The Golgi apparatus in chick corneal epithelium: changes in intracellular position during development.

The intracellular position of the Golgi apparatuses in the basal cell layer of the corneal epithelium in embryonic and hatched chicks has been studied in the light microscope by impregnating the Golgi apparatus with silver. During two distinct periods in development the Golgi apparatuses in the basal cells shift from an apical to basal position. Each of these periods correlates in time with the appearance of an acellular collagenous matrix beneath the epithelium. Examination of the basal epithelial cells in the electron microscope confirms the intracellular shifts in position of the Golgi apparatus. The results suggest that the Golgi apparatus shifts to the basal cell pole of the corneal epithelium in order to excrete connective tissue materials into the developing corneal stroma.

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 Our interpretation is that this third compartment forms as cell processes retract and cytoplasm is withdrawn between bundles.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.

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.

Morphogenesis of the collagenous stroma in the chick cornea.

The embryonic chick corneal epithelium produces a highly structured acellular matrix beneath its basal surface during early development. This matrix, which contains collagen, serves as a morphogenetic template for subsequent stromal development in that the three-dimensional architecture of the adult corneal stroma is initially established, in miniature, in this epithelially derived connective tissue. Examination of the early corneal epithelium and matrix in both the light and electron microscope suggests that self assembly of the matrix may be one of several important factors in the morphogenesis of this early connective tissue.

Epithelial collagens and glycosaminoglycans in the embryonic cornea. Macromolecular order and morphogenesis in the basement membrane.

The embryonic corneal epithelium synthesizes both collagen and chondroitin sulfate and excretes them across the basement membrane into the subepithelial space where they assemble into a spiraling orthogonal matrix of fibrils. The assembly of collagen into fibrils is first apparent at the outer face of the basement membrane in a region of ordered chondroitin sulfate molecules. Hyaluronate, another morphogenetically important corneal macromolecule, is produced at these early stages only by the inner endothelium. These correlated biosynthetic and ultrastructural data demonstrate discrete macromolecular products of the two corneal epithelia with differing morphogenetic properties and functions.

Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.

Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19--22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells.

Vitreous body collagen. Evidence for a dual origin from the neural retina and hyalocytes.

Two different cells types have been shown to synthesize embryonic chick vitreous collagen (vitrosin) at different stages of development. Identification of vitrosin was established by labeling the embryos in ovo [3H]proline at stages 23 and 28 and separating the extracted vitreous collagen alpha-chains by carboxymethylcellulose chromatography. The labeled collagen consisted predominately of alpha 1 chains, indicating a molecule in the form of a trimer of identical chains designated (alpha 1)3. The molecular weight of the labeled chains measured approximately 95,000 daltons by molecular sieve chromatography, and contained 41% of their imino acid as 4-hydroxyproline. To establish which eye tissues synthesize vitrosine, the collagens produced in organ culture by the isolated neural retina, lens and vitreous body from stages 26-27, 29-30, and 40 were examined. At the two earlier stages, only the neural retina synthesized large quantities of (alpha 1)3 collagen whereas the lens and the cells within the vitreous body itself synthesized relatively small amounts of collagen characterized by an alpha 1:alpha 2 ratio of about 2:1. At stage 40, however, the cells of the vitreous body itself synthesized the greatest quantities of collagen, which now was predominantly an (alpha 1)3 type molecule. Stage 40 neural retina and lens synthesized lesser amounts of collagen with an alpha 1:alpha 2 ratio of 2 to 3:1. Chick vitrosin thus appears to be synthesized by the neural retina in early embryonic stages, whereas the major contribution derives from cells within the vitreous body in later development.

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes.

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.

Cartilage collagen: a staggered substructure in reconstituted fibrils.

In contrast to the typical transverse banding pattern of native and reconstituted skin collagen fibrils, reconstituted fibrils of cartilage collagen have an oblique banding pattern that results from a regular axial shift (89 angstroms) of component "subfibrils." The 89-angstrom shift may be related to the major helix of the collagen molecule.

Collagen synthesis in vitro by embryonic spinal cord epithelium.

