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Expression of DA11, a Neuronal-Injury–InducedFatty Acid Binding Protein, CoincidesWith Axon Growth and Neuronal DifferentiationDuring Central Nervous System DevelopmentYi Liu, 1Carlos A. Molina,1Andrew A. Welcher,2 Lawrence D. Longo,3and Marino De Leon1*1Department of Physiology and Pharmacology, Center for Molecular Biology and Gene Therapy, Department ofPathology and Human Anatomy, and Center for Perinatal Biology, Loma Linda University School of Medicine,Loma Linda, California2Department of Immunology, Amgen, Inc., Thousand Oaks, California3Center for Perinatal Biology, Department of Physiology and Pharmacology, Loma Linda University School ofMedicine, Loma Linda, California

DA11 is the first fatty acid binding protein (FABP) forwhich gene expression has been shown to be upregu-lated following neuronal injury in the adult peripheralnervous system. To understand better the potentialregulatory role(s) of this unique FABP in axonalgrowth and neuronal differentiation, we undertook atemporal and spatial study of DA11 gene expression inthe developing rat central nervous system (CNS).Transient upregulation of DA11 mRNA and proteinlevels in CNS tissues were quantified by Northern blothybridization and Western immunoblot analyses atdifferent developmental ages. Homogenates of embry-onic and neonatal cerebral cortex, cerebellum, brain-stem, and hippocampal tissues contained 100-foldmore DA11 mRNA and protein than correspondingadult tissues. Significant increase in DA11 mRNAwasobserved as early as embryonic day (E) 14 in cerebralcortex and cerebellum and E19 in brain stem andhippocampus. Postnatal levels of DA11 remainedelevated through postnatal day (P) 10 in cerebralcortex, P14 in brain stem and hippocampus, and P20in cerebellum. Localization of DA11-like immunoreac-tivity to specific CNS tissues, cell types, and intracellu-lar compartments at P9 revealed a spatial pattern ofneuronal expression different than that reported forother FABPs. DA11 protein was detected in thenucleus, cytoplasm, axons, and dendrites of differenti-ating neurons in cerebral cortex, hippocampus, cerebel-lum, brain stem, spinal cord, and olfactory bulb. Thestrong association of DA11 gene expression withdevelopment throughout the CNS suggests that thisunique FABP plays an important role in axonal

growth and neuronal differentiation in many differentneuronal populations. J. Neurosci. Res. 48:551–562,1997. r 1997 Wiley-Liss, Inc.

Key words: DA11; fatty acid binding protein; FABP;CNS development; axon growth; neuron differentia-tion; fetal and newborn brain; nerve regeneration;neuronal injury

INTRODUCTIONA major focus of developmental neuroscience is to

determine the molecular events underlying the growthand differentiation of the central nervous system (CNS).These events require the regulated expression of genesthat coordinate important cellular processes, i.e., neuro-genesis, neuron and glial proliferation and differentiation,neurite extension and synaptogenesis (reviewed by Patter-son and Nawa, 1993; Bronner-Fraser, 1994; Simpson,1994).Analysis of rat CNS has identified when and wherethese processes occur during development (Mangin et al.,1989; Zheng et al., 1996). The gene regulatory eventsinvolved in these processes must now be identified.

Considerable evidence suggests that gene regula-tory events that occur during development are recapitu-

Contract grant sponsor: National Institutes of Health; Contract grantnumber: HD03807-Supplement.

*Correspondence to: Dr. Marino De Leo´n, Department of Physiologyand Pharmacology, Loma Linda University, Loma Linda, CA, 92350.E-mail: mdeleon@ccmail.llu.edu

Received 14 January 1997; Revised 18 February 1997; Accepted 3March 1997

Journal of Neuroscience Research 48:551–562 (1997)

r 1997 Wiley-Liss, Inc.

lated following neuronal injury (Bixby, 1991; Aubert etal., 1995; Brecknell and Fawcett, 1996). Peripheral nerveregeneration can be considered a developmental processin which the axotomized neuron rebuilds its own axonand reestablishes its connection to its target tissues(synaptogenesis; Fawcett and Keynes, 1990). Genesinduced during both development and regeneration (Levi-Montalcini, 1987; Skene, 1989; Thoenen, 1995) are likelyto be involved in axonal growth and synapse formation(Skene, 1989; Gispen et al., 1990). Genes encodingApoE(Ignatius et al., 1986), growth-associated protein 43(B50; Skene, 1989; Mahalik et al., 1992), andmembers ofthe neurotrophins family and their receptors (reviewed bySnider, 1994; Thoenen, 1995) meet this criterion.

