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Intermediate filaments (IFs) are involved in many important physiological functions, such as the distribution of organelles, signal transduction, cell polarity and gene regulation. However, little information exists on the structure of the IF networks performing these functions. We have clarified the existence of seven kinds of IF networks in the cytoplasm of diverse polarized cells: an apex network just under the terminal web, a peripheral network lying just beneath the cell membrane, a granule-associated network surrounding a mass of secretory granules, a Golgi-associated network surrounding the Golgi apparatus, a radial network locating from the perinuclear region to the specific area of the cell membrane, a juxtanuclear network surrounding the nucleus, and an entire cytoplasmic network. In this review, we describe these seven kinds of IF networks and discuss their biological roles.

The expression of intermediate filaments is sensitively reflected in cell function. To examine the involvement of keratin in a secretory function, 15 kinds of keratin (keratin-2, 3, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, 18, 19, 20) were detected immunohistochemically and immunoelectron microscopically in the rabbit duodenum. Four types of secretory cells existed in the rabbit duodenum: enteroendocrine cells and goblet cells in the epithelium and mucous and serous cells in the duodenal glands. Among the 15 kinds of keratin, keratin 20 was selectively expressed in all these secretory cells. However, localization of keratin 20 in the endocrine cells differed from that in three types of exocrine cells. In the enteroendocrine cells, keratin 20-containing filaments formed a juxtanuclear network from which they extended to the apical cell membrane. These filaments may play a role in intracellular signal transduction, since the apical cell membrane contains some receptors for binding a specific extracellular signal. In the exocrine cells, on the other hand, keratin 20-containing filaments existed just beneath the cell membrane. These filaments may play some role in maintaining cell shape, which is remarkably changed during the secretory cycle.

Keratins (Ks) expressed in the absorptive cells of rabbit duodenal epithelium were studied immunohistochemically and immunoelectron microscopically. These keratins were classified into three groups based on their distribution: keratins forming an apex network just under the terminal web, those forming a Golgi-associated network surrounding the Golgi apparatus, and those forming an entire cytoplasmic network. The apex network was composed of K5/K18 keratin filaments. The Golgi-associated network at first consisted of K8/K14 keratin filaments alone. When the cell migrated out of the crypt to the mid-villus, actin and K7/K17 keratin filaments successively entered this network. The entire cytoplasmic network was detected at the villus base, and consisted of K7/K17 keratin filaments. Thereafter, K5/K18 keratin filaments entered this network during migration of the cell. The apex network may serve to maintain cell-cell contact, since its filaments are tightly anchored to desmosomes. The Golgi-associated network may be involved in the maintenance of the complex structure of the Golgi apparatus, since its filaments constitute a three-dimensional network around the organelle. The entire cytoplasmic network may play a major role in the maintenance of cell structure and the distribution of cell organelles.

Enucleation is the last event in the development of a definitive erythroid line, and extruded nuclei are phagocytosed by macrophages. Both colchicine and cytochalasin have been known to exert a great influence on the enucleation process, but the relationship between enucleation and these agents has not yet been clearly revealed in vivo. Our aim was to clarify the significance of the enucleation in liver erythropoiesis and macrophage phagocytosis by colchicine and cytochalasin administration to embryonic mice. Pregnant mice were intraperitoneally injected with colchicine or cytochalasin at 13 days of gestation. Embryonic livers were removed at intervals of 3, 6 and 12 h after injection for processing for light and electron microscopy, and, to obtain three-dimensional morphology of erythroids at enucleation, computer-aided reconstructions were performed by light microscopy. Colchicine injections had cytolytic effects on hepatocytes and macrophages, and numerous erythroblasts were observed in the process of enucleation after colchicine injection. However, the extruding nuclei were irregularly shaped, and some erythroblasts at mitosis showed extreme peripheralization of their chromosomal masses and cell membrane constriction. Enucleation behavior could also be observed in immature erythroblasts. Liver macrophages engulfed extruded nuclei and erythroblasts in mitosis. Cytochalasin injections, on the other hand, had no significant effect on embryonic livers. The progress of erythroblast mitosis was clearly stopped by colchicine injection, and numerous erythroblasts at mitosis were extruding their nuclear compartment. Following colchicine injection, erythroid enucleation also took place in immature erythroblasts, and mitotic erythroids were phagocytosed. In enucleation, more attention should be paid to hematopoietic environmental factors than to hemopoietic cell factors. Anat. Rec. 251:290–296, 1998. © 1998 Wiley-Liss, Inc.

