CA2372559A1 - Neurite outgrowth and guidance by tenascin-c - Google Patents

Abstract

We developed an assay to quantify neurite behavior at sharp substrate boundaries and found that neurites demonstrated a strong preference for fnA-D
when given a choice at a poly-L-lysine/fnA-D interface. Furthermore, neurites preferred cells that over expressed the largest but not the smallest tenascin-C splice variant when given a choice between control cells and cells transfected with tenascin-C. The permissive guidance cues of large tenascin-C
expressed by cells were mapped to fnA-D. We demonstrate that neurite outgrowth and guidance are facilitated by distinct sequences within fnA-D. Hence, neurite outgrowth and neurite guidance mediated by the alternatively spliced region of tenascin-C are separable events which can be independently regulated.

Description

Neurite Outgrowth and Guidance by Tenascin-C
This work was supported by NIH RO 1 NS24168 to H.G and NIEHS Exploratory Research Award RQ 1610 to S.M.
This application incorporates material from and cites priority of U.S.
provisional application serial no. 60/132,137 filed May 1, 2000.
BACKGROUND OF THE INVENTION
Tenascin-C has been implicated in regulation of both neurite outgrowth and neurite guidance. We have previously shown that a particular region of tenascin-C has powerful neurite outgrowth promoting actions in vitro. This region consists of the alternatively spliced fibronectin type-III (FN-III) repeats A-D and is abbreviated fnA-D.
Development of the nervous system is absolutely dependent upon targeted growth of axons and dendrites. Not only must neuronal processes elongate to reach their correct destination (neurite outgrowth), but they must navigate in the proper direction (neurite guidance). Both neurite outgrowth (Smith et al., 1986) and neurite guidance (Letourneau et al., 1994) are thought to be regulated by astrocyte-derived surface molecules. Amongst these molecules is tenascin-C, an extracellular matrix protein which is transiently expressed at the boundaries of migratory pathways in the developing cortex (Steindler et al., 1989) and is re-expressed on glial scars in the adult central nervous system (CNS) (McKeon et al., 1991, Laywell et al., 1996;
Lochter et al., 1991 ). Based upon its localization, tenascin-C was originally thought to form barriers to advancing neuronal processes by stunting their outgrowth and/or deflecting them elsewhere (Steindler et al., 1989). However, functional studies in vivo (Gates et al., 1996; Gotz et al., 1997;
Zhang et al., 1997) and in vitro (Faissner and Kruse, 1990; Lochter et al., 1991; Meiners and Geller, 1997) have demonstrated that tenascin-C can provide permissive as well as inhibitory cues for neuronal growth.
Tenascin-C is not a single molecule, but is instead a family of alternatively spliced variants with potentially diverse actions (Chung et al., 1996; Gotz et al., 1997; Meiners and Geller, 1997). Tenascin-C splice variants differ only in their number of FN-III domains; for example. the largest splice variant of human tenascill-C has seven alternatively spliced FN-III
domains (designated fnA-D. Figure 1 ) that are missing in the smallest splice variant. Phases of increased cell migration and axonal growth in the developing CNS have been closely correlated with expression of large but not small tenascin-C (Crossin et al., 1989;
Steindler et al., 1989;
Kaplony et al., 1991; Bartsch et al., 1992), suggesting that fi~A-D might facilitate cell and neurite motility during embryogenesis. Our ovm structure-fimction studies of tenascin-C splice variants demonstrated that fnA-D avidly promoted neurite outgrowth in vitro from a variety of neuronal types, both by itself as a bacterial expression protein and as part of large tenascin-C (Meiners and Geller, 1997; Meiners et al.. 1999).
SUMMARY OF THE INVENTION
It is an object of the invention to develop a method of stimulating axonal and/or dendritic growth and guidance. Other objects and advantages of the invention will become apparent to those skilled in the art from the accompanying description of the invention.

In one general aspect, the invention is a peptide comprising the 8-amino acid sequence VFDNFVLK. as defined by the one-letter amino acid code, said peptide consisting of not more than 75 amino acids in particular embodiments adapted for situations where it is more appropriate to use smaller peptides, the peptide consists of not more than 50 amino acids, more preferably not more than 20 amino acids, even more preferably not more than 10 amino acids.
The 8-amino acid peptide unlinked to other amino acids is itself an invention.
In a related aspect, the invention is a peptide comprising a tenascin-C region selected from the group consisting of fnA-D, fnD, and fnC, said peptide free of any tenascin-C region, other than the selected tenascin-C region, exceeding 100 amino acids in length. (More preferably said peptide is free of any tenascin-C region, other than the selected tenascin-C region, exceeding 10 amino acids in length).
In another aspect, the invention is a method of stimulating axonal or dendritic growth and/or guidance, said process comprising administering a peptide described in the preceding 2 paragraphs to a neuron. In one embodiment, the axon or dendrite is in a human nervous system.
For example, the peptide is delivered to the spinal cord. In one delivery mode the peptide is delivered by infusion. In a related aspect of the invention. a vector is adminstered to an area of injury to the nervous system. the vector being nucleic acid comprising a base sequence coding for the peptide. In an alternative approach, nucleic acid molecule is in a virus at the time of administration.
In a related aspect, the invention is a method of stimulating axonal or dendritic growth or guidance, said method comprising administering a peptide to an axon or dendrite, said peptide being at least 7 amino acids in length, said peptide comprising all or part of a tenascin-C region, said tenascin-C region selected from the group consisting of fnA-D, fnD, and fnC. In a particular embodiment of this aspect. the peptide comprises the 8-amino acid peptide -, VFDNFVLK. For example, the peptide is free of any sequence of tenascin-C amino acid seqence that is both outside the tenascin-C region and exceeds 100 amino acids. In a further related aspect of the method, the peptide comprises a homologous peptide sequence identical in length to the tenascin-C region such that, if the homologous amino acid sequence and the tenascin-C
region are aligned and consecutively numbered from the same end, and like numbered amino acids from the two sequences are compared, there is at least N percent identity between the amino acids of the homologous sequence and the amino acids of the tenascin-C
sequence. N
being 70. N moi°e preferably being 80, N even more preferably being 90.
To illustrate the calculation, consider the following two aligned hypothetical 20-mer peptides:
AAAAAAAAAAVVVVVVVVVV
AAAAAAAGGGGGGVVVVVVV
At the 20 positions along the sequences, there is identity at 14. Therefore N
is 70.
In one general aspect the pL~rpose of the methods of the invention is to stimulate axonal and/or dendritic growth and. in particular embodiments, to stimulate axonal growth independent of axonal and/or dendritic ;guidance. Conversely. in another general aspect.
the purpose is to stimulate axonal and/or dendritic guidance and in particular embodii-c.ents to stimulate axonal and/or dendritic guidance independent of axonal and/or dendritic ~y-owth.
Anv of the methods of the invention can utilize any of the peptides of the invention.
In another aspect, the invention is any DNA molecule codin<~ for any peptide of the invention.
For all the peptides of the invention, a preferred embodiment is one in which the peptide is a purified peptide or in purified preparation of that peptide, for example.
as an isolated peptide.
Synthetically made peptides are examples of such prefen-ed embodiments, as are ones synthesized in a non-human cell, especially a non-mammalian cell.
Similarly, for all the DNA molecules of the invention. a preferred embodiment is one in which the DNA molecule is a purified DNA molecule or in a purified preparation of that DNA
molecule, for example as an isolated DNA molecule. Examples of such preferred embodiments are synthetically made DNA molecules, ones made by recombinant DNA technology.
or ones synthesized by replication in a non-human cell. especially a non-mammalian cell.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Multidomain structure of human tenascin-C. This diagram is adapted from Aukhil et al. (1993, ,1. Biol. Chem. 268:2542-2553). The N termini of three arms are joined to fomn a trimer, and two trimers are connected via a disulfide bond to form a hexamer. Each arm consists of 14 EGF domains_ 8-15 FN-III domains depending on alternative RNA
splicing. and a single fibrinogen domain. The universal FN-III domains (fnl-5 and fn6-8) are present in all tenascin-C splice variants. The largest tenascin-C splice variant contains 7 alternatively spliced FN-III domains (designated A1, A2, A4, B, C, and D, or fnA-D) which are missing in the shortest splice variant.
Figure 2. Schematic diagram illustrating the neurite guidance assay. A drop of the protein of interest in solution was placed in the center of a PLL-coated glass coverslip and allowed to bind. Excess protein solution was washed away, creating a protein/PLL interface.
Cep°ebellar granule neurons were cultured for 48 hours on the coverslip. and double immunocvtochemisti°y was performed at this time using an antibody against the protein in the drop and an antibody against neurofilament. Neurite behavior at the interface was then analyzed for neurites originating on fLL as well as for neurites originating on the protein spot. The followings criteria were established for the guidance assay: only single, nonfasiculated neurites within 10 ym of the interface were considered. In addition. only neurites moving toward the interface were coLmted, and no neurite whose soma was sittin<~ on the interface was counted.

Figure 3. The alternatively spliced region of tenascin-C provides permissive neurite guidance cues. (A) Cerebellar granule neurons were cultured for 48 hours on PLL-coated coverslips containing spots offnl-5, fnA-D, fn6-8, large tenascin-C (TN.L) or small tenascin-C
(TN. S). The percentage of neLUites that crossed from PLL to the protein spot and vice versa was S then assessed. Bars represent the mean ~ SEM (n= 4). In control experiments, 51 ~ 4% of the neurites crossed from PLL to a fluorescein-labeled BSA control and 50 ~ 2%
crossed from BSA
to PLL. Nem°ite behavior at fnl-5 or fn6-8/PLL interfaces did not vary significantly from the control. The percentage of neurites crossing from PLL to fnA-D was significantly higher than control (asterisk), and the percentage of neurites crossing from fnA-D to PLL
was significantly lower than control (double asterisk) (p < 0.05; Student-Newman-Keuls test). In contrast to fnA-D, the percentage of neurites crossing from PLL to large or small tenascin-C
(crosses) was significantly lower than control (~~ < 0.05; Student-Newman-Keuls test). A
polyclonal antibody (pAb) a~~ainst fnA-D fiuther reduced tile percentage of ncurites crossing to lar<~e tenascin-C
(double cross); the reduction was significant (p < 0.05; Student-Newman-Keuls test). (B ) Double I 5 immunocytochemistry was performed using a polvclonal antibody against full-length tenascin-C
followed by a fluorescein-conj ugated secondary antibody to detect filA-D
spots. and monoclonal antibody RT97 followed by a rhodamine-conju~~ated secondary antibody to detect neurons.
Neurites on both sides of the PLL/fnA-D inter°face showed a preference for fnA-D. Bar, 10 Om.
Figure 4. FnA-D overcomes tenascin-C boundaries to neurite advance. Cerebellar granule newon s were cultured for 48 hours on PLL-coated coverslips containing spots of p~°otein comprised of both small tenascin-C and fnA-D (A) or large tenascin-C and fnA-D
(B) (n = p).
The concentration of tenascin-C was held constant at 100 nM while that of fnA-D was increased from 100 nM (a 1:l ratio of iilA-D to tenascin-C) to 400 nM (a 4:1 ratio). The percentage of neurites crossing from PI.L to tile tenascin-C/fnA-D spot increased with increasing h concentrations of fnA-D. The maximal effect was observed with 300 nM fnA-D for small tenascin-C and 200 nM fnA-D for large tenascin-C.
Figure 5. The neurite outgrowth promoting site in fnD does not mediate neurite guidance. (A) Cerebellar granule neurons were cultured for 48 hours on PLL-coated coverslips containing spots of fnA-D, a mixture of fnA-D and monoclonal antibody (mAb) J1/tn2, or a mixture of fnA-D and monoclonal antibody tenascin III-B (mAb III-B) (n = 4).
Jl/tn2, which reacts in fiiD, did not change the percentage of neurites that crossed from PLL to fnA-D, nor did mAb III-B, which reacts in filB. (B) Cerebellar granule neurons were allowed to extend neurites for 48 hours on PLL-coated glass coverslips or PLL-coated glass coverslips to which fnA-D or mixtures of fnA-D and J 1 /tn2 or mAb III-B had been adsorbed. Distributions of the total neurite length are presented as a box-and-whisker plot. One representative experiment of 4 is shown.
Boxes enclose 25th and 75th percentiles of each distribution and are bisected by the median;
whiskers indicate 5th and 95th percentiles. FnA-D facilitated neurite outgrowth in comparison to PLL alone. J1/tn2 eliminated the neurite outgrowth promoting qualities of fnA-D, whereas mAb III-B had no effect.
Figure 6. Neurite guidance is localized to the C terminal portion of fnA-D.
Cerebellar granule neurons were cultured for 48 hours on PLL-coated coverslips containing spots of fnA 1-A4, fnB-D, or a mixture of fnA 1-A4 and fnB-D (n = 3 ). FnA 1-A4 formed an iWibitory boundary to neurites originating on PLL; behavior for neurites originating on fnAl-A4 was more or less random. (The dashed line indicates random neurite behavior at a BSA/PLL
interface.) On the other hand, neurites originating on either PLL on fnB-D showed a preference for fnB-D. A
mixture of fnAl-A4 and fnB-D also mimicked the actions of fnA-D; thus fnB-D
overcame the boundary formed by fnAl-A4.
Figure 7. FnC is implicated in mediation of neurite guidance. (A) Cerebellar granule neurons were cultured for 48 hours on PLL-coated coverslips with containing spots of fnA-D or fnA-D (-) C) (n = 3). Neurites on the PLL side of the interface preferred fnA-D but avoided fnA-D (-) C. Neurites on the protein side of the interface preferred fnA-D but demonstrated random behavior (dashed line) on fnA-D (-) C. (B) Cerebellar granule neurons were cultured for 48 hours on PLL-coated coverslips to which fnA-D or fnA-D (-) C had been adsorbed. One representative experiment of 4 is shown. FnA-D and fnA-D (-) C both facilitated neurite outgrowth in comparison to PLL; distributions of neurite length on fnA-D and fnA-D (-) C were the same.
Figure 8. FnA-D guides neurites in the context of cellular tenascin-C. (A) Cerebellar granule neurons were cultured for 48 hours on a mixed monolayer of BHK cells and BHK-TN.L
or BHK-TICS cells. Double immunocytochemistry was performed using a polyclonal antibody against full-length tenascin-C followed by a fluorescein-conjugated secondary antibody, and monoclonal antibody RT97 followed by a rhodamine-coyjugated secondary antibody. NeL~rites crossed from BHK cells to BHK-TN.L cells but avoided BHK-TICS cells. Bar, 12 Vim. (B) Neurite behavior at cellular interfaces was quantified (n = 4). In control experiments, 45-50%
of the neurites crossed from BHK cells to PKH26-labeled BHK cet~~. and vice versa. The percentage of neL~rites that crossed from BHK cells to BHK-TN.L cells was significantly higher than control (asterisk), and the percentage that crossed from BHK-TN.L cells to BHK cells was significantly lower (double asterisk) (p < 0.05; Strident-Newman-Keuls test).
In contrast, the percentage of neurites that crossed onto BHK-TICS cells was significantly lower than control (cross), and the percentage that crossed off was significantly higher (double cross) (h < 0.0~;
Student-Newman-Keuls test).
Figure 9. Neurite guidance in the presence of tenascin-C antibodies. (A) Cerebellar granule neurons were cultured for 48 hours on a mixed monolayer of BHK cells and BHK-TN.L

