Formation of the basement membrane during regeneration of the corneal epithelium

Summary The epithelium regenerating after a surface lesion of the cornea forms a new basement membrane. This process begins 6–8 days after the lesion when the wound is completely covered, and epithelial cells have ceased to migrate. Only that part of the epithelial cells facing the stroma is involved. First, tufts of fine filamentous structures (about 30 Å thick) appear on the internal side of the plasma membrane, and apparently penetrate it reaching the extracellular space where they form a loose network. This then differentiates into two discrete layers, a very thin discontinuous one, restricted to areas with tufts, very close to the plasma membrane (juxtamembranous layer), and a thick continuous layer, the basement membrane proper, parallel to and much further away from the plasma membrane. The basement membrane appears to be the product of cytoplasmic secretion by epithelial cells, and there is no evidence for connective tissue cells taking part in this process.Supported by the Deutsche Forschungsgemeinschaft.     

Structure and motility of primary cilia in the follicular epithelium of the human thyroid

In order to clarify contradictory reports concerning ciliary structure and function, follicular epithelium from macroscopically normal portions of 37 surgical specimens of human thyroid were processed for video-microscopy and/or transmission electron microscopy. The cilia of living cells were immotile. In transverse sections the cilia revealed a 9 + 0 pattern at the base of the shaft, whereas towards the distal end the number of microtubular doublets diminished. Dynein arms, radial spokes and central microtubules were absent. The immotility and structure of these primary cilia implies that their function is not related to motility. The phylogenetic and ontogenetic development of the thyroid suggests that tumor cells of follicular origin displaying abnormal secondary cilia may represent a pathological variant of differentiation.     

Structure and motility of primary cilia in the follicular epithelium of the human thyroid

In order to clarify contradictory reports concerning ciliary structure and function, follicular epithelium from macroscopically normal portions of 37 surgical specimens of human thyroid were processed for video-microscopy and/or transmission electron microscopy. The cilia of living cells were immotile. In transverse sections the cilia revealed a 9 + 0 pattern at the base of the shaft, whereas towards the distal end the number of microtubular doublets diminished. Dynein arms, radial spokes and central microtubules were absent. The immotility and structure of these primary cilia implies that their function is not related to motility. The phylogenetic and ontogenetic development of the thyroid suggests that tumor cells of follicular origin displaying abnormal secondary cilia may represent a pathological variant of differentiation.     

Ultrastructural development features of the human glomerular basement membrane

In the human embryo, the basement glomerular membrane appears early, at about ten weeks of age. The study of 36 human embryos aged 8-20 weeks revealed many arguments suggesting the epithelial origin of the basement membrane of the visceral capsula.     

Collagen types XII and XIV are present in basement membrane zones during human embryonic development

The collagens constitute a large group of proteins in the extracellular matrix that can be divided into several distinct families. Collagen types XII and XIV belong to a subgroup of non-fibrillar-collagens termed (fibril-associated collagens with interrupted triple-helices) (FACIT) and may be involved in basement membrane regulation providing specific molecular bridges between fibrils and other matrix components. However, the tissue distribution of the two proteins during human embryogenesis is still unclear. As a first step toward the elucidation of their possible cell biological functions, we compared the distribution of the two collagens during human organogenesis at the light microscopical level. We detected specific differences between the expression patterns of the two molecules, which may be related to their respective function within the basement membrane zones during human embryonic development. For example, in the developing intestine, collagen type-XII was present in the basement membrane zones of epithelia and endothelia. However, collagen type-XIV was restricted to the mesothelial basement membrane zones. We conclude that both collagens might well be able to serve different functions during human embryonic development although their structures are highly similar.     

Distribution of integrins and their ligands in the trunk of Xenopus laevis during neural crest cell migration

We have examined the distribution in Xenopus embryos of beta 1 subunits of integrin, as recognized by cross-reactive antibodies against the avian integrin beta 1 subunit. These antibodies recognize a doublet of bands of approximately 120 kD in Xenopus embryos. The distribution pattern of these integrin cell surface receptors was compared with that of two possible ligands, fibronectin and laminin, in the extracellular matrix during the time of neural crest cell migration. Integrin immunoreactivity in the early neurula was observed lightly outlining somite and epidermal cells and the notochord. The integrin immunostaining increased with developmental age and was observed on most cell types in the embryo but was particularly notable in the intersomitic clefts through which motoraxons grow. The immunoreactivity in this region was not, however, wholly on the axon surfaces, since intersomitic integrin remained detectable in embryos in which the neural tube had been ablated. Fibronectin and laminin were more extensively distributed than integrin at all stages examined. Immunoreactivity for both was observed around the neural tube, notochord, somites, epidermis, dorsal mesentery, and lateral plate mesoderm. The distribution of laminin and fibronectin around the somites was particularly interesting since it was non-uniform and similar to that of integrin. Strongest staining was observed in the intersomitic clefts, and weakest staining was observed on the medial surface of the somites, which faces the neural tube and notochord. The major differences in distribution pattern between the fibronectin and laminin immunoreactivities were that only fibronectin was detected in the mesenchyme of the dorsal fin. Our results demonstrate that a molecule homologous to avian integrin is present in Xenopus embryos during neural crest cell migration and motoraxon outgrowth. Its presence in the intersomitic clefts and on the surface of many embryonic cell types together with the abundant distribution of its ligands are consistent with a potentially important developmental function in neurite outgrowth and/or muscle development.     

Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains.     

The embryonic rat parietal yolk sac. Changes in the morphology and composition of its basement membrane during development.

The basement membrane (Reichert's membrane) of the entire capsular portion of the parietal yolk sac of rat embryos was examined both morphologically and chemically at various stages of gestation. The overall microscopic and compositional analyses showed Reichert's membrane to be typical of basement membranes isolated from other tissues and species. However, with increasing gestational age (from 11.5 to 17.5 days) a number of changes involving Reichert's membrane were noted: 1. The thickness increased rapidly then declined, while the surface area increased tenfold; 2. The total protein content increased twenty-fold while the collagen content increased eight-fold. As a result, the relative collagen content declined significantly; 3. The changes in the amino acid and carbohydrate composition were consistent with the latter finding.The observations listed above were evaluated in light of their possible relevance to an understanding of the morphogenesis of basement membranes during development, and to the possible mechanisms involved in pathogenesis of basement membrane dysfunction.     

Changes in the basement membrane zone components during skeletal muscle fiber degeneration and regeneration

The basement membrane of skeletal muscle fibers is believed to persist unchanged during myofiber degeneration and act as a tubular structure within which the regeneration of new myofibers occurs. In the present study we describe macromolecular changes in the basement membrane zone during muscle degeneration and regeneration, as monitored by immunofluorescence using specific antibodies against types IV and V collagen, laminin, and heparan sulfate proteoglycan and by the binding of concanavalin A (Con A). Skeletal muscle regeneration was induced by autotransplantation of the extensor digitorum longus muscle in rats. After this procedure, the myofibers degenerate; this is followed by myosatellite cell activation, proliferation, and fusion, resulting in the formation of new myotubes that mature into myofibers. In normal muscle, the distribution of types IV and V collagen, laminin, heparan sulfate proteoglycan, and Con A binding was seen in the pericellular basement membrane region. In autotransplanted muscle, the various components of the basement membrane zone disappeared, leaving behind some unidentifiable component that still bound Con A. Around the regenerated myotubes a new basement membrane (zone) reappeared, which persisted during maturation of the regenerating muscle. The distribution of various basement membrane components in the regenerated myofibers was similar to that seen in the normal muscle. Based on our present and previous study (Gulati, A.K., A.H. Reddi, and A.A. Zalewski, 1982, Anat. Rec. 204:175-183), it appears that some of the original basement membrane zone components disappear during myofiber degeneration and initial regeneration. As a new basement membrane develops, its components reappear and persist in the mature myofibers. We conclude that skeletal muscle fiber basement membrane (zone) is not a static structure as previously thought, but rather that its components change quite rapidly during myofiber degeneration and regeneration.     

Localization of basement membrane glycoproteins in rat kidney during foetal development

The distribution of basement membrane glycoproteins (type IV collagen, laminin, fibronectin, and proteoglycans) was studied in foetal rat kidney by immunohistochemical techniques using polyclonal antibodies. From the first stages of nephron differentiation, all these glycoproteins were detectable by immunofluorescence in the tubular and glomerular basement membranes and in the mesangial matrix. As differentiation proceeded, labelling of glycoproteins progressively intensified, except for that of fibronectin, which gradually decreased in the glomerular basement membrane (GBM) and was barely observable at full differentiation. With immunoperoxidase staining in electron microscopy, all glycoproteins were seen to be widely dispersed in the spaces between the epithelial and endothelial glomerular cells so long as the GBM remained a loose structure. However, after it became a compact, 3-layered formation, type IV collagen and laminin were distributed throughout the GBM, whereas proteoglycans and anionic sites appeared as 2 rows of granules confined to the laminae rarae.     

The receptor tyrosine phosphatase Dlar and integrins organize actin filaments in the Drosophila follicular epithelium.

