Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery

Hop, an abundant and conserved protein of unresolved function, binds concomitantly with heat shock protein 70 (Hsp70) and Hsp90, participates with heat shock proteins at an intermediate stage of progesterone receptor assembly, and is required for efficient assembly of mature receptor complexes in vitro. A largely untested hypothesis is that Hop functions as an adaptor that targets Hsp90- to Hsp70-substrate complexes; if true, then loss of either Hsp70 binding or Hsp90 binding by Hop should equally disrupt its ability to promote assembly of mature receptor complexes. To generate Hop mutants that selectively disrupt heat shock protein interactions, highly conserved amino acids in the previously mapped Hsp70 and Hsp90 binding domains of Hop and in a conserved C-terminal domain were targeted for small substitutions and deletions. In co-precipitation assays, these mutants displayed selective loss of association with heat shock proteins. In assays using Hop-depleted rabbit reticulocyte lysate for the cell-free assembly of receptor complexes, none of the Hop mutants inhibited Hsp70 binding to receptor, but all mutants were defective in supporting Hsp90-receptor interactions. Thus, Hop has a novel role in the chaperone machinery as an adaptor that can integrate Hsp70 and Hsp90 interactions.     

Effect of herbimycin A on hsp30 and hsp70 heat shock protein gene expression in Xenopus cultured cells

Glucose intolerance and diabetes mellitus are both prevalent in patients with chronic liver diseases. We examined the efficacy and systemic safety of therapy with an alpha-glucosidase inhibitor, acarbose, in diabetes mellitus associated with chronic liver diseases. Twenty patients with chronic hepatitis or liver cirrhosis and overt diabetes mellitus received acarbose (taken orally) for 8 weeks. The initial dosage of acarbose was 50 mg three times daily, taken before meals; this was increased to 100 mg three times daily after 2 weeks. The mean fasting plasma glucose level was 173.7 +/- 18.6 mg/dl (mean +/- SE) at entry, and was significantly decreased to 132.9 +/- 7.5 mg/dl (P < 0.05) after 8 weeks of acarbose treatment. The improved glycemic control was reflected by a significant decrease in glycosylated hemoglobin (HbA1c) from 7.2 +/- 0.3% at entry to 6.3 +/- 0.2% (P < 0.05) after 8 weeks. Serum levels of both aspartate and alanine aminotransferases fluctuated during acarbose treatment, probably due to the natural course of chronic liver diseases, but the mean values had decreased after 8 weeks of treatment. Plasma ammonia levels increased, from 61.3 +/- 10.7 micrograms/dl to 71.1 +/- 9.6 micrograms/dl after 8 weeks of acarbose treatment but the increase was not significant. Clinically significant elevation of plasma ammonia concentration was seen in 2 cirrhotic patients (121 and 124 micrograms/dl); this was asymptomatic and gradually returned to the normal range despite continuous acarbose treatment in one patient, and was reversed after the withdrawal of acarbose with the concomitant administration of lactulose in the other patient. No other blood tests results, including albumin, cholinesterase, and prothrombin time, or lipid profile and nutritional status, in terms of rapid turnover proteins, prealbumin, retinol binding protein, and transferin, were altered throughout the study period. These results indicate that diabetes mellitus associated with chronic liver diseases may be safely and effectively treated with acarbose. However, clinicians must be aware of the possibility of hyperammonemia when they prescribe acarbose for patients with diabetes mellitus and advanced liver cirrhosis.     

The different expression of proteins recognized by monoclonal anti-heat shock protein 70 (hsp70) antibody in human colonic diseases

Analysis of the expression of hsp70 (70 kDa heat-shock proteins) in normal and pathological tissues might prove their potential diagnostic and prognostic values. In the present study, we combined high-resolution two-dimensional polyacrylamide gel electrophoresis with immunoblotting to study hsp70 expression in normal, preneoplastic and neoplastic colonic mucosa. By monoclonal anti-hsp70 antibody, recognizing both the constitutive and inducible forms of hsp70, we have detected six charge isoforms localized in the pH 5.1-5.3 range and in the molecular mass range of 68-69 kDa in normal colonic mucosa. Immunostaining of hsp70 in polypous and malignant tissues revealed qualitative as well as quantitative changes in the expression of more acidic isoforms of hsp70 in comparison with normal tissue. Furthermore, the different basic isoforms of hsp70 were detected in chronically inflamed colonic mucosa from patients suffering from ulcerative colitis (UC) or Crohn's disease (CD). Besides the standard hsp70 protein pattern, additional proteins with molecular masses of about 39, 40, 74 and 75 kDa were variably immunostained in normal and pathological specimens. These observations suggest that hsp70 expression may be closely linked to disease etiology and/or pathophysiology.     