Previously, we reported the cloning of DA11 from arat dorsal root ganglion (DRG) cDNA library (De Leo´n etal., 1996). The upregulation of DA11 gene expression,which occurred after axotomy (De Leo´n et al., 1996), wastemporally associated with sciatic nerve regeneration(Molina et al., 1996), thus predicting DA11 participationin axonal growth. DA11 appears to be an isoform of afatty acid binding protein (FABP) isolated from keratino-cytes from human psoriatic patients, rat skin (Madsen etal., 1992;Watanabe et al., 1994), or multistage carcinogen-esis in mouse skin (Krieg et al., 1993). FABPs are afamily of small (14–15 kDa) cytosolic proteins that bindhydrophobic ligands, such as long-chain free fatty acids(FFAs) and retinoic acids (RA; reviewed by Veerkampand Maatman, 1995). Some FABPs regulate access ofthese ligands to their respective cellular receptors (Knud-sen, 1990; Minucci et al., 1996) and facilitate thetransport of FFAs to intracellular organelles (Fournier andRichard, 1988, 1990). The function of FABPs expressedin the nervous system, such as DA11, may be required forneuronal development.

In the present study, we investigated the temporaland spatial expression of DA11 during development ofthe rat CNS. Northern blot hybridization and Westernimmunoblot analyses quantified mRNA and protein lev-els in selected tissues at specific developmental ages.Immunocytochemistry localized DA11-like immunoreac-tivity (DA11-LI) within selected tissues, cell types, andintracellular compartments. The correlation of DA11gene expression with development throughout the CNSsuggests that this unique FABP plays an important role inaxonal growth and neuronal differentiation in manydifferent neuronal populations.

METHODSAnimal Manipulations

The animal protocol was reviewed and approved bythe Animal Care and Use Committee of Loma Linda

University. Timed pregnant Sprague-Dawley rats werekilled in a CO2 atmosphere to isolate embryos at differentstages of gestation (embryonic days 14–21; E14–E21).Postnatal day (P) 1 is the day of birth. Brain tissues weredissected on ice into cerebral cortex, cerebellum, hippo-campus, brain stem, and spinal cord specimens and thenfrozen quickly on dry ice. Specimens were stored at280°C until use for Northern blot hybridization andWestern immunoblot experiments. Postnatal rats (P9)used for immunocytochemistry (ICC) were anesthetizedwith ketamine (80 mg/kg body weight) and xylazine (60mg/kg body weight) and perfused intracardially with 4%paraformaldehyde in 0.1 M potassium phosphate buffer(pH 7.4). Brains were dissected out and postfixed for anadditional 2 hr in the same fixative solution used forperfusion (4% paraformaldehyde in phosphate bufferedsaline; PBS).

Northern Blot Hybridization AnalysisTotal RNAwas isolated from tissue homogenates of

brain specimens according to the method of Chomczyn-ski and Sacchi (1987), and the Northern blots procedureswere done as described by De Leo´n et al. (1995, 1996).Twenty-five micrograms of each total RNA (isolatedfrom the same brain region of either one or two rats at thesame developmental age) were loaded per lane in chrono-logical order from left to right in a 1% formaldehydeagarose gel (IBI Technical). Following gel electrophore-sis, fractionated RNAs were transferred to a Hybond-Nnylon membrane (blot; Amersham, Arlington Heights,IL) and ultraviolet cross-linked (IBI UltraLinker). Four tosix independent blots were prepared for quantitativecomparison of DA11 mRNA levels in each tissue. Eachblot was hybridized for 16–24 hr with a32P-labeledcDNA DA11 probe (0.7 kb) and washed at a highstringency (final wash: 0.13 saline sodium phosphate/EDTA (pH 7.4)/0.1% sodium dodecyl sulfate at 65°C).The blots were exposed to either a phosphor screen andanalyzed by a phosphorimager (BioRad, Hercules, CA)or X-ray films and analyzed by a densitometer (Bioimage, B.I. Systems Corporation, AnnArbor, MI).

