system and apparently promotes the early transformation of radial glia into astrocytes. J.Comp. Neurol. 460:410–424, 2003. © 2003 Wiley-Liss, Inc.

Indexing terms: alcohol; development; fetal alcohol syndrome; genetics; receptors; ventricular

zone

Transforming growth factor-� (TGF�1) affects multipledevelopmental processes, including cell proliferation, migra-tion, and neurite outgrowth. TGF�1 is a potent antiprolif-erative agent. It inhibits the mitogen-stimulated prolifera-tion of primary cultured neurons (Miller and Luo, 2002a),astrocytes (Ryken et al., 1992; Hunter et al., 1993; Vergeli etal., 1995; Luo and Miller, 1996; Miller and Luo, 2002b), andoligodendrocyte progenitors (McKinnon et al., 1993). Like-wise, TGF� inhibits the proliferation of transformed neuralcells, e.g., B104 neuroblastoma cells (Luo and Miller, 1999)and C6 glioma cells (Miller and Luo, 2002b).

There are no direct studies of the CNS showing thatTGF�1 affects neuronal migration or process (axonal or

Grant sponsor: Department of Veterans Affairs; Grant sponsor: NationalInstitute of Alcohol Abuse and Alcoholism; Grant number: AA06916; Grantnumber: AA07568; Grant number: AA09611.

Correspondence to: Michael W. Miller, Department of Neuroscience andPhysiology, SUNY-Upstate Medical University, 750 East Adams Street,Syracuse, NY 13210. E-mail: millermw@upstate.edu

Received 14 February 2002; Revised 18 October 2002; Accepted 17 Jan-uary 2003

DOI 10.1002/cne.10658Published online the week of April 14, 2003 in Wiley InterScience (www.

interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 460:410–424 (2003)

© 2003 WILEY-LISS, INC.

dendritic) outgrowth. On the other hand, TGF�1 can up-regulate cell adhesion molecule (CAM) expression by cul-tured, immature neural cells (Tsuzuki et al., 1998; Luoand Miller, 1999; Miller and Luo, 2002a), and CAMs arecritical regulators of neuronal migration and axonalgrowth (see, e.g., Lindner et al., 1983; Crossin et al., 1989;Miura et al., 1992; Asou et al., 1994; Zhu et al., 1995; Longand Lemmon, 2000). Isoforms of bone morphogenic pro-tein (BMP), also members of the TGF� superfamily, like-wise promote CAM expression (Lein et al., 1995; Le Rouxet al., 1999; Wilkemeyer et al., 1999, 2000).

Three isoforms of TGF� (TGF�1, TGF�2, and TGF�3)have been isolated from mammalian tissues (see, e.g.,Roberts and Sporn, 1990; Polyak, 1996). TGF� ligandsbind to specific receptors, TGF�rI and TGF�rII. Thesereceptors 1) bind TGF� with high affinity, 2) have serine/threonine kinase activity (Scott and Soderling, 1992), and3) must form a heterodimer to initiate signal transduction(Boyd and Massague, 1989; Laiho et al., 1990a; Wrana etal., 1992, 1994; Massague, 1998). There are few anatomi-cal studies of the distribution of components of the TGF�1system in the developing nervous system (Flanders et al.,1991; Pelton et al., 1991; Unsicker et al., 1991; Bottner etal., 1996; Galter et al., 1999). Accordingly, ligand expres-sion first appears on gestational day (G) 15. TGF�1 isexpressed only by cells in the meninges, and TGF�2 andTGF�3 are expressed by radial glia and astrocytes. In theadult, TGF�2 and TGF�3 are expressed by neurons incortical layers III and V. The appearance of receptor ex-pression coincides with ligand expression and may belocalized to radial glia.

Ethanol affects various events during early neuronaldevelopment. It inhibits cortical cell proliferation (in vivo:see, e.g., Kennedy and Elliott, 1985; Miller, 1989; Millerand Nowakowski, 1991; Miller and Kuhn, 1995; in vitro:see, e.g., Kennedy and Mukerji, 1986; Snyder et al., 1992;Luo and Miller, 1999; Jacobs and Miller, 2001), inducesmigration defects (in vivo: Clarren et al., 1978; Miller,1986, 1988a, 1993), and alters axonal and dendritic devel-opment (in vivo: see, e.g., Phillips, 1989; Phillips et al.,1991; Miller et al., 1990, 1999; Pentney and Miller, 1992;Miller and Al-Rabiai, 1994; in vitro: Pinazo-Duran et al.,1993; Clamp and Lindsley, 1998). Furthermore, ethanolaffects CAM expression (Minana et al., 1998, 2000; Luoand Miller, 1999; Wilkemeyer et al., 1999, 2000). Inas-much as cell proliferation, neuronal migration, and pro-cess outgrowth are regulated by TGF�, it is reasonable topredict that ethanol affects the expression of the TGF�system.

Recent in vitro data show that the actions of TGF�superfamily proteins on B104 neuroblastoma cells (Luoand Miller, 1999) and primary cultures of cortical neurons(Miller and Luo, 2002a) are affected by ethanol. BMP-mediated cell–cell adhesion among 3T3 fibroblasts andNG108 neuroblastoma cells is inhibited by ethanol (Wilke-meyer et al., 1999, 2000). Apparently, this effect is trans-duced through the CAM L1. Thus, TGF�1 has dual effects,to inhibit their proliferation and to promote their expres-sion of neural CAM (nCAM). Each activity is mediated bya distinct signal transduction pathway (Luo and Miller,1999).

The present study examines the effects of ethanol on theTGF� system in the developing cortex in vivo, exploringthe idea that the TGF� system (of ligands and receptors)moves cells along the developmental conveyer belt; i.e.,

the ligands encourage cells to end their cycling behaviorand to migrate and eventually elaborate their cell-specificmorphology.

MATERIALS AND METHODS

Subjects

The subjects were the offspring of pregnant Long-Evanshooded rats obtained from Harlan Sprague-Dawley (Indi-anapolis, IN). All procedures used in the present studywere approved by the Institutional Animal Care and UseCommittees at SUNY-Upstate and the Syracuse VeteransAffairs Medical Center (VAMC). The animals were main-tained at an AAALAC-accredited facility at the SyracuseVAMC.

The experimental paradigm for care and feeding of therats was the same as that used in earlier studies (e.g.,Miller, 1988a, 1992). Rats were fed one of two diets, a 6.7%(v/v) ethanol-containing diet (Et) or a control diet (Ct). TheEt and Ct were isocaloric liquid diets (Lieber and DeCarli,1986; Bio-Serv, Frenchtown, NJ). Rats were weaned ontothe liquid diets over a 6-day period beginning on G6 andmaintained on the diet until G20. Throughout the study,all animals were maintained in a room with a fixed 12hour light/12 hour dark cycle (light between 06:00 and18:00). It was essential that a regular light/dark cycle wasmaintained; cell proliferation has a distinct diurnalrhythm (Miller, 1992). The amount of liquid ingested bythe Et- and Ct-fed females was determined at 17:00 eachday, at which time fresh daily food allotments were given.On G21, the liquid diets were removed and all rats weregiven chow and water ad libitum. After birth, Et- andCt-treated neonates were surrogate fostered by lactatingCh-fed dams.

Blood ethanol levels were determined in the Et-fed ratson G16. Peak blood ethanol concentrations (of about 150mg/dl) are attained within 4 hours of feeding (Vavrousek-Jakuba et al., 1991; Miller, 1992). Hence, blood sampleswere taken at 22:00. Venous blood was taken from thetails of rats. The samples were spun, and the serum eth-anol levels were determined (Diagnostics Kit No. 332UV;Sigma, St. Louis, MO).

Biochemical studies

The immunoblotting protocol used samples of fresh (i.e.,unfixed) tissue from anesthetized (60 mg/kg ketamine and7.5 mg/kg xylazine) offspring. Fetuses were removed byCesarean section. All offspring were killed by cervicaldislocation. The brains were removed, and the cortex fromeach fetus and pup was dissected.

