Differentiated activities of decorin and biglycan in the progression of post-traumatic osteoarthritis.

OBJECTIVE: To delineate the activities of decorin and biglycan in the progression of post-traumatic osteoarthritis (PTOA). DESIGN: Three-month-old inducible biglycan (BgniKO) and decorin/biglycan compound (Dcn/BgniKO) knockout mice were subjected to the destabilization of the medial meniscus (DMM) surgery to induce PTOA. The OA phenotype was evaluated by assessing joint structure and sulfated glycosaminoglycan (sGAG) staining via histology, surface collagen fibril nanostructure and calcium content via scanning electron microscopy, tissue modulus via atomic force microscopy-nanoindentation, as well as subchondral bone structure and meniscus ossification via micro-computed tomography. Outcomes were compared with previous findings in the inducible decorin (DcniKO) knockout mice. RESULTS: In the DMM model, BgniKO mice developed similar degree of OA as the control (0.44 [-0.18 1.05] difference in modified Mankin score), different from the more severe OA phenotype observed in DcniKO mice (1.38 [0.91 1.85] difference). Dcn/BgniKO mice exhibited similar histological OA phenotype as DcniKO mice (1.51 [0.97 2.04] difference vs control), including aggravated loss of sGAGs, salient surface fibrillation and formation of osteophyte. Meanwhile, Dcn/BgniKO mice showed further cartilage thinning than DcniKO mice, resulting in the exposure of underlying calcified tissues and aberrantly high surface modulus. BgniKO and Dcn/BgniKO mice developed altered subchondral trabecular bone structure in both Sham and DMM groups, while DcniKO and control mice did not. CONCLUSION: In PTOA, decorin plays a more crucial role than biglycan in regulating cartilage degeneration, while biglycan is more important in regulating subchondral bone structure. The two have distinct activities and modest synergy in the pathogenesis of PTOA.

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts.

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.

Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation.

The formation of collagen fibrils, fibril bundles, and tissue-specific collagen macroaggregates by chick embryo tendon fibroblasts was studied using conventional and high voltage electron microscopy. During chick tendon morphogenesis, there are at least three extracellular compartments responsible for three levels of matrix organization: collagen fibrils, bundles, and collagen macroaggregates. Our observations indicate that the initial extracellular events in collagen fibrillogenesis occur within narrow cytoplasmic recesses, presumably under close cellular regulation. Collagen fibrils are formed within these deep, narrow recesses, which are continuous with the extracellular space. Where these narrow recesses fuse with the cell surface, it becomes highly convoluted with folds and processes that envelope forming fibril bundles. The bundles laterally associate and coalesce, forming aggregates within a third cell-defined extracellular compartment. Our interpretation is that this third compartment forms as cell processes retract and cytoplasm is withdrawn between bundles. These studies define a hierarchical organization within the tendon, extending from fibril assembly to fascicle formation. Correlation of different levels of extracellular compartmentalization with tissue architecture provides insight into the cellular controls involved in collagen fibril and higher order assembly and a better understanding of how collagen fibrils are collected into structural groups, positioned, and woven into functional tissue-specific collagen macroaggregates.

Collagen type I and type V are present in the same fibril in the avian corneal stroma.

The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.

The spatial organization of Descemet's membrane-associated type IV collagen in the avian cornea.

The organization of type IV collagen in the unconventional basement membrane of the corneal endothelium (Descemet's membrane) was investigated in developing chicken embryos using anti-collagen mAbs. Both immunofluorescence histochemistry and immunoelectron microscopy were performed. In mature embryos (greater than 15 d of development), the type IV collagen of Descemet's membrane was present as an array of discrete aggregates of amorphous material at the interface between Descemet's membrane and the posterior corneal stroma. Immunoreactivity for type IV collagen was also observed in the posterior corneal stroma as irregular plaques of material with a morphology similar to that of the Descemet's membrane-associated aggregates. This arrangement of Descemet's membrane-associated type IV collagen developed from a subendothelial mat of type IV collagen-containing material. This mat, in which type IV collagen-specific immunoreactivity was always discontinuous, first appeared at the time a confluent endothelium was established, well before the onset of Descemet's membrane formation. Immunoelectron microscopy of mature corneas revealed that the characteristic nodal matrix of Descemet's membrane itself was unreactive for type IV collagen, but was penetrated at intervals by projections of type IV collagen-containing material. These projections frequently appeared to contact cell processes from the underlying corneal endothelium. This spatial arrangement of type IV collagen suggests that it serves to suture the corneal endothelium/Descemet's membrane to the dense interfacial matrix of the posterior stroma.

Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal-specific fibril morphology.

A number of factors have been implicated in the regulation of tissue-specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta-galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large-diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino-terminal domain of type V collagen was associated with the small-diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.

Differential expression of lumican and fibromodulin regulate collagen fibrillogenesis in developing mouse tendons.

Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.

Type V collagen: molecular structure and fibrillar organization of the chicken alpha 1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis.

Previous work from our laboratories has demonstrated that: (a) the striated collagen fibrils of the corneal stroma are heterotypic structures composed of type V collagen molecules coassembled along with those of type I collagen, (b) the high content of type V collagen within the corneal collagen fibrils is one factor responsible for the small, uniform fibrillar diameter (25 nm) characteristic of this tissue, (c) the completely processed form of type V collagen found within tissues retains a large noncollagenous region, termed the NH2-terminal domain, at the amino end of its alpha 1 chain, and (d) the NH2-terminal domain may contain at least some of the information for the observed regulation of fibril diameters. In the present investigation we have employed polyclonal antibodies against the retained NH2-terminal domain of the alpha 1(V) chain for immunohistochemical studies of embryonic avian corneas and for immunoscreening a chicken cDNA library. When combined with cDNA sequencing and molecular rotary shadowing, these approaches provide information on the molecular structure of the retained NH2-terminal domain as well as how this domain might function in the regulation of fibrillar structure. In immunofluorescence and immunoelectron microscopy analyses, the antibodies against the NH2-terminal domain react with type V molecules present within mature heterotypic fibrils of the corneal stroma. Thus, epitopes within at least a portion of this domain are exposed on the fibril surface. This is in marked contrast to mAbs which we have previously characterized as being directed against epitopes located in the major triple helical domain of the type V molecule. The helical epitopes recognized by these antibodies are antigenically masked on type V molecules that have been assembled into fibrils. Sequencing of the isolated cDNA clones has provided the conceptual amino acid sequence of the entire amino end of the alpha 1(V) procollagen chain. The sequence shows the location of what appear to be potential propeptidase cleavage sites. One of these, if preferentially used during processing of the type V procollagen molecule, can provide an explanation for the retention of the NH2-terminal domain in the completely processed molecule. The sequencing data also suggest that the NH2-terminal domain consists of several regions, providing a structure which fits well with that of the completely processed type V molecule as visualized by rotary shadowing.

Altered wound healing in mice lacking a functional osteopontin gene (spp1).

Osteopontin (OPN) is an arginine-glycine-aspartate (RGD)- containing glycoprotein encoded by the gene secreted phosphoprotein 1 (spp1). spp1 is expressed during embryogenesis, wound healing, and tumorigenesis; however, its in vivo functions are not well understood. Therefore, OPN null mutant mice were generated by targeted mutagenesis in embryonic stem cells. In OPN mutant mice, embryogenesis occurred normally, and mice were fertile. Since OPN shares receptors with vitronectin (VN), we tested for compensation by creating mice lacking both OPN and VN. The double mutants were also viable, suggesting that other RGD-containing ligands replace the embryonic loss of both proteins. We tested the healing of OPN mutants after skin incisions, where spp1 was upregulated as early as 6 h after wounding. Although the tensile properties of the wounds were unchanged, ultrastructural analysis showed a significantly decreased level of debridement, greater disorganization of matrix, and an alteration of collagen fibrillogenesis leading to small diameter collagen fibrils in the OPN mutant mice. These data indicate a role for OPN in tissue remodeling in vivo, and suggest physiological functions during matrix reorganization after injury.

Nuclear ferritin protects DNA from UV damage in corneal epithelial cells.

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3'-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.

Corneal and scleral collagen fiber formation in vitro.

We have investigated the role of structural differences in collagen molecules and the effect of proteoglycan preparations on the control of collagen fibril formation. Collagen and proteoglycans were extracted, purified and characterized from two structurally and functionally different connective tissues, rabbit corneal stroma and sclera. Corneal collagen was found to form fibers 6- to 7-times more slowly than scleral type I collagen. Proteoglycans from both sources retard fibrillogenesis, with corneal proteoglycans having approximately 3-times the effect observed with scleral proteoglycans. The morphology of the fibers formed was normal in all cases. Therefore, the changes observed may reflect a true control mechanism related to the strict morphological arrangement associated with corneal transparency.

Development and roles of collagenous matrices in the embryonic avian cornea.

