John M. Fitch

Dual origin of glomerular basement membrane.

The histogenesis of renal basement membranes was studied in grafts of avascular, 11-day-old mouse embryonic kidney rudiments grown on chick chorioallantoic membrane (CAM). Vessels of the chick CAM invade the mouse tissue during an incubation period of 7-10 days and eventually hybrid glomeruli composed of mouse epithelium and chick endothelium form. Formation of basement membranes during this development was followed by immunofluorescence and immunoperoxidase stainings using polyclonal and monoclonal antibodies against mouse and chick collagen type IV and against mouse laminin. These antibodies were species-specific as shown in immunochemical and immunohistologic analyses. The glomerular basement membrane contained both mouse and chick collagen type IV, demonstrating its dual cellular origin. All other basement membranes were either exclusively of chick origin (mesangium, vessels) or of mouse origin (tubuli, Bowmans capsule).

Temporal expression of types XII and XIV collagen mRNA and protein during avian corneal development.

Using immunohistochemistry and competitive PCR for collagen types XII and XIV, we have followed the expression of these fibril‐associated molecules during development of the avian cornea. By immunofluorescence histochemistry, both molecules are found in the acellular primary stroma and are therefore presumably of epithelial origin. During formation and development of the secondary corneal stroma, which is populated by mesenchymal cells, the molecules generally appear to be spatially segregated from each other. Type XIV collagen is found throughout most of the stroma, and therefore is predominantly a product of stromal fibroblasts. During subsequent compaction of the cornea, an event necessary for corneal transparency, the collagen XIV mRNA level increases dramatically, suggesting that this molecule may play a role in this event.

Are collagen fibrils in the developing avian cornea composed of two different collagen types? Evidence from monoclonal antibody studies.

In the avian corneal stroma, the collagen is organized in orthogonal lamellae, each of which is composed of numerous striated collagen fibrils of uniform, thin (25 nm) diameter.’.’ This is true for both the epithelially derived primary stroma of the early embryo, and the mesenchymally derived secondary stroma of the more mature embryo and adult. In most other connective tissues, such as the adjacent sclera, the diameters of the collagen fibrils are thicker and more variable. It has been suggested that thin, uniform diameter fibrils are requisite for corneal transparency; a sequella of severe corneal injury may be the local deposition of large, randomly oriented collagen fibrils resulting in an opaque scar. The collagen fibril, then, is a major functional unit of the corneal stroma, as well as of the stromal matrices of other organs. These units form by a process of “molecular morphogenesis” termed fibrillogenesis.’ In this process, individual collagen molecules or their procollagen precursors become assembled into supramolecular structures having the proper diameters, lengths, and spatial relationships to each other. How is this assembly achieved and controlled? Results from molecular self-assembly and

Collagen fibril assembly by corneal fibroblasts in three-dimensional collagen gel cultures: Small-diameter heterotypic fibrils are deposited in the absence of keratan sulfate proteoglycan

Extracellular matrix assembly is a multistep process and the various steps in collagen fibrillogenesis are thought to be influenced by a number of factors, including other noncollagenous matrix molecules. The synthesis and deposition of extracellular matrix by corneal fibroblasts grown within three-dimensional collagen gel cultures were examined to elucidate the factors important in the establishment of tissue-specific matrix architecture. Corneal fibroblasts in collagen gel cultures form layers and deposit small-diameter collagen fibrils (approximately 25 nm) typical of the mature corneal stroma. The matrix synthesized contains type VI collagen in a filamentous network and type I and type V collagen assembled as heterotypic fibrils. The amount of type V collagen synthesized is relatively high and comparable to that seen in the corneal stroma. This matrix is deposited between cell layers in a manner reminiscent of the secondary corneal stroma, but is not deposited as densely or as organized as would be found in situ. No keratan sulfate proteoglycan, a proteoglycan found only in the corneal stroma, was synthesized by the fibroblasts in the collagen gel cultures. The assembly and deposition of small-diameter fibrils with a collagen composition and structure identical to that seen in the corneal stroma in the absence of proteoglycans typical of the secondary corneal stroma imply that although proteoglycan-collagen interactions may function in the establishment of interfibrillar spacing and lamellar organization, collagen-collagen interactions are the major parameter in the regulation of fibril diameter.

