Cell-matrix interactions and cell adhesion during development.
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Animals↗
Biomedical subjects
P Ekblom
Publications and source records attributed to P Ekblom.
Growth factors and the embryonic kidney.
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Angiogenesis Inducing Agents↗
Iron delivery during proliferation and differentiation of kidney tubules.
Proliferation during kidney development can be stimulated with an iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH). Neither the starting products nor the intermediary in FePIH synthesis stimulated proliferation. Thus, the growth-promoting effects of FePIH are due to the iron ion. Some other low molecular weight, saturated iron chelators such as glycyl-histidyl-lysine acetate, nitrilotriacetic acid, ascorbate, citrate, and unchelated ferrous sulfate could not support as high a degree of proliferation as FePIH or transferrin. FePIH delivered just slightly less radioactive iron into the trichloroacetic acid-precipitable fraction than transferrin. The octanol/saline partition coefficients of radioactive iron in solution with transferrin, nitrilotriacetic acid, or chloride were all less than 0.06. Thus, these compounds cannot efficiently traverse the lipid membrane. On the other hand, Fe3+ carried by PIH had a partition coefficient of 0.96. Hence, FePIH can stimulate proliferation because it can carry iron through the lipid membrane. Transferrin is not lipophilic but it delivers iron by receptor-mediated endocytosis.
Animals↗
Control of kidney differentiation by soluble factors secreted by the embryonic liver and the yolk sac.
Since transferrin is necessary for the differentiation of the embryonic kidney in organ culture, we have suggested that the component is a growth factor for in vivo development as well. In the present study we demonstrate that transferrin is present in the serum of 11-day-old mouse embryos, at the time when kidney differentiation starts. We have also tested whether various embryonic tissues can replace transferrin as stimulators of the differentiation and proliferation of the metanephric mesenchyme. We used a transfilter model system where nephrogenic mesenchymes are cultured with spinal cord, a known inductor of kidney tubules. The embryonic liver could not replace the spinal cord as an inducer of tubular differentiation. However, when the kidney mesenchymes were cultured together with both the spinal cord and the liver, the mesenchymes proliferated and differentiated also in the absence of exogenous transferrin. In such cocultures the spinal cord had to be in close contact with the mesenchyme while the embryonic liver could be located several cell layers apart. The liver-mediated stimulation of proliferation of the induced mesenchyme could be inhibited by anti-transferrin antibodies. Immunoprecipitation and immunoblotting with these antibodies of the liver-conditioned medium demonstrated that the 11-day mouse liver produces transferrin. Other potential mitogens produced by liver cells, alpha-fetoprotein, or multiplication stimulating activity, did not in any way stimulate the proliferation of induced mesenchymes. These studies suggest that the mitogen in the liver medium is transferrin. This is supported by data which show that another embryonic transferring producer, the visceral yolk sac, can replace the effect of the liver, whereas a tissue not producing transferrin, the salivary mesenchyme, cannot. In conclusion, an essential function of the inducer is to make the mesenchyme responsive to transferrin. The liver and the yolk sac stimulate early kidney differentiation by producing the soluble factor, transferrin, but they are ineffective as inductors of the transferrin responsiveness.
Amniotic Fluid↗
Cell-adhesion molecule uvomorulin during kidney development.
We studied the expression of a cell adhesion molecule during morphogenesis of the embryonic kidney. The 120-kDa glycoprotein, called uvomorulin, is known to be present on a number of epithelia. During the development of the kidney, a mesenchyme is converted into an epithelium when it is properly induced. The uninduced mesenchyme did not express uvomorulin, as judged by immunofluorescence and immunoblotting using previously characterized antibodies. Uvomorulin does not appear in the mesenchyme as a direct consequence of induction. Rather it becomes detectable approximately 12 hr after completion of induction, at 30-36 hr in vitro when the cells adhere to each other. Distinct differences in uvomorulin expression were seen in the different parts of the nephron. In the mesenchymally derived epithelia (glomeruli, tubules), uvomorulin could be detected only in the tubules, whereas the epithelium of the glomeruli remained negative at all stages of development. Our embryonic studies show that these differences arise very early, as soon as the different parts of the nephron can be distinguished morphologically. It is likely that uvomorulin plays a role in the initial adhesion of the differentiating tubule cells. However, we failed to disrupt histogenesis by applying antibodies to the organ cultures of developing tubules although the antibodies penetrated the tissues well and bound to the differentiating cells.
Animals↗
Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth.