or BHK-TICS cells in the presence of a polyelonal antibody against full-length tenascin-C. The antibody sigtuficantly reduced the percentage of neurites that crossed from BHK cells to BHK-TN.L cells from about 70 to 50% (asterisk) and si'~niticantly increased the percentage of neurites that crossed to BHK-TICS cells from about 20 to 50% (cross) (p < 0.05; Student-Newman-Keuls test). (B) Neurons were also cultured on a mixed monolayer of BHK cells and BHK-TN.L cells in the presence of polyclonal antibodies against i-n 1-5 or fi~A-D, or monoclonal antibody (mAb) ,I1 /tn2. The fnl-5 antibody did not effect the percentage of neurites that crossed to BHK-TN.L
cells. nor did J1/tn2. The fnA-D antibody significantly reduced the percentage of neurites that crossed to about 20% (double asterisk) p < 0.0>; Student-Newman-Keuls test).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following embodiments and Examples are intended to illustrate not limit the invention.
I S Abbreviations Abbrevicrtion.s zrsed irr tl7i.c aphlicatiorr inclzrde: CFDA. carboxv fluorescein diacetate: CNS, central nervous system: CSPG, chondroitin sulfate proteoglvcan: EGF, epidermal ~~rowth factor; fb~, fibrinogen;
FN-I I I, fibronectiu type I I l; fn. I~N-III domain: mAb. monoclonal antibody: pAb, polyclonal, TN.L, large tenascin splice variant: TICS, small tenascin splice variant.
The following one letter codes are used to i°epresent amino acids:
S-scrine. T-threonine. N-asparagine, Q-glutamine. K-lysine. IZ-arginine, H-histidine. E-glutamic acid. D-aspartic acid, C-cystine, G-glycine.
P-proline, A-alanine, I-isoleucine, L-leucine, M-methionine, c) F-phenylalanine, W-tryptophan, V-valine, Y-tyrosine, X-any amino acid.
The following one letter codes are used to represent nucleic acids:
A-adenine, C-cytosine, G-guanine, T-thymidine, R represents A or G, Y
S represents T or C, N represents any nucleic acid.
Amino acid sequence of Tenascin-C and DNA coding for it The following amino acid and nucleic acid sequences are human tenascin-C
sequences from Genbanlc, Accession Number X78S6S Version X78S6S.1, the nucleic acid sequences including designations LOCUS, HSTENAS3; 7560 by mRNA; H.sapiens mRNA for tenascin-C, 7S60bp.
/translation="MGAMTQLLAGVFLAFLALATEGGVLKKVIRHKRQSGWATLPEE
NQPWFNHWNIKLPVGSQCSVDLESASGEKDLAPPSEPSESFQEHTVDGENQIVFTH

CSGRGNFSTEGCGCVCEPGWKGPNCSEPECPGNCHLRGRCIDGQCICDDGFTGEDCSQ
LACPSDCNDQGKCWGVCICFEGYAGADCSREICPVPCSEEHGTCVDGLCVCHDGFAG
DDCNKPLCLNNCYNRGRCVENECVCDEGFTGEDCSELICPNDCFDRGRCINGTCYCEE
GFTGEDCGKPTCPHACHTQGRCEEGQCVCDEGFAGLDCSEKRCPADCHNRGRCVDGRC

EGKCVCEQGFKGYDCSDMSCPNDCHQHGRCWGMCVCDDGYTGEDCRDRQCPRDCSNR
GLCVDGQCVCEDGFTGPDCAELSCPNDCHGQGRCWGQCVCHEGFMGKDCKEQRCPSD
CHGQGRCVDGQCICHEGFTGLDCGQHSCPSDCNNLGQCVSGRCICNEGYSGEDCSEVS
PPKDLWTEVTEETWLAWDNEMRVTEYLWYTPTHEGGLEMQFRVPGDQTSTIIQEL
ZS EPGVEYFIRVFAILENKKSIPVSARVATYLPAPEGLKFKSIKETSVEVEWDPLDIAFE
TWEIIFRNMNKEDEGEITKSLRRPETSYRQTGLAPGQEYEISLHIVKNNTRGPGLKRV

TTTRLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDEN
QYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTTGLDAPRNLRRVSQTDNSITLEWR
NGKAAIDSYRIKYAPISGGDHAEVDVPKSQQATTKTTLTGLRPGTEYGIGVSAVKEDK
ESNPATINAATELDTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPTGQWVGVQ
S LPRNTTSWLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTEQAPELENLTVTEVGWD
GLRLNWTAADQAYEHFIIQVQEANKVEAARNLTVPGSLRAWIPGLKAATPYTVSIYG
VIQGYRTPVLSAEASTGETPNLGEVWAEVGWDALKLNWTAPEGAYEYFFIQVQEADT
VEAAQNLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVEVLTEEVPDMGNL
TVTEVSWDALRLNWTTPDGTYDQFTIQVQEADQVEEAHNLTVPGSLRSMEIPGLRAGT

IQVQEWKVEAAQNLTLPGSLRAVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEASTA
KEPEIGNLNVSDITPESFNLSWMATDGIFETFTIEIIDSNRLLETVEYNISGAERTAH
ISGLPPSTDFIWLSGLAPSIRTKTISATATTEALPLLENLTISDINPYGFTVSWMAS
ENAFDSFLVTVVDSGKLLDPQEFTLSGTQRKLELRGLITGIGYEVMVSGFTQGHQTKP

LAPERTRDLTGLREATEYEIELYGISKGRRSQTVSAIATTAMGSPKEVIFSDITENSA
TVSWRAPTAQVESFRITWPITGGTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKG
FEESEPVSGSFTTALDGPSGLVTANITDSEALARWQPAIATVDSWISYTGEKVPEIT
RTVSGNTVEYALTDLEPATEYTLRIFAEKGPQKSSTITAKFTTDLDSPRDLTATEVQS
O ETALLTWRPPRASVTGYLLVYESVDGTVKEVIVGPDTTSYSLADLSPSTHYTAKIQAL
NGPLRSNMIQTIFTTIGLLYPFPKDCSQAMLNGDTTSGLYTIYLNGDKAQALEVFCDM
TSDGGGWIVFLRRKNGRENFYQNWKAYAAGFGDRREEFWLGLDNLNKITAQGQYELRV
DLRDHGETAFAVYDKFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSA
ITNCALSYKGAFWYRNCHRWLMGRYGDNNHSQGWWFHWKGHEHSIQFAEMKLRPSN
ZS FRNLEGRRKRA" (SEQ ID N0:2) polyA signal 7522..7527 ORIGIN
1 accggccaca gcctgcctac tgtcacccgc ctctcccgcg cgcagataca cgcccccgcc 61 tccgtgggca caaaggcagc gctgctgggg aactcggggg aacgcgcacg tgggaaccgc 121 cgcagctcca cactccaggt acttcttcca aggacctagg tctctcgccc atcggaaaga 181 aaataattct ttcaagaaga tcagggacaa ctgatttgaa gtctactctg tgcttctaaa 241 tccccaattc tgctgaaagt gaatccctag agccctagag ccccagcagc acccagccaa 301 acccacctcc accatggggg ccatgactca gctgttggca ggtgtctttc ttgctttcct 361 tgccctcgct accgaaggtg gggtcctcaa gaaagtcatc cggcacaagc gacagagtgg 421 ggtgaacgcc accctgccag aagagaacca gccagtggtg tttaaccacg tttacaacat 481 caagctgcca gtgggatccc agtgttcggt ggatctggag tcagccagtg gggagaaaga 541 cctggcaccg ccttcagagc ccagcgaaag ctttcaggag cacacagtag atggggaaaa 601 ccagattgtc ttcacacatc gcatcaacat cccccgccgg gcctgtggct gtgccgcagc 661 ccctgatgtt aaggagctgc tgagcagact ggaggagctg gagaacctgg tgtcttccct 721 gagggagcaa tgtactgcag gagcaggctg ctgtctccag cctgccacag gccgcttgga 781 caccaggccc ttctgtagcg gtcggggcaa cttcagcact gaaggatgtg gctgtgtctg 841 cgaacctggc tggaaaggcc ccaactgctc tgagcccgaa tgtccaggca actgtcacct 901 tcgaggccgg tgcattgatg ggcagtgcat ctgtgacgac ggcttcacgg gcgaggactg 961 cagccagctg gcttgcccca gcgactgcaa tgaccagggc aagtgcgtga atggagtctg 1021 catctgtttc gaaggctacg ccggggctga ctgcagccgt gaaatctgcc cagtgccctg 1081 cagtgaggag cacggcacat gtgtagatgg cttgtgtgtg tgccacgatg gctttgcagg 1141 cgatgactgc aacaagcctc tgtgtctcaa caattgctac aacr_gtggac gatgcgtgga 1201 gaatgagtgc gtgtgtgatg agggtttcac gggcgaagac tcrr_agtgagc tcatctgccc ?0 1261 caatgactgc ttcgaccggg gccgctgcat caatggcacc tgctactgcg aagaaggctt 1321 cacaggtgaa gactgcggga aacccacctg cccacatgcc tgccacaccc agggccggtg 1381 tgaggagggg cagtgtgtat gtgatgaggg ctttgccggt ttggactgca gcgagaagag 1441 gtgtcctgct gactgtcaca atcgtggccg ctgtgtagac gggcggtgtg agtgtgatga 1501 tggtttcact ggagctgact gtggggagct caagtgtccc aatggctgca gtggccatgg 1561 ccgctgtgtc aatgggcagt gtgtgtgtga tgagggctat actggggagg actgcagcca 1621 gctacggtgc cccaatgact gtcacagtcg gggccgctgt gtcgagggca aatgtgtatg 1681 tgagcaaggc ttcaagggct atgactgcag tgacatgagc tgccctaatg actgtcacca 1741 gcacggccgc tgtgtgaatg gcatgtgtgt ttgtgatgac ggctacacag gggaagactg 1801 ccgggatcgc caatgcccca gggactgcag caacaggggc ctctgtgtgg acggacagtg WO 00/66628 PCTlUS00/11647 1861 cgtctgtgag gacggcttca ccggccctga ctgtgcagaa ctctcctgtc caaatgactg 1921 ccatggccag ggtcgctgtg tgaatgggca gtgcgtgtgc catgaaggat ttatgggcaa 1981 agactgcaag gagcaaagat gtcccagtga ctgtcatggc cagggccgct gcgtggacgg 2041 ccagtgcatc tgccacgagg gcttcacagg cctggactgt ggccagcact cctgccccag 2101 tgactgcaac aacttaggac aatgcgtctc gggccgctgc atctgcaacg agggctacag 2161 cggagaagac tgctcagagg tgtctcctcc caaagacctc gttgtgacag aagtgacgga 2221 agagacggtc aacctggcct gggacaatga gatgcgggtc acagagtacc ttgtcgtgta 2281 cacgcccacc cacgagggtg gtctggaaat gcagttccgt gtgcctgggg accagacgtc 2341 caccatcatc caggagctgg agcctggtgt ggagtacttt atccgtgtat ttgccatcct 2401 ggagaacaag aagagcattc ctgtcagcgc cagggtggcc acgtacttac ctgcacctga 2461 aggcctgaaa ttcaagtcca tcaaggagac atctgtggaa gtggagtggg atcctctaga 2521 cattgctttt gaaacctggg agatcatctt ccggaatatg aataaagaag atgagggaga 2581 gatcaccaaa agcctgagga ggccagagac ctcttaccgg caaactggtc tagctcctgg 2641 gcaagagtat gagatatctc tgcacatagt gaaaaacaat acccggggcc ctggcctgaa 2701 gagggtgacc accacacgct tggatgcccc cagccagatc gaggtgaaag atgtcacaga 2761 caccactgcc ttgatcacct ggttcaagcc cctggctgag atcgatggca ttgagctgac 2821 ctacggcatc aaagacgtgc caggagaccg taccaccatc gatctcacag aggacgagaa 2881 ccagtactcc atcgggaacc tgaagcctga cactgagtac gaggtgtccc tcatctcccg 2941 cagaggtgac atgtcaagca acccagccaa agagaccttc acaacaggcc tcgatgctcc 3001 caggaatctt cgacgtgttt cccagacaga taacagcatc accctggaat ggaggaatgg 3061 caaggcagct attgacagtt acagaattaa gtatgccccc atctctggag gggaccacgc 3121 tgaggttgat gttccaaaga gccaacaagc cacaaccaaa accacactca caggtctgag 3181 gccgggaact gaatatggga ttggagtttc tgctgtgaag gaagacaagg agagcaatcc 3241 agcgaccatc aacgcagcca cagagttgga cacgcccaag gaccttcagg tttctgaaac 3301 tgcagagacc agcctgaccc tgctctggaa gacaccgttg gccaaatttg accgctaccg 3361 cctcaattac agtctcccca caggccagtg ggtgggagtg cagcttccaa gaaacaccac 3421 ttcctatgtc ctgagaggcc tggaaccagg acaggagtac aatgtcctcc tgacagccga 3481 gaaaggcaga cacaagagca agcccgcacg tgtgaaggca tccactgaac aagcccctga 3541 gctggaaaac ctcaccgtga ctgaggttgg ctgggatggc ctcagactca actggaccgc ~J

