From the Department of Neurobiology, University of
Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
and § Laboratoire de Neurobiologie du Développement et
de la Régénération, UPR 1352, Centre de
Neurochimie du CNRS et Université Louis Pasteur, F-67084
Strasbourg, France
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ABSTRACT |
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The extracellular matrix glycoprotein tenascin-C
(TN-C) displays a restricted and developmentally regulated distribution
in the mouse central nervous system. Defined modules of the molecule have been shown to mediate specific functions, such as neuron migration, neurite outgrowth, cell adhesion, and cell proliferation. The smallest TN-C form contains a stretch of eight fibronectin type III
(FNIII) domains, which are common to all TN-C isoforms. Unrestricted
and independent alternative splicing of six consecutive FNIII cassettes
between the fifth and sixth constitutive FNIII domain bears the
potential to generate 64 different combinations that might code for
TN-C proteins with subtly different functions. To explore TN-C isoform
variability in mouse brain, the alternatively spliced region of TN-C
mRNAs was examined by the reverse transcription-polymerase chain
reaction technique. Polymerase chain reaction products of uniform size
were subcloned and analyzed using domain-specific probes to reveal the
expression of particular combinations of alternatively spliced FNIII
domains. 27 TN-C isoforms were identified to be expressed in mouse
central nervous system, of which 22 are novel. Furthermore, during
development, specific TN-C isoforms were found to occur in distinct
relative frequencies, as demonstrated for isoforms containing two
alternatively spliced FNIII domains. We conclude that TN-C is expressed
in a complex and regulated pattern in mouse central nervous system.
These findings highlight the potential role of TN-C in mediating
specific neuron glia interactions.
Tenascin-C (TN-C)1 is a
glycoprotein of the extracellular matrix. Similar to many other
extracellular matrix molecules, it is composed of serially arranged
structural units called protein domains (1). The amino-terminal
sequence contributes to the central knob, a cysteine-rich structure
that assembles six TN-C monomers via disulfide bridges to form the
native hexameric protein (the hexabrachion; Ref. 2). This region is
followed by 14.5 epidermal growth factor-type repeats and eight FNIII
domains in the shortest TN-C variant. The sequence is terminated by a
domain homologous to the TN-C is expressed in a variety of tissues and is thought to play a role
in mesenchymal-epidermal interactions during organ development but also
under conditions of tissue remodeling in the adult organism such as
wound healing and tumor growth (7-9). In the central nervous system,
TN-C is predominantly expressed by astrocytes and radial glia at early
stages of development, whereas its expression is down-regulated during
tissue differentiation (10-12). In the adult animal, the protein is
restricted to a few brain regions, e.g. the molecular layer
of the cerebellum, the olfactory bulb, the retina, and the optic nerve
head (10, 13-15). The expression of TN-C in the central nervous system
(CNS) has attracted special interest because of the inhibitory (16) and stimulatory actions the molecule can exert in different in
vitro assays (summarized in Ref. 9). The diverse functions of the molecule could be localized to distinct functional domains within the
protein using proteolytic fragments or fusion proteins encompassing defined structural units of TN-C (17-20). In this context, the alternatively spliced FNIII domains have attracted particular interest.
Fusion proteins comprising all alternatively spliced domains have been
found to down-regulate focal adhesion integrity of aortic endothelial
cells (21) and to support short-term adhesion of embryonic and early
postnatal neurons, but not long-term adhesion (19). Fusion proteins
consisting of the FNIII domains A1, A2, and A4 (TNfnA124) exhibited
repulsive properties, whereas the fusion proteins TNfnBD and, to a
lesser extent, TNfnD6 showed a pronounced neurite outgrowth-promoting
effect on hippocampal neurons (19).
The functional role of TN-C in vivo might be modified either
by alternative splicing of the primary transcript or by
post-translational modifications. Interestingly, most of the potential
glycosylation sites are found within the FNIII domains that are subject
to alternative splicing (22). With six alternatively spliced FNIII
domains, each of which can potentially be absent or present, up to 64 isoforms can theoretically be generated. Of these, only seven have been identified thus far in mouse tissue or cell lines (3, 4, 6).
Previous studies have primarily focused on combinations of adjacent
domains. These investigations do not take into account that nonadjacent
FNIII domains also might be combined by alternative splicing, thereby
possibly generating novel recognition sites for TN-C receptors and
mediating specific functions of the molecule. In this study, we show
the expression of a large variety of TN-C isoforms in mouse brain, most
of which are novel. Furthermore, we provide evidence that specific TN-C
isoforms are developmentally regulated.
Animals--
NMRI mice were used for the preparation of RNA. The
day a vaginal plug was observed was designated embryonic day 0 (E0),
and the day of birth was designated postnatal day 0 (P0). All animals were raised in the animal facility of the University of Heidelberg (INF
345; Heidelberg, Germany).