Differences in RNA loading between lanes on thesame blot were estimated by reprobing the blot with32P-labeled cyclophilin cDNA. To compare the differ-ences between blots, normalized DA11 levels for eachdevelopmental age were expressed as a percentage of themaximal expression level observed for that blot. Themean (6SEM) for the percentage of maximal expressionin each region of the brain (N5 4–6 blots) was thencalculatedandplotted for eachdevelopmental age.Significantdifferences between percentage of maximal values of DA11mRNA at different developmental ages were determined byusing one-way analysis of variance (ANOVA) with the post

552 Liu et al.

hoc Fisher multiple comparison method.P , 0.05 wasconsidered significant.

Western Immunoblot AnalysisProtein was extracted from brain specimens as

previously described (De Leo´n et al., 1995), and proteinconcentrations were determined according to the methodof Lowry et al. (1951). Twenty micrograms of proteinwere loaded per lane in 4–20% sodium dodecyl phosphate-polyacrylamide gel electrophoresis gels (NOVEX SanDiego, CA) under reducing conditions. Following gelelectrophoresis, the fractionated proteins were transferredin blotting buffer (NOVEX, San Diego, CA; 20% metha-nol, 20 mM Tris, and 150 mM glycine) to Immobilonmembranes (NitroBind, Millipore, Bedford, MA) for 1 hrby using a semidry blotter (Labconco, Kansas City, MI).The membranes (immunoblots) were allowed to dry for10–15 min and blocked for 4 hr with 5% bovine serumalbumin in 0.1% Tween-20 in PBS. The blocked mem-branes were washed three times with PBS/0.05%Tween-20 and then incubated with DA11 antiserum (DeLeon et al., 1996) (1:500 dilution) overnight at 4°C. Thewashed membranes were incubated with the biotinylatedsecondary antibody (1:1,000 dilution, 30 min at roomtemperature) followed by streptavidin and horseradishperoxidase [1:1,000 dilution in PBS/0.1% Tween-20(Fisher Biotech), 30 min at room temperature] (Vec-tastain ABC Kit, Vector Laboratories, Inc., Burlingame,CA). The reaction was developed with ECL reagents(Amersham). Immunoblots were exposed to film andDA11-LI quantified with a densitometer (Bio image, B.I.Systems Corporation).

ImmunocytochemistryThe ICC procedure used the free-floating technique,

as described previously (De Leo´n et al., 1995, 1996).Tissue sections were treated as described in the ABC kit(Vector Laboratories). Secondary antibodies were used ata 1:200 dilution and the avidin reagents at a 1:400dilution in PBS. Tissue sections were incubated withfreshly prepared 3,38-diaminobenzidine tetrahydrochlo-ride (DAB) in 0.1 M Tris buffer for 5 min for colordevelopment. Sections used for the double-labeling experi-ments were incubated with the glial fibrillary acid protein(GFAP) antiserum overnight, as described for the DA11antibody. The ABC kit with the Vector VIP substrate(Vector Laboratories) was used to develop a purple colorindicative of GFAP-like immunoreactivity (GFAP-LI).Cells positive for DA11 exhibited a brown color; thoseimmunoreacting with GFAP exhibited a distinct purplecolor. Control tissues were incubated without DA11antibody or with DA11 antibody preabsorbed with thesame DA11 peptide used to generate the antibody.

RESULTS

Temporal Expression of DA11 mRNA and Proteinand Localization of DA11-LI in the DevelopingCerebral Cortex

Embryonic and early postnatal rat cerebral cortex(CX) contained higher levels of DA11 mRNA than adult(A) CX (Fig. 1A,B). Expression of DA11 mRNA wasupregulated as early as E14, peaked around E19, signifi-cantly dropped after P10, and was at the lowest level inadult tissues (,5% of maximal expression). The relativelevels of DA11 mRNA observed from E14 to P10 weresignificantly greater (P, 0.05) than those observed fromP12 to adulthood. As expected from the temporal expres-sion of DA11 mRNA, embryonic and postnatal rat CXcontained considerably higher levels (.100-fold) ofDA11 protein (ca. 15 kDa) than adult rat CX (Fig. 3A).DA11 protein was detected as early as E16, and highlevels were observed from E21 through P12. After P3,DA11 protein levels progressively decreased with age tonegligible levels in the adult CX.

