Organization of Collagen Types I and V in the
Embryonic Chicken Cornea
David E. Birk,* John M. Firch,-f and Thomas F. Linsenmayert
The distribution and organization of type I and type V collagens were studied in the embryonic chicken
cornea using anti-collagen, type specific, monoclonal antibodies and immunoelectron microscopy. These
studies were performed on lathyritic 17-day corneas treated at 4°C or 37°C. At the lower temperature,
collagen fibril structure is disrupted; at the higher temperature, normal fibril structure is maintained.
Corneas from non-lathyritic 17-day chick embryos, reacted at the two different temperatures, were
studied for comparison. In Bowman's membrane, the thin (20 nm) fibrils were labelled by antibodies
against both type I and type V collagen under all conditions studied. In the corneal stroma, the striated
collagen fibrils (25 nm) were labelled with the antibodies against type I collagen in all cases, and by
antibodies against type V collagen under conditions where fibril structure was disrupted. These results
are consistent with the concept of heteropolymeric fibrils consisting of both type I and type V collagen
molecules assembled such that the epitopes on the type V molecule are unavailable to antibody unless
thefibrillarstructure is disrupted. We suggest that the interaction of type V collagen with type I collagen
may be responsible for the small diameter fibrils and the rigid control of fibril structure found in the
cornea. Invest Ophthalmol Vis Sci 27:1470-1477, 1986
Immunocytochemical studies of the chick cornea,
using anti-collagen monoclonal antibodies, have demonstrated that type I collagen is distributed throughout
Bowman's membrane and "the corneal stroma.12"14 In
contrast, in the normal chick cornea, type V collagen
appeared to be confined to Bowman's membrane, a
subepithelial, acellular structure containing small (20
nm) collagen fibrils.12"14 However, further analysis
showed that the pretreatment of corneas with agents
which disrupt collagen fibril structure (i.e. dilute acid),
or which remove type I collagen (i.e. vertebrate collagenase) permitted the visualization of type V collagen
throughout the corneal stroma.13"15
In addition, it was observed that the immersion of
lathyritic corneas in cold buffer also made stromal type
V collagen accessible for antibody binding.15 The in
ovo administration of the lathyrogen /3-aminopropionitrile (/3APN), which prevents the formation of
crosslinks through the inhibition of lysyl oxidase16 results in the deposition of collagen fibrils whose structure
is sensitive to temperature manipulations. 17 In such
tissues, normal fibril structure is maintained at 37°C
while at 4°C fibrils dissociate.18'19 This alteration of
fibril structure in the cold can be reversed when the
tissue is returned to 37 °C.17
When taken together, these studies indicate that type
V collagen is masked within the corneal stroma due
to some structural arrangement within fibrils. The unmasking of type V collagen by digestion with vertebrate
The size and organization of collagen fibrils within
the extracellular matrix are important determinants of
tissue structure and function. The embryonic chick
corneal stroma is a well-organized collagenous matrix
composed of uniform, small diameter collagen fibrils
(25 nm) with a relatively constant interfibrillar spacing.
The collagen fibrils are arranged as lamella with adjacent layers oriented at approximately 90° to one another.1"3 The rigid control of both collagen fibril microarchitecture and matrix macro-architecture is necessary
for corneal transparency.
Type I collagen is the predominant collagen in the
cornea,4 with type V collagen being a quantitatively
minor component.5"9 However, the content of type V
collagen in the cornea (5-20% of the total collagen) is
high when compared to other tissues containing predominantly type I collagen such as sclera, tendon, bone,
anddermis. 8 1 0 1 1
From the *Department of Pathology, University of Medicine and
Dentistry of New Jersey, Rutgers Medical School, Piscataway, New
Jersey, and the fDepartment of Anatomy and Cellular Biology, Tufts
University School of Medicine, Boston, Massachusetts.
Supported by National Institutes of Health Grants EY 05129 and
EY 05191 and a Research Career Development Award (EY 00254)
to Dr. Birk.
Submitted for publication: November 1, 1985.
Reprint requests: David E. Birk, Department of Pathology, University of Medicine and Dentistry of New Jersey, Rutgers Medical
School, Piscataway, NJ 08854.
1470
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
COLLAGEN TYPES I AND V IN THE CHICK CORNEA / DirK er ol.
