Myelination and behaviour of tenascin-C null transgenic mice

Myelination and behaviour of tenascin-C null transgenicmice

B. W. Kiernan,1 E. Garcion,1,2 J. Ferguson,1 E. E. Frost,1,2 E. M. Torres,2,3 S. B. Dunnett,2,3 Y. Saga,6 S. Aizawa,7

A. Faissner,8 R. Kaur,4 R. J. M. Franklin4 and C. ffrench-Constant1,2,5

1Wellcome/CRC Institute of Developmental Biology and Cancer, Tennis Court Road, Cambridge CB2 1QR, UK2MRC Cambridge Centre for Brain Repair, The E.D. Adrian Building, Forvie Site, Addenbrooke's Hospital, CambridgeCB2 2PY, UK3Department of Experimental Psychology, University of Cambridge, Cambridge, UK4Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge, UK5Department of Medical Genetics, University of Cambridge, Cambridge, UK6National Institute of Health Science, Tokyo 158, Japan7Kumamoto University Medical School, Japan8Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

Keywords: hyperlocomotion, lamina cribrosa, Morris water maze, migration, oligodendrocyte precursor, passive avoidance

Abstract

The extracellular matrix glycoprotein tenascin-C is widely expressed during development and repair, making it surprising that fewabnormalities have been found in transgenic mice lacking this molecule. We have therefore re-examined the transgenic micedescribed by Saga et al. [Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T. & Aizawa, S. (1992) Genes Dev., 6, 1821±1831] in whichtenascin-C was knocked-out by homologous recombination, focusing on two aspects of the nervous system likely to reveal anyabnormalities that might follow the loss of tenascin-C. First, we have determined the pattern of myelin and distribution ofoligodendrocyte precursor cells in those areas, such as the optic nerve and retina where local concentrations of tenascin-C havebeen proposed to act as barriers to oligodendrocyte precursor migration and so prevent inappropriate myelination. Secondly, wehave examined the behaviour of the mice in a number of well-characterized tests, e.g. beam-walking, passive avoidance and theMorris water maze. We ®nd no abnormalities of myelination or oligodendrocyte precursor distribution in adult mice, showing that localconcentrations of tenascin-C are not the sole mechanism responsible for the pattern of myelination in these regions of CNS.However, we do ®nd a number of behavioural abnormalities in these mice and show that hyperlocomotion and de®cits in coordinationduring beam walking can be ascribed to tenascin-C de®ciency. The effects on coordination are, however, not seen on a 129 geneticbackground. Taken together, these results signi®cantly extend the phenotype associated with tenascin-C de®ciency but argueagainst a role in myelination.

Introduction

Transgenic mice in which genes encoding extracellular matrix

molecules have been deleted by homologous recombination represent

an attractive means of examining the function of these molecules.

One molecule for which this approach has been used is the

extracellular matrix molecule tenascin-C (Saga et al., 1992;

Forsberg et al., 1996). This molecule is widely expressed during

development and following injury in the CNS, and is antiadhesive for

a range of neural cell types with inhibition of neurite outgrowth or

cell migration (reviewed in Chiquet-Ehrismann, 1991; Faissner et al.,

1995). These observations suggest an important role for tenascin-C in

the modulation of cell behaviour during neural development.

Surprisingly, however, tenascin-C-de®cient mice do not have an

obvious developmental phenotype (Saga et al., 1992; Forsberg et al.,

1996). While these studies did not examine individual developmental

programmes in detail, it is clear that the mice do not suggest any

essential role for tenascin-C and have raised the provocative question

as to whether this extracellular matrix molecule is redundant for

normal development (Erickson, 1993a). Rather than having no

function, however, it is possible that de®ciency of tenascin-C has

effects too subtle to detect by histological analysis of adult mice.

These studies, therefore, make it important to re-examine the

tenascin-C-de®cient mouse in more detail to resolve the controversy

as to whether there is an essential function for this molecule.

These further studies can be done in two ways. First, we can

undertake more detailed analyses of areas of CNS development

where previous experiments have led to speci®c hypotheses as to the

function of tenascin-C. Two such areas are the optic nerve and

cerebellum. Tenascin-C is expressed at high levels in the lamina

cribrosa, the point at which the optic nerve head pierces the sclera of

the eye, and the molecular layer of the cerebellum (Bartsch et al.,

1992; 1994). Based on experiments showing that tenascin-C inhibits

oligodendrocyte precursor cell adhesion and migration, it has been

suggested that tenascin-C de®nes the pattern of myelination in these

regions by acting as a barrier to oligodendrocyte precursor migration

(Bartsch et al., 1992; 1994; Frost et al., 1996; Kiernan et al., 1996). If

Correspondence: Dr C. ffrench-Constant, Wellcome/CRC Institute ofDevelopmental Biology and Cancer, Tennis Court Road, CambridgeCB2 1QR, UK. E-mail: [email protected]

Received 16 September 1998, revised 29 March 1999, accepted 21 April 1999

European Journal of Neuroscience, Vol. 11, pp. 3082±3092, 1999 Ó European Neuroscience Association

so, one would predict that tenascin-C-de®cient mice will have

abnormal patterns of oligodendrocyte precursor cell distribution and

myelination in the retina and cerebellum. Secondly, we can undertake

more sophisticated analyses of the CNS using behavioural and

cognitive testing. These will test function rather than anatomical

structure, and would therefore be expected to provide a more

sensitive assay of correct neural development.

To re-examine the question of tenascin-C function, we have taken

both approaches to analyse the transgenic mouse strain originally

described by Saga et al. (1992). Our results provide evidence for

strain-speci®c abnormalities of behaviour in tenascin-C-de®cient

mice but provide no support for an essential role of the molecule in

determining patterns of myelination.

Materials and methods

Animals

Histological analyses were based on young adult male and female

TN±/± mice derived from a homozygous F5 generation tenascin-C-

de®cient mice cross, compared with sex and age matched C57BL/6 J

mice.

Behavioural analyses were based on four sets of animals, using

different breeding regimes designed to progressively control for

differences in the genetic background on which the tenascin-C null

genotype was expressed.