Amplification of Tenascin-C Fragments by Reverse
Transcription-Polymerase Chain Reaction (RT-PCR)--
For the
amplification of two FNIII domain fragments of TN-C, RNA from mouse
brain tissues of different ages was reverse-transcribed using hexameric
random primers (Amersham Pharmacia Biotech). 4 µg of total RNA and
200 ng of random primer in a total volume of 13 µl were incubated at
70 °C for 10 min and then cooled slowly to room temperature. After
the addition of 4 µl of 5× First Strand Buffer (Life Technologies,
Inc.), 2 µl of 0.1 M dithiothreitol, and 1 µl of 10 mM deoxynucleotide triphosphates, the reactions were heated
to 37 °C, and 1 µl of SuperScriptTM reverse transcriptase (Life
Technologies, Inc.) was added. The reactions were incubated for 15 min
at 37 °C, 15 min at 40 °C, 15 min at 42 °C, 15 min at
45 °C, and 15 min at 50 °C. Finally, the reverse polymerase was
heat denatured during a 5-min incubation at 95 °C. The reactions were diluted with 30 µl of TE buffer (10 mM Tris/HCl, pH
7.2, and 1 mM EDTA). 0.5 µl of the reverse transcription
reaction was used per 25 µl of PCR reaction with 10 pmol of the
appropriate sense and antisense primers (see Table
I). The primers were designed to
hybridize to the 5' and 3' ends of the respective domains. The reaction
conditions were as follows: 60 mM Tris/HCl, 15 mM (NH4)2SO4, 2 mM MgCl2, 0.2 mM deoxynucleotide
triphosphates, and 1 unit of Taq polymerase (AGS GmbH,
Heidelberg, Germany) per reaction. Cycling was performed using a
robocycler (Stratagene) starting with a 3-min denaturation step at
95 °C followed by 30 cycles of 30 s at 95 °C, 30 s at
57 °C, and 90 s at 72 °C and a final extension step at
72 °C for 7 min.
For the amplification of isoform-specific TN-C fragments, RNA from
mouse brain tissues of different ages and oligonucleotides hybridizing
to the 5' end of the fifth and the 3' end of the sixth conserved FNIII
domain of mouse TN-C were used (see Table I, primers 5forw and 6rev).
Before reverse transcription, 5 µg of RNA were denatured at 85 °C
for 10 min in the presence of 40 pmol of 6rev in a volume of 7 µl.
TN-C-specific cDNAs were generated with 4 units of avian
myeloblastosis virus reverse transcriptase (Promega Corp.) in a final
concentration of 60 mM Tris/HCl, 15 mM
(NH4)2SO4, 5 mM
MgCl2, 1 mM deoxynucleotide triphosphates, 5 mM dithiothreitol, and 0.25 µl of RNasin RNase inhibitor
(Promega) in a 20 µl reaction at 42 °C for 1 h. After
denaturation of the enzyme at 93 °C for 3 min, PCR was performed in
25 µl reactions using 5 µl of the reverse transcription product
with the addition of oligonucleotides and buffer components to achieve
a final concentration of 60 mM Tris/HCl, 15 mM
(NH4)2SO4, 2 mM
MgCl2, 0.2 mM deoxynucleotide triphosphates, 1 mM dithiothreitol, 0.4 µM 5forw, 0.4 µM 6rev, and 1 unit of Taq polymerase (AGS)
per 50 µl reaction. Cycling conditions were the same as those
outlined above; the only modification was that 20 cycles were run. To
obtain a sufficient amount of amplification product for cloning,
reverse transcription was upscaled to 100 µl, and PCR was performed
in 20 parallel 25 µl reactions.
Generation of FNIII Domain-specific Probes--
The TN-C FNIII
domains A1, A2, A4, B, C, D, and 6 were amplified by RT-PCR using mouse
brain RNA and the primers specified in Table I. The primers were
designed to hybridize to the 5' and 3' ends of the respective domains
and contained restriction sites for directional cloning. RT-PCR was
performed as outlined above. The resulting fragments were cloned into
pBluescript II KS+ (Stratagene) yielding the plasmids pA1, pA2, pA4,
pB, pC, pD, and p6. The plasmids were sequenced to confirm that they
contained the respective FNIII domains (Sequenase Version 2.0; Amersham Pharmacia Biotech). Fluorescein-labeled probes were generated by PCR
using 10 pg of the respective plasmid, 0.2 µM the
domain-specific sense and antisense primer, 20 µM dATP,
20 µM dCTP, 20 µM dGTP, 17.5 µM dTTP, 2.5 µM fluorescein-11-dUTP
(Amersham Pharmacia Biotech), 2 mM MgCl2, 60 mM Tris/HCl, 15 mM
(NH4)2SO4,, and 1 unit of
Taq polymerase (AGS) per reaction. Cycling conditions were
as follows: 60 s at 94 °C and 30 cycles of 30 s at
94 °C, 30 s at 57 °C, 90 s at 72 °C, and 8 min at
72 °C.
