Cell, Vol. 61, 157-170,

April 6, 1990, Copyright

0 1990 by Cell Press

The Axond Glycoprotein TAG1 Is an ImmunogiobuMn SuperfamHy Member with Neurite Outgrowth-Promoting Activity Andrew J. Furley,’ Susan B. Morton: Dominador Manalo: Domna Karagogeos: Jane Dodd,* and Thomas M. Jessell’ Howard Hughes Institute ’ Department of Biochemistry and Molecular Biophysics *Department of Physiology and Cellular Biophysics Columbia University College of Physicians and Surgeons New York, New York 10032

Summary Pathfinding of axons in the developing nervous system is thought to be mediated by glycopmteins expressed on the surface of embryonic axons and growth cones. One molacule suggested to play a role in axonal growth is TAGl, a 135 kd glycopmteln expressed transiently on the surface of subsets of neurons in the developing mammalian nervous system. We isolated a full-length cDNA clone encoding rat TAO-l. TAG-1 has six immunogkbulln-like domains and four fibronectin type Ill-like repeats and is structumlly similar to other immunogkbulin-IIke proteins expressed on deveioping axons. Neurons maintained in vitro on a substrate of TAG1 extend long neurites, suggesting that this protein plays a mle in the initial growth and guidance of axons in vivo. TAG1 is anchored to the neuronal membrane via a glycosyl phoaphatidylinosltol linkage and is also released from neurons, suggesting that TAG1 also functions as a substrate adhesion molecule when mieased into the extracellular environment. Introduction The navigation of axons to their targets is a critical step in the patterning of neuronal projections. The growth cones of developing axons are thought to select appropriate pathways by recognizing several distinct guidance cues present in their environment. Some of these cues appear to be diffusible tropic factors that are released by restricted populations of target cells (Dodd and Jessell, 1988). In addition, there is substantial evidence that axonal guidance depends on molecules expressed on cells and in the extracellular matrix along the pathway of advancing axons (Dodd and Jessell, 1988; Harrelson and Goodman, 1988). The response of neurons to these guidance cues is likely to be mediated by a variety of glycoprotein receptors present on the surface of the axon and growth cone (Jessell, 1988; Tomaselli and Reichardt, 1989). One region of the vertebrate central nervous system in which it has been possible to analyze the surface properties and axonal trajectory of newly differentiated neurons is the embryonic spinal cord. Several studies have examined the mechanisms of guidance of a subset of spinal

cord relay neurons, termed commissural neurons (Holley and Silver, 1987; Dodd et al., 1988). These neurons differentiate in the dorsal region of the spinal cord and extend axons ventrally through the neural epithelium toward the floor plate, a specialized group of cells located at the ventral midline (Jesse11 et al., 1989) (Figure 1A). Commissural axons cross the floor plate and then turn orthogonally and project toward the brain (Figure 1B). Commissural growth cones may be guided to the ventral midline of the spinal cord, in part, by a diffusible chemoattractant factor released by the floor plate (TessierLavigne et al., 1988). In addition, several glycoproteins that have been suggested to play a role in axonal growth are expressed by commissural axons (Dodd et al., 1988). For example, during the period that the axons of commissural neurons extend through the neuroepithelium toward the floor plate, they express the glycoprotein TAG-l (Yamamoto et al., 1988; Dodd et al., 1988). However, TAG-l disappears from the surface of commissural axons when they reach the floor plate and is replaced by a distinct glycoprotein, Ll, which appears only after these axons have crossed the floor plate (Dodd et al., 1988) (Figure 1B). Other surface glycoproteins (for example, the neural cell adhesion molecule, NCAM) do not change their expression at the midline (Dodd et al., 1988). The transition in TAG-l and Ll expression coincides with the change in trajectory and the onset of fasciculation of commissural axons (Holley and Silver, 1987). Thus, the spatial regulation of axonal glycoprotein expression may contribute to the ability of commissural growth cones to migrate through the varied environments they encounter in different regions of the spinal cord. In this study, we have isolated a full-length cDNA clone encoding TAG-l. The sequence of TAG-l indicates that it is a new member of the immunoglobulin superfamily with six immunoglobulin C2 domains and four domains that are homologous to the type III repeats of fibronectin (FNIII). TAG-l has a high degree of structural homology with other vertebrate immunoglobulin superfamily members expressed on developing axons, in particular, Fll, Ll, and NCAM. TAG-l is anchored to the surface of neurons via a glycosylphosphatidylinositol (GPI) linkage and, in addition, is released from neurons. Moreover, when immobilized as a substrate, TAG-l promotes the extension of axons in vitro. Although the functional role of TAG-l in vivo has not been established, our studies, together with the expression pattern of the protein, suggest that TAG-l plays a role in the initial extension of developing axons within the rat spinal cord. Results Isolation and Nucleotide Sequence of cDNAs Encoding TAG1 Rabbit antibodies generated against affinity-purified TAG-l (Dodd et al., 1988) were used to screen an embryonic day 13 rat spinal cord cDNA library constructed in the hgtll

Cell 159

A.

Figure 1. Schematic Diagram Showing the Axonal Trajectory and Expression of Axonal Glycoproteins on Commissural Neurons in the Developing Spinal Cord (A) The trajectory of commissural axons before they reach the floor plate. At this stage axons express TAG-l and NCAM, but not Ll. (B) Transition in axonal glycoprotein expression when commissural axons cross the floor plate and turn orthogonally. TAG-l expression ceases as axons emerge on the contralateral side of the floor plate. At the same time Ll is expressed on the surface of the contralateral segment of commissural axons. The expression of NCAM is not changed (for details, see Dodd et al., 1999).

expression vector (Young and Davis, 1983). Approximately 200,000 clones were screened, and a single immunoreactive plaque containing a 3.6 kb insert (TAG-301) was isolated. DNA probes derived from the TAG-301 insert were used to isolate several additional cDNA clones, one of which, TAG-564, contains a 7.6 kb cDNA insert that includes the entire coding region of TAG-l (Figure 2A). The N-terminal peptide sequence of affinity-purified TAG-l matches closely the predicted N-terminus of the TAG-564 coding region (see Experimental Procedures). Moreover, cells transfected with a mammalian expression vector (Kirschmeier et al., 1988) containing the coding region of TAG-564 synthesize a 135 kd protein that reacts with monoclonal and rabbit anti-TAG-l antibodies (see below). Taken together, these findings establish that TAG-564 encodes the TAG-l protein. The nucleotide sequence of TAG-564 contains a single

long open reading frame that starts with a methionine codon at nucleotide 224 with a conventional translation initiation sequence (Kozak, 1984) and ends with a TGA stop codon at nucleotide 3344 (Figure 3). No in-frame methionine codons are found upstream of the putative start site, and sequences 5’of the start site contain stop codons in all three frames. Translation of the open reading frame of TAG-564 predicts a protein of 1040 amino acids (Figure 3) with a mass of 112,985 daltons. The observed molecular mass of TAG-l determined by SDS-PAGE (135 kd; Dodd et al., 1988) may result from extensive glycosylation of the protein backbone, since there are 13 N-linked glycosylation sites in the predicted TAG-l sequence. In support of this, N-glycanase treatment of TAG-l reduces its size to 115-120 kd (as determined by SDS-PAGE; not shown), which is in reasonable agreement with that predicted from the cDNA sequence. Hydrophobicity analysis (Kyte and Doolittle, 1982; see Figure 26) suggests that the protein has an N-terminal hydrophobic leader sequence, and there is also atypical signal peptide cleavage site (von Heijne, 1985). TAG-l also contains a second nonpolar region at its extreme C-terminus (Figure 28) which is characteristic of a group of proteins known to be linked to the cell surface via a GPI linkage (Ferguson and Williams, 1988; Low and Saltiel, 1988) (see below). RNA blot analysis using a probe derived from the coding region of TAG-564 detected three major transcripts of m9.5 kb, ~7.5 kb, and -5.3 kb in embryonic spinal cord RNA (Figure 4). These distinct transcripts appear to result from the use of alternative polyadenylation sites. Three different poly(A) tails were found among the cDNAs sequenced, and probes derived from these regions were used to determine the relationship between these cDNA clones and the RNA transcripts. Probes derived from the 3’ noncoding region of TAG-329 (3293’ in Figure 2A) detected only the 9.5 kb transcript, whereas a probe from the 3’ noncoding region of TAG-564 (564-3’ in Figure 2A) detected both the 9.5 and the 7.5 kb transcripts (data not shown). Probes derived from TAG-312 or any coding region probe detected all three transcripts. These findings indicate that the TAG-312, TAG-564, and TAG-329 cDNAs correspond to the 5.3 kb, 7.5 kb, and 9.5 kb transcripts, respectively. We have not found any evidence for alternative exon splicing in any of the regions sequenced, including the carboxyl region of the coding region, which has been completely sequenced in cDNAs representing each of the three major transcripts (see Figure 2A). TAG-l Has Structural Features of Proteins involved in Cell Adhesion Analysis of the deduced amino acid sequence of TAG-l reveals that the protein can be divided into two distinct regions: an N-terminal domain containing six repeats with conserved features typical of immunoglobulin-like domains of the C2 set (Williams and Barclay, 1988; Figure 5A) and a C-terminal domain containing four repeats with homology to FNIII repeats (Kornblihtt et al., 1985; Figure 5A). The immunoglobulin-like domains of TAG-l are separated from the FNIII domains by the sequence GPPGPPG

st6rrgucture and Adhesive

Function

of TAG-l

A.

