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Skeletal Development in Transgenic Mice Expressing
a Mutation at Gly574Ser of Type II Collagen
1Research Department, Shriners Hospital for Children, Portland, Oregon 97201
2Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon 97201
3Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
Skeletal development of transgenic mice with a type II collagen mutation was
analyzed and compared with wild-type littermates. The single base substitution in Col2a1
resulted in a glycine to serine mutation within
the helical domain and corresponded to one previously identified in a patient with the lethal
human chondrodysplasia, hypochondrogenesis
(Horton et al. [1992] Proc. Natl. Acad. Sci. U.S.A.
89:4583–4587). Skeletal staining of embryos from
14.5 through 18.5 days of gestation demonstrated
a dwarf phenotype in the transgenic embryos,
most notably short limb bones and vertebral
column that was first detected at 15.5 days postcoitus. In addition to the reduced length, the
extent of ossification was less in the transgenic
mice. The architecture of the long bone growth
plate was abnormal in the transgenic tissue, in
particular there was no discernible proliferative
zone. There were few stacks of characteristically
flattened cells and the overall length of the growth
plate in the mutant embryos was reduced. At the
ultrastructural level, there were fewer collagen
fibrils present in the transgenic mouse cartilage
compared to that of wild-type littermates. Ultrastructural localization of collagen types II, IX and
XI revealed a similar pattern between the transgenic and wild-type pups, suggesting that the
collagen fibrils present in the matrix of littermates with both phenotypes had a similar composition. Skeletal analysis and cartilage histochemistry indicated that effect of the type II collagen
mutation was to reduce the density of the collagen fibrils within the cartilage matrix which was
associated with delayed bone formation and resulted in a short-limbed phenotype. Dev. Dyn. 208:
170–177, 1997. r 1997 Wiley-Liss, Inc.
Key words: chondrodysplasia; cartilage; limb development; collagen
zone at the outermost area of the growth plate at the
epiphysis, the proliferative zone which contains groups
of flattened chondrocytes arranged in columns, and the
hypertrophic region immediately adjacent to the underlying bone (for a review, see Horton, 1993). The hypertrophic zone is degraded and replaced by bone during
the process of endochondral bone formation. Cycles of
chondrocyte proliferation, hypertrophy, and replacement by bone result in skeletal growth that continues
until the growth plate closes after puberty. By poorly
understood mechanisms, this process of bone lengthening is disturbed in a group of dwarfing conditions
known as the chondrodysplasias.
The histological findings commonly associated with
these skeletal disorders include a shortened proliferative zone and sparse extracellular matrix (Horton and
Hecht, 1993). In normal growth plate cartilage, a
network of collagen fibrils is intertwined with large
aggregated proteoglycans. The fibrils are heteropolymers of collagen types II, IX, and XI that are covalently
cross-linked in the molar ratio of 8:1:1, respectively
(Vaughan et al., 1988; Mendler et al., 1989). From
immunolocalization studies type IX collagen appears to
be aligned along the fibril surface (Vaughan et al., 1988)
where it is speculated to interact with other matrix
components through a large globular domain that
projects out into the extracellular space. Type XI collagen also contains a globular domain that is exposed to
the surface of the fibril and is thought to be responsible
for regulation of fibril diameter (Thom and Morris,
1991; Keene et al., 1995).
Genes coding for the cartilage matrix components are
good candidates for mutations in chondrodysplasias.
Indeed, mutations of genes for all three cartilagespecific collagens have been characterized in distinct
but related skeletal conditions (Briggs et al., 1994;
Spranger et al., 1994; Vikkula et al., 1995; Muragaki et
al., 1996). Mutations in COL2A1, the gene for type II
collagen, have been identified in chondrodysplasias of
the spondyloepiphyseal dysplasia class (Spranger et al.,
Linear growth of long bones occurs within the growth
plate, a narrow zone of specialized cartilage that forms
the interface of epiphyseal cartilage and developing
bone. It is organized into three regions from the resting
Contract Grant sponsor: National Institutes of Health; Contract
Grant number: HD20691; Contract Grant sponsor: Shriners Hospital
for Children; Contract Grant number: 15956.
*Correspondence to: W.A. Horton, Research Department, Shriners
Hospital for Children, Portland, OR 97201.