In experiment I, six male tenascin-C-de®cient mice were

compared with seven age-matched male control mice. One

tenascin-C-de®cient animal was killed during the analysis follow-

ing a fall during bridge testing. Control and experimental mice

were derived from different stocks. Both had genetic backgrounds

comprising an equal mixture of F1 C57BL/6 J-CBA and BALB/c,

with selection against inheritance of the albino (c) allele. The

origins of the TN±/± stock are described elsewhere (Saga et al.,

1992). The control stock was designed to replicate the breeding

protocol adopted for the TN±/± mice.

In experiment II, ®ve heterozygous and eight tenascin-C-

de®cient animals from a mixed sex litter from a backcross

between an F6 generation homozygote tenascin-C-de®cient mouse

and a heterozygote derived from a cross between the F6

generation homozygote tenascin-C-de®cient mouse and an F1

C57BL/6 J-CBA hybrid (TN±/± 3 [TN±/± 3 F1 C57BL/6 J-CBA])

were examined. This litter comprised both heterozygotes and

homozygotes with equivalent genetic backgrounds. The behaviour-

al tests on the ®rst and second sets of animals were conducted

sequentially over a period of 7 weeks.

In experiment III, eight wild-type, 12 heterozygous and six

tenascin-C-de®cient mice from mixed sex, age-matched litters from

intercrosses between the offspring of an F6 generation homozygote

tenascin-C-de®cient mouse and a 129 mouse ([TN±/± 3 129] 3

[TN±/± 3 129]) were examined. This litter comprised homozygotes,

heterozygotes and wild-type animals with equivalent genetic back-

grounds, and was tested over a period of 3 weeks.

In experiment IV, eight wild-type, nine heterozygous and eight

tenascin-C-de®cient age-matched female mice from a strain carrying

the tenascin-C null transgene on a largely 129 genetic background

were examined. The strain was derived from tenascin-C null

homozygotes from the [TN±/± 3 129] 3 [TN±/± 3 129] intercross

litters bred back onto the parental 129 strain for four generations.

This litter comprised homozygotes, heterozygotes and wild-type

animals on an » 94% 129 strain genetic background, and was tested

over 1 week.

Histological analyses

For Sudan black staining of myelin lipids, complete brains, eyes and

optic nerves were dissected from animals perfused transcardially with

2% paraformaldehyde in phosphate-buffered saline (PBS). Samples

were left in ®xative for 1±3 h, then in 34% sucrose in PBS for a

further 1±3 h (eyes) or until they sank to the bottom of the tube

(brains). Samples were mounted in Tissue-Tekq O.C.T. compound

blocks at ±37 °C, and transverse sections of retina, longitudinal

sections of optic nerve and sagittal sections of cerebellum cut on a

cryostat at ±24 °C. Sections (10±20 mm) were placed in 70% ethanol,

followed by Sudan black [prepared by dissolving 7 g of Sudan black

B (Sigma) in 500 mL 70% ethanol] for 7±10 min. Sections were

rinsed in 70% ethanol, washed in water for 3±4 min and mounted in

aqueous mounting medium.

For immunohistochemistry (IHC) and in situ hybridization (ISH),

brains were surgically removed and snap frozen at ±30 or ±40 °C in

isopentane cooled by liquid nitrogen. Optic nerves or cerebellum

were dissected in paraformaldehyde 4% in PBS and then embedded

in Tissue-Tek O.C.T. compound (Agar Scienti®c, Stansted, Essex,

UK) frozen on dry ice. Specimens were stored at ±80 °C until use.

Longitudinal optic nerve sections (10 mm for IHC and 15 mm for

ISH), to show where the optic nerve leaves the retina, and cerebellum

sections were placed onto Superfrost slides (Scienti®c Laboratory

Supplies, Nottingham, UK).

For ISH, frozen sections were thawed at room temperature for

30 min, ®xed with 4% paraformaldehyde in PBS, pH 7.4, for 10 min,

acetylated in a solution of 4 3 standard sodium citrate (SSC), pH 8.0,

containing 0.25% acetic anhydride and 0.1 M triethanolamine for

10 min at room temperature, dehydrated in ethanol, delipidated in

chloroform, and air-dried. The cRNA PDGF alpha-receptor probe

was transcribed from a 1637-bp EcoRI cDNA fragment encoding

most of the extracellular domain of mouse PDGF alpha-receptor

cloned into Bluescript KS (a kind gift of W. Richardson). RNA

polymerases and DIG labelling mix were obtained from Boehringer,

Lewes, Sussex, UK, and the transcription reaction was run as

recommended by the manufacturers. The labelled cRNA probe (1±

2 mg/mL) was dissolved in the hybridization solution (50% v/v

formamide, 4 3 SSC, 1 3 Denhardt's solution, 100 mg/mL herring

sperm DNA, 100 mg/mL polyA, 10% w/v dextran sulphate), and

applied onto each slide under coverslips. Hybridization was allowed

to proceed for 14±18 h at 65 °C. Slides were dipped into 4 3 SSC and

then coverslips were carefully removed. Slides were washed for

10 min in 1 3 SSC, incubated for 30 min with RNAase A

(Boehringer) and 30 min in RNAase A buffer alone at 37 °C, washed

three times for 40 min at 65 °C in 1 3 SSC, 50% formamide, 0.1%

Tween-20, and twice for 30 min in 100 mM maleic acid, 150 mM

NaCl, 0.1% Tween-20, pH 7.5. RNA hybridization was visualized by

IHC with alkaline phosphatase-conjugated anti-DIG antibody

(Boehringer) according to the manufacturer's instructions, except

that polyvinyl alcohol (PVA; 10% w/v) was included in the ®nal

colour reaction to increase sensitivity. Slides were then dehydrated in

ethanol and xylene, and mounted in Eukitt medium (Agar Scienti®c).

