Intermediate filament typing of the human embryonic and fetal notochord

In order to characterize human notochordal tissue we investigated notochords from 32 human embryos and fetuses ranging between the 5th and 13th gestational week, using immunohistochemistry to detect intermediate filament proteins cytokeratin, vimentin and desmin, the cytokeratin subtypes 7, 8, 18, 19 and 20, epithelial membrane antigen (EMA), and adhesion molecules pan-cadherin and E-cadherin. Strong immunoreactions could be demonstrated for pan-cytokeratin, but not for desmin or EMA. Staining for pan-cadherin and weak staining for E-cadherin was found on cell membranes of notochordal cells. Also it was demonstrated that notochordal cells of all developmental stages contain the cytokeratins 8, 18 and19, but not 7 or 20. Some cells in the embryonic notochord also contained some vimentin. Vimentin reactivity increased between the 8th and 13th gestational week parallel to morphological changes leading from an epithelial phenotype to the chorda reticulum which represents a mesenchymal tissue within the intervertebral disc anlagen. This coexpression reflects the epithelial-mesenchymal transformation of the notochord, which also loses E-cadherin expression during later stages. Our findings cannot elucidate a histogenetic germ layer origin of the human notochord but demonstrate its epithelial character. Thus, morphogenetic inductive processes between the human notochord and its surrounding vertebral column anlagen can be classified as epithelial-mesenchymal interactions.     

Transient coexpression of cytokeratin and vimentin in differentiating rat Sertoli cells

The expression of cytokeratin and vimentin type intermediate filaments were studied in fetal, postnatal, and adult rat testes. Immunocytochemical observations were correlated with the light and electron microscopic analysis of the developing organs. The Sertoli cell precursors in 15-day-old fetal testes contained both cytokeratin and vimentin. A gradual reorganization of both filaments, accompanied by a decrease of cytokeratin-positivity, was observed toward the end of the fetal period. The simultaneous presence of cytokeratin and vimentin in the same cells was shown by double immunofluorescence of newborn testes and the primary culture of dissociated testicular cells. In postnatal Sertoli cells, cytokeratin-positivity continued to decrease and disappeared by the age of 14 days. The increase in vimentin content and the appearance of axially oriented vimentin filaments coincided with the acquisition of the columnar shape of the Sertoli cells. The presence of cytokeratin and vimentin in fetal and newborn testes, and only vimentin in the adult testes was confirmed by immunoblotting. The present results suggest that major qualitative changes in the expression of intermediate filament proteins can take place during the embryonic development. The expression of cytokeratin in developing Sertoli cells, although only transient, supports the epithelial origin of these cells and can be applied as a marker for embryonic and early postnatal Sertoli cells.     

Transient coexpression of desmin and cytokeratins 8 and 18 in developing myocardial cells of some vertebrate species

During myogenesis the intermediate-sized filament (IF) cytoskeleton is characterized by increasing proportions of desmin. While skeletal and smooth muscle formation occurs in free mesenchymal cells containing vimentin-type IFs, myocardial development starts from a polar epithelium containing cytokeratin IFs and desmosomes. Therefore, we have studied the formation of the epicardium and the myocardium in different vertebrate species, combining light and electron microscopic immunolocalization techniques with gel-electrophoretic analyses of cytoskeletal proteins of microdissected myocardial tissue at differing developmental stages. In this report, we describe results obtained from advanced stages of myocardial differentiation. In all species studied the myocardial cell possess IFs abundant in desmin, often together with smaller amounts of vimentin, and the mesothelial layer of the epicardium contains cytokeratin IFs. However, we have observed remarkable interspecies differences with respect to the occurrence of cytokeratins in embryonic myocardial cells. In fetal human myocardium, from week 10 of pregnancy on, but not in juvenile and adult myocardium, and in chicken myocardium of all stages examined (until several days after hatching) specific immunostaining was seen with certain broad-range cytokeratin antibodies as well as with antibodies specific for cytokeratins 18 (in both species) and 8 (showing significant reaction only in human). This cytokeratin immunoreaction, however, did not appear in IFs extending throughout the cytoplasm or at Z-lines, but was localized in punctate arrays representing aggregates of dense material. The aggregates were often enriched at, but not restricted to, the desmosomal plaques of the intercalated discs. These observations were supported by gel-electrophoretic demonstration of small but significant amounts of cytokeratins 18 (in both species) and 8 (detected only in human) in microdissected myocardial tissue. We also observed cytokeratins in smooth muscle cells of some cardiac blood vessels. In contrast, bovine myocardium of advanced fetal age as well as rat and mouse myocardium (from fetal day 12 on) were negative for cytokeratins with all methods, although epicardial cytokeratin IFs were demonstrable. These observations are discussed in relation to myocardial histogenesis and to general problems of cytokeratin gene expression control in epithelial and nonepithelial cells.     