Erythroblastic islands were examined by ultrastructure and ultrastructural histochemistry in fetal and early neonatal livers of the mouse. The liver primordium of day 11 embryos contained not only immature hemopoietic cells in the hepatic cords but also macrophages in expanded sinusoids. At 12 and 13 days of gestation, macrophages bearing large cytoplasmic inclusions increased in number, and some of them moved from the sinusoids into the hepatic cords. A ring of erythroblasts surrounded the macrophages, and erythroblastic islands could be identified at 14 days of gestation. Fetal livers contained two kinds of macrophages: sinusoidal macrophages and central macrophages of the erythroblastic islands. These macrophages exhibited a similar binding pattern to Griffonia simplicifolia isoagglutinin-IB4 (GS-IB4) and soybean agglutinin (SBA). Fetal hepatocytes, however, did not appear to engage in active phagocytosis, and the binding patterns of GS-IB4 and SBA differed significantly between hepatocytes and the two kinds of macrophages. In the early postnatal mouse, a marked decrease in the number of erythroblastic islands occurred. Erythroblasts left the central macrophages, and the macrophages subsequently underwent degeneration. The erythroblastic islands finally disappeared at the end of liver hemopoiesis, and the degenerated central macrophages were removed by scavenger macrophages in the perisinusoidal space. Our data demonstrate that scavenger macrophages play an essential role in the development of hepatic hemopoiesis, with special reference to the formation and dissolution of erythroblastic islands. Microsc. Res. Tech. 39:398–405, 1997. © 1997 Wiley-Liss, Inc.

To clarify the origin of the central cells in hepatic erythroblastic islands, glycoconjugates on the surface of cellular constituents in fetal mice liver were ultrahistochemically examined using lectin staining. At 11 days of gestation, the cells derived from mesenchyme in fetal liver, including sinusoidal macrophages, endothelial cells, and erythropoietic cells, bound Griffonia simplicifolia isoagglutinin I-B4 (GS-I-B4), but hepatocytes lacked binding sites for the isolectin. Scavenger macrophages in the hepatic cords at 13 days of gestation and the central cells in the erythroblastic islands at 15 days of gestation also bound GS-I-B4. Hepatocytes, however, exhibited no GS-I-B4 binding site at any gestational day. At 11 days of gestation, none of the cells in fetal liver had binding sites for soybean agglutinin (SBA), but cells derived from mesenchyme acquired these binding sites at 13 days of gestation. The central cells in the erythroblastic islands also bound SBA, but hepatocytes did not bind the lectin at all. The central cells in the erythroblastic islands can be considered to belong to a mesenchymal cell lineage, and primitive sinusoidal macrophages at 11 days of gestation are possible precursors of these central cells.

Using lectin and colloidal iron (CI) stainings in combination with neuraminidase digestion, glycoconjugates on the surface of erythropoietic cells of the yolk sac and liver in fetal mice were examined. Fetal hepatic macrophages were capable of distinguishing between phagocytozed and non-phagocytozed erythroid elements as described in our previous study. Marked differences between these two elements could be ultrahistochemically detected on their cell surface. The phagocytozed elements, such as nuclei expelled from erythroblasts and degenerating primitive erythroblasts, faintly bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a weak peanut agglutinin (PNA) binding. In contrast, erythroblasts at various maturation stages, erythrocytes and normal primitive erythroblasts heavily bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a moderate PNA binding. No differences in binding of either concanavalin agglutinin, Ricinus communis agglutinin-I or PNA were noted between phagocytozed and non-phagocytozed erythroid elements. Desialylation appears to be one of the most important signs for the recognition mechanism of fetal macrophage phagocytosis. During maturation of hepatic erythroblasts, sialic acid changes its affinity for Limax flavus agglutinin from strong to weak, and soybean agglutinin binding sites disappear at the basophilic erythroblast stage. Glycoconjugates on polychromatophilic erythroblasts acquire similar compositions to those of erythrocytes.