Isolated spinal cords from chick embryos (stages 12 to 15) were incubated in vitro with radioactive proline. The proteins synthesized were fractionated by coprecipitation with added carrier collagen, followed by molecularsieve and ion-exchange chromatography. A portion of the isotopically labeled proteins were found to be collagen molecules consisting only of alpha1 chains.

A collagen defect in homocystinuria.

The biochemical mechanism accounting for the connective tissue abnormalities in homocystinuria was explored by examining the effects of various amino acids known to accumulate in the plasma of patients with this disease on cross-link formation in collagen. Neutral salt solutions of purified, rat skin collagen, rich in cross-link precursor aldehydes, were polymerized to native type fibrils by incubating at 37 degrees C in the presence of homocysteine, homocystine, or methionine. After the polymerization was completed, each sample was examined for the formation of covalent intermolecular cross-links, assessed indirectly by solubility tests and directly by measuring the cross-link compounds after reduction with tritiated sodium borohydride and hydrolysis. Collagen solutions containing homocysteine (0.01 M-0.1 M) failed to form insoluble fibrils. Furthermore, much less of the reducible cross-links, Delta(6,7) dehydrohydroxylysinonorleucine, Delta(6,7) dehydrohydroxylysinohydroxynorleucine, and histidino-dehydrohydroxymerodesmosine were formed in the preparations containing homocysteine as compared with the control and the samples containing methionine or homocystine. The content of the precursor aldehydes, alpha-aminoadipic-delta-semialdehyde (allysine) and the aldol condensation product, was also markedly diminished in tropocollagen incubated with homocysteine. It is concluded that homocysteine interferes with the formation of intermolecular cross-links that help stabilize the collagen macromolecular network via its reversible binding to the aldehydic functional groups. Analysis of the collagen cross-links in skin biopsy samples obtained from three patients with documented homocystinuria showed that the cross-links were significantly decreased as compared with the age-matched controls, supporting the tentative conclusions reached from the in vitro model studies. In addition, the solubility of dermal collagen in non-denaturing solvents was significantly increased in the two patients examined, reflecting a functional defect in collagen cross-linking. Although the concentration of homocysteine used in this study to demonstrate these effects in vitro is clearly higher than that which is observed in homocystinuric's plasma, the data do suggest a possible pathogenetic mechanism of connective tissue defect in homocystinuria.

Epidermolysis bullosa dystrophica recessive fibroblasts altered behavior within a collagen matrix.

Normal human fibroblasts incorporated into a collagen lattice reduce the size of that lattice over a period of time. Lattice size reduction or lattice contraction is directly related to initial cell number. When equal numbers of fibroblasts derived from patients with epidermolysis bullosa dystrophica recessive, (EBdr), are used, there is delayed lattice contraction. The EBdr fibroblasts have an altered cellular shape, when compared to normal cells, in that the EBdr cells fail to flatten out and elongate, but do attach to collagen fibers like normal fibroblasts. EBdr fibroblasts maintain a rounded shape with numerous filopodia radiating from the cell periphery and such filopodia are attached to the collagen fibers of the lattice. In monolayer tissue culture on glass surfaces, EBdr fibroblasts are three times more likely to grow over neighboring fibroblasts. EBdr cell filopodia structures are attached to the cell surfaces lying beneath them, which demonstrates another condition of altered anchorage attachment of EBdr fibroblasts.

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.

Age-dependent effects of glutamate on photoreceptor cells of the isolated chick embryo retina.

Neurotoxic and gliotoxic effects of glutamate were studied in isolated chick embryo retinas of various ages. Employing a short-term incubation system, we found that initial cellular changes in retinas from 15- and 21-day chick embryos were localized to glial Müller cells as previously shown in retinas from 12-day embryos. In the older retinas, however, an additional selective lesion was consistently found in the presumptive photoreceptor cells. Similar photoreceptor damage was not seen in 12-day retinas, even after treatment with relatively high glutamate doses. Autoradiographic uptake of tritiated glutamate in retinas at 12 and 14 days was localized to the glial Müller cells; in the 14-day retinas, however, there was also uptake of the labeled amino acid into the developing inner segments. Uptake of tritiated glutamate in retinas at younger (8-day) and older (21-day) ages did not show any obvious localization of the label.

