Cell, Vol. 80, 423-430, February10, 1995,Copyright© 1995by Cell Press

A Fibrillar Collagen Gene, Collla 1,
Is Essential for Skeletal Morphogenesis
Y. Li, 1 D. A. Lacerda, 1 M. L. Warman, ~ D. R. Beier, 2
H. Yoshioka,3 Y. Ninomiya,3 J. T. Oxford, 4 N. P. Morris, 4
K. Andrikopoulos, s F. Ramirez, s B. B. Wardell, 6
G. D. Lifferth, e C. Teuscher, 6 S. R. Woodward, 6
B. A. Taylor, ~ R. E. Seegmiller, 6 and B. R. Olsen ~1Department of Cell Biology
Harvard Medical School
Boston, Massachusetts 02115
2Division of Genetics
Brigham and Women's Hospital
Boston, Massachusetts 02115
3Department of Molecular Biology and Biochemistry
Okayama University Medical School
Okayama 700
Japan
4Shriners' Hospital for Crippled Children
Research Department
Portland, Oregon 97201
SBrookdale Center of Molecular Biology
Mount Sinai Medical Center
New York, New York 10029-6504
6Departments of Microbiology and Zoology
Brigham Young University
Provo, Utah 84602~The Jackson Laboratory
Bar Harbor, Maine 04609

Summary
Mice that are homozygous for the autosomal recessivechondrodysplasia (cho) mutation die at birth with abnormalities in cartilage of limbs, ribs, mandible, andtrachea. Limb bones of newborn cho/cho mice arewider at the metaphyses than normal bones and only
about half the normal length. By linkage analysis, thecho gene and the gene encoding the ~I(XI) chain of
cartilage collagen XI were mapped to the same regionof chromosome 3. Deletion of a cyUdine residue about
570 nt downstream of the translation initiation codon
in cho el(XI) mRNA causes a reading frame shift andintroduces a premature stop codon. The data demonstrate that collagen XI is essential for normal formationof cartilage collagen fibrils and the cohesive propertiesof cartilage. The results also suggest that the normal
differentiation and spatial organization of growth platechondrocytes is critially dependent on the presenceof type XI collagen in cartilage extracellular matrix.
Introduction

Autosomal recessive chondrodysplasia (cho) in mice ischaracterized by skeletal defects caused by abnormalities
in the cartilage of limbs, ribs, mandible, and trachea (Seegmiller et al., 1971). Homozygous (cholcho) mice are bornwith shortened snouts, cleft palate, protruding tongue, andshort limbs. The airway is reduced, and tracheal cartilage

rings are soft (Seegmiller et al., 1972). The thoracic cageis small, causing lung hypoplasia (Seegmiller et al., 1986;
Hepworth et al., 1990), and the spine is shortened (Figure1A). The homozygotes die at birth, probably as a resultof tracheal collapse, lung hypoplasia, or both. Previous
studies of cholcho cartilage have shown that collagen fibrils in the matrix are unusually thick and that proliferating
epiphyseal chondrocytes do not show the normal columnar arrangement (Seegmiller et al., 1971; Monson andSeegmiller, 1981; Figure 1B). It has also been found that
proteoglycans are more easily extracted from cholcho
than from normal cartilage and that mutant cartilage lacks
cohesiveness (Stephens and Seegmiller, 1976; Seegmiller et al., 1981).
Identification of the cho mutation should lead to an understanding of the molecular basis for the cohesive properties of normal cartilage. Also, since the features of cho
resemble those of certain human chondrodysplasias,
identification of the cho mutation is likely to provide insights into the molecular basis for human disorders. In thepresent paper, we report the mapping of the cho locus to
the Amyl-Amy2 region of mouse chromosome 3. Thisregion corresponds to the p21 region of human chromosome 1 (Seldin et al., 1993). The human gene encoding~I(XI) collagen, a component of cartilage collagen fibrils,
had previously been localized by in situ hybridization tothis region (Henry et al., 1988), making al(Xl) collagena candidate for the cho mutation. We demonstrate that
Co111al indeed is linked to the cho locus of mouse chromosome 3. We also show that ~I(Xl) collagen chains areundetectable in cholcho cartilage and that the cho mutation is deletion of a cytidyl residue, causing a prematuretranslational stop about 570 nt downstream of the initiation
codon of ~I(Xl) collagen mRNA. Finally, analysis of (~1(XI)
collagen transcripts in chol+ heterozygous cartilage revealed lower levels of the cho allele than the wild-typeallele transcripts. We conclude, therefore, that the cho
mutation affects the synthesis, stability, or both of ~I(Xl)collagen mRNA and suggest that cholcho mice are functionally equivalent to homozygous knockouts for Coll la 1.
Results
Mapping of the cho Locus to Mouse
Chromosome 3
To map the cho locus, C57BL/6FrSe (chol+) mice werecrossed with the MEV retroviral linkage testing strain (Taylor and Rowe, 1989), F1 animals were intercrossed, and
51 F2 (cholcho) homozygotes were collected. An additional 54 first generation backcross homozygotes wereobtained by backcrossing (C57BL/6FrSe x MEV) chol+heterozygotes with the chol+ parents. Analysis of Southern blots of DNA from the 105 cholcho mice, the DNA ofwhich had been hybridized with a probe for ecotropic
MuLV proviruses, showed absence of the 9.4 kb Emv27derived Pvull band in all samples except one, thus placing
the cho locus about 0.6 cM from the Emv27 provirus on