3601 ggctgaccag gcctatgagc actttatcat tcaggtgcag gaggccaaca aggtggaggc 3661 agctcggaac ctcaccgtgc ctggcagcct tcgggctgtg gacataccgg gcctcaaggc 3721 tgctacgcct tatacagtct ccatctatgg ggtgatccag ggctatagaa caccagtgct 3781 ctctgctgag gcctccacag gggaaactcc caatttggga gaggtcgtgg tggccgaggt 3841 gggctgggat gccctcaaac tcaactggac tgctccagaa ggggcctatg agtacttttt 3901 cattcaggtg caggaggctg acacagtaga ggcagcccag aacctcaccg tcccaggagg 3961 actgaggtcc acagacctgc ctgggctcaa agcagccact cattatacca tcaccatccg 4021 cggggtcact caggacttca gcacaacccc tctctctgtt gaagtcttga cagaggaggt 4081 tccagatatg ggaaacctca cagtgaccga ggttagctgg gatgctctca gactgaactg 4141 gaccacgcca gatggaacct atgaccagtt tactattcag gtccaggagg ctgaccaggt 4201 ggaagaggct cacaatctca cggttcctgg cagcctgcgt tccatggaaa tcccaggcct 4261 cagggctggc actccttaca cagtcaccct gcacggcgag gtcaggggcc acagcactcg 4321 accccttgct gtagaggtcg tcacagagga tctcccacag ctgggagatt tagccgtgtc 4381 tgaggttggc tgggatggcc tcagactcaa ctggaccgca gctgacaatg cctatgagca 4441 ctttgtcatt caggtgcagg aggtcaacaa agtggaggca gcccagaacc tcacgttgcc 4501 tggcagcctc agggctgtgg acatcccggg cctcgaggct gccacgcctt atagagtctc 4561 catctatggg gtgatccggg gctatagaac accagtactc tctgctgagg cctccacagc 4621 caaagaacct gaaattggaa acttaaatgt ttctgacata actcccgaga gcttcaatct 4681 ctcctggatg gctaccgatg ggatcttcga gacctttacc attgaaatta ttgattccaa 4741 taggttgctg gagactgtgg aatataatat ctctggtgct gaacgaactg cccatatctc 4801 agggctaccc cctagtactg attttattgt ctacctctct ggacttgctc ccagcatccg 4861 gaccaaaacc atcagtgcca cagccacgac agaggccctg ccccttctgg aaaacctaac 4921 catttccgac attaatccct acgggttcac agtttcctgg atggcatcgg agaatgcctt 4981 tgacagcttt ctagtaacgg tggtggattc tgggaagctg ctggaccccc aggaattcac 5041 actttcagga acccagagga agctggagct tagaggcctc ataactggca ttggctatga 5101 ggttatggtc tctggcttca cccaagggca tcaaaccaag cccttgaggg ctgagattgt 5161 tacagaagcc gaaccggaag ttgacaacct tctggtttca gatgccaccc cagacggttt 5221 ccgtctgtcc tggacagctg atgaaggggt cttcgacaat tttgttctca aaatcagaga 5281 taccaaaaag cagtctgagc cactggaaat aaccctactt gcccccgaac gtaccaggga 5341 cttaacaggt ctcagagagg ctactgaata cgaaattgaa ctctatggaa taagcaaagg 5401 aaggcgatcc cagacagtca gtgctatagc aacaacagcc atgggctccc caaaggaagt 5461 cattttctca gacatcactg aaaattcggc tactgtcagc tggagggcac ccacggccca 5521 agtggagagc ttccggatta cctatgtgcc cattacagga ggtacaccct ccatggtaac 5581 tgtggacgga accaagactc agaccaggct ggtgaaactc atacctggcg tggagtacct 5641 tgtcagcatc atcgccatga agggctttga ggaaagtgaa cctgtctcag ggtcattcac 5701 cacagctctg gatggcccat ctggcctggt gacagccaac atcactgact cagaagcctt 5761 ggccaggtgg cagccagcca ttgccactgt ggacagttat gtcatctcct acacaggcga 5821 gaaagtgcca gaaattacac gcacggtgtc cgggaacaca gtggagtatg ctctgaccga 5881 cctcgagcct gccacggaat acacactgag aatctttgca gagaaagggc cccagaagag 5941 ctcaaccatc actgccaagt tcacaacaga cctcgattct ccaagagact tgactgctac 6001 tgaggttcag tcggaaactg ccctccttac ctggcgaccc ccccgggcat cagtcaccgg 6061 ttacctgctg gtctatgaat cagtggatgg cacagtcaag gaagtcattg tgggtccaga 6121 taccacctcc tacagcctgg cagacctgag cccatccacc cactacacag ccaagatcca 6181 ggcactcaat gggcccctga ggagcaatat gatccagacc atcttcacca caattggact 6241 cctgtacccc ttccccaagg actgctccca agcaatgctg aatggagaca cgacctctgg 6301 cctctacacc atttatctga atggtgataa ggctcaggcg ctggaagtct tctgtgacat 6361 gacctctgat gggggtggat ggattgtgtt cctgagacgc aaaaacggac gcgagaactt 6421 ctaccaaaac tggaaggcat atgctgctgg atttggggac cgcagagaag aattctggct 6481 tgggctggac aacctgaaca aaatcacagc ccaggggcag tacgagctcc gggtggacct 6541 gcgggaccat ggggagacag cctttgctgt ctatgacaag ttcagcgtgg gagatgccaa 6601 gactcgctac aagctgaagg tggaggggta cagtgggaca gcaggtgact ccatggccta 6661 ccacaatggc agatccttct ccacctttga caaggacaca gattcagcca tcaccaactg 6721 tgctctgtcc tacaaagggg ctttctggta caggaactgt caccgtgtca acctgatggg 6781 gagatatggg gacaataacc acagtcaggg cgttaactgg ttccactgga agggccacga 6841 acactcaatc cagtttgctg agatgaagct gagaccaagc aacttcagaa atcttgaagg 6901 caggcgcaaa cgggcataaa ttggagggac cactgggtga gagaggaata aggcggccca 6961 gagcgaggaa aggattttac caaagcatca atacaaccag cccaaccatc ggtccacacc 7021 tgggcatttg gtgagaatca aagctgacca tggatccctg gggccaacgg caacagcatg ]i 7081 ggcctcacct cctctgtgat ttctttcttt gcaccaaaga catcagtctc caacatgttt 7141 ctgttttgtt gtttgattca gcaaaaatct cccagtgaca acatcgcaat agttttttac 7201 ttctcttagg tggctctggg atgggagagg ggtaggatgt acaggggtag tttgttttag 7261 aaccagccgt attttacatg aagctgtata attaattgtc attatttttg ttagcaaaga 7321 ttaaatgtgt cattggaagc catccctttt tttacatttc atacaacaga aaccagaaaa 7381 gcaatactgt ttccatttta aggatatgat taatattatt aatataataa tgatgatgat 7441 gatgatgaaa actaaggatt tttcaagaga tctttctttc caaaacattt ctggacagta 7501 cctgattgta tttttttttt aaataaaagc acaagtactt ttgaaaaaaa accggaattc ~~ (SEQ ID N0:3) Subregions of Tenascin-C
the peptide VFDNFVLK is amino acids 1646-1653 (SEQ ID NO:l);
filA-D is amino acids 1072-1078 (SEQ ID N0:8);
fnA-D being alternatively spliced FN-III domain D is amino acids1618-1708 (SEQ
ID
N0:9);
fnA-D being alternatively spliced domain C is amino acids 1527-1617 (Seq ID
NO:10);
filD is another term for alternatively spliced FN-II1 domain D; and fnC is another term for alternatively spliced FN-II1 domain C.
Integrin alpha 7 precursor sequence This amino acid sequence and related information and sununary was obtained through the NCBI Entrez protein database.
LOCUS NP 002197 l 137 as fRI O1-OCT-1999 DEFINITION integrin alpha 7 precursor [Homo sapiensJ.

VERSION NP 002197.1 GI:4504753 ITGA7 encodes integrin alpha chain 7. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. Alpha chain 7 undergoes post-translational cleavage within the extracellular domain to yield disulfide-linked light and heavy chains that join with beta 1 to form an integrin that binds to the extracellular matrix protein laminin-1.
ORIGIN
1 magarsrdpw gasgicylfg sllvellfsr avafnldvmg alrkegepgs lfgfsvalhr 61 qlqprpqswl lvgapqalal pgqqanrtgg lfacplslee tdcyrvdidq gadmqkeske 121 nqwlgvsvrs qgpggkivtc ahryearqrv dqiletrdmi grcfvlsqdl airdeldgge 181 wkfcegrpqg heqfgfcqqg taaafspdsh yllfgapgty nwkgllfvtn idssdpdqlv 241 yktldpadrl pgpagdlaln sylgfsidsg kglvraeels fvagapranh kgavvilrkd 301 sasrlvpevm lsgerltsgf gyslavadln sdgwpdlivg apyfferqee lggavyvyln 361 qgghwagisp lrlcgspdsm fgislavlgd lnqdgfpdia vgapfdgdgk vfiyhgsslg 421 vvakpsqvle geavgiksfg yslsgsldmd gnqypdllvg sladtavlfr arpilhvshe 481 vsiaprsidl eqpncagghs vcvdlrvcfs yiavpssysp tvaldyvlda dtdrrlrgqv 541 prvtflsrnl eepkhqasgt vwlkhqhdrv cgdamfqlqe nvkdklraiv vtlsyslqtp 601 rlrrqapgqg lppvapilna hqpstqraei hflkqgcged kicqsnlqlv harfctrvsd 661 tefqplpmdv dgttalfals gqpviglelm vtnlpsdpaq pqadgddahe aqllvmlpds 721 lhysgvrald paekplclsn enashvecel gnpmkrgaqv tfylilstsg isiettelev 781 elllatiseq elhpvsarar vfielplsia gmaipqqlff sgvvrgeram qserdvgskv 841 kyevtvsnqg qslrtlgsaf lnimwpheia ngkwllypmq veleggqgpg qkglcsprpn 901 ilhldvdsrd rrrreleppe qqepgerqep smswwpvssa ekkknitldc argtancvvf 961 scplysfdra avlhvwgrlw nstfleeysa vkslevivra nitvkssikn lmlrdastvi 1021 pvmvyldpma vvaegvpwwv illavlagll vlallvlllw kmgffkrakh peatvpqyha 1081 vkipredrqq fkeektgtil rnnwgsprre gpdahpilaa dghpelgpdg hpgpgta // (SEQ ID N0:4) This nucleotide sequence and related information and summary was obtained through the NCBI Entrez nucleotide database.
LOCUS NM-002206 4079 by mRNA PRI O1-OCT-DEFINITION
Homo Sapiens integrin, alpha 7 (ITGA7) mRNA.

ACCESSION
NM_002206 VERSION
NM 002206.1 GI:4504752 ORIGIN

1 ggagcggcgggcgggcgggagggctggcggggcgaacgtctgggagacgtctgaaagacc 61 aacgagactttggagaccagagacgcgcctggggggacctggggcttggggcgtgcgaga 121 tttcccttgcattcgctgggagctcgcgcagggatcgtcccatggccggggctcggagcc 181 gcgacccttggggggcctccgggatttgctacctttttggctccctgctcgtcgaactgc 241 tcttctcacgggctgtcgccttcaatctggacgtgatgggtgccttgcgcaaggagggcg 301 agccaggcagcctcttcggcttctctgtggccctgcaccggcagttgcagccccgacccc 361 agagctggctgctggtgggtgctccccaggccctggctcttcctgggcagcaggcgaatc 421 gcactggaggcctcttcgcttgcccgttgagcctggaggagactgactgctacagagtgg 481 acatcgaccagggagctgatatgcaaaaggaaagcaaggagaaccagtggttgggagtca 541 gtgttcggagccaggggcctgggggcaagattgttacctgtgcacaccgatatgaggcaa 601 ggcagcgagtggaccagatcctggagacgcgggatatgattggtcgctgctttgtgctca 661 gccaggacctggccatccgggatgagttggatggtggggaatggaagttctgtgagggac 721 gcccccaaggccatgaacaatttgggttctgccagcagggcacagctgccgccttctccc 781 ctgatagccactacctcctctttggggccccaggaacctataattggaaggggttgcttt 841 ttgtgaccaacattgatagctcagaccccgaccagctggtgtataaaactttggaccctg 901 ctgaccggctcccaggaccagccggagacttggccctcaatagctacttaggcttctcta 961 ttgactcggggaaaggtctggtgcgtgcagaagagctgagctttgtggctggagcccccc 1021 gcgccaaccacaagggtgctgtggttatcctgcgcaaggacagcgccagtcgcctggtgc 1081 ccgaggttatgctgtctggggagcgcctgacctccggctttggctactcactggctgtgg 1141 ctgacctcaacagtgatggctggccagacctgatagtgggtgccccctacttctttgagc 1201 gccaagaagagctggggggtgctgtgtatgtgtacttgaaccaggggggtcactgggctg 1261 ggatctcccctctccggctctgcggctcccctgactccatgttcgggatcagcctggctg 1321 tcctgggggacctcaaccaagatggctttccagatattgcagtgggtgccccctttgatg 1381 gtgatgggaaagtcttcatctaccatgggagcagcctgggggttgtcgccaaaccttcac 1441 aggtgctggagggcgaggctgtgggcatcaagagcttcggctactccctgtcaggcagct 1501 tggatatggatgggaaccaataccctgacctgctggtgggctccctggctgacaccgcag 1561 tgctcttcagggccagacccatcctccatgtctcccatgaggtctctattgctccacgaa 1621 gcatcgacctggagcagcccaactgtgctggcggccactcggtctgtgtggacctaaggg l~

