Fine structure of the respiratory organs of the Climbing perch, Anabas testudineus (Pisces: Anabantidae)

An electron microscopic study has been made of the three respiratory organs of climbing perch. The gill structure is similar to that of the other telcosts but the thickness of the water/blood barrier is much greater, being as great as 20 μm in some specimens. The increased thickness is due to a multilayered epithelium which is thinner (3.5–7 μm) over the marginal channel of the secondary lamellae. The other two main layers, basement membrane and pillar cell flange, are relatively thin (about 1 μm).
The pillar cells have a typical structure, but in certain regions they are contiguous with one another and line well-defined blood channels. Some of the columns of basement membrane material in such regions may be common to adjacent pillar cells.
The air-breathing organs are (a) the lining of the suprabranchial chambers , and (b) the labyrinthine plates attached to the dorsal region of branchial arches. Electron microscopy showed that their structure is well adapted for gas exchange, the air/blood barriers being only 0.12–0.3 μm, comprising an epithelial layer, basement membrane, and thin capillary endothelium. The many parallel blood channels of the respiratory islets of both organs are separated by pillar-like structures which differ from the pillar cells of the secondary lamellae. Thus the hypothesis that the air-breathing organs represent modified gills is not supported by this study.
The fine structure of the non-respiratory region of the air-breathing organs is similar to that of the skin, and includes chemoreceptor-like cells. Evidence concerning the possible homology of pillar cells with plain muscle cells is discussed.     

Immunohistochemical localization of fibronectin in the human placentas at their different stages of maturation

Summary The distribution of fibronectin in the human placenta was studied by the aid of the immunoperoxidase technique using specific antibodies against it. In the early chorionic tissue, fibronectin was distributed along the trophoblastic basement membrane, on the wall of fetal blood vessels, in the counective tissue core, and in the cytotrophoblastic cell columns. In the term placenta, this glycoprotein was detected mainly on the fetal blood vessels and less intensely in the stroma, but not along the trophoblastic basement membrane. Endothelial cells of the blood vessels, fibroblastic cells in the stroma, and unidentified cells in the cytotrophoblastic cell columns were immunostained positively for fibronectin. These data suggest that fibronectin of the placenta is produced locally and retained in the tissue, if not all.     

Gross and fine structure of the pseudo branch of the climbing perch, Anabas testudineus (Bloch)

The structure of the glandular pseudobranch of the air-breathing fish Anabas testudineus is described on the basis of light and electron microscopy. It is shown that the pseudobranch has the same basic structure as a typical gill, although in this case it is not freely exposed to the water.
Individual secondary lamellae can be recognized in which well-defined, but narrow, blood channels are present between typical pillar cells. The epithelial layer is mainly represented by enlarged mitochondria-rich cells which constitute a large proportion of the whole organ. Mitochondria-rich cells contain an abundant endoplasmic reticulum which is in close contact with the mitochondria and becomes concentrated near the vascular borders of the cell and opens directly into the basement lamina.
The presence of numerous pinocytotic vesicles in the enlarged pillar cell flanges may transfer material to the blood channels. The precise nature and role of any materials released in this way remains to be investigated.     

A simple, versatile, nondisruptive method for the isolation of morphologically and chemicaly pure basement membranes from several tissues.

A simple procedure has been developed for the isolation of ultrastructurally pure, intact basement membranes from bovine retinal and brain blood vessels, rabbit renal tubules and rat renal glomeruli. By this procedure, cell membranes and intracellular materials are selectively solubilized with 4% sodium deoxycholate to yield morphologically and chemically intact basement membrane preparations. Therefore, this method appears to be a versatile, nondisruptive procedure for the isolation and characterization of basement membranes from a variety of tissues. Its applicability has been demonstrated by the preparation for the first time of isolated basement membranes from non-renal mammalian blood vessels.     