The amount of the different probes used in the hybridization reaction
was adjusted to obtain comparable signals for identical amounts of
target sequence using dot blots with defined concentrations of target
plasmid. Dot blots were prepared using a 96-well dot blot device
(Schleicher & Schuell). In brief, Hybond N+ membrane (Amersham
Pharmacia Biotech) was prewetted in 10× SSC buffer (1.5 M
NaCl and 0.15 M sodium citrate, pH 7.0) and mounted in the
dot blot machine according to the manufacturer's instructions. 400 µl of 10× SSC buffer were applied to each well and filtered through the membrane with very low suction. Dilution series of the plasmids were prepared in 10× SSC buffer, the samples were heat denatured, and
100 µl/dilution step were loaded per well. After disassembly of the
device, the DNA loaded on the membrane was denatured on a stack of
filter paper soaked with 0.5 M NaOH and 1.5 M
NaCl for 10 min and was subsequently renatured for 5 min on filter paper soaked with 0.5 M Tris/HCl, pH 7.5, 1.5 M
NaCl, and 1 mM EDTA and dried at room temperature. The DNA
was fixed to the membrane by incubation at 80 °C for 2 h.
Hybridization and detection were performed using the Fluorescein Gene
Images Detection System (Amersham Pharmacia Biotech). In brief, the
membrane was prehybridized in 5× SSC, 0.1% (w/v) SDS, 5% (w/v)
dextran sulfate, and 5% (v/v) liquid block (Amersham Pharmacia
Biotech) for 30 min at 72 °C, and the labeled probes were denatured
at 95 °C for 5 min, quick chilled on ice, and added to the
prehybridization solution. After the first round of adjustment
experiments, the amount of probes used in 3 ml of hybridization
solution was 1.5 µl for A1, 2.5 µl for A2, 2 µl for A4, 0.22 µl
for B, 7 µl for C, 0.7 µl for D, and 10 µl for 6. The labeled
probes were stored at Screening Tenascin-C Isoform-specific RT-PCR
Fragments--
RT-PCR bands of defined size representing multiples of
FNIII domains were ligated into the plasmid pBluescript II KS+
(Stratagene) using restriction sites that had been included at the 5'
ends of the oligonucleotide primers used for amplification and
transformed into the bacterial strain XL1-blue (Stratagene). The
resulting transformants were checked by PCR for TN-C fragments of the
expected size. The PCR products were blotted onto Hybond N+ membrane
(Amersham Pharmacia Biotech) using the dot blot procedure outlined
above. Seven equivalent blots were produced and hybridized in parallel to the fluorescein-labeled FNIII domain specific probes. The signals obtained with specific probes revealed the FNIII domain composition of
single bacterial clones (as outlined in Fig. 7).
Southern Blots--
RT-PCR reaction products were separated in
seven parallel sets of lanes on 1.5% agarose gels, capillary blotted,
and fixed to Hybond N+ membrane (Amersham Pharmacia Biotech) to obtain
seven analogous blots. These were hybridized separately to the
fluorescein-labeled FNIII domain-specific probes. Hybridization and
detection were carried out as described for dot blots.
Alternatively Spliced FNIII Domains of Tenascin-C in the Murine
CNS--
A restricted number of TN-C isoforms had been described for
mouse TN-C (summarized in Fig. 1). Five
of these were found to be expressed in mouse brain (Fig. 1; Ref. 6).
All known isoforms of the mouse are generated by the insertion of up to
six alternatively spliced FNIII domains, namely, A1, A2, A4, B, C, and
D, between the fifth and sixth FNIII domain of the shortest TN-C
variant.
The derived amino acid sequence of FNIII domain C has been published
previously (6), but its nucleotide sequence was not available from
databases. The sequence, as demonstrated here (Fig. 2), displays a 95% identity with human
FNIII domain C (23) and therefore represents the mouse homologue of
this domain.
A search for additional FNIII domains corresponding either to the A3
domain found in human TN-C or to the newly identified domains AD1 and
AD2, which have been shown to be present in both human and chick TN-C
(24-26), was performed using RT-PCR techniques (Fig.
3). Experiments designed to amplify sets
of two adjacent FNIII domains from mouse brain of different
developmental stages did not yield products larger than two domains.
The amplification of the domain pairs A2A4 and BD did not reveal larger
products that might hint at the expression of mouse homologues of the
human domains A3, AD1, or AD2. Therefore, we conclude that additional domains, if present at all in the mouse TN-C gene, are not expressed in
brain tissue.
Tenascin-C Isoforms of All Possible Sizes Are Expressed in Mouse
Brain--
Differential splicing of mouse TN-C in the CNS was studied
using brain tissue of postnatal day 6 (P6) mice, comparing cerebellar tissue with brain tissue without cerebellum. The cerebellum has been
previously shown to contain high amounts and different isoforms of TN-C
mRNA and protein at this developmental stage (10). RT-PCR performed
with oligonucleotide primers flanking the site of alternative splicing
yielded six products that corresponded in size to one to six FNIII
domains (Fig. 4). This result indicates
that TN-C isoforms of the sizes expected for multiples of maximally six FNIII cassettes are expressed in mouse brain. Interestingly, variants that contain two or three alternatively spliced FNIII domains that had
not previously been described in mouse brain were also detected.