Received 7 May 1996; Accepted 11 September 1996
1994). Heterozygous glycine substitutions have been
reported in three cases of hypochondrogenesis, which
lies at the severe end of the clinical spectrum (Bogaert
et al., 1992; Horton et al., 1992; Freisinger et al., 1994).
This lethal disorder is characterized by short limbs, a
shortened trunk, cleft palate, and reduced ossification
of the vertebral column.
We recently reported a heterozygous mutation in the
gene for type II collagen in an infant with hypochondrogenesis (Horton et al., 1992). The mutation resulted in
an amino acid substitution of serine for glycine at
residue 574 (Gly574Ser), slightly carboxy-terminal to
the middle of the triple helical domain of the collagen
molecule. Towards understanding how it disrupts bone
formation, we introduced the Gly574Ser mutation into
transgenic mice. The murine Col2a1 promoter was used
to produce cartilage-specific expression of this mutation
which was expected to act in a dominant negative
fashion. In this paper, we examine the temporal aspects
of how the phenotype is produced. The data demonstrate that skeletal differences between the wild-type
and transgenic littermates are not apparent before
endochondral bone formation begins around day 15.5 of
embryonic development. In older embryos, histological
and ultrastructural analysis revealed an abnormal
growth plate architecture and a reduced abundance of
fibrils in the cartilage matrix from the transgenic mice
in conjunction with a short-limbed phenotype.
Generation of Transgenic Mice
PCR mutagenesis was used to introduce a point
mutation in the mouse type II collagen gene that
resulted in a Gly574Ser amino acid substitution slightly
carboxy-terminal to the middle of the triple helical
domain. The genomic clone has been previously described and successfully used to generate other type II
collagen transgenic mouse strains (Garofalo et al.,
1991; Metsaranta et al., 1992; Garofalo et al., 1993).
One male founder was generated using conventional
transgenic technology and subsequent breeding produced offspring, 25–50% of which were born dead and
had an abnormal phenotype. These pups presented
with short limbs, short snouts, cleft palate, and distended abdomen when compared to the wild-type littermates.
Skeletal Clearing of Embryos
Embryos were removed at days 13.5 through 18.5
post-coitus (p.c.) by Caeserian section. The identification of wild-type or transgenic embryos was verified by
Southern analysis (data not shown). They were skinned,
fixed, and stained using alcian blue to identify the
cartilaginous areas and alizarin red which stains the
mineralized bone tissue. The skeleton was revealed
following alkali clearing. At least two litters at each
stage were analyzed; one representative wild-type and
transgenic embryo from each litter at the respective
stage of development is shown (Fig. 1). At 13.5 days p.c.,
there was no discernable difference between the transgenic and the wild-type littermates (data not shown).
There was no ossified tissue present at this stage. At
14.5 days there was a small site of ossification above the
shoulder, presumably the clavicle, as indicated by the
alizarin red stain in both embryos (Fig. 1A, 14.5 arrow).
The length of the limbs and overall size appeared equal
at this stage of development.
By day 15.5, transgenic embryos were distinguished
from the wild-type pups based on a difference in the
pattern of ossification of the vertebral column, the rib
cage, and the limbs while overall size appeared similar.
The cranium and mandible, derived from membranous
bone, were similarly stained with alizarin red while the
limbs and vertebral column, derived by the process of
endochondral bone formation, were different between
littermates. Ossification of the rib cage proceeds from
the vertebral column towards the sternum. All of the
ribs in the wild-type embryo had begun to ossify (Fig.
1A, 15.5 arrowheads) and the vertebral bodies of the
thoracic region also showed mineral deposition indicating bone formation. Only five of the thoracic and sacral
ribs in the transgenic embryo had begun to mineralize
and these varied in the extent of alizarin red staining.
There was no indication of vertebral ossification and
the cartilaginous vertebral bodies appeared smaller
and the pedicles less prominent. In the wild-type
embryos, bone formation of the hindlimb had begun
within the midshaft of the femur, the tibia, and fibula to
an approximately equal degree. A similar pattern was
seen in the bones of the forelimb. Interestingly, only the
bones of the middle segments, the radius and ulna in
the forelimb, and likewise the tibia and fibula in the
hindlimb, had begun to ossify in the transgenic mouse.
There was no visible staining of bone in either the
femur or the humerus. Where ossification had occurred
in these mice, the bones appeared somewhat wider
compared to the normal embryos.
