Chicken Embryo Kidney Research Articles

Microvascular growth, development, and remodeling in the embryonic avian kidney: The interplay between sprouting and intussusceptive angiogenic mechanisms

Embryonic development is associated with extensive vascular growth and remodeling. We used immunohistochemical, light and electron microscopical techniques, as well as vascular casting methods to study the developing chick embryo kidney with special attention to the interplay between sprouting and intussusceptive vascular growth modes. During inauguration at embryonic day 5 (E5), the early mesonephros was characterised by extensive microvascular sprouting. By E7, the vascular growth mode switched to intussusception, which contributed to rapid kidney vasculature growth up to E11, when the first obvious signs of vascular degeneration were evident. The metanephros underwent similar phases of vascular development inaugurating at E8 with numerous capillary sprouts and changing at E13 to intussusceptive growth, which was responsible for vascular amplification and remodeling. A phenomenal finding was that future renal lobules arose as large glomerular tufts, supplied by large vessels, which were split into smaller intralobular feeding and draining vessels with subsequent formation of solitary glomeruli. This glomerular duplication was achieved by intussusception, i.e., by formation of pillars in rows and their successive merging to delineate the vascular entities. Ultimately, the maturation of the vasculature was achieved by intussusceptive pruning and branching remodeling. An interesting finding was that strong VEGF expression was associated with the sprouting phase of angiogenesis while bFGF was upregulated during the phase of intussusceptive microvascular growth. We conclude that microvascular growth and remodeling in avian kidney follows an adroitly crafted pattern, which entails a precise spaciotemporal interplay between sprouting and intussusceptive angiogenic growth modes supported partly by VEGF and bFGF.

A high throughput single-stranded conformation polymorphism assay for detection, subtyping and genotyping of influenza viruses

We have developed a high throughput single-stranded conformation polymorphism/capillary electrophoresis genotyping assay (SSCP/CE) to detect the presence of influenza virus and to determine its serotype, subtype and genotype. We configured the assay in two slightly different formats to suit the two applications: (i) To detect the presence of influenza virus and to determine its serotype and subtype in the clinical samples and (ii) to determine the genetic configuration of influenza virus reassortants generated by co-infection of two influenza virus strains in chicken embryo kidney (CEK) cells. We applied this newly developed assay to screen a large number of reassortants for a specific 6:2 genetic configuration characteristic of the FluMist Vaccines which exhibits two unique features: (i) the HA and NA segments from the circulating wild-type strains to confer immunogenicity and (ii) the internal segments of the master donor virus (MDV) to impart the attenuation phenotype. To evaluate the accuracy of the SSCP/CE assay, we analyzed 400 gene segments from approximately 50 different reassortant viruses and compared the SSCP/CE results with those obtained by the more conventional restriction fragment length polymorphism (RFLP) analysis. We found a very high concordance (98%) between the two sets of data.

The effect of influenza B virus M segment on the replication of reassortant vaccine viruses

Flumist™ is a cold-adapted (ca), live attenuated influenza virus vaccine containing H1N1, H3N2, and B strains. The vaccine strains have a 6:2 gene constellation. Six gene segments, PB1, PB2, PA, NP, M and NS, are derived from the master donor viruses, ca A/Ann Arbor/6/60(MDV-A) and ca B/Ann Arbor/1/66(MDV-B), and two gene segments, the hemagglutinin (HA) and neuraminidase (NA), are derived from circulating wild-type strains. We observed that the M gene segment of influenza B virus reassortants can influence the replication of these viruses in CEK cells. Replication curves revealed that a 6:2 reassortant B virus (wt HA and wt NA) replicated less well, by approximately 2 log 10, than the corresponding 5:3 strain (wt HA, wt NA and wt M) at 33 °C in chicken embryo kidney (CEK) cells; this difference was not observed at 25 °C.

The hydropericardium syndrome in poultry--a current scenario.