Cell
424

( C 5 7 B L / 6 F r S e - c h o / + x MEV) x ( C 5 7 B L / 6 F r S e - c h o / + x MEV)

A

Locus

cho

LOD

D3M[t54

0.1

D3M]t6

5.2

D3MJt26

16.0

Tshb

27.7

Ernv2727.5

D3Mit17

Genes

Locations of human homologues

Hsd3b
Gja5
Od2, Atplal, Ampdl
Cd53
Ngfb, Nras, Tshh, Rapla
Kcna2, Kcna3
Gnat2, Gnat3
Kcnal-rs3
Csfm
Amy1, Amy2, Co]1l a l
Cf3

lp11-p13
tp13, lp13, lp13
lp13, lp13, lp13, lp12-p13-, lp13
lp13-21
lp21, lp21, 1p21
lp22-p21

2.9

Figure 2. Partial LinkageMap of MouseChromosome3
The maximumlikelihoodpositionestimatesfor the locationof the cho
locus on chromosome3 are given based on analysisof the F2 intercross population.

chromosome 3 (95% confidence interval: 0.02-3.5 cM).
To confirm this location of the cho gene, we also screened
the F2 and first generation backcross DNAs using five
microsatellite markers that distinguished between C57BL/
6FrSe and MEV mice. These analyses placed the cho
locus in the interval between Tshb and Emv27on chromosome 3 (Figure 2).

B

Mapping of Co111al to Mouse Chromosome 3
Linkage mapping of Coll l a l was accomplished using asequence length polymorphism between different inbred
strains in the 3' untranslated region of ~l(Xl) collagenmRNA. Flanking primers were used to amplify this polymorphic region with a panel of DNAs from the BXD recombinant inbred series as template for polymerase chain reaction (PCR). Strains BXD-5, BXD-6, BXD-9, BXD-14,
BXD-16, BXD-18, BXD-19, BXD-20, BXD-23, BXD-24,
BXD-27, BXD-29, BXD-31, and BXD-32 exhibited thelarger fragment of C57BL/6J, while strains BXD-2, BXD-8,
BXD-11, BXD-15, BXD-21, BXD-22, BXD-25, BXD-28, and
BXD-30 exhibited the smaller DBA/2J fragment. The strain
distribution pattern was concordant in all 23 strains with
the Amy1 locus on chromosome 3, suggesting a genetic
distance between Co111al and Amy1 of less than 3.7 cM(95% upper confidence limit).

Figure 1. Comparisonof Normaland Mutant SkeletalTissues(A) Comparisonof normal(left)and cho/cho(right)specimensreveals
the shortenedspine and markedlyshortenedbones with flared metaphyses of the append!cularand thoracic skeleton. Calcifiedbone
and cartilage are differentiallystained with Alizarin red and Alcian
blue, respectively.
(B) Sectionsof tibial epiphysesfrom 19-day-oldcho/cho mutant(right)
and wild-type(left) embryosstained with toluidine blue. The growthplate region,while showingan orderedarrangementof chondrocytes
in differentstagesof differentiationin thewild-typetissue,is completely
disorganizedin cho/cho mutanttissue.

Abnormal Expression of ~I(XI) Collagenin cholcho Mice
The expression of the cho abnormality in cartilage andcolocalization of the cho gene with Coll l a I made Coll l a 1
a candidate for the cho mutation. To test this possibility,
we used polyclonal antibodies against a peptide fromwithin the carboxyl telopeptide of al(Xl) collagen chains(Figure 3A) to examine extracts of normal and cho/cho
cartilage by Western blotting. The antibodies were madeagainst rat ~l(Xl) sequences but cross-react with mouse~I(XI) chains. As shown in Figure 3B, extracts of normal
costal cartilage contained a positive band with the mobility
expected for ~ l(Xl) collagen chains, while extracts of cho/Colllal Is Essential for Skeletal Morphogenesis
425

A
C O L L A G E N C O M P O N E N T S OF C A R T I L A G E F I B R I L S

Interaction with proteoglycans?