1681 tctgtttcagctacattgcagtccccagcagctatagccctactgtggccctggactatg 1741 tgttagatgcggacacagaccggaggctccggggccaggttccccgtgtgacgttcctga 1801 gccgtaacctggaagaacccaagcaccaggcctcgggcaccgtgtggctgaagcaccagc 1861 atgaccgagtctgtggagacgccatgttccagctccaggaaaatgtcaaagacaagcttc 1921 gggccattgtagtgaccttgtcctacagtctccagacccctcggctccggcgacaggctc 1981 ctggccaggggctgcctccagtggcccccatcctcaatgcccaccagcccagcacccagc 2041 gggcagagatccacttcctgaagcaaggctgtggtgaagacaagatctgccagagcaatc 2101 tgcagctggtccacgcccgcttctgtacccgggtcagcgacacggaattccaacctctgc 2161 ccatggatgtggatggaacaacagccctgtttgcactgagtgggcagccagtcattggcc 2221 tggagctgatggtcaccaacctgccatcggacccagcccagccccaggctgatggggatg 2281 atgcccatgaagcccagctcctggtcatgcttcctgactcactgcactactcaggggtcc 2341 gggccctggaccctgcggagaagccactctgcctgtccaatgagaatgcctcccatgttg 2401 agtgtgagctggggaaccccatgaagagaggtgcccaggtcaccttctacctcatcctta 2461 gcacctccgggatcagcattgagaccacggaactggaggtagagctgctgttggccacga 2521 tcagtgagcaggagctgcatccagtctctgcacgagcccgtgtcttcattgagctgccac 2581 tgtccattgcaggaatggccattccccagcaactcttcttctctggtgtggtgaggggcg 2641 agagagccatgcagtctgagcgggatgtgggcagcaaggtcaagtatgaggtcacggttt 2701 ccaaccaaggccagtcgctcagaaccctgggctctgccttcctcaacatcatgtggcctc 2761 atgagattgccaatgggaagtggttgctgtacccaatgcaggttgagctggagggcgggc 2821 aggggcctgggcagaaagggctttgctctcccaggcccaacatcctccacctggatgtgg 2881 acagtagggataggaggcggcgggagctggagccacctgagcagcaggagcctggtgagc 2941 ggcaggagcccagcatgtcctggtggccagtgtcctctgctgagaagaagaaaaacatca 3001 ccctggactgcgcccggggcacggccaactgtgtggtgttcagctgcccactctacagct 3061 ttgaccgcgcggctgtgctgcatgtctggggccgtctctggaacagcacctttctggagg 3121 agtactcagctgtgaagtccctggaagtgattgtccgggccaacatcacagtgaagtcct 3181 ccataaagaacttgatgctccgagatgcctccacagtgatcccagtgatggtatacttgg 3241 accccatggctgtggtggcagaaggagtgccctggtgggtcatcctcctggctgtactgg 3301 ctgggctgctggtgctagcactgctggtgctgctcctgtggaagatgggattcttcaaac 3361 gggcgaagcaccccgaggccaccgtgccccagtaccatgcggtgaagattcctcgggaag 3421 accgacagcagttcaaggaggagaagacgggcaccatcctgaggaacaactggggcagcc 3481 cccggcgggagggcccggatgcacaccccatcctggctgctgacgggcatcccgagctgg 3541 gccccgatgggcatccagggccaggcaccgcctaggttcccatgtcccagcctggcctgt 3601 ggctgccctccatcccttccccagagatggctccttgggatgaagagggtagagtgggct 3661 gctggtgtcgcatcaagatttggcaggatcggcttcctcaggggcacagacctctcccac 3721 ccacaagaactcctcccacccaacttccccttagagtgctgtgagatgagagtgggtaaa 3781 tcagggacagggccatggggtagggtgagaagggcaggggtgtcctgatgcaaaggtggg 3841 gagaagggatcctaatcccttcctctcccattcaccctgtgtaacaggaccccaaggacc 3901 tgcctccccggaagtgccttaacctagagggtcggggaggaggttgtgtcactgactcag 3961 gctgctccttctctagtttcccctctcatctgaccttagtttgctgccatcagtctagtg 4021 gtttcgtggtttcgtctatttattaaaaaa tatttgagaa caaaaaaaaa aaaaaaaaa //

(SEQ ID NO:S) Integrin beta 1 sequence This amino acid seqL~ence and related information and summary was obtained through the NCBI Entrez nucleotide database.
LOCUS HSFNRB X014 by mRNA PRI 12-APR-1999 DEFINITION HLllTlan mRNA for integrin beta 1 subunit.

