DEVELOPMENTAL DYNAMICS 204:41-47 (1995)

Developmental Pattern of Expression of the Mouse al(X1)Collagen Gene (CoZlIal)
HIDEKATSU YOSHIOKA, KEN-ICHI IYAMA, KAZUHITO INOGUCHI, MOHAMMED KHALEDUZZAMAN,YOSHLFUMI NINOMIYA, AND FRANCESCO RAMIREZ
Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama 700 (H.Y.,KJ., M.K.,
Y.N.), and Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University
Graduate School of Medical Sciences, Kumamoto 862 (K.-i.I.), Japan; Brookdale Center for Molecular Biology, Mount Sinai
School of Medicine, New York, New York 10029 (F.R.1

paired development of mice harboring defective
collagen genes. This large body of work includes transdominant overexpression of mutated versions of thegenes coding for the al(I), al(II), al(IX), and a l ( X )collagen chain (Schnieke et al., 1983; Garofalo et al.,
1991; Vandenberg et al., 1991; Nakata et al., 1993;
Jacenko et al,, 1993);inactivation of the genes coding
for the al(I),al(IX),and al(X) collagen chain (Stacy etal., 1988; Fassler et al., 1994; Rosati et al., 1994); andtargeted mutagenesis of the gene coding for the a2(V)collagen chain (Andrikopoulos et al., 1995). Additionally, the craniofacial and skeletal abnormalities characteristic of the mouse cho (for chondrodysplasia) havebeen recently associated with a frame-shift mutation in
the gene coding for the a1 chain of type XI collagen( C o l l l a l ) (Li et al., 1995).
Earlier indirect evidence and the genetic studies
cited above indicate that types V and XI collagen regulate the growth and tridimensional assembly of thetypes I and I1 fibrillar networks in non-cartilaginousand cartilaginous matrices, respectively (Birk et al.,
1988, 1990; Mendler et al., 1989; Andrikopoulos et al.,
1995;Li et al., 1995).In addition to serving comparable
roles in fibrillogenesis, types V and XI collagen cangive rise to hybrid trimers whose functional significance remains obscure (reviewed by Brewton andMayne, 1994). Our cloning work provided the first evidence against the previously held belief that type XI is
exclusively produced by cartilaginous cells, since weidentified al(X1) transcripts in cells of non-cartilaginous origin (Bernard et al., 1988). A t about the sametime a biochemical analysis of adult bovine bone demonstrated assembly of the al(X1) protein with the a1
and a2 subunits of type V (Niyibizi and Eyre, 1989).
0 1995 Wiley-Liss, Inc.
Hybrid trirners were later reported in the bovine vitKey words: Type XI collagen, Extracellular ma- reous and in a human rhabdomyosarcoma cell line
(Mayne et al., 1993; Kleman et al., 1992). Co-exprestrix, Gene expression
sion of the al(X1) and a2(V)genes was also observed inbovine arterial smooth muscle cells and other tumor
INTRODUCTION
and virally transformed human cells (Brown et al.,
In addition to being responsible for the biochemical 1991; Yoshioka and Ramirez, 1990).
properties of most tissues, collagenous networks arealso intimately involved in a variety of morphogenetic
Received March 21, 1995; accepted April 25, 1995.
processes (reviewed by Linsenmayer, 1991). TransAddress reprint requests/correspondence to Hidekatsu Yoshioka,
genic and gene targeting experiments have dramati- Department of Molecular Biology and Biochemistry, Okayama Unically illustrated the latter role by documenting im- versity Medical School, Okayama 700, Japan.

ABSTRACT
Fibrillar networks are intimately involved in several morphogenetic processes which underlie the harmonious development of the vertebrate embryo. Recent genetic
evidence has demonstrated that the minor types V
and XI collagen are key regulators of types I and
XI fibrillogenesis in non-cartilaginous and cartilaginous matrices, respectively. A comprehensive
understanding of the expression and regulation of
the genes coding for the chains of the minor collagen types is therefore relevant to animal morphogenesis and development. The present study
was undertaken to elucidate the embryonic pattern of expression of the gene coding for themouse a1 chain of type XI colagen ( C o l l l a l )using
the technique of in situ hybridization. Transcriptsof the C o l l l a l gene were detected as early as 11days of gestation. The oll(X1) transcripts were
found to accumulate mostly in cartilaginous tissues, such as the chondrocranium and the developing limbs. Like the major cartilage-specific collagen (type XI), Colllal expression was also notedin the neuro-epithelium of the brain. However,al(X1) transcripts accumulated in several othernon-cartilaginous sites. They include odontoblasts, trabecular bones, atrioventricular valve of
the heart, the tongue, the intestine, and the otic
vesicle. Altogether, the data confirm that C o l l l a l
has a broader spectrum of expression than previously thought. This finding raises the possibility
that the (ul(X1) chain may participate in the formation of stage- and tissue-specific trimers with
distinct functional properties.