For immuno¯uorescent staining of myelin basic protein (MBP), the

cryostat sections were ®xed for 10 min in 4% paraformaldehyde in

PBS, followed by three rinses in PBS. They were then blocked for 1 h

at room temperature in gelatin 2 g/L in PBS, after which the ®rst

antibody incubation (rat antimouse MBP, IgG MCA 409, Serotec, 1/

100 in the blocking solution) was carried out overnight at 4 °C. After

washing in PBS, the sections were then incubated for 1 h with the

secondary antibody (biotin-conjugated antirat, 1/75 in the blocking

solution) followed by the streptavidin±¯uorescein complex (1/75 in

Myelination and behaviour in TN-C±/± mice 3083

Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 3082±3092

PBS) for 1 h. After further washes in PBS, sections were mounted

under coverslips in ¯uorostab medium (Eurodiagnostica, MalmoÈ,

Sweden) and visualized in a confocal microscope.

For the electron microscopy studies, mice were killed under

anaesthesia by transcardiac perfusion with 4% glutaraldehyde in

phosphate buffer with 0.1% calcium chloride (pH 7.2±7.4). The ®xed

brains were removed and post®xed in 4% glutaraldehyde for 5±24 h.

All tissue blocks were washed with phosphate buffer, post®xed with

2% osmium tetroxide, dehydrated through increasing concentrations

of alcohol and embedded in TAAB resin. Semi-thin sections (1 mm)

were cut from each block onto plain glass slides and stained with

toluidine blue for examination by light microscopy. Areas of interest

from selected blocks were trimmed for examination at the EM level.

Ultrathin (70 nm) sections were stained with uranyl acetate and lead

citrate, and examined using a Hitachi-H600 electron microscope.

Images of myelinated axons, obtained from the EM, were projected

on a computer monitor screen. The outline of axon diameter and the

®bre diameter were traced using the Digit semiautomatic image

analysis system (Institute of Ophthalmology, London) coupled to a

high-resolution Summagraphics Bit Pad 2 digit tablet (model 1103,

Advanced Microcomputer Applications, Nottingham, UK) and a

Acorn Archimedes compatible computer (Acorns Computer,

Cambridge, UK). The mean Feret diameter calculated from the

digitized shapes represented arbitrary units. The correlation between

®bre diameter and axon diameter was tested using Prism software.

Behavioural analyses

Behavioural tests were designed to assess a range of behaviours

relevant to neurological function. General locomotor ability, postural

changes, hearing, activity and exploratory behaviour were assessed

by observation. Finer motor coordination and balance were tested on

a variety of bridges, and swimming ability was tested in a water tank.

Finally, more complex behaviours involving learning were judged

using the Morris water maze and a step-through test of passive

avoidance. All experiments were undertaken blind to the genotype of

the subjects. All animals were between 2 and 6 months of age when

tested. For genotyping, the tip of each animal's tail was docked after

the completion of all behavioural tests and used in a PCR assay as

described (Saga et al., 1992) in order to identify wild-type,

heterozygous and homozygous littermates.

Open ®eld

Open ®eld behaviour assesses levels of spontaneous locomotor

activity and exploratory behaviour. The open ®eld was a 1-m2 square

arena with 50-cm-high walls. The ¯oor was divided into 25 equal

squares by black lines. The mice were placed in the centre square

facing away from the experimenter, and monitored by direct

observation and video cassette recording for 5 min. Entries into the

total, centre, side and corner squares were recorded with a line

crossing to enter a new square scored when a mouse crossed a line

with all four legs. The time spent in the centre nine squares and the

number of attempts made by the experimenter to catch the animal at

the end of the test period were recorded. Bouts of rearing (raising

forepaws off the ground), bouts of grooming and fecal boli were

counted. Each mouse received four tests in the open ®eld on

consecutive days. The data were aggregated over the four days for

analysis.

Hindlimb re¯ex

Changes in posture and limb position were examined by lifting

animals up by the tail. The mice were gently lifted by the tail until all

four paws were off the ground and held suspended. Positions of limbs

were noted. The mice were then permitted to contact a surface with

their forepaws and the position of the hindlimbs again noted.

Flinch hearing test (Preyer re¯ex)

Two metal objects were knocked together in order to produce a single

audible sound. The two objects were held out of sight of the test

animals and the mice were observed during the test for ¯inching. A

¯inch was de®ned as any action evoked by the stimulus ranging from

an ear twitch to a jump. Any ¯inch was taken as evidence that the test

animal was capable of hearing.

Balance and rod walking

The ability of rodents to traverse a stationary horizontal rod is

assumed to measure both sensorimotor coordination and the integrity

of the vestibular senses (Dean et al., 1981). Mice were trained to walk

across a 60-cm-long wooden bridge into a dark box. After training,

the complexity and dimensions of the bridge were altered in order to

assess motor coordination and the integrity of the vestibular system.

Each beam was made of a single wooden rod with either square (28 or

23 mm wide) or round (28, 18, 12 or 9 mm diameter) cross-section,

raised 45 cm above the bench surface. The mice were placed on top of

each bridge at one end facing the dark box for four consecutive trials,

between each trial the animal was returned to its home cage for a

minimum of 15 min. The time taken to traverse the bridge and the

number of occasions on which the animal fell, clung or one or other

of its feet slipped were recorded ®rst for the bridges with square

cross-sections and then for the four circular ones, in each case

progressing from the widest to the narrowest. One cling was de®ned

as the mouse freezing for 5 s or more.

Two days after the last trial, the mice were placed at one end of the

narrowest beam, and their balance was classi®ed as either success or

failure. Criterion for success was standing or clinging horizontally to

the top of the beam for 10 s, failure was scored in the event of

slipping sideways, hanging or falling.

Morris water maze

Spatial navigation abilities were evaluated using the Morris water

maze test (Morris & O'Shea, 1983; Morris et al., 1986), conducted in

a black circular pool, 100 cm in diameter and 40 cm deep. The pool

was positioned in a small test room ®lled with objects visible from

within and external to the pool (experimenter, door, lights, shelving,

equipment). The pool was ®lled with 19 °C water to a depth of 30 cm

made opaque by the addition of non-toxic white paint. A 10 3 10 cm

Perspex escape platform was positioned in the pool with its surface

1 cm below the water surface. A camera was ®xed above the pool and

used to record each trial.