VERSION X07979.1 GI:31441 /translation="MNLQPIFWIGLISSVCCVFAQTDENRCLKANAKSCGECIQAGPN
S CGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGT
AEKLKPEDIHQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLE
NVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTTPFS
YKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVF
STDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTI
FAVTEEFQPVYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEG
VTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGF
TEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVN
SEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGL
ICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPK
FQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQP
VQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDIIPIVAGVVAGI
VLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK
(SEQ ID N0:6) ORIGIN
1 gtccgccaaa acctgcgcgg atagggaaga acagcacccc ggcgccgatt gccgtaccaa 61 acaagcctaa cgtccgctgg gccccggacg ccgcgcggaa aagatgaatt tacaaccaat 121 tttctggatt ggactgatca gttcagtttg ctgtgtgttt gctcaaacag atgaaaatag 181 atgtttaaaa gcaaatgcca aatcatgtgg agaatgtata caagcagggc caaattgtgg 241 gtggtgcaca aattcaacat ttttacagga aggaatgcct acttctgcac gatgtgatga 301 tttagaagcc ttaaaaaaga agggttgccc tccagatgac atagaaaatc ccagaggctc 361 caaagatata aagaaaaata aaaatgtaac caaccgtagc aaaggaacag cagagaagct 421 caagccagag gatattcatc agatccaacc acagcagttg gttttgcgat taagatcagg 481 ggagccacag acatttacat taaaattcaa gagagctgaa gactatccca ttgacctcta 541 ctaccttatg gacctgtctt attcaatgaa agacgatttg gagaatgtaa aaagtcttgg 601 aacagatctg atgaatgaaa tgaggaggat tacttcggac ttcagaattg gatttggctc 661 atttgtggaa aagactgtga tgccttacat tagcacaaca ccagctaagc tcaggaaccc '721 ttgcacaagt gaacagaact gcaccacccc atttagctac aaaaatgtgc tcagtcttac 781 taataaaggagaagtatttaatgaacttgttggaaaacagcgcatatctg gaaatttgga 841 ttctccagaaggtggtttcgatgccatcatgcaagttgcagtttgtggat cactgattgg 901 ctggaggaatgttacacggctgctggtgttttccacagatgccgggtttcactttgctgg 961 agatgggaaacttggtggcattgttttaccaaatgatggacaatgtcacctggaaaataa 1021 tatgtacacaatgagccattattatgattatccttctattgctcaccttgtccagaaact 1081 gagtgaaaataatattcagacaatttttgcagttactgaagaatttcagcctgtttacaa 1141 ggagctgaaaaacttgatccctaagtcagcagtaggaacattatctgcaaattctagcaa 1201 tgtaattcagttgatcattgatgcatacaattccctttcctcagaagtcattttggaaaa 1261 cggcaaattgtcagaaggagtaacaataagttacaaatcttactgcaagaacggggtgaa 1321 tggaacaggggaaaatggaagaaaatgttccaatatttccattggagatgaggttcaatt 1381 tgaaattagcataacttcaaataagtgtccaaaaaaggattctgacagctttaaaattag 1441 gcctctgggctttacggaggaagtagaggttattcttcagtacatctgtgaatgtgaatg 1501 ccaaagcgaaggcatccctgaaagtcccaagtgtcatgaaggaaatgggacatttgagtg 1561 tggcgcgtgcaggtgcaatgaagggcgtgttggtagacattgtgaatgcagcacagatga 1621 agttaacaggaagacatggatgcttactgcaggaaaaaaaacagttcagaaatctgcag t 1681 taacaatggagagtgcgtctgcggacagtgtgtttgtaggaagagggataatacaaatga 1791 aatttattctggcaaattctgcgagtgtgataatttcaactgtgatagatccaatggctt 1801 aatttgtggaggaaatggtgtttgcaagtgtcgtgtgtgtgagtgcaaccccaactacac 1861 tggcagtgcatgtgactgttctttggatactagtacttgtgaagccagcaacggacagat 1921 ctgcaatggccggggcatctgcgagtgtggtgtctgtaagtgtacagatccgaagtttca 1981 agggcaaacgtgtgagatgtgtcagacctgccttggtgtctgtgctgagcataaagaatg 2041 tgttcagtgcagagccttcaataaaggagaaaagaaagacacatgcacacaggaatgttc 2101 ctattttaacattaccaaggtagaaagtcgggacaaattaccccagccggtccaacctga 2161 tcctgtgtcccattgtaaggagaaggatgttgacgactgttggttctattttacgtattc 2221 agtgaatgggaacaacgaggtcatggttcatgttgtggagaatccagagtgtcccactgg 2281 tccagacatcattccaattgtagctggtgtggttgctggaattgttcttattggccttgc 2341 attactgctgatatggaagcttttaatgataattcatgacagaagggagtttgctaaatt 2401 tgaaaaggagaaaatgaatgccaaatgggacacgggtgaaaatcctatttataagagtgc 2961 cgtaacaactgtggtcaatccgaagtatgagggaaaatgagtactgcccgtgcaaatccc 2521 acaacactgaatgcaaagtagcaatttccatagtcacagttaggtagctttagggcaata 2581 ttgccatggttttactcatgtgcaggttttgaaaatgtacaatatgtataatttttaaaa 2641 tgttttattattttgaaaataatgttgtaattcatgccagggactgacaaaagacttgag 2701 acaggatggttattcttgtcagctaaggtcacattgtgcctttttgaccttttcttcctg 2761 gactattgaaatcaagcttattggattaagtgatatttctatagcgattgaaagggcaat 2821 agttaaagtaatgagcatgatgagagtttctgttaatcatgtattaaaactgatttttag -2881 ctttacatagtcagtttgcagttatgcagaatccaaa<xtaaatgtcctgctagctagtt t 2941 aaggattgttttaaatctgttattttgctatttgcctgttagacatgactgatgacatat 3001 ctgaaagacaagtatgttgagagttgctggtgtaaaatacgtttgaaatagttgatctac 3061 aaaggccatgggaaaaattcagagagttaggaaggaaaaaccaatagctttaaaacctgt 3121 gtgccattttaagagttacttaatgtttggtaacttttatgccttcactttacaaattca 3181 agccttagataaaagaaccgagcaattttctgctaaaaagtccttgatttagcactattt 3241 acatacaggccatactttacaaagtatttgctgaatggggaccttttgag ttgaatttat 3301 tttattattt ttattttgtttaatgtctggtgctttctatcacctcttct aatcttttaa 3361 tgtatttgtttgcaattttggggtaagacttttttatgag tactttttct ttgaagtttt 3921 agcggtcaat ttgccttttt aatgaacatg tgaagttata ctgtggctat gcaacagctc 3481 tcacctacgc gagtcttact ttgagttagt gccataacag accactgtat gtttacttct 3541 caccatttga gttgcccatc ttgtttcaca ctagtcacat tcttgtttta agtgccttta 3601 gttttaacag ttca // (SEQ ID N0:7) EXAMPLES
Example 1 The purpose of this study was to investigate whether fnA-D also provides neurite guidance cues and whether the same or different sequences mediate outgrowth and guidance.
We developed an assay to quantify neurite behavior at sharp substrate boundaries and found that neurites demonstrated a strong preference for fnA-D when given a choice at a poly-L-lysine/fnA-D interface. Furthermore, neurites preferred cells that over expressed the largest but not the smallest tenascin-C splice variant when given a choice between control cells and cells transfeeted with tenascin-C. The permissive guidance cues of large tenascin-C expressed by cells were mapped to fnA-D. Using a combination of bacterial expression proteins corresponding to specific alternatively spliced FN-III domains and monoclonal antibodies against neurite outgrowth promoting sites, we demonstrated that neurite out~~rowth and guidance were facilitated by distinct sequences within fi~A-D. Hence neurite outgrowth and neurite guidance mediated by tile alternatively spliced region of tenascin-C are separable events which can be independently regulated.
Our previous work suggests that neurite outgrowth and guidance may be separable events (Powell and Geller, 1999). We therefore used two complementary choice assays to investigate the hypothesis that fnA-D imparts distinct outgrowth and guidance cues. In one, growing neurites were allowed to choose between poly-L-lysine and purified expression pi°oteins corresponding to alternatively spliced and universal tenascin-C FN-III
domains. as well as large and small tenascin-C splice variants. The other assay was designed to investigate the actions of fnA-D in cellular tenascin-C. which more closely approximates the in i~ioo environment.
~s Neurites were allowed to choose between transfected cells that over expressed either large or small tenascin-C in heterogenous monolayers with untransfected cells. Neurites demonstrated a strong preference for fnA-D which was masked in large tenascin-C on inert substrates and only revealed in large tenascin-C on cellular substrates (Meiners and Geller, 1997:
Meiners et al., 1999). Guidance and outgrowth promotion were further localized to different sequences within fnA-D using monoclonal antibodies against neurite outgrowth promoting sites and expression proteins corresponding to specific alternatively spliced FN-III domains. Hence neurite outgrowth and guidance can be independently regulated by the alternatively spliced region of tenascin-C.
Materials and Methods Proteins and Antibodies Transfected baby hamster kidney (BHK) cells, bactei°ial expression proteins, and rabbit polyclonal tenascin-C antibodies were gifts of Dr. Harold Erickson (Department of Cell Biology, Dulce University Medical Center, Durham, NC). Splice variants of human tenascin-C were produced in the transfected cells (Aukhil et al.. 1993). Native large and small tenascin-C were purified from culture super°natants of these cells by gelatin-sepharo~s~and hydroxyapatite cluomatography (Aukhil et al., 1990; Ericlcson and Briscoc. 1995) followed by eleetroelution from nondenaturillg gels (Ho, S.-Y.. Palnitkar. S.. and Meincrs, S., unpublished data). Bacterial expression proteins (AVclul et al., 1993) corresponded to universal FN-III
domains 1-5 and 6-8 (fnl -5 and fn6-8), fi1A-D, the alternatively spliced FN-III domains of large tenascin-C, and fnA-D (-) C, the alternatively spliced domains minus FN-III domain C (fnC). Fnl-5.
fn6-8, and fnA-D were produced using the polymerase chain reaction (PCR) and cDNA isolated from BHK cells transfected with large tenascin-C as the template. FnA-D (-) C was produced using PCR and cDNA isolated from U251-MG glioma cells as the template. (U251-MG cells produce ~ ~l alternatively spliced transcripts of tenascin-C that contain fnA-D as well as fnA-D (-) C, although the species that contains fnA-D predominates (Erickson and Bourdon, 1989).) Rabbit polyclonal full-length tenascin-C antibody was prepared against highly purified tenascin-C from U251-MG
cells, which is almost entirely large tenascin-C (Erickson and Bourdon, 1989).
Rabbit polyclonal antibodies against fnl-5 and fnA-D were prepared against the corresponding expression protein.
All reagents cited correspond to the human protein.
Bacterial expression proteins corresponding to fnA 1-A4, the N terminal region of fnA-D, and fnB-D. the C terminal region of fnA-D, were gifts of Drs. Harold Erickson and Frans~oise Coussen (University of Bordeaux, Bordeaux, France). Both of these correspond to the human protein.
Monoclonal antibody J 1 /tn2 against mouse tenascin-C was a gift of Dr.
Andreas Faissner (Department ofNeurobiology, University of Heidelberg, D-X9120 Heidelberg, Germany). The epitope for J1/tn2 is contained on fnD of mouse tenascin-C (Gotz et al., 1996).
CSPG mixture isolated from embryonic chick brain (consisting primarily of neurocan, phosphacan, versican, and aggrecan) was obtained from Chemicon International Inc. (Temecula, CA). Aggrecan was from Sigma Chemical Co. (St. Louis. MO). and laminin-1 was from GIBCO
BRL (Rockville, MD). Monoclonal antibody CS-56. which reacts with the glycosaminoglycaa portion of native chondroitin sulfate proteoglycans, was from Sigma Chemical Co. Monoclonal antibody RT97 against neurofilament was from the Developmental Studies Hybridoma Banlc (Iowa City. IA), and a polyclonal antibody against neurofilament 200 was from Sigma Chemical Co. Monoclonal antibody tenascin-IIIB, which recognizes an epitope in fnB of human tenascin-C. was from Chemicon.
Neuronal Cell Culture Cerebellar granule neuronal cultures were prepared as described by Levi et al.
(1984).
Neuronal cultures were cultivated from postnatal day 8 (P8) rat pups. Brains were removed into a Petri dish containing 5 ml of BME with 2M HEPES buffer (BME-HEPES).
Cerebella were removed, and meninges and blood vessels were peeled off and discarded to ensure minimal contamination from endothelial cells. Cerebella were then minced into fine pieces (<0.5 mm) with dissecting knives and incubated in BME-HEPES containing 0.025% trypsin for 10 minutes at 37°C. Following incubation, the trypsinization was halted by adding one ml of BME
containing 0.025% soy bean trypsin inhibitor and 0.05% DNase I. The tissue was then gently triturated through a fire-polished pasteur pipette until it was dispersed into a homogeneous suspension. The suspension was transferred into a fresh tube. DMEM-25 mM
KCI/10% heat inactivated FCS (3-4 ml) was added to any remaining tissue clumps, and the trituration was repeated. Cells were then filtered through an ethanol-sterilized 40 yn mesh and centrifuged for 10 minutes at 1500 rpm. The pellet of cerebellar grmule neurons was resuspended in DMEM-25 mM KCl/10% FCS and used for neurite guidance and neurite outgrowth assays as described below.
Neurite Guidance Assay Neurite guidance is operationally defined as directed neurite movement which is significantly different from chance. The two most fi°equentlv used guidance assays are the stripe assay (Vielmetter et al., 1990) and the spot assay (Snow et al., 1991 ).
However, in neither has neurite behavior been quantified. We therefore modified the spot assay to quantify the behavior of neurites at an interface created between PLL and tenascin-C FN-III
expression proteins, native tenascin-C splice variants, or CSPGs. The PLL-protein interface was created by placing a 5 ~l drop of the protein of interest (300 nM in Hanl<'s buffered salt solution, HBS) in the center of a 12 mm PLL-coated glass coverslip. Coverslips in 24-well trays were incubated with the protein drop for 2 hours at 37°C, and excess protein solution was rinsed away with HBS. Similar coating efficiencies between the tenascin-C splice variants and expression proteins (about 5 pmole/cm') were verified by incubating emir a coverslips with proteins conjugated to NHS-fluorescein (Pierce Chemical, Roclcford, IL). Coated proteins were removed after 2 hours by adding 2% SDS. The fluorescence of proteins bound to PLL-coated glass was then assessed in a Cytofluor II
fluorescence microplate reader (PerSeptive Biosystems, Framingham, MA) as we have described previously for proteins bound to cellular monolayers (Meiners et al., 1999).
In agreement with the results of others (Dorries et al., l 996; Fischer et al., 1997), no major differences in coating efficiencies could be observed.
Cerebellar granule neurons were plated onto the coverslips at a density of 60,000 neurons/well and cultured for 48 lours in DMEM-25 mM KCl/10% FCS. At this time, coverslips were fixed with acetic acid/ethanol (~'%/95%) for 5 minutes at -20°C. Following fixation. coverslips were rinsed in PBS (pH 7.4. 0.14 M NaCI, 2.7 mM KCI. 1.5 mM KH~P04, 4.3 mM NaHP04) and incubated with the appropriate primary antibody against the protein in the drop (polyclonal full-length tenascin-C antibody for native tenascin-C splice variants and expression proteins, or monoclonal antibody CS-56 for CSPGs) diluted 1:100 in PBS containing 10% FCS (PBS-serum). After rinsing in PBS, the coverslips were incubated with fluorescein-coyjugated secondary antibodies diluted 1:100 in PBS-serum (goat anti-rabbit secondary antibodies for tenascin-C spots and goat anti-mouse secondary antibodies for CSPG spots) (Organon-Technilca Cappel. Durham, NC). The coverslips were again rinsed in PBS, and those containing tenascin-C spots were incubated with monoclonal antibody RT97 against neurofilament followed by a rhodamine-conjugated ~~oat anti-mouse secondary antibody, whereas those containing CSPG spots were incubated with polyclonal antibody against neurofilament 200 followed by a rhodamine-conjugated goat anti-rabbit secondary antibody. All primary and secondary antibody incubations were for 30 minutes at 4°C. Coverslips were rinsed in PBS
followed by ddH~O and then mounted in Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) on microscope slides. Nonspecific binding of secondary antibodies was controlled for by omitting the appropriate primary antibody in parallel cultures.
Cultures were examined using a Zeiss Axioplan microscope equipped with an epifluorescence illuminator with appropriate filter sets to visualize the fluorochromes. Images of the cultures were captured using a Macintosh Quadra 700 with a Scion LG-3 frame grabber board. Images were analyzed by counting tile number of neurites on both sides of the PLL/protein interface that either remained on their substrate (by virtue of either stopping or turning at the interface) or crossed to the other side. A sample of 75 neurites was considered for each side of the interface for each condition. Figure 2 is a schematic diagram presenting our modified neurite guidance assay. Only single, nonfasiculated neurites within 10 qm of the protein/PLL interface were considered for the analysis. This distance was chosen because filopodia have been shown to extend 10-50 ym ((iomez and Letourneau, I 994).
In addition, only neurites moving toward the interface were counted (the angle between the neurite and the interface was less than 90°). and no neurite whose soma was sitting on the interface was counted.
The percentage of neurites that crossed from PLL to the protein of interest or from the protein to PLL was then assessed.
Neurite Outgrowth Assay To investigate the neurite outgrowth promoting properties of fnA-D vs. filA-D
(-) C, PLL-coated glass coverslips in 24-well trays were incubated with expression proteins (250 nM
in HBS) for 2 hours at 37°C. In some experiments. coverslips were incubated with a mixture of fnA-D and monoclonal antibody J1/tn2 (75 qg/ml). Excess protein solution was rinsed away with HBS, and cerebellar granule neurons were plated onto the coverslips at a density of 60,000 neurons/well and allowed to extend neurites for 48 hours in DMEM-25 mM KC1/10%
FCS. The extent of neurite outgrowth was then determined via carboxyfluorescein diacetate (CFDA) labeling (Petroski and Geller, 1994). CFDA (Sigma Chemical Co.) intensely stains the soma and all processes of cultured, living neurons. Images of the cultures were captured using a Macintosh Quadra 700 and analyzed with the NIH Image Software (available at http://rsb.info.nih.gov/).
A sample of 100 neurons with processes equal to or greater than one cell soma was considered for each condition. The length of each primary process and its branches was measured for each neuron, and the total neurite length was calculated as the sum of the lengths of individual neurites.
Neurite Guidance Assay on Cellular Substrates To investigate regulation of neurite guidance in a cellular context, we generated cellular interfaces between untransfected BHK cells, which express no tenascin-C, and transfected BHK
cells, which over express either the largest or su~allest tenascin-C splice variant (Aukhil et al., 1993), according to a modified method of Powell et al. (1977). First, transfected BHK cells were labeled with the red fluorescent cell linker PKH26 (Sigma Chemical Co.) according to the manufacturer's instructions. This dye binds irreversibly within the membranes of cells by selective partitioning with no apparent transfer of the label to Luzlabeled cells (Ford et al., 1996).
Single cell suspensions of transfected cells and untransfected cells were then mixed in a 1:10 ratio. The cell mixture was plated onto PLL-coated glass coverslips in 24-well trays at a density of 1 x 10' cells per coverslip. This density yielded confluent monolayers 24 hours later with readily distinguishable "islands" of individual PICH26-labeled, transfected cells interspersed amongst the untransfected cells. The transfected cells were also readily distinguished from untransfected cells by tenascin-C immunoreactivity.
Cerebellar granule neurons were plated onto BHK monolayers in DMEM-25 mM
KCl/10% FCS and were allowed to extend neurites for 48 hours. At this time, neurons and their processes were labeled with CFDA. Images of the cultures were captured, and neurite behavior was analyzed on both sides of the interface formed between an untransfected cell and a transfected cell. The number of neurites that originated on an untransfected cell and either remained on the untransfected cell or crossed to a transfected cell was assessed, as was the number of neurites that originated on a transfected cell and either remained on the transfected cell or crossed to an untransfected cell. A sample of 75 neurites was considered for each of these conditions. Only neurites within 10 ~m of the interface were included in the analysis.
Antibody Blocking Experiments on Cellular Substrates To investigate the role of specific FN-1IT sequences in the regulation of neurite guidance by cellular tenascin-C. blocking experiments were conducted using polyclonal antibodies against full-length tenascin-C, alternatively spliced domains fnA-D, and universal domains fnl-5.
Monoclonal antibody JI/tn2, which reacts within fnD of fnA-D, was also employed in blocking experiments. Mixed monolayers containing untransfected and transfected BHK
cells were incubated with 75 ~ghnl of antibody in DMEM-25 mM ICCI/10% FCS for 1 hours at 37°C.
Cerebellar granule neurons were plated onto the cells and cultured for 48 hours in the presence ~0 of antibodies. Neurite behavior at the interface Uetween transfected and untransfected cells was then evaluated.
JO

Results The Alternatively Spliced Region of Tenascin-C Provides Permissive Neurite Guidance Cues.
FnA-D avidly promotes neurite outgrowth from a variety of CNS neurons (Meiners and Geller, 1997). We therefore investigated whether fnA-D can also provide guidance cues to growing neurites. Cerebellar granule neurons were cultured for 48 hours on PLL-coated glass coverslips containing spots of alternatively spliced or universal tenascin-C
FN-III domains. The behavior of the neurites was then analyzed at the protein/PLL interface.
Figure 3 A shows that cerebellar <granule neurites demonstrated a strong preference for fi1A-D when encountering an interface between fnA-D and PLL. The neurites (which showed with a red color), and the fnA-D/PLL interface (the fnA-D region showed as a green color) are visualized in the black and white Figure 3 B. More than 80% of the neurites originating on PLL crossed to fnA-D, and less than 20% of the neurites originating on fnA-D crossed to PLL. This was significantly different from results obtained with neurites growing across a control fluorescein-labeled BSA/PLL
interface, where about 50% of the neurites originating on l'LL crossed to BSA
and vice versa.
The same 50/50 crossing ratio was observed for neurites growing across an imaginary interface, created by drawing an ink circle approximating the size of a 5 ~l protein drop on the back of the PLL-coated coverslip (data not shown). The increased number of neurites crossing onto fnA-D
indicates that the alternatively spliced region pi°ovides permissive neurite guidance cues. In contrast, universal FN-III domains fnl-~ and fn6-8 did not elicit neurite behavior which differed significantly from the control.
Because fnA-D promotes neurite outgrowth as an expression protein and as a part of the largest tenascin-C splice variant (Meiners and Geller. 1997). we investigated its ability to guide neurites in the context of native tenascin-C. Neurites were allowed to choose between PLL and either the largest or smallest tenascin-C splice variant. We only assessed neurite behavior on the PLL side of the interface as very few neurons adhered to purified tenascin-C.
Neurites consistently avoided both splice variants (Figure 3 A). This is in agreement with qualitative results of others showing cerebellar granule neurite deflection by spots of tenascin-C
(representing a mixture of splice variants) isolated from neonatal mouse brain (Gotz et al., 1996;
Dorries et al., 1996). Therefore, the permissive guidance properties of fnA-D
were masked by other parts ofthe tenascin-C molecule, indicating that tenascin-C was more iWibitory on a molar basis than fizA-D was permissive. This observation was also reflected in dose-response curves obtained for tenascin-C and tiiA-D actions: the inhibitory effect of both tenascin-C splice var iants and the permissive effect of fiiA-D were dose-dependent with a tendency toward saturation at 100 and 300 nM, respectively (data not shown). On the other hand, the smallest tenascin-C
splice variant was always more repellant than the largest tenascin-C splice variant, with only 1-2% of the neurites crossing ti~om PLL to small tenascin-C as opposed to about 10% for large tenascin-C. Blocking large tenascin-C with a polyclonal antibody against fnA-D
reduced the percentage of neurites that crossed. This suggests that fnA-D included only in large tenascin-C
may partially overcome the boundary formed by the rest of the molecule. Given that homogenous substrates of fn6-8 (Meiners and Geller, l 997) and large and small tenascin-C splice variants (Chiquet and Wehrle-Halter, 1994; Meiners and Geller, 1997) all promote neurite outgrowth, the results of this experiment indicate that the ability to facilitate neurite extension does not necessarily correlate with the ability to provide permissive neurite guidance cues.
The Alternatively Spliced Region Overcomes Tenascin-C Boundaries to Neurite Advance.
We next investigated the hypothesis that a molar excess ofthe alternatively spliced region 3~

