Matrix Biology 20 Ž2001. 53᎐61

Differential expression of two exons of the ␣1ž XI/ collagengene ž Col11a1/ in the mouse embryo 夽
Ken-ichi Iyamaa , Hideaki Sumiyoshi b, Mohammed Khaleduzzamanc , Noritaka
Matsuo c , Yoshifumi Ninomiyad, Hidekatsu Yoshiokac,UbaDepartment of Surgical Pathology, Kumamoto Uni¨ ersity School of Medicine, Kumamoto 860-8556, Japan
Brookdale Center for De¨ elopmental and Moleculer Biology, Mount Sinai School of Medicine, New York, NY 10029, USAc
Department of Biochemistry, Oita Medical Uni¨ ersity, Oita 879-5593, Japand
Department of Molecular Biology and Biochemistry, Okayama Uni¨ ersity Medical School, Okayama 700-8558, Japan

Received 19 June 2000; received in revised form 19 October 2000; accepted 02 November 2000

Abstract
The amino terminal domain of collagen XI has a unique structure, which is believed to participate in the regulation of
matrix assembly. Interestingly, several distinct isoforms of the amino terminal domain of ␣1ŽXI. and ␣ 2ŽXI. collagen chains
exist as a result of alternative splicing. Here we report the analysis of the alternative splicing pattern of the mouse ␣1ŽXI.
collagen gene Ž Col11a1.. Like other vertebrate species, the mutually exclusive expression of exons 6A and 6B of Col11a1
results in the inclusion in the ␣1 chain of either an acidic peptide ŽpI 3.14. or a basic peptide ŽpI 11.66.. Expression of these
two exons was monitored in several tissues of the 16.5-day mouse embryo by in situ hybridization and immunohistochemistry,
with exon-specific cDNA probes and peptide-specific antibodies, respectively. The results documented that isoforms containing
the exon 6B-encoded peptide accumulate predominantly in the vertebrae, skeletal muscles and intestinal epithelium. By
contrast, exon 6A products were found to be most abundant in the smooth muscle cells of the intestine, aorta and lung. The
results using in situ hybridization confirmed those using immunohistochemistry. Albeit correlative, the evidence suggests
distinct contributions of the two peptides to the differential assembly of tissue-specific matrices. 䊚 2001 Elsevier Science
B.V.rInternational Society of Matrix Biology. All rights reserved.
Keywords: Alternative splicing; Col11a1 gene; Differential gene expression; Extracellular matrix; Mouse development; Immunohistochemistry;
In situ hybridization

Abbre¨ iations: d.p.c., days post coitum; bp, base pairŽs.; ELISA, enzyme-linked immunosorbent assay; MMLV, Moloney murine leukemia
virus; PARP, proline- and arginine-rich peptide; PBS, phosphate buffered saline; RT-PCR, reverse transcription-polymerase chain reaction
夽
The nucleotide sequence reported in this paper has been submitted to the DDBJrEMBLrGenBank under accession number,
AB044888᎐AB044898.
U
Corresponding author. Tel.: q81-97-586-5670; fax: q81-97-549-6302.
E-mail address: hidey@oita-med.ac.jp ŽH. Yoshioka..
0945-053Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved.
PII: S 0 9 4 5 - 0 5 3 X Ž 0 0 . 0 0 1 3 0 - X

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K. Iyama et al. r Matrix Biology 20 (2001) 53᎐61