(K)LSPKKTRRHTESlQA~~~](Xl)TYPE Xl COLLAGEN~TYPE IX C'OLLA,3EN J "w..~
TYPE II COLLAGEN

Bn

200 - -

c

n

c

: ~

116-97-66 - !! i!!! i200

Figure 3. Diagram of Collagen Xl Molecules in Cartilage Fibrils andDetection of ~I(Xl)Chains in Extracts(A) Diagram of a cartilage collagen fibril, composed of the fibrillar
collagens II and X l and the FACIT component type IX (Shaw and Olsen,1990). Type IX molecules are localized on the surface of the fibrils,
while type Xl collagen molecules may be partially embedded within
the fibrillar structure. The peptide sequence (K)LSPKKTRRHTESIQA
from within the carboxyl region of ~I(Xl) collagen chains, used togenerate polyclonal antibodies, is shown above the diagram.
(B) Western blot showing the reactivity of cartilage extracts with polyclonal anti-~l(XI) antibodies (left panel) and monoclonal antibodies
against al(ll) collagen (right panel). Before incubation with the primary
antibodies, the filters were stained for protein with Ponceau red. As
shown in the insets below the Western blots, the amount of protein
loaded in each lane was similar since a band with the mobility of myosin
heavy chain appeared with about the same intensity in all lanes. The
positions of molecular mass markers (in kilodaltons) are indicated to
the left of the figure, n, extract of wild-type C57BLJ6FrSe rib plates;c, extract of cholcho rib plates.

cho cartilage did not. In contrast, when immunoblots were
stained with an antibody specific for the major collagenof cartilage, (zl(ll), both normal and cholcho extracts
contained positive bands of equal i~tensity. This observation suggests that (zl(Xl) collagen i~ absent from cholcho
cartilage. The conclusion was supported by immunohistochemical analyses with the (~I(Xl / collagen antibodies.
The antibodies stained normal tracheal, costal, and epiphyseal cartilage intensely, but did not stain cholcho cartilage (Figure 4 shows the results foriepiphyseal cartilage).
Both types of cartilage showed positive staining, however,Lwith antibodies against type II collagen.

Figure 4. Immunohistochemistry of Normal and Mutant Cartilage
Sections of knee joints from cholcho mutants (right) and normal littermates (left) stained with antibodies against al(XI) collagen (a andb) and al(ll) collagen (c and d). For sections shown in (e) and (f), only
secondary antibody was used.

Identification of the cho Mutation
To identify the cho mutation, we used reverse transcription(RT)-PCR with mRNA isolated from homozygous cholcho
embryos to generate a set of cDNAs spanning about 5700
nt of ~I(XI) collagen mRNA (Figure 5A). These cDNAs,
labeled with [32P]dCTP, were digested with restriction endonucleases into smaller fragments and were comparedwith similar fragments obtained from wild-type ~1 (Xl) collagen cDNAs by single strand conformational polymorphism(SSCP) analysis. Only one cDNA (YLIO-2) showed a difference between wild-type and cho (Figure 5B); a difference
was observed even when the fragments of YLIO-2 wereanalyzed on a polyacrylamide sequencing gel under denaturing conditions (Figure 5C). Direct nucleotide sequencing of YLIO-2 demonstrated that this difference was due
to a single nucleotide deletion in the cho RT-PCR product(Figure 6). To confirm the presence of this deletion in genomic DNA from cho mice, we also performed PCR analysis
of genomic DNA, using oligonucleotide primers that flankthe deletion. As shown in Figure 7, this analysis showed
amplification of the expected 96 nt and 95 nt bands withwild-type and cholcho DNA, respectively, and a double
band with heterozygous chol+ DNA.
Analysis of cho mRNA Levels
To examine the relative levels of cho and wild-type
mRNAs, we used RT-PCR to amplify a portion of the 3'

Cell
426

B
YLIO-2A single nucleotide
deletioncausesprematureterminationof cd(X[) collagenin choA
c D N A of a lchain of mouse type

CIaI
EcoR1
HincIIC]aI
E~RI
Hiadll[
MspI
StyI

i!