Samples were homogenized rapidly in cold electrophore-sis buffer (pH 8.5), i.e., 25 mM Tris, 192 mM glycine, 100mM sodium vanadate, 3.0% aprotinin, and 0.10% sodiumdodecyl sulfate (SDS). The homogenates were centrifugedat 25,000g for 1 hour, and the supernatants were dividedinto aliquots containing 25 �g protein. The DNA contentof each sample was determined spectrophometrically(A

260 nm; Sambrook et al., 1989). The proteins in three ali-

quots were separated by a standard one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) tech-nique (Laemmli, 1970; Mooney and Miller, 2000).Electrophoresis was performed using 12% or 5–15% gra-dient minigels. Samples were loaded onto a gel, one sam-ple per age per treatment group. In addition, a set of

411DEVELOPMENTAL EXPRESSION OF TGF� AND ITS RECEPTORS IN RAT CORTEX

biotinylated proteins of known molecular weights, range13–205 kDa (Bio-Rad, Hercules, CA), and an internalstandard (tissue from whole brains of pooled 12-day-oldrats) were added to two or three lanes of each gel.

Immunoblots of the gels (containing the samples andstandards) were produced. The proteins were transferredto nitrocellulose at 200 V and 10°C (Towbin et al., 1979).After transfering the proteins, the blots were immuno-stained. Nonspecific labeling was blocked with bovine se-rum albumin. The blots were incubated for 24–48 hr at4°C in a diluted (1:75–250 in phosphate buffer; PB) pri-mary antibody. The various antibodies used included anti-human (Upstate Biotechnology Inc., Lake Placid, NY; gen-erously provided by K. Flanders, NCI, Bethesda, MD) andanti-mouse (Oncogene, Boston, MA) TGF�1 antibodies,anti-chick (R&D Systems, Minneapolis, MN) and anti-human (Santa Cruz Biotechnology, Santa Cruz, CA)TGF�2 antibodies, an anti-mouse TGF�Ir antibody (On-cogene), or anti-rabbit (Santa Cruz Biotechnology) andanti-goat (Oncogene) TGF�IIr antibodies. Biotinylatedsecondary antibodies, streptavidin-horseradish peroxi-dase complex, and enhanced chemiluminescence detectionreagents (ECL; Amersham, Arlington Heights, IL) wereused to visualize the immunoreaction.

The amount of a protein on the immunoblots was deter-mined densitometrically (Mooney and Miller, 2000). Den-sitometric readings of target protein contents were nor-malized first against the actinin immunolabeling (tocontrol for equiloading of the samples) and then againstthe ligand or receptor expression in the internal stan-dards. The averaged value for the internal standards runon each gel was equated with that for internal standardsrun on other gels. Use of such a procedure permitted directcomparisons among animals within and among treatmentgroups.

Various controls for the immunochemical reactionswere performed. Nonspecific binding was assessed on im-munoblots that were processed without the primary orsecondary antibody. In addition, blots were prepared witha preabsorbed primary antibody that had been bound withspecific antigen. The results of these controls were consis-tently negative. After completion of the immunolabelingand imaging of the blots, the filters were stripped of im-munolabeling and reprobed with an anti-�-actinin anti-body (Upstate Biotechnology Inc.). Ethanol does not affect�-actinin expression. These preparations were used toensure that equal amounts of protein were loaded ontoeach lane.

Anatomical studies

TGF�1, TGF�2, and receptor immunoreactivity in thedeveloping somatosensory cortex was determined using aprocedure that is routine in our laboratory (see, e.g., Pe-ters et al., 1983; Miller, 1985; Pitts and Miller, 1995; Kuhnand Miller, 1996). The offspring of pregnant Et-, Ct-, andCh-fed rats were anesthetized with ketamine (60 mg/kgbw) and xylazine (7.5 mg/kg bw) and perfused transcardi-ally with 4.0% paraformaldehyde in 0.10 M PB (pH 7.4).Fetuses (on G16 or G20) and pups [on postnatal day (P) 0,P3, P6, P9, P12, P15, P30, or P60] were killed. Theirbrains were removed, cryoprotected, and cut into consec-utive 10-�m sections. The sections were divided into fiveseries consisting of every fifth section.

One series of sections was mounted and stained withcresyl violet. These sections were used to identify the

somatosensory cortex and its lamination (Miller and Vogt,1984; Miller, 1987a). Four series were prepared immuno-histochemically. The second and third series of sectionswere processed for TGF�1 and TGF�2 immunoreactivity,and the fourth and fifth series were immunoreacted forTGF� receptor expression using an avidin-biotin method.Sections were incubated for 30 min in a solution of 0.40%Triton X-100, 0.030% hydrogen peroxide, and 3.0% goatserum in PB to quench endogenous hydrogen peroxidaseactivity and to block nonspecific staining. After a quickwash in PB, sections were incubated with the same pri-mary antibodies used in the Western immunoblots dilutedat 1:1,000–3,000 with a solution of 3.0% goat serum in PB.Incubations were at 4°C for 16–20 hours. Then, the sec-tions were washed in 0.45% biotinylated anti-rabbit anti-body (Vector, Burlingame, CA) for 45 minutes, in 1.8%avidin-bound peroxidase complex (Vector) for 30 minutes,and finally in a solution of 0.15% diaminobenzidine (Sig-ma type VI) and 0.017% hydrogen peroxide for 7 minutes.The processed samples were dehydrated, cleared, and cov-erslipped. Micrographs of the slides were taken with aZeiss Axioplan photomicroscope, and images were com-piled and annotated with Abode Photoshop 5.5 and CorelDraw 9.0, respectively.

Controls for nonspecific binding (omission of the pri-mary or secondary antibodies) were performed. In addi-tion, two controls for specific binding were executed. Inone, a primary antibody was preabsorbed with an excessof full-length antigen and then used in the immunohisto-chemical processing. For the primary antibodies that werepolyclonal (i.e., anti-rabbit and anti-goat antibodies), somesections were processed with a preimmune serum. In allcases, no immunostaining was detected.

Statistical analysis

The data were analyzed by a Tukey B test for changesover time or by an analysis of variance followed by a posthoc Newman-Keuls test for analyses of ethanol effects.

RESULTS

Ligands

TGF�1.

Biochemical studies. TGF�1 was expressed through-out the period from G16 through P30 (Fig. 1). In controlrats, expression rose consistently and significantly (P �0.05), trebling over this period. Similar data were gener-ated with the human and mouse antibodies from UBI andOncogene, respectively; however, the signal with themouse antibody was the most robust.

Rats were treated with ethanol through the period ofneocortical neuronogenesis, i.e., G12–G21. Blood ethanolconcentrations on G16 were 138 � 18 mg/dl (n � 8). Thepattern of TGF�1 expression in Et-treated rats differedfrom that in the controls. TGF�1 expression was low fe-tally, peaked at the end of the first postnatal week, andthen dropped to barely detectable amounts during thesucceeding weeks. Ethanol induced a significant (P �0.05) depression in TGF�1 expression between G16 andP0 and after P12.

Anatomical studies. TGF�1-immunoreactive cellswere evident in the cerebral wall from G16 to P30 (Fig. 2,Table 1). Similar results were obtained with the two an-tibodies used. The data presented relied on the Flandersantibody.

412 M.W. MILLER

In the Ct-treated fetus, expression was weak and waslargely confined to the surface of the ventricular zone (VZ)and cells in the cortical plate (CP) and marginal zone(MZ). Labeling in the subventricular zone (SZ) appearedon G19 and was evident through much of the early post-natal period. At fetal and neonatal ages, it was difficult todetermine whether the labeled cells in the CP were neu-rons or glia, but, by P6, neuronal expression was discrim-inable. Immunolabeled cells were broadly distributedthrough cortex, and many had long processes that arosefrom the apex of the cell body and were directed towardthe pial surface. These are hallmarks of pyramidal neu-rons. It is noteworthy, however, that some small somata inthe white matter were TGF�1 immunolabeled. Presum-ably, these were glia. In the mature cortex, TGF�1 immu-nohistochemistry was expressed by neurons in all corticallayers and by scattered cells in the white matter.