No. 10
collagenase, an enzyme which degrades type I but not
type V collagen, further indicates that a fibrillar organization involving type I collagen is responsible for the
masking of type V collagen. This suggests that, within
extracellular matrices in situ, type I and type V collagens
may be co-assembled within a single heteropolymeric
fibril.
In this paper, we have employed collagen type-specific monoclonal antibodies and immunoelectron microscopy to directly visualize collagen type I and type
V in the normal chick embryo cornea and lathyritic
corneas in which fibril structure has been altered. The
results support the presence of heteropolymeric fibrils
composed of collagen types I and V.
Materials and Methods
White leghorn chick embryos were incubated at
37.5°C in a humidified atmosphere and staged according to Hamburger and Hamilton. 20
Tissue Preparation
Chick embryos were made lathyritic beginning on
day 14 by injection of /?-aminopropionitrile fumerate
(0APN; Sigma Chemical Co., St. Louis, MO). Eggs were
injected with 1 mg 0APN in 0.1 ml of phosphate buffered saline (PBS) on day 14, 1-2 mg on day 15, and 2
mg on day 16. On day 17 of development, normal or
lathyritic corneas were carefully dissected with a ring
containing the scleral ossicles. The corneas were washed
in PBS, incubated in 10% dimethyl sulfoxide (DMSO)
in PBS, and embedded in OCT compound (TissueTek; Miles Scientific, Naperville, IL), all at room temperature. The embedded tissue was frozen in liquid
nitrogen and 8-10 /im cryostat sections were cut perpendicular to the corneal surface. Sections were picked
up onto albumin-coated microscope slides, air dried,
washed with PBS, and incubated with PBS for approximately 16 hr at either 4°C or 37°C. Following
this pre-incubation, the sections were routinely fixed
in 4% paraformaldehyde in PBS, pH 7.4, for 15 min
at 4°C. This was followed by washing in PBS, incubation in sodium borohydride (50 mg/100 ml PBS) for
1 hr,21 and three washes in PBS, all done at 4°C.
Antibody Labelling
The sections were incubated with 2% normal goat
serum for 2 hr at room temperature. The serum was
decanted and the sections were incubated with the affinity purified monoclonal antibodies (35 fig/m\) at 4°C
for 8-12 hr. The antibodies were isolated by protein
A chromatography of the hybridoma culture supernatants. Monoclonal antibodies against type I and type
V collagens were prepared and characterized as de-
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
1471
scribed previously.13'1422 Two antibodies against type
V collagen (V-DH2 and V-AB12) and two antibodies
against type I collagen (I1B6 and I-BA1) were used.
Affinity purified non-immune mouse IgG (Cappel
Laboratories, Malvern, PA) was used in control preparations. The sections were washed five times over 1
hr with PBS, and processed for either immunofluorescence or immunoelectron microscopy.
In addition, a variety of controls and variations on
this experimental protocol were performed. These included substitution of the primary antibody with nonimmune mouse IgG, anti-type IV collagen antibodies,
or normal mouse serum; two different well-characterized monoclonal antibodies were used against each
collagen type. We have unmasked type V collagen using
temperature manipulation of lathyritic tissue as well
as pretreatment with dilute acetic acid, examined penetration into the cornea by looking at unwashed preparations and by serially sectioning heavily labelled
blocks, used unfixed tissues and those fixed under a
variety of conditions, and used a ferritin bridge method
as well as indirect labeling with a secondary ferritin or
gold conjugated antibody. These experiments and other
combinations have been done over 40 different times.
Immunofluorescence Microscopy
For indirect immunofluorescence microscopy, the
sections were incubated with rhodamine-conjugated
goat anti-mouse IgG (Cappel Laboratories, Malvern,
PA), diluted at 1:150 to 1:250 with PBS. Incubation
was for 4 hr at room temperature followed by five
washes in PBS over 1 hr. The sections were examined
and photographed using a Zeiss Photomicroscope III
with epifluorescence optics. The film was developed
and micrographs were printed under identical conditions to permit comparison.