Although local epithelial-mesenchymal tissue interactions which are presumably mediated by extracellular matrix molecules are important regulators of tooth morphogenesis and differentiation, our studies have indicated that these developmental processes also depend on circulating molecules. The iron-carrying serum protein transferrin is necessary for the early morphogenesis of mouse tooth in organ culture (A-M. Partanen, I. Thesleff, and P. Ekblom, 1984, Differentiation 27, 59-66). In the present study we have examined the effects of other growth factors on mouse tooth germs grown in a chemically defined medium containing transferrin. Fibroblast growth factor and platelet derived growth factor had no detectable effects but epidermal growth factor (EGF) inhibited dramatically the morphogenesis of teeth, and prevented odontoblast and ameloblast cell differentiation. EGF stimulated cell proliferation in the explants measured as [3H]thymidine incorporation in DNA. However, when the distribution of dividing cells was visualized in autoradiographs, it was observed that cell proliferation was stimulated in the dental epithelium but was inhibited in the dental mesenchyme. The inhibition of cell proliferation in the dental mesenchyme apparently caused the inhibition of morphogenesis. We do not know whether the dental epithelium or mesenchyme was the primary target for the action of EGF in the inhibition of morphogenesis. It is, however, apparent that the response of the dental mesenchymal cells to EGF (inhibition of proliferation) is regulated by their local environment, since EGF enhanced proliferation when these cells were disaggregated and cultured as monolayers. This indicates that the organ culture system where the various embryonic cell lineages are maintained in their original environment corresponds better to the in vivo situation when the roles of exogenous growth factors during development are examined.
Animals↗
The role of transferrin receptors and iron delivery in mouse embryonic morphogenesis.
The iron-carrying serum protein transferrin is required for the proliferation and differentiation of embryonic tissues in culture. We studied the expression and role of transferrin receptors in two model systems using a monoclonal antibody against the transferrin receptor of mice. The addition of 20-100 micrograms/ml antibody to a chemically defined culture medium containing transferrin (10 micrograms/ml) inhibited morphogenesis and cell proliferation in kidneys and teeth. However, the antibody did not inhibit development when iron was delivered to the cells by a lipophilic iron chelator i.e., by-passing the receptor-mediated pathway. Hence, the binding of the receptor antibody to the receptor apparently did not affect cell proliferation, and the antibody was not toxic to the tissues. Our results suggest that the antibody to the transferrin receptor inhibits development by blocking the normal endocytotic route of iron delivery. Cells derived from embryonic kidneys and teeth expressed the transferrin receptor when cultured as monolayers. However, using immunofluorescent techniques, we were unable to detect the receptor in frozen tissue sections. It is possible that the seeding of cells in monolayer cultures affects the expression of the transferrin receptor, since it is known that all types of cells require transferrin for continued proliferation in culture. Organ-cultured kidney mesenchymal cells are not initially responsive to transferrin, but they acquire responsiveness as a consequence of an inductive tissue interaction. Although it remains unknown as to whether the acquisition of transferrin responsiveness is directly related to the expression of transferrin receptors, our results suggest that transferrin and its receptors play a role in embryonic morphogenesis.
Animals↗
Malignant transformation in human colorectal mucosa as monitored by distribution of laminin, a basement membrane glycoprotein.
The distribution of laminin, a basement membrane glycoprotein, was studied by indirect immunofluorescence technique using rabbit anti-laminin serum in fifty-seven samples of normal, hyperplastic, adenomatous and carcinomatous colorectal tissue. As judged by the expression of laminin normal, hyperplastic and adenomatous colorectal mucosa were characterized by a continuous, linear basement membrane. A thick band-like basement membrane was seen under the surface epithelium and at the mouths of the crypts in normal and hyperplastic mucosa. The adenomatous epithelium was uniformly characterized by a thin, linear basement membrane. In contrast, marked alterations were constantly observed in colorectal carcinomas and their metastases, which showed a discontinuous, linear or patchy basement membrane. Our study suggests that the expression of laminin is related to cellular differentiation, and the thickness of the laminin-positive basement membrane parallels with the epithelial maturation of normal and hyperplastic colorectal mucosa and with the failure of the epithelial differentiation in adenomas. In colorectal carcinomas and their metastases the distribution of laminin is constantly altered and the severity of the alterations correlates with the histological grade of these tumours.
Adenocarcinoma↗
Extracellular matrix and epithelial differentiation of Wilms' tumor.
