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P Ekblom

Publications and source records attributed to P Ekblom.

At least 37 records · Page 2Linked to original sources
PubMed1997

Characterization of monoclonal antibodies against tenascin-C: no apparent effect on kidney development in vitro.

Tenascin-C is an extracellular matrix glycoprotein found in embryonic mesenchyme. The precise biological function of tenascin-C is unknown, but different parts of the molecule have effects on cell adhesion and other cellular activities. We studied the expression and role of tenascin-C in the embryonic mouse kidney. By Northern blots, no tenascin-C was detectable in uninduced mesenchyme from day 11 embryonic kidneys, but after 24 hours of in vitro culture both major splice variants of tenascin-C were detected. The larger variant was the predominant form. By in situ hybridization tenascin-C mRNA in 13-day old embryonic kidneys was detected in the mesenchyme surrounding newly formed epithelial structures. In 17-day old embryonic kidneys, tenascin-C mRNA was detected in mesenchyme around the forming epithelial structures in the cortex, and expression was also seen in mesenchyme surrounding the capsular epithelium of glomeruli. In newborn kidneys, expression had shifted to the medulla but was still confined to mesenchymal areas. We have characterized 6 new monoclonal antibodies against mouse tenascin-C, which all stain embryonic kidneys from different stages in a pattern consistent with earlier reports and with the mRNA data. The binding sites of the monoclonal antibodies on the tenascin-C molecule were mapped to discrete regions of tenascin-C. These six and five previously described antibodies against tenascin-C were tested in antibody perturbation experiments. Three of these have been shown by in vitro assays to perturb function of other cell types. Despite this, none of them inhibited development of mouse kidneys in organ culture, although they were tested at 1 mg/ml. It raises the possibility that tenascin-C is not crucial for kidney development. Alternatively, tenascin-C has more subtle functions which could not be identified with the assays used here.

Animals↗
PubMed1996

Skin wounds and severed nerves heal normally in mice lacking tenascin-C.

A large number of functions have been demonstrated for tenascin-C by antibody perturbation assays and in vitro cell culture experiments. However, these results contrast sharply with the lack of any apparent phenotype in mice with a genetic deletion of tenascin-C. A possible explanation for the lack of phenotype would be expression of some altered but functional tenascin-C in the mutant. We report the generation of an independent tenascin-C null mouse and conclude that the original tenascin-C knockout, which is genetically very similar to ours, is also a true null. As found previously, the absence of tenascin-C has no influence on development, adulthood, life span, and fecundity. We have studied in detail two models of wound healing. After axotomy, the regeneration of the sciatic nerve is not altered without tenascin-C. During healing of cutaneous wounds, deposition of collagen I, fibulin-2, and nidogen is identical in mutant and wild-type mice. In contrast. fibronectin appears diminished in wounds of tenascin-C-deficient mice. However, the lack of tenascin-C together with the reduced amount of fibronectin has no influence on the quality of the healing process.

Animals↗
PubMed1996

Integrin subunit expression associated with epithelial-mesenchymal interactions during murine tooth development.

The initial information for patterning of early tooth development resides in the epithelium. Later, this is shifted to the mesenchyme. The process is governed by multiple epithelial-mesenchymal interactions. Integrins are cell surface receptors for extracellular matrix components. Expression of the beta 5 integrin subunit alternates between epithelium and mesenchyme during early tooth development (Yamada et al. [1994] Int. J. Dev. Biol. 38: 553-556). By immunofluorescence and in situ hybridization we show here a remarkably similar oscillating expression pattern of the alpha v integrin subunit. This subunit is known to associate with beta 5, and we therefore suggest that integrin alpha v beta 5 is involved in epithelial-mesenchymal interactions during tooth development. We also demonstrate that the developing tooth epithelium expresses the alpha 6, beta 1 and beta 4 subunits. The laminin receptors alpha 6 beta 1 and alpha 6 beta 4 may thus in part mediate the effect of basement membranes on tooth epithelial development. Interestingly, the enamel knot region expressed very little alpha 6 integrin subunit, whereas some expression was seen transiently in the condensing mesenchyme. During early tooth development, integrins possessing the alpha 6 subunit might also be involved in cell-cell interactions independently of laminins.