Glutamate-induced cellular injury in isolated chick embryo retina: Müller cell localization of initial effects.

The neurotoxic and gliotoxic effects of glutamate and several glutamate analogues were studied in isolated chick embryo retinas. To facilitate examination of initial pathological events, a short-term incubation system was developed and used for light microscopic and autoradiographic investigation. Low-dose, short-term glutamate treatment of 12-day retinas resulted in a glial-specific lesion in the Müller cells, characterized by extensive cellular edema; at higher concentrations and/or longer treatment times, neurotoxic as well as gliotoxic effects were seen. The early glial damage was identical in appearance to that seen after incubation with DL-alpha-aminoadipate and other reported gliotoxins. No evidence of a similar glial-specific action was seen after administration of kainic acid, although extensive neuronal degeneration did result. Incubation of retinas with tritiated glutamate (3H-glu) revealed a selective uptake of the label by Müller cells. Autoradiographic grains were localized over Müller foot processes at the inner limiting membrane, and by 30 minutes labeled the entire glial system. Prior treatment with neurotoxic levels of glutamate did not alter the autoradiographic localization to glial cells. Possible glial-neuronal interactions and their effect on cytotoxic patterns are discussed.

Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study.

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.

Immunocytochemical localization of Mullerian inhibiting substance in the rough endoplasmic reticulum and Golgi apparatus in Sertoli cells of the neonatal calf testis using a monoclonal antibody.

Mullerian Inhibiting Substance (MIS) has been localized in the Sertoli cells of the neonatal calf testis using preembedding immunoperoxidase techniques and a monoclonal antibody which almost completely blocks the biological activity of MIS. Both the peroxidase-labeled antibody method using a peroxidase-conjugated F(ab')2 fragment of IgG as a second antibody and the unlabeled antibody peroxidase-antiperoxidase (PAP) method using Fab fragments of the PAP complex were employed. With both methods, MIS was demonstrated within the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus. In the Golgi, MIS was concentrated in the transmost cisternae especially at their peripheral expansions. This study indicates that MIS is synthesized in the RER and transported to the Golgi apparatus, presumably for glycosidation, before secretion from Golgi derived vacuoles.

Omphalocele and multiple severe congenital anomalies associated with osteodysplasty (Melnick-Needles syndrome).

Osteodysplasty (Melnick-Needles syndrome, MNS), a severe bone dysplasia with presumed autosomal dominant inheritance, has now been described in 24 individuals, with a predominance of females (21:3). We report an affected woman who gave birth to a male infant with omphalocele, hypoplastic kidneys, and the skeletal changes of this disorder; he died soon after birth. Histologic studies of the calvaria and long bones showed normal maturational sequences, but suggest that remodeling was not normal. This is the second known instance of a male infant with omphalocele and this skeletal dysplasia born to a woman with MNS. We suggest that the gene for the MNS may also cause a syndrome of multiple abnormalities that can be lethal and that this more severe phenotype in males may account for the altered sex ratio among reported cases. Both X-linked dominant and autosomal-dominant sex-limited inheritance are feasible interpretations of the existing information.

Nanophthalmic sclera. Ultrastructural, histochemical, and biochemical observations.

Scleral tissue from two cases of nanophthalmos was examined by amino acid analysis, light microscopy, histochemistry, and transmission electron microscopy. Perifibrillar aggregates, similar to proteoglycans, were prominent in the nanophthalmic sclera. The sclerae were thicker than normal and the bundles of collagen fibrils were less ordered. The clinical features of vortex vein compression seem causally related to the disordered and thickened sclera, which, in turn, may be caused by dysfunctional proteoglycans, or interaction with the scleral collagen, or both.

Fibrous histiocytoma of the trachea.

The light and electron microscopic features of a fibrous histiocytoma of the trachea that occurred in a 15-year-old Caucasian girl are presented. Emphasis is placed on the aggresive behavior and the importance of early recognition of the lesion in an unusual location.