1. Introduction
Collagen XI is a relatively minor fibrillar collagenthat regulates the assembly of cartilaginous matrices
by co-polymerizing with collagen II trimers ŽMendler
et al., 1989; Li et al., 1995.. Collagen XI consists ofthree subunits, one of which Ž ␣ 3 chain. corresponds
to the unprocessed form of ␣1ŽII. collagen ŽWu andEyre, 1995.. Following our initial suggestion, evidence
has rapidly accumulated to indicate a broader thanpreviously thought expression of collagen XI in cartilaginous and non-cartilaginous tissues ŽBernard et al.
1988; Niyibizi and Eyre, 1989; Yoshioka and Ramirez,
1990; Yoshioka et al., 1995a; Brown et al., 1991; Nahet al., 1992; Kleman et al., 1992; Mayne et al., 1993..
Subsequent analyses revealed the exsistence of ␣1ŽXI.
and ␣ 2ŽXI. collagen isoforms resulting from alternative splicing of the primary transcripts ŽOxford et al.,
1995; Yoshioka et al., 1995b; Zhidkova et al., 1995;
Tsumaki and Kimura, 1995; Davies et al., 1998..
The pattern of alternative splicing of the ␣1ŽXI.
chain has been studied in some detail in the rat andchicken genes and, to a lesser extent in the humangene, and found to involve the exons coding for the
N-terminal globular domain of the protein ŽOxford etal., 1995; Zhidkova et al., 1995; Davies et al., 1998..
Based on the original cloning of the human transcript,
the N-terminus of ␣1ŽXI. collagen consists of a proline- and arginine-rich peptide ŽPARP., an acidic segment and a short helical sequence ŽYoshioka andRamirez, 1990.. A similar arrangement has been also
reported for the ␣ 2ŽXI. chain ŽTsumaki and Kimura,

1995.. Alternative splicing results in the generation of
six obligatory combinations of the exons coding forthe acidic segment, thus termed the variable regionŽOxford et al., 1995; Zhidkova et al., 1995; Davies etal., 1998.. The variable region exons are called I, IIA,
IIB, III, IV and V in the chick, and C1, V1a, V1b, C2,
V2 and C3 in the rat ŽZhidkova et al., 1995; Oxford etal., 1995.. No information is yet available for themouse ␣1ŽXI. collagen gene Ž Col11a1., where alternative splicing has only been detected by the RT-PCR
analysis of RNA from different embryonic tissuesŽYoshioka et al., 1995b..
The present study was designed to extend the analysis of the Col11a1 isoforms by focusing on two mutually exclusive expressed exons using peptide-specificantibodies. Aside from characterizing alternative
splicing in another vertebrate species, the analysis wasundertaken to eventually aid the interpretation ofphenotypic abnormalities associated with the loss ofeither of the exons by homologous gene targeting.
The results of our descriptive survey corroborate theidea that predominant expression of different peptides of ␣1ŽXI. collagen may be involved in the different assembly of tissue-specific matrices.

2. Materials and methods
2.1. Genomic and RNA analyses
Two mouse genomic libraries ŽStratagene, CA; Andrikopoulos et al., 1995. were screened with cDNAs

Fig. 1. Genomic clones organization of the region coding for PARP and the variable domain of N-propeptide of ␣1ŽXI. chain. The locationsof the clones are shown at the top. The exons are indicated by black boxes in the middle. Oval-shaped circles show regions that are notcovered by these clones. The striped box, closed box and open box at the bottom show PARP region, the variable domain and the short helicaldomain, respectively. The vertical arrow at the C-terminal end of PARP indicates the N-protease cleavage site.

K. Iyama et al. r Matrix Biology 20 (2001) 53᎐61

encoding the PARP and the variable domain of themouse ␣1ŽXI. collagen chain ŽYoshioka et al., 1995b..
The screening was performed according to the standard procedure ŽSambrook et al., 1989.. Positive clones
were characterized by genomic mapping, Southernblot hybridization and sequencing of both DNA
strands ŽSambrook et al., 1989..
RNA samples were prepared from different tissuesof 18-d.p.c. mouse embryos ŽChirgwin et al., 1979. andsubjected to RT-PCR as previously describedŽYoshioka et al., 1995b; Sumiyoshi et al., 1997.. The
nucleotide sequence of the primers used in thesereactions were:Žforward. 5⬘-CTCGATAGAAGTGAGAGATC-3⬘
Žnt number 565᎐584.