TAG-564 TAG-329 TAG-301 TAG-31 2 TAG-541 lkb TAG-556

0.

Figure

2. Schematic

Representation

of TAG-l

mRNA

and Hydrophobicity

Plot of TAG-l

Amino

Acid Sequence

(A) TAG-l mRNA (top) is shown colinear with the six cDNA clones (lower) used to derive the TAG-I sequence. The extent of TAG-l mRNA is depicted by the thin line with the coding region expanded: six immunoglobulin-like domains and four FNIII repeat domains are shown as open and stippled boxes, respectively. The extended hydrophobic regions of the TAG-l sequence are shown in solid boxes. Potential sites of N-linked glycosylation are marked above with daggers. The three sites of polyadenylation in the 3’ noncoding region are marked AAAAA. Arrows prior to the poly(A) stretch indicate the presence of an AAUAAA consensus polyadenylation sequence. The regions of cDNA clones used for Northern blot and in situ analyses are shown above the restriction map of TAG-1 cDNA clones. Bold lines indicate the regions of each cDNA clone that were sequenced. The only differences in nucleotide sequence found were in TAG-312 where 3 nucleotides differed from those in corresponding positions in TAG-301 and TAG-564 (positions: 3626, A to G; 3663, G to A; and 3736, T to C). These changes lie in the 3’ untranslated region and may be the result of errors made by reverse transcriptase during cDNA synthesis. TAG-301 ends with the same 260 bp of 3’ sequence, including the poly(A) site, as TAG-329, but is missing about 4.0 kb of adjacent 5’ sequence. Although there is a consensus splice donor site at the 5’ end of this deletion, the 3’ boundary lies in a long poly(GT) stretch that cannot form a splice acceptor (the sequences of the unspliced boundaries were determined from TAG-329 and TAG-664, which are identical in this region). Thus, this deletion probably represents an aberrant splice or an artifact generated during cloning. Restriction sites marked are: N, Notl; H, Hindlll; R, EcoRI; X, Xhol; 6, BamHI; S, Sacl. (B) Analysis of hydrophobicity of TAG-l amino acid sequence. The plot was generated using parameters given in Kyte and Doolittle (1962). Positive values indicate hydrophobic residues with the NH? terminus on the left. Positive peaks representing the N-terminal signal peptide and a C-terminal hydrophobic sequence can be seen.

(see Figure 3), which is present many times in the a chain of collagen (Miller and Gay, 1987). In addition to TAG-l, several other glycoproteins expressed on the surface of developing axons are members of the immunoglobulin superfamily (Figure 58). In vertebrates, these include NCAM (Cunningham et al., 1987) Ll (Moos et al., 1988) and three closely related proteins: contactin (Ranscht, 1988) Fll (Brummendorf et al., 1989) and F3 (Gennarini et al., 1989). The chick molecules contactin and Fll are nearly identical in sequence and may be encoded by the same or closely related genes (Ranscht, 1988; Briimmendorf et al., 1989). The murine protein F3 is 80% identical to and may be the mouse homolog of Fll (Gennarini et al., 1989). In invertebrates, two axonal glycoproteins, neuroglian (Bieber et al., 1989) and fasciclin II (Harrelson and Goodman, 1988) have been identified as

immunoglobulin superfamily members with structural features in common with Ll and NCAM, respectively (Figure 56). We compared the predicted protein sequence of TAG-l with these and other immunoglobulin-like molecules (Lipman and Pearson, 1985; see Experimental Procedures). The highest degree of sequence similarity was to immunoglobulin-like molecules expressed in the nervous system (see Figures 5A and 58; Table 1 and Experimental Procedures). TAG-l exhibits over 50% identity with the Fll set of proteins in an alignment extending over 95% of the TAG-l protein (comparison of Fll and TAG-l is shown in Figure 5). Moreover, comparison of the individual immunoglobulin-like and FNIII domains of TAG-l and Fll indicates that each TAG-l domain is most closely related to its colinear domain in Ml (see Table 1). Although there is

Cell 160

1 v~attcccvcc~vctvccvcc~cvcc~vv~c~vcc~gtqgct~~vvccgvcggggc~~gc~gccctq~qgctgqc~gc~qqgtctqctc~cc~qqcgqccgcagc~gtgccccagccaac 121 acccttcccgcactct~g~gtqcctg~gtctcc~gttq~ttctcccggaqcgg~gctgcqqctcctctcttttqg~ctctqcctct~~ctga~agacccac~~qg~~~~~*~q~~~q -30

)IG*H*8

241 qaaaaaggcaagcttgctgctgctq~gctqgcc~c~~ggccctq~ctcctctcc~qg~tgg~gttttgcccagqgaaccccll(yctacctttgq~cccatctt~v~aqagca~~~~~t -24 ~KASLLLL"LAT"AL"SSPG"SF~ .OilX~tiXTSiF.4FEEQP' .I.. 11.1,.*, 11.1...I. .I.. .**.* I... *w*. II.. II 361

+17

tqQcctgct~ttcccaqagga~ctgc~g~gqatcaq~tg~c~ctqqc~gcc~gcccgt0tggaagatqaatqgcacaqatatgaacctggaacc

GLLFPEESAEDQVTLA@R

ARASPPITYRWKM~GTDMNLEP

481 tqgctccc~cSccaqctqatggggggcSScctgqtc~tcatgSgccccScca~q~c~~SgqStqctgqtgt~t~cc*gtgcct~q~~t~~aaccca~aqq~~~tgtggt~~q~~~gq~ 51 GSRHQLMGGNL"I"SPTITQG~G"XQ~L~s~P~GT""s~S 601 97

gQCtgtCCtCCQCtttQQCtttCt~C(IQQ~~ttCtCC~~gg~QQ~Q~g~Q~CCCt~Q~~~~CCC~tQ~QggCtQQQQaQtQatQCtQCCCtQtaaCCCQCCtQCCClttaCCCaQQttt

721

qtcctaccqctgqctcctc~~cgagttcccc~acttc~tccca~cqg~tqgqcg~c~cttcgtgtccc~~~~t~~bqq~aacctgt~~~t~gcccqqacca~tq~~tcagacct~gqcaa

137

AVLRFGPLQEPSKEERDP”KTHEGnGYnlPONPPAHYeGL

SYRWLLNEFPNFIPTDGRHFVSQTTGNLYIA'RTNASDLGN

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.

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tttqqctaccagccScatqgacttttccSccSSga~qtcttcsgc~a*tttgcgcaqctca~cctggctgcggaagatcccCqactcttcqctcccagtatcaaaqctcg LATSHMDFSTKSVFSKFAQLNLAAEDPRLFAPSIKAR 6

QttCCCCCCgQ~Q~CCt~CQC8Ct~~tQQQC~gC~~gtC~CCCtQQ~QtQCtttQCCtttQQQ~~CCCQQttCCCCQQ~tC~~QtQQCQC~~~gtQQ~tQQttCCttgtCCCCtC~QtQ

FPPETYAL"GQQVTLE@P

AFGNPVPRIKWRKVDGSLSPQW

1081 qgccacaqctqagcccac==tg=~g~t~=~=Sq=qtq~g=tttg~~gS=gSggqtS==tStq~~tgtg~qq=~q~q=~=t=~~=qqqt=qtg~=~==qt~=~qqq~=g==t==t=qtq~~ 257 ATAEPTLQIPS"SFEDEGTIE@E AENSKGRDTVQGRI

