Mesenchyme cells degrade epithelial basal lamina glycosaminoglycan

We investigated whether turnover of basal lamina glycosaminoglycan (GAG), an active process during epithelial morphogenesis, involves the mesenchyme. Fixed, prelabeled, isolated mouse embryo submandibular epithelia were prepared retaining radioactive surface components, as determined by autoradiographic and enzymatic studies, and a basal lamina, as assessed by electron microscopy. Recombination of mouse embryo submandibular mesenchyme with these epithelia stimulates the release of epithelial radioactivity when the labeled precursor is glucosamine or glucose but not when it is amino acid. The release is linear with time during 150 min incubation. Augmented release of epithelial label requires living mesenchyme which must be close proximity with the epithelia. Although heterologous mesenchymes, including lung, trachea, and jaw, stimulate the release of submandibular epithelial label, epithelial tissues do not. The label released by intact submandibular mesenchyme from prelabeled epithelia is in GAG and in two unique fractions: heterogeneous materials of tetrasaccharide or smaller size and N-acetylglucosamine. Enzymatic treatment of the heterogeneous materials revealed the presence of glycosaminoglycan-derived oligosaccharides. These unique products were not obtained by incubating prelabeled epithelia with a mesenchymal cell extract, suggesting that intact mesenchymal cells are required. N-Acetylglucosamine was also released when mesenchyme was recombined with living prelabeled epithelia which contained labeled basal laminar GAG. Our results establish that submandibular epithelial basal lamina GAGs are degraded by submandibular mesenchyme. We propose that one mechanism of epithelial-mesenchymal interaction is the degradation of epithelial basal laminar GAG by mesenchyme.     

Fenestrations of the basal lamina of intestinal villi of the rat

Fenestrations of the basal lamina of rat intestinal villi were revealed by scanning electron microscopy after removal of the overlying epithelial cells by osmic acid maceration. These fenestrations are circular to oval in shape and are 0.5 micron to 5 microns in diameter. They are richly distributed at a density of 1-2 X 10(4)/mm2 in the upper two thirds of the villi, except at the very tips. Roughly 500 fenestrations are found on each side of an average sized tongue-shaped villus. Transmission electron-microscopic observations showed that these fenestrations were passages for migrating cells of the immune system such as lymphocytes, eosinophils and macrophages. Protrusions from the basal parts of epithelial cells were also observed passing through these fenestrations. These findings are discussed with respect to their immunological implications and to the passage of nutrients.     

Type I collagen reduces the degradation of basal lamina proteoglycan by mammary epithelial cells

When mouse mammary epithelial cells are cultured on a plastic substratum, no basal lamina forms. When cultured on a type I collagen gel, the rate of glycosaminoglycan (GAG) synthesis is unchanged, but the rate of GAG degradation is markedly reduced and a GAG-rich, basal lamina-like structure accumulates. This effect of collagen was investigated by comparing the culture distribution, nature, and metabolic stability of the 35S-GAG-containing molecules produced by cells on plastic and collagen. During 48 h of labeling with 35SO4, cultures on collagen accumulate 1.4-fold more 35S-GAG per microgram of DNA. In these cultures, most of the extracellular 35S-GAG is immobilized with the lamina and collagen gel, whereas in cultures on plastic all extracellular 35S-GAG is soluble. On both substrata, the cells produce several heparan sulfate-rich 35S-proteoglycan fractions that are distinct by Sepharose CL-4B chromatography. The culture types contain similar amounts of each fraction, except that collagen cultures contain nearly four times more of a fraction that is found largely bound to the lamina and collagen gel. During a chase this proteoglycan fraction is stable in cultures on collagen, but is extensively degraded in cultures on plastic. Thus, collagen-induced formation of a basal lamina correlates with reduced degradation and enhanced accumulation of a specific heparan sulfate-rich proteoglycan fraction. Immobilization and stabilization of basal laminar proteoglycan(s) by interstitial collagen may be a physiological mechanism of basal lamina maintenance and assembly.     

The turnover of basal lamina glycosaminoglycan correlates with epithelial morphogenesis