The pattern of TGF�1 immunoreactivity in the Et-treated rats was like that in the controls. TGF�1 expres-sion in the fetus, neonate, and adult appeared to beweaker in the Et-treated rats. The most notable excep-tions were the increased expression in the fetal VZ and therelative dearth of TGF�1 immunolabeling in the SZ.

TGF�2.

Biochemical studies. The cortices of Ct-treated ratswere examined for TGF�2 expression; the strongest mes-sage in the immunoblots was obtained with an anti-human antibody. This expression was greatest during thelast week of gestation and the first postnatal week (Fig. 1).

The temporal expression of TGF�2 in the Et-treated ratswas identical to that in the controls. TGF�2 was most evi-dent during the week before and after birth. Expressionduring this time was significantly (P � 0.05) greater than itwas in 3- and 4-week-old animals. This time-dependent pat-tern of TGF�2 expression was particularly evident in theEt-treated rats. Early expression was threefold greater thanit was in the more mature pups. Interestingly, TGF�2 ex-pression was significantly (P � 0.05) greater in the Et-treated rats during this period of peak expression.

Anatomical studies. The pattern of TGF�2 expression(with use of an anti-human antibody) contrasted with thatfor TGF�1, both in timing and in the identity of immuno-reactive cells. During the last fetal week, TGF�2 immu-nolabeling in the neocortical VZ of Ct-treated fetuses wasweak (Fig. 3, Table 1). In neonates (i.e., on P0 and P3),however, TGF�2 positivity was richly expressed by radialfibers. That is, expression was detected in ventricular cellsand in processes of radial glia that extended well into thedeepest third of the CP. By P6, the radial arrays haddisappeared, and TGF�2 immunoreactivity was commonamong cells that exhibited an astrocytic morphology; i.e.,they had small cell bodies and short, branched processes(Fig. 4). These glia first appeared in the deep CP, and overtime their distribution included progressively more super-ficial strata. By the end of the second postnatal week,these cells were distributed through the cortical depth andin the subcortical white matter. Furthermore, neurons inadult rats exhibited TGF�2 immunoreactivity.

Prenatal exposure to ethanol induced three conspicuouseffects on TGF�2 immunolabeling (Fig. 3, Table 1). 1)Labeling in the VZ was more robust in Et-treated fetusesand neonates than in the age-matched controls. 2) TGF�2immunolabeling of the radial glia disappeared in Et-treated rats earlier than in controls. No TGF�2-positiveradial glia were detected in the intermediate zone of3-day-old Et-treated pups, whereas, in age-matched con-trols, the processes of such cells could be traced into theCP. 3) TGF�2-positive glia were evident in the CP earlierand at more superficial positions in the Et-treated con-trols. By P3, TGF�2-expressing cells were distributed inthe outermost segment of the CP.

Receptors

TGF�Ir.

Biochemical studies. The effects of prenatal exposureto ethanol on two receptors for the TGF� ligands (TGF�Irand TGF�IIr) were examined. Two anti-TGF�Ir antibod-ies revealed similar patterns of expression. TGF�Ir ex-pression followed a bimodal pattern in the cortices ofCt-treated rats (Fig. 5). That is, expression in the fetusand in 3- and 4-week-old pups was significantly (P � 0.05)greater than it was in neonates.

In the Et-treated rats, TGF�Ir expression continued toincrease over the period from G16 to P30 so that, by P30,expression was �150% of that detected in the fetus. Infact, TGF�Ir expression in the Et-treated rats was consis-tently and significantly (P � 0.05) higher in Et-treatedrats than in the controls.

Fig. 1. Expression of two TGF� ligands in developing cortex.Top: Pregnant dams were treated with an ethanol-containing dietor pair fed an isocaloric, isonutritive control diet. The expression ofTGF�1 and TGF�2 in the cortices of fetal and preweanling off-spring was examined with Western immunoblots. Bottom: Therelative amount of a protein (25 kDa, TGF�1; 12 kDa, TGF�2) wasdetermined microdensitometrically in five individual animals perage per treatment group. Each point (and bar) represents the meanthe five animals, each of which was taken from a different litter(�SEM). Asterisks denote statistically significant (P � 0.05) dif-ferences between treatment groups on a particular day. G, gesta-tional day; P, postnatal day.

413DEVELOPMENTAL EXPRESSION OF TGF� AND ITS RECEPTORS IN RAT CORTEX

Fig. 2. Effect of ethanol on the distribution of TGF�1-expressingelements in developing cerebral wall. Pregnant rats were fed a liquiddiet with ethanol (Et) or a liquid control diet (Ct). Sections of thecortices of their offspring were obtained on gestational day (G) 16 andpostnatal day (P) 0, P6, or P60. Ligand immunohistochemical labelingwas detected in the various compartments of the cerebral wall: the

ventricular zone (VZ; examples are indicated by straight open ar-rows), the subventricular zone (SZ; open arrowheads), the intermedi-ate zone (IZ), and the white matter (wm; curved arrows), cortical plate(CP), and CP derivatives (layers II–VI; straight solid arrows), and themarginal zone (MZ) and its derivative, layer I (solid arrowheads).Scale bars � 100 �m.

Anatomical studies. TGF�Ir was expressed in the de-veloping cerebral wall. In the fetus, immunolabeling wasmarked in somata in the VZ and within the CP (Fig. 6,Table 2). The labeling in the VZ was largely confined torounded cells that line the ventricular surface and bipolarcells oriented perpendicularly to the ventricular surfacethat are located within the inner third of VZ.

In the neonate, radial glia were TGF�Ir immunoposi-tive. These cells had labeled somata in the proliferativezones and processes that radiated toward and through theintermediate zone and could be traced into the deep CP.Only the rudiments in the vicinity of the proliferativezones were evident on P6. A similar pattern was evident inthe Et-treated rats; however, the radial glia were lessextensive and had almost totally regressed by P3.

At the end of the first postnatal week, immunopositivecells were distributed through all cortical laminae and inthe white matter. It appeared, based on somatic size, thatboth glia and neurons were labeled. By the third week,TGF�Ir-positive glia were no longer evident, and neuronalimmunolabeling had become largely restricted to layer Vpyramidal neurons. These were characterized by large cellbodies (among the largest in somatosensory cortex) andapical dendrites that extended toward the pial surface.

Ethanol treatment affected the character of corticalTGF�Ir expression during cortical development. As withthe controls, TGF�Ir positivity was evident in the VZ ofEt-treated fetuses. TGF�Ir continued to be expressed inEt-treated neonates; however, the pattern differed fromthat in the controls in three respects. 1) Fetal TGF�Irexpression in the IZ and CP was reduced by Et. 2) Et-treated rats did not exhibit TGF�Ir-immunoreactive ra-dial glial fibers. 3) Cell bodies in the intermediate zoneand the CP were TGF�Ir positive. This appeared to be anaccelerated expression of TGF�Ir by cortical cells, whichwas evident by the end of the first postnatal week. Thepattern of TGF�Ir-positive elements during the secondand ensuing postnatal weeks was similar in Et- and Ct-treated rats.

TGF�IIr.

Biochemical studies. As with TGF�Ir, TGF�IIr ex-pression rose steadily over the 5 weeks of cortical devel-opment from G16 to P30. This pattern was evident withboth antibodies used. Expression more than quadrupled inthe Ct-treated rats over this period. The same sequenceoccurred in the Et-treated rats. There were no significantdifferences between the two treatment groups.