I

V

Q

1201 agCtClgCCtq~qtqqCtaaagqtg~tctc*qacacaqaqqccqac~ttqg~tccaacttacqttqqq*ctgtgcagcagc~qq~~~~~~~~qq~~~~tq~g~g~tgqctqaqaaacqq 297 AQPEWLl"ISDTEADIGSNLRWG~AAAGKP~P""SWL~NG 1321 ggaacctctggcctcccagSaccg*gtgqag~tcttggctggggacct*cg~ttctctaagctqaqcctggagqactctqq~~tqt~~~~gt~~gg~tq~~~~~~aqcatqgcaccat 337 EPLASQNR"E"LAGDLRFSILSLEDSG"YQ@V~ENKHGTI 1441 Ct~tq~c~qtgCtgagctggCtgtS~ILSqCtCtggCCCC*qS~tt~Sqq~~q~~~~~tqtgaqacqg~tqatccctqcagctcqaqqcqqagaq~t~~q~~t~~t~ 377 YASAELAVQALAPDFRQNPVRRLIPAIROGEISILCaPRA

ccaqcctcqcgc 6

1561 ~qcccc~~~~gct~c~~t~CttttggSg~~~*qgt~~tg~q~ttttqgqq~~~~qt~~~~g*gtg~~tgt~~~tt~~q~tgg~~~~ttq~t~~t~~q~~~~~t~~q~~g~t~~q~tq*~qg 417 APKATILWSIGTEILGNSTRVTVTSDOTLI IRNISRSDEG . . 1681 caaatlltacctqctttqctqaqaactt=~tggg=SSSg=~S~=~~===gqq~t==tgtccgtgcqcqatqcaaccaaqatcaccctWctccctccagtqctqacatcaacqtqggtqa 457 K Y T@F AENFMGKINSTGILSVRDATKITLAPSSAD . lSO1 caacctqaccctacaatgt~~tg~~t~g~~~q~~~~~~~t~tgg~~~t~~~gtt~~~~t**~~~~tgq~tg~ttt~~~t~ttqS~tttq~tS~g~~tgg~qqt~~~t~~~gg~g~g~~~q As"DPT"DLTFTWTLDDFP IDFDKPGGHYRRAS 497 :LTLQ@H

1

N

V

G

0

v

R

N

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1921 t*cgaaqqagaccattggggS~~tgS~tSt~~t~SS~q~~~~~qt~~q~~~tggSqgg~~gtS~S~~tg~~tqq~~~~g*~tgtqqtS*~tg~~~~t~~~~gqS**~~~~~qt~~tqqt 537 ARETIGDLTILNAHVRHGGKYTOn AQTVVDGTSKEATYL" 2041 ccqaq~tcccccag~tcccc=~gggggt~gqtgqtqaqaqacatcqgagacaccaccgttcaqcttaqctgqagtcgtgqctttqacaaccacaqccccattgccaaqtacacqctgca 577 RGPPGPPGGVVVRDIGDTTVQLS~SRGFD~HSPIAKYTLQ 2161 .gCtCgt~~t~~~~~~t~ggqg~~Stqq~~q~~g~tcggaccaatcctqtqaatatcqagqgtaatgccgagactqcccaq~gctgqgtctcatqccttqqatqqactatgagtttcq 617 ARTPPSGKWIQ"RTNP"NIEGNAETAQ"LGL"PWMD~EFR 2281 gOtttCIIgCtSqCII(IC~tCttgggC.CtggggSgCCCSq~qqq===t==~q=~~~~tccqcactaagqaaqcaqtcccctcagtqq~accatcqqq~=t=~~qg~gggqq=qq~q=~~~ 657 "SASNILGTGEPSGPSSRIRTSEA"PS"APSGLSGGGGAP 2401 tqg=qaqctcstcatcil~=tggS=t==~gt~~S=qqqSqt~==~q~~=gq~q~=qg=ttcqqctacctqctqtccttccqcaggcaaqqcaqftccaqctqgcaqactqcccqqgtqcc 697 GELIIN~TPVSREYQNGDGFGYLLSFRRQGSSSWQTRRVP . 2521 tgqcqctg.tqcqcsqtacttc~tct~cggcSStgScagcatccaqccctacacaccctttgag~caaqatccqaagctacaatcqccgggqq*atqqqcccqagaqcctcactqcqtt 137 GADIQYFVYGNDSIQPYTPOEVKIRSYNRRGOGPESLTAL . 2641 aqtqtactcaqcagaggaag~g===*gggtgg~~~~tq~=~=q~=tggg==~~ggq~~=t=~t=tt=~q~q~tq~~=gtq~g=tqqq~q~=tqtq~t~=~~g~==tq=~=qq=~tt=t 777 "YSAEEEPRVAPIlVWAlGSSSSEM~VS~SPVLQ~~NG~L 2761 cctgggatatgaqattcgctactqqaaag~~gggS~~~~gSSg~Sg~~g~tgaccqaqtqaqqacaqcaqqg~tSgS~S~~~qtq~ccqaqtcactqqcctqaaccccaacaccaaata 817 LGYEIRYWIAGDNEAI~DR"RTAGLDTSARVTGL~TS*~"TGLNPNTK~ 2881 ccacpt~actgtg*gggcctac~accggqccqgcactqq~~~~q~t~q~~~tt~~g~tq*tq~~~tq~~~qtq~~q~~~~~q~~~cgga*a~ctcctgqcaacatctcctqqactttctc 857 ""TVRAYNRAGTGPLSPSADAHTVKPPPRRPPGN

.

ISWTFS

3001 ..gCtCC.gtCtC.gCCtt*Sgtqgg*~~~t*tggtt~~t~t~~q~~Stq~~t~t~~qgt~~~tgg~t~~~~q~tg~tqt~t~~q~~tq~tttq~~~~~~~~t~~t~~g~t~~~~~t~~~ 897 SSSLSLK~DPVYPLR~ESTVTGYKMLYaNDLHPTPTLHLT 3121 caqcaaqaactqqataqa~~t~~~~qt*~~~q~*q~~*ttqq~~~~q~t~tqqt~~~q~tt~q~~~~~~~qgg~~tqq~qgqq~tqqq~t~~~~q~~q~~qt~~~~~ttqtq~q~~~tqq 937 SKNWIEIPYPEDIGHALVQIRTTGPGGDGIPAEVH 3241 977

I

aqgcacaaqcatQatq~QgaqagCQc~CCgCCCQCCCtqCcCatCCCqQaCCtQCgttCtCCtgcatqgtgatattQatQCtCgCtQQCt~CC~QaagCtC~tCtC~~CaCtgCCC

GTSYWVESbAARPAHPGPAFSCMVILnLAGYQKL

3361 qccacqcccaaqctqg~acac~~~~~~t~~~q~~~~~g~gq~tg~~~~~~q~t~~~ttt~~~~~~q~qqt~~~~~~~tQtqcctqaqc~QgttQQcttaQacacct~ctcccaacaq 3481 t.cccttt.t~Sgg.qqtaqq~tattcctattctgcc*cSqqStaqaaccStqcqaqq~aattttctttaagtcaaqagqcactgqqca~qacttccatqataatarJtactagqccta 3601 atqcctqqaccccttgggqtcttq~cgSa~gg~Scgggcctttgattaagcag~tqqtcctttgqggccacaagtgqcactgccatctgagatcaqaqtaccaqgcccaqcaqqaacat 3121 qqgcaqca~ggqgt~tt~tttCC~ct~tg~~gcag~ggg~CCtCttCta~CCtcactqqaga~qc~Cc~tggttggtCCCgaCaCqgtCttCCatq~CtCCCtgqCttCCtCq~a 3841

gccaaqg~c~aggccctgg~t~ctgggqataq~~qctc~~a~~q~tq~q~qgct~cccc~ccc~~tgg*~aggggcacc~qcctaaqcccattggccatcctggtgqcactqccctct

3961 ~.q~~.q~~~tq~~.~qCC~St~~tqt~~~~t~~SqStgq..tggtgqSqtqSC.q~q~~~~tt~~q~qg~tatgtgactaaaqqqcttgcct=q~qq~gttqccttqcctcatcaaq 4081 atgCttCCttcatggaccctcca*qgtacqqgcaggsgatcfcc~tctqaacgctactctcttcccttca*ctctqctgcaaacttgtqcctqcctccacctcccacaactqcaqqcccc 4201 .g...tc.gctCtC..C.C.gC.tCC.ttCttCtttqtCctggg~t~Q~gaQQC~tCCQ~g~~gqQCC~QC~tC~~aqtQQCCCtQCCtQCttCC~QQ~~t~tCCtCC~tC~CCtQQCCaCaC 4321 ctgctcccCagsaCtgCCtQg~CtaCtctCttCaQtcCCCaC~~Q~~a~~QQ~ta~taaQQQQQQQgQqQ~QgCCtQCCttqeQttCtQQQt~Qtt~CCaQqQataQ~CC~Q~CtaCg 4441 qgagctgaagaagccttataacttg~cttatcc~~cccacacttaac~gacgaqqaastggaq*t*cagaagqgttagggacttcttg***~Cacatq*tctgtaa*qacaa**cat 4561 g~caqcacaqq~CtCCtcCCcacCt~qgq~gqctct~t~q~q~ga~qq~qq~t~ttq~VC~~C~C~gCCtqtCCtCtagqaCtCtgqaqV~CtCtVqaqqagqaqCCCtCtVCttC 4681

aagaqgttCtgqCtqgtqaq~tqqaC~~atg~gCtCC~~CCa~qqC~t~~qC~q~ttCC~~~~VtC~~tq~CCtq~qqC~qCCttCt~Ctqq~~~CtCqqC~qq~~qC~CtQtCt~~~~q