The mouse embryonic submandibular epithelium begins as a single bud from the floor of the mouth which, under the influence of its surrounding mesenchyme, grows and forms lobules that subsequently branch repetitively. The lobular morphology of the 13-day epithelium is maintained by its basal lamina which is a continuous layer on the interlobular clefts but is interrupted on the distal aspects of the lobules. The structural integrity of this lamina is dependent upon its glycosaminoglycan (GAG) which, by histochemistry, is more abundant on the interlobular clefts than on the distal lobules. We have investigated the basis for these regional differences in the lamina by examining the synthesis and degradation of total GAG as well as the accumulation and loss of laminar GAG during the morphogenesis of the 13-day gland. Autoradiography and histochemistry show that laminar GAG is rapidly turning over. Although it is relatively stable in the interlobular clefts, GAG is rapidly degraded on the distal lobules. This difference can account for the regional variation in basal laminar GAG accumulation. The results of incorporation kinetics and precursor pool specific activities of total epithelial GAG show that the rate of GAG synthesis is greater than its rate of degradation in the base of the lobules, which includes the interlobular clefts. In contrast, during morphogenesis, the rate of GAG degradation becomes greater than its rate of replacement in the distal lobules. The epithelial stalk appears to be in the steady state regarding GAG metabolism. We propose (a) that the rapid laminar GAG degradation on the distal lobules produces the interruptions in the lamina, allowing epithelial growth and expansion, and (b) that the metabolic stability of laminar GAG on the interlobular clefts maintains the integrity of this lamina which serves as a cellular constraint. The results are consistent with a model for epithelial morphogenesis in which the mesenchyme remodels the lamina, which in turn, dictates epithelial morphology. Regulation of basal lamina turnover may be a general mechanism for controlling the behavior of epithelial cell populations.     

Carbohydrate moieties of the basal lamina: their role in attachment and spreading of basal corneal epithelial cells

Summary Three lectins, Wheat germ agglutinin, succinyl Concanavalin A and Ricinus communis agglutinin were used to block specific sugar moieties in the basal lamina. Corneal epithelial basal cells were plated onto freshly denuded basal lamina. Attachment was studied by quantifying the adherence of prelabeled cells and by examining attachment sites using transmission electron microscopy. Spreading was examined using scanning electron microscopy. Attachment of the cells occurred within 15 min and spreading was apparent after 45 min. Both Wheat germ agglutinin and -N-acetylglucosaminidase inhibited cellular attachment. Succinyl Concanavalin A and Ricinus Communis agglutinin permitted attachment, but inhibited extensive cellular spreading. The results indicate that the attachment of basal cells is dependent on N-acetylglucosamine residues, and spreading is mediated by alpha methylmannoside, glucose, and galactose residues.     

The epithelial basal lamina of the isolated colonic mucosa: scanning and transmission electron microscopy

The morphology of the rat colonic basement membrane was studied under control conditions and after induction of secretion by distension in the Ussing chamber. The basement membrane was visualized by removing the epithelium with the aid of Ca2+ chelation combined with vibration. Specimens with and without metallic coating were studied with SEM and TEM, respectively. The surface of the basement membrane in the surface region of the epithelium, i.e. between the mouths of the crypts, is rough. In contrast, the deeper parts of the crypts have a rather smooth basal lamina, which is finely pleated. Fenestrations of the basal lamina with a diameter of 0.5-1 microns were found frequently at the surface region, but less frequently in the crypts. Fenestrations with diameters larger than 2 microns were never observed. After the distension experiments and long-time incubation in vitro (5 h), the size of the fenestrations remained unchanged, however, their number increased compared to the controls.     

Retention of epithelial basal lamina allows isolated mandibular mesenchyme to form bone

The initiation of bone formation in the avian mandible requires that neural crest-derived cells undergo an inductive interaction with mandibular epithelium. To examine the role of the epithelial basal lamina in that interaction, mandibles were separated into their epithelial and mesenchymal components following exposure to the chelating agent, EDTA. Transmission and scanning electron microscopy was used to show that the basal lamina was retained as a continuous layer over the mesenchyme. Osteogenesis was initiated when such EDTA-isolated mesenchyme was grafted to the chorioallantoic membranes of host embryos. In contrast, mesenchyme isolated using trypsin and pancreatin failed to form bone. It is concluded that the property of mandibular epithelium which permits osteogenesis resides within the basal lamina.     

A simple mechanical method to isolate the basal lamina of insect midgut epithelial cells

In mealworms (Tenebrio molitor) the midgut epithelium is surrounded by a 1.6mum thick basal lamina of low electron density with a framework of high electron density imbedded in a part of it. The lamina can be isolated by ultrasonication followed by repeated filtrations and high-speed centrifugations, making large-scale preparation of the lamina for further analyses possible. The isolation of the basal lamina is confirmed by electron microscopy.     