Anatomical studies. In the fetus, TGF�IIr was ex-pressed by cells in the VZ and by scattered cells in the SZand the CP (Fig. 7, Table 2). In the neonate, immunola-beled cells were common through the full depth of the CPand the proliferative zones. By the end of the first postna-tal week, TGF�IIr-immunolabeled cells were distributedthrough the full depth of cortex. It appeared, based on thesize of their cell bodies and the morphology of their pro-cesses, that both neurons and glia were TGF�IIr positive,although the preponderance of the labeled cells appearedto be neurons. This perception is supported by the weaklabeling among glia in the white matter. Both the rabbitand the goat antibodies generated similar data, but themost robust expression was obtained with the rabbit an-tibodies.

Few ethanol-induced differences in the pattern ofTGF�IIr positivity were detected. Ethanol-inducedchanges in TGF�IIr expression included 1) a decrease inexpression in the VZ and 2) an increase in expressionwithin the fetal SZ; in addition, 3) the frequency of therounded cells at the ventricular surface appeared to behigher in Et-treated fetuses than in the controls, and 4)the numbers of immunoreactive cells in the IZ and CP ofthe perinate also appeared to be greater in Et-treated rats.By the end of the first postnatal week, however, no differ-ences in TGF�IIr positivity were evident.

DISCUSSION

The TGF� system changes dynamically in the develop-ing neocortex. The changing spatiotemporal patterns ofligand and receptor expression are consistent with thenotion that TGF� ligands affect both neuronal prolifera-tion and neuronal migration in vivo. Furthermore, prena-tal exposure to ethanol profoundly affects the expressionof TGF� ligands and receptors; these changes may under-lie Et-induced defects in early cortical development.

Effects of TGF� on cell proliferation

Most neocortical neurons are generated prenatally inzones lining the lateral ventricles, the VZ and SZ (Miller,1992; Luo and Miller, 1998). The present immunoblottingstudy shows that expression of TGF�1 and TGF�2 isconsiderable in the fetal rat cerebral wall. The comple-mentary immunohistochemical preparations show thatthe ligands are expressed in the fetal VZ and SZ. Further-more, the two TGF� receptors have overlapping distribu-

TABLE 1. Relative Expression of TGF� Ligands in the Developing Cerebral Wall1

Segment

Ct rats Et-treated rats

G16 P0 P6 P12 P60 G16 P0 P6 P12 P60

TGF�1Marginal zone/layer I �� �� �� � � � �� � � �Cortical plate/layers II–VI ���� ���� ���� ���� ���� ���� ���� ��� ��� ���Intermediate zone/white matter �� �� � � �� �� � �Subventricular zone �� �� Ventricular zone � ��� �� � � ��� ��� ��� � �

TGF�2Marginal zone/layer I �� �� �� �� �� �� ��Cortical plate/layers II–VI � �� �� �� �� �� �� ��Intermediate zone/white matter �� �� � � ��� � � �Subventricular zone �� � �� �� Ventricular zone � � �� � � ��� ���� ���� ��� ���

1Effects of prenatal exposure to a control (Ct) or ethanol-containing (Et) diet on ligand immunolabing are described. Higher relative amounts of labeling are indicated withincreasing numbers of plus signs. G, gestational day; P, postnatal day.

415DEVELOPMENTAL EXPRESSION OF TGF� AND ITS RECEPTORS IN RAT CORTEX

tions. Thus, the components of the TGF� system are ex-pressed by cells in the cortical proliferative zones.

Our data challenge those of previous immunohisto-chemical and in situ hybridization studies of the develop-ing murine CNS (Flanders et al., 1991; Pelton et al., 1991;Bottner et al., 2000). Such studies show that TGF� li-gands are not expressed in the neocortical VZ. Moreover,TGF�1 is not expressed in the developing cortex, andTGF�2 and TGF�3 expression is confined to glia. Other

investigators concluded, based on these findings, thatTGF� ligands do not affect neural cell proliferation. Sucha conclusion is contradicted by various evidence.

First, the present study and other studies (Flanders etal., 1991; Pelton et al., 1991) show that TGF�2, andTGF�3 are richly expressed in the SZ. The SZ is not just azone through which postmitotic VZ cells pass on theirmigration to the CP; it is an active proliferative zone fromwhich neurons are derived (Miller, 1989, 1992; Reznikov

Fig. 3. Fetal and neonatal expression of TGF�2. Fetal expressionof TGF�2 was restricted to the ventricular zone (VZ; straight openarrows). In Ct- and Et-treated neonates, radial glial fibers in thesubventricular zone (SZ; open arrowheads) and intermediate zone (IZ;curved arrows) were the most conspicuous TGF�2-positive elements.Immunostaining was also present in the VZ, particularly in Et-

treated neonates. Three days later, TGF�2-labeled cells were distrib-uted in the cortical plate (CP; straight solid arrows) and marginalzone (MZ; solid arrowheads). In the Et-treated rats, TGF�2-immunoreactive radial glial fibers in the IZ were no longer evident onP3. Scale bars � 100 �m.

416 M.W. MILLER

Fig. 4. Cortical expression of TGF�2 on postnatal days 6 and 60.Regardless of the prenatal treatment, TGF�2-positive cells on P6 areglia distributed through layer I (arrowheads) and layers II–VI(straight arrows) into the white matter (wm; curved arrows). Most

cells exhibited the morphology of the astrocytes shown in the enlarge-ments below (scale bars � 10 �m). On P60, not only were glia immu-nolabeled but neuronal perikarya also expressed TGF�2. Scale bars �100 �m.

417DEVELOPMENTAL EXPRESSION OF TGF� AND ITS RECEPTORS IN RAT CORTEX

et al., 1997; Luo and Miller, 1998; Doetsch et al., 1999;Temple and Alvarez-Buylla, 1999; Martens et al., 2000).

Second, the various in vitro studies show that prolifer-ation of neural precursors is affected by TGF� ligands.TGF�1 affects the proliferation of primary cultured corti-cal neurons (Miller and Luo, 2002a) and astrocytes (Luoand Miller, 1999) derived from rat neocortex. TGF�1 de-creases the growth in the numbers of cultured cells and intheir [3H]thymidine incorporation. TGF�2 also depressesthe proliferation of neurons, specifically, cerebellar gran-ule cells (Kane et al., 1996). It is appealing to generalizethat TGF�1 and TGF�2 act similarly on all cycling cells;however, the parallel inhibitory actions of TGF�1 andTGF�2 are not universal. These two ligands differentiallyaffect cell proliferation in the peripheral nervous system.Whereas TGF�1 inhibits cell proliferation of neural crest-derived cells (e.g., ciliary ganglion neurons) and has noeffect on the survival of these neurons, TGF�2 has theinverse effects (Flanders et al., 1991; Zhang et al., 1997).Nevertheless, the in vivo and in vitro evidence that TGF�ligands regulate the proliferation of cortical neurons iscompelling.

Ethanol affects the expression of the TGF�system in cortical proliferative zones

Proliferative zones are differentially affected by prena-tal exposure to ethanol. In the VZ, fetal expression of thetwo TGF� ligands is increased by prenatal exposure to

ethanol. Ethanol exposure also increases the expression ofthe TGF�Ir in the VZ. Expression of the TGF� system inthe SZ is less conspicuous than in the VZ. Nevertheless,ethanol reduces TGF�Ir expression in the SZ, whereasTGF�IIr expression is increased; possibly these effects arecompensatory. The two cortical proliferative zones aredifferentially affected by ethanol exposure (Miller, 1989,1995, 1996; Miller and Nowakowski, 1991). Cell prolifer-ation in the VZ is depressed by ethanol, whereas prolifer-ative activity in the SZ is increased. Insight into the basisof these differential effects comes from cell culture studiesof the effects of ethanol on TGF�-regulated cell prolifera-tion.