Immunocytochemical analysis of the high levels ofDA11 protein in CX at P9 further localized DA11 tospecific cell types (Fig. 4A–C). Control tissues incubatedwithout DA11 antibody or with DA11 antibody preab-sorbed with the same DA11 peptide used to generate theantibody showed no DA11-LI (data not shown). TheDA11 antibody reacted with the cell body (cytoplasm andnucleus) and to a lesser extent with dendrites and axons ofneurons in the cortical plate (Fig. 4C). Furthermore, theDA11 antibody showed differential staining in the CX,i.e., not all immunoreactive neurons exhibited the samedegree of labeling and not all neurons exhibited DA11-LI(Fig, 4A,B). Double-labeling experiments using DA11and GFAP antibodies failed to show coexpression of bothproteins in the same cells. This observation suggests thatDA11-LI is distinctly associated with neurons (Fig.4A,B).

The immunoreactive pattern exhibited by the DA11antibody identified several waves of migrating neurons(Fig. 4A) along the glial fibers (Fig. 4B) across theintermediate zone (IZ; Fig. 4A) and in the cortical plate(CP; Fig. 4A). DA11-LI was observed in more cells in theCP region than in the IZ and molecular layer (ML; Fig.4A). Interestingly, the DA11 antibody immunoreactedwith more cells in the CP region contiguous to the regionadjacent to the ML than in the region bordering the IZ(Fig. 4A). The pyramidal cells, identified by their longapical dendrites pointing to the surface of the CX, werethe major cell type detected with the DA11 antibody (Fig.4C).

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Temporal Expression of DA11 mRNA and Proteinand Localization of DA11-LI in the DevelopingCerebellum

The level of DA11 mRNA in cerebellum (CB)showed a temporal expression pattern similar to CXduring development (Fig. 1C,D). Relative DA11 mRNAlevels observed from E14 to P20 were significantly higher(P , 0.05 by ANOVA with post hoc comparisons) thanthose observed after P20 to adulthood. The DA11 mRNAreached its highest level between E18 and E21, followedby a gradual decline through the first 20 days of postnatalmaturation and resulting in very low levels by P27 and

adult. Adult CB contained less than 5% of the amount ofDA11 mRNA detected at E18. Relative protein levelsparalleled mRNA levels with higher DA11 protein inembryonic and early neonatal CB as opposed to adult CB(Fig. 3B;.100-fold greater than in the adult). In contrastto the CX, high levels of DA11 protein was still present atP14 (Fig. 3B).

DA11-LI in the CB was associated predominantlywith the Purkinje cell layer (Fig. 4D). Only occasionalcellular staining was observed in the granular layer, theML, and the white mater (Fig. 4D). The DA11-LI wasalso observed in the leptomeningeal cells of the pia and

Fig. 1. Temporal analysis of DA11 mRNA expression incerebral cortex and cerebellum during rat CNS development.A: Representative Northern blot of cerebral cortex.B: Quantifi-cation of DA11 mRNA in the cerebral cortex.C: Representa-tive Northern blot of cerebellum.D: Quantification of DA11mRNA in the cerebellum. E, embryonic day; P, postnatal day;A, adult. P1 is the day of birth. The32P-labeled DA11 cDNAprobe recognized a single 0.6–0.7-kb mRNA band characteris-tic for DA11 (upper band). Hybridization of a second probe to

the cyclophilin mRNA (lower band) verified RNA integrity andprovided for normalization of DA11 mRNA levels (to accountfor variation in the loading of 25 µg total RNA/lane). Therelative amount of DA11mRNAat each developmental age wasmeasured in units of percentage of maximal value for each blot.Significant differences (*) between groupmean values (6SEM)for four to six independent blots was determined by ANOVAwith post hoc Fisher multiple comparisons (P, 0.05).