Animals↗
PubMed1996

Fibulin-1 and fibulin-2 expression during organogenesis in the developing mouse embryo.

Fibulin-1 and fibulin-2 are extracellular matrix proteins with unique structural features. We used in situ hybridization and immunofluorescence staining to examine the expression of fibulin-1 and fibulin-2 during mouse embryogenesis. Both fibulins have previously been shown to be deposited at sites where polarized cells convert into mesenchyme, during early stages of development of endocardial cushion tissue and in neural crest cells. By Northern blots we confirm that expression of fibulin-2 is particularly high in the developing and newborn heart. We also show that fibulin-2 mRNA and protein remained highly expressed during organogenesis in tissues derived from neural crest mesenchyme. In addition, a locally restricted expression pattern of fibulin-1 and fibulin-2 mRNA and protein at sites of epithelial-mesenchymal interactions was detected in two tissues, the developing tooth and hair follicles. In other tissues where epithelial-mesenchymal interactions occur, fibulin-1 mRNA and its corresponding protein were detected rather uniformly around mesenchymal cells, and no expression of fibulin-2 was noted. Fibulin-1 protein was located in some embryonic epithelial basement membranes. Fibulin-1 mRNA was also expressed in the epidermal layer of brain and in the mesenchyme of choroid plexus and the meninges which surround the spinal cord. Overall, fibulin-2 expression was much more limited than fibulin-1 expression. A very prominent expression of fibulin-2 was seen during early stages of chondrogenesis in all cartilages analyzed. These studies show that the differential expression of the fibulin family contributes to the formation of molecularly distinct extracellular matrices already during early developmental stages of a large number of tissues.

Animals↗
PubMed1996

Expression of laminin alpha 1, alpha 5 and beta 2 chains during embryogenesis of the kidney and vasculature.

Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of alpha, beta and gamma chain subunits. To resolve conflicting data in the literature concerning coexpression of alpha 1 and beta 2 chains, expression of alpha 1 chain was studied with two different antisera against the E3 fragment of laminin alpha 1 chain. Expression of the alpha 1 chain was seen in several types of epithelial basement membranes throughout development, but its expression in rat glomerular basement membranes and some other types of epithelial basement membranes occurred only during early stages of development. By contrast, beta 2 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, beta 2 chains were detected somewhat earlier, but in situ hybridization revealed that beta 2 chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of alpha 1 and beta 2 chains was mutually exclusive. To explore whether the newly described alpha 5 chain is expressed in locations lacking alpha 1 chain, expression of alpha 5 chain was studied by Northern blots and in situ hybridization. The alpha 5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little alpha 1 chain. There also appeared to be a developmental trend, with alpha 1 chain appearing early and alpha 5 later, in maturing epithelial sheets. The alpha 5 chain could be a major alpha chain of the adult glomerular basement membrane.

Aging↗
PubMed1996

Receptors for laminins during epithelial morphogenesis.

Laminin-1 is expressed by many embryonic epithelial cell types. It binds to receptors on the epithelial cell surface. The integrin alpha6beta1 is a well known laminin-1 receptor that is expressed on many embryonic epithelial cells. More recently, dystroglycan was discovered as a high-affinity receptor for laminin-1 and laminin-2. It is expressed not only by muscle cells but also by embryonic epithelial cells. In embryonic epithelia, dystroglycan may act by binding to the E3 fragment of laminin-1. Integrins and the dystroglycan complex seems to be important for epithelial morphogenesis, but the relative roles of these two receptor systems for epithelial cells are still unclear.