Structural proteins in sexual differentiation of embryonic gonads

Sexual differentiation of embryonic gonads was studied by immunocytochemical analysis of cytoskeleton, basement membrane and extracellular matrix. The epithelial cells of the prospective gonadal region in both sexes contained vimentin and desmin intermediate filament proteins but not cytokeratin. Basement membrane components laminin, collagen types IV and V, heparan sulfate proteoglycan, and fibronectin were seen in an unorganized form in the extracellular space. The development of the gonads started by proliferation of the pregonadal epithelial cells, which formed separate clusters and loose mesenchyme. In the male gonad the clusters joined together into elongated cords, outlined by basement membrane components. The cord cells became polarized epithelial cells, and cytokeratin appeared with the disappearance of desmin in their cytoplasm. Desmin and vimentin remained in the interstitial cells. In the female gonad the clusters were smaller, and the cords were irregular in shape and size. Desmin disappeared from the cord cells and cytokeratin appeared, but more slowly and less well polarized than in the testis. The present results show that after common early development, the sexual differences in gonads emerge as different organization of the internal epithelial tissue with different timing of changes in intra- and extracellular components.     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a pan-epithelial monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation     

Modulation of cytokeratin expression during in vitro cultivation of human hepatic stellate cells: evidence of transdifferentiation from epithelial to mesenchymal phenotype

The embryonal origin of hepatic stellate cells (HSCs), the principal cells in hepatic fibrogenesis, is still intriguing. To explore the origin and the differentiation of HSCs, we studied the expression of cytokeratin 18 (CK18) and 19 (CK19), the standard markers of simple epithelial cells, in cultured human HSCs. Hepatic stellate cells were isolated from five normal human livers. In immunofluorescence staining, both clone C-51 anti-CK18 antibody and clone RCK108 anti-CK19 antibody labeled almost all stellate cells in the primary culture. Double immunofluorescence staining for cytokeratin/vimentin and cytokeratin/alpha-smooth muscle actin detected by confocal laser scanning microscopy clearly demonstrated the localization of cytokeratin immunoreactivity in human HSCs. During subsequent cultivation of human HSCs to the tenth passage, immunocytochemical staining and western blot analysis demonstrated gradually decreasing profiles of CK18 and CK19 expression. The progressive reduction of cytokeratin expression was further confirmed in a culture of clone cells originated from a single HSC. In conclusion, both CK18 and CK19 are expressed in cultured human HSCs, and the extent of their expression decreases gradually during prolonged cultivation. Our results suggest that HSCs may be of epithelial origin, and that they undergo the transdifferentiation from epithelial to mesenchymal phenotype during an activation process in vitro.     

An unusual type of cytokeratin filament in cells of a human cloacogenic carcinoma derived from the anorectal transition zone

Epithelia-derived tumors (carcinomas) can be distinguished from mesenchymally derived tumors by the presence of intermediate-sized filaments of the cytokeratin type, which usually coincides with the absence of other types of intermediate-sized filaments such as vimentin filaments. In the course of diagnostic examinations of human tumors, using immunofluorescence microscopy, we have come across a case of an unusual carcinoma (Primary tumor and lymph node metastasis) positively stained not only with cytokeratin antibodies but also with immunoglobulins present in vimentin antisera. Therefore, this tumor, a cloacogenic carcinoma apparently derived from the rectal-anal transitional region, has been examined in greater detail using both immunofluorescence microscopy and immuno-electron microscopy as well as gel electrophoretic analysis of cytoskeletal polypeptides from total tumor tissue and from microdissected nodules enriched in carcinoma cells. The unusual reaction of the carcinoma cells with immunoglobulins present in seven different (rabbit or guinea pig) antisera raised against vimentin, has been found to be diminished after absorption on purified cytokeratin or total epidermal cytoskeletal material, but not after absorption on purified vimentin. Gel electrophoretic analysis of tumor cytoskeletons showed an unusual complex pattern of cytokeratin polypeptides containing relatively large (Mr 68,000 and Mr 58,000) neutral-to-slightly basic cytokeratins, as are typically found in epidermis and other stratified squamous epithelia, as well as several smaller acidic cytokeratins, including a Mr 40,000 polypeptide found in certain nonstratified epithelial such as colon and small intestine. Total tumor also showed the inclusion of some vimentin which, however, was significantly decreased in analysis of excised carcinoma nodules. Examining antibody binding to polypeptides separated by gel electrophoresis and blotted on nitrocellulose paper, we have found that antisera raised against vimentin contained not only vimentin antibodies but also immunoglobulins which specifically bound to the largest cytokeratin component. We conclude that the unusual reaction of immunoglobulins present in vimentin antisera with cytokeratin filament bundles does not represent specific binding to vimentin in these carcinoma cells, but is due to a component obviously widespread in vimentin antisera which binds specifically to a cytokeratin present in this type of tumor but not in most other carcinomas. It is proposed that use is made in diagnostic examinations of vimentin antisera or affinity-purified vimentin antibodies that have been pre-absorbed on cytokeratin protein, in order to eliminate such disturbing reactions.     