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arachnoid mater (Fig. 4D). The double-labeling experi-ments confirmed the presence of DA11-LI associatedwith the Purkinje cells and only occasionally withgranular cells. Glial cells, which showed GFAP-LI,exhibited a distinct purple color (Fig. 4D). We did notobserve any overlap between the brown color of the DABsubstrate used for DA11 antibody and the purple color ofthe substrate used for the GFAP antibody. The DA11immunostaining was associated primarily with the somaof the Purkinje cell; however, there was a moderate levelof DA11-LI associated with the initial part of the dendritetrees and the axonal projections (Fig. 4E).

Temporal Expression of DA11 mRNA and Proteinand Localization of DA11-LI in the Developing BrainStem

DA11 mRNA (Fig. 2A,B) and protein levels (Fig.3C) in the brain stem (medulla oblongata and pons) werealso upregulated in embryonic and early postnatal ratbrain, and the mRNA dropped signicantly by P20 (Fig.2B). The DA11 antibody immunoreacted with the somaand processes of neurons in brain stem nuclei, such as themesencephalic tract of the trigeminal nerve, the motornucleus of the trigeminal (V) nerve, the lateral vestibularnucleus, the facial nerve (VII), and the acoustic nerve(VIII; data not shown). Neurons in the superior olivarycomplex nuclei showed a prominent nuclear localizationof DA11 (Fig. 5A). Other nuclei exhibiting DA11-LIinclude the nucleus of the spinal tract of the trigeminal(V) nerve, superior and inferior vestibular nuclei, thelateral parabrachial nucleus, and the dorsal cohlear (datanot shown). In addition to DA11-LI associated with cellbodies, a strong DA11-LI was also observed in thereticular formation, which is composed mostly of anetwork of nerve fibers (Fig. 5B). The nerve fiber stainingwas also observed in other areas such as the medial andinferior cerebellar peduncles, the medial and laterallemniscus, the inferior colliculus, and the spinal tract ofthe trigeminal nerve (V; data not shown).

Temporal Expression of DA11 mRNA andLocalization of DA11-LI in the DevelopingHippocampus

DA11 mRNA was significantly upregulated in em-bryonic and early postnatal hippocampus as comparedwith the same tissues in P20 and adult rats (Fig. 2C,D).Temporal expression of DA11 protein in the hippocam-pus exhibited a pattern similar to that observed in the CX(data not shown). DA11 antibody immunoreacted withthe pyramidal cell layer of Ammon’s horn (Fig. 5C), inwhich CA1 and CA2 showed the strongest staining ascompared with CA3. A few pyramidal cells stained

heavily in the soma and axons (Fig. 5D), but mostpiramidal cells exhibited light DA11-LI. The dentategyrus exhibited the lowest level of DA11-LI, but granulecells showed a similar DA11-LI in pyramidal cells inAmmon’s horn (Fig. 5E). Interestingly, the DA11-LIobserved in the hippocampus was less pronounced thanthat observed in either the CX or the CB (cf. Fig. 5C withFig. 4A,D). DA11-LI in neurons of the spinal cord wasobserved in all regions, but it was particularly prominentin the ventral horn (Fig. 4F). The motor neurons exhibitedan intense DA11-LI associated with the soma, particu-larly in the nuclear region. In the olfactory bulb (OB), astronger DA11-LI was associated with the mitral andglomerular cell layers than with the granular layer (Fig.5F). The DA11-LI was distinctly observed in granulecells (granular layer), mitral cells (mitral layer), and tuftand juxtaglomerular cells (glomerular layer 1).

DISCUSSION

The present study showed that DA11 gene expres-sion was significantly higher during prenatal and postna-tal CNS development than in the adult brain. Theimmunocytochemical studies showed the expression ofDA11 protein in neurons. The reduction of DA11 proteinin homogenates during postnatal maturation suggests arapid downregulation in the expression of DA11 gene inthe cell body and processes of these DA11-positiveneurons. This downregulation of DA11 protein wascorrelated with a significant reduction of DA11 mRNAduring the third week after birth. The dramatic (.100-fold) reduction of DA11 protein during brain maturationis consistent with a decrease in the expression of DA11protein in the neuron during differentiation and matura-tion. For example,Western and Northern blots only foundvery low levels of DA11 protein and mRNA in the adultbrain, even though more than 60% of adult rat brainvolume consists of gray matter (Eayrs and Goodhead,1959; Brizzee et al., 1964), Furthermore, in situ andimmunocytochemical data from our laboratory show thatneurons in the adult brain express very low levels ofDA11 mRNA and protein (Liu and De Leo´n, manuscriptin preparation) vs. neurons found in embryonic andneonatal brain. Only a small portion of the reductionDA11 protein in the adult brain might be due to a‘‘dilution of neuronal tissues’’ that normally occursduring brain maturation. This phenomenon may play asignificant contribution in situations where there are onlysmall alterations (two–threefold) in the level of geneexpression because adult rat brain exhibits a glia/neuronindex only two–fourfold higher than that found in neona-tal animals (Brizzee et al., 1964).