Inclusion-body hepatitis hydropericardium syndrome (IBH-HPS) is an important, recently emerged, disease of poultry, particularly of 3- to 6-week-old broiler chicks, characterized by its sudden onset, with high mortality ranging from 20% to 70%, typical hydropericardium and enlarged mottled and friable livers, with intranuclear inclusion bodies in the hepatocytes. The causative agent is a non-enveloped icosahedral fowl adenovirus (FAV) serotype 4, belonging to the Adenovirus genus of the family Adenoviridae, which can be propagated or cultivated in chicken embryo liver and kidney primary cell cultures. The transmission of disease occurs vertically and laterally by the oral-faecal route. The liver of infected birds shows necrotic foci and basophilic intranuclear inclusion bodies in the hepatocytes. The disease can be diagnosed from its gross and histopathological changes in the liver and by various serological tests, such as agar gel immunodiffusion, counterimmunoelectrophoresis, indirect haemagglutination, the fluorescent antibody technique, enzyme-linked immunosorbent assay and the polymerase chain reaction. The disease has been brought under control by the use of formalin-inactivated vaccines, prepared from infected liver homogenate, and of inactivated cell culture vaccines. The vaccines are effective in the face of natural outbreaks or experimental challenge and significantly reduce the mortality.

Interactions between escherichia coli and Newcastle disease virus in chickens.

We investigated the interaction between Newcastle disease virus (NDV) and Escherichia coli in cell cultures, embryonated eggs, and 8-wk-old chickens. We measured the interactions on the basis of bacterial adherence and NDV hemagglutination titer in chickens, chicken embryos, and chicken embryo cell culture. Depending on the inoculation order of E. coli, a significant alteration of the growth of NDV was observed in both chickens and chicken embryos. When certain strains of E. coli were given before NDV exposure, the virus titers were lowered. In chickens, the mean virus titer was significantly (P < 0.05) lowered in the crop, the proventriculus, the gizzard, and the jejunum. However, there were no significant differences (P < 0.05) between the two groups for NDV titers in the duodenum, ileum, and cecum. In chicken embryos, when E. coli serotypes O78 and O119:B14 were inoculated before NDV exposure, the mean NDV titers were significantly (P < 0.5) lowered. However, there were no significant differences (P < 0.05) in NDV titer between the two groups when E. coli serotypes O78:K80:NM and O1ab:K NM were inoculated 24 hr before NDV exposure. When NDV was given prior to E. coli exposure, NDV titer was higher in both chickens and chicken embryos. In chickens, when NDV was given 48 hr before E. coli inoculation, NDV was detected in the proventriculus, gizzard, jejunum, ileum, and cecum, whereas no virus was detected in the control groups (NDV only). In the crop, NDV was detected at a significantly (P < 0.05) higher titer in the E. coli-inoculated group when compared with the control group that received NDV alone. In chicken embryos, virus titer was significantly (P < 0.05) higher when NDV was given 24 hr before E. coli inoculation for all three NDV strains used (Ulster and V4 strains). Adherence of E. coli to chicken embryo kidney (CEK) cells was significantly higher (P < 0.05) when the CEK cells were infected first with NDV and then by E. coli. The mean bacterial count per microscopic field in NDV-uninfected monolayers was eight compared with 112 for the NDV-infected monolayers. In approximately 10% of the fields in NDV-infected monolayers, the bacteria were too numerous to count.

Preparation and characterisation of attenuated cold-adapted influenza A reassortants derived from the A/Leningrad/134/17/57 donor strain

The development of a rapid cell culture method for the preparation of cold-adapted (ca) influenza A reassortant viruses is described and compared with a currently used egg method. Mixtures of the ca donor A/Leningrad/134/17/57-ca (A/Len/17) and A/Beijing/32/92 (A/Beij/32), a recent H3N2 epidemic strain, were used to co-infect chicken embryo kidney (CEK) cell cultures; reassortant progeny were selected using an infectious centre assay. The assay was capable of detecting interference where the infectivity ratio for A/Len/17 and A/Beij/32 was 1:7. Progeny viruses were characterised genetically by amplification of defined regions within each of the six internal genes by PCR and identification of the products by restriction enzyme analysis. Reassortants were also tested for ca and temperature-sensitive (ts) phenotype and the identity of the surface antigens. The infectious centre assay was shown to be an effective method for isolating reassortant progeny that possessed the haemagglutinin and neuraminidase surface antigens of A/Beij/32. Reassortants with the six internal genes derived from the donor strain and possessing the ca and ts phenotype were readily obtained when (a) an infectivity ratio of 1:49 was used and (b) two plaque-to-plaque isolations of progeny virus were made after growth at 25 °C and one at 34 °C, both in the presence of antiserum to the donor strain.