Immuno-electron-microscopic localization of laminin and collagen type IV in normal and denervated tooth pulp of the cat

Summary The distribution of laminin-like immunoreactivity in adult normal and denervated cat mandibular tooth pulps was studied by the use of fluorescence microscopy and pre-embedding immunogold electron microscopy. Immunoreactivity to collagen IV was also assessed in order to distinguish basement membranes. In normal pulps, light-microscope laminin-like immunoreactivity was strong along blood vessels and Schwann cell sheaths, and a faint immunoreactivity was seen also in the odontoblast layer. Electron microscopy confirmed the laminin-like immunoreactivity of endothelial and Schwann cell basement membranes at all pulpal levels. In the odontoblast layer and the predentine, nerve-like structures lacking basement membranes but possessing strong membrane laminin-like immunoreactivity were encountered. In addition, a clear-cut laminin-like immunoreactivity of plasma membranes of the somata and processes of odontoblasts was seen. Observations on denervated pulps as well as pulps in which nerve regeneration had taken place did not reveal any changes in the pattern of laminin-immunoreactivity in basement membranes or odontoblasts. Distribution of collagen IV-like immunoreactivity was very similar to laminin-like immunoreactivity in basement membranes of blood vessels and Schwann cells, and appeared unaffected by denervation. The odontoblasts and nerve-like profiles in the odontoblast layer were devoid of collagen IV-like immunoreactivity. We propose that odontoblast-associated laminin could be of significance as guidance for regenerating terminal pulpal nerve fibers to appropriate targets.     

Biomechanical properties of native basement membranes

Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.     

Heterogeneity of basement membranes of the human genitourinary tract revealed by sequential immunofluorescence staining with monoclonal antibodies to laminin

We used monoclonal antibodies specific for human laminin to analyze immunohistochemically the heterogeneity of the basement membranes in various parts of the genitourinary tract. By indirect immunofluorescence microscopy we show that antibody 3H11 reacts with all epithelial basement membranes in the kidneys, testes, epididymis, prostate, uterus, oviduct, and ovary, as well as the smooth muscle cells, blood vessels, and nerves. Antibody 4E10 reacted with most epithelial basement membranes in these organs but was unreactive with the basement membranes of peripheral glomerular capillary loops and the basement membranes of the oviductal mucosa, seminiferous tubules, straight tubules, and rete testis. Hilar seminiferous tubules were reactive with 4E10. In contrast to 3H11, which reacted with all vascular, subendothelial, and muscular basement membranes, 4E10 reacted only with the subendothelial basement membrane of capillaries and veins. The difference in the distribution of epitopes could be demonstrated in tissue sections sequentially reacted with two monoclonal antibodies, but only if the antibody of restricted reactivity (4E10) was used first. These data show that the heterogeneous expression of distinct epitopes of laminin in basement membranes can be demonstrated in the same tissue section by sequential staining. This heterogeneity of basement membranes most likely reflects conformational differences in the expression of epitopes on the laminin molecule in various anatomic structures.     

Bioimaging of Fluorescence-Labeled Mitochondria in Subcutaneously Grafted Murine Melanoma Cells by the “In Vivo Cryotechnique”

The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes.     

Nervous system of the snail Helix aspersa

Summary The fine structure of vascular channels and amebocytes associated with the sheath of the infraesophageal ganglion of Helix aspersa, is described. The extracellular stroma of the sheath, together with the hemocoel and blood vessels, forms an interconnected system of pathways which appears to be involved in the transport of metabolites, amebocytes, hemocyanin and experimentally introduced opaque tracers. The hemocoel, blood capillaries and precapillaries are lined by a discontinuous layer of single muscle cells whose luminal aspect is covered by a lamina of extracellular material named the vascular coat. This coat consists of a ground substance that forms a basement membrane and filamentous elements some of which are collagenous. Gaps in the blood vessel wall seem to provide the main routes for the movement of cells and large molecules to the hemocoel. Tracer experiments have given support to the idea that a diffusion barrier may be absent at the sheath-ganglion junction. Amebocytes have phagocytic properties; they appear associated in groups or scattered singly within the extracellular space of the sheath and the lumen of blood vessels. Single amebocytes have features of mobile cells and may function in the transport of hemocyanin as well as other proteins.This work has been supported by the Rockefeller Foundation and grants NB 06662 (from the U.S. Public Health Service) and N-105 (from Conicyt, Santiago, Chile). The continuous advice and encouragement of Drs. R. W. Guillery and D. B. Slautterback are gratefully acknowledged.     

Electron microscopy of the mucosal plexus of the rat colon.

The ultrastructure of neurons, glial cells and axons of the mucosal plexus of the rat colon descendens was studied. Serial semithin sections and a re-embedding technique were used in order to localize the ganglia. The ganglia are free of blood vessels and connective tissue. The ratio of neurons to glial cells is approximately 1. Ganglia and nerve strands are enclosed by a basement membrane, without a well-defined perineural connective tissue. The neurons show a structure similar to other enteric plexus. Synaptic contacts were observed frequently in the neuropil, where nerve endings and varicosities show a diverse outfit in vesicles. The glial cells, which contain immunocytochemically detectable glial fibrillary protein, possess the same ultrastructural attributes in the intra- and extraganglionic localizations. In the nerves, axonic profiles and varicosities appear in close relation with glial cells or their processes. The distance between the nerves and their target cells, i.e. the enterocytes, is 0.5 microns or more with interposed basement membranes and fibroblasts.     