Embryonic corneal epithelial interaction with exogenous laminin and basal lamina is F-actin dependent

Between the third and sixth day of embryonic development, the avian corneal epithelium produces both a basal lamina and the primary corneal stroma composed of 20 orthogonally arranged layers of collagen fibrils. If the epithelium is removed by enzyme treatment from the basal lamina and stroma, the basal cell surface extends cell processes (blebs) which contain disorganized actin filaments and the epithelium decreases production of collagen. When placed on extracellular matrix or on Millipore filters in media containing soluble matrix molecules, the epithelium retracts the blebs, forms an organized basal actin cortical mat, and doubles its production of collagen. In the current investigation, we provide evidence for the hypothesis that organization of the RER by the actin cytoskeleton mediates this stimulation of collagen production. Laminin-treated epithelia and epithelia isolated with the basal lamina intact were treated with an actin-disrupting drug, cytochalasin D. Actin aggregates occur throughout the epithelium, the RER becomes disorganized, and the increase in collagen production expected to result from addition of laminin does not take place. Morphometrical analysis of the distribution of RER in the basal compartment of control and cytochalasin-treated epithelia shows that the decrease in collagen production is accompanied by displacement of the RER from the basal area of the cells, suggesting that attachment of RER to the intact actin cytoskeleton is essential to maintenance of normal RER organization and function. We also found that laminin-mediated bleb retraction requires intact actin microfilaments, whereas bleb extension does not, and that nocodazole does not inhibit bleb extension or retraction.     

Activity-dependent accumulation of basal lamina by cultured rat myotubes

Myoblasts from 20-day rat embryos fuse and differentiate in culture to form spontaneously active myotubes. The myotubes acquire an extracellular matrix that includes a patchy basal lamina (BL) and a layer of fibrils that runs among and above the cells. Several antibodies that bind to muscle fiber basement membrane in vivo were used to study the organization of the extracellular matrix and the effect of muscle activity on the accumulation of its components. Light and electron microscopic immunohistochemical methods showed that the composition and organization of myotube BL in vitro resemble those seen in vivo. Antibodies that bind to both synaptic and extrasynaptic muscle fiber BL, in vivo stain the entire myotube BL in vitro, while antisera that bind preferentially to synaptic BL in vivo stain small patches of myotube BL, which are usually associated with regions rich in acetylcholine receptors. The effects of activity on accumulation of BL were studied by comparing control myotubes to myotubes paralyzed with tetrodotoxin or lidocaine. Immunohistochemical and 125I-antibody binding experiments with three antisera that stain the entire BL showed that paralyzed myotubes accumulate less BL than active myotubes. The effects of activity and inactivity are reversible: new BL forms if toxin is removed from cultures and BL is lost if active myotubes are paralyzed. Thus, accumulation of BL by myotubes is dependent, at least in part, on activity. In contrast, the number of patches stained by synapse-specific BL antibodies is increased in inactive cultures. Thus, immunologically distinguishable components of BL are differentially affected by activity.     

Microthread-like filaments connecting the epithelial basal lamina with underlying fibrillar components of the connective tissue in the rat trachea

Summary The structural relationship between the basal lamina and the underlying reticular tissue was studied, with special attention to the relationship among basal lamina-associated anchoring fibrillar (AF) arcs (Kawanami et al. 1978, 1979) and other fibrillar components, in the epithelium-denuded trachea of the rat. Quantitative analysis of a large number of AF arcs reveals that the majority of the AF arcs has no other fibrillar components of passage. This suggests that most AF arcs do not serve as a real anchoring device, connecting the basal lamina with the underlying reticular tissue, as has so far been suggested by Kawanami et al. (1978). Ruthenium-red staining reveals the presence of a unique meshwork of microthread-like filaments connecting the undersurface of the basal lamina or the AF arcs with the underlying fibrillar components with a remarkable continuity, suggesting that the filaments act as a real anchoring device; these filaments link, instead of the AF arcs, the basal lamina, to the subjacent reticular tissue. Various enzymatic treatments of the filaments indicate that their chemical nature is probably non-collagenous (glyco)protein without glycosaminoglycan moieties.     

Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis

During mammalian development, a pair of shelves fuses to form the secondary palate, a process that requires the adhesion of the medial edge epithelial tissue (MEE) of each shelf and the degeneration of the resulting medial epithelial seam (MES). It has been reported that epithelial-mesenchymal transformation (EMT) occurs during shelf fusion and is considered a fundamental process for MES degeneration. We recently found that cell death is a necessary process for shelf fusion. These findings uncovered the relevance of cell death in MES degeneration; however, they do not discard the participation of other processes. In the present work, we focus on the evaluation of the processes that could contribute to palate shelf fusion. We tested EMT by traditional labeling of MEE cells with a dye, by infection of MEE with an adenovirus carrying the lacZ gene, and by fusing wild-type shelves with the ones from EGFP-expressing mouse embryos. Fate of MEE labeled cells was followed by culturing whole palates, or by a novel slice culture system that allows individual cells to be followed during the fusion process. Very few labeled cells were found in the mesenchyme compartment, and almost all were undergoing cell death. Inhibition of metalloproteinases prevented basal lamina degradation without affecting MES degeneration and MEE cell death. Remarkably, independently of shelf fusion, activation of cell death promoted the degradation of the basal lamina underlying the MEE ('cataptosis'). Finally, by specific labeling of periderm cells (i.e. the superficial cells that cover the basal epithelium), we observed that epithelial triangles at oral and nasal ends of the epithelial seam do not appear to result from MEE cell migration but rather from periderm cell migration. Inhibition of migration or removal of these periderm cells suggests that they have a transient function controlling MEE cell adhesion and survival, and ultimately die within the epithelial triangles. We conclude that MES degeneration occurs almost uniquely by cell death, and for the first time we show that this process can activate basal lamina degradation during a developmental process.     