Corneal development requires the production, assembly and sometimes replacement of a number of collagenous matrices. The embryonic chick cornea is well-characterized and offers certain advantages for studying the assembly and roles of these matrices. We will first describe the matrices to be examined. These include the corneal stroma proper, first formed as the primary stroma and subsequently as the secondary (mature) stroma; Bowman's Membrane; Descemet's Membrane; and the hemidesmosome of the epithelial cell attachment complex. We will then describe the characteristics of the collagen types involved, including: the fibrillar collagens (types I, II and V), the fibril-associated collagens (types IX, XII and XIV), and the transmembrane collagen of the hemidesmosome (type XVII). Then, in each subsequent section we will examine in detail the structure, assembly and development of each collagenous matrix, and how each specific collagen and/or combination of collagens are thought to provide the matrices with their unique properties. The work and views presented here are largely from our own laboratories. Thus, this article is not meant to be a comprehensive review of the literature. For pertinent references by others, when possible, we will cite recent reviews.

Development of tendon structure and function: regulation of collagen fibrillogenesis.

In the tendon, the development of mature mechanical properties is dependent on the assembly of a tendon-specific extracellular matrix. This matrix is synthesized by the tendon fibroblasts and composed of collagen fibrils organized as fibers, as well as fibril-associated collagenous and non-collagenous proteins. All of these components are integrated, during development and growth, to form a functional tissue. During tendon development, collagen fibrillogenesis and matrix assembly progress through multiple steps where each step is regulated independently, culminating in a structurally and functionally mature tissue. Collagen fibrillogenesis occurs in a series of extracellular compartments where fibril intermediates are assembled and mature fibrils grow through a process of post-depositional fusion of the intermediates. Linear and lateral fibril growth occurs after the immature fibril intermediates are incorporated into fibers. The processes are regulated by interactions of extracellular macromolecules with the fibrils. Interactions with quantitatively minor fibrillar collagens, fibril-associated collagens and proteoglycans influence different steps in fibrillogenesis and the extracellular microdomains provide a mechanism for the tendon fibroblasts to regulate these extracellular interactions.

Achilles tendons from decorin- and biglycan-null mouse models have inferior mechanical and structural properties predicted by an image-based empirical damage model.

Achilles tendons are a common source of pain and injury, and their pathology may originate from aberrant structure function relationships. Small leucine rich proteoglycans (SLRPs) influence mechanical and structural properties in a tendon-specific manner. However, their roles in the Achilles tendon have not been defined. The objective of this study was to evaluate the mechanical and structural differences observed in mouse Achilles tendons lacking class I SLRPs; either decorin or biglycan. In addition, empirical modeling techniques based on mechanical and image-based measures were employed. Achilles tendons from decorin-null (Dcn(-/-)) and biglycan-null (Bgn(-/-)) C57BL/6 female mice (N=102) were used. Each tendon underwent a dynamic mechanical testing protocol including simultaneous polarized light image capture to evaluate both structural and mechanical properties of each Achilles tendon. An empirical damage model was adapted for application to genetic variation and for use with image based structural properties to predict tendon dynamic mechanical properties. We found that Achilles tendons lacking decorin and biglycan had inferior mechanical and structural properties that were age dependent; and that simple empirical models, based on previously described damage models, were predictive of Achilles tendon dynamic modulus in both decorin- and biglycan-null mice.

Localization of collagen types I, III and V during tendon development. Changes in collagen types I and III are correlated with changes in fibril diameter.

Collagen types I, III and V were localized at different stages of tendon development: a stage when tendon architecture is established, but immature (14-day), a mature tendon (hatchling) and an intermediate point where there is a rapid growth of tendon fibrils (17-day). The tendon fascicles and their connective tissue investments (endotendenium) were studied. The data show a changing pattern of type III collagen expression in the developing metatarsal tendon. In the immature tendon at 14 days of development, collagen types I and III are codistributed throughout the fascicles and their connective tissue investments. At this stage all of the fibrils regardless of the site are small. With development the regions segregate and become easily recognizable. As this occurs, the fibril diameter distributions diverge; those in the fascicle become larger while those in the endotendenium remain small. During this period, the fascicle loses type III collagen expression, while the endotendenium becomes type III collagen rich. At the same time, the connective tissue investments develop, and the fibrils of the endotendenium remain small during this period, but then become larger in the mature tendon. The increases in diameter are associated with a decrease in type III collagen reactivity. At hatching, both significant collagen type III reactivity and small diameter fibrils are restricted to the outer sheaths. During all stages of tendon development there is a constant small, but detectable amount of type V collagen. However, no correlation between type V reactivity and fibril diameter was observed at any stage of development. These results indicate an inverse relationship between type III collagen reactivity and fibril diameter in the developing tendon.