4801 cctctcgqgcttgctcatttcaaqaaq.ggcca*agcaaqg~c~qagttccttaqacgaqgaacctqcaqcaqcacgaccagaaaaccccaptgtccacqccctcaqcccacgggggcag 4921 cagagcagqcatttcaaqatgcacttg==~tq=tq=t==tt~gq==~ttt=tqt~qttt~==qtt~q~q=t=t~ttttqtt~tqqqttttt~~==tt~=~q==ttq=t=tqttttt=tqq

Figure

3. cDNA

and Predicted

Amino

Acid

Sequence

of TAG-l

The numbering of amino acids starts at the presumptive N-terminal line at the C-terminal indicates the potential residues cleaved during corresponding to the protein sequence of TAG-l obtained by Edman lin domains. Boxed amino acids indicate residues highly conserved located W-15 amino acids after the conserved tryptophan residue.

glutamine of the mature protein with the signal sequence underlined. The thin processing and transfer of the GPI anchor. The dashed line indicates the region degradation. Circled cysteines indicate conserved residues Of the immunoglobuin FNIII domains. For clarity we have not boxed the additional conserved tyrosine Dots indicate potential sites of N-linked glycosylation.

Structure 161

and Adhesive

Function

of TAG-l

9.5 1 7.5 b 5.3 b

0.65

h

Figure 4. Neural-Specific Expression of TAG-l mRNA Poly(A)+ RNA was isolated from the embryonic and adult brain. RNA (5-10 9g) was separated on 1% agarose-formaldehyde gels and blotted to nylon membranes. The blot was probed with cDNA probe derived from the TAG-l coding region (region P in Figure 2A) labeled by random priming. Three major transcripts (5.2 kb, 7.5 kb, and 9.5 kb) are detected in embryonic and adult neural tissues but not in nonneural tissues (also including adult heart, liver, spleen, lung, and intestine; not shown). Two fainter bands of ~4 kb also appear inconsistently in some RNA samples. The relationship of these bands to the cloned cDNAs has not been determined. The blot was reprobed with cDNA coding for the S-16 ribosomal RNA (Wagner and Perry, 1965) to determine the amount of RNA loaded in each lane.

a striking conservation in primary sequence and domain organization, TAG-l is not the rat homolog of Fll or F3. We have isolated a distinct rat cDNA clone that exhibits over 80% nucleotide identity with F3 (F. J. Casano, A. J. F., and T. M. J., unpublished data) and thus is likely to encode the rat homolog of these proteins. Significant sequence identity is observed with other members of the neural immunoglobulin-like group of proteins: Ll (Moos et al., 1988), neuroglian (Bieber et al., 1989) and, to a lesser extent, NCAM (Cunningham et al., 1987; Small et al., 1987) and fasciclin II (Harrelson and Goodman, 1988). These proteins are between 20% and 30% identical to TAG-l and contain both immunoglobulin and FNIII domains. The individual domains of these molecules, in particular Ll and neuroglian, are arranged colinearly with those of TAG-l, although this alignment is less rigid than that found between TAG-l and Fll (Table 1). Other molecules with multiple immunoglobulin domains are expressed in the nervous system (Williams and Barclay, 1988) but exhibit even less sequence identity with this group and do not have FNIII domains. Within the FNIII domains of TAG-l there are characteristic aromatic residues (tryptophan and tyrosine) at conserved positions in all but the fourth repeat, which lacks the C-terminal tyrosine. Fibronectin itself contains many type Ill repeats that comprise a large contiguous domain in the center of the molecule (Komblihtt et al., 1985; Schwarz-

bauer et al., 1987). The FNIII repeats of TAG-1 exhibit the closest sequence conservation to repeats 8-10 of fibronectin, which form the cell attachment domain of this molecule (Pierschbacher and Ruoslahti, 1984). The cell binding activity of fibronectin depends on an ArgOly-Asp (RGD) sequence present in the 10th FNIII repeat of fibronectin (Figure 5A) (Pierschbacher and Ruoslahti, 1984). Strikingly, an RGD sequence is found in the second FNIII repeat of TAG-l in the same position as that of the RGD in fibronectin. Hydropathy analysis and prediction of the setondary structure of this region of TAG-l (not shown) indicate that the RGD sequence of TAG-l is in a highly charged region within an exposed 8 turn similar to that of the RGD sequences in the cell binding domains of fibronectin, vitronectin, and von Willebrand’s factor (Ruoslahti and Pierschbacher, 1987). Other neural immunoglobulin-like molecules show some sequence conservation in the region corresponding to that of the RGD sequence in TAG-l (Figure 5A) (see below). However, TAG-l is the only neural immunoglobulin-like molecule that contains an RGD sequence within the FNIII domains. TAG-l Is a GPI-Anchored Membrane Protein The hydrophobic@ analysis of TAG-1 reveals a stretch of nonpolar amino acids at its extreme C-terminus, raising the possibility that it is attached to the membrane via a GPI lipid anchor (Ferguson and Williams, 1988). In a first set of experiments to test this possibliity, membranes isolated from El5 brain were incubated in the presence or absence of phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that releases lipid-anchored surface membrane proteins by cleavage of the GPI linkage (Low and Saltiel, 1988). Released TAG-l was detected by immunoblotting after separation by SDS-PAGE. PI-PLC treatment enhanced the release of TAG-l from embryonic brain membranes (Figure 8A). However, a significant amount of TAG-l was removed from the membranes simply by incubation with the control buffer (Figure 8A), suggesting that there is a form of TAG-l that is loosely bound to membranes. After treatment of membranes with pH 11.0 buffer, which removes peripherally associated but not GPI-linked membrane proteins (Neubii et al., 1979; Ferguson and Williams, 1988), no additional TAG-l was released during subsequent incubations in control buffer (Figure 8A). A substantial fraction of TAG-1 remained associated with the membranes after treatment with pH 11.0 buffer, and this form of TAG-l was released by incubation with PI-PLC (Figure 8A). In control experiments there was no release of the 140 kd and 180 kd transmembrane isoforms of NCAM, either by incubation with control or pH 11.0 buffer or after PI-PLC treatment (Figure 8B). These findings provide evidence that there are two forms of TAG-l associated with embryonic brain membranes, one form attached via a GPI anchor and a second form that behaves like a peripheral membrane protein. We next examined the mode of attachment of TAG-l to the surface of neuronal cells in vitro. A TAG-l cDNA containing the entire TAG-584 coding region was cloned into the expression vector pMV7 (Kirschmeier et al., 1988) to generate the construct pMV7TAG. This construct was

Cell 162

Figure 5. Alignment of the Sequence munoglobulin Superfamily Members

of TAG-l

and Related

Neural

Im-

(A) Each of the six immunoglobulin C2 domains (C2 I-VI) of TAG-1 is aligned with the corresponding immunoglobulin domains of chick Fll (Fll; Eriimmendorf et al., 1989) mouse Ll (Ll; Moos et al., 1988). Drosophila neuroglian (NGL; Bieber et al., 1989) and rat NCAM (NCAM; Small et al., 1987, which has only five immunoglobulin domains). Residues identical in all or all but one of the proteins are shaded. Similarly, each of the four FNIII domains in TAG-l is aligned with the corresponding domains of Fil, Ll, neuroglian, and NCAM (note that FNIII domain V of Drosophila neuroglian and Ll are not shown). The position of the RGD sequence present in the second FNIII repeat of TAG-1 is shown in relation to the conserved RGD present in the tenth type III repeat of rat fibronectin (Schwarzbauer et al., 1987). (B) Schematic diagram showing the structural features of TAG-1 and other neural immunoglobulin superfamily members. lmmunoglobulin C2 domains are shown as loops, with the size of each loop corresponding to the length of the immunoglobulin domain. FNIII repeats are shown as open boxes. The absence of transmembrane or cytoplasmic domains in TAG-l and Fll is shown. The position of ROD sequences in TAG-l and Ll is indicated by the black triangles. NGL. neuroglian; Fascll. fasciclin II.