0 1995 WILEY-LISS.INC

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YOSHIOKA ET AL.

A

7.3kb +
6.3kb -+

B

origin +

1

2

3

4

28S+

Fig. I . Northern blot analysis of a1(XI) expression in the mouse embryo. A: Total RNA (20 bg) isolated from human A-204 cells (lane I)and
from limbs (lane Z ) , vertebrae (lane 3), heart (lane 4), brain (lane 5), liver(lane 6), tail (lane 7), skin (lane 8 ) , kidneys (lane 9), calvaria (lane 10) of
18 d.p.c. mouse embryos was hybridized to probe mHYl . Arrows indicate
transcripts of mouse a1 (XI) collagen gene. B: Poly (A)+ RNA (1 pg) from18 d.p.c. embryonic mouse limbs was hybridized to cDNA probes coding
mouse a1 (V) (lane 2), human a2(XI) (lane 3),
for mouse a1 (XI) (lane l),
and mouse a 2 ( V ) (lane 4) chains, respectively. The estimated sizes oftranscripts are: 7.3 and 6.3 kb in a1(XI); 7.7,7.4,6.3, and 4.8 kb in a1( V ) :6.1 and 4.8 kb in al(XI): 6.3and 5.0 kb in a2(V). Arrows indicate therelative positions of the 28 and I8 S ribosomal RNA.

Based on this evidence, Nah et al. (1992) recently
performed a preliminary survey of the pattern of theal(X1) collagen gene expression during vertebrate embryogenesis using as a model the chick embryo. The
study revealed that some of the al(X1)-positive tissuesof the chick coincide with the sites known to produceal(I1) collagen in the mouse (Cheah et al., 1991).Moreover, al(X1) transcripts were found to accumulate inorgans of the chick, which in the mouse have beenshown to contain a2(V) collagen-producing sites (Andrikopoulos et al., 1992). The informative value ofthese correlations is unfortunately limited by two major differences in the experimental designs of the(rl(X1) compared to the ul(I1) and a2(V) studies. First,

embryonic expression of c1 l(X1) collagen was examinedin the chick and not in the mouse, the organism where
the patterns of the ul(I1) and a2(V) collagen genes
have been described in the greatest details (Nah et al.,
1992; Cheah et al., 1991; Andrikopoulos et al., 1992).
Second, the al(X1) analysis could not determine theprecise sites of gene expression within each of the positive organs because it relied mostly on Northernrather than in situ hybridization data (Nah et al.,1992). The present study was therefore undertaken toprovide a more reliable picture of the overall pattern ofexpression of the fibrillar collagene genes during thedevelopment of the same vertebrate embryo, notably
the mouse.

EMBRYONIC al(X1) COLLAGEN EXPRESSION

43

Fig. 2. Developmental expression of mouse collagen genes. Semi-serial sections are from a 14.5 d.p.c.
embryo (top panels) and from a 16.5 d.p.c. embryo (bottom panels). From left to right, the probes are for al(l)(A,E), al(ll) (B,F), and ul(XI) (C,G) collagen. In D is the negative control section which was pre-treated with
RNase. Bar = 1 mm.

RESULTS AND DISCUSSION
As a first step toward defining the developmental
pattern of Colllal gene expression, we initially evaluated the presence of oll(X1) transcripts in the whole
embryo a t 11, 12, and 14 days post coitum (d.p.c.1, as
well as in different tissues of 18 d.p.c. mice by amplifying reverse transcribed RNA with the polymerase
chain reaction technique (RT-PCR). Colllal transcripts were readily detectable a t all stages of development; they were also present in limbs, vertebrae, heart,brain, liver, tail, kidney, calvaria, lung, muscle, andintestine of 18 d.p.c. embryos (data not shown). A subsequent Northern analysis identified oll(X1) mRNA in
the limbs, vertebrae, brain, tail, skin, and calvaria, but
not in the heart, liver, and kidney, of 18 d.p.c. embryos(Fig. 1A). As a point of comparison, we hybridized
mRNA from 18 d.p.c. embryonic limbs to al(XI),
a2(XI), al(V), and a2W) collagen probes (Fig. 1B). Sizedifferences among the different collagens are mostly
due to the variation in the length of the amino-terminal peptide-codingregions, whereas the multiple bands