Immunoelectron Microscopy
The corneal sections were prepared for immunoelectron microscopy using a modified ferritin bridge
technique. 23 After incubation with the primary antibody, the sections were incubated for 8-12 hr at 4°C,
with goat anti-mouse IgG (anti H & L chain) gamma
globulin fraction from whole antiserum (Jackson Laboratories, Avondale, PA), diluted 1:100 with PBS, followed by five washes in PBS over 1 hr. This was followed by incubation with affinity purified mouse antihorse spleen ferritin (20 /ug/ml; Jackson Laboratories,
Avondale, PA) for 8-12 hr at 4°C. The tissues were
washed and incubated in horse spleen ferritin (10 ng/
ml PBS) for 8-12 hr at 4°C. The tissues were washed
five times over 1 hr in PBS, one time with 0.1 M sodium
cacodylate pH 7.4, and fixed in 4% paraformaldehyde,
2.5% glutaraldehyde, 0.1 M sodium cacodylate pH 7.4
1472
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Ocrober 1986
for 1 hr, washed with 0.1 M cacodylate buffer, postfixed
with 1% osmium tetroxide in 0.1 M cacodylate pH 7.4,
dehydrated in ethanol, cleared in propylene oxide, and
embedded in epon-araldite. An epon-araldite filled
Beem capsule was inverted on the section and polymerized at 68°C for 18-36 hr. The slides and Beem
capsules were removed from the oven and allowed to
cool. The slide was then placed on a 100°C hot plate
for 10-15 sec and the Beem capsule "popped off' the
slide with the tissue section.
Sections with a pale gold interference color were cut
for electron microscopy. The sections were picked up
onto mesh grids, stained with 2% aqueous uranyl acetate, examined, and photographed using either a Philips 300 or Philips 420 transmission electron microscope.
Results
As a control and a confirmation for the specificity
of the immunoelectron microscopy studies, in each experiment parallel tissue sections were processed for
immunofluorescence microscopy. Representative results are presented in Figure 1. Types I and V collagen
were localized by indirect immunofluorescence microscopy in non-lathyritic corneas and in lathyritic
corneas after pretreatment in PBS at 37°C and 4°C.
In all cases, type I collagen was distributed throughout
Bowman's membrane and the corneal stroma (Fig. 1).
In non-lathyritic corneas and lathyritic corneas incubated at 37°C, type V collagen was localized to Bowman's membrane with very little, if any, staining of
the corneal stroma (Fig. 1). However, in lathyritic corneas pretreated at 4°C, type V collagen was now detected throughout the corneal stroma as well as Bowman's membrane, giving a pattern identical to that of
type I collagen (Fig. 1). In all cases, the control preparations, non-immune mouse IgG substituted for the
primary antibody, showed no reactivity (Fig. 1).
The structure and relationship of collagen types I
and V within Bowman's membrane and the corneal
stroma was then visualized by immunoelectron microscopy using a ferritin bridge technique. Bowman's
membrane of both non-lathyritic and lathyritic 17-day
corneas treated at 4°C and 37°C was uniformly labelled
with antibodies against type I and type V collagen (Fig.
2-4). Both collagen types were localized to the small
diameter (20 nm) collagen fibrils comprising Bowman's
membrane. In no case did any of the antibodies against
type V collagen label the corneal epithelial basement
membrane. The control preparations were not reactive
(Figs. 2-4).
In the corneal stroma, type I collagen was localized
to the striated collagen fibrils in all preparations (Figs.
2-4). Labelled fibrils were found throughout the entire
stroma. In the non-lathyritic corneas and lathyritic
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
Vol. 27
corneas pretreated at 37°C, the stromal fibrils were
typically cross-striated; however, when lathyritic corneas were cooled in PBS to 4°C, numerous areas
within the stroma were observed in which collagen fibrillar striations were not apparent or in which the fibrils had partially dissociated into thin strands. These
fine fibrillar structures were derived from striated fibrils
as evidenced by the continuity between striated and
dissociated portions of fibrils in some regions
(Fig. 4).
When reacted with anti-type V collagen antibodies,
the stroma of non-lathyritic corneas or lathyritic corneas maintained at 37°C (Figs. 2, 3) showed labelling
of the typical striated collagen fibrils which was only
slightly greater than the level seen in the control preparations. The pretreatment of the non-lathyritic cornea
at 4°C (data not shown) or 37 °C gave identical results.
When the lathyritic corneas were pretreated at 4°C
prior to localization of type V, the corneal stroma was
now heavily labelled by the anti-type V collagen antibodies (Fig. 4). The label was localized chiefly to collagen fibrils that appeared to be at least partially dissociated. The regions containing typical, compact,
striated collagen fibrils were not as heavily labelled.