Animals↗
PubMed1996

Tenascin-C contains distinct adhesive, anti-adhesive, and neurite outgrowth promoting sites for neurons.

The glia-derived extracellular matrix glycoprotein tenascin-C (TN-C) is transiently expressed in the developing CNS and may mediate neuron-glia interactions. Perturbation experiments with specific monoclonal antibodies suggested that TN-C functions for neural cells are encoded by distinct sites of the glycoprotein (Faissner, A., A. Scholze, and B. Götz. 1994. Tenascin glycoproteins in developing neural tissues--only decoration? Persp. Dev. Neurobiol. 2:53-66). To characterize these further, bacterially expressed recombinant domains were generated and used for functional studies. Several short-term-binding sites for mouse CNS neurons could be assigned to the fibronectin type III (FNIII) domains. Of these, the alternatively spliced insert TNfnA1,2,4,B,D supported initial attachment for both embryonic day 18 (E18) rat and postnatal day 6 (P6) mouse neurons. Only TNfn1-3 supported binding and growth of P6 mouse cerebellar neurons after 24 h, whereas attachment to the other domains proved reversible and resulted in cell detachment or aggregation. In choice assays on patterned substrates, repulsive properties could be attributed to the EGF-type repeats TNegf, and to TNfnA1,2,4. Finally, neurite outgrowth promoting properties for E18 rat hippocampal neurons and P0 mouse DRG explants could be assigned to TNfnB,D, TNfnD,6, and TNfn6. The epitope of mAb J1/tn2 which abolishes the neurite outgrowth inducing effect of intact TN-C could be allocated to TNfnD. These observations suggest that TN-C harbors distinct cell-binding, repulsive, and neurite outgrowth promoting sites for neurons. Furthermore, the properties of isoform-specific TN-C domains suggest functional significance of the alternative splicing of TN-C glycoproteins.

Amino Acid Sequence↗
PubMed1996

Genetics of kidney development.

Genetic approaches have clarified the molecular basis of many different stages of kidney development with considerable clarity. By introducing targeted mutations in mice, a number o transcription growth factors have been shown to be required for early kidney tubulogenesis. Most recently, the transcription factor Pax-2, and bone morphogenetic protein-7, a growth factor, were added to the list of factors required for the early stages of kidney tubulogenesis. Compared with the defects seen in mice lacking Pax-2 or bone morphogenic protein-7, the formation of cysts in tubules is morphologically a very mild defect. Cysts are seen in many transgenic mice with overexpression of a gene in the kidney, and in some 'knockout' mice lacking a gene. Some of these genes might be involved in human cystic diseases. However, it was recently shown that the gene affected in 85% of patients with autosomal dominant polycystic kidney disease encodes a novel protein, called polycystin. This protein is very large and has a sequence suggesting multiple transmembrane domains. It extracellular domains suggest that polycystin is involved in cell-cell and cell-matrix interactions.

Aging↗
PubMed1996

Integrin alpha 6B beta 1 is involved in kidney tubulogenesis in vitro.

Laminin-1 has previously been shown to be of major importance for the development of kidney tubules. Antibodies against fragments E8 and E3 of laminin-1 perturb kidney development in vitro. We here studied expression of integrins alpha 6 beta 1 and alpha 6 beta 4, two known laminin receptors, during kidney development. Integrin beta 1 subunit could be detected by immunofluorescence on all cell types of embryonic mouse kidney, but we could not detect integrin beta 4 subunit in embryonic kidney by immunofluorescence or by in situ hybridization. The presence of integrin alpha 6 subunit in all epithelia of embryonic kidney was demonstrated by immunofluorescence and by in situ hybridization. RT-PCR showed that alpha 6B is the major splice variant in embryonic kidney. During in vitro conversion of nephrogenic mesenchyme to epithelial tubules, a strong increase in the expression of the 6 kb mRNA for alpha 6 integrin subunit was seen by northern blotting at the onset of epithelial morphogenesis, on day two of culture. Immunoprecipitation of extracts from embryonic kidney with antibodies against alpha 6 subunit yielded bands corresponding to the expected size of beta 1 integrin subunit but not of beta 4 subunit. Monoclonal antibodies against either alpha 6 or beta 1 subunit but not against E-cadherin blocked kidney tubulogenesis in vitro. This suggests that integrin alpha 6B beta 1 is involved in kidney tubulogenesis in vitro. Another possibility is that the antibodies against integrin alpha 6 and beta 1 subunit cause abnormal signalling by the integrin.