12

Xl c o l l a g e n

1 176

5587
TAA

ATG

YL3-4
-AccI
Apal
Aval

wt

TGT ~ G ~ G ~ ATC ACA ~ ~ ~ C ~ T AGA AGT GAG A ~ ~ G ATA G ~ GATC K K K I
T K P
L D R S E R
S I V Dcho

TGT AAG AAG ~_2C

YLI0-21031
IS0 S4~bp

K

K

K

I

T

K

PIS

I

E

V

I~

D

R

*1"

ATC ACA AAA CCC TCG ATA GAA GTG AGA C-C-~TCGA TAG ~ G AT=

YL3-4 1735

93S sozbp

1711

iL12=13
1995b"~'~

3706¥L9G7 4534

C

3637 ~697b

4446

iLl
14SIbpIF=(i!li!iiii

SgZ7

Figure 5. ElectrophoreticAnalysis of al(Xl) Collagen cDNA Fragments(A) Diagramshowingthe 5927 nt long mouseCLI(XI)collagenmRNA(Yoshiokaet al., submitted)(top) and five DNA fragmentsgenerated
by RT-PCR. The lengthof each PCR product(in basepairs)obtainedwith mRNAfrom wild-typemiceis indicatedbeloweachfragment.For
each fragment,the first and last nucleotides(as countedfrom the 5'
end of the mRNAsequence)are indicatedat the beginningand theend of eachfragment.Withinthe mRNAsequence,the initiationcodon
starts with nucleotide176;the TAA stop codonstarts with nucleotide
5587.
(B) The resultsof SSCPanalysisof fragmentsYLIO-2 and YL3-4 (see[A])obtainedwith wild-typemRNA(lanes1)and cholcho mRNA(lanes
2). For the analysis,the 32P-labeledcDNAsweredigestedwith restriction endonucleases,as indicatedabovethe lanes.All cDNAs shownin (A) (except YL1; this was completelysequenced)were analyzed,
but only YLIO-2 showeddifferencesbetweenthe wild-typeand cholcho products. Therefore,only the results with YL3-4 are shown as acontrol. As indicated by the arrows fragments of YL10-2 generated
by two different sets of restriction endonucleasesshowed different
mobilitiesbetweenwild-typeand cholcho lanes.
(C) When fragmentsobtainedfrom YLIO-2 were analyzedon a 6%
polyacrylamidesequencinggel, a differencein mobilitybetweenwildtype and cholcho DNA was observed(arrows), indicatinga singlenucleotidedeletionin the cholcho product. In contrast,fragmentsderived
from YL3-4 showed no difference.

untranslated region of al(Xl) mRNA. Since this region
gave rise to products of different size with RNA from DBA/
2J and C57BL/6FrSe mice, RNA was extracted from animals derived from a cross between DBA/2J (+/+) and
C57BL/6FrSe (chol+) mice and used as tem plate for PCR.
As shown in Figure 8, RT-PCR of (C57BL/6FrSe x DBA/
2J) c h o l + RNA produced mostly the wild-type product,
while RT-PCR of (C57BL/6FrSe x DBA/2J) +/+ RNA produced bands of equal intensity. In contrast, when genomic
DNA was used as template for PCR, no difference wasobserved between products obtained with DNA from
(C57BL/6FrSe x DBA/2J) chol+ and +/+ animals.

Discussion
The cho Phenotype Is Caused by an Absenceof al(XI) Collagen
Based on the colocalization of the cho gene with (?,o/11al,the absence of immunodetectable al(XI) collagen in cholcho cartilage, and the presence of a single nucleotide deletion in the coding region of cho al(XI) collagen mRNA,
we conclude that the cho phenotype is caused by the absence of al(XI) collagen in extracellular matrices. The mu-

Figure 6. Single NucleotideDeletionin al(Xl) CollagenCodingSequence
Partial nucleotidesequenceof YLIO-2 (see Figure 5A) from wild-type(lanes 1) and cholcho (lanes2) mice, showingthe deletionof a single
C residuein the choloho sequence(arrow). This causes prematureterminationof ~I(Xl) collagenin cho.
tation would allow the synthesis of a truncated a l (Xl) collagen chain, containing 195 amino acid residues (including
the signal peptide), but the low level of cho mRNA as compared with wild-type m RNA suggests that such a truncatedpolypeptide would be present at very low levels. We conclude, therefore, that cho represents a loss-of-function
phenotype and is not caused by the accumulation of anabnormal protein. The reason for the low level of cho al(Xl)transcripts is not clear, but frameshift mutations in manyother proteins frequently lead to a decreased synthesisor reduced stability of the mRNA. For example, fibrillin
mutations in Marfan patients leading to a premature stopcodon have been shown to reduce the expression levelof the mutant allele (Dietz et al., 1993).