Different histologic types of 82 Wilms' tumors were graded on the basis of the histologic pattern. Representative tumors of each group were analyzed by the immunoperoxidase method for evaluation of the histogenesis of Wilms' tumor and the value of antibodies against extracellular matrix (ECM) components (collagens I and III, laminin, fibronectin) in differential diagnosis of different types of Wilms' tumors. The tubules of classic Wilms' tumor expressed laminin, which could be seen also in and around some blastemal cells. Blastema and tubules were negative for interstitial collagens, but Type I and III collagen were prominent in the fibrovascular stroma. The monomorphous tubular, psammomatous and rosetting tumors expressed laminin, but no interstitial collagens. In sarcomas, only the blastemal variant of spindle-cell sarcomas was negative for interstitial collagens, which were abundantly seen in all other sarcomas. While spindle-cell sarcomas were devoid of laminin, the highly malignant rhabdoid and clear-cell sarcomas expressed laminin in a characteristic dotted fashion. Staining for fibronectin gave varying results and had therefore only a limited value in distinguishing different types of Wilms' tumors. However, the antibodies against interstitial collagens and against the basement membrane glycoprotein laminin turned out to be a useful adjunct in differential diagnosis and classification, especially of sarcomatoid Wilms' tumors. The basement membrane of normal nephrons is similar to that in tubules of triphasic Wilms' tumor, but the ECM of blastemas is different. This transformed phenotype might represent a maturation arrest of the blastemal cell when compared with the expression of proteins during normal nephrogenesis.
Adenocarcinoma↗
Basement membrane and organogenesis.
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Animals↗
Histogenesis of granular cell tumour--an immunohistochemical and ultrastructural study.
Fifteen cases of granular cell tumour of superficial soft tissues or tongue were immunohistochemically evaluated for different types of intermediate filament proteins and for laminin, a glycoprotein of basal laminae. Four of the tumours were studied ultrastructurally. The tumour cells appeared to contain only vimentin-type of intermediate filament protein. The lobules of tumour cells were surrounded by laminin-positive material, but in contrast to schwannomas and neurofibromas, the individual tumour cells were not covered by laminin. In line with the immunohistochemical observations, by electron microscopy basal lamina-like material could not be demonstrated between individual cells, but only surrounding groups of cells. Lysozyme, a histiocytic marker, was absent in the tumour cells. Our results do not confirm any particular cell type for the histogenetic origin of granular cell tumour, but suggest that it may rather be derived from uncommitted possibly nerve-related mesenchymal cells.
Adolescent↗
Dual origin of glomerular basement membrane.
The histogenesis of renal basement membranes was studied in grafts of avascular, 11-day-old mouse embryonic kidney rudiments grown on chick chorioallantoic membrane (CAM). Vessels of the chick CAM invade the mouse tissue during an incubation period of 7-10 days and eventually hybrid glomeruli composed of mouse epithelium and chick endothelium form. Formation of basement membranes during this development was followed by immunofluorescence and immunoperoxidase stainings using polyclonal and monoclonal antibodies against mouse and chick collagen type IV and against mouse laminin. These antibodies were species-specific as shown in immunochemical and immunohistologic analyses. The glomerular basement membrane contained both mouse and chick collagen type IV, demonstrating its dual cellular origin. All other basement membranes were either exclusively of chick origin (mesangium, vessels) or of mouse origin (tubuli, Bowman's capsule).
Allantois↗
Pathology of fetal congenital nephrosis: immunohistochemical and ultrastructural studies.
The kidneys of four human fetuses aged 18 to 20 weeks of gestation with congenital nephrosis of the Finnish type (CNF) were studied by immunohistochemistry and electron microscopy to elucidate the pathogenesis of the disease. The immunohistochemical stainings for laminin, type IV collagen, fibronectin, brushborder antigens (BBA), Tamm-Horsfall protein (TH), and binding of wheat germ agglutinin (WGA) did not reveal changes as compared to the age-matched control kidneys. Proximal tubules of the CNF kidneys showed excessive accumulation of coarse granular alpha fetoprotein (AFP) and large absorption droplets with paracrystalline and membraneous structures were seen in electron microscopy. The dilated tubules were both of proximal and distal origin as judged from the BBA and TH stainings. It is suggested that the formation of the dilated tubules results from degeneration caused by obstruction and excessive protein loading of the tubular epithelium. Demonstration of the coarse granular AFP in the proximal tubular epithelium and lumina indicates heavy fetal proteinuria and serves as an additional diagnostic marker for prenatal CNF.
Female↗
A lipophilic iron chelator can replace transferrin as a stimulator of cell proliferation and differentiation.
Of the different growth supplements used in chemically defined media, only transferrin is required for differentiation of tubules in the embryonic mouse metanephros. Since transferrin is an iron-carrying protein, we asked whether iron is crucial for tubulogenesis. Differentiation of metanephric tubules both in whole embryonic kidneys and in a transfilter system was studied. The tissues were grown in chemically defined media containing transferrin, apotransferrin, the metal-chelator complex ferric pyridoxal isonicotinoyl hydrazone (FePIH), and excesses of ferric ion. Although we found that apotransferrin was not as effective as iron-loaded transferrin in promoting proliferation in the differentiating kidneys, excess ferric ion at up to 100 microM, five times the normal serum concentration, could not promote differentiation or proliferation. However, iron coupled to the nonphysiological, lipophilic iron chelator, pyridoxal isonicotinoyl hydrazone, to form FePIH, could sustain levels of cell proliferation and tubulogenesis similar to those attained by transferrin. Thus, the role of transferrin in cell proliferation during tubulogenesis is solely to provide iron. Since FePIH apparently bypasses the receptor-mediated route of iron intake, the use of FePIH as a tool for investigating cell proliferation and its regulation is suggested.
