Cellular/Molecular
Versican V2 Assembles the Extracellular Matrix Surrounding
the Nodes of Ranvier in the CNS
María T. Dours-Zimmermann,
1
Konrad Maurer,
2
Uwe Rauch,
3
Wilhelm Stoffel,
4
Reinhard Fa¨ssler,
5
and
Dieter R. Zimmermann
1
Institutes of
1
Surgical Pathology and
2
Anesthesiology, University Hospital Zurich, CH-8091 Zurich, Switzerland,
3
Vascular Wall Biology, Department of
Experimental Medical Science, University of Lund, S-221 00 Lund, Sweden,
4
Center for Biochemistry, Medical Faculty, University of Cologne, D-50931
Cologne, Germany, and
5
Department of Molecular Medicine, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany
The CNS-restricted versican splice-variant V2 is a large chondroitin sulfate proteoglycan incorporated in the extracellular matrix sur-
rounding myelinated fibers and particularly accumulating at nodes of Ranvier. In vitro, it is a potent inhibitor of axonal growth and
therefore considered to participate in the reduction of structural plasticity connected to myelination. To study the role of versican V2
during postnatal development, we designed a novel isoform-specific gene inactivation approach circumventing early embryonic lethality
of the complete knock-out and preventing compensation by the remaining versican splice variants. These mice are viable and fertile;
however, they display major molecular alterations at the nodes of Ranvier. While the clustering of nodal sodium channels and paranodal
structures appear in versican V2-deficient mice unaffected, the formation of the extracellular matrix surrounding the nodes is largely
impaired. The conjoint loss of tenascin-R and phosphacan from the perinodal matrix provide strong evidence that versican V2, possibly
controlled by a nodal receptor, organizes the extracellular matrix assembly in vivo.
Introduction
Neuronal and glial cells of the adult CNS are surrounded by
specialized extracellular matrices (ECMs) that assemble during
maturation and replace the loosemeshwork of the late embryonic
and early postnatal phase [for review, see Bandtlow and Zimmer-
mann (2000), Yamaguchi (2000), Rauch (2004), and Zimmer-
mann and Dours-Zimmermann (2008)]. Both juvenile and ma-
ture forms of the CNS matrix are composed of chondroitin
sulfate proteoglycans (CSPGs), tenascins, link proteins, and hya-
luronan. Themajority of theCSPGs belongs to the lectican family
that includes brevican, neurocan, aggrecan, and versican. Lecti-
cans share N- and C-terminal globular structures (G1 and G3,
respectively) separated by unique glycosaminoglycan (GAG) car-
rying middle portions of variable size. Moreover, alternative in-
corporation of the two glycosaminoglycan attachment regions,
GAG-
and GAG-, results in four versican splice variants (V0
contains the GAG-
and GAG- domain, V1 only GAG-,V2
only GAG-
, and V3 neither of these domains) (Dours-
Zimmermann and Zimmermann, 1994; Zako et al., 1995).
All lecticans bind via their G1 domain to hyaluronan. This
interaction is stabilized by one of four link proteins (HAPLN1–4)
(Spicer et al., 2003). In addition, the lectin-like element in the
C-terminal G3 domain displays in vitromoderate-to-high bind-
ing affinities toward tenascin-R (TnR) (Aspberg et al., 1997),
tenascin-C (TnC) (Day et al., 2004), and sulfated glycolipids
(Miura et al., 1999). The trimeric TnR and the hexameric TnC
glycoproteins may cross-link the G3 domains of lecticans and,
thus, tie up the extracellular network (Lundell et al., 2004). Fi-
nally, phosphacan, a secreted CSPG form of the receptor-like
protein tyrosine phosphatase  (RPTP) (Maurel et al., 1994),
joins the complex.
In the adult CNS, versican V2 andHAPLN2/Bral1 are the prom-
inent components of the white matter ECM and particularly accu-
mulate at the nodes of Ranvier (Schmalfeldt et al., 1998, 2000;
Oohashi et al., 2002; Melendez-Vasquez et al., 2005). Conversely,
aggrecan, HAPLN4/Bral2, and neurocan associate with perineuro-
nal nets (Bru¨ckner et al., 2000; Bekku et al., 2003). Brevican, hyaluronan,
TnR, and phosphacan are observed in both of these meshworks.
Since lecticans strongly inhibit axonal growth in vitro, these
specialized ECMs might participate in consolidating myelinated
fiber tracts, limiting structural plasticity, and restricting regener-
ation in thematuratingCNS (Yamaguchi, 2000; Zurn andBandt-
low, 2006; Galtrey and Fawcett, 2007; Zimmermann and Dours-
Zimmermann, 2008). Furthermore, they may regulate the
assembly of axoglial complexes and facilitate the induction and
propagation of action potentials at the axon initial segments
(AISs) and the nodes of Ranvier (Bru¨ckner et al., 1993, 2006;
Poliak and Peles, 2003; Salzer, 2003; Sherman and Brophy, 2005;
Hedstrom and Rasband, 2006), or they could fulfill neuroprotec-
tive functions (Morawski et al., 2004).
To further explore these potential roles in vivo, we have now
suppressed the expression of versican V2, one of the main con-
Received Sept. 1, 2008; revised March 27, 2009; accepted April 23, 2009.
This work was financed in part by grants from the Swiss National Science Foundation and the Velux Foundation
to D.R.Z. We thank Anders Aspberg and Takako Sasaki for the generous gift of antibodies, Marie-Therese Abdou,
Belinda Senn, and Karin Hansen for preparing tissue sections, Mathias Hoechli and Nicole Schaeren-Wiemers for
technical advice, and Holger Moch for support.
This article is freely available online through the J Neurosci Open Choice option.
Correspondence should be addressed to Dieter R. Zimmermann, Institute of Surgical Pathology, University Hos-
pital Zurich, Schmelzbergstrasse 12, CH-8091 Zu¨rich, Switzerland. E-mail: dieterzi@pathol.uzh.ch.
DOI:10.1523/JNEUROSCI.4158-08.2009
Copyright © 2009 Society for Neuroscience 0270-6474/09/297731-12$15.00/0
The Journal of Neuroscience, June 17, 2009 • 29(24):7731–7742 • 7731