During training in the hidden platform test, the platform was

maintained in a constant position in the middle of one quadrant of the

pool. On each trial the animal was placed at one of four positions

around the wall of the pool in a random order and allowed to swim

until it found the platform. Four trials per day were performed. Each

trial ended when the mouse reached the platform. Each trial was

permitted a maximum of 60 s, after which mice were placed on the

platform. The animal remained on the platform for 15 s before being

returned to the home cage. The mice were tested at approximately the

same time every day but in a random order. After initial training,

mice were tested in a probe trial to determine whether or not they had

learnt the spatial position of the platform using external cues. On the

probe trial day, the platform was removed for the third trial and the

mice were placed in the pool for 30 s. The time spent in the quadrant

previously holding the platform was recorded. The platform was

replaced and the mice permitted to locate the platform in the fourth

3084 B. W. Kiernan et al.


trial. After the probe trial, mice were tested in a reversal trial to

determine whether or not they were capable of learning a new

platform position. During the reversal trial, the platform was placed

in a new location (in the opposite quadrant to the previous) and

training restarted. Finally, during visible platform training the water

level in the pool was dropped by 15 mm allowing the surface of the

platform to break the surface of the water by 5 mm. For each trial, the

platform was placed in one of four positions in a random order.

Passive avoidance

The passive avoidance test measures an animal's ability to learn to

avoid and retain the memory of a noxious stimulus by inhibiting

normal behaviour (Robichaud et al., 1973; Bartus et al., 1980).

Passive avoidance was evaluated in a 50-cm-high two-compartment

step-through test chamber. One 80 3 50 cm compartment was open

and exposed, with a transparent Perspex wall. The other compartment

(80 3 30 cm) was dark and covered. The ¯oor in both compartments

was made of 5-mm-diameter stainless steel rails spaced 15 mm apart.

A 10-cm-wide by 5-cm-high guillotine door separated the two

compartments. Each animal received a familiarization trial in the

apparatus on day 1, in which they were placed in the exposed

compartment opposite and facing away from the open door. The

latency to enter the dark compartment was recorded for each trial. On

the second day, the animals were once again placed opposite and

facing away from the open door in the light compartment. As soon as

the mouse entered the dark side the guillotine door was closed and a

0.5-s 0.2-mA scrambled footshock was applied to the grid ¯oor. After

15 s the animal was removed from the dark chamber and returned to

its home cage. Each mouse was trained every 1.5 h until they all

avoided the dark compartment for 5 min. On each successive day

thereafter the mice were given two trials, and the latency to enter the

dark compartment was recorded, with no further footshocks given.

Results

Myelination patterns

To examine the pattern of myelination in optic nerve and cerebellum,

we cut sections for light and electron microscopy studies from adult

mouse brains of the tenascin-C stock described by Saga et al. (1992),

and from age-and sex-matched control animals.

In the retina, the tenascin-C null mice exhibited a normal

distribution of myelin when visualized by Sudan black (Fig. 1A±C)

or anti-MBP immuno¯uorescent staining (Fig. 1D and E). As in wild-

type (control) animals, no myelin was detected in transverse sections

of the retina of tenascin-C-de®cient animals (Fig. 1A±E). The extent

of myelin stain along the optic nerve was identical in both the control

and tenascin-C-de®cient animals: in both sets of animals, myelination

ended abruptly at the retina±optic nerve junction (Fig. 1A±E). The

absence of myelin in the optic nerve ®bre layer was con®rmed by

further light and electron microscopy of semi- and ultrathin sections

of the retina in the region of the lamina cribrosa (Fig. 1F and G). The

distribution of myelin in the cerebellum of tenascin-C-de®cient mice

also appears identical to that present in control animals. Dense

myelination, as visualized by Sudan Black, is con®ned to the white

matter layer (Fig. 2A±F), and MBP expression is present in the white

matter layer and, to a lesser extent, in the granule cell layer (Fig. 2G

and H). No abnormal myelination or MBP expression was seen in the

molecular layer.

These data suggest that any barriers to oligodendrocyte precursor

migration that de®ne the pattern of myelination in optic nerve and

cerebellum are present in both control and tenascin-C-de®cient

animals. To examine this more directly, we performed in situ

hybridization experiments using cRNA probes complementary to the

PDGF alpha-receptor, a marker for oligodendrocyte precursor cells in

vivo (Pringle et al., 1992). These studies showed the pattern of

oligodendrocyte precursor cells to be identical in control and

tenascin-C-de®cient animals. In the optic nerve, precursor cells were

abundant in the nerve but were not present in the region of the lamina

cribrosa in either control or transgenic animals (Fig. 1H±J). In the

cerebellum, precursors were present in the white matter layer with

occasional cells seen within the granular layer. Precursors were not

seen in the molecular layer in either control or transgenic animals.

To examine the architecture of myelinated tracts in the tenascin-C-

de®cient mice in more detail, we performed electron microscopy

studies of the dorsal funiculus in wild-type and transgenic mice. No

differences in the morphology of the myelinated tracts was noted

(Fig. 3A and B), with the overall architecture being the same in the

presence or absence of tenascin-C. We then examined the relationship

between axon diameter and overall axon/myelin sheath diameter in

these sections. As shown in Fig. 3C, there is no signi®cant difference

in the slope of the graph (P = 0.28, NS), showing that the relationship

between increasing axon diameter and myelin sheath thickness is not

altered in the transgenic mice.

Behavioural testing

Four different strains were used for these experiments, as described in

Materials and methods. Initially, we performed a comparison of the

original tenascin-C-de®cient stock with an appropriate wild-type

control (experiment I). Having documented a number of abnormal-

ities, we performed further studies on a backcross (using a C57BL/

CBA parent ± experiment II) or an intercross (using a 129 parent ±

experiment III) litter so as to determine which abnormalities were

related to tenascin-C de®ciency and to examine the effect of genetic

background. Finally, we focused on a novel abnormality related to

tenascin-C de®ciency that we observed in beam walking by

examining motor and inner ear function in more detail in a congenic

129 strain of mice (experiment IV).

Open ®eld

When placed in an open ®eld, mice move along the walls with short

stops during which they often rear. Low levels of locomotion,

restriction to the sides and corners, and frequent defecation are taken

to indicate fear or anxiety. High levels of locomotion, rearing and

time spent in the centre are considered to be indicators of activation

and exploration.