Structure 163

and Adhesive

Function

of TAG-l

Table 1. Homologies between the lmmunoglobulin and FNlll Domains of TAG-l, Fi 1, neuroglian, Ll, and NCAM

i4ao2Y

A. TAGcZl TAGc211 TAGc2111 TAGc2lV TAGc2V TAGCNI

69.10 lOA3 6.70 12m 1265 944

FllC21 F1lC21, Fllc2111 FllC2N FllC2v FllCZVI

b72b 0.50 4.w 5.96 12.56

mb4 12.79 II.34 2.11

37.97 5.57 935 4.96 13.77 6.33

5.10 1647 1.52 1.69 6.7b 7.97

3.72 3% 12.81 11.74 5.52

NGLc21 NGLc21 NGLc2111 NGLc21V NGLc2V NGLc2Vl

16.12 Mb 83 134) 9.15 am

7.97 a.w I& 8.H Ma 8.31

5.10 I.22 11.94 1l.P 16.V3 4.93

LlC2i LlC2u LlCzlll LlC2lV LlC2V LlC2VI

16.11 721 a97 llD1 6.55 Il.35

4.22 13.M 8.91 198 6.16 bA2

543 2.71 l&70

4.57 5.w

9.53 692 727

5.31 5.54 as 311 7.m

NCAMc21 NCAMcZII NCAMc2lll NCAMcZlV NCAMcZV

B.

1% 7Dl

.E 9m

mm

1238 3m

67.77 446

8.18 341 17.15 WA3 law 5.91

1143 1% 4A3 wm 10.59 Il.22 5.41 14.35

6ms 1007 12s 4.35 254 4.12 mm

mA7 a.25

502 139 242 bA3 555 ,620

8.15 6.15 16.18 1274 las3 7.M

9m 7m 14% 5.85 15.20 11.M

4.43 329 447 522 3Dl 11.11

961 6.37 1556 6.29 5.70

4.39 13.54 15.72 5.28 8.90

ll*p 266 PA 8.15 072

iim

IAWU

_

TAGFNI TAGFNII TAGFNIII TAGFNN

Km 4.18 b.07 0.42

63.91 lbbl 3.77

a.14 3.78

s7.51

Fl IFNll Fl IFMI FllFNN

Wa6 3.27 6.61 1.Ol

227 wm 6.W 3Al

bB 12.27 !27M 5b7

a55 1.51 2.31 21.69

NGLW NGLFNII NGLFNtl NGLFNN NGLFNV

l4.W 1.92 0.03 5.12 5.55

4.9I 1636 2.31 0.52 0.m

866 3.w 1205 7.77 2.37

1.15 3.al 1.79 5.19 0.42

LlFNl LlFNll LIFNIII LIFNN LIFNV

MA3 0.73 3.b7 5.78 cl91

5.86 l&SU 5.5) 5.73 0.42

6.73 7.75 lSD7 5.11) 141

1.19 OAb 475 bw -1.03

4w 2.28

2m 560

523 I .51

-1.46 1M

.,

NCAMFN NCAMFNII

Each value represents the number of standard deviations between optimized similarity score of each pairwise companion and the mean score of 1OOCl shuffled sequences generated by the RFD2 program (Pearson and Lipman, 1986). The first amino acid sequence of each domain is as shown in Figure 6. High scores indicate greater homology.

transfected into the neuroblastoma-dorsal root ganglion (DRG) neuron hybrid line ND7, and one transfected line, ND7TAG3x, which expresses high levels of TAG-l on the cell surface, was selected for analysis. Treatment of these cells with PI-PLC removed virtually all TAG-l from the cell surface, as assessed by indirect immunofluorescence labeling and flow cytometry (Figure 6C). In contrast, the level of NCAM, which is expressed on the surface of the parental and transfected ND7 cells, was not affected by PI-PLC treatment (Figure 6C). PI-PLC treatment similarly removed TAG-l from the surface of NIH 3T3 and embryonic kidney 293 cells transfected with pMV7XAG (not shown) and from

primary neurons (D. K. and T. M. J., unpublished data). These findings provide additional evidence that the cell surface form of TAG-l is attached to neuronal membranes via a PI-PLC-sensitive linkage. The detection of a peripherally associated form of TAG-l in preparations of embryonic neuronal membranes raises the possibility that TAG-l is released from living neuronal cells. To examine this, we collected media that had been conditioned overnight by ND7TAG3x cells and assayed the presence of TAG-l by SDS-gel electrophoresis and Western blotting. ND7TAG3x cells released large amounts of TAG-l into the medium (Figure 6D), whereas the 140 kd and 180 kd forms of NCAM were not released (not shown). High levels of TAG-l are also released from primary cultures of rat spinal cord and DRG neurons (D. K. and T M. J., unpublished data). Collectively, these findings show that TAG-l is attached to the surface of neuronal cells via a GPI linkage and is also released from neurons in vitro. Neuronal-Specific Expression of TAG-l mRNA TAG-l is expressed transiently by subsets of neurons in the developing nervous system (Yamamoto et al., 1986; Dodd et al., 1988). To determine the mechanisms responsible for transient expression of TAG-l protein, we localized TAG-l mRNA in the developing spinal cord and cerebellum by in situ hybridization. At embryonic day 10-10.5, TAG-l mRNA is localized exclusively in the ventral region of the spinal cord (not shown) over the area known to contain differentiating motoneurons (Dodd et al., 1988). By embryonic day 11-11.5, TAG-l mRNA was still detectable over the cell bodies of developing motoneurons but not over undifferentiated neuroepithelial cells (Figures 7A and 78). In the dorsal spinal cord at this stage, hybridization was observed over the lateral region containing the cell bodies of TAG-l immunoreactive commissural neurons (Figures 7A and 78). Intense hybridization was also observed over dorsal root ganglia (Figure 78) but not over sympathetic ganglia (not shown). By embryonic day 15-16, the expression of TAG-l protein and mRNA is markedly reduced (Figures 7C and 7D). TAG-l mRNA is detected only over differentiating neurons in the ventricular region of the dorsal spinal cord (Figure 7D), whereas hybridization to the DRG was maintained (Figure 7D). We also compared TAG-l mRNA and protein expression in postnatal cerebellum. The patterns of TAG-l mRNA and protein expression in postnatal day 13 cerebellum were similar, with the highest level of labeling in the internal region of the external granular layer (Figures 7E and 7F). These observations provide evidence that the transient and restricted expression of TAG-l protein results largely from spatial and temporal restrictions in high-level expression of TAG-l mRNA. RNA blot analysis confirmed immunohistochemical studies indicating that TAG-l is expressed solely by neural tissues in developing embryos (see Figure 4). We also detected TAG-l RNA transcripts in adult brain, spinal cord, and cerebellum (see Figure 4), but not in nonneuronal tissues. Western blot analysis also reveals that there is a 130-135 kd immunoreactive protein detectable at low levels in adult brain and spinal cord (not shown). This protein

Cell 164

has a slightly lower (by m2.5 kd) apparent molecular weight than the embryonic form of TAG-l, but is likely to be a modified form of TAG-l since it is detected by both rabbit antisera and by monoclonal antibodies to TAG-l (not shown). The detection of TAG-l in adult brain and spinal cord contrasts with immunocytochemical studies showing that TAG-l is expressed transiently by neurons in the developing central and peripheral nervous systems (Yamamoto et al., 1986; Dodd et al., 1988; J. D., unpublished data). The levels of TAG-l in the adult nervous system may be below the sensitivity for detection by immunocytochemistry. Alternatively, the adult form of TAG-1 may be present in a released, posttranslationally modified or bound form that is undetectable in situ.