Ultrastructure of the neural basal lamina in loop-tail mice

Ultrastructural features of the neural basal lamina were studied by means of the tannic acid and ruthenium red techniques in normal and abnormal dysraphic loop-tail mice at 9-11 days of gestation. With ruthenium red, the configuration of the neural basal lamina is similar in both normal and abnormal embryos at 9-11 days. However, differences were detected in the abnormal 9-day embryos processed with tannic acid, as compared with normal littermates. These include irregularities in the lamina rara externa, as well as differences in the staining pattern of the neuroepithelial cell plasma membrane. By 11 days of gestation, the lamina rara externa of the normal embryos shows features similar to those observed in the 9-day abnormal embryos.     

Structure and regeneration of the planarian basal lamina: An ultrastructural study

The structure and regeneration of the planarian subepidermal basement membrane or basal lamina have been electron microscopically examined, particularly in relation to the changes of extracellular products at the wounded area. The intact basal lamina consists of three structural elements; namely, an electron-lucent zone, a limiting layer and a microfibrillar layer. Ultrastructural changes during wound healing have suggested that the amorphous material secreted in the interspace between the epidermal cells and blastema contains precursors of the basal lamina. Within the amorphous zone two distinct phases of the basal lamina regeneration are observed: one is a reconstitution of the limiting layer and the other is a polymerization of the microfibrils. The limiting layer arises from areas subjacent to newly developed hemidesmosomes of epidermal cells. The unit microfibrils are formed from an accumulation of the precursors through transitional smaller microfibrils. At the late stage, individual mature microfibrils are regularly lined with the limiting layer and cell membranes of the newly differentiated muscle fibres. On the basis of these observations we suggest that the planarian basal lamina is regenerated by the interaction between epidermal cells and myoblasts.     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

Preparation and characterization of the lateral and basal plasma membranes of the rat intestinal epithelial cell

A technique is described for the isolation of a plasma-membrane fraction from the rat intestinal epithelial cell which is distinct from the microvillus membrane of that cell. The isolated fraction contains only about 0.2% of the sucrase activity in the original homogenate and negligible quantities of nuclear and mitochondrial membrane markers. It contains 12% of the total Na(+),K(+)-dependent adenosine triphosphatase and 7% of the alkaline phosphatase, with significant increments in specific activity of these enzymes. Multiple membrane preparations were highly reproducible with respect to the specific activities of the markers studied. The small intestine of one rat yields material containing about 1.3mg of protein. In addition an assay is described suitable for determining 5'-nucleotidase in the small intestine.     

Nerve-induced remodeling of muscle basal lamina during synaptogenesis

To identify mechanisms that regulate the deposition of the junctional basal lamina during synaptogenesis, immunocytochemical experiments were carried out on cultured nerve and muscle cells derived from Xenopus laevis embryos. In some experiments successive observations were made on individual muscle cells after pulse-labeling with a fluorescent monoclonal antibody specific for a basal lamina proteoglycan. In others, old and new proteoglycan molecules were differentially labeled with antibody conjugated to contrasting fluorochromes. These observations revealed that surface deposits of antibody-labeled proteoglycan remain morphologically stable for several days on developing muscle cells. Over the same period, however, new sites of proteoglycan accumulation formed that contained primarily those antigenic sites recently exposed at the cell surface. When muscle cells became innervated by cholinergic neurites, new proteoglycan accumulations were induced at the developing neuromuscular junctions, and these too were composed almost exclusively of recently deposited antigen. In older muscle cultures, where many cells possessed relatively high background concentrations of antigen over their surfaces, developing neuromuscular junctions initially showed a markedly reduced proteoglycan site-density compared with the adjacent, extrajunctional muscle surface. Much of this perineural region eventually became filled with dense, nerve induced proteoglycan plaques at later stages of synapse development. Motoneurons thus appear to have two, superficially paradoxical effects on muscle basal lamina organization. They first cause the removal of any existing, extrajunctional proteoglycan from the path of cell contact, and then induce the deposition of dense plaques of recently synthesized proteoglycan within the developing junctional basal lamina. This observation suggests that the proteolytic enzyme systems that have already been implicated in tissue remodeling may also contribute to the inductive interaction between nerve and muscle cells during synaptogenesis.     

Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans

Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production.