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. KEY WORDS small intestine epithelioid cell cultures cell-specific antigensSmall intestinal epithelial cells represent a rapidly renewing cell population characterized by a precise segregation between mitotically active cells, present in the crypt region, and mature differentiated villus cells, mitotically inactive. Intestinal cells have a rapid cell turnover, with a mean cell duration time of 2-3 d in most animals (5,6,8,27). The differentiation of the mitotically active crypt cells is accompanied by dramatic changes in enzyme and transport activities and in cell morphology, including the appearance of a well-organized brush border at the luminal surface and a more columnar cell shape. It is of interest that, although intestinal crypt cells have one of the shortest cell cycle times in vivo (5), the occurrence 248 J, CELL BIOLOGY 9 The Rockefeller University Press

Abstract. The formation of collagen fibrils, fibril bundies, 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 extraceUular 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.

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 p,m 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.The rigid control of collagen fibril structure and the arrangement of fibrils into a specific three-dimensional architecture is necessary for the development of a transparent corneal stroma. In this paper we demonstrate how the corneal fibroblast exerts control over collagen fibrillogenesis and the positioning of newly formed fibrils into a highly ordered lamellar matrix.The mature chick corneal stroma is composed of collagen fibrils arranged as lamellae parallel to the corneal surface. Collagen fibrils within a lamella have the same orientation; collagen fibrils in adjacent layers are oriented approximately at fight angles, forming an orthogonal grid. The orthogonal corneal lamellae are composed of small diameter (25 nm) collagen fibrils equidistantly spaced (1, 2). The orthogonal lamellae describe a gradual clockwise shift from epithelium to endothelium of approximately 220 °. This clockwise spiral pattern resembles a cholesteric liquid crystal and has the same handedness in both eyes (2, 3); in contrast, all other ocular features, such as the pattern of overlap of the scleral ossicles, demonstrate mirror symmetry (4).Morphogenesis of the chick corneal stroma occurs in a sequence of relatively well-described stages (for review, see reference 5). Initially, the corneal epithelium deposits the primary corneal stroma beneath its basal surface. The pattern of this epithelially derived acellular stroma is i...

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epitheial cells bears both heparan and chondroitin sulfate chains and is recognized by the monodonal 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 ofplasma cells and Leydig cells. Squamous and transitional epitheia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface ofthe basal cells. Within squamous and transitional

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