Intermediate filament proteins in nonfilamentous structures: Transient disintegration and inclusion of subunit proteins in granular aggregates

The intermediate filament cytoskeleton of cultured bovine kidney epithelial cells and human HeLa cells changes dramatically during mitosis. The bundles of cytokeratin and vimentin filaments progressively unravel into protofilament-like threads of 2–4 nm diameter, and intermediate filament protein is included in numerous, variously sized (2–15 μm) spheroidal aggregates containing densely stained granular particles of 5–16 nm diameter. We describe these mitotic bodies in intact cells and in isolated cytoskeletons. In metaphase to anaphase of normal mitosis and after colcemid arrest of mitotic stages, many cells contain all their detectable cytokeratin and vimentin material in the form of such spheroidal aggregate bodies, whereas in other mitotic cells such bodies occur simultaneously with bundles of residual intermediate filaments. In telophase, the extended normal arrays of intermediate filament bundles are gradually reestablished. We find that vimentin and cytokeratins can be organized in structures other than intermediate filaments. Thus, at least during mitosis of some cell types, factors occur that promote unraveling of intermediate filaments into protofilament-like threads and organization of intermediate filament proteins into distinct granules that form large aggregate bodies. Some cells, at least certain epithelial and carcinoma cells, may contain factors effective in structural modulation and reorganization of intermediate filaments.     

K562 erythroleukemia cells express cytokeratins 8, 18, and 19 and epithelial membrane antigen that disappear after induced differentiation.

The effects of differentiation-modulating drugs were studied on the expression of intermediate filaments (IFs) in the human K562 erythroleukemic cell line. The untreated cells contained typical cytoplasmic coiling bundles, positive for both vimentin and cytokeratin as judged by indirect immunofluorescence microscopy with monoclonal antibodies (Mabs). Some of the cells also showed bright immunoreactivity for epithelial membrane antigen (EMA), as revealed with a Mab and polyclonal antiserum. When exposed to hemin or to sodium butyrate, most of the cells became cytokeratin negative within 3 days and showed dispersion of vimentin fibrils. Upon exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the amount of both vimentin and cytokeratin appeared to be greatly increased within 3 days and was found both in dispersed cytoplasmic fibrils, in large spherical, eccentric aggregates, as well as in cytoplasmic fibrils in cells spreading on fibronectin. TPA induced a complete loss of proliferation, as judged by immunostaining with the Mab Ki-67. The effects of TPA were found to be irreversible and could be induced by only a short exposure to the drug. Western blotting analysis and monoclonal antibodies to individual cytokeratins revealed that untreated K562 cells expressed Mr 52,000 (No. 8), 46,000 (No. 18), and 40,000 (No. 19) cytokeratin polypeptides, which disappeared when the cells were exposed to hemin or to sodium butyrate to induce erythroid differentiation but were greatly enhanced when exposed to TPA. The monoclonal anti EMA antibody reacted in K562 cells with a single Mr 320,000 polypeptide that was also revealed in MCF-7 breast carcinoma cells. Human bone marrow cells or other leukemic cell lines with erythroid differentiation capacity (HEL and KG-1) did not contain cytokeratin- or EMA-immunoreactive cells, suggesting that in K562 cells these properties may rather represent abnormal cytodifferentiation or retrodifferentiation toward early embryonic mesenchymal cells, than a more general expression of epithelial features in human leukemic cells.     

Fibrillin-1 and fibrillin-2 in human embryonic and early fetal development.