TCDD Induces CYP1A4 and CYP1A5 in Chick Liver and Kidney and Only CYP1A4, an Enzyme Lacking Arachidonic Acid Epoxygenase Activity, in Myocardium and Vascular Endothelium

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other Ah receptor ligands, species differences in sensitivity and the relationship of CYP1A induction to the toxicity, are poorly understood. Ah receptor ligands induce formation of CYP1A1 and 1A2 in mammals and of a different set of enzymes, CYP1A4 and 1A5, in chicks. We examined induction by TCDD of CYP1A4 and 1A5 mRNA and protein in chick embryo liver, heart, kidney, lung, intestine, bursa, spleen, thymus, brain, and muscle by in situ hybridization and immunohistochemistry and verified the histochemical findings by CYP-specific assays, 7-ethoxyresorufin deethylase for CYP1A4 and arachidonic acid epoxygenation for CYP1A5. CYP1A4 alone was extensively induced in the cardiovascular system, in cardiac myocytes, in perivascular cells having the same location as impulse-conducting Purkinje cells, and like CYP1A1, in vascular endothelium in every organ examined. Unlike mammalian CYP1A, CYP1A4 and 1A5 were both substantially induced in kidney proximal tubules as well as liver, and neither enzyme was induced in kidney glomeruli or lung or brain parenchymal cells. The findings demonstrate (a) a route for CYP1A4 to affect cardiac function, (b) that vascular endothelium is a major site of CYP1A induction across species, and (c) that CYP1A induced in heart or endothelial cells cannot affect cardiac or vascular function via generation of arachidonic acid epoxides because the CYP1A enzymes induced in those organs are not arachidonic acid epoxygenases. Further, the specificity of CYP1A induction sites and of the catalytically active enzymes induced at each site support a significant role for CYP1A induction in Ah receptor ligand toxicity and species differences in sensitivity.

Transcriptional Activation of AvianCYP1A4andCYP1A5by 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Differences in Gene Expression and Regulation Compared to MammalianCYP1A1andCYP1A2

The toxicity, carcinogenicity, and biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are dependent upon activation of theAhreceptor, a ligand-activated transcription factor. Ah receptor activation leads to the induction of cytochrome P450 (CYP) 1A enzymes, which include CYP1A1 and 1A2 in mammals and CYP1A4 and 1A5 in chickens. CYP1A induction is a major effect of TCDD exposure although its relationship to TCDD toxicity and carcinogenicity are not understood. In these studies we investigated by nuclear run-on transcription assays along with Northern and Western blotting in chick embryo liver, kidney, and heart whether avian CYP1A4 and 1A5, like mammalian CYP1A1 and 1A2, are transcriptionally induced by TCDD and whether the chick CYP1A enzymes exhibit differences analogous to mammalian CYP1A enzymes in organ expression. We report that CYP1A4 and 1A5, like CYP1A1 and 1A2, are transcriptionally induced by TCDD in liver. However, whereas CYP1A1 is not constitutively expressed in liver, CYP1A2 and both CYP1A4 and 1A5 are constitutively expressed. Further, whereas TCDD induces only CYP1A1 and not CYP1A2 in extrahepatic organs, TCDD induces both CYP1A4 and 1A5 in chick kidney. Also, TCDD induced CYP1A4 but not 1A5 in both myocardium and heart vessels whereas CYP1A1 induction has only been found in endocardium. Further, liver CYP1A4 and 1A5 mRNAs had the same half lives and were both superinduced by cycloheximide, whereas mRNA half lives differ for CYP1A1 and 1A2, and cycloheximide superinduces only CYP1A1. We suggest that there are species differences in the effects of TCDD on CYP1A gene expression, organ distribution, and regulation that are likely to be accompanied by differences in CYP1A function and that this diversity may contribute to the large differences in sensitivity to TCDD among species.

The development and evaluation of two tissue culture‐grown Marek's disease challenge viruses

Standardized challenge viruses are essential for the evaluation of Marek's disease (MD) vaccines with many MD challenge preparations consisting of lymphocytes or whole blood from infected birds. Virus present in these preparations is difficult to quantify by tissue culture assays and, therefore, the infectious bird dose and long‐term storage viability cannot be assured. We report on the properties of two low‐passage virulent Australian MD viruses, the Woodlands No. 1 strain and strain MPF 57. Both strains were isolated in chicken embryo kidney cultures and adapted to grow in chicken embryo fibroblast cultures for a maximum of 14 passages. Both strains could be readily assayed in tissue culture and produced titres of 103 to 104 50% tissue culture infectious doses per ml (TCID50). Birds inoculated at three different doses were observed over 10 weeks, and tissues examined for gross and histological lesions, bursarbody weight ratios and the presence of viraemia. Tissue culture‐grown preparations of both strains were only slightly less virulent than the original lymphocyte challenge material and produced similar pathological responses and around 80% death or gross lesions. From bursarbody weight ratios strain MPF 57 appeared to be more virulent than the Woodlands No. 1 strain.