D. TAG-l -

Log

Relative

TAG-3X

-

pM’J7

Fluorescence

Figure 6. Spontaneous and Phospholipase C-Evoked TAG-l from Isolated Embryonic Rat Brain Membranes I-Transfected Neuronal Cell Lines

Release of or from TAG-

Membranes were prepared from embryonic day 15 rat brain and used, untreated or after exposure to 0.1 NaOH (pH ll.O), to remove peripheral membrane proteins (A and 8). Membranes were incubated for 30 min at 37% in 50 mM Tris-HCI (pH 7.5) in the presence or absence of PI-PLC. pelleted, and the supernatant was subjected to SDS-gel electrophoresis, transferred to nitrocellulose, and probed with antibodies against TAG-l (A) or NCAM (6). Lane 1 in each set of three is the original membrane fraction (membrane) (this lane is underloaded by J-fold to prevent distortion of the band). Lane 2 (SN) shows protein released spontaneously from membranes in the absence of PI-PLC, and lane 3 (SN + PLC) shows protein released after PI-PLC treatment. Each sample was divided and probed either with anti-TAG-l or anti-NCAM antibodies. Similar results were obtained in four additional experiments. Molecular size markers are indicated in kilodaltons. Flow cytometric(C) and immunoblot (D) analysis of PI-PLC-evoked release of recombinant TAG-l expressed in neuronal cell lines. ND7 cells transfected with control pMV7 plasmid do not express TAG-1 on their surface (C, top panel). In contrast, cells transfected with the pMV7-TAG construct (ND7-TAG3x; see text) express high levels of TAG-1 immunoreactivity on the cell surface (C, shaded peak in the middle panel). Treatment of ND%TAG3x cells (for 30 min at 37%) with PI-PLC removes TAG-l from the cell surface (C, open peak in the middle panel) without affecting expression of NCAM (C. bottom panel). TAG-l can be detected in the supernatant after treatment of ND7TAG3x cells with PI-PLC for 30 min (D, lane 2) when analyzed by immunotransfer as described above (for comparison, lane 1 shows the presence of the TAG-l protein in the ND7-TAG3x cells). In contrast, very little TAG-l is detected after a similar period without PI-PLC treatment (D, lane 3). However, if the supernatant is analyzed after exposure overnight to ND7-TAG3x cells, substantial quantities of TAG-1 are detected (D, lane 4).Control experiments do not detect the release of 140 kd and 160 kd forms of NCAM, which are also expressed by these cells (not shown). TAG-l immunoreactivity is not detected in the supernatant of the control cell line ND7-pMV7 (D. lane 5).

TAG-l Promotes Neurite Outgrowth Thd presence of multiple immunoglobulin and FNIII domains combined with the expression pattern of the protein suggests that TAG-l, either in its cell surface or released form, may play a role in axonal growth and guidance. As one test of this possibility, we have used an in vitro assay to examine whether a substrate of TAG-l can support the extension of neurites. TAG-l was released from embryonic day 15 rat brain membranes by treatment with PI-PLC and then isolated on a monoclonal anti-TAG-l affinity column. Purified TAG-l was immobilized on a nitrocellulosecoated tissue culture dish (Lemmon et al., 1989) and tested for its ability to promote the extension of neurites from developing neurons. For comparison, laminin, bovine serum albumin, and two monoclonal rat IgGs were also used as substrates. The cell bodies and neurites of neurons were visualized by expression of the neuron-specific antigen 3AlO. We examined neurite extension using two classes of neurons: embryonic DRG neurons, virtually all of which express TAG-l on their axons, and superior cervical ganglion sympathetic neurons, which do not express TAG-l (Dodd et al., 1988). Each of the substrates used supported the adhesion of both classes of neurons, although to different extents. The density of DRG neurons on TAG-l (17 neurons per mm2) was similar to that on laminin (22 neurons per mm2) and about 2- to 3-fold greater than that on bovine serum albumin (7 neurons per mm2) or IgG (5 neurons per mm2). There were more striking differences in the ability of the different substrates to promote neurite outgrowth from DRG neurons. Laminin was the most effective substrate. Over 95% of DRG neurons extended neurites on laminin, and 50% had neurites with a length in excess of 170 pm (Figures 8A and 88). TAG-l was also a highly effective substrate, with over 85% of neurons extending neurites (Figure 8A). The length of neurites on a substrate of TAG-l was consistently less than that on laminin (Figure 8C), although over 50% of neurons had neurites in excess of 100 Wm. In contrast, IgG and bovine serum albumin were very poor substrates for DRG neurite extension (Figures 8A and 8D). On IgG substrates fewer than 40% of neurons extended neurites, and only 10% had neurites longer than 100 pm (Figure 8A). Recombinant TAG-l isolated from kidney 293 cells transfected with pMV7-TAG is also an effective substrate for DRG neurite outgrowth (not shown). These experiments provide

$Jcture

Figure

and Adhesive

7. Correlation

Function

between

of TAG-l

Expression

of TAG-l

Protein

and mRNA

in Developing

Rat Spinal

Cord

and Postnatal

Cerebellum

(A) lmmunoperoxidase localization of TAG-l in embryonic day 11.5 rat spinal cord showing expression of the protein on the axons of DRC neurons (d), commissural neurons (c), and motoneurons (m). (B) Localization of TAG-l mRNA in a corresponding section of embryonic day 11.5 rat spinal cord by in situ hybridization using an antisense probe from region P indicated in Figure 2A. Intense hybridization is detected in cells in the dorsal root ganglia, in the dorsolateral region of the spinal cord that contains the cell bodies of commissural neurons, and a lower hybridization signal is present in the ventral horn corresponding to the localization of motoneuron cell bodies. No hybridization was apparent when corresponding sense probe was used. (C) lmmunoperoxidase localization of TAG-l in embryonic day 16 rat spinal cord. TAG-1 is still detectable on the axons of sensory neurons in the DRG (d) and dorsal root entry zone (dz), but the protein has disappeared from motoneurons and commissural axons. There is faint labeling of axons in the dorsal horn at this age. (D) Localization of TAG-l mRNA in a corresponding section of embryonic day 16 rat spinal cord. Hybridization is detectable in the DRG and in the medial region of the dorsal horn, but the grain density over the rest of the spinal cord is no greater than background. (E and F) Localization of TAG-l protein (E) and mRNA (F) in postnatal day 13 rat cerebellum. Both protein and mRNA are prominent in the inner region of the external granular layer, with lower or background levels in other regions. Scale bar: (A) and (8) = 100 pm; (C) and (D) 3 70 pm; (E) and (F) = 200 Mm.

Cell 166

__

TAG.1

---.--

LMl”O I@

Figure

8. TAG-I

Promotes

Neurite

Extension

Neurons isolated from embryonic day 15 rat dorsal root ganglia were plated on TAG-l, laminin, IgG, or bovine serum albumin substrates for 12-18 hr and then fixed and labeled with the neuron-specific monoclonal antibody 3AlO (see Experimental Procedures) and visualized by indirect immunofluorescence. The length of the longest neurite of each 3AlO positive cell was measured (or recorded as 0 urn if no neurite was seen). The percentage of neurons (ordinate) with neurites longer than a given length in urn (abscissa) is plotted (A). The combined results from the separate experiments is shown. Similar results were obtained in five separate experiments. The length of neurites on both the TAG-1 and laminin substrates is underestimated since many neurites contacted and fasciculated with the neurites of adjacent neurons (see Experimental Procedures). Only nonfasciculated neurites were counted to generate the plot shown in (A). Representative fluorescence micrographs of neurons grown on these different preparations show the extent of neurite outgrowth on TAG-l (B), laminin (C), and bovine serum albumin (D). Scale bar = 100 urn.

evidence that TAG-l can promote the adhesion and extension of neurites from embryonic DRG neurons in vitro. In contrast, superior cervical ganglion sympathetic neurons did not extend neurites on a substrate of TAG-l, although extensive neurite extension was observed on a laminin substrate (not shown). These findings establish that TAG-l promotes neurite extension from a subset of developing neurons. However, our findings do not indicate whether the capacity of DRG neurons to extend neurites on a TAG-l substrate depends on the presence of TAG-l on the axonal surface or on the presence of a distinct receptor for TAG-l. Studies to map the domains of the TAG-1 molecule responsible for neurite outgrowth should help clarify the contribution of homophilic and heterophilic interactions to TAG-l function. Discussion TAG-l is a neuronal-specific glycoprotein that is transiently expressed on a subset of axons in the developing rat