The Role of Type Xl Collagen in Cartilage
Type XI collagen molecules are heterotrimers composedof al(XI), (~2(XI), and a3(XI) chains (Morris and Bachinger,
1987). Their genes, including Coil l a l , belong to the fibrillar class of collagen genes (Jacenko et al., 1991) and encode precursor chains, procollagens, that are processed
to mature collagen XI chains after secretion from cells.
Type XI molecules copolymerize with molecules of types
II and IX collagen to form the collagen fibrils of cartilage(Figure 3A) (Mendler et al., 1989). In the vitreous of the
mammalian eye in which type II collagen is also the major
fibrillar collagen, a unique hybrid form of collagen XI isfound, consisting of al(XI), a2(V), and a3(Xl) chains (Mayne
et al., 1993).
It has been hypothesized that collagen XI plays a role
in regulating the diameter of collagen fibrils, similar to themanner in which type V collagen is thought to regulate
the diameter of type I-containing fibrils (Eyre and Wu,
1987; Linsenmayer et al., 1993). The molecular basis forsuch a role is thought to be the incomplete processingof the amino propeptide domains of these two types ofprocollagens (Linsenmayer et al., 1993; Sheren et al.,
1986; Broek et al., 1985). The presence of amino-terminal
extensions on the processed molecules would sterically
restrict the addition of type II collagen molecules to thesurface of fibrils in which type XI molecules are incorporated. Thus, the diameter of cartilage collagen fibrils would
be determined by the ratio between type Xl and type II
molecules in the fibrils. The hypothesis predicts that anincreased ratio leads to thinner fibrils, while a decl'eased
ratio results in thicker fibrils. The data reported here on theCo111al Is Essential for Skeletal Morphogenesis

427

Co111a1-18

1234

mR.

5678

CAATGA]3-G13GATTGTAAG~AG-12nt(wt) COOCTC............................

.......................
(Cho)CCCTC 33nt- CCTTAGTACCATAAACCTTG
C57BL/6FrSe
DBA/2J

Co111a1-15

123

123

RT-PCRof RNA

PCRof DNA

Figure 7. Mutation Detection in Genomic DNA

Figure 8. Reduced Levels of cho (~I(XI) Transcripts

Gel electrophoresis of PCR products generated with primers C o l l l a 118 and Co111a1-15 and genomic DNA from wild-type mice (lanes 1),
chol+ heterozygotes (lanes 2), and cholcho homozygotes (lanes 3).
For clarity, we show results obtained with two different amounts ofsample loaded on the gel. The wild-type product is 96 nt in length;
the cholcho product is 95 nt.

Gel electrophoresis of PCR products with primers flanking a portionof the 3' untranslated region of the a I(Xl) collagen gene. As describedin the text, this PCR product is larger with template cDNA and genomic
DNA from C57BL/6FrSe than from DBA/2J mice (lanes 1, 2, 5, and
6). As expected, with DNA from C57BL/6FrSe × DBN2J mice, bothproducts were generated (lane 7). RT-PCR also generated two bands
with RNA from (C57BL/6FrSe x DBN2J) +/+ mice (lane 3). With RNA
from (C57BL/6FrSe x DBN2J) chol+ mice (lane 4) mostly the smaller
product was generated, while the use of DNA from (C57BL/6FrSe x
DBA/2J) cho/+ mice (lane 8) as tern plate produced two bands of almost
equal intensity.