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Temporal Expression of DA11Within the Developing CNS

Upregulation of DA11 mRNA and protein wasinitially observed during the prenatal period of neurogen-esis (E14–E19; Altman and Bayer, 1978, 1980a,b; Bayer,1980; Bayer and Altman, 1991). The upregulation ofDA11 mRNA continued after birth into early postnataldevelopment in CX (P10) and hippocampus (P14). Dur-ing the first 2 weeks after birth, most pyramidal cells inthe CX and hippocampus have already finished theirneurogenesis and are undergoing terminal differentiation(Bayer, 1980; Altman and Bayer, 1990; Bayer andAltman, 1991). In contrast, the CB exhibited a prolongedupregulation of DA11 mRNA that was still significant byP20 and corresponded with measurable level of DA11protein. This prolonged expression of DA11 mRNA

correlates with the longer maturation period in thePurkinje cells of the CB (Altman 1972). Although thesecells form the monocellular Purkinje layer by E19, mostof them do not reach terminal differentiation until afterP20 (Altman, 1972).

Localization of DA11-LI Within the Developing CNS

Localization of DA11 protein to the nucleus andcytoplasm of differentiating neurons in the developingbrain supports the potential regulatory role for DA11 firstsuggested by DA11 induction in DRG neurons ipsilateralto sciatic nerve transection (De Leo´n et al., 1996; Molinaet al., 1996). The DA11 antibody showed a strongDA11-LI associated with projection neurons (‘‘macroneu-rons’’; Altman et al., 1966) such as pyramidal cells in CX

Fig. 2. Temporal analysis of DA11 mRNA expression in brain stem and hippocampus duringCNS development.A: Representative Northern blot of brain stem.B: Quantification of DA11mRNA in the brain stem.C: Representative Northern blot of hippocampus.D: Quantification ofDA11 mRNA in the hippocampus. See Figure 1 for description of experiment design andanalysis.

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and hippocampus, Purkinje cells of the CB, and mitralcells of the OB. The genesis of these neurons occursduring prenatal development (Altman, 1966), but theirdifferentiation and maturation last until the first 2 weeksafter birth (Walsh and Cepko, 1988). The DA11-LIassociated with neurons migrating through the CP isconsistent with the ‘‘inside-out model’’ (reviewed bySidman and Rakic, 1973). This model proposes thatneurons migrate from the ventricle through the IZ and CPbefore arriving and positioning in the CP. The CP seemsto contain postmitotic neuronal precursors undergoingterminal differentiation (Luskin, 1994). This migration ofneurons in the CX is a significant process in the prenatal

brain but is also observed in postnatal brain (Walsh andCepko, 1988).

Interestingly, neurons lying deep in the CP within acolumn in the CX exhibited a lower level of DA11-LIthan those lying superficially. This finding is consistentwith reports showing that neurons lying deep in thecolumn structure of the CP are more differentiated thanthose neurons lying at the top of the column (Berry andRogers, 1965). Purkinje cells of the CB also showed astrong DA11-LI. These cells undergo maturation of thesynaptic domain in the soma region during the third phase(P8–P12) of development, and the maturation of theirfan-shaped dendritic arborizations occur afterward (Alt-man, 1972). The DA11-LI associated with the soma of thePurkinje cell and in the base of the dendrites suggests thatDA11 may be involved in the formation of the dendritetree.