When equivalent amounts of cerebellar RNA and of RNA from brain tissue
devoid of cerebellum were subjected to RT-PCR, less product was
obtained for the latter (Fig. 4). This is in agreement with recent
observations suggesting that at P6 the cerebellum is the main source of
TN-C mRNA in mouse
brain.2 In both tissues, the
most intense amplification pertained to the inserts comprising one and
six cassettes, whereas the three-FNIII domain PCR product proved to be
the weakest. This result is indicative of differential expression of
TN-C isoforms of distinct sizes.
Design and Adjustment of FNIII Domain-specific
Probes--
Following the assumption that the six alternatively
spliced FNIII domains of TN-C are independently combined to form
different TN-C isoforms, only the largest product assembling six FNIII
domains is of unambiguous composition because it must include all
available domains: A1, A2, A4, B, C, and D. In contrast, the amplified
inserts of smaller size might represent various combinations of these domains. For example, RT-PCR products consisting of three FNIII domains
could potentially encompass up to 20 possible combinations (Fig.
5). To further examine the composition of
these inserts, specific fluorescein-labeled probes for the FNIII
domains A1, A2, A4, B, C, and D and the constitutively expressed FNIII
domain 6 were produced using PCR. The concentrations of the probes used for hybridization were adjusted to ensure comparable target detection sensitivity (Fig. 6A).
Furthermore, the conditions for probe hybridization and the subsequent
washing steps were modified to obtain a high specificity of target
recognition (Fig. 6B). This is particularly important
because of the considerable sequence similarities that prevail between
the alternatively spliced FNIII domains.
Identification of Tenascin-C Isoforms--
To unravel the exact
domain compositions of the TN-C isoform-specific products (Fig. 4), the
six RT-PCR amplified bands were separately cloned into the plasmid
pBluescript II KS+. The resulting bacterial colonies were screened
using the fluorescein-tagged FNIII domain-specific probes. A
representative example of original screening data is shown in Fig.
7.
Starting with RT-PCR products of RNA from P6 cerebellum, screening of
more than 200 transformant colonies for the products containing one
FNIII domain, screening of more than 100 colonies for each of the
products containing two to five FNIII domains, and screening of 22 colonies for the six FNIII domain product resulted in the detection of
25 combinations of alternatively spliced FNIII domains expressed in
mouse CNS (Fig. 8). Of these, 19 are
entirely novel, 2 have as yet only been described in tumor cell lines
(4), and 4 combinations have been previously detected in mouse brain
(6).
TN-C isoforms in P6 cerebellum possessing one alternatively spliced
FNIII domain predominantly carried the FNIII domain D (98%), whereas
only 2% contained the domain A1. None of the four other alternatively
spliced domains was found to be exclusively inserted between the fifth
and sixth constitutive FNIII domain.
Of the two-FNIII domain RT-PCR products, about two-thirds consisted of
the domains C and D, and one-third consisted of the domains A1 and D. The two other combinations found (A1A2 and A4B) represented only 10%
of this RT-PCR product.
The combination of three FNIII domains implies the highest potential
for variation, i.e. 20 different combinations are possible, as shown in Fig. 5. The detection of eight combinations for cerebellar tissue at a specific time point during development indicates that this
potential is used to a high extent in vivo. However, taking into account the very low intensity of the three-FNIII domain RT-PCR
product (Fig. 4), these forms probably represent only a minor portion
of brain TN-C isoforms. Nevertheless, the expression of these isoforms
seems to be regulated, as demonstrated by their distinct frequencies.
Seven combinations of four alternatively spliced FNIII domains were
identified. However, only three of these made up 87% of the
four-domain RT-PCR product. This again points to a regulated expression
of TN-C isoforms. Interestingly, the two most frequent combinations
A1A2A4B (36%) and A4BCD (31%) consisted of a series of FNIII domains
following the order of the respective exons on the TN-C gene.
The major combination with five domains consisted of A1A2A4BD (90%).
This TN-C splice variant had previously been documented in fibroblasts,
tumor cell lines and brain tissue (3, 4, 6). The two other five-domain
splice variants detected in this screen, one missing domain A1 and the
other missing domain A4, represented only about 10% of the colonies
subcloned from this RT-PCR product.
Screening of 22 transformant colonies with six FNIII domains led to
unambiguous hybridization signals with all six probes, consistent with
the assumption that no alternatively spliced FNIII domains other than
A1, A2, A4, B, C, and D are expressed in mouse brain.
Within the subset of TN-C isoforms that was found to be expressed in P6
cerebellum, none of the alternatively spliced FNIII domains was
restricted to only one specific isoform. For example, FNIII domain C
that had previously been attributed to the largest TN-C isoform only
(Fig. 1; Ref. 6) was also found in shorter TN-C variants,
preferentially in those containing two alternatively spliced domains
(Figs. 4 and 9). Interestingly, FNIII domain C was never found to be
spliced directly to the sixth FNIII domain, thereby omitting domain D. In addition, no isoforms were identified where the FNIII domain A4 was
directly linked to the C domain. In contrast, all other possible links
between pairs of alternatively spliced FNIII domains were found to be
realized within the 25 TN-C isoforms identified in the P6 cerebellum.