absence of ~1 (Xl) collagen in cholcho cartilage, combinedwith the previous electron microscope demonstration ofabnormally thick collagen fibrils in mutant cartilage (Seegmiller et al., 1971), provide strong and direct support forthis hypothesis. Consistent with the hypothesis is also thefinding of thicker than normal fibrils in the cartilage oftransgenic mice overexpressing type II collagen (Garofalo
et al., 1993).
Smith et al. (1985) reported that type Xl collagen binds
with high affinity to proteoglycans and speculated thatsuch binding may be important in vivo for anchoring cartilage proteoglycans to the collagen fibrillar network. The
unusual extractabilityof proteoglycans from cholcho cartilage (Stephens and Seegmiller, 1976) clearly indicatesthat type XI collagen is essential for the interaction between the large protoglycan aggregates and the collagen,but this interaction may not involve a direct molecular association of proteoglycan components and collagen XI.
An alternative explanation for the increased extractability
and the lack of cohesiveness is that the presence of fewer,albeit thicker, fibrils in cholcho cartilage provides less ofa physical entrapment of large proteoglycan aggregates.
Potential Effect of Co111al Haploinsufficiency
Heterozygous carriers for the cho mutation do not manifest
an obvious skeletal phenotype. This is somewhat surprising, since haploinsufficiency for structural matrix components, such as collagens I, II, and X, have been associatedwith clinical phenotypes in humans. Several explanations
for the lack of obvious abnormalities in heterozygous
cho/+ mice can be postulated. First, the phenotypic effects
in heterozygotes may be quite subtle. For example, highfrequency hearing loss or myopia may not be detected in
the absence of specialized testing. Second, humans andmice differ substantially with respect to growth, development, and lifestyle. It is reasonable to assume, therefore,that stresses upon bones and joints effecting cartilage
might differ between the two species. Consequently, provocative testing may be required to elicit a phenotype inmice. Third, the regulatory pathways responsible for control of collagen Xl synthesis may be able to compensate for
haploinsufficiency states. Collagen XI is a quantitatively
minor component of collagen fibrils, accounting for lessthan 10% of total cartilage collagen content. Therefore,
up-regulation of the wild-type allele could negate the effectof haploinsufficiency.
Murine Chondrodysplasia as a Modelfor Human Chondrodysplasias
The cho mice provide an animal model for human disorders due to mutations in type Xl collagen chains. Several
human chondrodysplasias share inheritance patterns andphenotypic similarities, such as abnormally wide collagen
fibrils, with cho (Spranger and Maroteaux, 1990; Burton
et al., 1986). The collagen XI genes can be consideredcandidates for these conditions. Also of interest is the identification of abnormal collagen fibrils in several types ofEhlers-Danlos syndrome, potentially implicating collagen
V, which is closely related to collagen XI, in the pathogenesis of these disorders (Hausser and Anton-Lamprecht,
1994).
As has previously been noted for other structural matrix
components, a spectrum of phenotypes may occur depending upon the effect of the gene mutation on the protein
product. For example, haploinsufficiency of collagen II
causes a form of Stickler syndrome, whereas dominant negative mutations cause a spectrum of phenotypes ranging
from mild chondrodysplasia with arthritis to lethal achondrogenesis (Kiviri kko, 1993). We anticipate a similar spectrumof phenotypes for collagen Xl mutations. This conclusion
is supported by the results reported in the accompanying
paper (Vikkula et al., 1995 [this issue of Cell]). In a familywith autosomal dominant Stickler syndrome linked to the
COL11A2 gene (Brunner et al., 1994), we demonstrate asplice junction mutation causing an in-frame deletion of
18 amino acid residues in ~2(Xl) collagen. Members of
this family do not show the ophthalmological abnormalities
usually associated with chondrodysplasia in the Stickler
syndrome, consistent with the absence of the ~2(XI) chain
in the mammalian vitreous form of type Xl collagen (Mayne
et al., 1993). In the accompanying paper (Vikkula et al.,1995), we further demonstrate linkage to COL11A2 in a

Cell
428

k i n d r e d with a n a u t o s o m a l r e c e s s i v e b o n e d y s p l a s i a , ass o c i a t e d with s e n s o r i n e u r a l d e a f n e s s a n d a Stickler-likefacial m o r p h o l o g y .

Experimental Procedures
Linkage Analysis to Map the cho and Co111al Genes
C57BLJ6FrSe mice heterozygous for the cho gene (cho/+) werecrossed with a linkage testing stock, MEV, containing multiple ecotropic proviruses (Taylor and Rowe, 1989). F1 animals were intercrossed, and 51 dwarfed, nonviable F2 homozygotes (cho/cho) werecollected. An additional 54 homozygotes were obtained from a backcross of C57BL/6FrSe/MEV (cho/+) heterozygotes with cho/+ mice ofthe parental stock. Genomic DNA was isolated from livers or bodytissue according to Bell et al. (1981). For Southern blotting, DNA digested with Pvull was electrophoresed through 0.7% Seakem ME
agarose (FMC) gels and blotted onto Biotrace HP membranes (Gelman
Biosciences). Hybridization was with a 32P-labeled MuLV probe (pEcB4) at 42°C.
For linkage analysis using simple sequence length polymorphisms,
primers for PCR were synthesized based on published data (Dietrich
et al., 1992) and from sequences available in the Whitehead Institute/
Massachusetts Institute of Technology Genome Center Genetic Database. The PCR products were electrophoresed through 4% agarose
gels. The maximum likelihood genetic map and positional estimate
for the cho locus were generated using the MAPMAKER program(Lander et al., 1987).
For linkage mapping of Co111al, we utilized a sequence length
polymorphism between different inbred strains in the 3' untranslated
region of ctl(XI) collagen mRNA. A panel of DNA from the BXD (C57BL/
6J x DBA/2J) recombinant inbred series was used as template for
PCR with primers flanking this polymorphic region, as described(Beier, 1993).