55Žreverse. 5⬘-GTGGTCCTTCAACAAGCATC-3⬘
Žnt number 1324᎐1305..
The specific cDNA probes for exon 6A and 6B weregenerated by PCR procedures. The cDNA that recognized all transcripts of ␣1ŽXI. and the procedures forin situ hybridizations were previously describedŽYoshioka et al., 1995a..
2.2. Antibodies and immunohistochemistry
The amino acid sequence of the peptides used toraise specific antibodies against the portions encodedby exon 6A, 6B and the ␣1ŽXI. carboxyl telopeptide
ŽCT. were:

Fig. 2. Ža. Partial sequences of the exonrintron junctions of Col11a1 with the size of the exon indicated. The nucleotide sequences ofexonrintron boundaries are shown by uppercase letters for exons and lowercase letters for introns. The 5⬘ gt and the 3⬘ ag of introns areshown in boldface. The total numbers of nucleotides are in parentheses. Žb. The nucleotide and amino acid sequence of exon 6B.

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K. Iyama et al. r Matrix Biology 20 (2001) 53᎐61

Fig. 3. RT-PCR detection of Col11a1 transcripts in embryonic tissues. Ža. RT-PCR analysis performed with primers that span between exon 5
and 9 in mouse ␣1ŽXI. gene. RNA samples were purified from intestine Žlane 1., brain Žlane 2., calvaria Žlane 3., heart Žlane 4., tongue Žlane
5. and vertebra Žlane 6. of 18-d.p.c. embryos. Amplified products were electrophoresed in 2.0% agarose gels. Five isoforms, 6A᎐7᎐8, 6A᎐7,
6B᎐7, 7᎐8 and 7 were identified; some of the bands in lanes 3 and 6 Žasterisks . were not identified by DNA sequencing. Žb. ␤-actin mRNA as
control. The size of the products is indicated on the left ŽM. with ␾X marker Ž1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118 and 72 bp.. ŽB.
Schematic representation of the exon structure of PARP and the variable region, and the splicing isoforms identified in the mouse. The
striped boxes and closed boxes show exons encoding basic and acidic peptides, respectively. The numbers in parenthesis under each exon show
the pI of the peptide encoded by each exon. The horizontal bars at exon 6A and 6B, and the horizontal arrows at exon 5 and 9 show the
support used to make the antibodies and the position of primers that used for RT-PCR analysis, respectively. Although the isoform 6B-7-8
could not be identified from the cDNA sequence, it was suggested by Southern blot analysis.

exon 6A: ETDYKEAESVTEMPTFC
exon 6B: KKKTKKFTSPKSEKFACCT:
CLSPKKTRRHTESIQG

The underlined cysteinyl residues were added forcoupling to a carrier protein. The synthesis and purification of the peptides was carried out according toK. Iyama et al. r Matrix Biology 20 (2001) 53᎐61

57

column chromatography. The titers of the antibodies
were measured with enzyme linked immunosorbent
assay ŽELISA. ŽMurata et al., 1990.. Immunohistochemical procedures were performed as previously
described ŽKitaoka et al., 1994.. The specific antibodies against 6A-, 6B- and CT-peptide were diluted
1:500, 1:10 and 1:10, respectively.

3. Results and discussion

Fig. 4. ELISA using: Ža. anti-6A; Žb. 6B; and Žc. C-telopeptide of
␣1ŽXI. chain. Each antibody reacts with corresponding antigen andnever cross-reacts with other antigens. The antigens coated on thewell are 6A Žclosed squares., 6B Žopen squares., C-telopeptide of
␣1ŽXI. Žclosed circles. and ␣1ŽV. Žopen circles. chains.

previously described procedures ŽNinomiya et al.,1995.. Approximately 200 ␮g of synthetic peptide
conjugated to KLH in 1 ml of 0.15 M NaCl was
emulsified with an equal volume of complete Freund’s
adjuvant ŽDifco, Detroit, MI., and then injected subcutaneously into a rabbit. A booster injection Ž200
␮g. was given with incomplete Freund’s adjuvantŽDifco, Detroit, MI. at 3 weeks after the first injection. The antibodies were purified using affinity

