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Anchoring of Epithelia to Underlying Connective Tissue: Evidence of Frayed Ends of Collagen Fibrils Directly Merging with Meshwork of Lamina Densa

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Anchoring of Epithelia to Underlying Connective Tissue: Evidence of Frayed Ends of Collagen Fibrils Directly Merging with Meshwork of Lamina Densa / Electron Mkrosc 43: 264-271 (1994) Anchoring of Epithelia to Underlying Connective Tissue: Evidence of Frayed Ends of Collagen Fibrils Directly Merging with Meshwork of Lamina Densa Eijiro Adachi and Toshihiko Hayashi* Department of Anatomy, Osaka University Medical School Sidta, 565 Japan 'Department of Chemistry, College of Arts and Sciences, The University of Tokyo, Tokyo, 153 Japan We examined the epithelial-subepithelial junction of mouse pancreas, human placenta and monkey oral mucosa. In mouse pancreas, in quick-freeze, deep-etch, rotary-replicated micrographs, collagen fibrils ran close to the lamina densa and frayed out into two or three subfibrils merging with lamina densa meshwork. In placenta! villi, some collagen fibrils ran toward trophoblasts and probably merging with lamina densa. In oral mucosa, some collagen Downloaded from https://academic.oup.com/jmicro/article/43/5/264/895801 by guest on 30 September 2021 fibrils curved to epithelial cells, passed through the anchoring fibril network and apparently merged with the basal surface of lamina densa. Together with the morphology of reconstituted collagen fibrils from type V collagen and hybrid fibrils from type V and type I collagen and the results presented here, we propose that a direct connection of collagen fibrils with lamina densa could be a ubiquitous anchoring system to stabilize epithelia] tissues. Key words: anchoring system, collagen fibrils, lamina densa, type V collagen, type IV collagen A great number of studies have been done on basement mucosa, which contain type V collagen,1 *' to see whether membranes and collagen fibrils in various tissues showing collagen fibrils in lamina fibroreticularis connect directly chemical composition, structure and role in biological with the lamina densa. processes such as morphogenesis and regeneration.12* Thin-section images show the banding pattern of Basement membranes are subdivided into three layers: collagen fibrils but give a very restricted two-dimensional lamina lucida, lamina densa, and lamina fibrore- view of the interface between the lamina densa and ticularis.3' The lamina densa is an electron-dense layer fibroreticularis. We compare these with quick-freeze, connected with cell membranes by thin filaments deep-etch, rotary-shadow images which give three- traversing the lamina lucida. Its major components are dimensional views. Preliminary reports on some of four type IV collagen, laminin, and heparan sulfate pro- findings have appeared in abstract form.12' teoglycans.4' Lamina fibroreticularis is essentially the part of the stroma closest to lamina densa and composed MATERIALS AND METHODS of collagen fibrils (approximately 30 nm in diameter), microfibrils, anchoring fibrils, and elastic fibers. The Freshly obtained pancreas from ddY mice was prepared collagen fibrils have a 67-nm periodic banding pattern for quick freezing. Small pieces of pancreas were placed and are ubiquitously distributed in the lamina fibro- on metal plates, then slammed with a Polaron slammer reticularis of any tissue. The other components vary in onto a copper-block precooled with liquid helium. Frozen proportion in different tissues. In vascular tissue, for 5) tissues were transferred to a Bakers BAF400, fractured, example, microfibrils are major components, in stratified deep-etched and rotary replicated with platinum-carbon. squamous epithelia anchoring fibrils,6' and in respiratory 7 The replicas were examined with a transmission electron epithelia elastic fibers. ' The connection between the microscope. collagen fibrils and the lamina densa remains unclear. It Pancreatic tissues for thin section were obtained from is believed that in human skin and cornea, the collagen ddY mice after perfusion with 2% paraformaldehyde, fibrils are entrapped by a network of anchoring fibrils 8 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. connecting them with the lamina densa. ' In dominant The tissues were dissected into small pieces with a razor dystrophic epidermolysis, however, even through anchor- blade and immersed in the same fixative for 2 hr at 4°C. ing fibrils are found along the epidermal-dermal junctions, 9 After rinsing with 0.1 M cacodylate buffer, pH 7.4, they blisters are formed after a slight scratching of the skin, ' were postfixed for 2hr in 0.1 M cacodylate buffer a finding which suggests that anchoring fibrils do not containing 2% 0s04. They were then immersed in the adequately connect the epidermis with the dermis. We same buffer containing 1% tannic acid for 1 hr at room suggest that a direct connection of collagen fibrils