could overcome the inhibition of the rest of the tenascin-C molecule. To address this issue, we incubated PLL-coated coverslips with spots of protein consisting of a mixture of fnA-D and small tenascin-C. The concentration of small tenascin-C was held constant at 100 nM while that of fnA-D was increased from 100 nM to 400 nM (Figure 4 A). As in Figure 3, only 1-2 % of the neurites crossed onto small tenascin-C. This number increased to 8-I 0 % for small tenascin-C
in combination with 100 nM fiZA-D, precisely the same percentage of neurites that crossed to large tenascin-C (Figure 3). The percentage of neurites that crossed onto mixtures of small tenascin-C and fnA-D increased as the concentration of fnA-D was increased and reached the maximum with 300 nM fi~A-D. This concentration resulted in 60-70% of the neurites crossin'.~;
larger concentrations of fnA-D did not further increase the percentage of neurites crossing.
Hence, the concentration of fnA-D that is the most efficacious at providing neurite guidance cues by itself (Figure 3) is also best to overcome the inhibitory guidance cues of small tenascin-C.
Neurites more readily crossed onto 300 nM fnA-D (Figure 3) than onto a mixture of 300 nM
fnA-D and 100 nM small tenascin-C, indicating that fnA-D largely mitigates.
but does not entirely abolish. the inhibitory properties of small tenascin-C.
We also investigated the ability of the alternatively spliced region to overcome the inhibitory guidance cues of lar<~e tenascin-C. Vve found that a molar excess of fnA-D weakened the boundary formed by this tenascin-C splice variant (100 nM); however. the maximal effect was observed with 200 nM foA-D (Figure 4 B) rather than 300 nM. The lower concentration of fnA-D necessary to overcome the boundaries formed by larvae as opposed to small tenascin-C
probably reflects the fact that large tenascin-C already contains one filA-D
sequence whereas small tenascin-C contains none.
The Alternatively Spliced Region Overcomes CSPG Boundaries.
., -, Our next objective was to investigate whether the permissive guidance cues of fnA-D
could also override inhibitory guidance cues provided by other types of molecules. We investigated its effects in combination with CSPGs. because CSPGs deflect neuronal processes in culture (Snow et al., 1990). and because tenascin-C and CSPGs are often co-regulated on astrocytes (Meiners et al., 1995; Powell et al.. 1997; McKeon et al., 1991).
We first assessed neurite behavior at an interface formed between PLL and a mixture of CSPGs (Table 1 ) consisting largely of neurocan. phosphacan, versican, and ag'~recan. Because native CSPGs with intact glycosaminoglycan side chains revealed a smear on SDS-PAGE gels and accurate molecular weights could not be assigned (data not shown), we used 10 ~g/ml of the CSPG
mixture in this experiment rather than a specified molar concentration.
Neurites avoided the CSPG mixture, with only 1-2'% crossing fiom PLL to the CSPGs. As with tenascin-C, neurons did not adhere to the CSPG mixture. and neurite behavior on the CSPG side of the interface was not assessed. When the CSPGs were combined with lilA-D ( 300 nM), about 60% of the neurites now crossed onto the mixture of CSPGs and fnA-D. Lar~~er concentrations of fi~A-D did not fiu-ther increase the percentage of neurites crossing.
TABLE I. FNA-D OVERCOMES CSPG BOUNDARIES TO NEURITE ADVANCE
of nenrites crossing to CSPG mix % of nenrites crossing to aggrecan Addition Concentration Concentration 300 nM I yM 300 nM 1 pM
None 1 ~ 1 2 ~ 1 FnA-D 58~d ~9~4 644 635 Laminin-1 9 t 1 25 ~ 4 10 ~ 2 23 ~ 3 fn6-8 1212 203 14=2 21 ~4 Data represent the mean ~ SEM (n = 4) 2~

We compared the effects of fnA-D with laminin-1, a potent promoter of neurite outgrowth. Laminin-1 was not nearly as effective in guiding neurites by itself (45% of the neurites crossed onto 300 nM laminin-1 (M.L.T. Mercado, unpublished data) as opposed to 80%
for fnA-D) or in overcoming the CSPG barrier (only about 10% of the neurites crossed onto the mixture of CSPGs and lamiW n-1). Increasing the concentration of laminin-1 to 1 q,M only increased the percentage of crossed neurites to 2~-30%. The experiment was repeated using a single CSPG, aggrecan, instead of a mixture, and similar results were obtained. FnA-D was more effective than laminin-1 in mitigating the inhibitory guidance cues of aggrecan. FnA-D was also more effective than fn6-8, another neurite outgrowth promoting molecule (Meiners and Geller, 1997) that does not provide guidance information to neurites (Figure 3 A).
Thus fnA-D
specifically overcomes boundaries to neurite advance that are formed by a variety of different CSPGs.
Neurite Guidance and Outgrowth Are Mediated by Different Sequences Within FnA-D.
Facilitation of neurite outgrowth by fnA-D bound to inert substrates has been mapped to filD (Gotz et al., 1996). We therefore explored the question, are neurite outgrowth and neurite guidance mediated by the same or different sequences within fnA-D? To do this, we evaluated the ability of monoclonal antibody Jl/tn2 to alter neurite behavior in both neurite guidance and neurite outgrowth assays. This antibody specifically blocks the neurite outgrowth promoting site within fnD (Gotz et al., 1996: Gotz et al., 1997; Meiners et al., 1999). Spots of fnA-D or a mixture of fnA-D and .11/tn2 were made in the center of PLL-coated coverslips, and neurite behavior was quantified at the interface. .T1/tn2 did not alter the percentage of neurites crossing from PLL onto fnA-D (Figure 5 A) or the percentage of neurites crossing from fnA-D to PLL
(data not shown). Neurite out~~rowth assays were then conducted to quantify process extension ;>

on PLL or homogenous substrates of fnA-D or a mixture of fnA-D and J1/tn2 adsorbed to PLL-coated coverslips. Box-and-whisker plots of total neurite length are shown in Figure 5 B. Boxes enclose 25th and 75th percentiles of each distribution and are bisected by the median; whiskers indicate 5th and 95th percentiles. As expected, neurites were considerable longer on fnA-D in comparison to PLL, and J1/tn2 eliminated the promotion of neurite outgrowth by fnA-D. In control experiments, monoclonal antibody tenascin III-B, which reacts within fnB (Chemicon International Inc.), failed to alter outgrowth or guidance by fnA-D. These results indicate that neurite guidance by fnD is regulated by a different sequence from that promoting neurite outgrowth.
To begin to localize neurite guidance sites) within fiiA-D to a particular region of the protein, cerebellar granule neurons were cultured for 4~ hours on PLL-coated coverslips containing spots of fnAl-A4, the N terminal portion of fnA-D; fnB-D, the C
terminal portion of fnA-D; or a mixture of fnA 1-A4 and fnB-D (s00 nM of each). Neurite behavior was then evaluated at the expression protein/PLL interface (Figure 6). Only 20% of the neurites originating on the PLL side of a PLL/fnAl-A4 interface crossed onto fnAl-A4, while neurites originating on the fnAl-A4 side of the interface showed no bias for either PLL
or fnAl-A4 (compare to neurite behavior at the BSA/PLL control interface in Figure 3). On the other hand, neurites showed a preference for fnB-D: the percentages of neurites crossing to fnB-D fi-om PLL
and to PLL from fnB-D were nearly identical to those observed for fiiA-D
(compare to Figure 3). Hence filB-D mimicked the actions of fnA-D. An equimolar mixture of fnAl-A4 + fnB-D
also mimicked the actions of filA-D, suggesting that the C terminal portion of fnA-D provides permissive neurite guidance cues and overcomes the inhibitory boundary formed by the N
terminal portion.
W

We next explored the hypothesis that fnC provides guidance cues to growing neurites.
The rationale for this hypothesis was based on published work demonstrating that cerebellar granule neurites avoid rodent fnA-D expression proteins which lack fnC (Gotz et al., 1996). We compared fnA-D to an fnA-D expression protein missing fnC (fnA-D (-) C) in neurite guidance and neurite outgrowth assays. Neurite behavior was quantified at fnA-D or fnA-D (-) C (300 nM)/PLL interfaces (Figure 7 A). As in Figure 3 A, more than 80% of the neurites crossed from PLL onto fnA-D. This was reduced to about 25% for neurites crossing onto fnA-D
(-) C. These data are consistent with the hypothesis that fnC provides permissive guidance cues which overcome the barrier to neurite advance formed by fnA 1-A4, the N terminal portion of fnA-D
(Figure 5). As with fnAl-A4, neurites originating on filA-D (-) C did not show a preference for either fnA-D (-) C or PLL. When neurite outgrowth assays were performed for neurons cultured on fnA-D or fnA-D (-) C adsorbed to PLL-coated coverslips (Figure 7 B), both proteins were found to be equally permissive to process extension in comparison to PLL
alone. These results along with those of Figure 6 imply that neurite ~~uidance and neurite promotion by fnA-D a1°e mediated through differ°ent alternatively spliced I~N-III domains: fnC
for neurite guidance and fnD for neurite promotion.
FnA-D Guides Neurites in the Context of Cellular Tenascin-C.
Work with purified substrates is informative but does not always predict the iu vivo situation, where many molecules are present in a biological matrix (Meiners and Geller, 1997).
We therefore investigated the ability of fnA-D to provide permissive neurite guidance cues in the context of tenascin-C expressed by a cell, where a neuron would normally encounter it. BHK
cells transfected with the largest or smallest splice variant of human tenascin-C (BHK-TN.L or BHK-TICS cells, respectively) were combined with control. untransfected BHK
cells in a mixed monolayer. Cerebellar granule neurons were cultured on the mixed monolayer for 48 h, and the behavior of neurites at the interface formed between transfected and control cells was assessed.
Figure 8 A presents an image of the neuron/BHK co-culture following double immunocytochemistry with antibodies against full-length tenascin-C to detect transfected cells and RT97 to detect neurons. Neurites crossed quite readily from control BHK
cells to BHK-TN.L cells, but they avoided crossing from BHK cells to BHK-TICS cells.
Similar results were obtained using cerebral cortical neurons (data not shown). This is in contrast to results obtained with purified substrates of tenascin-C, which always formed barriers to neurites regardless of the splice variant present (Figure 2).
We then quantified neurite behavior at the interface formed between transfected and control BHK cells (Figure 8 B). BHK-TN.L or BHK-TICS cells were labeled with the membrane marker PKH26 to ensure that we were examining a cellular rather than a matrix boundary. Immunocytochemistry and Western blotting demonstrated that PKH26 labeling did not interfere with the expression of tenascin-C by the transfected cells (data not shown). Neurites 1 > demonstrated a preference for BHK-TN.L cells in comparison to control BHK
cells. About 70%
of the neurites that originated on a BHK cell crossed to a BHK-TN.L cell, and only 20% of the neurites that originated on a BHK-TN.L cell crossed to a BHK cell. This was significantly different fiom neurite behavior observed at a control BHK/BHK interface created between BHK
cells and PKH26-labeled BHK cells, where the percentage of neurites that crossed to and from a PKH26-labeled cell was 45-50%. On the other hand. neurites demonstrated a preference for BHK cells over BHK-TICS cells. The percentage of neurites that crossed from a BHK cell to a BHK-TICS cell (20%) was significantly lower than control, while the percentage of neurites that crossed from a BHK-TICS cell to a BHK cell (60-6~%) was significantly higher than control. Therefore, only small tenascin-C provides iWibitory neurite guidance cues when J

expressed by a BHK cell. This suggests that the alternatively spliced region included in large tenascin-C overcomes the barrier formed by the rest of the molecule by providing permissive neurite guidance cues of its own.
To asceuCain that fnA-D does indeed provide permissive guidance cues in the context of a cellular matrix, a panel of antibodies against tenascin-C was tested for interference with neurite behavior at cellular interfaces (Figure 9). The selection included polyclonal antibodies against full-length tenascin-C, frZA-U, and fnl-5, and monoclonal antibody J1/tn?. All of these antibodies cross-react with the large tenascin-C' splice variant on transfected BHK cells. As I 0 expected. the polyclonal antibody against fizA-D and monoclonal antibody J
1 /tn2 do not cross react with the small tenascin-C' splice variant, the fill-5 antibody does not cross-react wiih fhA-D, and the fnA-D antibody does not cross-react with fnl-5 (Meiners and Geller, 1997).
The first antibody tested was a polyclonal antibody against full-length tenascin-C. In the presence of this antibody. the percentage of neurites that crossed from a BHK
cell to a BHK-I5 TICS or a BHK-TN.L cell was indistin~~uishable -Ii~om the control value obtained for neurites crossing from BHK cells to BHK cells (compare to Figure R B). as was the percentage of neurites that crossed from a BHK-TN.L or BIIK-TICS cell to a BHK cell (data not shown).
This confirms that large and small tenascin-C were directly responsible for the permissive and inhibitory nenrite guidance properties of BHK-TN.L and BHK-TICS cells, as opposed to some ?0 other factor produced by the transfected cells. ~f~he polyclonal antibody against fnl-~ did not alter the percentage of neuritcs that crossed to BHK-TN.L c>r BHK-TICS cells_ which was to be expected because an expression protein corresponding to this sequence did not provide neurite guidance cues (Figure 3).