Discussion
Monoclonal antibodies against collagen types I and
V, an immunoferritin technique, and electron microscopy were used to localize these collagens and to study
their morphology within the embryonic chicken
cornea.
Both collagen types are found within the small diameter fibrils of Bowman's membrane, as well as the
larger diameter, striated fibrils of the stroma. These
results are the first direct evidence that collagen types
I and V are deposited together within the same heteropolymeric fibril. This conclusion is based on the assumption that morphologically identical fibrils are also
chemically homogeneous. Electron microscopic analysis of corneas doubly labelled with antibodies against
type I and V collagen, now underway, should demonstrate unequivocally the spatial relationship between
these collagen types.
Type V collagen could be detected within the small
diameter fibrils (20 nm) of Bowman's membrane without any pretreatment of the tissue. However, to detect
immunohistochemically the type V collagen within the
stroma, it was necessary to disrupt collagen fibril structure to "unmask" type V collagen. This was true for
both of the anti-type V collagen monoclonal antibodies
we used. As previously described, "unmasking" can
be effected by pretreatment of tissue sections with dilute
acid or vertebrate collagenase, or by the pre-incubation
of lathyritic tissues in cold saline.13"15 These treatments
are all known to disrupt collagen fibril structure, either
by swelling/dissociating the fibrils, or by selectively re-
COLLAGEN TYPES I AND V IN THE CHICK CORNEA / Dirk er QI.
No. 10
NORMAL CORNEA
Antil
LATHYRITIC CORNEA - 3 7 ^
1473
LATHYRITIC CORNEA-4°C
.
Anti Y
Control
Fig. 1. Immunofluorescent localization of collagen types I and V. Normal chick embryos and those made lathyritic beginning on day 14 of
incubation were dissected on day 17 and the corneas were prepared for immunocytochemical localization of collagen types I and V using typespecific monoclonal antibodies and a Rhodamine-conjugated goat anti-mouse IgG. Pre-treatment of lathyritic tissues in cold PBS (C, F, and I)
disrupts type I fibril structure, while at 37°C (B, E, and H) fibril structure is maintained. The pre-incubation has no effect on the normal cornea
(A, D, and G). In A, B, and C, the corneas were incubated with a monoclonal antibody against type I collagen; in D, E, and F, the corneas
were incubated with an antibody against type V collagen; and in G, H, and I, the primary antibody was replaced with affinity purified nonimmune mouse IgG. The corneal stroma is labelled (S) while Bowman's membrane is indicated by the arrows.
moving the type I collagen. In lathyritic tissues, only
those fibrils formed in the presence of /3APN, in this
case during day 14-17 of development, would be susceptible to temperature manipulation. In the present
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
series of immunoelectron microscopic studies, we employed both the dilute acetic acid pretreatments and
the cold saline pretreatment of lathyritic corneas. The
latter was chosen for routine use, since the preservation
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / October 1986
1474
Vol. 27
Normal Cornea
BOWMAN'S MEMBRANE
I.
*JT*
STROMA
Anti £
Fig. 2. Localization of collagen types I and V in normal
chick embryo corneas. Normal 17-day chick embryo
corneas were dissected, and
frozen sections were prepared. Collagen types were
localized by immunoelectron
microscopy with type specific
monoclonal antibodies and a
ferritin bridge method. A, C,
and E are of Bowman's
membrane, while B, D, and
F are from the corneal
stroma. In A and B, the corneas were incubated with a
monoclonal antibody against
type I collagen; in C and D,
the corneas were incubated
with an antibody against type
V collagen; and in E and F,
the primary antibody was
replaced with affinity purified non-immune mouse
IgG. The epithelial basement
membrane is indicated (*).
Bar = 100 nm.
of ultrastructural detail was considerably better. We
observed that good antibody labelling of fibrils for type
V collagen can be achieved in fixed tissues if the fixation
is performed after these "unmasking" procedures. If
the order is reversed, and the tissues are fixed before
the "unmasking" is performed, no type V collagen labelling is observed in the stroma. This is consistent
with our interpretation that unmasking requires alterations in fibril structure. Prefixed fibrils apparently are
unable to undergo these requisite changes.