In experiment I (the stock comparison), no differences between the

two groups were observed in locomotor activity (number of squares

entered), time spent in the centre squares or grooming (Table 1, left

columns). However, the tenascin-C-de®cient stock reared more often

and defecated less often, suggesting that the mice were less anxious

and more exploratory than the control strain.

In con®rmation of this, increased rearing and decreased defecation

were also seen in experiment II comparing TN±/± 3 [TN±/± 3 F1

C57BL/6 J-CBA] backcross heterozygotes and homozygotes

(Table 1, right columns). In addition, and unlike the stock

comparison, differences were seen in general locomotor activity

due to an increase in the number of side and corner squares entered by

the tenascin-C-de®cient homozygotes (Table 1).

Hindlimb re¯ex and postural changes

Normal mice hung by the tail adopt a posture with their hindlimbs

splayed and their forelimbs tucked underneath their bodies. When

lowered towards a surface, the forelimbs are extended for contact and

placing, and the hindlimbs become stationary. This pattern of



FIG. 1. (A±C) Sudan black-stained sections of adult mouse optic nerve and retina from control (A and C) and tenascin-C null transgenic (B) mice. Magni®cation:10 3 (A and B) and 20 3 (C). Scale bar, 0.1 mm. Note that the distribution of staining is unaltered in the tenascin-C null animals compared with the controls, withdense staining of the myelin in the nerve but not at the retina±optic nerve junction in the region of the lamina cribrosa. Some non-speci®c staining of the retinallayers is seen in both A and B. (D and E) Sections of adult mouse optic nerve labelled with antibodies against MBP and visualized using immuno¯uorescence. Notethat in both wild-type (D) and mutant (E) MBP, labelling stops abruptly at the lamina cribrosa and there is no labelling of the retina. (F) Toluidine blue-stainedsemithin section of retina taken near the optic nerve head, at 2.5 3 the magni®cation of A±C. The surface of the retina is arrowed. Note the absence of myelin in theoptic nerve ®bre layer (ONFL). RGC, retinal ganglion cell layer. (G) Electron micrograph of the optic nerve ®bre layer near to the optic nerve head. Note theabsence of myelin around the axons cut in transverse sections in this micrograph. Magni®cation, 18 900 3. (H±J) In situ hybridization studies using a probe to thePDGF alpha-receptor to visualize oligodendrocyte precursor cells. Note that these cells are present in the optic nerve but not the region of the lamina cribrosa orretina in both wild-type (H) and mutant (I) nerves. There is non-speci®c labelling of the retina, as shown in the sense probe control illustrated in J.



behaviour was seen with the control stock. In contrast, animals from

the tenascin-C-de®cient stock ¯exed their limbs whilst suspended and

swayed from side to side by ¯exing ¯ank muscles. When allowed to

contact a surface with their forepaws, their hindlimbs continued to

kick. However, in the three sets of test animals on equivalent genetic

backgrounds (experiments II±IV), no signi®cant differences in hang-

splaying or posture were observed (not shown).

Hearing tests

All animals from the tenascin-C-de®cient stock failed to demonstrate

a Preyer re¯ex on exposure to a sharp noise, in contrast to those from

the control stock (Table 2; Fisher's exact probability, P = 0.0046),

indicating that they may have been deaf. However, as many

laboratory mouse strains carry hereditary deafness genes, this could

merely be a defect of the strain rather than a result of tenascin-C

de®ciency. In the [TN±/± 3 129] 3 [TN±/± 3 129] intercross litters

(experiment III), equivalent proportions of heterozygotes and

homozygotes failed the test, and all animals from the 129 partial

congenic strain (experiment IV) demonstrated a Preyer re¯ex

(Table 2; all comparisons NS). This suggests that tenascin-C

de®ciency per se does not lead to a loss of hearing, and it is more

likely that the original strain carries a separate locus causing hearing

de®ciency.

Bridge tests

In experiment I, comparing separate strains of tenascin-C null and

wild-type mice, falls occurred in ®ve out of 260 trials. All falls were

by tenascin-C-de®cient animals, but this occurred too infrequently to

be statistically signi®cant. No differences were observed in the

number of clings. The tenascin-C-de®cient stock suffered more

footslips than the control strain on all bridges, and the effect was even

more marked on the more dif®cult narrow bridges than the wider

bridges (Fig. 4A; genotype F1,11 = 85.17; genotype 3 bridge

F4,44 = 15.50; both P < 0.001). In the subsequent test of balance on

the narrowest beam, six out of seven control strain mice were able to

maintain balance for 10 s, whereas all but one of ®ve animals from

the tenascin-C-de®cient stock failed the test (Fisher's exact

probability, P = 0.045). This suggests that mice of the tenascin-C-

de®cient strain have a balance defect.

In experiment II, comparing heterozygotes and homozygotes from

the TN±/± 3 [TN±/± 3 F1 C57BL/6 J-CBA] backcross, no differences

were observed in the number of clings or crossing times between the

FIG. 2. Sudan black-stained sagittal sections of adult mouse cerebellum from control (A±C) or tenascin-C null (D±F) animals. Magni®cation: 2.5 3 (A and D),10 3 (B and E) and 20 3 (C and F). Scale bar, 1 mm. Note that the distribution of staining is unaltered in the tenascin-C null animals compared with the controls.(G and H) Sections of cerebellum labelled with antibodies against MBP as in Fig. 1. Note that the white matter layer (WML) and, to a lesser extent, the granule celllayer (GCL) are labelled in both wild-type (G) and mutant (H) cerebellum but that there is no labelling in the molecular layer (ML). (I and J) In situ hybridizationstudies using a probe to the PDGF alpha-receptor to visualize oligodendrocyte precursor cells as in Fig. 1. Labelled cells are shown by arrows in the wild-type (I)and mutant (J) cerebellum. They are present in an identical distribution in both the white matter layer and granule cell layer, but not the molecular layer.



two groups. Out of 312 trials there were two falls, both by tenascin-C-

de®cient animals, but this was not statistically signi®cant. The

homozygotes did make signi®cantly more footslips than hetero-

zygotes on the narrowest bridge (Fig. 4B; genotype 3 bridge,

F5,55 = 3.62, P < 0.01), con®rming the bridge-crossing de®cit seen in

the initial experiment. However, no signi®cant differences were seen

in the balance test (3/5 heterozygotes versus 5/8 homozygous mice

failed, NS).