The absence of nidogen 1 does not affect murine basement membrane formation

Nidogen 1 is a highly conserved protein in mammals, Drosophila melanogaster, Caenorhabditis elegans, and ascidians and is found in all basement membranes. It has been proposed that nidogen 1 connects the laminin and collagen IV networks, so stabilizing the basement membrane, and integrates other proteins, including perlecan, into the basement membrane. To define the role of nidogen 1 in basement membranes in vivo, we produced a null mutation of the NID-1 gene in embryonic stem cells and used these to derive mouse lines. Homozygous animals produce neither nidogen 1 mRNA nor protein. Surprisingly, they show no overt abnormalities and are fertile, their basement membrane structures appearing normal. Nidogen 2 staining is increased in certain basement membranes, where it is normally only found in scant amounts. This occurs by either redistribution from other extracellular matrices or unmasking of nidogen 2 epitopes, as its production does not appear to be upregulated. The results show that nidogen 1 is not required for basement membrane formation or maintenance.     

Ancestral Vascular Lumen Formation via Basal Cell Surfaces

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.     

Gill ultrastructure of the Pacific hagfish Eptatretus stouti

At the gross anatomical level, hagfish gills show unusual features not seen in any other fish gills. Our study was undertaken to determine if peculiarities also characterize the microscopic anatomy and ultrastructure of hagfish gills. To the contrary, branchial respiratory lamellae of Pacific hagfish were found to resemble the lamellae of lampreys, elasmobranchs, and teleosts, often down to the finest subcellular details. As in other fish, hagfish lamellae are lined by epithelium containing pavement cells with organelles indicative of a secretory function, basal cells showing undifferentiated cell features, and branchial ionocytes. The ionocytes are identical to chloride cells of teleosts in cytostructure, distribution, and abundance. There are pillar and marginal capillaries in hagfish gill lamellae. Pillar cells contain bundles of 5-nm microfilaments, and they associate with collagen columns as in other fish. Hagfish pillar cells do exhibit odd features, however: They cluster (groups of up to nine were seen), and their extracellular collagen columns are rarer than in other fish gills (averaging only two columns per three pillar cells). Other special features of hagfish gills are the following: lipid droplets and smooth endoplasmic reticulum are well developed in all cell types; pavement cells secrete a lipomucous product (stains with periodic acid-Schiff, Alcian blue, and Sudan black B); and goblet cells are absent. The presence of "chloride cells" in hagfish is puzzling, as hagfish body fluids are iso-osmotic to seawater and there is no need to osmoregulate for sodium chloride; the ionocytes contain carbonic anhydrase, suggesting a function in acid/base regulation.     

A morphological study on gills of the brown shrimp, Penaeus aztecus

The gills of Penaeus aztecus were examined by light and electron microscopy. They are dendrobranchiate, consisting of a central axis with biserially arranged branches that subdivide into bifurcating filaments. A septum divides the lumina of these structures into afferent and efferent channels. Hemolymph from the sternal sinus flows through the afferent channels into the filaments where it is directed into the efferent channels and finally to the pericardial cavity. In addition to these channels, numerous blood vessels permeate the gill. The cuticle covering the gill overlies a thin epithelium which is separated from hemolymph by a basal lamina. The epithelium, which is active in cuticle secretion, has a series of pillar processes that form subcuticular lacunae. The apical membranes of epithelial cells become folded in shrimp exposed to hypo- and hyperosmotic salinities. Granular cells that contain elaborate Golgi apparati and several types of granules are present throughout the gill. Nephrocytes resembling glomerular podocytes line the efferent channels. A large nerve traverses the septum in the axis.     