Effects of N-nitrosodimethylamine on glutathione levels during development of chick-embryo

Glutathione content in the liver, kidney and eye of chick embryo increased during development, having the maximum at day 17 after incubation of eggs. Activity of enzymes involved in glutathione metabolism changed during development. γ-Glutamyltransferase activity increased gradually until day 17 in the liver and eye, but decreased thereafter, while the enzyme activity increased continuously in the kidney. Glutathione reductase and glutathione peroxidase activities did not change significantly during development. Administration of N-nitrosodimethylamine (NDMA) to the fertile eggs 2 days after incubation resulted in an increase of glutathione levels in the liver and kidney at a dose of 0.01 mmol NDMA/egg, but the effect was not significant for glutathione levels by increasing the dose of NDMA. Decrease in viability and increase in formation of thiobarbituric acid (TBA) reactive substance were observed in the liver and kidney by administration of NDMA at 0.1 mmol/egg. By administration of buthionine sulfoximine (BSO) alone, viability and glutathione levels were decreased and TBA reactive substance was increased in the liver. Fatal toxicity of NDMA was observed especially when BSO was administered together with NDMA. These results indicate that glutathione plays an important role in protecting chick embryos against toxic effects induced by administration of NDMA.

Isolation of serotype 1 Marek's disease viruses from vaccinated Australian flocks

Field samples were received for Marek's disease virus (MDV) isolation from clinically affected flocks from several regions of Eastern Australia. Lymphocytes were fractionated in Ficoll-Paque and passaged once or twice in chicken embryo kidney cultures. Serotype-specific virus was detected in infected cultures by indirect immunofluorescence with monoclonal antibodies. Serotype 1 MDV was isolated from 10 flocks. In samples from 5 of these flocks, serotype 2 and 3 vaccine viruses were isolated from the same specimen. In a parallel study, plasmas obtained during lymphocyte isolation were tested for antibodies to MDVs by agar gel precipitin (AGP) tests using serotype 1 and 3 antigen extracts. No correlation was observed between the rate of virus isolation and AGP positivity. The AGP test was incapable of discriminating between the different MDV serotypes.

The immunogenicity of reassortants of the cold-adapted influenza A master strain A/Ann Arbor/6/60 is determined by both the genes for cold-adaptation and the haemagglutinin gene.

Two surface antigen segregants were prepared by co-infection of chicken embryo kidney cell cultures with reassortants of the cold-adapted influenza A master strain A/Ann Arbor/6/60-ca (H2N2) possessing the surface antigens of A/Queensland/6/72 (H3N2) and A/Hong Kong/123/77 (H1N1) and other genes that were common to the master strain. The segregants were shown by serological tests to possess H3N1 and H1N2 surface antigens but it was not possible to determine the presence of H1 or N1 genes by single-stranded RNA polyacrylamide gel electrophoresis. The immunogenic properties of A/Queensland/6/72-ca and H3N1 segregant CR6/35/2/9 were compared by immunising mice intranasally with graded doses of each virus twice at an interval of 3 weeks and then challenging with the wild-type A/Queensland/6/72 (H3N2). Clearance of the challenge virus occurred in mice immunised with the same vaccinating dose, indicating that the immunogenicity of both the ca H3N2 and H3N1 viruses was identical and similar findings were obtained for mice immunised with A/Hong Kong/123/77-ca and the H1N2 segregant CR6/35/1/19 and challenged with the wild-type A/Hong Kong/123/77. Therefore, there appears to be a good correlation between immunogenicity and the inheritance of the haemagglutinin gene.