Thermotolerance and heat shock protein (HSP 70) in vascular endothelial cells

Recently, it was determined that the endothelial cells of blood vessel play a very important physiological role in the regulation of blood coagulation and selective permeability. To study the thermotolerance of vascular endothelial cells, human umbilical vein endothelial cells (HUVEC) were heated at 40, 43, 45 or 50 degrees C for various lengths of time with or without preheating at 40 or 43 degrees C for 30 min. The cell viability (CV) of HUVEC decreased gradually according to heating time. However, the CV of preheated HUVEC decreased slightly or not at all. Heat shock protein (HSP) in HUVEC heated at 37, 40, 43, or 45 degrees C was examined by immunoblotting. A new HSP 70 band was detected in HUVEC by heating at 40, 43 or 45 degrees C. HUVEC revealed thermotolerance with induction of HSP by heat stress.     

Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers

1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated. 2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii. 3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h. 4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals.     

Folding of the glucocorticoid receptor by the heat shock protein (hsp) 90-based chaperone machinery. The role of p23 is to stabilize receptor.hsp90 heterocomplexes formed by hsp90.p60.hsp70

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor.hsp90 heterocomplexes. We have reconstituted a minimal receptor.hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833-12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR.hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). In this work, we show that addition of p23 to native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR.hsp90 heterocomplexes prepared with the minimal (hsp90.p60.hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR.hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90.p60.hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR.hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90.p60.hsp70 forms a GR.hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR.hsp90 heterocomplex to inactivation and disassembly.     

Experimental immunization with anti-rheumatic bacterial extract OM-89 induces T cell responses to heat shock protein (hsp)60 and hsp70; modulation of peripheral immunological tolerance as its possible mode of action in the treatment of rheumatoid arthritis (RA)

High intracellular 1,2,-sn-diacylglycerol (DAG) usually activates protein kinase C (PKC). In choline-deficient Fischer 344 rats, we previously showed that fatty liver was associated with elevated hepatic DAG and sustained activation of PKC. Steatosis is a sequelae of many liver toxins, and we wanted to determine whether fatty liver is always associated with accumulation of DAG with activation of PKC. Obese Zucker rats had 11-fold more triacylglycerol in their livers and 2-fold more DAG in their hepatic plasma membrane than did lean control Zucker rats. However, this increased diacylglycerol was not associated with translocation or activation of PKC in hepatic plasma membrane (activity in obese rats was 897 pmol/mg protein X min(-1) vs. 780 pmol/mg protein X min(-1) in lean rats). No differences in PKC isoform expression were detected between obese and lean rats. In additional studies, we found that choline deficiency in the Zucker rat did not result in activation of PKC in liver, unlike our earlier observations in the choline deficient Fischer rat. This dissociation between fatty liver, DAG accumulation and PKC activation in Zucker rats supports previous reports of abnormalities in PKC signaling in this strain of rats.     

Abundance of heat shock proteins (hsp89, hsp60, and hsp27) in malignant cells of Hodgkin's disease

Heat shock proteins (HSPs) or stress proteins are synthesized by cells in response to environmental stress. Expression of HSPs by cells may have important physiological or pathological implications. In this study, we carried out an immunohistochemical and biochemical examination of low (hsp27), intermediate (hsp60), and high (hsp89) molecular weight HSP expression in reactive lymph nodes and in lymph nodes of patients with various types of lymphomas. In normal or reactive lymphoid tissues, hsp89 is abundant in large "transformed" lymphoid cells and immunoblasts. Hsp60 is widely distributed in lymphoid tissues, whereas hsp27 is absent in all lymphoid cells and histiocytes. Among lymphomas, the Hodgkin's Reed-Sternberg (H-RS) cells in Hodgkin's disease (HD) had the greatest abundance of hsp89 and hsp60 and, in 20% of cases, hsp27, in contrast to a much weaker staining of anti-hsp89 and -hsp60 in the background reactive lymphoid cells. The large lymphoid cells in small lymphocytic lymphoma are also rich in hsp89, but not hsp60 and hsp27. In contrast, the malignant cells in anaplastic large cell lymphoma and most high-grade tumors, including immunoblastic lymphomas, expressed minimal amounts of hsp89 and hsp60 and virtually no hsp27. Thus, the cellular level of HSPs was neither correlated with the proliferative capacity nor with the aggressiveness of the lymphomas. Hsp89, hsp60, and hsp27, as well, serve critical roles in the chaperoning of cellular proteins (e.g., a Mr 43,000 protein) in H-RS cells. The known interactions of HSPs with Rb, p53, peptide-MHC class II complexes, and cofactors of the glucocorticoid hormone receptor have further broadened the importance of HSPs in cell metabolism and in response to extracellular signals for proliferation, differentiation, or growth suppression (or apoptosis) of H-RS cells. Abundant HSP expression is seen only in HD, but not in other lymphomas. Such expression could have vital roles in the pathogenesis of HD.     