The effects of ethanol on the expression of TGF�1 mayunderlie the differential effects of ethanol on the two neo-cortical proliferative zones. Prenatally, TGF�1 was ex-pressed in the VZ of Et-treated rats. In primary cultures ofcortical neurons, the inhibitory effects of TGF�1 and eth-anol on neuronal precursors are additive (Miller and Luo,2002a). Therefore, the structure and function of TGF�1expression in fetal cortex are consistent with evidencethat ethanol exposure depresses cell proliferation in theVZ. In contrast, the Et-induced loss of TGF�1 expressionin the SZ of the perinate is consistent with findings thatethanol promotes cell proliferation in the SZ.

Role of TGF� in neuronal migration

Two components of the TGF� system, one ligand(TGF�2) and one receptor (TGF�Ir), are expressed byradial glia during the perinatal period, the period of neu-ronal migration in rat cortex (Berry and Rogers, 1965;Miller, 1988b). Radial glia are cells that span the distancebetween the ventricular and pial surfaces. They coordi-nate the inside-to-outside sequence of migration by whichyounger cortical neurons take positions in progressivelysuperficial positions (Angevine and Sidman, 1961; Berryand Rogers, 1965; Rakic, 1971; Miller, 1988b).

Radial glia are dynamic cells. They are present through(and even beyond) the period of neuronal migration (Choiand Lapham, 1978; Schmechel and Rakic, 1979; Pixleyand DeVellis, 1984; Voigt, 1989; Miller and Robertson,1993). It is generally believed, based on the paired disap-pearance of the radial glia and the emergence of astro-cytes, that radial glia transform into astrocytes. The ex-pression of both TGF�2 and TGF�Ir in radial glia andastrocytes is consistent with this notion. TGF�2 andTGF�Ir immunoreactivities are lost in radial glia about 6days before the fiber system disintegrates (present re-sults; Miller and Robertson, 1993). The concomitant ap-pearance of TGF�2- and TGF�Ir-positive astrocytes alsooccurs 3–6 days before they express glial fibrillary acidprotein (GFAP). The appearance of TGF�2 and TGF�Irimmunolabeling in the superficial cortex is particularlyprecocious. These data indicate 1) that the TGF� system isunrelated to the morphological transformation of radialglia to astrocytes and 2) that TGF�2 is a better marker forastrocytes than GFAP.

An alternative mechanism by which TGF� ligands canaffect neuronal migration is through nCAM. TGF�1 in-duces nCAM expression by primary cultured cortical as-trocytes (Luo and Miller, 1999) and by neurons (Miller andLuo, 2002a). nCAM facilitates the interaction betweenneurons and their glial guides (Hatten, 1990; Edelman,1992, 1994).

Fig. 5. In vivo expression of two TGF� receptors in developingcortex. Top: Western immunoblots show that the developing corticesof control and Et-treated rats express both TGF�Ir and TGF�IIr.Bottom: Relative expression of the two receptors in the immunoblotswas quantified. Notations as in Figure 1.

418 M.W. MILLER

Fig. 6. Ethanol affects the distribution of TGFIr expression indeveloping cortex. Cortices from the offspring of pregnant dams fedethanol (Et) or a control (Ct) diet. Samples were obtained on gesta-tional day (G) 16 and postnatal day (P) 0, 6, and 60. In the fetus,regardless of the prenatal treatment, immunolabeling was detectedalong the ventricular surface of the ventricular zone (VZ), in the

intermediate zone (IZ), and in the cortical plate (CP); noted bystraight open arrows, curved arrows, and straight solid arrows, re-spectively. On P0, TGF�Ir was expressed by radial glial fibers in theIZ (curved arrows). On P60, the principal type of cell labeled waspyramidal neurons in layer V (straight solid arrows) and their apicaldendrites (solid arrowheads). wm, White matter. Scale bars � 100 �m.

Effects of Ethanol on TGF�1-mediatedneuronal migration

Ethanol causes 1) an early disappearance of TGF�2-positive and TGF�Ir-positive radial glia and 2) an earlyappearance of immunolabeled astrocytes. These data con-cur with previous work showing that ethanol induces thepremature 1) loss of nestin-positive fibers and 2) appear-ance of GFAP-immunoreactive astrocytes (Miller and Rob-ertson, 1993). Inasmuch as the expressions of TGF�2 andTGF�Ir are lost from radial glial fibers days before thefibers disappear, it is likely that the ethanol-inducedchanges in the TGF� system are secondary to more globaleffects of ethanol on glial development. This does notmean that the TGF� system is not involved in neuronalmigration; quite the opposite.

Anatomical examination of control perinates shows thatTGF�2 and TGF�Ir immunoreactivity are strongest in theproliferative zones and the IZ. This suggests that theTGF� system is involved in the initiation and early mi-gration of cortical neurons. Studies on the effects of pre-natal exposure to ethanol on the kinetics of neuronal mi-gration support this conclusion. Ethanol 1) increases thetime during which postmitotic cells remain in the prolif-erative zones before commencing their migrations and 2)decreases the rate of neuronal migration (Miller, 1993).These facets of migration rely on neuron–radial glia inter-actions occurring in the proliferative zones and the IZ.Insofar as these locations are coincident with the distri-bution of TGF�2 and TGF�Ir, it is reasonable to speculatethat ethanol-induced changes in migration kinetics aretransduced through the TGF� system.

Ethanol can affect TGF�-regulated expression of CAMsthat mediate interactions between migrating neurons andradial glia. nCAM expression is increased by eitherTGF�1 or ethanol (Miller and Luo, 2002a, b). In contrast,combined treatment with TGF�1 and ethanol inhibitsnCAM expression. It is this inhibition that likely modelsthe ethanol-induced damage in vivo. In addition to TGF�1and TGF�2, ethanol affects other TGF� superfamily pro-teins that are implicated in cell adhesion and migration.These proteins include BMP and glial-derived neurotro-phic factor (GDNF). As with TGF�1, BMP (see, e.g., Per-ides et al., 1994; Ramanathan et al., 1996) and GDNF(Tang et al., 1998; Gattei et al., 1999; Murakami et al.,1999) also induce CAM expression. Ethanol affects thisCAM induction, although it is a manner that is oppositethe effect of ethanol on TGF�1-induced nCAM expression.Ethanol reduces BMP-mediated expression of L1, which

underlies the self-adhesion of neuroblastoma cells andfibroblasts (Ramanathan et al., 1996; Wilkemeyer et al.,1999) and decreases the translation of GNDF (McAlhanyet al., 1999).

In summary, ethanol can affect neuronal migrationthrough at least two mechanisms, through the physicalguidance system and through CAMs. TGF� ligands ap-pear to be involved in each of these processes.

Process outgrowth

The TGF� system is richly expressed in segments of theCP containing postmigratory neurons. As early as G16,neurons in the CP express TGF� ligands and receptors.Although we know that TGF�1 can promote nCAM ex-pression in dissociated neuronal (Miller and Luo, 2002a)or glial (Luo and Miller, 1999) cultures, we do not knowwhich of the cultured cells produce the nCAM, the prolif-erating or differentiating cells. Immunohistochemicalpreparations of untreated and ethanol-treated corticalslice cultures show that nCAM is expressed in postprolif-erative compartments (Seigenthaler, unpublished data).Thus, it appears that the part of the CP containing differ-entiating neurons coexpresses TGF�1, TGF�IIr, andnCAM. It is likely that this colocalization is more thancasual. Aside from mediating neuronal migration, nCAMcan promote neurite outgrowth (Long and Lemmon, 2000).Thus, the rich expression of the TGF� system in the CPmay be related to TGF�1 regulation of process outgrowthamong differentiating neurons.