When using an independent RNA preparation, the RT-PCR products of
distinct sizes were found to consist of the same combinations of FNIII
domains that, by and large, were present in the same relative
proportions as in the experiment shown in Fig. 7. One additional
combination (A2BD) was detected but was found to represent a minor
portion of the three-domain amplification products.
Tenascin-C Isoform Expression Changes during Development--
To
assess variations of TN-C isoform expression during brain development,
RNA of embryonic day 13 to adult mouse CNS was prepared and subjected
to RT-PCR, using the same primers and cycling conditions described
above. Southern blots of the resulting amplification products were
hybridized with the probes for the alternatively spliced domains (Fig.
9). Overall expression of differentially sized TN-C isoforms clearly peaked around birth. Isoform expression decreased substantially at the end of the second postnatal week, and
only splice variants with a single D domain were detected in the adult
mouse brain. Considering the isoforms that contain one alternatively
spliced FNIII domain, the Southern blot results further substantiated
the previous observation that domains A1 and D are present in
single-domain isoforms, whereas only extremely weak signals were
obtained with probes for A2 and A4, and no signal was detected with
probes for B and C.
When the signal intensities obtained with the different probes were
compared for specific RT-PCR products, a similar developmental pattern
was observed for five of the six bands, i.e. low signal intensity in early and late development, but high intensity at P0. A
notable exception was detected for the two-FNIII domain product that
developed the strongest signal for A1 and A2 at P0, whereas the signals
obtained using probes C and D peaked at E16, with probe C showing a
more prominent decrease toward P0 than probe D. This observation
suggested a shift of expression for the TN-C isoforms comprising two
alternatively spliced FNIII domains from CD at E16 toward an A1- and/or
A2-containing combination at P0.
This interpretation was explored using the screening method described
above for brain tissue of E16, P0, and P6 mice (Fig. 10). The predominant domain composition
of the two-FNIII domain TN-C isoforms detected at the late embryonic
state E16 was CD (91%), but its expression was found to be decreased
toward P0 (38%). The combinations A1A2 and A1D represented only a
minor portion of the two-FNIII domain isoforms at E16, but their
expression levels increased to 38% and 20%, respectively, at P0. At
P6, the combination A1A2 only represented 7% of the isoforms of this
size, whereas the expression of A1D was slightly increased (32%), and the expression of CD was clearly increased (59%).
The study of TN-C isoforms on the mRNA level in mouse brain
revealed the expression of six alternatively spliced FNIII domains. In
contrast, a total of nine alternatively spliced domains has been
identified in human TN-C (23, 24, 26, 27). These are, following the 5'
to 3' arrangement on the gene, the domains A1, A2, A3, A4, B, AD2, AD1,
C, and D. TN-C sequence motifs in different species display a high
degree of homology, which reaches 95% for analogous FNIII domains. On
this basis, the six alternatively spliced FNIII domains found in mouse
TN-C were identified as the mouse homologues of human A1, A2, A4, B, C,
and D (5, 6). Studies on the expression of the FNIII domains AD1 and
AD2 in E10 chick spinal cord revealed low expression of AD1 in a
restricted subpopulation of TN-C-expressing cells, e.g. a
subpopulation of cells within the ependymal layer, but no expression by
radial glia. In contrast, expression of AD2 was not detected (28). In
E14 brain, only cells located in the ventricular zone of the optic
tectum showed expression of AD1, which was not included in TN-C
mRNA in other brain areas (28). It is unclear whether these
findings can be transferred to the mouse nervous system, because the
territories of TN-C expression vary in different species (29). Along
these lines, FNIII domain C was concluded to be CNS-specific according
to in situ hybridization studies carried out on E15 mouse
(6), but it was found to be completely absent from spinal cord in
chicken E10 embryos (28). It has been proposed that in the human the
expression of particular splice variants comprising AD1 is
tumor-associated (24), and domain AD2 was documented in two of four
specimens of squamous cell carcinomas studied (26). In the present
study using a RT-PCR-based strategy, no evidence was obtained for the
expression of the mouse homologues of FNIII domains A3, AD2, or AD1 in
brain tissue at any of the developmental stages studied. Furthermore,
no hints at the expression of alternatively spliced FNIII domains other
than A1, A2, A4, B, C, and D were obtained by screening more than 1,000 RT-PCR products spanning the splice site of mouse TN-C. Nevertheless, the expression of FNIII domains equivalent to human TN-C domains A3,
AD1, and AD2 in other mouse tissues or in the CNS at developmental stages different from those investigated in this study cannot be
excluded. From an evolutionary point of view, the existence of mouse
TN-C domains equivalent to the recently discovered AD1 and AD2 is
highly probable because both domains were identified in avian and human
TN-C. In contrast to these, at present, domain A3 has only been
detected in human TN-C. In the well-studied chicken TN-C molecule, only
one A domain has been described (25). These findings may suggest that
the domains AD1 and AD2 arose from duplication events that occurred
before the avian and mammalian species diverged and should thus be
inherited by rodents as well. In contrast, the different A domains
found in mammals presumably evolved later; therefore, the A3 domain
might be unique for primate TN-C. A careful analysis of the genomic
sequence is required to ultimately identify the subset of alternatively
spliced FNIII domains present in the mouse TN-C gene.