The co-localization of type V with type I collagen
within the corneal stroma suggests that one role for
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
type V collagen may be in the regulation of collagen
fibril structure. Matrices with less type V collagen, such
as the sclera, contain fibrils with larger and more
variable diameters.8 Bowman's membrane, on the
other hand, contains smaller diameter fibrils in which
type V collagen is normally exposed ("unmasked"). It
is possible that the smaller fibril diameters are due to
the incorporation of a greater proportion of type V
collagen into these heteropolymeric fibrils. The interaction of type V collagen with type I collagen within
heterotypic fibrils may therefore, be responsible for the
small, rigidly controlled diameters of the corneal fibrils.
No. 10
1475
COLLAGEN TYPES I AND V IN THE CHICK CORNEA / Dirk er ol.
Lathyritic Cornea
Pretreated 37°C.
BOWMAN'S MEMBRANE
STROMA
f'-'
Afitil.
Fig. 3. Localization of collagen types I and V in lathyritic corneas pretreated at
37°. Chick embryos were
made lathyritic beginning on
day 14 of incubation by administration of jSAPN. The
corneas were dissected on day
17, and the sections were incubated in phosphate buffered saline at 37°C, a condition in which fibril structure is maintained. Collagen
types were localized by
immunoelectron microscopy
with type specific monoclonal antibodies and a ferritin bridge method. A, C,
and E are of Bowman's
membrane, while B, D, and
F are from the corneal
stroma. In A and B, the corneas were incubated with a
monoclonal antibody against
type I collagen; in C and D,
the corneas were incubated
with an antibody against type
V collagen; and in E and F,
the primary antibody was replaced with affinity purified
non-immune mouse igG. The
basement membrane is indicated at (*). Bar = 300 nm.
A
AntiV.
c
Control
Evidence exists that other collagens may interact in
a similar manner. It has been shown in vitro that types
I and III collagen can interact with one another to influence the diameter of fibril bundles.24 Also, cyanogen
bromide peptides of collagen from certain tissues have
been isolated which are composed of one peptide from
the type I collagen and one peptide from the type III
molecule.25 These data suggest the existence of heterotypic fibrils composed of type I collagen and type III
collagen molecules. It seems likely that interstitial collagen is commonly deposited in heteropolymeric form.
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
rol
Type V collagen also has been described in association with basement membranes,26-27 and in the pericellular environment.28"30 Although we did not observe
a similar pattern of distribution, it is possible that different investigators are identifying different molecules. Type V collagen is known to exist in at least
three distinct molecular forms; al(V)2,a:2(V))31"33
al(V) 3 , 33 - 36 and al(V),«2(V),a3(V).35-37 Our antibodies were produced against the cd(V) 2 ,a2(V) form of
the molecule, and are likely to be specific for it. Possibly, it is molecular diversity which has prompted
1476
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Ocrober 1986
Vol. 27
Lathyritic Cornea-Pretreated 4°C.
BOWMAN'S MEMBRANE
STROMA
A
B
AntiY.
Anti ¥.
c
Control
Con
some of the considerable controversy surrounding the
localization and function of type V collagen.
In one study, type V collagen has been localized to
12 nm fibrils in the subepithelial region of the amnion
using immunoelectron microscopy.30 This, coupled
with the observations that type V collagen is sometimes
seen to be associated with basement membranes, suggests that another possible function of type V collagen,
or at least one species of type V collagen, may be to
act as a transitional molecule, anchoring basement
membranes to the interstitium. In this case, the mol-
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
Fig. 4. Localization of collagen types I and V in lathyritic corneas pretreated at
4°C. Chick embryos were
made lathyritic by administration of jSAPN beginning
on day 14 of incubation. On
day 17 of development, the
corneas were dissected and
the sections were incubated
in phosphate buffered saline
at 4°C, a condition which
disrupts fibril structure. Collagen types were localized by
immunoelectron microscopy
with type-specific monoclonal antibodies and a ferritin bridge method. A, C,
and E are of Bowman's
membrane, while B, D, and
F are from the corneal
stroma. In A and B, the corneas were incubated with a
monoclonal antibody against
type I collagen; in C and D,
the corneas were incubated
with an antibody against type
V collagen; and in E and F,
the primary antibody was replaced with affinity purified
non-immune mouse IgG.
The epithelial basement
membrane is indicated (*).