Studies on a new enterovirus‐like virus isolated from chickens

During investigations into an outbreak of respiratory distress in broilers chicks, a small round virus was isolated following inoculation into chicken embryos. The isolate, designated 612, was identified as an enterovirus-like virus on the basis of its size and morphology, resistance to chloroform and to treatment at pH 3.0, and intracytoplasmic replication in cell culture. The virus produced a partial cytopathic effect following inoculation into chick embryo kidney cell cultures and viral antigens could be detected by immunostaining. The preferred culture method for 612 virus was by inoculation onto the CAM of chick embryos. Cross-immunofluorescence indicated that the virus is not antigenically related to five previously identified chicken enterovirus/enterovirus-like virus serogroups. Following experimental inoculation of 1-day-old male broilers a number of which had maternal antibody to 612, growth retardation ranging from 9.6 to 20.4% was detected. Serological studies demonstrated antibody to 612 virus was widespread in commercial chicken flocks in N. Ireland.

Biological characterisation of an Indian isolate of egg drop syndrome-76 virus

An isolate of egg drop syndrome-76 virus replicated best in primary chicken embryo liver cells and less well in duck embryo liver cells, duck embryo fibroblast cells and chicken embryo kidney cells. The cytopathic effect in chicken embryo liver cells was marked by the presence of round and refractile cells and detachment of cells from the glass surface. The intranuclear eosinophilic inclusion bodies were observed by 24 to 48 hours after infection. No virus multiplication was observed in primary quail embryo fibroblast cells, chicken embryo fibroblast cells or mammalian cells like Vero, BHK-21 and MDBK. Duck embryos supported the maximum growth of the virus, with allantoic fluid having the highest haemagglutinin titre, followed in order by chorioallantoic membrane, skin and internal organs. Chicken and quail embryos did not support the growth of the virus.

Further Evidence That the Nucleic Acid of Avian Encephalomyelitis Virus Consists of RNA

The nucleic acid of the Van Roekel strain of avian encephalomyelitis virus (AEV) was determined to be RNA, according to the inability of the nucleoside analog 5-bromo 2'-deoxyuridine (BUdR) to inhibit its growth in chicken embryo kidney cell cultures. The test was carried out using known DNA and RNA viruses as controls, and the results are consistent with classification of AEV as a member of the family Picornaviridae within the genus Enterovirus.

Localization of capping protein in chicken epithelial cells by immunofluorescence and biochemical fractionation.

We have localized capping protein in epithelial cells of several chicken tissues using affinity-purified polyclonal antibodies and immunofluorescence. Capping protein has a distribution in each tissue coincident with proteins of the cell-cell junctional complex, which includes the zonula adherens, zonula occludens, and desmosome. "En face" views of the epithelial cells showed capping protein distributed in a polygonal pattern coincident with cell boundaries in intestinal epithelium, sensory epithelium of the cochlea, and the pigmented epithelium of the retina and at regions of cell-cell contact between chick embryo kidney cells in culture. "Edge-on" views obtained by confocal microscopy of intact single intestinal epithelial cells and of retinal pigmented epithelium showed that capping protein is located in the apical region of the epithelial cells coincident with the junctional complexes. These images do not resolve the individual types of junctions of the junctional complex. Immunolabeling of microvilli or stereocilia was faint or not detectable. Capping protein was also detected in the cytoplasm of intact intestinal epithelial cells and in nuclei of cells in the pigmented retina and in the kidney cell cultures, but not in nuclei of cells of the intestinal epithelium or sensory epithelium. Biochemical fractionation of isolated intestinal epithelial cells shows capping protein in the brush border fraction, which contains the junctional complexes, and in the soluble fraction. These results are consistent with the results of the immunolabeling experiments. Highly purified microvilli of the brush borders also contained capping protein; this result was unexpected based on the low intensity of immunofluorescence staining of microvilli and stereocilia. The microvilli were not contaminated with junctional complexes, as defined by the absence of several markers for cell junctions. The cause and significance of this discrepancy is not certain at this time. Since capping protein binds the barbed end of actin filaments in vitro, we hypothesize that capping protein is bound to the barbed ends of actin filaments associated with one or more of the junctions of the junctional complex.