with temperature to enhance the contrast of collagen fibrils the lamina densa is possible. It should be possible that under an electron microscope.13' type V collagen fibrils, when reconstituted, fray out into subfibrils at their ends in tissues.10' We have examined Normal term placentae were obtained immediately tissues from mouse pancreas, placental villi, and oral after delivery. Pieces of placental tissue were immersed in 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M 264 / Electron Miarosc Anchoring of Collagen Fibrils into Lamina Densa 265 cacodylate buffer, pH 7.4 for 4hr at 4°C. They were mucosae were dissected into small pieces and immersed postfixed for 2 hr in 0.1M cacodylate buffer containing in the above fixative for 2 hr at 4°C, and then post-fixed 2% OsO4. Oral mucosae were obtained from snow in 2% OsOv Pancreatic, placenta!, and mucosal tissues monkeys (Macacafuscata) after perfusion with 0.2% picric were embedded in epoxy resin, sectioned, and the thin- add, 4% parafonnaldehyde and 0.5% glutaraldehyde in sections were examined with an electron microscope. 0.1 M phosphate buffer (pH 7.4) under anesthesia. The Downloaded from https://academic.oup.com/jmicro/article/43/5/264/895801 by guest on 30 September 2021 Fig. 1. Qutck-freerc deep-etching electron micrograph of extracellular space in mouse pancreas. Lamina densa (D) is found on the basal surface of adnar cells (A). Periodical ridges and furrows are observed on the collagen fibrils. Collagen fibrils (30-40 nm in diameter) gather In bundles (B). Many fibrils subdivide into two or more subfibrtls (arrows) and run individually in various directions in lamina fibroreticularis. Mlcrofibrils (arrowheads) are also seen under the lamina densa. Bar=100nm. Vol. 43, No. 5, 1994 266 E. Adachi and T. Hayashi Downloaded from https://academic.oup.com/jmicro/article/43/5/264/895801 by guest on 30 September 2021 Fig. 2. Quick-freeze, deep-etching electron micrographs of lamina densa in mouse pancreas, a) Anchoring filaments (arrow) traverse lamina lucida to connect lamina densa with the cell membrane. Collagen fibrils (arrowheads) and microfibrils (double arrow) run underneath the lamina densa (D). One collagen fibril (asterisk), dislocated by the fracture, merges with lamina densa (D). Acinar cell (A). Bar 100 nm. b) Higher magnification of the framed area in Fig. 2a, showing collagen fibril (asterisk) subdividing into two subfibrils and merging with meshwork (arrowheads) of lamina densa (D). Bar=50nm. RESULTS approximately 15nm in width. Occasionally, collagen fibrils approached the lamina densa, and divided into two In quick-freeze, deep-etch electron micrographs, of or more subfibrils to merge with the strands of the lamina mouse pancreas extracellular matrix, periodical furrows densa. Even when the fibrils were obviously dislocated and ridges, corresponding to the 67-nm periodic banding by the fracture, they merged smoothly into the lamina pattern, were shown on individual fibrils of 40 nm densa. This image suggested that some collagen fibrils diameter, clearly identifying these as collagen fibrils. In connected with the meshwork of lamina densa smoothly the lamina fibroreticularis, collagen fibrils tended to run in spite of dislocation. Collagen fibrils piercing the lamina densa and running in lamina lucida as reported by at random, and occasionally to subdivide into two or 14 more. In deeper regions, they formed bundles. In the Campbell and co-workers, ' however, were not observed three-dimensional, deep-etch, electron micrograph im- (Fig. 2a, b). ages, the lamina densa and lamina lucida were clear, In thin-section, merging of collagen fibrils into lamina also microfibrils, and the absence of banding pattern was densa was suggested although the images were not as clear as in the thin-section images. Microfibrils formed absolutely certain (Fig. 3). Again in deep-etch, electron three-dimensional networks in the lamina fibroreticularis. micrograph images, collagen fibrils (approximately 40 nm Some collagen fibrils divided into subfibrils to form in width) approached the lamina densa and merged with collagen networks in the lamina fibroreticularis (Fig. 1). the meshwork of the lamina densa. The fibrils appeared Lamina densa appeared as a continuous sheet of fine to grow flat (approximately 50 nm in width) just before meshwork lining acinar cells, and was connected with merging with the fine strands in lamina densa (Fig. 4a, cell membrane by anchoring filaments, approximately b). lOnm in diameter, traversing the lamina lucida. The Concerning other tissues, in placental villi, simple meshworks in lamina densa is composed of fine strands squamous epithelia, from a loosely woven network of / Electron Microsc Anchoring of Collagen Fibrils into Lamina Densa Downloaded from https://academic.oup.com/jmicro/article/43/5/264/895801 by guest on 30 September 2021 Fig. 3; Thin section of acinar cefls (A) shows collagen fibril (arrow-
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