At day 16.5, the differences in skeletal development
were more noticeable. The wild-type embryos were
larger due to the longer vertebral column and the tail,
which was quite short in the transgenic embryo. In
regards to the rib cage, the distance between ribs,
reflecting the height of the vertebral body, looked
slightly smaller in the transgenic embryo. In addition,
there was a thickening in the ossified region of several
ribs (Fig. 1A, 16.5 arrow), reminiscent of the thickening
seen in the lower limbs at 15.5 days p.c. The pedicles on
the sacral vertebrae had begun to ossify in the wildtype skeleton, unlike the transgenic skeleton where the
staining of the vertebral bodies and pedicles indicated
only cartilage. The limbs were distinctly shorter as
well: the long bones were shorter and paws were
underdeveloped in both size and ossification. The femur
and humerus had begun the process of endochondral
bone formation by this time point, 16.5 days p.c.
Profiles of the skulls of the embryos showed differences
as well. The mandible, the premaxilla and nasal cavity
were not as prominent in the transgenic mouse, result-
Fig. 1. Skeletal analysis of wild-type and transgenic embryos from 14.5 to 18.5 days of gestation. Embryo skeletons were removed by Caesarean
sections at time-points indicated; the skeletons were stained for cartilage (alcian blue) and bone (alizarin red). A: Lateral view of intact skeletons from
wild-type (WT), upper row, and transgenic (TG) embryos, lower row. At 14.5 days p.c. there is a small ossified site above the shoulder in each embryo
(arrow). At 15.5 days p.c. there is less mineralization of the ribs in the TG than WT embryo (arrowheads) and only the tibia and fibula of the hind limb had
ossified in the TG (arrows). At 16.5 days p.c. the skulls of the WT have a larger developed occipitus and otic capsule than the TG (arrowheads); the
bone-portion of the TG ribs are irregularly thickened (arrow). At 17.5 days p.c. the delay in ossification of the vertebral column of the TG embryo is
evident (arrowhead) and persists through day 18.5 p.c. B: The vertebral columns were removed and aligned with the WT above its TG littermate in
increasing order of gestational age, from 16.5 to 18.5 days p.c. The proportional difference in the length of the ossified region of the vertebral columns
between the WT and TG decreases with increasing days of development (bars delineate the ossified region). C: Hind limbs were removed and aligned
as in B. The TG limbs are very short and the reduced ossification in the limb bones and paws is evident Bar 5 5 mm.
ing in a shortened snout. The posterior region of the
skull which included the occipitus and the otic capsule
were smaller in the transgenic skeleton so that the
parietal and more frontal structures were closer to the
cervical spine (Fig. 1A, 16.5 arrowheads).
The differences noted earlier persisted through days
17.5 and 18.5 of embryonic development. The transgenic limb length was short and the bone looked thicker
than the wild-type littermate due to the shorter length.
However, the carpals and metacarpals of the paws had
begun to ossify and by 18.5 days p.c., the transgenic
paws looked like the paws of wild-type 16.5 or 17.5 days
p.c. embryos. There was no ossification of the transgenic lower spine at 17.5 days p.c., although by 18.5
days p.c., there appeared to be a small amount of
alizarin red staining in the pedicles. The vertebral
bodies of the tail did not ossify during the period of this
study and the length of the tail continued to be disproportionally short. In addition, the facial features of the
transgenic embryos remained flat and the posterior
portion of the skull undeveloped.
The abnormal skeletal features noted above, the
short limbs, vertebral column and odd-shaped skull
were observed at 15.5 days p.c. when endochondral
ossification became apparent. Discrepancy in the relative length of the vertebral columns and limbs increased between the wild-type and transgenic embryos
as embryonic development proceeded (Fig. 1B,C); however, the proportional difference in the length of the
ossified region of the vertebral column between the
transgenic and wild-type embryos was lessened as
development proceeded (Fig. 1B; bars delineate the
ossified vertebral column). The same phenomenon of
initial delay of ossification followed by tendency to
normalize occurred at other sites, such as the carpal
and metacarpal bones of the paws. Unfortunately, the
Gly574Ser mutation was lethal so that post-natal development was not studied.