Ethanol-induced reduction in TGF�1-promoted nCAMexpression may underlie ethanol-induced alterations inaxonal and dendritic growth. Ethanol does not affect theexpression of TGF�IIr and TGF�Ir is not expressed inneurons during the first postnatal week, when neurites ofcortical neurons are growing (Miller, 1988b). Hence, what-ever effects there are must result from changes in ligandexpression. Prenatal exposure to ethanol does reduceTGF�1 expression among CP neurons. These data are bestinterpreted in the context of studies of neuronal cultures(Miller and Luo, 2002a) in which TGF�1 is absent andonly ethanol is present. Under these circumstances,nCAM expression is increased. Potentially, therefore, pre-natal exposure to ethanol in vivo results in an ethanol-induced profusion of neurite outgrowth. There are, in fact,in vivo (Miller et al., 1990; Miller and Al-Rabiai, 1994;Miller et al., 1999) and in vitro (Clamp and Lindsley,1998) data indicating that ethanol induces the overpro-duction of both axons and dendrites.

TABLE 2. Relative Expression of TGF� Receptors in the Developing Cerebral Wall1

Segment

Ct rats Et-treated rats

G16 P0 P6 P12 P60 G16 P0 P6 P12 P60

TGF�IrMarginal zone/layer I � � � � � � � � � �Cortical plate/layers II–VI �� �� ���� ��� � �� �� ���� ��� �Intermediate zone/white matter � �� �� �� �� �� �� �� �� ��Subventricular zone � �� ��� �� � � �� ��� �� �Ventricular zone � �� �� � � � ��� ��� �� ��

TGF�IIrMarginal zone/layer I � � � � � � � � � �Cortical plate/layers II–VI � � ��� ��� ��� � ��� ��� ��� ���Intermediate zone/white matter � � � � � � � �Subventricular zone � ��� � Ventricular zone � � � � � � ��� ��� �� ��

1Notations as in Table 1.

420 M.W. MILLER

Fig. 7. Expression of TGF�IIr immunoreactivity in the developingcerebral wall. TGF�IIr-positive cells were detectable as early as ges-tational day (G) 16. Labeled cells were in the ventricular zone (VZ;straight open arrows) and cortical plate (CP; straight solid arrows). Asimilar pattern was evident on postnatal day (P) 0. In older pups,

TGF�IIr-positive cells were distributed in layer I (arrowheads), layersII–VI (straight arrows), and white matter (wm; curved arrows). Notreatment-induced differences were discernible at any age. SZ, sub-ventricular zone; MZ, marginal zone. Scale bars � 100 �m.

Relevance to fetal alcohol syndrome

The most profound effects of early exposure to ethanolare on the nervous system. These include hyperactivityand learning and memory deficits; in fact, prenatal expo-sure to ethanol is a leading cause of mental retardation(Abel and Sokol, 1992; Abel and Hannigan, 1995). Thebrain exhibits multiple abnormalities; the proliferation ofcortical neurons is hindered (Miller, 1989, 1997; Millerand Nowakowski, 1991), neurons are in the wrong place(Clarren et al., 1978; Miller, 1986, 1988a, 1993, 1997), andaberrant connections are formed among surviving neu-rons (see, e.g., Volk et al., 1981; Stoltenburg-Didinger andSpohr, 1983; Shapiro et al., 1984; Miller, 1987b, 1997;Al-Rabiai and Miller, 1989; Miller et al., 1990; Smith andDavies, 1990). TGF� regulates cell proliferation, neuronalmigration, and process outgrowth. The constellation ofethanol-induced effects on the developing nervous systemsuggests that the TGF� system is key to the damageassociated with fetal alcohol syndrome.

ACKNOWLEDGMENTS

Many thanks are due to Shirley Knapp, and JeffreyStaedler for processing the samples examined in thisstudy.

LITERATURE CITED

Abel EL, Hannigan JH. 1995. Maternal risk factors in fetal alcohol syn-drome: provocative and permissive factors. Neurotoxicol Teratol 17:445–462.

Abel EL, Sokol RJ. 1992. A revised conservative estimate of the incidenceof FAS and economic impact. Alcohol Clin Exp Res 15:514–524.

Angevine JB, Sidman RL. 1961. Autoradiographic study of cell migrationduring histogenesis of cerebral cortex in the mouse. Nature 192:766–768.

Asou H, Miura M, Kobayashi M, Uyemura K. 1994. The cell adhesionmolecule L1 has a specific role in neural cell migration. Neuroreport3:481–484.

Berry M, Rogers AW. 1965. The migration of neuroblasts in the developingcerebral cortex. J Anat 99:691–709.

Bottner M, Krieglstein K, Unsicker K. 2000. The transforming growthfactor-�s: structure, signaling, and roles in nervous system develop-ment and functions. J Neurochem 75:2227–2240.

Boyd F, Massague J. 1989. Transforming growth factor � inhibition ofepithelial cell proliferation linked to the expression of a 53 kDa mem-brane receptor. J Biol Chem 264:2272–2278.

Choi BH, Lapham LW. 1978. Radial glia in the human fetal cerebrum: acombined Golgi, immunofluorescence and electron microscope study.Brain Res 148:295–311.

Clamp PA, Lindsley TA. 1998. Early events in the development of neuronalpolarity in vitro are altered by ethanol. Alcohol Clin Exp Res 22:1277–1284.

Clarren SK, Alvord EC, Sumi SM, Streissguth AP, Smith DW. 1978. Brainmalformations related to prenatal exposure to ethanol. J Pediatr 92:64–67.

Crossin KL, Hoffman S, Tan SS, Edelman GM. 1989. Cytotactin and itsproteoglycan ligand mark structural and functional boundaries in so-matosensory cortex of the early postnatal mouse. Dev Biol 136:381–392.

Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A. 1999.Subventricular zone astrocytes are neural stem cells in the mammalianbrain. Cell 97:703–716.

Edelman GM. 1992. Mediation and inhibition of cell adhesion by morpho-regulatory molecules. Cold Spring Harbor Symp Quant Biol 57:317–325.

Edelman GM. 1994. Adhesion and counter adhesion: morphogenetic func-tions of the cell surface. Prog Brain Res 101:1–14.

Flanders KC, Ludecke G, Engels S, Cissel DS, Roberts AB, Kondaiah P,

Lafyatis R, Sporn MB, Unsicker K. 1991. Localization and actions oftransforming growth factor-�s in the embryonic nervous system. De-velopment 113:183–191.

Galter D, Bottner M, Unsicker K. 1999. Developmental regulation of theserotonergic transmitter phenotype in rostral and caudal raphe neu-rons by transforming growth factor-betas. J Neurosci Res 56:531–538.

Gattei V, Degan M, Rossi FM, De Iuliis A, Mazzocco FT, Cesa E, AldinucciD, Zagonel V, Pinto A. 1999. The RET receptor tyrosine kinase, but notits specific ligand, GDNF, is preferentially expressed by acute leukae-mias of monocytic phenotype and is up-regulated upon differentiation.Br J Haematol 105:225–240.

Hatten ME. 1990. Riding the glial monorail: a common mechanism forglial-guided neuronal migration in different regions of the developingmammalian brain. Trends Neurosci 13:179–184.

Hunter KE, Sporn MB, Davies AM. 1993. Transforming growth factor-�sinhibit mitogen-stimulated proliferation of astrocytes. Glia 7:203–211.

Jacobs JS, Miller MW. 2001. Proliferation and death of cultured fetalneocortical neurons: effects of ethanol on the dynamics of cell growth.J Neurocytol 30:391–401.

Kane CJ, Brown GJ, Phelan KD. 1996. Transforming growth factor-beta 2both stimulates and inhibits neurogenesis of rat cerebellar granulecells in culture. Dev Brain Res 96:46–51.

Kennedy LA, Elliott MJ. 1985. Cell proliferation in the embryonic mouseneocortex following acute maternal alcohol intoxication. Intl J DevNeurosci 3:311–315.

Kennedy LA, Mukerji S. 1986. Ethanol neurotoxicity. I. Direct effects onreplicating astrocytes. Neurobehav Toxicol Teratol 8:11–17.

Kuhn PE, Miller MW. 1996. Developmental expression of c-neu proteins incerebral cortex: relationship to epidermal growth factor receptor.J Comp Neurol 372:189–203.