We then examined the effects of the two antibodies that react within the alternatively spliced region, polyclonal antibody against fnA-D and monoclonal antibody J 1 /tn2. Neither of these antibodies altered the percentage of neurites that crossing from BHK
cells to BHK-TICS
cells (data not shown). However, the polyclonal antibody against fnA-D
dramatically reduced the percentage of neurites that crossed to BHK-TN.L cells ti~om 70% to 20%. In the presence of this antibody, BHK-TN.L cells repelled neurites to the same extent as BHK-TICS cells.
Therefore the permissive guidance cues of large tenascin-C expressed by transfected BHK cells could be mapped to the alternatively spliced region, suggesting that fnA-D's ability to guide neurites is masked in purified tenascin-C (Figure 3) but revealed in the BHK
cell matrix.
Monoclonal J1/tn2 had no effect on the percentage of neurites crossing from BHK cells to BHK-TN.L cells. Hence neurite promoting sequences within fnA-D do not provide guidance cues to neurites. indicating that neurite outgrowth and ~~uidance facilitated by the alternatively spliced region of tenascin-C are distinct events which can he independently regulated on cellular as well as inert substrates.
Disc~ission The alternatively spliced FN-III region of tenascin-C. designated fnA-D, promotes neurite outgrowth as a substrate-bound molecule and also facilitates neurite guidance.
Its permissive actions can be seen whether liiA-D is presented to neurons as a purified expression protein or as part of cellular tenascin-C in a biological matrix. Other molecules, such as the netrins (Kennedy et al., I 994; Serafini et al., 1994), also have strong effects oa both the outgrowth and orientation of axons. However, in the case of the netrins. both processes are mediated through the same neuronal receptor. which probably interacts with the same functional domain of the netrin molecule (de la Torre et al.. 1997). To our knowledge. fnA-D is the first molecule that independently facilitates neurite outgrowth and guidance through different sequences (located within alternatively spliced FN-III domains D and C, respectively), providing strong evidence that outgrowth and guidance are separable events.
The fact that hwnan lizA-D provided permissive guidance cues was somewhat surprising at first given published data with purified substl-ates of mouse fnA-D (Gotz et al., 1996). Mouse fnA-D facilitates process extension to the same extent as human filA-D due to a common neurite outgrowth promoting site within fiiD (Gotz et al.. I 996), but has been reported to forms barriers to neurites. This implies that some sequence unique to human but not mouse InA-D facilitates neurite ;~uidauce and that the common neurite outgrowth promoting site is not involved. In agreement with this hypothesis, monoclonal antibodies against the neurite outgrowth promoting site within fnD did not alter the ability of human fnA-D to guide neurites. We also found that neurites demonstrated a preference for human fn B-D in guidance assays but avoided rat fnB-D
(data not shown). The rat fnB-D expression protein used in our assay and the mouse fnA-D
1 s expression protein used in the Gotz et al. study were both obtained via PCR using rodent cDNA
as the template. However, it appears both of these proteins lack the alternatively spliced FN-III
domain C. in agreement with reports demonstratin~~ that cDNA encoding the lamest splice variant of rodent tenascin-C does not contain this domain (LaFleur et al., 1994). We found that a naturally occurring variant of human fnA-D lacking fnC also formed barriers to neurites instead ?0 of attracting them. Taken together. these data suggest that fnC is not only responsible for the permissive guidance cues of human fnA-D, but overcomes inhibitory guidance cues provided by the rest of the molecule. In contradiction to the earlier reports (LaFleur et al., 1994; Gotz et al..
1996), PCR data have now demonstrated that alternatively spliced transcripts of mouse tenascin-C can contain fnC (Dorries et al., 1996; Gotz et al., 1997), although they are presumably less common than mouse transcripts lacking fnC. Hence, it is of great interest to determine if mouse fnC also imparts permissive guidance cues to neurites, or if the guidance properties of the alternatively spliced region are unique to the human protein.
In contrast to the permissive guidance cues provided by human fnA-D, purified substrates of all splice variants of human (see Figure 3) and mouse (Gotz et al., 1996;
Dorries et al., 1996) tenascin-C are repulsive to advancing growth cones (Dorries et al., 1996). The growth cone repelling properties have been attributed to the EGF domains (Gotz et al., 1996; Dorries et al., 1996). Therefore, on a molar basis, the EGF domains are moi°e inhibitory than the alternatively spliced region is permissive. However, a 2-3 fold molar excess of fnA-D
significantly overcomes the boundary formed not only by tenascin-C, but also by a variety of CSPGs.
Much larger concentrations of laminin-1 were not nearly as effective. This is significant in that tenascin-C
and CSPGs are up regulated on glial scars after injury (McKeon et al., 1991;
Pindzola et al., 1993), where they have been strongly implicated in failed axonal regeneration (Gates et al., 1996;
Davies et al., 1997). Full recovery cannot occur following CNS injury unless axons are guided across the iWibitory terrain of the glial scar, su<~gesting a potential therapeutic role for fiiA-D.
We have previously suggested that functions of the EGF domains of tenascin-C
are obscured in cellular as opposed to purified tenascin-C, perhaps by cell-derived molecules binding to them or due to conformational restraints on cellular tenascin-C (Meiners and Geller, 1997).
Specifically, the EGF domains promoted neurite outgrowth as purified expression proteins (Dorries et al., 1996; Gotz et al., 1996) but had no effect on outgrowth in the context of cellular tenascin-C (Gotz et al., 1997: Meiners and Geller, 1997). We found that antibodies directed against tenascin-C FN-III domains 6-8 and A-D blocked al l regulation of neurite outgrowth by 4?

cellular tenascin-C; the EGF domains did not contribute (Meiners and Geller, 1997). We therefore reasoned that the boundary-forming properties of the EGF domains, in addition to the neurite promoting properties, might be similarly attenuated in the cellular tenascin-C. If the EGF
boundary was weakened in a biological matrix, the permissive guidance cues of fnA-D might then be revealed in the large tenascin-C splice variant. In support of this hypothesis, transfected BHK cells which over expressed small tenascin-C formed a barrier to neurites whereas cells which over expressed large tenascin-C were attractive to neurites. However, more neurites crossed onto BHK-TICS cells than onto purified small tenascin-C, and fewer neurites crossed onto BHK-TN.L cells than onto purified fnA-D (compare Figs. 3 and 8). At the same time, in early experiments, neurites preferred BHK cells to which thA-D was bound over BHK-TN.L
cells. This suggests that the boundary-forming properties of tenascin-C's EGF
domains were partially but not totally eliminated in the BHK cell environment. Monoclonal antibodies against neurite outgrowth promoting) sites did not affect the percentage of neurites that crossed to either BHK-TN.L or BHK-TICS cells, demonstrating that neurite guidance and outgrowth facilitated by fnA-D were separable phenomena on cellular as well as inert substrates.
While our results were obtained using BHK cells, it seems quite conceivable that guidance ofneuronal processes by tenascin-C splice variants could vary with cell type. Different cell type-specific molecules might bind and mask different active sites for neurite guidance within the tenascin-C molecule, as we have seen for neurite outgrowth promoting sites within ?0 the alternatively spliced region (Meiners et al., 1999). Alternatively, cell-type specific molecules might provide neurite guidance cues of their own which compete with or over ride those of tenascin-C. For example, when the ratio of CSPGs to fnA-D was low, neurites were deflected, but as the ratio of fnA-D was increased, neurites crossed. Hence the neuronal growth regulatory properties of tenascin-C or any other matrix protein can at best be discussed in a relative sense, and coordinated expression of specific tenascin-C splice variants by particular subsets of cells may provide appropriate micro environments for regulated changes in neuronal process outgrowth.
In summary, we have shown that the alternatively spliced region of human tenascin-C
contains independent domains that promote either neurite outgrowth or neurite guidance.
Extension of neurites is facilitated through alternatively spliced FN-III
domain D, and orientation of growth is influenced by alternatively spliced FN-III domain C. Each of these processes can be regulated without affecting the other, indicating that neurite outgrowth and neurite guidance are distinct fundamental mechanisms of neuronal growth. Moreover, the ability of fnA-D to promote guidance was stoichiometric, and fnA-D could overcome inhibitory actions of both tenascin-C and CSPGs. Thus. fnA-D on its own might find applicability as a reagent to promote neurite growth in otherwise inhibitory environments.
References 1. AL~Ichil, L, P. Joshi, Y. Yan, and H.P. Erielcson. 1996. Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression p,~~~oteins.
.l.I3iol.Chen~.
268:2542-2553.
2. Aulchil, I., C.C. Slemp, V.A. Lightner, K. Nishilnura, G. Briscoe, and H.P.
Erickson.
1990. Purification of hexabrachion (tenascin) from cell culture conditioned medium, and separation from a cell adhesion factor. ~ldcrtriv 10:98-111.
3. Bartsch, S., U. Bartsch, U. Dorries, A. Faissner. A. Weller, P. Ekblom, and M.
Schaclmer. 1992. Expression of tenascin in the developing and adult cerebellar cortex.
.LNeurosci. 12:736-749.
4. Chiquet, M. and B. Welu-le-Haller. 1994. Tenascin-C' in peripheral nerve moiphogenesis.

Peg°spec. Dev. Neu~obiol. 2:67-74.

5. Chung, C.-Y., J.E. Murphy-Ullrich, and H.P. Erickson. 1996. Mitogenesis, cell migration, and loss of focal adhesions induced by tenascin-C interacting with its cell surface receptor, annexin II. Mol.Biol.C'ell 7:883-892.

6. Crossin, K.L., S. Hoffman, S.S. Tan, and G.M. Edelman. 1989. Cytotactin and its proteoglycan ligand mark structural and functional boundaries in somatosensory cortex of the early postnatal mouse. Dev.Biol. 136:381-392.

7. Davies, S. J., M.T. Fitch, S.P. Memberg, A.K. Hall. G. Raisman, and J.
Silver. 1997.
Regeneration of adult axons in white matter tracts of the central nervous system. Nature 390:680-683.

8. de la Torre, J.R., V.I I. Hopker, G.L. Ming, M.M. Poo, M. Tessier-Lavigne, Hemmati-Brivanlou, and C.E. Holt. 1997. Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neunorr 19:121 1-1244.

9. Dorries, U., J. Taylor. Z. Xioa, A. Lochter, D. Montage. and M. Schaclmer.
1996. Distinct effects of recombinant tenascin domains on neuronal cell adhesion, growth cone guidance, and neuronal polarity. .LNeuro,s~ci.Re.s. 4 3:420-438.

10. Erickson, H.P. and M.A. Bourdon. 1989. Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu.Rev.Cell Biol.
5:71-92.
l l . Ericlcson, H.P. and Briscoe, G. (1995) Tenascin, laminin, and fibronectin produced by cultured cells. In: Extracellular matrix: a practical approach. M.A. Haralson and J.R.
Hassell, eds. Oxford Univ. Press, Oxford, pp. 187-198.

12. Faissner, A. and J. Kruse. 1990. J1/tenascin is a repulsive substrate for central nervous system neurons. Neuron 5:627-637.
13. Fischer, D., M. Brown-Ludi, T. Schulthess, and R. Chiquet-Ehrismann. 1997.
Concerted action of tenascin-C domains in cell adhesion, anti-adhesion and promotion of neurite outgrowth. J. Cell Sci. 110:1513-1522.
14. Ford, J.W., T.H.3. Welling, J.C. Stanley, and L.M. Messina. 1996. PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitt°o and in vivo endothelial cell localization and tracking. J.Sur~.Res. 62:23-28.
15. Gates, M.A., H. Fillmore, and D.A. Steindler. 1996. C'hondroitin sulfate proteoglycan and tenascin in the wounded adult mouse neostl~iatum in vitro: dopamine neuron attachment and process outgrowth. JNeuro.sci. 16:8005-8018.
16. CJomez, T.M. and P.C. Letourneau. 1994. Filopodia initiate choices made by sensory neuron growth cones at laminin/fibronectin borders in vitro. .LNeurosci.
14:5959-5972.
17. Gotz, B., A. Scholze. A. Clement, A. Joester. K. Schutte, F. Wigger, R.
Frank, E. Spiess, P. Ekblom, and A. Faissner. 1996. Tenascin-C contains distinct adhesive. anti-adhesive, and neurite outgrowth promoting sites for neurons. .LC'ell Biol. 132:681-699.
18. Gotz, M., J. Bolz, A. .loester, and A. Faissner. 1997. Tenascin-C
synthesis and influence on axon growth during rat cortical development. Em.J.Neuro,sci. 9:496-506.
19. Kaplony. A., D.R. Zimmermam, R.VV. Fischer, 13.A. Imhof, B.F. Odermatt, K.H.
Winterhalter, and L. Vaughan. 1991. Tenascin Mr 220 000 isoform expression correlates with corneal cell migration. Devclopmem 112:605-614 20. Kennedy, T.E., T. Serafini, J.R. de la Torre. and M. Tessier-Lavigne.
1994. Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78:425-435.
21. LaFleur, D.W., J.A. Fagin, J.S. Forrester, S.A. Rubin, and B.G. Sharifi.
1994. Cloning and characterization of alteryatively spliced isoforms of rat tenascin.
Platelet-derived growth factor-BB markedly stimulates expression of spliced variants of tenascin mRNA
in arterial smooth muscle cells. J.Biol.C'hem. 269:20757-20763.
22. Laywell, E.D., P. Friedman, K. Harrington, J.T. Robertson, and D.A.
Steindler. 1996.
Cell attachment to frozen sections of injured adult mouse brain - Effects of tenascin antibody and lectin perturbation of wound- related extracellular matrix molecues.
J.Nem°o.S~ci.Meth. 66:99-108.
23. Letourneau, P.C., D.M. Snow, and T.M. Gomez. 1994. Regulation of growth cone motility by substratum bound molecules and cytoplasmic [Ca2+~. Prog.Brair7 Rea~.
103:85-98.
24. Levi, G., F. Aloisi, M.T. Ciotti, and V. Gallo. 1984. Autoradiograpl>ic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures. B~°ain Re.s. 290:77-86.
25. Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A.
Faissner.
1991. J1 /tenascin in substrate-bound and soluble form displays contrary effects on neurite outgrowth. .LC.'ell Binl. I 13:1159-1171.
26. McKeon. R.J., R.C. Schreiber, ,1.S. Rud;~e, and J. Silver. 1991. Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes. .LNem°osci.