Elevated ovarian follicular apoptosis and heat shock protein-70 expression in white sucker exposed to bleached kraft pulp mill effluent

Exposure of feral fish populations to bleached kraft pulp mill effluent (BKME) results in a variety of negative impacts on reproductive fitness including reduced ovarian development, reduced egg size, decreased fecundity with age, delayed sexual maturation, and alterations in reproductive endocrine homeostasis at multiple sites along the pituitary-gonadal axis. The present study provides evidence of elevated apoptotic DNA fragmentation and increased expression of the 70-kDa heat shock protein (HSP70) in ovarian follicular cells from prespawning white sucker (Catostomus commersoni) exposed to BKME. Apoptosis is the molecular mechanism responsible for ovarian follicular atresia which is involved in various stages of vertebrate ovarian development such as follicular recruitment, growth, differentiation, and regression. In mammals, induction of HSP70 is associated with inhibition of hormone-sensitive steroidogenesis and mediation of luteal regression. The 3'-end labeling of isolated ovarian follicular cell DNA revealed a 10-fold increase in the extent of apoptosis in BKME-exposed white sucker in comparison to follicles collected from a nearby reference site. Western blotting for ovarian follicular HSP70 levels showed increased expression of this protein in fish exposed to BKME. The elevated ovarian cell apoptosis and increased HSP70 expression in BKME-exposed fish were associated with reduced ovary size, decreased plasma testosterone, and increased plasma 17 beta-estradiol concentrations, but not induction of hepatic ethoxyresorufin O-deethylase activity. It is not known whether increased ovarian HSP70 expression in BKME-exposed fish is related to elevated apoptosis or represents a general response to environmental stress. Since apoptosis is regulated by several hormonal factors and conserved gene products, these data suggest that certain components of BKME increase ovarian cell apoptosis in fish via stimulation of cell death signaling. However, it is unclear whether BKME stimulates ovarian cell apoptosis directly or if this response occurs secondarily as a result of altered reproductive endocrine homeostasis.     

Hormonal regulation and germ cell-specific expression of heat shock protein 60 (hsp60) in the testis of macaque monkeys (Macaca mulatta and M. fascicularis)

FNA smears from six histologically documented cases of tubular adenoma of breast were critically analysed and compared with 10 histologically confirmed cases of fibroadenoma (five pericanalicular and five intracanalicular). Initially a cytological diagnosis of tubular adenoma was rendered only in one case. On review, two cases could be characterized as tubular adenoma, while the findings were suggestive in two others. The features helpful in diagnosis of tubular adenoma were the presence of benign ductal cells as three-dimensional cohesive balls and tubular structures in highly cellular smears. Stroma was conspicuously scanty or absent. Myoepithelial cells were present along with sheets of ductal cells as well as bipolar naked nuclei. Confusion with fibroadenoma occurred in two cases due to presence of a stag-horn pattern of ductal cells.     

Heterogeneous patterns of constitutive and heat shock induced expression of HLA-linked HSP70-1 and HSP70-2 heat shock genes in human melanoma cell lines

The heat shock response, which is characterized by the induction of heat shock proteins, is known to affect the ability of tumour cells to cope with potentially adverse conditions such as hypoxia, glucose starvation and cytotoxic immune reactions. To assess the heat shock response of melanoma cells, spontaneous and heat shock induced expression of heat shock proteins was analysed in a panel of 17 human melanoma cell lines. Constitutive expression of HSP27, HSP70, HSC70, HSP90alphabeta and GRP94 proteins was found in all the melanoma cell lines, and HSP70 and HSC70 were also induced by heat shock. The major heat inducible HLA-linked HSP70-1 and HSP70-2 genes were analysed at the mRNA level. Basal expression and inducibility varied between the different melanoma cell lines. In addition, in situ hybridization demonstrated heterogeneous expression of these genes among single cells of a given cell line. In general, each melanoma cell line appears to exhibit an individual type of HSP70 expression that might reflect selection during tumour progression and therapy.     