Ultrastructure of type VI collagen in human skin and cartilage suggests an anchoring function for this filamentous network

An mAb was used in conjunction with immunoelectron microscopy to study the ultrastructure and distribution of the type VI collagen network. Type VI collagen in femoral head and costal cartilage was found distributed throughout the matrix but concentrated in areas surrounding chondrocytes. Three-dimensional information gained from high voltage stereo pair electron microscopy showed that the type VI collagen network in skin was organized into a highly branched, open, filamentous network that encircled interstitial collagen fibers, but did not appear to interact directly with them. Type VI collagen was also found concentrated near basement membranes of nerves, blood vessels, and fat cells although in a less organized state. Labeling was conspicuously reduced close to the epithelial basement membrane in the region of the anchoring fibrils. No labeling of basement membranes was seen. Based on these observations it is suggested that the type VI collagen forms a flexible network that anchors large interstitial structures such as nerves, blood vessels, and collagen fibers into surrounding connective tissues.     

Characterization of the column and autocellular junctions that define the vasculature of gill lamellae.

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.     

New aspects of the possible sites of ultrafiltration in annelids (oligochaeta)

Electron microscopic investigations of blood vessels were conducted to show sites of filtration such as podocytes or fenestrated endothelia. The endothelia of the blood vessels of Aelosoma hemprichi, Nais elinguis, Dero obtusa and Enchytraeus buchholzi consist of myoendothelial cells, chloragocytes and podocytes. The podocytes form large archs over a considerable area of the vessels. On the lumen side of the vessel there are several columnar processes which split into numerous small pedicels. The gaps between the adjacent pedicles are bridged by slit membranes. The podocytes are restricted to the front part of the ventral vessel. They are presumed to form a filtration surface. Furthermore, some parts of the ventral vessel are formed by a fenestrated endothelium, mainly in Enchytraeus buchholzi. In the vascular system of E. buchholzi two separate filtration sites were found. Additionally to the filtration site between ventral vessel and coelomic cavity a second filtration site was found in the front part of the body between blood sinus and coelomic cavity. In such areas the basement membrane is the only continuous layer between the blood vessel and the coelomic cavity. Its thickness is in the range of 40 nm. Possible filtration sites in the form of podocytes and irregular fenestrations could be localized at the border between the blood compartment and the coelomic compartment. It can be presumed that the primary urine may be formed by ultrafiltration of blood.     

The connective tissue of the rat lung: electron immunohistochemical studies

The ultrastructural distribution of specific connective-tissue components in the normal rat lung was studied by electron immunohistochemistry. Three of these components were localized: type I collagen, fibronectin and laminin. Type I collagen was present not only in major airways and vascular structures, but also in alveolar septa. Laminin was found in all basement membranes, and only in basement membranes, demonstrating once more that this glycoprotein is an intrinsic component of the basement membrane. Fibronectin was found free in the interstitium and on the surfaces of collagen fibers. The basement membranes of bronchial, glandular and endothelial cells of large vessels lacked fibronectin; however, capillary endothelial and occasionally epithelial alveolar basement membranes contained some fibronectin in an irregular, spotty distribution. This localization suggests that in the lung, as in other tissues, fibronectin is not an intrinsic component of the basement membrane, but rather a stromal and plasma protein. Only basement membranes in the alveolar parenchyma contained "trapped" plasma fibronectin.     

Distinct distribution of laminin and its integrin receptors in the pancreas.

Tissue function is regulated by the extracellular microenvironment including cell basement membranes, in which laminins are a major component. Previously, we found that laminin-1 promotes differentiation and survival of pancreatic islet cells. Here we characterize the expression pattern of laminins and their integrin receptors in adult pancreas. Although they are expressed in the basement membrane of acinar cells and duct epithelium, no laminin chains examined were detected extracellularly in the pancreatic islets. In contrast to laminin beta(1)- and gamma(1)-chains, the alpha(1)-chain, unique to laminin-1, was not detected. Laminin-10 (alpha(5)beta(1)gamma(1)) was expressed in acinar tissue, whereas laminins-2 (alpha(2)beta(1)gamma(1)) and -10 were expressed in the blood vessels. The laminin connector molecule, nidogen-1, had a distribution similar to that of laminin beta(1) and gamma(1), whereas fibulin-1 and -2, which compete with nidogen-1, were mostly confined to blood vessels. Integrin subunits alpha(6) and alpha(3) were detected in acinar cells and duct epithelial cells, but alpha(6) was absent in islet cells. Integrin alpha(6)beta(4) was detected only in duct cells, alpha(6)beta(1) in both acinar and ductal cells, and alpha(3)beta(1) in acinar, duct, and islet cells. These findings are a basis for further investigation of the role of extracellular matrix molecules and their receptors in pancreas function.     

A Novel Mechanism of Bacterial Toxin Transfer within Host Blood Cell-Derived Microvesicles

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.