Collagen fibrillogenesis in tissues, in a solution and from modeling: a synthesis.

Collagen fibril formation has been studied in tissues by light and electron microscopy; in solution by light scattering and microscopy; and from modeling based on the amino acid sequence of type I collagen. Taken together these studies indicate that collagen fibril assembly involves a stepwise formation of intermediate aggregates in which each intermediate is formed from earlier aggregates. In this sequence, monomeric collagen contributes only to the formation of early aggregates; and fibrils grow in length by the addition of intermediate aggregates to the end of a subfibril and in width by lateral wrapping of subfibrils. Modeling based on amino acid sequence data of possible intermolecular charge-charge interactions indicate 2 different kinds, one which promotes linear aggregation and the other which promotes linear aggregation. The effects of different collagens and coprecipitants such as glycoproteins and proteoglycans can begin to be explained by their influence on the character of intermediate subassemblies. Ultrastructural data from 2 tissues, embryonic cornea and tendon, indicate that the site of fibril growth and assembly is at the cell surface.

Characterization of collagen fibril segments from chicken embryo cornea, dermis and tendon.

The cornea, dermis and tendon have extracellular matrix architectures with differences in fibril diameter, packing and organization. An early step in fibril assembly is the formation of a striated fibril of discrete length (segment). Fibril segments were isolated from developing chicken cornea, dermis and tendon by physical disruption and the structure characterized. In all three tissues, intact but relatively short fibril lengths were isolated. These segments were asymmetric, having long (alpha) and short (beta) tapered ends. They were also centrosymmetric with respect to molecular packing. Segments isolated from 12- to 16-day corneas, dermis and tendons had identical structures, but their lengths and diameters were distinct. We propose that the increase in length is, at least in part, the result of lateral associations of adjacent segments. In the developing tendon, there is a rapid increase in length and diameter between day 16 and 17, while in the dermis the increase is more linear with respect to time. In the cornea, the fibril segments grow longer, but their diameters remain constant. Disruption of corneas in phosphate-buffered saline yielded larger diameter segments than seen in situ, while tendon or dermis maintained tissue-specific diameters. When corneas were disrupted in buffers that stabilized the water layer associated with the collagen molecules or containing the corneal proteoglycans, then tissue-specific diameters were maintained. These data suggest differences in the stabilization of segments during growth in tissues where diameter increases versus those where diameter remains constant, and this may be related to collagen-proteoglycan interactions.

Complete primary structure of the chicken alpha1(V) collagen chain.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

Differences in integrin expression during avian corneal stromal development.

PURPOSE: The purpose of this study was to determine whether there are changes in integrin expression associated with the spatial and temporal variations in matrix expression that occur during specific stages in corneal stromal development. METHODS: Immunofluorescence techniques were used to analyze beta 1-containing integrins and alpha v beta 3 localization both in situ and in cell cultures. RESULTS: In situ, beta 1 and alpha v beta 3 were present with different patterns of localization, and these varied with developmental stage. beta 1-containing integrins were present on most cells, whereas alpha v beta 3 was present on cells at the corneal-scleral epitheliomesenchymal interface during migration of keratocyte precursors; very little alpha v beta 3 was localized in keratocytes. Keratocytes and undifferentiated periocular mesenchyme cells grown in vitro also exhibited differences in localization of beta 1-containing integrins and alpha v beta 3. All focal adhesions contained beta 1, whereas a subset contained both beta 1 and alpha v beta 3, indicating potential functional differences in focal adhesions. In addition, most periocular mesenchyme cells exhibited alpha v beta 3-containing focal adhesions throughout, but the majority of keratocytes contained only peripherally located alpha v beta 3-positive focal adhesions. The localization of both beta 1-containing integrins and alpha v beta 3 was modulated by time allowed for attachment and spreading. CONCLUSIONS: Keratocytes and undifferentiated periocular mesenchyme cells exhibit developmental differences in integrin localization in situ. These two cell types also exhibit different patterns of alpha v beta 3 localization in vitro, possibly as a result of developmental differences in ligand-binding properties. beta 1-containing integrins and alpha v beta 3 define different types of focal adhesions, implying different functions. These differences in expression may be important in the initiation of cellular migration in the early stages of corneal development, as well as in the transition from the undifferentiated to the differentiated keratocyte phenotype.

Organization of collagen types I and V in the embryonic chicken cornea.

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 degrees C or 37 degrees 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 the fibrillar structure 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.