Identification and characterization of a heat shock protein 70 family member in channel catfish (Ictalurus punctatus)

The primary objective of this study was to evaluate the electrophysiologic effects of large-dose propofol, used as the sole anesthetic in patients with epilepsy. Nine patients with medically intractable complex partial epilepsy undergoing a three-stage approach to the surgical management of epilepsy were recruited. State I involved placement of the intracranial electrode array, while Stage II consisted of extraoperative localization of the seizure focus. The patients were studied during induction of anesthesia for Stage III (removal of electrodes and resection of seizure focus). Unpremedicated patients were induced with a propofol infusion (0.5 mg.kg-1.min-1) until one of the following occurred: 1) electrical seizure activity, 2) burst suppression, or 3) total dose of 10 mg/mg. Electrocorticography (ECoG) was recorded continuously during this period. Two patients were excluded from the study after experiencing delayed awakening after the Stage I procedure. Both had received propofol along with other anesthetics. No ECoG evidence of seizure activity was detected in the seven patients completing the study. Burst suppression was attained in six patients using a mean dose of 5.7 mg/kg +/- 2.6. We conclude that large dose propofol alone does not trigger electrical epileptiform activity on the ECoG of seizure patients.     

Pressure-induced expression of heat shock protein 70 mRNA in adult rat heart is coupled both to protein kinase A-dependent and protein kinase C-dependent systems

BACKGROUND: Production of heat shock protein 70 (HSP70) in the heart is induced by hemodynamic stress, but its intracellular signal transduction system has not been elucidated well. OBJECTIVE: To investigate the hypothesis that protein kinase A (PKA)-dependent and protein kinase C (PKC)dependent systems are involved in the pressure-induced expression of HSP70 mRNA in perfused adult rat heart METHODS: Isolated tetrodotoxin-arrested Sprague-Dawley rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. Aortic pressure in rats of the pressure-overloaded group was elevated from 60 to 120 mmHg for 2-120 min. cAMP contents and rates of synthesis of protein were measured by radioimmunoassay and the incorporation of [14C]-phenylalanine into total heart protein, respectively. Expression of HSP70 mRNA was determined by Northern blot analysis. RESULTS: Elevation of aortic pressure significantly increased cAMP content after 2 min of perfusion (by 41%), significantly increased rates of synthesis of protein during the second hour of perfusion (by 41%), and induced expression of HSP70 mRNA maximally after 60 min of perfusion (2.7-fold the control value). Exposure to glucagon, forskolin or 1 -methyl-3-isobutylxanthine mimicked increases in these parameters caused by elevation of aortic pressure. Administration of a selective PKA inhibitor, H-89, significantly prevented induction of increases in expression of HSP70 mRNA and rates of synthesis of protein by a high pressure overload and exposure to agents that increase cAMP content. Furthermore, administration of phorbol ester induced expression of HSP70 mRNA. Administration of a PKC inhibitor, calphostin C, significantly prevented induction of increases in expression of HSP70 mRNA by a pressure overload and by exposure to phorbol ester. CONCLUSIONS: These results suggest that the pressure-induced induction of production of HSP70 is regulated both by PKA-dependent and by PKC-dependent systems during periods of active synthesis of protein in adult rat heart.     

Effect of ischemia--reperfusion on heat shock protein 70 and 90 gene expression in rat liver: relation to nutritional status

p53 gene mutations occur in most human cancers and result in an altered protein product that accumulates within the cell. Although the observed endogenous human CTL response to p53 is weak, high-affinity, human p53-specific CTLs have been generated from HLA A2.1 transgenic mice immunized with human CTL epitope peptides. In this study, we examine the ability of HLA A2.1-restricted and human p53-specific CTLs from HLA A2.1 transgenic mice to suppress the growth of p53-overexpressing human tumors in severe combined immunodeficient (SCID) mice. In vitro, murine p53(149-157)-specific CTLs selectively lysed the p53-overexpressing pancreatic carcinoma cell line Panc-1 but did not recognize HLA A2.1- tumor cells or HLA A2.1+ normal human fibroblasts. Furthermore, in vivo, the growth of established human tumor xenografts in SCID mice was significantly reduced and survival was prolonged after the administration of p53-specific CTLs but not after the administration of control CTLs or PBS alone. Following treatment with p53(149-157)-specific CTLs, regressing Panc-1 tumors were infiltrated by the CD8+ CTLs, as demonstrated by immunohistochemistry. These findings suggest that p53(149-157)-specific and HLA A2.1-restricted murine CTLs suppress the growth of established Panc-1 tumors following adoptive transfer into SCID hosts and prolong their survival.     