In experiments III and IV, with the [TN±/± 3 129] 3 [TN±/± 3 129]

intercross litters and the 129 partial congenic tenascin-C-de®cient

strain, no differences were observed on any of the bridges. The

number of footslips in experiment IV was greater (3.3 6 0.9,

3.4 6 0.8 and 3.1 6 0.3 for wild-type, heterozygous and homozygous

de®cient animals, respectively, on the narrowest beam) than that

observed in experiment III (1.3 6 0.6, 1.8 6 0.5 and 1.6 6 0.5). These

results suggest that the 129 congenic mice used in experiment IV

perform poorly on the bridge compared with the 129 hybrids in

experiment III. However, as the number of footslips in the wild-type

animals in experiment III is no greater than that seen in experiments I

and II (see Fig. 4), we can conclude from these experiments that the

introduction of a 129 background in the hybrid mice abolished the

bridge-crossing defect rather than simply masking the abnormality.

Morris water maze

The ability of mice in experiments I and III to ®nd a submerged

platform using extra-maze visual cues is shown in Fig. 5. Because

water maze latencies typically yield markedly skewed data, which

was also true for the present experiments (note the high variance in

the ®rst day of testing in all groups, Fig. 5A and B), the latency results

were all subjected to logarithmic transformation for analysis.

In experiment I, the transgenic tenascin-C null mice were able to

learn to ®nd the platform, with training over several days, but they

showed modest de®cits in the latency to escape when compared with

the wild-type controls both during task acquisition (Fig. 5A, D1±6)

and during the reversal trials (Fig. 5A, R1±5; genotype, F1,10 = 6.12,

P < 0.05, and 16.47, P < 0.01, respectively). The de®cit was not due to

a swimming de®cit per se because there were no differences in the

latencies to locate the visible platform (Fig. 5A, V1±2; F1,10 = 0.04,

NS). Moreover, the de®cits in acquisition and reversal were mild and,

by the end of training, all animals had reached a similar asymptote

(Fig. 5A, R4±5). In the probe trial (when the platform is removed),

both strains of mice spent similar periods of time searching the

training quadrant (wild, 17.6 6 4.7 s; tenascin-C null mice

17.8 6 5.2 s, NS).

FIG. 3. (A and B) Electron micrographs of dorsal funiculus from wild-type (A) and tenascin-C-de®cient (B) animals. Note the similar architecture in both animals.Magni®cation, 7000 3. (C) Graphs showing the relationship between increasing axon diameter and myelin sheath thickness. Note that the slope of both graphsdoes not differ signi®cantly.



In experiment III, both heterozygote and homozygote animals from

the [TN±/± 3 129] 3 [TN±/± 3 129] intercross litters exhibited a de®cit

in swim latencies even to the visible platform (Fig. 5B, V1±2;

genotype, F2,23 = 3.42; P < 0.05) which was re¯ected in de®cits in the

acquisition and reversal tests when compared with wild-type (Fig. 5B,

D1±6, R1±5; genotype 3 days, F12,138 = 2.01, P < 0.05). While we

cannot explain the presence of de®cits in both heterozygous and

homozygous animals compared with wild-type, as heterozygotes

express tenascin-C these de®cits must be unrelated to tenascin-C

de®ciency. In this experiment there was no difference between the

homozygotes and the heterozygotes in either the acquisition or reversal

FIG. 4. The average number of footslips made by each animal traversing a 60-cm length bridge of differing complexity. (A) A tenascin-C-de®cient strain ofmice (experiment I) is signi®cantly less able to traverse all bridges comparedwith a control strain. (B) Animals from a TN±/± 3 [TN±/± 3 F1 C57BL/6 J-CBA] backcross homozygous for a disruption of the tenascin-C gene(experiment II) are less able to traverse the most complex bridge comparedwith heterozygote littermates. An asterisk (*) notes the bridge on which asigni®cant difference was seen in this experiment. Vertical bars indicate SEM.

FIG. 5. Morris water maze hidden platform test. (A) A comparison of thetenascin-C-de®cient strain with a control strain (experiment I). Days 1±6(D1±6) represent the initial acquisition trial, which was followed by thereversal trial on days 7±11 (R1±5). Finally, a test of swimming to a visibleplatform was performed for 2 days (V1±2). (B) A comparison of wild-type,heterozygote and homozygous littermates in experiment III, with the differentcomponents of the test identical to A. Vertical bars represent SEM. See text forstatistical comparison and discussion.

TABLE 1. Open ®eld performance

Experiment I Experiment II

Control Tenascin-C- Heterozygotes HomozygotesParameter de®cient (+/±) (±/±)

Time in central squares 21.9 6 2.0 21.1 6 3.0 9.1 6 0.8 9.3 6 1.5Attempts to capture 7.5 6 0.8 1.6 6 0.2* 4.6 6 0.7 7.0 6 1.6Entries into central squares 24.9 6 3.4 19.2 6 3.7 9.9 6 1.5 13.8 6 2.0Entries into side squares 70.9 6 11.2 65.9 6 8.8 81.1 6 3.5 106.6 6 10.9*Entries into corner squares 22.6 6 3.5 20.9 6 2.5 26.5 6 1.0 34.6 6 3.5*Total entries 118.4 6 17.3 105.9 6 14.6 117.5 6 4.7 154.3 6 15.7*Rears 19.7 6 3.3 36.7 6 5.9** 40.7 6 3.8 57.7 6 6.3*Grooming bouts 1.3 6 0.2 0.9 6 0.3 1.8 6 0.7 1.1 6 0.1Fecal boli 2.6 6 0.8 1.1 6 0.3** 0.6 6 0.1 0.3 6 0.1*

*P < 0.05 and ** P < 0.01, by main effect of genotype by ANOVA, with repeated measures over trials.



tests (Fig. 5B, D16, R1±5; genotype, F2,23 = 1.84; genotype 3 days,

F8,92 = 0.71; both NS), showing that tenascin-C de®ciency on the 129

background had no effect on the performance in the water maze test.