A few neurons of the adult rabbit spinal ganglion express keratin. To examine the characters of these keratin-positive neurons, six kinds of intermediate filament proteins, namely keratin 8, keratin 14, nestin, vimentin, neurofilament 68 (NF-L) and glial fibrillary acidic protein (GFAP), were investigated immunohistochemically in developing and adult rabbit spinal ganglia. At 15 days of gestation, the spinal ganglion increased rapidly in volume and mainly consisted of three kinds of cells: small cells expressing vimentin, spindle-shaped cells co-expressing vimentin and nestin, and ovoid cells with an eccentric nucleus expressing nestin. Since some ovoid cells co-expressed nestin with either NF-L or GFAP, the ovoid cell may be considered to be an embryonic neural stem cell of the ganglion. In addition, a few keratin-positive polymorphic cells could be observed among these three kinds of cells. These polymorphic cells expressed five kinds of intermediate filament proteins, namely keratin 8, keratin 14, nestin, NF-L and GFAP. These cells were also detected in newborn and adult ganglia. A few neurons in the adult ganglion also expressed these five kinds of proteins as a Golgi-associated network. However, neurons expressing these proteins could not be detected in embryonic and newborn ganglia. Therefore, it may be considered that the keratin-positive polymorphic cell is a postnatal neural stem cell of the ganglion and that neurons transiently express keratin when polymorphic cells differentiate into neurons.

The duct epithelial cells of rabbit submandibular glands expressed keratin 8, keratin 14, and actin in the supranuclear region, and these cytoskeletal proteins formed ring structures, approximately 1-2.5 microm in diameter. Ultrastructurally, these ring structures were observed as a 'Golgi-associated filament network' surrounding Golgi apparatuses. Double immunofluorescent studies showed that keratin 8 and keratin 14 formed keratin 8/14 filaments, and that these filaments and actin filaments colocalized as components of the Golgi-associated filament network. Our studies suggested that the Golgi-associated filament network maintains the complex structure and location of the Golgi apparatus of the duct epithelium of rabbit submandibular glands. Tubulin was distributed diffusely throughout the cytoplasm of columnar cells, but no special relationship was found between tubulin and the Golgi apparatus.

In rabbit intestinal epithelium, vimentin intermediate filaments are selectively expressed in the M cells of follicle-associated epithelium (FAE). To find intestinal epithelial cells belonging to the M cell lineage, vimentin was detected immunohistochemically in the rabbit small and large intestines. Vimentin-positive columnar cells were scattered throughout the villus epithelium of the small intestine. In their cytoplasm, vimentin was located from the perinuclear region to the cell membrane touching intraepithelial lymphocytes. These cells had microvilli shorter than those of absorptive cells, and the alkaline phosphatase activity of the microvilli was markedly weaker than that of absorptive cell microvilli. Glycoconjugates on the surface of the microvilli were alcian blue positive and periodic acid-Schiff negative. The morphological and histochemical features of these vimentin-positive villus epithelial cells differed from those of adjacent absorptive cells and closely resembled those of the M cells in FAE covering Peyer's patches and solitary lymphatic nodules. These results suggest that the vimentin-positive cells in the villus epithelium belong to the M cell lineage.