BACKGROUND: Regulation of actin structures is instrumental in maintaining proper cytoarchitecture in many tissues. In the follicular epithelium of Drosophila ovaries, a system of actin filaments is coordinated across the basal surface of cells encircling the oocyte. These filaments have been postulated to regulate oocyte elongation; however, the molecular components that control this cytoskeletal array are not yet understood. RESULTS: We find that the receptor tyrosine phosphatase (RPTP) Dlar and integrins are involved in organizing basal actin filaments in follicle cells. Mutations in Dlar and the common beta-integrin subunit mys cause a failure in oocyte elongation, which is correlated with a loss of proper actin filament organization. Immunolocalization shows that early in oogenesis Dlar is polarized to membranes where filaments terminate but becomes generally distributed late in development, at which time beta-integrin and Enabled specifically associate with actin filament terminals. Rescue experiments point to the early period of polar Dlar localization as critical for its function. Furthermore, clonal analysis shows that loss of Dlar or mys influences actin filament polarity in wild-type cells that surround mutant tissues, suggesting that communication between neighboring cells regulates cytoskeletal organization. Finally, we find that two integrin alpha subunits encoded by mew and if are required for proper oocyte elongation, implying that multiple components of the ECM are instructive in coordinating actin fiber polarity. CONCLUSIONS: Dlar cooperates with integrins to coordinate actin filaments at the basal surface of the follicular epithelium. To our knowledge, this is the first direct demonstration of an RPTP's influence on the actin cytoskeleton.     

Basement membrane stimulates the polarized distribution of integrins but not the Na,K-ATPase in the retinal pigment epithelium.

The basement membrane stimulates the differentiation and polarity of simple transporting epithelia. We demonstrated for the retinal pigment epithelium (RPE) of chicken embryos that polarity develops gradually. Although the RPE and an immature basement membrane are established on embryonic day 4 (E4), the distribution of the Na,K-ATPase and a family of basement membrane receptors containing the beta 1 subunit of integrin is nonpolarized. The percentage of polarized cells increases gradually until cells in all regions of the epithelium are polarized on E11. During this time, the basement membrane increases in size and complexity to form Bruch's membrane. To study the ability of the basement membrane to stimulate the polarized distribution of the beta 1 integrins or the Na,K-ATPase, RPE was harvested from E7, E9, or E14 embryos and cultured on Bruch's membrane isolated (in association with the choroid) from E14 embryos. As a control, the RPE was plated on the side of the choroid lacking a Bruch's membrane. The distribution of the beta 1 integrins and the Na,K-ATPase was determined by indirect immunofluorescence. Bruch's membrane stimulated the polarized distribution of the beta 1 integrins regardless of the developmental age of the RPE even though E7 RPE is nonpolarized in vivo. To examine the role of individual matrix components, RPE was plated on matrix-coated filters. The polarized distribution of the beta 1 integrins was stimulated by laminin, collagen IV, and Matrigel but not by fibronectin. Interestingly, laminin and collagen IV are present in the basement membrane on E4 when RPE is not polarized in vivo. Under no circumstances was the distribution of the Na,K-ATPase polarized. These data indicate that the basement membrane influences the distribution of a subset of plasma membrane proteins but that other factors are required for full polarity.     

Changes in thyroid hormone levels during zebrafish development

The zebrafish (Danio rerio) has been used as a model for the study of endocrine disrupting chemicals. This study set out to determine the profiles of whole-body thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels during the development of zebrafish from embryo to adult. Enzyme-linked immunoassay was used to analyze whole-body T4 and T3 contents. The results showed that whole-body T4 and T3 levels remained stable during the pre-hatching period (0-3 d) and increased significantly during early development after hatching. The T3 level peaked at 0.28 ± 0.01 ng g(-1) body weight at 10 days post-fertilization (dpf), and T4 peaked at 0.58 ± 0.09 ng g(-1) body weight at 21 dpf. Both thyroid hormones subsequently declined during later development. This study establishes a baseline for thyroid hormones in zebrafish, which will be vital for the understanding of thyroid hormone functions and in future studies of thyroid toxicants in this species.     

Changes in membrane phospholipid distribution during platelet activation

(1) Exposure of phospholipids at the outer surface of activated and control platelets was studied by incubation with a mixture of phospholipase A₂ from Naja naja and bee venom, solely or in combination with sphingomyelinase from Staphylococcus aureus, using conditions under which cell lysis remained below 10%. (2) Incubation with phospholipase A₂ alone revealed a markedly increased susceptibility of the phospholipids in platelets activated by a mixture of collagen plus thrombin, by the SH-oxydizing compound diamide, or by calcium ionophore A23187, as compared to control platelets or platelets activated separately by collagen or thrombin. (3) Collagen plus thrombin, diamide, and ionophore treated platelets revealed an increased exposure of phosphatidylserine at the outer surface accompanied by a decreased exposure of sphingomyelin, as could be concluded from incubations with a combination of phospholipase A₂ and sphingomyelinase. These alterations were much less apparent in platelets activated either by thrombin or by collagen alone. (4) The increased exposure of phosphatidylserine in activated platelets is accompanied by an increased ability of the platelets to enhance the conversion of prothrombin to thrombin by coagulation factor Xa, in the presence of factor Va and calcium. (5) It is concluded that the altered orientation of the phospholipids in the plasma membrane of platelets activated by collagen plus thrombin, by diamide, or by calcium ionophore, is the result of a transbilayer movement. Moreover, the increased exposure of phosphatidylserine in platelets stimulated by the combined action of collagen and thrombin might be of considerable importance for the hemostatic process.