the proliferating zone between the transgenic and
wild-type cartilage, the cells within the resting zone of
the mutant cartilage had a normal rounded morphology
(top of TG panel, Fig. 2). Morphological differences were
even more apparent between the hypertrophic zones of
the respective littermates. The hypertrophic zone of the
wild-type growth plate was distinguished by enlarged
cells that were organized in clusters and contained
large vacuoles. They were much larger than the transgenic cells which did not contain similar vacuoles. In
addition, the hypertrophic cells were well-separated by
extracellular matrix in contrast to the tightly packed
hypertrophic cells of the wild-type growth plate. An
interesting observation was made concerning the provisional calcification of the hypertrophic zone. In the TG
growth plate there were deposits of calcium scattered
irregularly throughout the hypertrophic area (Fig. 2
TG, arrowheads) while they tended to be in longitudinal septa near the interface with bone in the wild-type
Ultrastructure of the Cartilage Matrix
Consistently through the growth plate, the density of
collagen fibrils was reduced in transgenic mice (Fig. 3,
Tg). This was observed in the matrix adjacent to the
cells as well as between cells. The fibrils from the
transgenic animals, while few in number, appeared to
be the same diameter as wild-type (Fig. 3,F).
Antibodies to the three known collagen components
of cartilage fibrils were used to assess the composition
of normal-appearing fibrils. Immunoelectron microscopy using antibodies specific for collagen types II, IX,
and XI demonstrated that the pattern of staining along
the individual fibrils was no different between the
wild-type and transgenic mouse cartilage, although
density of the fibrils differed (Fig. 4, compare A-B, C-D,
Growth Plate Histology
The architecture of the growth plate from the right
distal humerus of mice from several different litters
was analyzed at 18.5 days p.c. by electron microscopy. A
typical growth plate was observed in the wild-type
embryos as indicated by the demarcation of the zones
represented by distinct cell morphologies (Fig. 2, WT).
The space normally occupied by the proliferative zone,
i.e., the space between the resting and hypertrophic
zones, was shortened in the transgenic tissue and
contained fewer cells. Therefore, the same distance
from the hypertrophic zone in the transgenic growth
plate extended further into the resting zone than the
wild-type. The proliferating chondrocytes in the wildtype growth plate were flattened and organized in
linear clusters, typical of normal cartilage, while the
corresponding cells in the transgenic growth plate were
more homogeneously dispersed. The transgenic cells
also appeared to be larger and less extended. While
there seemed to be a difference in shape of cells within
We have described a transgenic mouse strain with a
mutation in type II collagen at Gly574Ser that resulted
in a short-limbed phenotype. This mutation was identical to one previously characterized in a patient who
presented with hypochondrogenesis (Horton et al.,
1992), a lethal human chondrodysplasia. There were
distinctive similarities in the abnormal skeletal features between the patient and the transgenic mouse,
especially the reduced limb length, the delay of ossification of the vertebral column, and the short rib cage. The
transgenic mouse model was subsequently used to
investigate how defective molecules implicated in these
disorders disturb the processes of chondrogenesis and
bone formation. From biochemical analyses, it was
apparent that the mutation caused a reduced amount of
type II collagen to be secreted and assembled into the
cartilage matrix. From these data, it appeared that the
pathology associated with the mutation was the result
of less type II collagen present which accounted for the
sparse fibrillar network shown here.
Fig. 2. Growth plate morphology from a WT and TG embryo at 18.5
day p.c. The dark lines to the left of the micrograph denote the regions of
the growth plate in the distal humerus as resting (R), proliferating (P) and
hypertrophic zone (H); the arrow adjacent to the (R) indicates that the
resting zone continues beyond the field shown. The proliferating zone of
the WT growth plate contains clusters of flattened, stacked cells (arrow)
that are missing in the TG embryos. There are unusual mineral depositions in the hypertrophic zone of the TG section (arrowheads). Bar 5
50 µm.
The onset of the abnormal skeletal features coincided
with the initiation of ossification of cartilaginous tissues. The molecular events that occur during endochondral bone formation are complex and poorly understood. For example, it is not clear if cartilage acts as a
scaffold upon which ossification occurs, if it provides the
molecular signals that lead to bone formation, or
whether it is simply degraded and replaced by bone in a
temporal manner. In terms of a specific mechanism, it is
difficult to explain the resulting short bones of the
Col2a1 mutation from the sparse fibrillar network.