nervous system. The isolation of cDNA clones encoding TAG-l reveals that this glycoprotein is a new member of the immunoglobulin superfamily. TAG-l shares overall structural features and exhibits extensive sequence conservation with other neuronal immunoglobulin superfamily members, in particular Ll, the Fll set of proteins, and neuroglian. TAG-l is anchored to the membrane via a GPI linkage and is released from neuronal cells. Moreover, when TAG-l is immobilized as a substrate, it promotes the extension of neurites from embryonic neurons in vitro. These findings suggest several possible roles for TAG-l in the early growth of developing axons. We discuss these functions in the context of commissural axon guidance. lmmunocytochemical studies have shown that TAG-l is expressed at the time of neuronal differentiation. In the developing spinal cord, TAG-l is expressed on commissural axons and growth cones as they first project through the neuroepithelium. These pioneer commissural axons may be guided by chemoattractant molecules secreted by the floor plate (Messier-Lavigne et al., 1988). At this stage, commissural growth cones are in contact with undifferentiated neuroepithelial cells and do not grow along other commissural axons (Holley and Silver, 1987). One function of TAG-l may therefore be to promote axon extension by mediating the binding of commissural growth cones to the neuroepithelial cell surface. Since TAG-l is not expressed by neuroepithelial cells, such interactions would have to be achieved via a heterophilic binding mechanism. Heterophilic interactions could involve the binding of the immunoglobulin domains of TAG-l to other immunoglobulin superfamily members expressed on the surface of neuroepithelial cells. Alternatively, the FNIII domains of TAG-l may be involved in interactions with members of the integrin family (Hynes, 1987) present on the neuroepithelial cell surface. We have found that a significant fraction of TAG-l is released from neuronal cells and is peripherally associated with embryonic brain membranes. TAG-l is also released by commissural neurons cultured in vitro (D. K. and T. M. J., unpublished data). Thus, it is likely that TAG-l is released in vivo from commissural axons into the surrounding neuroepithelial environment. TAG-l released from the surface of early developing commissural axons may be retained in the extracellular matrix of the neuroepithelium and provide a local adhesive substrate pathway that promotes the growth and guidance of later developing commissural growth cones. In support of this, our studies indicate that TAG-l can act as a substrate to promote the growth of axons. The released form of TAG-l could promote commissural axon growth by a homophilic interaction with the GPI-linked form of TAG-l on the axon surface or by heterophilic binding to integrins or other axonal receptors for TAG-l. Ultrastructural studies have shown that commissural axons are not fasciculated during the time that they migrate through the neuroepithelium to the floor plate (Holley and Silver, 1987). Thus, another possible function of the released form of TAG-l may be to block interactions between growth cones and axons that

$ryture

and Adhesive

Function

of TAG-l

express TAG-l on their surface and in this way prevent the fasciculation of commissural axons. Other axonal glycoproteins with adhesive function also exist in both surface-associated and released forms. For example, axonin-1, a 135-149 kd chick glycoprotein with biochemical properties similar to TAG-l, is present on axons and is also released in large amounts from the surface of embryonic neurons (Ruegg et al., 1989). Similarly, NCAM exists in distinct transmembrane, GPI-linked and released forms (Gower et al., 1988). Both the GPI-linked and transmembrane isoforms of NCAM expressed on 3T3 cells are effective in promoting the extension of neurites (Doherty et al., 1989). One mechanism for generating the GPI-linked and released forms of TAG-l may involve alternative splicing of TAG-l RNA. For example, the GPI-linked and secreted forms of NCAM are generated by alternative splicing in the region near the transmembrane domain (Gower et al., 1988). Although we detect three distinct TAG-l mRNA transcripts, our sequence analysis of TAG-l cDNAs has not revealed any evidence for alternative splicing. We cannot exclude the existence of alternatively spliced forms of the molecule not represented in the cDNAs that we have isolated. However, the demonstration that neuronal cells transfected with a single TAG-l cDNA produce both the cell surface and released form of TAG-l shows that differential RNA processing is not required to produce the two forms of the molecule. The released form of TAG-l may be generated by cleavage of the C-terminal hydrophobic sequence without subsequent transfer of the GPI linkage. Alternatively, there may be a rapid cleavage of the GPI linkage, either before or after the molecule reaches the cell surface. The structural features that contribute to differences in the efficiency of transfer or cleavage of the GPI anchor may reside in sequences adjacent to the cleavage site (Cams et al., 1989; Ferguson and Williams, 1988). As commissural axons cross their intermediate target in the spinal cord, the floor plate, they stop expressing TAG-l (Dodd et al., 1988). Our in situ hybridization studies suggest that the brief duration of the TAG-l protein on the axon surface reflects, in part, the transient expression of high levels of TAG-l mRNA. However, the abrupt loss of TAG-l expression on commissural axons as they emerge from the floor plate (Dodd et al., 1988) may not depend solely on the temporal regulation of TAG-l mRNA levels. It is possible that the precise spatial regulation of expression of TAG-l on different regions of a developing axon results from cleavage of the protein from the axonal surface, conceivably by phospholipases present in or released by the floor plate. Thus, TAG-l expression may be regulated in two distinct ways. The overall developmental period of expression may be controlled at the level of TAG-l mRNA synthesis or stability. In contrast, the spatial distribution of TAG-l on subsegments of axons may be brought about by removal of the protein from the neuronal surface. The loss of TAG-l on commissural axons as they cross the floor plate is accompanied by the onset of expression of another neural immunoglobulin superfamily member, Ll (Dodd et al., 1988). The appearance of Ll on commis-

sural axons after crossing the floor plate coincides with the onset of axon fasciculation. Ll is known to mediate axon-axon interactions via a homophilic binding mechanism (Fischer et al., 1988; Chang et al., 1987; Lemmon et al., 1989). Thus, the switch in expression of TAG-l and Ll on commissural neurons as they cross the floor plate may provide neurons with a mechanism for adapting to a changing neural environment. TAG-l may be involved in axon extension over neuroepithelial cells, whereas growth along other axonal surfaces may depend on the related protein Ll . More generally, our findings demonstrate that a subset of vertebrate neurons can regulate the expression of several closely related members of the neural immunoglobulin family in quite distinct ways during the initial phases of axon extension. Although the functions of these different immunoglobulin-like proteins are not well defined, the precise temporal and spatial regulation of these proteins on a single axon provides a plausible mechanism for enhancing the specificity and accuracy of axonal pathfinding within the developing vertebrate nervous system. Exf~~rfmental

Pmteln

Procedures

Sequence

Detefmlnatlon

Gel slices containing affinity-purified TAG-1 were loaded onto a preparative SDS-PAGE gel, overlaid with 0.125 M TM-HCI (pH SB), buffer containing 16% glycerol, 0.7% P-mercaptoethanol, and 0.1 pg of V8 protease (Soehringer Mannheim), and run into the stacking gel where digestion was allowed to proceed for 40 min. The digest products were then separated by SDS-PAGE, blotted to polyvinylidene difluoride membrane (Immobilon, Millipore), and subjected to Edman degradation as described by Matsudaira (1997). The location of TAG-l peptides was detected by developing test strips with anti-TAG-l antiserum as described (Dodd et al., 1999). The largest TAG-I immunoreactive band (M30 kd) was used for sequence determination. The sequence determined was: GGFPAITTFGEIF.

lmmunoeffinlty

Puffflcatlon

of AM-TAG-1

Antfbodlee

Polyclonal antibodies to TAG-l (Dodd et al., 199S) were affinity purified on TAG-1 immobilized on nitmcellulose (Smith and Fisher, 1994). TAG-1 protein was affinity purified from NP40 homogenates of embryonic day 15 brain and spinal cord by passage over an anti-TAG-l MAb (lCl2) affinity column, separated by SDS-PAGE, and transferred to nitrocellulose (Dodd et al., 1999). After blocking in low fat milk, the nttrocellulose-bound protein was incubated with anti-TAG-1 antiserum for 12 hr at 4%. Sound antibody was recovered from the 135 kd region by elution with 0.2 M glycine (pH 23) and neutralized with 3 M Tns (pH 9.7). The purified antibody was used directly to screen cDNA libraries.

Ptwphollpeee

C matment

of Ret Bmln

Membranes

Membrane fractions were prepared from embryonic day 15-16 brain and spinal cord and treated for 30 min at 37X with or without 1 U/ml PI-PLC (isolated from Bacillus thuringiensis; a kind gift of hf. Low) in 50 mM Bis-HCI (pH 75). Membranes were then repelleted at 100,000 x g and the supernatants assayed for the presence of TAG-l by immunoblotting as described above. Llbrery Construction and Screening An embryonic day 13 rat spinal cord cDNA library was constructed in Qtll (Statagene) using a variation of the method of Gubler and Hoffman (1993) as described by Safzer et al. (1997). The library was screened using affinity-purified anti-TAG-l antiserum (see above) essentially as detailed by Huynh et al. (1995) except that the nitrocellulose filters were developed with horseradish peroxidase-conjugated second antibody as described for I&stern blots by Dodd et al. (1999). The TAG301, TAG-312, and TAG-329 clones were isolated from this library. To

Cell 166

obtain clones covering the entire coding sequence, we constructed a second library from embryonic day 13 rat brain mRNA in mP (Stratagene), using equimolar amounts of two 17 mer oligonucleotides corresponding to sequences at .the 5’ end of TAG-312 and oligo(dT) to prime cDNA synthesis from embryonic day 13 brain RNA. This library was screened using DNA probes derived from TAG-312 and TAG-301 and yielded several overlapping clones of which TAG-541, TAG-556, and TAG-564 were analyzed further.