Summary For the study of the differentiation process of optic vesicle epithelium into neural retina, pigment epithelium and pars caeca retinae, vimentin intermediate filament protein in retinal epithelial cells was detected immunohistochemi- cally in chick embryo at stages 11-21. In the late stage of optic vesicle development (stage 14), optic vesicle epithelium was classified into the following 3 different portions on the basis of vimentin staining intensity: latero-central epithelium under the lens placode, medio-central epithelium facing the latero- central epithelium, and peripheral epithelium connecting the latero-central and medio-central epithelia. Latero-central epithelium, the future neural retina, exhibited strongest staining of vimentin of the 3 portions. In contrast, medio-central epithelium, the future pigment epithelium, showed weakest staining. Moderate staining was observed in peripheral epithelium, the future pars caeca retinae. These differences in levels of vimentin expression were observed during optic cup formation. The present results clearly demonstrate that differentiation of retinal epithelium into neural retina, pigment epithelium and pars caeca retinae occurs in the late stage of the optic vesicle, and that retinal differentiation is reflected by the amount of vimentin in epithelial cells.

The aim of this study was to establish whether or not mononuclear cells which appear in both the vitelline vessels and embryonic coelom in mice prior to liver hemopoiesis are specialized scavengers. Before the initiation of liver hemopoiesis, the majority of the embryonic blood cells were primitive erythroblasts derived from yolk sac hemopoietic foci. In addition, the peripheral blood contained a few free phagocytes as early as 10 days of gestation. The phagocytes devoured various cell elements such as degenerating erythroblasts and cell fragments. Ultrastructurally, they had long filamentous cytoplasmic projections on their cell surface, clear subsurface vacuoles or vesicles, lipid droplets, a few lysosomal granules, large heterogeneous phagolysosomes and residual bodies. Mononuclear phagocytes with ultrastructural features similar to those of the intravascular phagocytes also could be observed in the intraembryonic peritoneal cavities at 10 days of gestation; they sometimes engulfed possible mesothelial cells undergoing degeneration. Based on fine structural criteria, these intravascular and coelomic phagocytes were considered to be specialized scavenger macrophages with the function of clearing the blood and tissue fluid of whatever has been ingested. In so doing, they serve as the most primitive discriminating filter set in embryonic circulation.

Glycoconjugates on the surface of pulmonary epithelial cells were ultrahistochemically examined in the fetal, neonatal and adult rat lung. Lectin and colloidal iron staining procedures were performed in combination with digestion using carbohydrate-degrading enzymes or methylation. The glycoconjugate composition of columnar cells at 16 days gestation was similar to that of cuboidal cells at 19 days gestation. Glycoconjugate differentiation on the cell surface occurred at 20 days gestation, and especially the loss of soybean agglutinin (SBA) binding sites could be detected on type II cells. The contents of Ricinus communis agglutinin-I (RCA-I) and Concanavalin A (Con A) binding sites on type II cells also began to decrease. On the contrary, the content of sulfated saccharides decreased on the surface of type I cells during development. Glycoconjugate differentiation on both type I and II cells was completed with the disappearance of hyaluronic acid and peanut agglutinin (PNA) binding sites; type I and II cells acquired a similar histochemical composition to that on adult type I and II cells at 5 days after birth. Both type I and II cells share a common early precursor cell, that is, the cuboidal epithelial cell at the canalicular stage.

The ultrastructural localization of mucopolysaccharide in the pulmonary alveolus of a rat was studied by means of the colloidal iron reaction, the ruthenium red staining and the phosphotungstic acid (PTA) staining.Colloidal iron-positive material was observed in a thin layer, 15-25nm in thickness, closely attached to the luminal surface of the alveolar epithelium. The superficial layer, the hypophase, and the tubular-myelin figures, however, lacked the colloidal iron reactivity. Ruthenium red-positive material was observed on the luminal surface of the alveolar epithelium as well as in the hypophase. In the ultrathin sections stained with PTA (pH 0.4 and 1.5), the microvillous surface of the type II cell was densely stained, whereas the luminal surface of the type I cell was not discernible. The results suggest that acid mucopolysaccharide is not involved in the surfactant lining, but may be in existence as a surface coat on the alveolar epithelium; the surface coat on the type II cell differs chemically from that on the type I cell.