The RT-PCR strategy using primers upstream and downstream of the splice
site located between the fifth and sixth FNIII cassette of TN-C yielded
products that represented integral multiples of one to six FNIII
domains. This result supports the conclusion that TN-C isoforms of all
possible sizes that can be generated with six domains are expressed in
mouse brain. Although RT-PCR is primarily a qualitative technique, it
can provide some insight into the relative abundance of alternatively
spliced transcripts of given sizes. Interestingly, the amount of
product obtained by RT-PCR for the different size classes differed in a
way that was not directly correlated with the lengths of the inserts.
This finding opposes the general experience that smaller products are amplified more readily than larger ones in PCR. In the case of TN-C,
the largest alternatively spliced RT-PCR product exhibited an intensity
second only to that of the shortest product, whereas the intermediately
sized products of three and four FNIII domains were amplified to a
lower extent. Thus, the RT-PCR results support the notion that
expression of differentially sized TN-C isoforms is regulated and is
not merely a reflection of a random production of variants due to the
failure of the splicing apparatus to include some or all of the
potentially available domains.
The FNIII domain-specific probes that were generated for the purpose of
this study proved to be efficient tools for decoding TN-C isoforms. Due
to the fluorescein label, the probes could reliably be used over a
prolonged time period without a substantial loss of activity.
Adjustment of the concentrations applied in hybridization procedures
was required due to the differential labeling efficiency of the various
probes. No clear relationship between sequence composition,
e.g. AT content, and labeling efficiency could be
established. After the adjustment of concentrations, the seven
different probes recognized the respective target sequences with
comparable sensitivities. High stringency conditions were chosen for
hybridization to avoid cross-reactions of the probes with nonspecific
target sequences. On average, the alternatively spliced FNIII domains
share 52% of their nucleotide sequence. The lowest degree of
similarity is recorded between A2 and D (41%), and the highest degree
of similarity was recorded between A1 and A4 (80%). Interestingly, the
constitutively expressed FNIII domains 1-8 only display an average
degree of sequence identity of 44%, whereas the sequence homology
between the constitutive and the alternatively spliced FNIII domains is
even lower, with an average value of 40%. This points to close
evolutionary relationships within these two groups of FNIII domains.
Screening of RT-PCR products of the alternatively spliced transcripts
revealed the expression of a complex but restricted repertoire of
different TN-C isoforms in the developing mouse brain. 27 TN-C isoforms
were identified, of which 22 had not been previously reported. The 27 TN-C isoforms identified in mouse brain represent 42% of the 64 possible combinatorial TN-C variants that can be achieved with six
independently arranged cassettes. The present study is the first that
combines the amplification of the complete alternatively spliced region
with a systematic analysis of representative numbers of single RT-PCR
products. Other investigations dealing with the isoform variability of
TN-C have used RT-PCR to amplify shorter stretches within the region of
alternative splicing, thereby identifying combinations of up to four
domains, but not entire isoforms (25, 26). Others have not taken into
account that TN-C isoforms of identical size might not be homogenous
with regard to domain composition (6). The screening strategy developed
in this study allowed the resolution of six populations of RT-PCR
products. Within each size class, substantial heterogeneity of FNIII
domain composition was revealed. Due to the screening procedure, the
relative frequencies of given isoforms could only be compared within a
given size category. For example, the CD variant made up two-thirds of
the two-domain group of TN-C isoforms in P6 cerebellum, whereas the
combination A1D represented the other third of these isoforms. Two
other variants, namely, A1A2 and A4B, were found to be relatively rare.
FNIII domain D was detected in most of the splice variants expressed in
mouse brain. Within each size class of isoforms, the D domain
containing splice variants made up more than 88% of the inserts. The
only exception was noted within the four-domain forms, where D was
present in only 65% of all the RT products analyzed, whereas domain A4
was found in 95% of these isoforms in P6 cerebellum. Considering the
general organizational features of TN-C isoforms, it is striking that
FNIII domain C was found to occur in conjunction with domain D in all
cases studied thus far, thereby excluding the direct transition between
domain C and 6. This observation is in agreement with analogous reports
for normal, malignant, and reactive oral mucosae (26). Interestingly,
of the 28 possible transitions between pairs of FNIII domains, only 2 were not realized in TN-C isoforms in the developing mouse brain:
(a) the transition between domains C and 6, and
(b) that between domains A4 and C. Total exclusion of these
junctions for any size class would decrease the number of potential
TN-C isoforms by 20. Additional studies have to show whether these
junctions are generally prohibited. If this proves to be the case,
these sequences, in comparison to those found frequently, might be
interesting candidates for the study of specific splicing factors.