Animals↗
PubMed1995

Extracellular matrix protein fibulin-2 is expressed in the embryonic endocardial cushion tissue and is a prominent component of valves in adult heart.

Fibulins-1 and -2 are two recently characterized extracellular matrix proteins of a novel protein family. They are found in many tissues in the adult stage, but several differences in their expression patterns were noted in cultured cells and adult tissues. Fibulin-1 is particularly well expressed in the endocardial cushion tissue (ECT) during embryogenesis and we therefore compared the expression patterns of fibulin-1 and -2 in the developing mouse heart. Antibodies against mouse fibulin-1 and -2 were used in immunofluorescence, and the expression of mRNAs were studied with synthetic oligonucleotides by in situ hybridization. A striking codistribution of fibulin-1 and -2 was seen at all stages of the development of the endocardial cushion tissue. Fibulin-2 was found to be a more specific marker for the embryonic ECT than fibulin-1, which was also transiently expressed in cardiac muscle. Immunofluorescence showed that the fibulins remained expressed in valves of the heart in the adult stage. The results suggest a role of the two fibulins in the development of the ECT and in the maintenance of the tensile strength of the cardiac valves.

Animals↗
PubMed1995

Evidence for a role of protein phosphatases 1 and 2A during early nephrogenesis.

Although most transcriptional events appear to be modulated by reversible protein phosphorylation, little is known about the role of this regulatory system during the development of mammalian organs. Here we have studied the serine/threonine protein phosphatases (PP) 1 and 2A in the early embryonic rat kidney with regard to expression and effects on growth and differentiation. All isoforms of PP-1 and PP-2A were ubiquitously expressed in 15-day embryonic (E15) kidneys (in situ hybridization studies). In contrast, mRNA for inhibitor-1 (I-1), an endogenous inhibitor of PP-1, was detected only in undifferentiated stem cells in the outer cortical area. I-1 is a novel marker for these cells. The abundance of the PP-1 protein, confirmed with immunoblotting, was high in the embryonic kidney. In organ culture of E13 kidneys, okadaic acid (OA), an exogenous inhibitor of PP-1 and PP-2A, dose-dependently inhibited growth and nephron formation (apparent half-maximal effect at 6 nM). OA 10 nM had little effect on the growth of cultured E15 kidneys, whereas nephron formation was disturbed and morphological evidence of apoptosis was seen. In summary, this study points towards important roles for protein phosphatases 1 and/or 2A in regulation of mitogenic activity in the early embryonic kidney.

Animals↗
PubMed1995

Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes.