Passive avoidance

Both of the original stocks of mice learnt equally quickly (generally

within one trial) to avoid the stimulus by avoiding the dark

compartment of the apparatus (Fig. 6), and showed good retention

by staying out the following day. However, the wild-type mice

progressively overcame their inhibition to re-enter on subsequent

days of testing (when no further shocks were given), whereas the

tenascin-C null mice were more prone to continue to avoid the dark

chamber even over 2 weeks later (Fig. 6A; genotype 3 days,

F9,90 = 3.62, P < 0.001). It should be noted that the difference is in a

direction opposite to that which would be predicted by a de®cit in

memory, and may re¯ect differences in activity, anxiety or

exploratory behaviour observed in the open ®eld tests.

The passive avoidance de®cit observed in the separate strains was

not replicated in the backcross experiment II (Fig. 6B; genotype,

F1,10 = 0.00; genotype 3 days, F14,140 = 0.51; both NS). However, in

this experiment, even the wild-type mice failed to re-enter even after

3 weeks of repeat testing, so any differences appear to be masked by a

ceiling effect under the training conditions that applied in this

experiment.

Discussion

Previous histological studies have failed to reveal any abnormalities

in two independently derived tenascin-C-de®cient transgenic mouse

lines during development or repair of the peripheral nervous system

and skin wounds (Saga et al., 1992; Forsberg et al., 1996). However,

our results show clear behavioural abnormalities in the animals

derived by Saga et al. (1992), and con®rmed as either lacking (Settles

et al., 1997) or expressing low levels of a truncated form (Mitrovic &

Schachner, 1995) of tenascin-C. These abnormalities comprise

hyperlocomotion, increased defecation frequency, abnormal postural

re¯exes, deafness, coordination abnormalities on the beam as

measured by footslips, cognitive de®cits in the Morris water maze

and prolonged passive avoidance. In order to con®rm the relationship

of these abnormalities to tenascin-C de®ciency and explore the

effects of different mouse genetic backgrounds, we performed a

series of breeding experiments. Two further conclusions arise from

these experiments. First, abnormalities of coordination on the beam,

hyperlocomotion and defecation frequency are related to tenascin-C

de®ciency on a C57BL/6 J-CBA background, as there was a

signi®cant correlation between these abnormalities and tenascin-C

de®ciency in the backcross experiment. Secondly, however, the lack

of any behavioural abnormalities in the experiments on a 129

background show that the manifestation of these effects is dependent

on the genetic strain used.

Our study represents the most comprehensive behavioural analysis

of genetic background effects in the tenascin-C-de®cient mice

performed to date, and the conclusions are important for three

reasons. First, they signi®cantly extend the phenotype associated with

tenascin-C de®ciency. In addition to the hyperlocomotion seen in

previous studies (Fukamauchi et al., 1996; 1997a,b), we have

observed two novel abnormalities in measures of coordination and

anxiety, and con®rmed their relationship to tenascin-C de®ciency.

Secondly, our experiments showing that the beam walking de®cit is

not seen in association with tenascin-C de®ciency following the

introduction of a 129 background emphasizes the importance of

analysing genetic background effects in behavioural and other studies

of transgenic mice. Thirdly, they show that breeding experiments to

con®rm that abnormalities in the original mouse strain described by

Saga et al. (1992) can be ascribed to tenascin-C de®ciency may need

to be performed on a number of different genetic backgrounds to

reveal an effect. The use of congenic mice may be inappropriate for

these further studies. Wolfer et al. (1997) observed that 129 backcross

mice performed poorly in the water maze and masked the effect of

introduced transgenes. They concluded that `creation of congenic

strains by backcrossing mutant hybrids to an inbred strain in order to

avoid genetic variability does not pay off' and our own observations

that the congenic mice performed poorly on the bridge tests supports

this conclusion.

The novel observation that tenascin-C de®ciency causes an

abnormality of the nervous system that results in dif®culty of

coordination on the beams adds to the evidence of a signi®cant role

for this molecule in neural development. The precise nature of this

abnormality remains unde®ned. One possibility we considered was a

balance/hearing de®cit caused by inner ear malfunction. However,

this was ruled out as homozygous tenascin-C-de®cient mice from the

TN±/± 3 [TN±/± 3 F1 C57BL/6 J-CBA] backcross litter suffered more

slips than their heterozygote littermates but did not have a balance

defect. Other putative causes, e.g. a weaker grip or muscle strength

are unlikely as homozygous tenascin-C-de®cient mice are capable of

suspending themselves upside-down on the wire roofs of their cages

and clinging onto vertical wire walls or pencils (unpublished

observations). This leaves defects in motor coordination or

proprioception as the most likely causes of the `footslip phenotype'.

Two recent studies have examined the neuromuscular junction

(NMJ) in tenascin-C-de®cient mice; while Moscoso et al. (1998)

found no abnormalities in the structure of the developing or adult

NMJ, Cifuentes-Diaz et al. (1998) found preterminal disorganiza-

tion and evidence for axonal overgrowth. This latter study raises

the possibility that NMJ abnormalities underlie the phenotype we

observe during beam walking. However, this study was performed

on the original strain of Saga et al. (1992), in contrast to that of

Moscoso et al. (1998) who outcrossed the mice with a C57BL/6

female. Further studies using different genetic backgrounds on the

NMJ and other areas involved in proprioception and balance are

clearly required.