Bar = 300 nm.
ecule would exist in situ as thin fibrils. Type V collagen
can exist in this form, since purified type V collagen
molecules can form thin fibrils (approximately 22 nm)
in vitro in the absence of other collagens." The subepithelial localization of type V collagen to thin (20
nm) fibrils in Bowman's membrane suggests that type
V may serve this function in the cornea. But, even in
this location, our data suggest that these thin fibrils
also contain type I collagen. The diameter of the fibrils
in this region may not be sufficient to mask the epitopes
on type V collagen.
No. 10
COLLAGEN TYPES I AND V IN THE CHICK CORNEA / Dirk er ol.
The presence of heteropolymeric collagen fibrils in
which fibril structure is responsible for the masking of
the type V epitopes and the variable chemistry of the
collagen molecules themselves is consistent with a view
of the extracellular matrix as being chemically and
structurally heterogeneous. This heterogeneity may
partially explain the diversity of extracellular matrices
composed of a limited number of macromolecules.
Key words: collagen type I, collagen type V, cornea, monoclonal antibodies, lathyrism, immunoelectron microscopy
Acknowledgments
We would like to thank Dr. Richard Mayne for his help
in the preparation of the monoclonal antibodies. The expert
technical assistance of Joanne Babiarz and Kathy Kloos is
gratefully acknowledged.
References
1. Hay E and Revel JP: Fine strucuture of the developing avian
cornea. In Monographs on Developmental Biology, Vol. I, Wolsky A and Chen P, editors. New York, S Karger, 1969,. pp. 1144.
2. Trelstad RL and Coulombre AJ: Morphogenesis of the collagenous stroma in the chick cornea. J Cell Biol 50:840, 1971.
3. Hay ED: Development of the vertebrate cornea. Intl Rev Cytol
63:263, 1980.
4. Hay ED, Linsenmayer TF, Trelstad RL, and von der Mark K:
Origin and distribution of collagens in the developing avian cornea. In Current Topics Eye Research, Zadunaisky J A and Davson
H, editors. New York, Academic Press, 1979, pp. 1-35.
5. Davison PF, Hong BS, and Cannon DF: Quantitative analysis
of the collagens in the bovine cornea. Exp Eye Res 29:97, 1979.
6. Welsh C, Gay S, Rhodes RK, Pfister R, and Miller EJ: Collagen
heterogeneity in normal rabbit cornea. I. Isolation and biochemical characterization of the genetically distinct collagens. Biochim
Biophys Acta 625:78, 1980.
7. Cintron C, Hong BS, and Kublin CL: Quantitative analysis of
collagen from normal developing corneas and corneal scars. Curr
Eye Res 1:1, 1981.
8. Freeman IL: The eye. In Collagen in Health and Disease, Weiss
JB and Jayson MIV, editors. London, Churchill Livingstone,
1982, pp. 388-403.
9. Lee RE and Davison PF: The collagens of the developing bovine
cornea. Exp Eye Res 39:639, 1984.
10. Hong BS, Davison PF, and Cannon DJ: Isolation and characterization of a distinct type of collagen from bovine fetal membranes and other tissues. Biochemistry 18:4278, 1979.
11. Broek DL, Madri J, Eikenberry EF, and Brodsky B: Characterization of the tissue form of type V collagen from chick bone. J
Biol Chem 260:555, 1985.
12. von der Mark K and Ocalan M: Immunofluorescent localization
of type V collagen in the chick embryo with monoclonal antibodies. Collagen Rel Res 2:541, 1982.
13. Linsenmayer TF, Fitch JM, Schmid JM, Zak NB, Gibney E,
Sanderson RD, and Mayne R: Monoclonal antibodies against
chicken type V collagen: production, specificity, and use for immunocytochemical localization in the embryonic cornea and
other organs. J Cell Biol 96:124, 1983.
14. Linsenmayer TF, Fitch JM, and Mayne R: Extracellular matrices
in the developing avian eye: Type V collagen in corneal and non
corneal tissues. Invest Ophthalmol Vis Sci 25:41, 1984.
15. Fitch JM, Gross J, Mayne R, Johnson-Wint B, and Linsenmayer
TF: Organization of collagen types I and V in the embryonic
chicken cornea: monoclonal antibody studies. Proc Natl Acad
Sci USA 81:2791, 1984.