Preparation of Antibody Specific for ¢¢1(Xl) CollagenThe synthetic peptide, KLSPKKTRRHTESIQA, corresponding to the
distal 15 amino acids of the rat c¢1(XI) carboxyl telopeptide domain(Bernard et al., 1988) was provided by J. Gambee of the Shriners'
Hospital analytical core facility. The proximal six amino acids of thisdomain, IQPLPI, were not included because they are shared by ~2(Xl)(Kimura et al., 1989) and ~I(V) (Greenspan et al., 1991). The aminoterminal lysine is noncoded and was used to enhance coupling to
keyhole limpet hemocyanin carrier. Details of the methods of antibody
production and characterization are presented elsewhere (Oxford etal., 1994).

Western Blotting and Immunohistochemistry
For Western blot analysis, rib plates of normal C57BL/6FrSe and cho/cho newborn mice were pulverized in liquid nitrogen. About 50 mg oftissue powder were transferred to 400 ~1 of hot sample buffer (2%SDS, 10% glycerol, 12.5 p_M Tris-HCI [pH 6.8], 0.1% bromophenol
blue) and immersed in boiling water for 5 rain. After spinning in a
microcentrifuge for 1 rain, the supernatant was used for gel electrophoreels.
Samples were reduced by addition of 5% 2-merceptoethanol andelectrophoresed through 6% polyacrylamide gels. For Western blots,
the nitrocellulose strips were blocked for 2 hr in 3% nonfat dry milk
in PBS and incubated overnight at 4°C with polyclonal al(XI) collagenantibodies or with monoclonal antibodies (2B1) against el(ll) collagen.
After washing in PBS, the blots were treated with secondary antibodies
conjugated to alkaline phosphatase (Promega; anti-rabbit IgG or antimouse IgG). The membranes were washed in PBS and developedwith the Bio-Rad alkaline phosphatase reagent kit.
From a C57BL/6FrSe cho/+ female in day 19 of pregnancy, cho/cho mutants and normal littermates were derived and processed for
microscopy. Eviscerated embryos were fixed for 48 hr in 100% ethanol
containing 3.2% (w/v) polyvinylpyrrolidone (MW 40,000) and 20o/0 dimethyl sulfoxide, washed in 100% ethanol, cleared in Histosol, andinfiltrated and embedded in paraffin wax (Paraplast, mp 56-57°C).
For immunohistochemistry, slides were deparaffinized and quenched
for endogenous peroxidase activity by soaking in 1% hydrogen peroxide in 100% methanol for 30 rain. After rinsing in PBS, sections weretreated with hyaluronidase (1,000 U/ml of Sigma type i-S from bovine
testis) in PBS and incubated in PBS containing 10% fetal bovine serumfor 2 hr at room temperature to reduce nonspecific background staining. Incubation with primary antibody (polyclonal anti-al(XI) collagen,diluted 1:50, or monoclonal anti-~l(ll) collagen antibodies (2B1), diluted 1:1000) was at 4°C overnight. After washing in PBS for 30 rain,
the sections were incubated with secondary antibodies (Sigma antirabbit or anti-mouse IgG conjugated with peroxidase) at room temperature for 2 hr. Following incubation with the secondary antibody, sections were washed in PBS and peroxidase activity was visualized byreacting with a diaminobenzidine solution containing hydrogen peroxide (kit from Sigma). Slides were washed, counterstained with Harris
Modified Hematoxylin, dehydrated, cleared, and mounted with Permount.
Controls included sections that had the primary antibody omitted.