However, it was apparent that the growth plate was
short and that the number of cells within this specialized region of cartilage was fewer than normal, possibly
reflecting a reduced ability of the chondrocytes to
proliferate. The morphology of the hypertrophic cells
was abnormal, an observation that raises the possibility of incomplete differentiation of the cells. Both of
these findings would affect bone length by reducing the
total number of cells turned over in a given period of
time in the growth plate and reducing the volume in the
hypertrophic cells.
There have been a number of other transgenic mouse
strains produced with Col2a1 mutations. These include
a single base substitution (Garofalo et al., 1991), deletions (Vandenberg et al., 1991; Metsaranta et al., 1992;
Helminen et al., 1993), and inactivation of the Col2a1
gene, discussed below (Li et al., 1995). The resulting
phenotypes varied, some of which had abnormal skeletal features similar to our dwarf mouse strain with the
Gly574Ser mutation while others survived and later
developed osteoarthritis. The fact that other Col2a1
mouse mutations resulted in a similar short-limbed
dwarfism suggests that they affect bone formation and
growth in a common manner which in this case involves
reduced secretion of type II collagen. The effect of
overexpression of the normal gene product was addressed (Garofalo et al., 1993) in which an increasing
level of expression of the transgene was correlated with
an increasing number of large diameter fibrils in the
cartilage matrix. At 43% expression of transgene to
endogenous type II mRNA, thick fibrils were evident. In
our mouse strain, the transgene was expressed at
approximately this same level and no thick fibrils were
present. This provides support that the phenotype of
the Gly574Ser strain was the result of the specific
mutation rather than the presence of the transgene.
We were surprised to discover that the cartilage
matrix of the Gly574Ser Col2a1 mutation did not
contain any detectable serine at that position because a
more severe phenotype is generally thought to result
from assembly of the mutant procollagen chains into
the extracellular matrix. Milder forms of osteogenesis
imperfecta are typically associated with mutations in
which the mutant gene products are not incorporated
into the extracellular matrix (Byers et al., 1991; Byers,
1993). One notable distinction between the Gly574Ser
and the other mouse strains is that the latter contained
distended rough endoplasmic reticulum (RER) in the
chondrocytes, indicating intracellular storage and presumably poor secretion of newly synthesized proteins.
We did not consistently observe enlarged RER in the
chondrocytes from the Gly574Ser transgenic mice.
A recent publication addressed the role of type II
collagen in cartilage and bone formation by inactivating its gene to produce type II collagen-null mice (Li et
al., 1995). Comparing the phenotype of the heterozygous and null mice with that of the Gly574Ser transgenic strain described in this report is perplexing. The
heterozgous mice showed little difference in skeletal
Fig. 3. Ultrastructure of cartilage matrix. Electron microscopy of an area within the growth plate resting
zone shows fewer fibrils (f) in the extracellular matrix of the transgenic (Tg) matrix to the right of wild-type (Wt).
Note the plasma membrane (pm) of a chondrocyte (C) at the lower periphery of the micrograph. Bar 5 50 nm.
features compared to their wild-type littermates. On a
superficial level, the null mice and our transgenic ones
presented with short-limbed chondrodysplasia. The
facial features and limb length of the null mouse were
consistent with the Gly574Ser mutant phenotype and
other previously described transgenic mice mentioned
above. However, other subtle differences were noted.
For example, the shape of the long bones of the null
mouse was quite distinct in that the diameter was
abnormally large, reducing the ratio of length to width.
The delay of ossification of the vertebral column was
much greater in the Gly574Ser mice. Interestingly,
there were fewer collagen fibrils in the null mouse
cartilage than in our transgenic mouse, and yet more
extensive ossification of their skeletons. While it is
difficult to explain the respective phenotypes based on
the information available, it raises intriguing questions
about the biological functions of type II collagen for
normal skeletal formation and linear growth of bones.
In conclusion, we have analyzed the developing skeleton of a mouse expressing a dominant negative mutation of Col2a1. We speculate that the reduced density of
collagen fibrils in the matrix and poorly differentiated
chondrocytes contribute to the profound delay of the
normal developmental sequence and suggest that this
phenomenon accounts for the similar developmental
delay seen in human hypochondrogenesis. Our observations raise the possibility that chondrocyte proliferation and differentiation is somehow perturbed by the
reduction of collagen fibrils.