The small heat shock protein hsp27 is correlated with growth and drug resistance in human breast cancer cell lines

An emerging body of evidence suggests that the heat shock proteins (hsp) may be involved in drug resistance. When hsp are induced by elevated temperatures, resistance to doxorubicin (Dox), but not to other commonly used chemotherapeutic agents, is induced in breast cancer cells. To evaluate the role of hsp27 in this phenomenon, we have transfected MDA-MB-231 breast cancer cells, which normally express low levels of hsp27, with a full-length hsp27 construct. These hsp27-overexpressing cells now display a 3-fold elevated resistance to Dox. Anchorage-dependent proliferation and anchorage-independent growth were also increased 2-4-fold in these transfectants. We have also derived a MCF-7 breast cancer cell line with amplified endogenous hsp27 which is highly resistant to Dox. When these cells are transfected with an antisense hsp27 construct, they are rendered sensitive to Dox (3-fold) with anchorage-dependent as well as anchorage-independent growth, similarly decreased. These results suggest that hsp27 specifically confers Dox resistance in human breast cancer cells and, furthermore, that hsp27 may be involved in the regulation of cell growth.     

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37 degrees C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.     

Age-related alteration of proline hydroxylase and collagen-binding heat shock protein (HSP47) expression in human fibroblasts

It is now well recognized that a disorder of left ventricular filling can be sufficient to account for congestive heart failure. Furthermore, evaluation of heart disease would not be complete if it did not include assessment of left ventricular filling, improvement of which probably ensures better control of the heart disease. An efficient and reliable tool for the study of diastolic function is therefore essential. The authors review the current state of knowledge and the more recent developments in Doppler echocardiography in the evaluation of left ventricular diastolic function. After revising the pathophysiology, the methods of studying ventricular filling are described. The recording technique is described, taking into account recent developments in transthoracic and transoesophageal approaches. This investigation provides parameters allowing semiquantitative estimation of filling pressures (mean left atrial pressure, end-diastolic pressure) and reliable evaluation of overall diastolic performance.     

Constitutive expression of heat shock protein 90 (HSP90) in neurons of the rat brain

Immunoblot analysis, immunocytochemistry and immuno-electron microscopy were employed to study the expression of HSP90 protein in the adult rat brain, using a specific polyclonal antiserum. Immunoblot analysis demonstrated equal levels of HSP90 in microdissected extracts from hippocampus, cortex, striatum and cerebellum. Immunocytochemistry and immuno-electron microscopy provided evidence that HSP90 is markedly expressed throughout all neuronal subpopulations of the CNS but not in non-neuronal cells except ependyma and choroid plexus. At the ultrastructural level, HSP90 immunoreactivity was predominantly found in perikarya but to a lesser extent also in dendrites and nuclei. The constitutive expression of HSP90 in widespread neuronal cell populations suggests a functional role in the physiological molecular program of CNS neurons.     

Involvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells

The EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have used in situ hybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8. Three of the receptors examined correspond to the novel chicken homologs of EphA2, EphA6, and EphA7. Unexpectedly, we found that many Eph receptors are expressed not only in retinal ganglion cells, but also in tectal cells, In particular, EphA3 mRNA is prominently expressed in the anterior tectum, with a pattern reciprocal to that of ephrin-A2 and ephrin-A5. Similarly, ephrin-A5 is expressed not only in tectal cells but also in the nasal retina, with a pattern reciprocal to that of its receptor EphA3 and partially overlapping with that of its other receptor EphA4. Consistent with the even distribution of EphA4 and the polarized distribution of EphA4 ligands in the retina, probing EphA4 immunoprecipitates from different sectors of the retina with anti-phosphotyrosine antibodies revealed spatial differences in receptor phosphorylation. These complex patterns of expression and tyrosine phosphorylation suggest that Eph receptors and ephrins contribute to establishing topography of retinal axons through multiple mechanisms, in addition to playing a role in intraretinal and intratectal organization.