Identification of the cho Mutation
Total RNA was isolated from whole newborn C57BIJ6FrSe +/+ andcho/cho mice, using the USB Rex Total RNA Extraction Kit. mRNA
was purified using the Promega PolyATract Kit.
The mRNAs served as templates for first-strand cDNA synthesis
using either oligo(dT)s or random primers and the SuperScript kit from
Bethesda Research Laboratories. Following removal of the mRNA
template with RNase H, the cDNAs were used for PCR amplificationof al(XI) collagen sequences, using the mouse el(XI) collagen cDNA
sequence as a basis for designing specific primers (Yoshioka et al.,submitted), Several fragments were amplified. Their designation andassociated sense and antisense primers are as follows: YL10-2(5'-GAGGTCCTTCAGCACCAGC-3' and 5'-GCATCTCTGTTACACTCTCAG-3~; YL3-4 (5'-GCCTCACA'I-rGATGAGTATGC-3' and 5'-CTGCTGAAGAATCGCCTGAGC-3~; YL12-13 (5'-CAAGCTCAGGCGATTC'I-FCAG-3' and 5'-AAACAI-rCCCTGCTGTCCTCG-3'); YLgG7 (5'-CCTGI-IGGTGCTCCTGGAATC-3' an d 5'-GAGGTCCI-ICAGCACCAGC-3');
YL1 (5'-AAAGGTGATGAGGGTGC-3' and 5'-ACTATATCTI-FGATGGACGGG-3'). Amplification reactions were in 25 p_lof 1 x PCR buffer containing 1 I~1of first strand cDNA, 1 pM of each primer, 0.2 mM of eachdNTP, 0.5 p_l of [32P]a-dCTP (3000 Ci/mmole), and 1 U of Amplitaq
Polymerase (Perkin Elmer Cetus). Amplification conditions were 95°C
for 5 min, followed by 30 cycles at 94°C for 1 rain, 56°C for 90 s,
72°C for 2 rain, and a final 10 rain extension at 72°C.
Before analysis of YLIO-2, YL3-4, and YL12-13 by SSCP, the threefragments were digested with several restriction endonucleases(YL10-2 with Clal, EcoRI, Hincll, Hindlll, Mspl, and Styl; YL3-4 with
Accl, Apal, and Aval; YL12-13 with Bgll and Sau3AI). The digested
PCR products were loaded on MDE gels (AT Biochem) and 6°/0 denaturing polyacrylamide sequencing gels. The cDNAs YLgG7 and YL1
were analyzed by direct nucleotide sequencing.
The fragment YLIO-2was subcloned into the TA cloning vector from
Invitrogen and sequenced by dideoxy sequencing. Genomic DNA fromwild-type (cho/+) and mutant (cholcho) animals was used as templatefor PCR. The 5' sense primer was 5'-CAATGATTGTTGATTGTAAGAAG-3' (Co111a1-18) and the 3' antisense primer was 5'-GTTCCAAATACCATGATTCC-3'(Coll lai-15). Each reaction contained 10~1 of 1 x PCR reaction buffer, 50 ng genomic DNA, 1 I~M of eachprimer, 0.2 mM of each dNTP, 0.2 id of [32P]~-dCTP (3000 Ci/mmole),
and 0.5 U of Amplitaq Polymerase (Perkin Elmer Cetus). Amplification
conditions were 30 cycles at 94°C for 90 s, 55°C for I rain, 72°C for
40 s, and a final 10 rain extension at 72°C. The PCR products wereanalyzed by electrophoresis through a 6% denaturing polyacrylamide
sequencing gel.
Analysis of cho and Wild-Type mRNA
Total RNA was isolated from whole newborn C57BL/6FrSe +/+ andcholoho mice and from costal cartilage of adult (C57BL/6FrSe x DBA/
2J) cho/+ heterozygotes using the USB Rex Total RNA Extraction Kit.
Total RNA, treated with RNase-free DNase I, served as templatefor first-strand cDNA synthesis using oligo(dT) priming and the SuperScript kit from Bethesda Research Laboratories. Following removalof the RNA template by digestion with RNase H, the cDNA was used
for PCR amplification of the 3' untranslated region of mouse al(XI)
collagen mRNA. The 5' sense primer (ST3) was 5'-ACCAACCATTTACGCACATGC-3' and the 3' antisense primer (RU2) had the sequence
5'-ACTATATCTTTGATGGACTGGG-3'. Amplification reactions wereCo111al Is Essential for Skeletal Morphogenesis429

in 50 i~1 of 1 x PCR buffer containing 1 I~M of each primer, 0.2 mMof each dNTP, 1 ~1of [s2P]a-dCTP (3000 Ci/mmole), and 2 U of Amplitaq
Polymerase (Perkin Elmer Cetus). Amplification conditions were 95°C
for 5 rain, followed by 30 cycles of 94°C for 1 min, 55°C for 1 rain,
and 72°C for 90 s, and a final 10 min extension at 72 °C. PCR products(2 ~1) were heat denatured at 95°C and electrophoresed through 6%
acrylamide sequencing gels.

Acknowledgments
This study was supported by grants from the National Institutes of
Health AR36819 and AR36820 (B. R. O.), GM38862 (N. P. M.),
HD21926 and HD27275 (C. T.), CA33093 (B. A. T.), AR38648 (F. R.),a grant from Shriners' Hospital, and funds from the Professional Development Fund of Brigham Young University (R. E. S.). M. Jakoulov
provided expert secretarial assistance. The monoclonal antibody
against al(ll) collagen (2B1) was generously provided by R. Mayne.
Received September 20, 1994; revised December 6, 1994.

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