None of the six alternatively spliced FNIII domains was confined
exclusively to one specific TN-C isoform. In particular, domain C,
which had been assigned earlier to the largest TN-C isoform only (6),
was also detected in smaller forms. The occurrence of this domain in
isoforms with two additional FNIII domains proved to be of special
interest in the context of developmental regulation. The expression of
the CD-containing isoform dominated during late embryonic development
(E16), representing more than 90% of the isoforms of this size. The
prevalence of this variant decreased toward birth and increased again
at the end of the first postnatal week. This might indicate the
involvement of this isoform in specific developmental processes. In
late embryonic development, neuron generation reaches a maximum, and
the neurons migrate toward their destination within the forming
cortical layers and finally differentiate. Neuronal migration and
neurite outgrowth are processes that have been found to be influenced
by TN-C in vitro (summarized in Ref. 30). During the first
two postnatal weeks, the cerebellum is generating its structure, being
one of the last parts of the brain to differentiate (31). In
situ hybridization has shown the cerebellum to be the major source
of TN-C at P6.2 Therefore, the CD isoform might be of
particular importance for the process of differentiation at E16 as well
as at P6.
Until now, studies on TN-C isoform expression, regulation, and function
have focused primarily on the comparison of the smallest TN-C isoform
with the largest TN-C isoform. Generally, high molecular weight TN-C
transcripts or proteins were found to be associated with motile cells
(32) and enriched in tissues undergoing dynamic remodeling (33).
Different factors have been described to influence the expression of
TN-C isoforms, for example, extracellular pH, TGF-
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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and
chains of fibrinogen (3, 4).
Larger isoforms of mouse TN-C are generated by the insertion of up to six additional alternatively spliced FNIII domains between the fifth
and sixth FNIII domain of the smallest TN-C variant. These alternatively spliced domains have been named A1, A2, A4, B, C, and D,
according to their counterparts in human TN-C (5, 6).
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Primers used for the amplification TN-C FNIII-domains
20 °C and showed a slight decrease in target
recognition sensitivity over a period of 18 months. This was most
probably due to degradation of the fluorescein group. For this reason,
the amount of probe used for hybridization was regularly readjusted. To
ensure specificity of target recognition, high stringency hybridization
and washing conditions were developed. In detail, hybridization was
performed at 72 °C for 2.5-16 h, depending on the amount of target
on the blot, followed by two washes at 72 °C with 0.5% (w/v) SDS
and 0.1× SSC for 15 min each. After stringency washes, the hybridized probes were visualized by binding an alkaline phosphatase-coupled anti-fluorescein antibody that was developed with a dioxetane-based substrate (Amersham Pharmacia Biotech). The resulting emission of light
was detected on autoradiographic films.
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MATERIALS AND METHODS
RESULTS
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Fig. 1.
Structure of mouse tenascin-C and its
published isoforms. The structure of a monomer of the shortest
TN-C splice variant is depicted at the top with the
amino-terminal cysteine-rich region, the 14.5 epidermal growth
factor-type repeats, the constitutively expressed FNIII domains 1-8
( ), and the fibrinogen homology region at the carboxyl terminus. Six
larger isoforms have been identified thus far. These are generated by
the insertion of alternatively spliced FNIII domains (
) between the
fifth and sixth FNIII domain of the smallest variant. The different
symbols indicate the TN-C isoforms that have been documented in mouse
brain (
; Ref. 6), in primary cultures of mouse fibroblasts (
;
Ref. 3), and in a mouse mammary tumor cell line (
; Ref. 4).
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Fig. 2.
Alignment of the nucleotide sequences of
FNIII domain C of mouse and human tenascin-C. The mouse sequence
reveals a 95% identity to the human FNIII repeat C.
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Fig. 3.
No evidence for A3, AD1, and AD2 in mouse
brain. RT-PCR was performed on RNA of E16 (a), P0
(b), and P7 (c) mouse brain using primer pairs
1-7, which are indicated with pairs of arrows
(see Table I for details). Sense primers were chosen to hybridize to
the 5' end, and antisense primers were chosen to hybridize to the 3'
end of the respective FNIII domains. RT-PCR with primer pair 8 was
performed to ensure that the experimental conditions allowed the
amplification of products larger than two FNIII repeats. The positions
of amplification products representing one to four FNIII domains (273, 546, 819, and 1092 base pairs, respectively) are depicted to the right
of the marker (100-base pair ladder). Note that the RT-PCR products
obtained with primer pairs 1-7 had the expected size of two FNIII
repeats, indicating that no supplementary FNIII repeats corresponding
to the human domains A3, AD2, and AD1 are expressed in mouse brain at
the developmental stages studied.
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Fig. 4.
RT-PCR reveals tenascin-C isoforms of all
predicted sizes. A diagram of the TN-C monomer is given at the
top, showing the site of hybridization of the primers used
in RT-PCR (fn5forw and fn6rev are shown as arrows). These
hybridize to the sequences directly adjacent to the site of alternative
splicing. The result of the RT-PCR is given below. Lane 1 represents the result from RNA prepared from P6 mouse brain without
cerebellum, and lane 2 represents the result of P6 mouse
cerebellar RNA. Note that the size of the resulting RT-PCR products
corresponds exactly to multiples of FNIII domains, as indicated on the
right.
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Fig. 5.
Possible tenascin-C isoforms with three FNIII
domains. 20 TN-C isoforms might be formed by unrestricted
combination of three of the six alternatively spliced FNIII repeats, as
shown here by depicting the possible variants between the fifth and
sixth FNIII domain of the TN-C monomer.