stituents of the mature neural ECM. We
avoided the early embryonic lethality of
the complete gene knock-out (Mjaatvedt
et al., 1998) by using an unconventional
isoform-specific gene targeting strategy.
The newly generatedmouse strain is viable
and fertile, but displays major aberrations
in the matrix assembly.
Materials and Methods
Generation of versican V0/V2 KO mice. Using a
targeting vector containing a floxed neomycin-
thymidine kinase (neo-tk) selection cassette
under the control of the HSV-tk promoter, we
introduced an ER-retention signal followed by
an early translational stop codon into the
GAG-
encoding exon VII of the mouse versi-
can gene (codon-insertion KDEL-stop after
E749, Swiss-ProtQ62059) (Fig. 1). The two 3.9-
kb-long genomic arms of the construct in-
cluded corresponding parts of exonVII plus ad-
jacent intron sequences, previously cloned
from syngeneic DNA by PCR (primer se-
quences and PCR conditions available on re-
quest). Electroporation of the linearized con-
struct into R1 129Sv embryonic stem (ES) cells
(Nagy et al., 1993), selection, and screening of
ES cell clones were done as previously described
(Talts et al., 1999). Homologous recombinants
were identified by Southern blot analysis using
a digoxigenin (DIG)-labeled probe (Roche Ap-
plied Science) downstream of the target site.
The ES cells were injected into C57BL/6J blas-
tocysts and transferred into a foster mother.
The chimeric offspring were cross-bred with
C57BL/6J WT mice. Germline transmission of
the mutated versican allele was verified by
Southern blotting and long-distance PCR.
The floxed neo-tk selection cassette was re-
moved in vivo by cross-breeding heterozygous
V0/V2 neo-tk animals with a mouse strain expressing the CRE transgene
under the control of the cytomegalovirus promoter (Schwenk et al.,
1995). Correct target integration and recombinationwas verified by PCR
and sequencing of the modified allele. The mutant mice, named
VCAN
(tm1Zim)
, were backcrossed to establish 129Sv inbred and
C57BL/6J outbred strains.
Northern blotting and quantitative RT-PCR. Primary fibroblasts were
isolated from E14.5 embryos and maintained in culture (Talts et al.,
1999). Total RNA extraction, Northern blotting, and DIG labeling of a
riboprobe hybridizing with the hyaluronan-binding region of the mouse
versican mRNA (HABR, positions 458–1203, GenBank D28599) were
done as described previously (Zimmermann et al., 1994).
To determine relative amounts of versican mRNA expression in the
mutant versus WT mouse brains, quantitative RT-PCR was performed
with an ABI PRISM 7700 Sequence Detection System using the Quanti-
Tect SYBR Green RT-PCR Kit (Qiagen). Total RNAs from brains of
littermates were extracted with RNeasy Protect kit (Qiagen). Primers
detecting the different splice variants of versican were designed to cover
the isoform-specific exon–exon boundaries (supplemental Table 1,
available at www.jneurosci.org as supplemental material). Threshold cy-
cle (CT) values from at least three animals per genotype and per devel-
opmental time point were normalized against glyceraldehyde-3-
phosphate dehydrogenase mRNA (mGAPDH, GenBank M32599) as
endogenous control. RelativemRNA amounts (2