Animals↗
Cellular origin of fibronectin in interspecies hybrid kidneys.
The cellular origin of fibronectin in the kidney was studied in three experimental models. Immunohistochemical techniques that use cross-reacting or species-specific antibodies against mouse or chicken fibronectin were employed. In the first model studied, initially avascular mouse kidneys cultured on avian chorioallantoic membranes differentiate into epithelial kidney tubules and become vascularized by chorioallantoic vessels. Subsequently, hybrid glomeruli composed of mouse podocytes and avian endothelial-mesangial cells form. In immunohistochemical studies, cross-reacting antibodies to fibronectin stained vascular walls, tubular basement membranes, interstitium, and glomeruli of mouse kidney grafts. The species-specific antibodies reacting only with mouse fibronectin stained interstitial areas and tubular basement membranes, but showed no reaction with hybrid glomeruli and avian vascular walls. In contrast, species-specific antibodies against chicken fibronectin stained both the interstitial areas and the vascular walls as well as the endothelial-mesangial areas of the hybrid glomeruli, but did not stain the mouse-derived epithelial structures of the kidneys. In the second model, embryonic kidneys cultured under avascular conditions in vitro develop glomerular tufts, which are devoid of endothelial cells. These explants showed fluorescence staining for fibronectin only in tubular basement membranes and in interstitium. The avascular, purely epithelial glomerular bodies remained unstained. Finally, in outgrowths of separated embryonic glomeruli, the cross-reacting fibronectin antibodies revealed two populations of cells: one devoid of fibronectin and another expressing fibronectin in strong fibrillar and granular patterns. These results favor the idea that the main endogenous cellular sources for fibronectin in the embryonic kidney are the interstitial and vascular cells. All experiments presented here suggest that fibronectin is not synthesized by glomerular epithelial cells in vivo.
Allantois↗
Expression of laminin in pancreatic neoplasms and in chronic pancreatitis.
The distribution of laminin, a basement membrane glycoprotein, was studied by immunohistological techniques in 10 samples of normal pancreatic tissue, in 15 samples of chronic pancreatitis, and in 33 pancreatic neoplasms. Sections of formalin-fixed, paraffin-embedded specimens were pretreated with pepsin and immunostained for laminin. As judged by the expression of laminin, normal pancreatic glands were surrounded by a continuous, intact basement membrane. In chronic pancreatitis the basement membrane was also mainly continuous, but focally weaker and thinner than around normal glands. In pancreatic adenocarcinomas laminin was irregularly distributed and in large areas totally absent. In anaplastic carcinomas no extracellular laminin was seen, but two cases showed some intracellular laminin in a punctate pattern. The findings suggest that these cancers have defects in the deposition of a basement membrane or that it is degraded. Our data suggest that the integrity of the basement membrane correlates with the degree of malignancy in ductal adenocarcinomas, but this is not the case for mucinous cystic neoplasms or for islet cell tumors. In these neoplasms a nearly intact basement membrane was seen both in malignant tumors and in their benign counterparts.
Adenocarcinoma↗
Transferrin is required for early tooth morphogenesis.
The role of circulating molecules during early tooth morphogenesis was studied in organ cultures of mouse embryonic molar-tooth germs. Special attention was focused on the effect of transferrin and insulin, which are necessary for the growth of most cells in culture. The requirement of serum factors for tooth morphogenesis was shown to diminish as the developmental stage advances from the bud stage in day-13 embryos to the cap stage at day 15. The day-15 teeth underwent morphogenesis and cell differentiation in unsupplemented basal culture medium, but the addition of transferrin (50 micrograms/ml) was necessary for the morphogenesis of day-14 tooth germs. We demonstrated, by using transferrin-depleted serum, that transferrin is also necessary for the morphogenesis of day-13 tooth germs. However, some still-unidentified serum components are also required for the morphogenesis of the bud-stage day-13 teeth. These factors apparently do not include insulin, since it was shown to inhibit tooth development. Analysis of the DNA content of tooth germs cultured in various culture media showed that the ability of transferrin to support tooth morphogenesis correlated with a stimulation of growth. The results support our earlier suggestions that transferrin functions as a fetal growth factor. The availability of the transferrin-containing chemically defined medium facilitates studies on the roles of other growth factors during tooth development.
Animals↗