Monoclonal antibody analysis of ocular basement membranes during development

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histological sections of ocular tissues from 4 1/2- to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemets membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowmans membrane and Descemets membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1/2- to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.

Ferritoid, a Tissue-specific Nuclear Transport Protein for Ferritin in Corneal Epithelial Cells

Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of ∼50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).

Multiple-reaction cycling: a method for enhancement of the immunochemical signal of monoclonal antibodies.

Most current studies using immunochemical and immunohistochemical procedures to detect antigen-antibody complexes employ some type of indirect method. Such procedures afford signal amplification because several marker-conjugate molecules can bind to each primary antibody molecule. We have observed that for monoclonal antibodies an even greater amplification can be afforded simply by performing two (or more) reaction cycles (i.e., primary antibody, secondary antibody-primary antibody, secondary antibody-etc). In the present report, we demonstrate the utility of this method for immunohistochemical (immunofluorescence) and immunochemical (ELISA: enzyme-linked immunosorbent assay) procedures employing well-characterized monoclonal antibodies directed against avian type IV (basement membrane) collagen.

Collagen Type IX and Developmentally Regulated Swelling of the Avian Primary Corneal Stroma

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5½–6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non‐crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1–2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP‐2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue‐specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling. Dev. Dyn. 1998;212:27–37.

Monoclonal Antibodies that Distinguish Avian Type I and Type III Collagens: Isolation, Characterization and Immunolocalization in Various Tissues

Monoclonal antibodies were prepared that were specific for chicken type I and type III collagens. The specificity of these antibodies was determined by ELISA, inhibition ELISA, and immunoblot assays. The results showed that the monoclonal antibodies were specific for their respective antigens without significant cross reactivity to other types of collagen. An analysis of the location of the epitopes by rotary shadowing that a monoclonal antibody for type I collagen (called DD4) recognized type I procollagen close to the large globular domain at the carboxyl terminus of the molecule. A monoclonal antibody for type III collagen (called 3B2) recognized both the intact type III molecule and also the TCA fragment of type III collagen after mammalian collagenase digestion. The epitope was located approximately one-fifth of the distance from the amino-terminus of the intact molecule. The monoclonal antibodies were used for immunolocalization of type I and type III collagens in cryosections of heart, aorta, kidney, liver, thymus, skin, gizzard and myotendinous junction. In heart, aorta, kidney, liver, thymus and skin, type I and III collagens were colocalized in the connective tissue of each organ. In contrast, gizzard and myotendinous junction showed distinctly different staining patterns for the distribution of type I and type III collagen. The two monoclonal antibodies reported here are potentially useful reagents to study fibril formation involving type I and type III collagens.

Cellular invasion of the chicken corneal stroma during development: Regulation by multiple matrix metalloproteases and the lens

Avian corneal development requires cellular invasion into the acellular matrix of the primary stroma. Previous results show that this invasion is preceded by the removal of the fibril‐associated type IX collagen, which possibly stabilizes matrices through interfibrillar cross‐bridges secured by covalent crosslinks. In the present study, we provide evidence for the expression of three matrix metalloproteinases (MMPs) in early corneas, two of which act cooperatively to selectively remove type IX collagen in situ. In organ cultures, MMP inhibitors (either TIMP‐2 or a synthetic inhibitor) resulted in arrested development, in which collagen IX persisted, and the stroma remained compact and acellular. We also show that blocking covalent crosslinking of collagen allows for cellular invasion to occur, even when the removal of type IX collagen is prevented. Thus, one factor regulating corneal invasion is the physical structure of the matrix, which can be modified by either selective proteolysis or reducing interfibrillar cross‐bridges. We also detected another level of regulation of cellular invasion involving inhibition by the underlying lens. This block, which seems to influence invasive behavior independently of matrix modification, is a transient event that is released in ovo just before invasion proceeds. Developmental Dynamics 232:106–118, 2005.