reflect the utilization of alternative polyadenylationsites (reviewed by Vuorio and de Crombrugghe, 1990).
Next, we performed a series of in situ hybridizations
in order to identify the precise sites of Colllal gene
expression. To this end, we compared the hybridization
pattern of the al(I), al(II),and al(X1) collagen genes
using sagittal sections of 14.5 and 16.5 d.p.c. embryos.
At both stages of mouse development, al(X1) signals
were located in the vertebral column, the nasal septum, Meckel's cartilage, the sternum, the primordia ofhyoid and thyroid cartilages, and the cartilage of thedeveloping feet (Fig. 2C,G). As expected, accumulationof al(1) and al(I1) collagen transcripts was mutually
exclusive, with the latter particularly strong in thesame cartilaginous sites that express high amounts ofal(X1) mRNA (Fig. 2).
Previous immunohistochemical and in situ hybridization experiments have revealed transient al(I1) collagen accumulation in a few non-cartilaginous tissuesof the mouse embryo, such as the developing heart andbrain (Thorogood and von der Mark, 1986; Kosher and44

YOSHIOKA ET AL.

Fig. 3. Dark field analysis of collagen expression in the neuro-epithelium,vertebral column and mandibularbone. Serial sections of the telencephalon (A,B) are from 16.5 d.p.c.embryo, and the lumbar vertebral column( G E ) and the mandibular bone (F-H) from an 18.5 d.p.c. embryo hybridized to probes for ~ r l ( l(C,F),)CXI(II)(A,D,G), and aI(XI) (B,E,H) collagen. In the mandibular arch (F-H), the odontoblasts are indicated witharrowheads. The letter "n" indicates neuro-epithelium. Bar = 200 pm.

Solursh, 1989;Cheah et al., 1991).Upon re-examining
the sections at higher magnification, we were able todetect al(X1)transcripts in the neuro-epithelium of thebrain (Fig. 3B). Aside from confirming the Northerndata of Nah et al. (19921,the finding localized the rwl(X1)transcripts in the same non-cartilaginous tissue and at
about the same developmental stage as the al(I1) collagen gene product (Fig. 3A) (Cheah et al., 1991).Since
the a3 chain of type XI is believed to be a post-translational variant of al(II), our result raises the possibility that al(I1) gene expression in the neuro-epitheliumof the brain might be related to the production of some
type XI collagen, in addition to type IT trimers.
Aside from confirming co-expression of al(X1) andal(I1) collagens in non-cartilaginous tissues, the highmagnification analysis revealed several sites in whichaI(X1) and al(1) collagen transcripts accumulate together. In addition to cartilage, strong al(X1) mRNAsignals were in fact noted in the perichondrium and thetrabecular bone of the lumbar vertebral column (Fig.
3E). The al(1) and al(X1) collagen genes were transcriptionally active in the mandibular bones too, with

EMBRYONIC al(X1) COLLAGEN EXPRESSION

45

odontoblasts containing significant amounts of bothtranscripts and lacking those of the al(I1) gene (Fig.
3F,G,H). The rul(X1) transcripts were also noted to accumulate in the atrioventricular valve, pulmonary
valve, and artery of the developing heart (Fig. 4A).
Furthermore, the al(X1) signal was strong in the
tongue, the intestine, and the otic vesicle of the mouseembryo (Fig. 4B,C,D). These last sites are the sameones in which we previously observed substantial accumulation of a2(V) mRNA (Andrikopoulos et al.,1992). This finding further strengthens the closer relationship between these two collagen chains comparedto other subunits of types V and XI collagen. Alongthese lines, the co-expression of the two genes in sometissues is consistent with our recent identification of acommon regulatory element in the promoter of thegenes coding for the human a2(V) and al(X1) chains(Yoshioka et al., 1995).
In conclusion, we confirmed and extended previous
data on the identity and spectrum of al(X1) collagenproducing tissues. In doing so, we elucidated for the
first time the spatiotemporal pattern of expression ofthis minor fibrillar collagen gene during mouse embryogenesis. We confirmed that transcription of the
Colllal gene is not restricted only to al(I1)collagenproducing tissues; we extended the number of al(X1)collagen positive sites to a larger variety of tissues
than previously recognized; and we interpreted some of
the in situ data as suggesting that the al(X1) collagenchain may participate in the formation of several tissue- and stage-specifictrimers with distinct functional
properties. One such property could be to expand theability of regulating the tridimensional assembly of
fibrillar macro-aggregates by varying the biochemicalcomposition of the minor collagen trimers.