The expression of DA11 in the OB resembles thepattern of DA11-LI observed in the CX. Neurons of theOB originate from progenitor cells of the anterior part ofthe subventricular zone (Luskin, 1993) before migratingand grouping in distinctive cell layers. The mitral andtufted cells are major output neurons that originateprimarily between E14 and E22, whereas the neurogen-esis of interneurons in the glomerular and granular celllayers occurs and continues after birth (Bayer, 1983). Thefinding that the cell bodies and processes of mitral andtuffed cells expressed more DA11-LI than interneuronsfound in the glomerular and granular cell layers providesadditional support for DA11 playing a significant role inneuronal differentiation.

FABPs in Other Tissues Suggest Potential Rolesfor DA11 in CNS Development

Because DA11 is a member of the FABP family (DeLeon et al., 1996), we anticipated that it would bind FFAsduring the development of the rat nervous system.Neonatal brain synthesizes lipid at higher rates than adultbrain (Bourre, 1980), and the cellular concentration ofFFAs or retinoids is significantly higher than that in theadult brain (Bazan et al., 1971; Maden and Holder, 1991).Cellular FFAs mediate a number of processes such asenzyme inhibition (Bass et al., 1984), regulation of cellgrowth and differentiation (Muller et al., 1989), andinduction of the expression of genes associated with lipidmetabolism (Distel et al., 1992; Amri et al., 1995).Therefore, the regulation of DA11 expression duringCNS development may be triggered in part by the actionof its putative ligand in responsive neurons.

Although DA11 shares a higher level of homologyto FABPs such as C-FABP, DA11 also has homology tothe subfamily of retinoic acid binding proteins (RABPs;De Leon et al., 1996). The RABPs are involved in the

Fig. 3. Temporal analysis of DA11 protein expression duringCNS development. Representative Western immunoblots (N52–3) are shown for the following regions of the brain.A:Cerebral cortex.B: Cerebellum.C: Brain stem. The DA11antibody recognized a protein of approximately 15.2 kDa inprotein extracts (20 µg total protein loaded/lane) of tissuescollected from animals at different developmental ages. For allthree tissues studied, DA11 protein level was high in prenataland neonatal tissues, decreased with age, and was negligible inadult tissues. E, embryonic day; P, postnatal day; A, adult. P1 isthe day of birth.

Fatty Acid Binding Protein Expression in CNS Development 557

Fig. 4. Localization of DA11-LI in cerebral cortex, cerebellum,and spinal cord on P9. DA11-positive neurons in the CX wereidentified in typical column structures containing successivewaves of migration across the IZ and cortical plate CP.A: Noimmunoreactive neurons were detected in the ML.B: Thedifferentiating neurons (solid arrows, brown) migrated alongglial fibers (open arrows, purple) extended from most glial cells(immnoreactive to the GFAP antibody) and distributed in thesubventricular zone (A, solid arrows, purple).C: The majorDA11-positive neurons were pyramidal cells (solid arrows) thatshowed an apical dendrite (open arrows) pointing to the surface.The axon (curved arrows) was clearly seen opposite to theapical dendrite. Some pyramidal cells (star) within the same

column did not exhibit DA11-LI in the nucleus.D: ThePurkinje layer (PL) was labeled heavily by DA11-LI (browncolor). The granule layer showed DA11-LI in a few isolatedcells (open arrows). DA11-LI was also observed in the leptomen-ingeal cells of the pia and arachnoid mater (solid arrows). Notethat most glial cells (purple) exhibit GFAP-LI in the whitematter area (WM).E: Purkinje cells show DA11-LI in thenuclear and cytoplasmic regions (solid arrows) and in the initialpart of dendrite tree (open arrows).F: DA11-LI was alsoobserved in motor neurons, with significant nuclear staining(arrows) in the ventral horn of spinal cord. Scale bars5 200 µmin A,D, 50 µm in B, 20 µm in C,E,F.