Previous work has documented the existence ofalternatively spliced transcripts of Col11a1 in themouse embryo ŽYoshioka et al., 1995b.. In order toextend this initial observation, we isolated and sequenced five genomic clones covering ; 70 kb of the5⬘ portion of Col11a1. As a result, 11 exons wereidentified; they encode the 5⬘ UTR of the mRNA andthe signal peptide of the protein Žexon 1., the PARP
Žexons 2᎐4., the variable region Žexons 5᎐9. and theshort helical sequence Žexon 10. ŽFig. 1.. Exons 5, 6A,
6B, 7, 8 and 9 correspond to exons C1, V1a, V1b, C2,
V2 and C3 of the rat gene, and to exons I, IIa, IIb,
III, IV and V of the chick gene, respectively ŽOxford
et al., 1995; Zhidkova et al., 1995.. Likewise, exons 5,
6A, 6B and 7 correspond to exons I, IIa, IIb and III of
the human gene, respectively ŽZhidkova et al., 1995..
Exon 6A codes for the acidic peptide ŽpI 3.14. foundin the original human cDNA, whereas exon 6B specifies the basic peptide ŽpI 11.66., later identified as analternatively spliced product in the human, rat andchick genes ŽYoshioka and Ramirez, 1990; Oxford etal., 1995; Zhidkova et al., 1995.. The exonrintron
boundaries of the Col11a1 exons are shown in Fig. 2,
along with the sequence of exon 6B.
Based on the above information, oligonucleotide
primers specific for exons 5 and 9 were synthesized
and used to amplify RNA isolated from a variety ofembryonic tissues by the RT-PCR technique. Multiple
bands were mainly seen in the RNA preparations
from calvaria and vertebrae ŽFig. 3a.. Upon sequencing, they were found to correspond to five specificexon combinations; they are exons 6A᎐7᎐8, exons
6A᎐7, exons 6B᎐7, exons 7᎐8 and exon 7 ŽFig. 3B..
These five combinations were identical to those previously obtained with the rat and chick genes, except
for the combination of exons 6B᎐7᎐8, which was
suggested by Southern blot analysis in the mouseŽdata not shown. ŽZhidkova et al., 1995; Davies et al.,1998..
In order to correlate these findings with the mutually exclusive expression of exons 6A and 6B in themouse embryo, peptide-specific antibodies were prepared for immunohistochemistry in the 16.5-d.p.c.
mouse embryo. Polyclonal antibodies were generated
against synthetic peptides corresponding to the 6A

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K. Iyama et al. r Matrix Biology 20 (2001) 53᎐61

Fig. 5.

K. Iyama et al. r Matrix Biology 20 (2001) 53᎐61

59

Fig. 6. Bright field micrographs demonstrating the expressions of 6A mRNA ŽA and D., 6B mRNA ŽB and E. and all-XI mRNA ŽC and F. byin situ hybridization with 35 S-labeled cDNA probes in the serial sections of vertebrae ŽA, B and C. and aorta ŽD, E and F. from 16.5-d.p.c.
mouse embryo. The expression of 6A mRNA is intensely observed in the perichondrium and in aortic tunica media. A high content of all XI
mRNA is detected in the developing chondrocytes, including hypertrophic chondrocytes. Bar: 100 ␮m.