Generation of the Transgenic Mice
The original transgene construct, cosmid pWE15
containing the murine wild-type Col2a1, has been
previously described (Garofalo et al., 1991) and used
successfully for the production of other transgenic
mouse strains (Garofalo et al., 1991; Metsaranta et al.,
1992; Garofalo et al., 1993). Two mutations were introduced into pWE15 using site-directed mutagenesis
to make the Gly574Ser transgene, one of which was
a G = A base substitution in exon 33 and coded for
the amino acid substitution; the second introduced a
silent mutation that destroyed a SacI site. The founder
mice were mated with B6D2F1 mice for transgenic F1
litters; founder mice and subsequent offspring were
identified and transgene copy number determined by
Southern blot analysis after digestion of tail genomic
DNA with NcoI as previously described (Garofalo et al.,
Preparation of Stained Skeletons
The method of staining and clearing the embryo
skeletons was based on two previously described protocols (McLeod, 1980; Martin et al., 1995). Briefly, following a Caesarean section, the embryos were removed
and eviscerated. The placentas were set aside for DNA
extraction and Southern analysis to identify which
carried the transgene. The embryos were placed in
Fig. 4. Immunolocalization of collagens to the cartilage fibrils. Antibodies to collagen types II, IX, and XI recognize epitopes along the fibrils in
tissue from WT (A,C,E) and TG (B,D,F) growth cartilage. The upper
panels have been incubated with antibodies to type II collagen (A,B); the
middle panels with antibodies to type IX collagen (C,D); the lower panels
with antibodies to type XI collagen (E,F). A secondary antibody conjugated to a 5 nm gold bead particle is directed to the collagen antigens.
While there are fewer fibrils present in the Tg tissue, the labeling of the
fibrils appears similar to the Wt. Bar 5 200 nm.
water overnight. The following day they were immersed in water at 65°C for 30 sec to 1 min (14.5 and
15.5 days p.c. embryos were immersed for 30 sec; the
larger embryos for up to 1 min). For the larger embryos,
the skin was removed prior to fixation in absolute
ethanol for 3 days. The cartilage was stained with an
acetic alcian blue solution overnight at 37°C (15 mg
alcian blue in 100 ml 75% ethanol containing 20% (V/V)
acetic acid). The mice were then placed in 100% ethanol
for another overnight period. The following day the
skeletons were cleared for 6 hr in 0.5% for the 14.5 and
15.5 days p.c. embryos and 1% for the 16.5, 17.5, and
18.5 days p.c. The bone was stained overnight in
alizarin red (50 mg in 1 L of potassium hydroxide, the
concentrations consistent for the relative stage of development as above). The clearing was continued in potassium hydroxide until only the skeletons remained.
They were stored in 50% glycerol.
Ultrastructural Analysis
For ultrastructural observation, right forearms, including growth plate and bone, were examined following fixation in 1.5% paraformaldehyde/1.5% glutaraldehyde and osmium tetroxide, dehydration in ethanol and
embedding in Spurrs epoxy.
Ultrastructural immunocytochemistry was performed
using the enbloc method previously described (Keene et
al., 1995) with some modification. Briefly, left forearms
were snap-frozen in hexanes, then thawed in PBS.
Following exposure to chondroitinase (0.25 units/ml
PBS, pH 7.4) for 2 hr at room temperature, the tissues
were rinsed in PBS for 30 min prior to immersion in
primary antibody diluted 1:5 in PBS overnight at 4°C.
Primary antibodies included a rabbit anti-XI antibody,
pAb16123 (Keene et al., 1995) a goat anti-type II
antibody (Southern Biotech 1320-01, Birmingham, AL),
and a rabbit anti-type IX antibody, pAb9264 (Keene et
al., 1995). Following an extensive rinse in PBS, the
samples were immersed in 5 nm gold conjugated to
appropriate secondary antibody (Amersham) diluted
1:3 in 1.0% BSA buffer (20 mM Tris-HCl, 0.9% NaCl, 1
mg/ml BSA, 20 mM NaN3) pH 8.0 for about 15 hr. The
tissues were again rinsed extensively in PBS then fixed,
dehydrated, and embedded as described previously.
The authors gratefully acknowledge the expert technical assistance of Catherine Ridgway and Brian Demings. This work was supported by grants from the
National Institutes of Health, HD20691, Shriners Hospital for Children, 15956, Fred Meyer Charitable Trust
Foundation, and the R. Blaine Bramble Medical Research Foundation.
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