The extracellular glycoproteins fibrillin-1 and fibrillin-2 are major components of connective tissue microfibrils. Mutations in the fibrillin-1 and fibrillin-2 genes are responsible for the phenotypical manifestations of Marfan syndrome and congenital contractural arachnodactyly respectively, which emphasizes their essential roles in developmental processes of various tissues. Consistent with this last notion, organ culture experiments have indirectly suggested morphogenic roles for fibrillins in lung and kidney development. In order to contribute to the understanding of the roles of fibrillins in developmental and morphogenetic events, we have investigated the distribution of fibrillin-1 and fibrillin-2 in human embryonic and early fetal tissues between the 5th and the 12th gestational week, i.e. at the beginning of organogenesis. Fibrillin-1 and fibrillin-2 were localized immunohistochemically using specific monoclonal antibodies, mAb 69 and mAb 48, respectively. Both fibrillins are widely distributed in various human anlagen, from early developmental stages. In most embryonic and early fetal human organs such as skin, lung, heart, aorta, central nervous system anlage, nerves, and ganglia, fibrillin-1 and fibrillin-2 follow the same temporo-spatial pattern of distribution. However, in other organs such as kidney, liver, rib anlagen, notochord fibrillin-1 and fibrillin-2 are distributed differentially. The present paper is focused on this aspect. These results suggest different roles for fibrillin-1 and -2 in the development of these structures.     

Inhibition of "spontaneous," notochord-induced, and collagen-induced in vitro somite chondrogenesis by the calcium lonophore, A23187.

The present study represents a first step in investigating the possible involvement of calcium (Ca2+) in the stimulation of somite chondrogenesis elicited by extracellular matrix components produced by the embryonic notochord. The ionophore, A23187, a drug that facilitates Ca2+ uptake leading to elevation of cytoplasmic Ca2+ levels, at concentrations of 0.25-1.0 microgram/ml severely impairs "spontaneous" somite chondrogenesis, i.e., inhibits the formation of the small amount of cartilaginous matrix normally formed by embryonic somites in vitro in the absence of inducing tissues. This inhibition is reflected in a considerable reduction in sulfated glycosaminoglycan (GAG) accumulation by A23187-treated somite explants. Furthermore, A23187 inhibits the striking stimulation of cartilaginous matrix formation and sulfated GAG accumulation normally elicited by the embryonic notochord and collagen substrates. In fact, 1.0 microgram/ml of A23187 reduces sulfated GAG accumulation by somites cultured in association with notochord or on collagen to a level even below that accumulated by somites cultured in the absence of these inductive agents. Although these results must be interpreted with caution, they provide incentive for considering a possible regulatory role for Ca2+ in the chondrogenic response of somites to extracellular matrix components produced by the embryonic notochord.     

Cytokeratins in certain endothelial and smooth muscle cells of two taxonomically distant vertebrate species, Xenopus laevis and man

Using immunolocalization techniques, electron microscopy, and gel electrophoresis combined with immunoblotting, we have noted remarkable interspecies differences in the expression of cytokeratins in certain nonepithelial cells. In the present study we describe, in two taxonomically distant vertebrate species, the African clawed toad Xenopus laevis and man, endothelial and smooth muscle cells which express cytokeratin intermediate filaments (IFs), in addition to vimentin and/or desmin IFs. In Xenopus, all endothelia seem to produce both vimentin and cytokeratin IFs. As well, certain smooth muscle bundles located in the periphery of the walls of the esophagus and the urinary bladder produce small amounts of cytokeratin IFs in addition to IFs containing vimentin or desmin or both. The amphibian equivalents of human cytokeratins 8 and 18 have been identified in these nonepithelial tissues. In human endothelial cells, immunocytochemical reactions with certain cytokeratin antibodies are restricted to a rare subset of blood vessels. Vessels of this type were first noted in synovial and submucosal tissues, but also occur in some other locations. Cytokeratins have also been detected in certain groups of smooth muscles, such as those present in the walls of some blood vessels in synovial tissue and umbilical cord. Here, the synthesis of low levels of cytokeratins 8 and 18, sometimes with traces of cytokeratin 19, has been demonstrated in smooth muscle cells by colocalization with myogenic marker proteins, such as desmin and/or the smooth-muscle-specific isoform of alpha-actin. Possible reasons for the differences in cytokeratin expression between adjacent endothelia in man, and smooth-muscle structures in both species, as well as biologic and histodiagnostic implications of these findings, are discussed.     

Presence of cilia in the chick embryonic notochord

Cilia have been observed in cells of the chick embryonic notochord in different developmental stages. The ciliated notochordal cells were present both at the center and at the periphery of the organ. A well outlined central cavity in the notochord was not observed.     

Intermediate filament protein expression in early developmental stages of the mouse

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.     