Abnormalities of dopaminergic transmission and reduced tyrosine

hydroxylase activity were observed in recent studies on the

TABLE 2. Preyer re¯ex

Experiment Animal set +/+ +/± ±/±

I Strains 7/7 (100%) N/A 0/6 (0%)**II Backcross: TN±/± 3 [TN±/± 3 F1 C57BL/6-CBA] N/A 5/5 (100%) 2/8 (25.0%)III Intercross: [TN±/± 3 129] 3 [TN±/± 3 129] 8/8 (100%) 9/12 (75%) 4/6 (66.7%)IV 129 partial congenic 8/8 (100%) 9/9 (100%) 8/8 (100%)

The number of animals within each set displaying normal Preyer re¯exes as a fraction (and percentage). Note that the trend in the homozygote animals istoward normality as the tenascin-C de®ciency is bred away from the original strain. ** P = 0.01



hyperlocomotion present in tenascin-C-de®cient mice bred from a

C57BL/6N stock. These abnormalities of neurotransmission may also

be responsible for the apparently decreased anxiety levels seen in the

open ®eld experiments in association with tenascin-C in our study.

Fukamauchi et al. (1996) also reported a swimming de®cit in the

tenascin-C-de®cient mice. We failed to ®nd any such de®cit related to

tenascin-C de®ciency in our studies, either in the Morris water maze

(see Results) or in direct swimming tests (B. W. Kiernan, 1995). We

conclude therefore that this represents another strain-dependent

tenascin-C-de®cient phenotype apparent on the C57BL/6N back-

ground used by Fukamauchi et al. (1996) but not on the hybrid

background used in our swimming experiments.

In our study of myelination, we found no abnormalities of myelin

pattern or architecture, and no differences in the distribution of

oligodendrocyte precursor cells in adult animals. Our study represents

the ®rst in vivo test of the hypothesis that tenascin C provides a

barrier to limit oligodendrocyte precursor migration, and so de®nes

the pattern of myelination in the optic nerve and cerebellum (Bartsch

et al., 1992; 1994; Kiernan et al., 1996). This hypothesis is not

supported by our results. While our data from the adult do not

preclude alterations in the behaviour of myelin-forming cells

occurring during development, our present study does allow the

important conclusion that inhibition of migration by tenascin-C is not

the only available mechanism for the regulation of myelin pattern in

the optic nerve head. Equally, our ®nding that the relationship

between axon diameter and myelin sheath thickness is not altered in

the tenascin-C-de®cient mice shows that there is no essential role for

tenascin-C in myelin formation or maintenance.

Given the widespread expression of tenascin-C during develop-

ment and the signi®cant effects of this glycoprotein in cell culture, the

subtlety of the phenotype of the knockout mice is surprising. Proteins

which are able to substitute for tenascin-C may normally be

coexpressed, making it functionally redundant. This concept of

redundancy has been used to explain the unexpected phenotypes in

many homozygous null mice. However, tenascin-C-like proteins have

been characterized from organisms as diverse as sponges (Humbert-

David & Garrone, 1993) and humans (Gulcher et al., 1986), and it is

unlikely that such a highly conserved protein is totally redundant

because it would otherwise revert to a pseudogene (Erickson, 1993a).

A gene conferring a selective advantage of as little as 1 in a 100 can

become ®xed in a model population (Kimura, 1962). The phenotype

we observe in the tenascin-C-de®cient mouse would represent a

signi®cant disability in the wild, and may be suf®cient to explain the

gene conservation.

A second mechanism to explain the lack of a severe phenotype

would be compensatory upregulation of other proteins. Tenascin-C is

a member of a multigene family (Erickson, 1993b) which includes

tenascin-R, J1-160/180, janusin (Pesheva et al., 1989) or restrictin

(Norenberg et al., 1992), tenascin-X (Matsumoto et al., 1992; Bristow

et al., 1993; Matsumoto et al., 1994) and tenascin-Y (Hagios et al.,

1996). The most obvious candidate for upregulation in the tenascin-

C-de®cient mice is tenascin-R, which is functionally related to

tenascin-C (Taylor et al., 1993; Pesheva et al., 1994). However, there

is at present no evidence for upregulation of tenascin-R in the

tenascin-C-de®cient mouse (Steindler et al., 1995). Another possible

compensatory tenascin is tenascin-X, de®ciency of which in humans

has been associated with one form of the connective tissue

abnormality Ehlers±Danlos syndrome (Burch et al., 1997). A study

of the distribution of other tenascins in the tenascin-C null mice could

yield important insights into the role of compensatory upregulation,

as could multiple `knockouts' of tenascin gene family members.

Any demonstration of compensation would suggest that individual

tenascin receptors were capable of being activated by different

tenascin family members. This might then explain a further

apparently paradoxical observation: comparative studies of tenas-

cin-C expression during CNS development show different patterns in

humans, rodent, chick and pig, despite the similar organization of the

CNS in these animals (Rettig et al., 1992; Perides et al., 1993). If the

members of the tenascin gene family can substitute function, then any

selection pressure during evolution would be on the overall pattern of

tenascin expression rather than the pattern of individual tenascins. As

a result, one would expect this overall pattern to remain constant

throughout evolution, while the expression of individual tenascins

may gradually shift as one tenascin replaces another. Testing of this

hypothesis will require a study of the distribution of all tenascins in a

range of vertebrates, with the prediction being that the summation of

the different expression patterns will be well conserved.

Acknowledgements

This work was funded by the Wellcome Trust, the Medical Research Counciland the Multiple Sclerosis Society of Great Britain and Northern Ireland.B.W.K. was funded by a Science and Engineering Research CouncilStudentship, R.J.M.F. is a Wellcome Trust Research Career DevelopmentFellow and C.ff.-C. was a Wellcome Trust Senior Clinical Fellow.

FIG. 6. Passive avoidance test. (A) A comparison of the tenascin-C-de®cientstrain with a control strain (experiment I). Both groups enter the darkcompartment rapidly on the acquisition trial (Acq) following which theyreceived footshocks. Retention of this aversive training was tested onsubsequent days (1±11) as measured by the time taken to enter the darkcompartment. (B) A comparison of homozygous and heterozygous littermatesin experiment II, tested as in A. Vertical bars represent SEM. See text forstatistical comparison and discussion.



Abbreviations

IHC, immunohistochemistry; ISH, in situ hybridization; MBP, myelin basicprotein; NMJ, neuromuscular junction; NS, not signi®cant; PBS, phosphate-buffered saline; PVA, polyvinyl alcohol; SSC, standard sodium citrate.

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Myelination and behaviour of tenascin-C null transgenic mice