Branching epithelial morphogenesis requires interactions between the surrounding mesenchyme and the epithelium, as well as interactions between basement membrane components and the epithelium. Embryonic submandibular gland was used to study the roles of two mesenchymal proteins, epimorphin and tenascin-C, as well as the epithelial protein laminin-1 and one of its integrin receptors on branching morphogenesis. Laminin-1 is a heterotrimer composed of an alpha 1 chain and two smaller chains (beta 1 and gamma 1). Immunofluorescence revealed a transient expression of laminin alpha 1 chain in the epithelial basement membrane during early stages of branching morphogenesis. Other laminin-1 chains and alpha 6, beta 1, and beta 4 integrin subunits seemed to be expressed constitutively. Expression of epimorphin, but not tenascin-C, was seen in the mesenchyme during early developmental stages, but a mAb against epimorphin did not perturb branching morphogenesis of this early epithelium. In contrast, inhibition of branching morphogenesis was seen with a mAb against the carboxy terminus of laminin alpha 1 chain, the E3 domain. An inhibition of branching was also seen with a mAb against the integrin alpha 6 subunit. The antibodies against laminin alpha 1 chain and integrin alpha 6 subunit perturbed development in distinct fashions. Whereas treatment with the anti-E3 resulted in discontinuities of the basement membrane at the tips of the branching epithelium, treatment with the mAb against alpha 6 integrin subunit seemed to leave the basement membrane intact. We suggest that the laminin E3 domain is involved in basement membrane formation, whereas alpha 6 beta 1 integrin binding to laminin-1 may elicit differentiation signals to the epithelial cells.

Animals↗
PubMed1995

Non-muscle alpha-dystroglycan is involved in epithelial development.

The dystroglycan complex is a transmembrane linkage between the cytoskeleton and the basement membrane in muscle. One of the components of the complex, alpha-dystroglycan binds both laminin of muscle (laminin-2) and agrin of muscle basement membranes. Dystroglycan has been detected in nonmuscle tissues as well, but the physiological role in nonmuscle tissues has remained unknown. Here we show that dystroglycan during mouse development in nonmuscle tissues is expressed in epithelium. In situ hybridization revealed strong expression of dystroglycan mRNA in all studied epithelial sheets, but not in endothelium or mesenchyme. Conversion of mesenchyme to epithelium occurs during kidney development, and the embryonic kidney was used to study the role of alpha-dystroglycan for epithelial differentiation. During in vitro culture of the metanephric mesenchyme, the first morphological signs of epithelial differentiation can be seen on day two. Northern blots revealed a clear increase in dystroglycan mRNA on day two of in vitro development. A similar increase of expression on day two was previously shown for laminin alpha 1 chain. Immunofluorescence showed that dystroglycan is strictly located on the basal side of developing kidney epithelial cells. Monoclonal antibodies known to block binding of alpha-dystroglycan to laminin-1 perturbed development of epithelium in kidney organ culture, whereas control antibodies did not do so. We suggest that the dystroglycan complex acts as a receptor for basement membrane components during epithelial morphogenesis. It is likely that this involves binding of alpha-dystroglycan to E3 fragment of laminin-1.

Animals↗
PubMed1995

Regulation of mesenchymal extracellular matrix protein synthesis by transforming growth factor-beta and glucocorticoids in tumor stroma.

We have here studied the composition and regulation of stromal extracellular matrix components in an experimental tumor model. Nude mice were inoculated with WCCS-1 cells, a human Wilms' tumor cell line. In the formed tumors the stroma was found to contain mesenchymal extracellular matrix proteins such as tenascin-C, fibulins-1 and 2 and fibronectin, but no nidogen. Nidogen was confined to basement membranes of tumor blood vessels. Since glucocorticoids have been shown to downregulate tenascin-C expression in vitro, we tested whether dexamethasone can influence biosynthesis of extracellular matrix components during tumor formation in vivo. A downregulation of tenascin-C mRNA and an upregulation of fibronectin mRNA expression by dexamethasone was noted. Transforming growth factor-beta 1 mRNA levels were unaffected by the dexamethasone treatment. Glucocorticoids can thus downregulate tenascin-C synthesis although local stimulatory growth factors are present. The competition between a negative and a positive extrinsic factor on synthesis of stromal extracellular matrix components was studied in a fibroblast/preadipocyte cell line. Transforming growth factor-beta 1 stimulated tenascin-C synthesis but did not affect fibronectin or fibulin-2 synthesis. Dexamethasone at high concentrations could completely suppress the effect of transforming growth factor-beta 1 on tenascin-C mRNA expression. Transforming growth factor-beta 1 could in turn overcome the downregulation of tenascin-C mRNA expression caused by a lower concentration of dexamethasone. We therefore suggest that the limited expression of tenascin-C in part is due to a continuous suppression by physiological levels of glucocorticoids, which can be overcome by local stimulatory growth factors when present in sufficient amounts.