DNA Sequencing

and Analysis

cDNA inserts from purified phage were subcloned into a Bluescript plasmid or, in the case of IZAP clones, excised directly as Bluescript plasmids (Stratagene). The nucleotide sequences of the inserts were determined by the dideoxy chain-termination method of Sanger et al. (1977) using both double-stranded and single-stranded,DNA as templates for T7 DNA polymerase (Sequenase; United States Biochemicals). The entire sequences of TAG-301 and TAG-312 and substantial parts of TAG-564 (and other clones as detailed in Figure 2A) were determined. The nucleotide sequence of the entire coding region was determined from at least two different cDNAs and confirmed by sequencing in both directions. Sequences were assembled on an Apple Macintosh computer using the DNA Strider (Marck, 1988) and IBI Pustell (Pustell, 1988) programs. The TAG-l sequence was compared as DNA using the Wordsearch program (Devereux et al., 1984) and also as protein to databases using the Pustell algorithm (Pustell, 1988). Databases searched were Genbank Release 60.0 and a local database containing immunoglobulin-like proteins (provided in part by A. N. Barclay and A. F. Williams). All protein comparisons used the PAM-250 scoring matrix (Dayhoff et al., 1983). Optimal alignment scores to TAG-l for the 12 most related immunoglobulin-like proteins were as follows: Fll, 2577; F3,2609; neuroglian, 1143; Ll, 1134, leukocytecommon antigenrelated protein (Streuli et al., 1988) 381; rat NCAM (140 kd), 306; rat myelin-associated glycoprotein, 272; Beta-l (carcinoembryonic antigen-like; Rooney et al., 1988), 230; amalgam (Seeger et al., 1988). 223; opiate binding CAM (Schofield et al., 1989) 215; fasciclin II, 172 (TAG-1 matched to itself gives a score of 5076). Domain comparisons were made using the RDFP program (Pearson and Lipman, 1988) with ktup = 1 and 1000 random shuffles performed. Figure 5 was generated with the multiple sequence alignment program of Lipman et al. (1989).

Expression of TAG-1 cDNA Neuronal Cell Lines

Transfected

Into

NIH 3T3 fibroblasts, ND7 neuroblastoma-DRG hybrid cells (generously provided by Dr. J. N. Wood) and human embryonic kidney 293 cells (ATCC collection; kindly provided by C. Gorman, Genentech, Inc.) were transfected with 5 ug of pMV7TAG or pMV7 (Kirschmeier et al., 1988) by calcium phosphate precipitation (Wigler et al., 1979) (Mammalian Cell Transfection kit; Speciality Media) and selection using 6418 as described (Kirschmeier et al., 1988). Stable transfectant lines expressing TAG-l were identified by indirect immunofluorescence, recloned, and expanded. In addition, some lines were enriched for high-level TAG-l expression by fluorescence-activated cell sorting on an Epics V flow cytometer.

RNA Transfer

Analysis

Total RNA was prepared from various tissues using LiCl/guanidinium isothiocyanate (Cathala et al., 1983) and then enriched for poly(A)+ containing transcripts by passage over an oligo(dT)-cellulose matrix. RNA transfer was performed as described by Thomas (1983). Probes were labeled by random priming (Feinberg and Vogelstein. 1983) and hybridized under standard conditions.

In Situ Hybridlzatlon Localization of TAG-l mRNA by in situ hybridization was performed according to a variation of the method of Clayton et al. (1988). Fresh frozen embryonic tissues were sectioned onto slides coated with 2% aminopropyltriethoxysilane (Pierce) and briefly fixed in 4% paraformaldehyde (in 0.12 M phosphate buffer [pH 7.61). Sections were then washed in 2x SSPE (lx SSPE = 150 mM NaCI, 10 mM phosphate [pH 7.51, 1 mM EDTA), exposed for 30 s to a UV transilluminator (Fotodyne), washed again with 2x SSPE, and then acetylated by a 5 min immersion in 0.1 M triethanolamine (pH 8.0) 0.25% acetic anhydride. After a further wash in 2x SSPE, the sections were dehydrated in in-

creasing concentrations of ethanol (containing 300 mM ammonium acetate) and then overlaid with hybridization mixture (hybridization mix = 5,000-20,000 cpmlml 35S-labeled RNA probe in 50% formamide, 2x SSPE with 10 mM dithiothreitol, 2 mgll Escherichia coli tRNA, 2 mgll bovine serum albumin, 0.4 mgll poly(A), coverslipped, and immersed in a 65OC oil bath for 8-12 hr. Cover slips were removed under 2x SSPE, and then the slides were washed twice for 30 min at 65OC in a solution containing 2 mM tetrasodium pyrophosphate, 1 mM sodium phosphate (pH 7.2) and 1 mM EDTA. The slides were then dehydrated in ethanol and subjected to emulsion autoradiography. Antisense or sense RNA probes were transcribed from TAG-l cDNA templates subcloned into the Bluescript vector (Stratagene) using either T7 or T3 polymerase according to the recommendations of the supplier (Promega). Adhesion and Neurlte Outgrowth Assays Embryonic day 15 rat brain membranes were treated with 1 U/ml PIPLC as described above. PI-PLC-cleaved proteins were affinity purified on a monoclonal anti-TAG-l MAb (lC12) affinity column (Dodd et al., 1988). The purified protein reacted with anti-TAG-l but not anti-L1 or anti-NCAM antisera. Affinity-purified TAG-l (lo-50 pglml in PBS) was absorbed onto nitrocellulose (Lemmon et al., 1969). For controls, laminin (Collaborative Research; 20 @ml), rat anti-mouse K light chain monoclonal IgG (Zymed; 50 uglml), or rat anti-mouse CD4 monoclonal IgG (GK14, Becton Dickinson; 50 uglml) were used as substrates on nitrocellulose. The nitrocellulose was then blocked with bovine serum albumin (10 mglml), which provided a further control for background neurite outgrowth. Embryonic day 15 DRG neurons or postnatal day 0 superior cervical ganglion neurons were isolated (Dodd and Jessell, 1985) plated on immobilized protein substrates at a density of 2-10 x lo4 cells per 35 mm tissue culture dish (Nunc; 35 mm diameter), and grown for 12-18 hr. Cultures were then fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained using MAb 3AlO (S. B. M., unpublished data; available from Develop mental Studies Hybridoma Bank), which recognizes a neuron-specific filament protein and serves as a marker for fine neurites. Neuronal cell bodies and neurites were visualized by indirect immunofluorescence (Dodd and Jessell, 1985) on a Zeiss Axiophot microscope. Neurite lengths were calculated using an Optimas digitized graphics system (Bioscan): neurite length was measured as the distance between the edge of the soma (sharply defined by 3AlO fluorescence) and the tip of its longest neurite. Neurite lengths were only measured if the entire length of the neurite could be unambiguously identified. About 25 neurites were measurable within each protein-coated area (3-4 mmz). This criterion for measurement underestimates the length of neurites extended on both TAG-l and laminin. 40% of neurons grown on TAG-1 and ~80% on laminin had neurites that fasciculated with neurites of adjacent neurons.

Acknowledgments We are grateful to David Colman for his expertise and advice in the preparation of cDNA libraries, to Martin Low for generously providing phospholipase C, to John Pintar for advice on in situ hybridization techniques, to Alan Williams for his analysis of the TAG-l sequence and for alignment programs, and to Richard Axel for continual consultations Barbara Han, Ira Schieren, and Caroline Kopec provided expert assistance with dissections, DNA transfections, cell sorting, and analy sis. We thank MaryAnn Gawinowicz for peptide sequencing. We also thank Dr. Corey Goodman and Dr. Fritz Rathjen for making the sequences of neuroglian and Fll available prior to publication, Eric Hubel for photography, and Karen Liebert for preparing the manuscript. Richard Axel, Marc Tessier-Lavigne. Gary Struhl, and David Colman provided helpful criticism of the manuscript. This work was supported by the Howard Hughes Medical Institute, and grants from the National Institutes of Health and Klingenstein Foundation to J. D. A. J. F. was supported by a Jane Coffin Childs Memorial Fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

December

28, 1989.

$rcture

and Adhesive

Function

of TAG-l

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recognized

Accession number

by

Number for the sequence

reported

in this

paper

is