Extracellular matrix in animal development. Role of extracellular matrix in animal development--an introduction.
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Biomedical subjects
P Ekblom
Publications and source records attributed to P Ekblom.
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.
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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.
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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.
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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.
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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.
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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.
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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.
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A new nomenclature for the laminins.
The authors have adopted a new nomenclature for the laminins. They are numbered with arabic numerals in the order discovered. The previous A, B1 and B2 chains, and their isoforms, are alpha, beta and gamma, respectively, followed by an arabic numeral to identify the isoform. For example, the first laminin identified from the Engelbreth-Holm-Swarm tumor is laminin-1 with the chain composition alpha 1 beta 1 gamma 1. The genes for these chains are LAMA1, LAMB1 and LAMC1, respectively.
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Role of mesenchymal nidogen for epithelial morphogenesis in vitro.
Recent biochemical studies suggested that the extracellular matrix protein nidogen is a binding molecule linking together basement membrane components. We studied its expression and role during development. By immunofluorescence and northern blotting, nidogen was found early during epithelial cell development of kidney and lung. Yet, in situ hybridization revealed that nidogen was not produced by epithelium but by the adjacent mesenchyme in both organs. Binding of mesenchymal nidogen to epithelial laminin may thus be a key event during epithelial development. This is supported by antibody perturbation experiments. Antibodies against the nidogen binding site on laminin B2 chain perturbed epithelial development in vitro in embryonic kidney and lung. Mesenchymal nidogen could be important for early stages of epithelial morphogenesis.
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Anti-beta 1 integrin IgG inhibits pulmonary macrometastasis and the size of micrometastases from a murine mammary carcinoma.
In the present report, we investigated the possible importance of beta 1 integrins in the growth and metastasis of a murine mammary carcinoma, SP1, and a metastatic variant, SP1-3M in vivo. CBA/J female mice bearing SP1 tumor transplants were injected with anti-beta 1 integrin IgG or control nonimmune IgG (200 micrograms per mouse; i.p.) every two days. Animals received anti-CD4 antibody (100 micrograms per mouse) at time zero to suppress immunity against rabbit IgG. Outgrowth of macroscopic metastases from SP1, but not from SP1-3M primary tumors, was markedly inhibited in animals receiving anti-beta 1 integrin IgG but not nonimmune IgG. To assess the stage(s) in the metastatic cascade affected, we examined the number and diameter of micrometastatic nodules in treated and untreated groups. The diameter of micrometastases was significantly reduced in SP1-tumor-bearing mice treated with anti-beta 1 integrin IgG compared to control IgG, although the number of nodules per cm2 of lung sections examined remained unchanged. No change in the number or size of micrometastases in SP1-3M tumor-bearing mice was observed. No difference in the binding, or complement-mediated and antibody-dependent cell-mediated cytotoxicity of anti-beta 1 integrin IgG with SP1 and SP1-3M cells was detected. The results suggest that under these conditions anti-beta 1 integrin inhibits metastatic tumor growth in lung tissue, but has minimal effect on intravasation, adhesion to target organs and extravasation.
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Induction of mouse tenascin expression by a human sarcomatoid Wilms' tumor cell line growing in nude mice.
A new cell line from a sporadic Wilms' tumor was established and extensively characterized. In nude mice, the tumor cells rapidly formed tumors which, in histological characteristics and extracellular-matrix (ECM) composition resembled sarcomatoid Wilms' tumor. The tumor cells produced B chains of laminin, but no A chain, and laminin was deposited into the ECM in a punctate pattern typical of sarcomatoid tumors. Strong expression of tenascin was detected within the stromal ECM of the tumors. Species-specific antibodies reacting either with human or with mouse tenascin showed that tenascin was exclusively derived from mouse host cells. The human Wilms' cell line thus induced a strong stromal response with increased deposition of tenascin. The cell line may be useful for studying the behavior of sarcomatoid Wilms' tumor cells and for identifying factors that stimulate synthesis of tenascin.
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Downregulation of tenascin expression by glucocorticoids in bone marrow stromal cells and in fibroblasts.
Tenascin, a predominantly mesenchymal extracellular matrix (ECM) glycoprotein has a rather restricted tissue distribution, but until now factors that inhibit its expression have not been identified. Glucocorticoids are known to be beneficial for establishment of myelopoiesis in long-term bone marrow cultures. Tenascin was found to be expressed in the bone marrow, and glucocorticoids were found to affect bone marrow tenascin expression. Both tenascin mRNAs and the mRNA of another ECM protein, laminin B1 chain, were drastically downregulated by glucocorticoids during initiation of bone marrow cultures. However, in already established long-term cultures glucocorticoids did not affect laminin B1 chain mRNA levels although tenascin mRNAs continued to be downregulated. Studies with a stromal cell line (MC3T3-G2/PA6) and fibroblasts (3T3) suggested that glucocorticoids act directly on the stromal cells that produce tenascin. In 3T3 cells this downregulation occurred within 12 h of glucocorticoid-treatment, suggesting that glucocorticoids acted through cis regulatory elements of the tenascin gene. We suggest that glucocorticoids in part regulate hematopoiesis by modifying the ECM. Furthermore, downregulation of tenascin expression by glucocorticoids may in part explain the restricted tissue distribution of tenascin in other tissues.