Cell type heterogeneity of intermediate filament expression in epithelia of the human pituitary gland

In the present study we have localized immunohistochemically the intermediate filament proteins of the human pituitary gland (adenohypophysis, pars intermedia and pars tuberalis) by an indirect immunoperoxidase technique or by double immunofluorescence methods and analysed the individual cytokeratin polypeptides using two-dimensional gel electrophoresis. We found that the expression of cytokeratins in different epithelial cells of the human anterior pituitary gland was heterogeneous. Whereas the endocrine cells only expressed cytokeratins 8 and 18, the folliculo-stellate cells exhibited a reactivity for cytokeratins 7, 8, 18 and 19 as well as for GFAP and vimentin. The squamous epithelial cells of the pars tuberalis and the Ratke's cysts showed a more complex cytokeratin pattern of both squamous and simple type. Whereas in may cystic epithelial cells including the "pseudo-follicles" a triple expression of cytokeratin, vimentin and GFAP could be observed, only some basal cells of squamous epithelial nests coexpressed cytokeratin and vimentin. The differences in the intermediate filament protein distribution are discussed in the light of embryological relationships of the different parts of the human pituitary gland.     

Expression of intermediate-sized filaments in developing and adult human kidney and in renal cell carcinoma.

We studied the distribution of intermediate-sized filaments in developing and adult kidneys and renal cell carcinoma (RCC) by indirect immunohistochemistry, using a pan-cytokeratin mouse monoclonal antibody (MAb), chain-specific anti-cytokeratin MAb, and anti-vimentin and anti-desmin MAb, to resolve controversy concerning intermediate-sized filament expression in the kidney. With the pan-cytokeratin MAb, cytokeratin expression was detectable in all stages of nephron development, starting with expression in the renal vesicles, the progenitors of the glomeruli, proximal tubules, Henle's loop, and part of the distal tubules. Using chain-specific anti-cytokeratin MAb, cytokeratin 8 and 18 expression was demonstrated in all developmental structures of the nephron, whereas cytokeratin 19 expression was more complex. None of the nephrogenic blastema cells from which the renal vesicles arise expressed cytokeratins. Transient expression of vimentin and cytokeratin 19 was observed in differentiating collecting ducts and proximal tubule cells at the S-shaped stage of nephron development, respectively. In RCC, cytokeratin expression closely resembled that of the mature proximal tubule, i.e., RCC cells expressed cytokeratins 8 and 18. However, in a subset of RCC additional cytokeratin 19 expression was noted. In addition, all except one RCC showed co-expression of cytokeratins and vimentin.     

Epithelial and mesenchymal cell differentiation in the fetal rat genital ducts: changes in the expression of cytokeratin and vimentin type of intermediate filaments and desmosomal plaque proteins

Mesonephric and paramesonephric ducts develop in different ways in male and female fetuses. We have analyzed the changes in the expression of cytokeratin and vimentin type of intermediate filaments and desmosomal plaque proteins in progressing and regressing genital ducts of rat fetuses. The concomitant changes in the basement membranes were detected by laminin antibody. Epithelial cells of the indifferent (Day 15) male and female mesonephric and paramesonephric ducts contained faint vimentin positivity which, however, later disappeared. Indifferent mesonephric duct epithelium stained strongly for cytokeratin, whereas in the corresponding paramesonephric duct only a weak and spotty positivity was seen. Immunocytochemical localization of cytokeratin filaments and desmosomal plaque proteins correlated with the ultrastructural differences in the apical junctional complexes of the mesonephric and paramesonephric ducts. Regardless of the ongoing regression of the male paramesonephric duct, cytokeratin positivity increased in the disorganizing epithelium; the most weak and a granular immunoreaction was seen in the cells found in the intensively vimentin-positive periductal mesenchyme. In the regressing female mesonephric duct cytokeratin positivity was lost before the final dissolution of the basement membrane. Immunoblotting analysis of cytokeratin and vimentin polypeptides of the individual genital ducts were in agreement with the immunocytochemical results obtained in 15- and 16-day-old fetuses. The results suggest that the expression of vimentin type intermediate filaments is an indication of the mesothelial origin of the genital ducts. The increase in cytokeratin positivity of the regressing paramesonephric duct epithelium suggests that the degenerative changes are initiated by the mesenchyme. Cytokeratin-positive cells found in the periductal mesenchyme of the male paramesonephric duct may be epithelial cells transforming into mesenchyme. The results emphasize a close relationship between the changes of the intermediate filament system and extracellular matrix upon differentiation of the fetal genital ducts.