11:3398-341 1.
27. Meiners, S. and H.M. Geller. 1997. Long and short splice variants of human tenascin differentially regulate neurite outgrowth. Mol.Cell.Neurosci. 10:100-116.
28. Meiners, S., E.M. Powell, and H.M. Geller. 1995. A distinct subset of tenascin/CSPG-rich astrocytes restricts neuronal growth in vitro. J.Neurosci. 15:8096-8108.
29. Meiners, S., E.M. Powell, and H.M. Geller. 1999. Neurite outgrowth promotion by the alteratively spliced region of tenascin-C is influenced by cell type-specific binding.
Matrix Biology 18:75-87.
30. Petroski, R.E. and H.M. Geller. 1994. Selective labeling of embryonic neurons cultured on astrocyte monolayers with 5(6)-carboxyfluorescein diacetate (CFDA).
J.Neurosci.Meth. 52:23-32.
31. Pindzola, R.R., C. Doper, and J. Silver. 1993. Putative inhibitory extracellular matrix molecules at the dorsal root entry zone of the spinal cord during development and after root and sciatic nerve lesions. Dev.Biol. 156:34-48.
32. Powell, E.M., J.W. Fawcett, and H.M. Geller. l x)97. Proteoglycans provide neurite <guidance at an astrocyte boundary. Mol. C.'ell.Neurn,w~i. 27-42.
33. Powell, E.M. and H.M. Geller. 1999. Dissection of astrocyte-mediated cues in neuronal guidance and process extension. Glia 26:73-83.
34. Serafini, T., T.E. Kennedy, M.J. Galko. C. Mirzayan, T.M. Jessell. and M.
Tessier-Lavigne. 1994. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78:409-424.
35. Smith, G.M., R.H. Miller, and J. Silver. 1986. Changing role of forebrain astrocytes during development, regenerative failure. and induced regeneration upon transplantation.
4~

J. Con2p.Neurol. 251:23-43.
36. Snow, D.M., V. Lemmon, D.A. Carrino, A.I. Caplan, and J. Silver. 1990.
Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro.
Exp.Neurol.
109:111-130.
37. Snow, D.M., M. Watanabe, P.C. Letourneau, and J. Silver. 1991. A
chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth.
DevelopJnent 1 13:1473-1485.
38. Steindler, D.A., N.G. Cooper, A. Faissner, and M. Schachner. 1989.
Boundaries defined by adhesion molecules during development of the cerebral cortex: the Jl/tenascin glycoprotein in the mouse somatosensory cortical barrel field. Dev.Biol.
131:243-260.
39. Vielmetter, J., B. Stolze, F. Bonhoeffer. and C.A. Stuermer. 1990. In vitro assay to test differential substrate affinities of growing axons and migratory cells.
Exp.BJ°ain Res 81:283-287.
1 ~ 40. Zhang, Y., J.K. Wintcrbottom. M. Schachner. A.R. Lieberman, and P.N.
Anderson. 1997.
Tenascin-C expression and axonal sprouting following injury to the spinal dorsal columns in the adult rat. J.Neurosci.Re.s. 49:433-450.
Example 2 We have found that the region of tenascin-C containing only alternately spliced fibronectin type-III domains A-D, called fiiA-D, when used by itself, dramatically increases neurite outgrowth from a variety of CNS neurons in culture. In addition, the fnA-D protein also can overcome the inhibitory actions of chondroitin sulfate proteoglycans, which are thought to inhibit neuronal regrowth after injury to the central nervous system. We have found that the fnA-D region of tenascin-C, when used by itself is able to promote:
( 1 ) growth of axons (2) extension of axons across inhibitory boundaries To further elucidate these phenomena, we used overlapping synthetic peptides to localize the neurite outgrowth promoting site within fnD to two 15 amino acid sequences, called D4 and D5.
An antibody against DS blocked promotion of neurite outgrowth from cerebellar granule neurons by fnD as well as tenascin-C, indicating that this peptide sequence is functional in the context of the native molecule. We then evaluated the overlapping region of D4 and DS
with testing of shorter synthetic peptides restricted the neurite outgrowth promoting site to 8 amino acids, VFDNFVLK (SEQ ID NO: l ), which represents AA 1646-1 C53 of the human tenascin-C cDNA
(Gherzi,R., Carnemolla,B.. Siri,A., Ponassi,M.. Balza,E. and Zardi,L, Human tenascin gene.
Structure of the 5'-region, identification, and characterization of the transcription regulatory sequences. .1. Biol. Chem. 270, 3429-3434. 1995: MEDLINE 95155442; Genbanlc ACCESSION: X78565) Ofthese, "FD" and "FV" are conserved intenascin-C sequences derived from all the species available in Genbank. To investigate the hypothesis that "FD" and "FV" are critical for the interaction with neurons, we tested a recomUinant fnD protein and a synthetic peptide in which "FD" and "FV" were changed to "SA" and"SS", respectively.
These molecules did not facilitate process extension, suggesting that the conserved amino acids are required for formation of the active site in fnD.
~0 We conclude that the growth promoting region of fnA-D is within fnD, and the minimal region we have identified is seven amino acids comprising AAs 1647-1653 of the human tenascin-C cDNA as included in Genbanlc.
Example 3 We also investigated whether VFDNFVLK could be used as a reagent to overcome the neurite outgrowth iWibitory properties of chondroitin sulfate proteoglycans (CSPGs), the major inhibitory molecules found in the central nervous system after iyjury. Neurons do not grow when they come in contact with CSPGs. When mixed with CSPGs, the peptide sigW
ficantly enhanced outgrowth on proteoglycans and was more efifiective than the neurite outgrowth promoting molecules laminin-1 or Ll-~c. thus demonstrating that fnn-D and its smaller derivatives might find utility as part of a regeneration cocktail to stimulate neuronal regrowth following CNS
trauma.
I
Example 4 We hypothesized that there would be a receptor for DS that would mediate the neurite-outgrowth promoting actions. (31 integrins have been shown to act as receptors for tenascin-C, mediatin~~ signals from the e~tracellular matrix to the cvtoskeleton. For this reason, we investigated the potential role of integrins as receptors for the neurite outgrowth promoting ~I

tenascin-C peptide VFDNFVLK. We found that a commercially-available blocking antibody against (31 integrin chain eliminated the enhancement of neurite outgrowth from cerebellar granule neurons by VFDNFVLK as well as fnA-D, supporting a role for a (31 integrin as a neuronal receptor for this sequence.
We then searched for the a chain partner for the (31 integrin chain. Because the a7 integrin knockout mice show defects at the myotendinous junction, which is rich in tenascin-C, we tested the hypothesis that the a7 integrin chain is the partner for (31 in the promotion of neurite outgrowth by the VFDNFVLK. An antibody against a7 integrin eliminated the enhancement of neurite outgrowth by VFDNFVLK and fiiA-D. These results cannot be explained by the monoclonal a7 antibody recognizing the VFDNFVLK sequence because the antibody did not cross react with the peptide on a dot blot. Thus, the a7(3l integrin is a neuronal receptor mediating neurite outgrowth promotion by VFDNFVLK and fnA-D. Our data is the first evidence that this receptor mediates a response to a matrix molecule other than laminin-1 and the first functional data on the role of the a7(31 integrin receptor in neurons. Peripheral neurons that successfully regenerate processes following traumatic inju~w dramatically increase their expression of a7(31 inte~m°in, which may then allow them to respond to permissive substrate molecules in their environment such as tenascin-C.
Example ~
In addition to inhibition of growth, injured areas of the central nervous system form boundaries to the entry of regrowing axons. This property has been attributed to the upregulation of the synthesis of proteoglycans. We created an assay for boundary formation and found that fnA-D could allow neurons to overcome proteoglycan boundaries. The fnC region is required for this action, since a tenascin fnA-D protein without fnC did not have this property. Thus, the permissive boundary crossing ability is likehyto be within fnC.
Example 6 We propose that fnA-D or subregions can be used therapeutically to stimulate regrowth of injured axons in the human central nervous system. This protein can be delivered therapeutically to the area of a spinal cord lesion. The smallest possible peptide can be delivered as an infusion directly into a lesion site via a catheter. Peptides can be delivered in combinations with trophic agents or other agents, like caspase inhibitors, that stimulate neuronal survival, and other agents that might stimulate neuronal growth. This can be done in combination with therapies to limit the expression of proteoblycans in the nervous system.
Alternately. one can use gene delivery technology to have cells within the region of a lesion express fnA-D a follows: The astrocytes or other glial cells within a lesion site are caused to express a transgene containing the fnA-D region or subregion. The transgene for filA-D is under the control of a cell-type specific promoter, such as the ghial fibrillary acidic acid gene promoter for astrocytes or other appropriate promoters for expression in other cell types in the wound area.
These constricts are placed into viral vectors and injected into a lesion area. The viral vectors can adeno-associated virus or lentivirus which can express genes in filial cells or fibroblasts. The construct would have fnA-D being under the control of the GFAP promoter to restrict expression to astrocytes.
5p References of interest are: Chen H, McCarty DM, Bruce AT, Suzuki, Oligodendrocyte-specific gene expression in mouse brain: use of a myelin-forming cell type-specific promoter in an adeno-associated virus. .1 Neurosci Res 1999 Feb 15;55(4):504-13; and Mitrophanous K, Yoon S, Rohll J, Patil D, Winces F, Kim V, Kingsman S, Kingsman A, Mazarakis N, Stable gene transfer to the nervous system using a non-primate lentiviral vector.Gene Ther 1999 Nov;6(11):1808-18.
Example 7 We propose that the gene delivery can be similarly used to express the a7(31 integrin receptor in neurons to increase the neuronal response to fnA-D. This can be accomplished with replication-deficient Sindbis vims vectors or Herpes virus vectors which are neurotropic.
References of interest include:Wan g X, Zhang GR, Yang T, Zhang W, Geller AI
Fifty-one kilobase HSV-1 plasmid vector can be packaged using ahelper virus-free system and supports expression in the rat brain. Biotechniques 2000 Jam;28(1 ):102-7; Coopersmith R, Neve RL
Expression of multiple proteins within single primary cortical neurons using a replication deficient HSV vector. Bioteclmiques 1999 Dec;27(6):11 ~6-60; Navarro V, Millecamps S, Geoffroy MC, Robert JJ, Valin A, Mallet J, Gal La Salle GLEfficient gene transfer and long-term expression in neurons usin g a recombinant adenovirus with a neuron-specific promoter. Gene Ther 1999 Nov;6(11):1884-92.

Claims (32)

We claim:

1. A peptide comprising the 8-amino acid sequence VFDNFVLK. as defined by the one-letter amino acid code, said peptide consisting of not more than 75 amino acids.

2. A peptide of Claim 1, said peptide consisting of not more than 50 amino acids.

3. A peptide of Claim 1, said peptide consisting of not more than 20 amino acids.

4. A peptide of Claim 1, said peptide consisting of not more than 10 amino acids.

5. A peptide of Claim 1, said peptide being the 8-amino acid sequence VFDNFVLK
unlinked to any other amino acids.

6. A method of stimulating axonal and/or dendritic growth and/or guidance said method comprising administering a peptide of Claim 1 to a neuron.

7. A method of Claim 6 wherein the neuron is in a human.

8. A method of Claim 7 wherein the peptide is delivered to the spinal cord.

9. A method of Claim 8 wherein the peptide is delivered by infusion.

10. A method of stimulating axonal and/or dendritic growth and/or guidance said process comprising administering a vector to an injured nervous system, said vector being nucleic acid comprising a base sequence coding for the peptide of Claim 1.

11. The method of Claim 10 wherein the nucleic acid molecule is in a virus at the time of administration.

12. A method of stimulating axonal and/or dendritic growth and/or guidance, said method comprising administering a peptide to a neuron, said peptide being at least 7 amino acids in length, said peptide comprising all or part of a tenascin-C region, said tenascin-C region selected from the group consisting of fnA-D, fnD, and fnC.

13. A method of Claim 12 wherein the peptide comprises the 8-amino acid peptide VFDNFVLK.

14. A method of Claim 13 wherein the peptide is free of any sequence of tenascin-C
amino acid sequence that is both outside the tenascin-C region and exceeds 100 amino acids.

15. A method of Claim 12 wherein the peptide comprises a homologous peptide sequence identical in length to the tenascin-C region such that, if the homologous amino acid sequence and the tenascin-C region are aligned and consecutively numbered from the same end, and like numbered amino acids from the two sequences are compared, there is at least N
percent identity between the amino acids of the homologous sequence and the amino acids of the tenascin-C
sequence, N being 70.

16. A method of Claim 15 wherein N is 80.

17. A method of Claim 16 wherein N is 90.

18. A method of Claim 12 wherein the neuron is in a human.

19. A method of Claim 12 wherein the neuron is human and the peptide is delivered to the spinal cord.

20. A method of Claim 12 wherein administration of the peptide is achieved by infecting the injured nervous system with a nucleic acid vector comprising a region coding for the peptide or a virus comprising nucleic acid comprising a region coding for the peptide.

21. A method of Claim 12 wherein the tenascin-C region is fnA-D.

22. A method of Claim 12 wherein the tenascin-C region is fnD.

23. A method of Claim 12 wherein the tenascin-C region is fnC.

24. A method of Claim 12 wherein the purpose is to stimulate axonal and/or dendritic growth.

25. A method of Claim 24 wherein the purpose is to stimulate axonal and/or dendritic growth independent of neurite guidance.

26. A method of Claim 12 wherein the purpose is to stimulate axonal and/or dendritic guidance.

27. A method of Claim 26 wherein the purpose is to stimulate axonal and/or dendritic guidance independent of axonal and/or dendritic growth.

28. A peptide comprising a selected peptide region selected from the group consisting of fnA-D, fnD, fnC, and homologs to fnA-D, fnD, and fnC, said peptide free of any tenascin-C
region not in the selected peptide region and exceeding L amino acids in length, a homolog of a region being identical in length to that region and if aligned end-to-end with that region having at least N percent identical amino acids with that region but less than 100 percent identical amino acids, N being 70, L being 100.

29. A peptide of Claim 28, L being 10.

30. A method of stimulating axonal and/or dendritic growth and/or guidance said method comprising administering a peptide of Claim 28 to a neuron.

31. A method of Claim 6, 12, or 28, wherein, prior to administration of the peptide, nucleic acid coding for the integrin receptor is added to the neuron via a vector or virus so as to cause the number of integrin receptors in the neuron to increase.

32. A method of malting a neuron more responsive to factors that promote growth or guidance, said method comprising altering the DNA content of the neurite so that it will contain more integrin receptors.