CT
) were calculated
on the basis of the comparative CT method, applying the formula
CT  (CT KO
CT WT). The CT of each measurement was
determined by subtracting the GAPDH CT from the versican CT value
(CT VC CT
GAPDH CT).
Antibodies. All polyclonal antibodies have been raised against recom-
binant core protein fragments as described previously (Zimmermann et
al., 1994). The GAG-
-specific antibodies (Schmalfeldt et al., 2000) rec-
ognize the N-terminal portion of this domain of mouse versican (amino
acids 362–585, Swiss-Prot Q62059), also present in the potentially trans-
lated truncated V0/V2 polypeptide of the mutant mice. The GAG- an-
tigen comprised amino acids 2750–3040. Both versican fragments were
used to generate rabbit and guinea pig antisera in parallel. Recombinant
portions of aggrecan (mAC-IGD: residues 368–481 and mAC-GAG:
1678–1896; Swiss-Prot Q61282) and neurocan (mNC-C: residues 645–
944; Swiss-Prot P55066) served as antigens for the immunization of
guinea pigs. Rabbit antisera against rat brevican (Thon et al., 2000) and
TnR (Day et al., 2004) were kind gifts of Takako Sasaki (University of
Erlangen, Erlangen, Germany) and Anders Aspberg (University of Lund,
Lund, Sweden), respectively.
The anti-phosphacan monoclonal antibody 3F8 (Rauch et al., 1991)
was purchased from the Developmental Studies Hybridoma Bank (Uni-
versity of Iowa). Other commercial antibodies used were as follows: goat
anti-human contactin-1 polyclonal antibodies (R&D Systems), mouse
monoclonal antibody K14/16 against the K
v
1.2
-subunit (Millipore),
rabbit anti-rat-voltage gated sodium channel 1.6 (Na
v
1.6) polyclonal
antibodies (Alomone Labs), mouse monoclonal antibody anti-Caspr
clone K65/35 (obtained from the UC Davis/NIH NeuroMab Facility,
University of California), rabbit anti-humanmyelin basic protein (MBP)
polyclonal antibodies (Dako), mouse anti-humanMBPmonoclonal an-
tibody 67–74 (Millipore Bioscience Research Reagents), rabbit poly-
clonal antibodies against bovine glial fibrillary acidic protein (GFAP)
(Dako), mouse (pan-)sodium channel monoclonal antibody K58/35
Figure 1. Generation of versican V0/V2 KO mice. A, Intron/exon organization of the mouse versican gene giving rise to four
different versican isoforms (V0–V3) due to alternative splicing of exons VII and VIII encoding the GAG-attachment domains
(GAG-
and GAG-). Gray box shows region of interest displayed in B. B, Locus diagram and targeting strategy: introduction of
an ER retention signal, a translational stop codon, and a floxed neo-tk cassette into exon VII of the versican gene (V0/V2 neo-tk).
Removal of selection cassette by cross-breedingwith a CRE-deleter strain leaves only the KDEL coding sequence and a stop codon
plus one loxP site in exon VII of themutated versican allele (V0/V2


). Corresponding restriction fragments detected in Southern
blot aredepictedbelow.C, Southernblot analysis of genomicDNA frommice andES cells: theDIGprobedownstreamof the targeting locus
hybridizes to the 17 kb EcoRI fragment of the WT allele, which converts to a 6.6 kb fragment in versican V0/V2-null mutants. BamHI
restriction allows a distinction betweenWT, V0/V2 neo-tk


, and V0/V2


alleles, confirming the successful targeting.
7732 • J. Neurosci., June 17, 2009 • 29(24):7731–7742 Dours-Zimmermann et al. • ECM Assembly at CNS Nodes of Ranvier