and 6B sequences and, as a control, the C-terminal
telopeptide of ␣1ŽXI. collagen, which is retained by
all isoforms. The antibodies were purified by affinity
column chromatography and their specificity was confirmed by ELISA ŽFig. 4..
Consistent with our earlier analysis ŽYoshioka etal., 1995a., the mHY 1 cDNA identified ␣1ŽXI. transcripts in several cartilaginous and non-cartilaginous
tissues at levels ranging from strongly positive to justabove background. As expected, the distribution ofthe ␣1ŽXI. chain monitored using the antibodies
against the C-terminal telopeptide of the protein
closely paralleled the mRNA pattern ŽFig. 5C, F, I, L,
and O.. A similar survey using the 6A- and 6B-specificantibodies revealed both quantitative and qualitative
differences in the distribution of the ␣1ŽXI. isoforms.
A few illustrative examples are presented here.
Isoforms containing the exon 6B-encoded sequences were found throughout the entire cartilaginous matrix of the developing vertebrae ŽFig. 5B..
Exon 6A products were detectable in the perichondrium, and in significantly lesser amounts than theexon 6B isoforms ŽFig. 5A.. The signal of the exon 6A
antibodies was very strong in the smooth muscle cellsof the intestine, where the exon 6B epitopes were lessevident, but more widely expressed, to include themucosal epithelium ŽFig. 5D, E.. A similar pattern
was observed in the lung, with nearly exclusive accumulation of 6A and 6B isoforms in the smooth muscle
cells and epithelium of the developing bronchi, respectively ŽFig. 5G, H.. Even more striking were thedifferences in the distribution of the two groups of
␣1ŽXI. isoforms in the skeletal muscle and aorta. The
6B isoforms were the prevalent species in the former,
whereas the 6A were the only detectable epitopes in
the aortic tunica media wall ŽFig. 5J, K, M, and N.. Inaddition, in situ hybridization analysis using exonspecific probes confirmed the data using immunohistochemistry in vertebrae and aorta, in which both
expressions are typically different ŽFig. 6..
Alternative splicing of genes coding for extracellular matrix proteins is a common occurrence that may
have functional implications ŽBreitbart et al., 1987..
Amongst others, the most extensively studied exam-

Fig. 5. Immunolocalization of 6A Žleft panels: A,D, G, J and M., 6B Žcenter panels: B, E, H, K and N., and all-XI Žright panels: C, F, I, L and
O. in serial sections of vertebrae ŽA, B and C., intestine ŽD, E and F., lung ŽG, H and I., skeletal muscle ŽJ, K and L. and aorta ŽM, N and O.
from 16.5-d.p.c. mouse embryo. Note the peptide of 6A exclusively localized in perichondorium and in the smooth muscle layer of theintestine, lung and aorta. The peptide of 6B and all-XI co-localized intensely in cartilage, the epithelial cells of intestine and lung, and skeletal
muscle. Bar: 100 ␮m.

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K. Iyama et al. r Matrix Biology 20 (2001) 53᎐61

ples include fibronectin and collagen II ŽSchwarzbauer
et al., 1983; Sandell et al., 1991.. Alternative splicingof ␣1ŽII. collagen has recently been linked with
TGF␤-1 and BMP-2 signaling in the developing cartilage ŽZhu et al., 1999.. Along these lines, the alternative splicing of Col11a1 may be indicative of differential functions of this collagen in collagenous andnon-collagenous extracellular matrices. Since the variable domain is retained in the mature trimer following extracellular processing of the N-terminal domain, peptides with very different pIs may conceivably
mediate distinct interactions between collagen II fibrils andror amongst them and other matrix components. Consistent with previous in vitro data by Brown
et al. Ž1991., 6A-containing isoforms might be involved in the formation of heterotypic collagen VrXI
fibrils by vascular smooth muscle cells. Homologous
gene targeting in the mouse will test the validity ofthis hypothesis, and provide genetic support to thenotion for stage- and tissue-specific assembly of collagen XI matrices.

Acknowledgements
We are indebted to Dr Y. Sado for preparing thesynthetic peptide and and to Dr F. Ramirez for the
support and review of the work. This work was supported by a Grant-In-Aid for Scientific Research
Ž09671497, 11470312 to H.Y.. from the Japanese Ministry of Education, Science and Culture, and The
Nakatomi Foundation Žto H.Y.., and from a grantfrom the National Institutes of Health ŽAR45881 toHS..
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