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The extracellular matrix glycoproteins BM-90 and tenascin are expressed in the mesenchyme at sites of endothelial-mesenchymal conversion in the embryonic mouse heart.
BM-90 is a novel glycoprotein initially isolated from the extracellular matrix of a mouse tumor. We here studied the expression of BM-90 during embryonic development of the mouse heart and compared its expression pattern with that of tenascin and laminin. Distribution was studied by immunofluorescence using antibodies specifically raised against mouse BM-90, laminin and tenascin. Some expression of BM-90 was seen in myocardial basement membranes at early developmental stages, but expression abruptly decreased from these sites at day 12 of embryogenesis. Laminin B chains were also found in the muscle basement membranes early but did not decrease with advancing development. The most striking observation was the markedly enriched expression of BM-90 in the endocardial cushion tissue (ECT). The ECT is derived from mesenchymal cells converted from endothelium and they will form the cardiac valves and septa. In the ECT, BM-90 showed considerable co-distribution with tenascin, but tenascin expression was more focal and did not mark all areas of the ECT. Northern blot data show that BM-90 and tenascin were produced by the developing heart. With antibodies detecting A, B1 and B2 chains of mouse laminin, no immunoreactivity was seen in the ECT. Our data thus show clear-cut differences in the molecular composition of the ECT and muscle basement membranes in the developing heart. The focal expression of BM-90 in the ECT suggests that BM-90 could be involved in epithelial-mesenchymal transitions.
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Expression of tenascin in developing and adult mouse lymphoid organs.
The extracellular matrix (ECM) is essential in regulating many cell functions in non-lymphoid cells, and the ECM may also play a role in the function of the immune system. Tenascin is a hexameric glycoprotein of the ECM. In mouse, two major polypeptides of MW 210 KD and 260 KD are formed by differential splicing. Northern blot screening of various mouse tissues showed that the short 6 KB tenascin message was strongly expressed in the adult thymus, whereas very little or no tenascin mRNA could be detected in spleen. In addition, immunoblotting and histological analysis with monoclonal anti-tenascin antibodies revealed the presence of tenascin in lymph nodes and spleen. In thymus, only a short-splice variant of tenascin was detected by immunoblotting, which supported the Northern blot results. Immunohistology showed that the epithelial reticular stroma in both embryonic and adult mouse thymus expressed tenascin, as did the postnatal mesenchymal reticular stroma in lymph nodes and spleen. The distribution of tenascin in the thymus was more restricted than that of fibronectin and laminin.
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Monoclonal antibodies against laminin A chain fragment E3 and their effects on binding to cells and proteoglycan and on kidney development.
Rat monoclonal antibodies were raised against fragment E3 of the mouse Engelbreth-Holm-Swarm (EHS) tumor laminin and selected according to their exclusive reaction with laminin A chain by immunoblotting and staining pattern in embryonic kidneys by immunofluorescence. Immunochemical studies of nine purified antibodies showed a comparable reaction with unfragmented laminin and fragment E3 but no cross-reaction with several other, unrelated laminin fragments including the major cell-binding fragment E8. Reduction or pepsin digestion of fragment E3 reduced or abolished antibody binding indicating that most of the epitopes involved are conformation dependent and do not include carbohydrates. They are, however, not identical as shown by different reactivities after proteolytic or chemical cleavage of E3. Four of the antibodies were highly active in inhibiting cell adhesion of the teratocarcinoma cell line F9 and the Schwannoma cell line RN22 on fragment E3 (IC50 approximately 1 microgram/ml), while the others were distinctly less active. No inhibition was observed for cell adhesion on unfragmented laminin, consistent with previous findings that this is largely mediated by binding of fragment E8 to alpha 6 beta 1 integrin. A distinct correlation was observed between cell adhesion inhibition and the inhibition of heparansulfate proteoglycan and heparin binding to fragment E3. Since heparin is not very efficient in inhibiting cell adhesion, it indicates that heparin- and cell-binding sites on fragment E3 are in close proximity but not identical. Two of the antibodies also showed partial inhibition of kidney tubule formation in organ culture of embryonic kidney mesenchyme while the other antibodies were inactive. It suggests some but probably minor involvement of the fragment E3 structure of laminin in this developmental process.
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Development of tubular and glomerular cells of the kidney.
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Laminin in the male germ cells of Drosophila.
To study genes that may be crucial for the male germ cell development of Drosophila we screened a cDNA expression library with a polyclonal antiserum against testis proteins of Drosophila hydei. We identified a cDNA fragment that exhibited a complete sequence similarity with the cDNA of the laminin B2 chain, an important component of the extracellular matrix. Transcripts of laminin B2 were detected in the RNA of male germ cells with the polymerase chain reaction and by in situ hybridization. We studied the reaction of different polyclonal antibodies including those against a Drosophila laminin B2-lac fusion protein, the entire Drosophila laminin complex, or against the mouse laminin complex and against laminin A and B1 chains with specific structures in developing male germ cells of Drosophila. Antigenic sites against laminin B2 were found in the lampbrush loops in primary spermatocyte nuclei, in nuclei of spermatids, and in heads of spermatozoa. The axonemes of elongating spermatids react with antibodies against the Drosophila laminin B1, B2 and laminin A chains. The possible biological functions of the laminin in the male germ cells of Drosophila are discussed.
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