Animals↗
PubMed1995

Mammary epithelial cell differentiation in vitro is regulated by an interplay of EGF action and tenascin-C downregulation.

Expression of the extracellular matrix glycoprotein tenascin-C in the mammary gland is associated with cellular proliferation and cell motility during organogenesis and tumorigenesis. Because the source and the regulation of tenascin-C in these tissues are unclear, we have used tenascin-C cDNA, FITC-immunofluorescence and immuno-precipitation to examine tenascin-C expression of mammary epithelial cells. Using several mammary epithelial cell lines we could show that tenascin-C can be produced and secreted by epithelial cells. However it was found that tenascin-C synthesis was inversely correlated with the polarized epithelial phenotype. Among three mouse mammary epithelial cell clones, tenascin-C expression was most abundant in HC-11 cells, the least differentiated cell type. Expression levels were high during the growth phase but were nearly abolished when cells were grown to confluence and induced to express milk proteins. Downregulation of tenascin-C by EGF apparently commits HC-11 cells to respond to lactogenic hormones and consequently, hormone induced levels of beta-casein mRNA decreased significantly when HC-11 cells were grown on a tenascin-C substrate. On the other hand, TGF-beta, another growth factor involved in coordinated growth and differentiation of the mammary gland in vivo was found to be a very potent inducer of tenascin-C. The generation of fully polarized and tight epithelium affected the levels of tenascin-C expression. In contrast to HC-11 cells, which do not form epithelial domes in vitro, highly polarized and dome forming EpH4 and Fos-ER cells nearly lacked tenascin-C. Similarly, induction of dome formation in the rat mammary stem cell line Rama 25 by the differentiation inducer dimethylsulfoxide caused a loss of TN-C-transcripts. The inability of Fos-ER cells to develop domes in the presence of soluble tenascin-C also suggests its interference with induction and maintenance of mammary epithelial cell differentiation.

Animals↗
PubMed1995

Differential distribution of laminin chains in the development and sex differentiation of mouse internal genitalia.

The distribution of laminin chains and basement membranes (BMs) in the ontogenesis and sex differentiation of male and female mouse gonads and mesonephros was studied by conventional and immunocytochemical light and electron microscopy. The alpha 1 (synonymous to A) chain was recognized with MAbs against fragment E3, and three chains of laminin with PAbs raised against EHS-laminin. BMs, which formed around the mesonephric duct, the mesonephric tubules, and the paramesonephric duct, contained the laminin alpha 1 chain. The alpha 1 chain appeared with epithelial differentiation in the developing gonads in both sexes. The alpha 1 chain was first evident around the embryonic gonadal cords and remained, after development, in the BMs of the testicular cords and ovarian follicles. The laminin alpha 1 chain was also detected in BMs of the myoid cells around the epithelial rete cords, and transiently in the surface epithelium and in the corpus luteum. Laminin beta-gamma chains were found in many locations where the alpha 1 chain was not detected. These included the mesenchyme of the early mesonephros, the BMs of blood vessels and surface epithelium in the differentiated testis and ovary, between the theca cells in the ovary, and in some corpora lutea. The morphological differentiation of the BMs of the embryonic testicular cords proceeded rapidly. In contrast, the BM of the ovarian cords remained relatively poorly differentiated during the prenatal phases, and developed concomitantly with the differentiation of the follicles. The results show that BMs in the differentiating internal genitalia are heterogeneous with respect to their laminin chains, and suggest that all known laminin chains must be analyzed in the differentiation of gonadal epithelia for a complete role of the BMs in gonadal sex differentiation.

Animals↗