Laemmli UK. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

Laiho M, DeCaprio JA, Ludlow JW, Livingston DM, Massague J. 1990.Growth inhibition by TGF-� linked to suppression of retinoblastomaprotein phosphorylation. Cell 62:175–185.

Le Roux P, Behar S, Higgins D, Charette M. 1999. OP-1 enhances dendriticgrowth from cerebral cortical neurons in vitro. Exp Neurol 160:151–163.

Lein P, Johnson M, Guo X, Rueger D, Higgins D. 1995. Osteogenicprotein-1 induces dendritic growth in rat sympathetic neurons. Neuron15:597–605.

Li W, Cogswell CA, LoTurco JJ. 1998. Neuronal differentiation of precur-sors in the neocortical ventricular zone is triggered by BMP. J Neurosci18:8853–8862.

Lieber CS, DeCarli LM. 1986. The feeding of ethanol in liquid diets. AlcoholClin Exp Res 10:550–553.

Lindner J, Rathjen FG, Schachner M. 1983. L1 mono- and polyclonalantibodies modify cell migration in early postnatal mouse cerebellum.Nature 305:427–430.

Long KE, Lemmon V. 2000. Dynamic regulation of cell adhesion moleculesduring axon outgrowth. J Neurobiol 44:230–245.

Luo J, Miller MW. 1996. Ethanol inhibits bFGF-mediated proliferation ofC6 astrocytoma cells. J Neurochem 67:1448–1456.

Luo J, Miller MW. 1998. Effects of ethanol on growth factor-mediatedproliferation of neural cells. Brain Res Rev 27:157–167.

Luo J, Miller MW. 1999. Transforming growth factor �1 (TGF�1) mediatedinhibition of B104 neuroblastoma cell proliferation and neural celladhesion molecule expression: effect of ethanol. J Neurochem 72:2286–2293.

Martens DJ, Tropepe V, van der Kooy D. 2000. Separate proliferationkinetics of fibroblast growth factor-responsive and epidermal growthfactor-responsive neural stem cells within the embryonic forebraingerminal zone. J Neurosci 20:1085–1095.

Massague J. 1998. TGF� signal transduction. Annu Rev Biochem 67:753–791.

McAlhany RE Jr, Miranda RC, Finnell RH, West JR. 1999. Ethanol de-creases glial-derived neurotrophic factor (GDNF) protein release butnot mRNA expression and increases GDNF-stimulated Shc phosphor-ylation in the developing cerebellum. Alcohol Clin Exp Res 23:1691–1697.

McKinnon RD, Piras G, Ida JA Jr, Dubois-Dalcq M. 1993. A role for TGF-�in oligodendrocyte differentiation. J Cell Biol 121:1397–1407.

Miller MW. 1985. Cogeneration of projection and local circuit neurons inneocortex. Dev Brain Res 23:187–192.

422 M.W. MILLER

Miller MW. 1986. Fetal alcohol effects on the generation and migration ofcerebral cortical neurons. Science 233:1308–1311.

Miller MW. 1987a. The origin of corticospinal projection neurons in rat.Exp Brain Res 67:339–351.

Miller MW. 1987b. Effect of prenatal exposure to ethanol on the distribu-tion and time of origin of corticospinal neurons in the rat. J CompNeurol 257:372–382.

Miller MW. 1988a. Effect of prenatal exposure to ethanol on the develop-ment of cerebral cortex: I. Neuronal generation. Alcohol Clin Exp Res12:440–449.

Miller MW. 1988b. Development of projection and local circuit neurons incerebral cortex. In: Peters A, Jones EG, editors. Cerebral cortex, vol 7.Development and maturation of cerebral cortex. New York: Plenum. p133–175.

Miller MW. 1989. Effect of prenatal exposure to ethanol on the develop-ment of cerebral cortex. II. Cell proliferation in the ventricular andsubventricular zones of the rat. J Comp Neurol 287:326–338.

Miller MW. 1992. Circadian rhythm of cell proliferation in the telence-phalic ventricular zone: effect of in utero exposure to ethanol. Brain Res595:17–24.

Miller MW. 1993. Migration of cortical neurons is altered by gestationalexposure to ethanol. Alcohol Clin Exp Res 17:304–314.

Miller MW. 1995. Generation of neurons in the rat dentate gyrus andhippocampus: effects of prenatal and postnatal treatment with ethanol.Alcohol Clin Exp Res 19:1500–1509.

Miller MW. 1996. Limited ethanol exposure selectively alters the prolifer-ation of precursors cells in cerebral cortex. Alcohol Clin Exp Res 20:139–143.

Miller MW. 1997. Effects of prenatal exposure to ethanol on callosal pro-jection neurons in rat somatosensory cortex. Brain Res 766:121–128.

Miller MW, Al-Rabiai S. 1994. Effects of prenatal exposure to ethanol onthe number of axons in the pyramidal tract of the rat. Alcohol Clin ExpRes 18:346–354.

Miller MW, Kuhn PE. 1995. Cell cycle kinetics in fetal rat cerebral cortex:effects of prenatal exposure to ethanol assessed by a cumulative label-ing technique with flow cytometry. Alcohol Clin Exp Res 19:233–237.

Miller MW, Luo J. 2002a. Effects of ethanol and basic fibroblast growthfactor (bFGF) on the transforming growth factor �1 (TGF�1) regulatedproliferation of cortical astrocytes and C6 astrocytoma cells. AlcoholClin Exp Res 26:671–675.

Miller MW, Luo J. 2002b. Effects of ethanol on transforming growth factor� (TGF�) mediated neuronal proliferation and nCAM expression. Al-cohol Clin Exp Res 26:1073–1079.

Miller MW, Muller SJ. 1989. Structure and histogenesis of the principalsensory nucleus of the trigeminal nerve: effects of prenatal exposure toethanol. J Comp Neurol 282:570–580.

Miller MW, Nowakowski RS. 1991. Effect of prenatal exposure to ethanolon the cell cycle kinetics and growth fraction in the proliferative zonesof the fetal rat cerebral cortex. Alcohol Clin Exp Res 15:229–232.

Miller MW, Robertson S. 1993. Prenatal exposure to ethanol alters thepostnatal development and transformation of radial glia to astrocytesin rat somatosensory cortex. J Comp Neurol 337:253–266.

Miller MW, Vogt BA. 1984. Direct connections of rat visual cortex withsensory, motor, and association cortices. J Comp Neurol 226:184–202.

Miller MW, Chiaia NL, Rhoades RW. 1990. Intracellular recording andlabeling study of corticospinal neurons in the rat somatosensory cortex:Effect of prenatal exposure to ethanol. J Comp Neurol 296:1–15.

Miller MW, Astley SJ, Clarren SK. 1999. Number of axons in the corpuscallosum of the mature Macaca nemestrina: effect of prenatal exposureto ethanol. J Comp Neurol 412:123–131.

Minana R, Sancho-Tello M, Climent E, Segui JM, Renau-Piqueras J,Guerri C. 1998. Intracellular location, temporal expression, and poly-sialylation of neural cell adhesion molecule in astrocytes in primaryculture. Glia 24:415–427.

Minana R, Climent E, Barettino D, Segui JM, Renau-Piqueras J, Guerri C.2000. Alcohol exposure alters the expression pattern of neural celladhesion molecules during brain development. J Neurochem 75:954–964.

Miura M, Asou H, Kobayashi M, Uyemura K. 1992. Functional expressionof a full-length cDNA coding for rat neural cell adhesion molecule L1mediates homophilic intercellular adhesion and migration of cerebellarneurons. J Biol Chem 267:10752–107528.

Mooney SM, Miller MW. 2000. Expression of bcl-2, bax, and caspase-3 inthe CNS of the developing rat. Dev Brain Res 123:103–117.

Moses HL, Tucker RF, Leof EB, Coffey RJ, Halper J, Shipley GD. 1985.Type-� transforming growth factor is a growth stimulator and a growthinhibitor. Cancer Cells 3:65–71.