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Fig. 6.
Sensitivity and specificity of FNIII
domain-specific probes. A, dot blots of dilution series
of plasmid DNA containing the appropriate target sequence for the
different probes were hybridized with the respective probes. The signal
intensity obtained with the different probes is highly comparable.
B, seven identical dot blots with defined amounts of A1, A2,
A4, B, C, D, 6, and pJT1# target sequence were hybridized with the
different FNIII domain-specific probes. Hybridization signals are only
detected with the appropriate target sequence. Plasmid pJT1#, which
contains the constitutive FNIII domain 2-8 but none of the
alternatively spliced domains, served as an additional control. Note
that specific detection is also achieved between FNIII domains A1, A2,
and A4, although they share 62-80% of their nucleotide
sequence.
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Fig. 7.
Screening for different tenascin-C
isoforms. To reveal the heterogeneity of equally sized RT-PCR
products, the single bands were cloned separately. The cloned inserts
of the resulting transformant bacterial clones were amplified by PCR,
and the resulting PCR products were dot blotted on Hybond N+ membrane
to create seven identical blots. These were hybridized separately to
the FNIII domain-specific probes. The probe for FNIII repeat 6 served
as a negative control. The composition of the RT-PCR products was read
directly from the resulting film. The example given here shows a
representative experiment depicting the variability found within the
three-FNIII domain RT-PCR product from mouse cerebellar RNA.
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Fig. 8.
Tenascin-C isoforms in P6 mouse
cerebellum. The shortest TN-C isoform is depicted at the
top, and the 25 additional isoforms identified in a first
screening experiment are listed underneath it. These are grouped
according to the number of FNIII domains they contain. At the
right, the number of bacterial clones screened per group and
the percentages represented by the single combinations are given. Note
that within one size group, the prevalence of different isoforms may
vary from 1% to 98%, thereby indicating regulated isoform expression.
In none of the isoforms is domain C found to be spliced directly to
domain 6 or domain A4, whereas all other possible two-domain pairs are
found to be realized in TN-C isoforms.
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Fig. 9.
Developmental variation of tenascin-C isoform
expression. RT-PCR was performed with the primer pair shown in
Fig. 4 on the basis of RNA prepared from E13, E16, P0, P7, P14, and
adult mouse brain. The products were separated in seven equivalent sets
on an agarose gel and blotted. The seven analogous blots thus obtained
were hybridized separately to probes specific for domains A1, A2, A4,
B, C, D, and 6. Hybridization with the probe recognizing FNIII domain 6 did not show any signal, as expected (data not shown). Overall, TN-C
isoform expression peaks around P0 and decreases toward the second
postnatal week. In adult brain, only small isoforms persist. Note that
for the two-FNIII domain RT-PCR product, the signals obtained with the
A1 and A2 probes peak at P0, whereas the signals for the C and D probe
reach their maxima at E16, suggesting that different isoforms of this
size might be expressed at different developmental stages.
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Fig. 10.
The expression of tenascin-C isoforms with
two FNIII domains is developmentally regulated. Two-FNIII domain
RT-PCR products obtained with the primer pairs described in Fig. 4 from
RNA of E16, P0, and P6 mouse brain were cloned, and the resulting
transformant bacterial clones were screened as outlined in Fig. 7. The
prevalence of the different two-domain TN-C isoforms is indicated as a
percentage of the total number of bacterial clones tested for each
developmental stage. Note that at E16, the combination CD accounts for
nearly all of the isoforms of this size. In contrast, at P0, the
prevalence of CD equals that of the combination A1A2, whereas the FNIII
domain pair A1D represents one-fifth of these isoforms. At P6, the
combination CD is dominant again (59%), whereas A1D accounts for
one-third of the two-domain isoforms.
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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1, and bFGF
(34-38). The present study shows that a large variety of TN-C isoforms
are expressed in the developing brain and that their expression is
developmentally regulated. With its many isoforms, TN-C has the
potential of encoding positional specificities of astrocytes and,
consequently, specific microenvironments for different neuronal
populations. Whether particular isoforms display topologically
restricted expression patterns will be the focus of additional studies.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. J. Hannah and J. Garwood for comments on the manuscript, D. Schnörr for technical assistance, and Prof. Dr. W. B. Huttner for ongoing support.
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FOOTNOTES |
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* This work was supported by German Research Council Grants DFG SFB317/A2 and RI 742/2-1 and by the DFG Graduate College "Molecular and Cellular Neurobiology."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ236880.
¶ A recipient of an H.-L. Schilling Professorship for Neuroscience during part of this work.
To whom correspondence should be addressed. Tel.:
49-6221-545467; Fax: 49-6221-548301; E-mail:
faissner{at}sun0.urz.uni-heidelberg.de.
2 A. Joester and A. Faissner, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TN-C, tenascin-C; FNIII, fibronectin type III; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; E13 and E16, embryonic day 13 and 16, respectively; P0, day of birth; P6 and P7, postnatal day 6 and 7, respectively; CNS, central nervous system.
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