Downloaded From: http://iovs.arvojournals.org/ on 06/18/2017
1477
16. Tanzer ML: Covalent crosslinking of collagen. Science 180:561,
1973.
17. Levene CI: Lathyrism. In Molecular Pathology of Connective
Tissues, Perez-Tamayo R and Rojkind M, editors. New York,
Marcel Dekker, 1973, pp. 175-193.
18. Levene CI and Gross J: Alterations in state of molecular aggregation of collagen induced in chick embryos by j8-aminopropionitrile (Lathyrus Factor). J Exp Med 110:771, 1959.
19. van den Hooff A, Levene CI, and Gross J: Morphologic evidence
for collagen changes in chick embryos treated with j8-aminoproprionitrile. J Exp Med 110:1017, 1959.
20. Hamburger V and Hamilton HL: A series of normal stages in
development of the chick embryo. J Morphol 88:49, 1951.
21. Martinez-Hernandez A: The hepatic extracellular matrix. I.
Electron immunohistochemical studies in normal rat livers. Lab
Invest 51:57, 1984.
22. Linsenmayer TF, Hendrix MJ, and Little CD: Production and
characterization of a monoclonal antibody against chicken type
I collagen. Proc Natl Acad Sci USA 76:3703, 1979.
23. Willingham MC: Electron microscopic immunocytochemical
localizaton of intracellular antigens in cultured cells: the ECS
and ferritin bridge procedures. Histochem J 12:419, 1980.
24. Lapiere CM, Nusgens B, and Pierard GE: Interactions between
collagen type I and type III in conditioning bundles organization.
Conn Tissue Res 5:21, 1977.
25. Henkel W and Glanville RW: Covalent crosslinking between
molecules of type I and type III collagen. Eur J Biochem 122:
205, 1982.
26. Madri JA and Furthmayr H: Isolation and tissue localization of
type AB2 collagen from normal lung parenchyma. Am J Pathol
94:323, 1979.
27. Roll FJ, Madri JA, Albert J, and Furthmayr H: Codistribution
of collagen types IV and AB2 in basement membranes and mesangium of the kidney. J Cell Biol 85:597, 1980.
28. Gay S, Rhodes RK, Gay RE, and Miller EJ: Collagen molecules
comprised of al(V)-chains (B chains): an apparent localization
in the exocytoskeleton. Collagen Rel Res 1:53, 1981.
29. Gay S, Martinez-Hernandez A, Rhodes RK, and Miller EJ: The
collagenous exocytoskeleton of smooth muscle cells. Collagen
Rel Res 1:377, 1981.
30. Modesti A, Kalebic T, Scarpa S, Togo S, Grotendorst G, Liotta
LA, and Triche J: Type V collagen in human amnion is a 12
nm fibrillar component of the pericellular interstitium. Eur J
Cell Biol 35:246, 1984.
31. Bentz H, Bachinger R, Glanville R, and Kuhn K: Physical evidence for the assembly of A and B chains of human placenta)
collagen in a single triple helix. Eur J Biochem 92:563, 1978.
32. Kumamoto CA and Fessler JH: Biosynthesis of al(V) 2 a2(V) A,
B procollagen. Proc Natl Acad Sci USA 77:6434, 1980.
33. Fessler LI, Robinson WJ, and Fessler JH: Biosynthesis of procollagen (pro «1(V)2 (pro «2V) by chick tendon fibroblasts and
procollagen (pro a 1 V)3 by hamster lung cell cultures. J Biol Chem
256:9646, 1981.
34. Haralson MA, Mitchell WM, Rhodes RH, Kresina TK, Gay R,
and Miller EJ: Chinese hamster lung cells synthesize and confine
to the cellular domain a collagen composed solely of B chains.
Proc Natl Acad Sci USA 77:5205, 1980.
35. Madri JA, Foellmer HG, and Furthmayr H: Type V collagens
of the human placenta: trimer a-chain composition, ultrastructural morphology and peptide analysis. Collagen Rel Res 2:19,
1982.
36. Rhodes RK and Miller EJ: Evidence for the existence of an
al(V)«2(V)a3(V) collagen molecule in human placental tissue.
Collagen Rel Res 1:337, 1981.
37. Niyibizi C, Fietzek PO, and van der Rest M: Human placenta
type V collagens. Evidence for the existence of an al(V),
«2(V)a3(V) collagen molecule. J Biol Chem 259:14170, 1984.