Murakami H, Iwashita T, Asai N, Iwata Y, Narumiya S, Takahashi M.1999. Rho-dependent and independent tyrosine phosphorylation of fo-cal adhesion kinase, paxillin and p130Cas mediated by Ret kinase.Oncogene 18:1975–1982.

Pelton RW, Saxena B, Jones M, Moses HL, Gold LI. 1991. Immunohisto-chemical localization of TGF�1, TGF�2, and TGF�3 in the mouseembryo: expression patterns suggest multiple roles during embryonicdevelopment. J Cell Biol 115:1091–1105.

Pentney R, Miller MW. 1992. Effects of ethanol on neuronal morphogene-sis. In: Miller MW, editor. Development of the central nervous system:effects of alcohol and opiates. New York: Wiley-Liss. p 47–69.

Perides G, Safran RM, Downing LA, Charness ME. 1994. Regulation ofneural cell adhesion molecule and L1 by the transforming growthfactor-beta superfamily. Selective effects of the bone morphogeneticproteins. J Biol Chem 269:765–770.

Peters A, Miller MW, Kimerer LM. 1983. Neurons with cholecystokinin-like immunoreactivity in rat neocortex. Neuroscience 8:431–448.

Phillips DE. 1989. Effects of limited postnatal ethanol exposure on thedevelopment of myelin and nerve fibers in rat optic nerve. Exp Neurol103:90–100.

Phillips DE, Krueger SK, Rydquist JE. 1991. Short- and long-term effectsof combined pre- and postnatal ethanol exposure (three trimesterequivalency) on the development of myelin and axons in rat optic nerve.Int J Dev Neurosci 9:631–647.

Pinazo-Duran MD, Renau-Piqueras J, Guerri C. 1993. Developmentalchanges in the optic nerve related to ethanol consumption in pregnantrats: analysis of the ethanol-exposed optic nerve. Teratology 48:305–322.

Pitts AF, Miller MW. 1995. Expression of nerve growth factor, p75, and trkin the somatosensory-motor cortices of mature rats: evidence for localcortical support circuits. Somatosens Motor Res 12:329–342.

Pixley SKR, DeVellis J. 1984. Transition between immature radial glia andmature astrocytes studies with a monoclonal antibody to vimentin. DevBrain Res 15:201–209.

Polyak K. 1996. Negative regulation of cell growth by TGF�. BiochimBiophys Acta 1242:185–199.

Rakic P. 1972. Mode of cell migration to the superficial layers of fetalmoneky neocortex. J Comp Neurol 145:61–83.

Ramanathan R, Wilkemeyer MF, Mittal B, Perides G, Charness ME. 1996.Alcohol inhibits cell–cell adhesion mediated by human L1. J Cell Biol133:381–390.

Reznikov K, Acklin SE, van der Kooy D. 1997. Clonal heterogeneity in theearly embryonic rodent cortical germinal zone and the separation ofsubventricular from ventricular zone lineages. Dev Dyn 210:328–343.

Roberts AB, Sporn MB. 1990. The transforming growth factor-betas. In:Sporn MB, Roberts AB, editors. Peptide growth factors and their re-ceptors. Handbook of experimental pharmacology. Berlin: SpringerVerlag. p 419–472.

Ryken TC, Traynelis VC, Lim R. 1992. Interaction of acidic fibroblastgrowth factor and transforming growth factor-beta in normal andtransformed glia in vitro. J Neurosurg 76:850–855.

Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratorymanual. Cold Spring Harbor, NY: Cold Spring Harbor LaboratoryPress.

Schmechel DE, Rakic P. 1979. A Golgi study of radial cells in developingmonkey telencephalon: morphogenesis and transformation into astro-cytes. Anat Embryol 156:115–152.

Scott JD, Soderling TR. 1992. Serine/threonine protein kinases. Curr OpinNeurobiol 2:289–295.

Shapiro MB, Rosman NP, Kemper TL. 1984. Effects of chronic exposure toalcohol on the developing brain. Neurobehav Toxicol Teratol 6:351–356.

Smith DE, Davies DL. 1981. Effect of perinatal administration of ethanolon the Ci pyramidal cells of the hippocampus and the Purkinje cell ofthe cerebellum: an ultrastructural survey. J Neurocytol 19:708–717.

Snyder AK, Singh SP, Ehmann S. 1992. Effects of ethanol on DNA, RNA,and protein synthesis in rat astrocyte cultures. Alcohol Clin Exp Res16:295–300.

Stoltenburg-Didinger G, Spohr HL. 1983. Fetal alcohol syndrome andmental retardation: spine distribution of pyramidal cells in prenatalalcohol-exposed rat cerebral cortex. A Golgi study. Dev Brain Res1:119–123.

423DEVELOPMENTAL EXPRESSION OF TGF� AND ITS RECEPTORS IN RAT CORTEX

Tang MJ, Worley D, Sanicola M, Dressler GR. 1998. The RET-glial cell-derived neurotrophic factor (GDNF) pathway stimulates migration andchemoattraction of epithelial cells. J Cell Biol 142:1337–1345.

Temple S, Alvarez-Buylla A. 1999. Stem cells in the adult mammaliancentral nervous system. Curr Opin Neurobiol 9:135–141.

Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of pro-teins from polyacrylamide gels to nitrocellulose sheets: procedure andsome applications. Proc Natl Acad Sci USA 76:4350–4354.

Tsuzuki T, Izumoto S, Ohnishi T, Hiraga S, Arita N, Hayakawa T. 1998.Neural cell adhesion molecule L1 in gliomas: correlation with TGF-betaand p53. J Clin Pathol 51:13–27.

Unsicker K, Flanders KC, Cissel DS, Lafyatis R, Sporn MB. 1991. Trans-forming growth factor beta isoforms in the adult rat central and pe-ripheral nervous system. Neuroscience 44:613–625.

Vavrousek-Jakuba E, Baker RA, Shoemaker WJ. 1991. Effect of ethanol onmaternal and offspring characteristics: comparison of three liquid dietformulations fed during gestation. Alcohol Clin Exp Res 15:129–135.

Vergeli M, Mazzanti B, Ballerini C, Gran B, Amaducci L, Massacesi L.1995. Transforming growth factor-beta 1 inhibits the proliferation ofrat astrocytes induced by serum and growth factors. J Neurosci Res40:127–133.

Voigt T. 1989. Development of glial cells in the cerebral wall of ferrets:

direct tracing of their transformation from radial glia into astrocytes.J Comp Neurol 289:74–88.

Volk B, Maletz J, Tiedemann M, Mall G, Klein C, Berlet HH. 1981.Impaired maturation of Purkinje cells in the fetal alcohol syndrome ofthe rat. Acta Neuropathol 54:19–29.

Wilkemeyer MF, Pajerski M, Charness ME. 1999. Alcohol inhibition of celladhesion in BMP-treated NG108-15 cells. Alcohol Clin Exp Res 23:1711–1720.

Wilkemeyer MF, Sebastian AB, Smith SA, Charness ME. 2000. Antago-nists of alcohol inhibition of cell adhesion. Proc Natl Acad Sci USA97:3690–3695.

Wrana JL, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, Wand XF,Massague J. 1992. TGF� signals through a heterodimeric protein ki-nase receptor complex. Cell 71:1003–1014.

Wrana JL, Attisano L, Wieser R, Venture F, Massague J. 1994. Mechanismof activation of the TGF� receptor. Nature 370:341–347.

Zhang JM, Hoffman R, Sieber-Blum M. 1997. Mitogenic and anti-proliferative signals for neural crest cells and the neurogenic action ofTGF�1. Dev Dyn 208:375–386.

Zhu H, Wu F, Schacher S. 1995. Changes in expression and distribution ofAplysia cell adhesion molecules can influence synapse formation andelimination in vitro. J Neurosci 15:4173–4183.

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