Comparison of Electron Microscopy and Polyacrylamide Gel Electrophoresis in the Diagnosis of Avian Reovirus and Rotavirus Infections

Electron microscopy (EM) and genome electropherotyping by polyacrylamide gel electrophoresis (PAGE) for the detection of avian rotaviruses and reoviruses in intestinal specimens and cell cultures were compared. Fifty-eight field samples of intestine with intestinal contents, referred to as direct specimens, from turkey and chicken flocks located in different regions of California and submitted during 1989 for virus isolation were randomly selected as test samples. Also, 38 field intestinal specimens with suspected viral infection that had been passaged three times in primary chicken embryo kidney (CEK) cell cultures were used in their third passage. The percentage of agreement and the Kappa statistic of positive and negative results between these two tests were calculated. In the comparison, EM was considered the standard test. By statistical analysis, an agreement of 87% was observed in cell-culture samples analyzed by the two virus-detection methods, as contrasted with an agreement of 72% for direct specimens. The analysis of the number of segments and band migration profiles of reference and field virus strains indicated that only reoviruses replicated in CEK cell cultures and mainly rotaviruses were detected by both tests in direct specimens. The Kappa statistic analysis indicated substantial agreement (0.69) between the two tests for CEK samples, with moderate agreement (0.45) for the direct specimens examined.

Detection of avian infectious bronchitis viral infection using in situ hybridization and recombinant DNA

A recombinant DNA probe with specificity for the 3′ end of genomic RNA from the Ark 99 strain of infectious bronchitis virus (IBV) was found to hybridize with extracted RNA of three strains with the Ark serotype, as well as the Mass41, Holl52, Gray, JMK, Conn, Fla and SE17 strains of IBV. Viral infection was detected in the cytoplasm of chicken embryo kidney cells inoculated with Mass41, Ark99, SE17 or two recent field isolates of IBV using in situ cytohybridization and a biotinylated probe. In vivo infections were detected in individual cells of tracheas and lungs 2, 4, and 6 days after inoculation of chicks with Mass41 and Ark99. In situ hybridization of Ark99 infected tissue sections using 32P-dATP labelled probe indicated that more viral replication was present in the trachea on day 4 than either days 2 or 6; whereas more viral RNA was found in the lungs on day 6 than days 2 or 4 after inoculation.

Association of Reoviridae Particles in an Enteric Syndrome of Poults Observed in Turkey Flocks during 1988

An enteric syndrome of turkey poults, characterized by enteritis, crop mycosis, intestinal changes (pale, thin-walled ballooning with watery contents), and rickets, occurred during 1988 in 74 turkey flocks from different farms belonging to 9 California turkey growers. The flocks ranged in size from 9,000 to 120,000 birds. Pools of intestine sections from 618 birds, representing 78 field cases, were examined. Histopathological examination of the intestines showed a mild to severe atrophy with a reduced depth of crypts, which was more prominent in the distal part of the small intestine. Viral isolation attempts with primary cell cultures of chicken embryo kidney cells were negative. Examination by electron microscopy of negatively stained intestinal specimens revealed the presence of Reoviridae particles of 58.8 to 80 nm in diameter. Enzyme-linked immunosorbent assay results on the intestinal pools for mammalian and group A avian rotaviruses were negative. A statistically significant relationship was found for the presence of Reoviridae particles in the intestines of 10-21-day-old birds. Of the 7 most common pathological conditions analyzed, 2, rickets and intestinal changes (thin-walled ballooning intestine with watery contents), showed a statistically significant association with the presence of Reoviridae particles.

Cingulin: characterization and localization.

Cingulin, a protein component associated with the tight junctions of chicken intestinal epithelium, has been purified to homogeneity by a new procedure and characterized. Purified cingulin is a heat-stable elongated dimer, composed of two polypeptides of Mr 108,000 (cingulin-108), with a Stokes' radius of approximately 15 nm, and a molecular length of 130 nm +/- 32 nm. Monoclonal antibodies were used to determine the tissue distribution and subcellular localization of cingulin in a variety of avian tissues and cultured cells. Indirect immunofluorescence analysis of semi-thin frozen sections demonstrated that cingulin is localized in the junctional complex of various polarized epithelia and in the endothelium, whereas it is essentially absent from mesenchymal and myogenic cells. In permeabilized and fixed cultured chick embryo kidney cells, the antibodies stained solely the regions of contacts between the epithelial cells. Double immunofluorescent labeling of these cells with anti-cingulin and anti-vinculin antibodies showed that cingulin is localized close to the vinculin-rich cytoskeletal belt associated with adherens junctions, but is absent from focal contacts and stress fibers. In cultured kidney cells, actin was detected mainly in stress fibers and in the peripheral junctional regions, where it showed a distribution similar to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton at the level of the tight junction. Indirect immunoelectron microscopic labeling of ultrathin frozen sections of chicken intestine showed that cingulin is localized along the endofacial surfaces of the tight junction (zonula occludens), and is apparently excluded from the more basal zonula adhaerens, and from the desmosomes.