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metabolism and trafficking of retinoic acids. For example,the cellular RABPs (e.g., CRABP-1) bind RAs, therebymodulating the accessibility of RA to its receptor in thecell nucleus (Wolf, 1991; Ross, 1993). RA is a potent

regulator of cellular differentiation in the developingnervous system (Langman and Welch, 1967) and is aninhibitor of cell proliferation in human neuroblastomacells (Sidell, 1982). Based on the fact that DA11 shares

Fig. 5. Localization of DA11-LI in brain stem, hippocampus,and the olfactory bulb on day P9.A: DA11-positive neurons inthe superior olivary complex exhibited a predominant nuclearand axon staining (arrows).B: The reticular formation area ofthe brain stem showed widespread DA11-LI associated withaxon fibers (arrows).C:Within the hippocampus, DA11-LI wasobserved in the CA1–3 region and in the dentate gyrus (DG).D:The DA11-L1 was localized mainly in pyramidal cells (CA1–3), which showed very intense soma (solid arrow) and axonal

(open arrow) immunoreactive staining.E: In DG, DA11-L1was observed in granule cells that also showed soma (solidar-row) and axonal (open arrow) staining.F: The DA11-LI in theolfactory bulb was distributed in the glomerular cell layer (GL),the ML, and the granule cell layer (GCL). Glial cells exhibitstrong GFAP-LI (open arrows). Among these three layers, theML showed the highest level of expression. Scale bars5 100µm in A,B,F, 200 µm in C, 20 µm in D,E.

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amino acid sequence homology with both FABPs and thesubfamily of RABPs, its functional roles in neurons mayalso be similar. The pleiotropic functions and regulatoryinteractions described for other FABPs and RABPs areconsistent with the finding of DA11-LI in both cytoplas-mic and nuclear compartments of the cell and in axons.

Other Brain FABPs and DA11 Differ in Localizationbut May Share Some Biological Activities

H-FABP (Schoentgen et al., 1989; Sellner et al.,1995) and BLBP (Feng et al., 1994; Kurtz et al., 1994) aretwo FABPS that are also upregulated during CNS devel-opment. In contrast to DA11, H-FABP and BLBP are notexpressed in the cerebellar Purkinje cells, and their spatialdistribution pattern in the brain differs with that of DA11.H-FABP and DA11 are expressed in both the nervoussystem and in other tissues (Schoentgen et al., 1989,1990; Sellner, 1993; Sellner et al., 1995, Veerkamp andMaatmann, 1995; De Leo´n et al., 1996). H-FABP isexpressed in a population of CNS neurons and is poten-tially involved in FFA transport during differentiation(Sellner, 1993; Sellner et al., 1995). BLBP is expressedexclusively in the nervous system of the mouse (Feng etal., 1994; Kurtz et al., 1994). BLBP is expressed predomi-nantly in glial cells and may participate in neuronaldifferentiation via the radial glial fiber system in thedeveloping brain (Feng et al., 1994; Kurtz et al., 1994).The distinct cellular expression of each FABP suggeststhat each may play a role uniquely relevant to theparticular cell type in which it is expressed.

The conservation of critical amino acids involved inFFAs binding and cellular differentiation in DA11 (Ross,1993; Yang et al., 1994; Veerkamp and Maatman, 1995;De Leon et al., 1996) suggests a mechanism of actionsimilar to other FABPs (Muller et al., 1989; Yang et al.,1994). For example, in vitro experiments have shown thatBLBP antiserum inhibits neuronal and glial differentia-tion (Feng et al., 1994). Furthermore, H-FABP and themammary-derived growth inhibitor (MDGI) proteinsinhibit cell growth (Huynh et al., 1995; Huynh et al.,1996) and stimulate cellular differentiation in normalmouse mammary epithelial cells (Yang et al., 1994).These findings support the possibility that MDGI may beacting as a tumor-supressor gene in epithelial mammarycells (Huynh et al., 1995, 1996). Interestingly, DA11shares more than 70% of homology to the H-FABP andMDGI protein at the region responsible for its differenti-ating effect (Yang et al., 1994; De Leo´n et al., 1996). Itwill be of great interest to determine whether DA11possesses similar biological activities.

In summary, DA11 mRNA and protein are dynami-cally regulated during development of the rat CNS.Analysis of the spatial and temporal patterns of expres-sion suggests that DA11 is involved in the process ofneuronal differentiation. Increased understanding of DA11

and its ligand will elucidate some of the molecular eventsunderlying development and regeneration of the nervoussystem.

ACKNOWLEDGMENTThis work was supported by grant HD03807-

Supplement (M.D.L. and L.D.L.) from the NationalInstitutes of Health. We acknowledge Dr. Sandy Hillikerfor her critical reading of the manuscript and Lie HongChen for her assistance in the statistical analysis.

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