EXPERIMENTAL PROCEDURES
RNA Analysis
RNA was isolated from different developmental
stages using the acid-guanidine method (Chirgwin etal., 1979). Poly (A)' RNA was purified by oligotex 30(Japan Synthetic Rubber Co., Tokyo, Japan) and subjected to Northern analysis as previously described(Yoshioka and Ramirez, 1990; Yoshioka et al., 1995).
The Northern analysis employed cDNAs coding formouse a l ( X I ) ,al(V), and a2(V) collagen [clones mHYl
(Yoshioka et al., 1995), mHY8 (Yoshioka, unpublisheddata), and KA311 (Andrikopoulos et al., 1992)l and forhuman a2(XI) collagen [clone HY9 (Yoshioka, unpublished data)]. RT-PCR amplifications were performedfor 35 cycles with 2.5 U of Taq DNA polymerase at 94°C
for 1 min, 60°C for 2 min, 72°C for 3 min, followed by
finalextension a t 72°Cfor 7 rnin (Wang et al., 1989).The
nucleotide sequences for the primers used in these reactions are:forward direction: 5'-GACACACTGAAAGCATCCAG-~'[CX~(XI)],
5'mAAGAGAGGTATCCTGACCCT-3'(P actin).

Fig. 4. Dark field analysis of al(Xl) expression in the heart, tongue,
intestine, and otic vesicle. A: Hybridization of the al(XI) probe to a 16.5
d.p.c. embryonic heart. Highlighted are the atrioventricular (arrowhead)and pulmonary (arrow) valves. 6,C.D: al(XI) hybridization in the tongue
and intestine of 16.5 d.p.c. embryo, and the otic vesicle (arrow in D) of
14.5 d.p.c. embryo. s, sternum; P, pulmonary artery; t, tongue; 0,oralcavity; m, metencephalon. Bar = 200 pm.

46

YOSRIOKA ET AL.

T.F. (1990) Collagen fibrillogenesis in vitro; Interaction of type I
reverse direction: 5’-GGCAGCAGTGATand V collagen regulate fibril diameter. J. Cell Sci. 95:649-657.
TCCTAAAC-3’[al(XI)I,
Brewton, R.G., and Mayne, R. (1994) Heterotypic type 11, IX and XI
5’-TACATGGCTGGGGTGTTGAAfibrils: comparison of vitreous and cartilage forms. In: “Extracellu3’(p actin).
lar Matrix Assembly and Structure,” Yurchenco, P.D., Birk, D.E.,

In Situ Hybridizations
Balb/C mouse embryos a t 14.5, 16.5, and 18.5 d.p.c.
were collected, and fixed in 4% paraformaldehyde in
0.01 M phosphate buffer saline (PBS,pH 7.4) overnightat 4°C. Whole embryos were dehydrated in a graded
series of ethanol and embedded in paraffin. Consecutive sections were cut at 5 bm and were then processed
for in situ hybridization. The hybridization procedures
used in this study were essentially the same as thosedescribed elsewhere (Hayashi et al., 1986; Iyama et al.,1991). In situ probes included mouse ul(X1) (Yoshioka
et al., 1995), al(I1) (Andrikopoulos et al., 1992), andal(1)collagen (Yoshioka,unpublished data); their sizes
are 1,810, 618, and 250 bp, respectively. The cDNAs
were labeled with 35S-thymidine 5’-[a-thiol triphosphates (TTP)(Dupont, Boston, MA) by nick-translation
to a specific activity of 1.8-4.5 x 10’ cpmlbg DNA.
After hybridization, slides were washed under conditions of high stringency as previously described (Hayashi et al., 1986; Iyama et al., 1991). The dried tissue
sections were dipped into Kodak NTB-2 emulsion andexposed for 3-7 days at 4°C.The sections were counterstained with hematoxylin. Differential expression ofeach collagen mRNA in comparable tissue sections provided a good internal control. As a negative control,
parallel sections were also digested before hybridization with 2 mg/ml of RNase for 1hr at room temperature.
ACKNOWLEDGMENTS
We thank Ms. M. Kohmoto and M. Sozomenu for typing the manuscript. This work was supported by aGrant-in-Aid for Scientific Research (03670178 to
H.Y., and 06670233 to K.I.) from the Japanese Ministry of Education, Science and Culture, and by the National Institutes of Health (AR-38648 to F.R.). This i sarticle 181 from the Brookdale Center for Molecular
Biology.
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