Biomedical subjects

R Timpl

Publications and source records attributed to R Timpl.

At least 19 recordsLinked to original sources

PubMed1995

Binding of mouse and human fibulin-2 to extracellular matrix ligands.

Recombinant mouse and human fibulin-2 were obtained as disulfide-bonded trimers from transfected kidney cell clones and used in solid phase, biosensor and radioligand binding assays. Strong binding occurred with fibronectin and required calcium. A distinct interaction was also observed with nidogen but this was only partially blocked by EDTA. Distinctly weaker affinities were detected for collagen IV, perlecan and the N-terminal globule of collagen VI alpha 3 chain, while no or only little binding activity could be detected for several other collagen types, laminin-1, BM-40, fibulin-1 and vitronectin. This weaker binding reactions were also dependent on calcium. Surface plasmon resonance assays demonstrated for fibulin-2 binding to nidogen and fibronectin high equilibrium dissociation constants (0.5 to 1 microM) due to a rapid initial dissociation of the complexes. This is apparently followed by a slower stabilizing reaction. The fibulin-2 binding site of nidogen could be localized to its C-terminal globular domain G3, which also possesses a high-affinity binding site for laminin-1. Several tests demonstrated competition between the two ligands, probably due to steric hindrance. Binding of nidogen to immobilized fibulin-2 allowed the formation of ternary complexes with collagen IV, perlecan and fibulin-1, which, as shown previously, bind independently of the G3 domain. This indicated multifunctional binding properties for fibulin-2 and several alternative routes for its integration into basement membranes and other extracellular structures.

Amino Acid Sequence↗

PubMed1995

Characterization of mutant forms of recombinant human properdin lacking single thrombospondin type I repeats. Identification of modules important for function.

Properdin is a serum glycoprotein that up-regulates the alternative pathway of complement by stabilizing the C3b-Bb complex. It also binds sulfated glycoconjugates, such as sulfatide, in vitro. Properdin is composed of cyclic dimers, trimers, and tetramers of a 53-kDa monomeric subunit. The monomer contains an N-terminal region of no known homology and six thrombospondin type 1 repeats (TSRs) of approximately 60 amino acids. To identify the regions of properdin important for function, we have expressed human properdin, and mutant forms each lacking a single TSR, in Chinese hamster ovary cells. In addition, limited tryptic digestion yielded "nicked" properdin by the cleavage of one peptide bond in TSR5. The structural and functional properties of these altered forms of properdin were investigated. Properdin "nicked" in TSR5 is unable to bind C3b but retains its overall structure and its ability to bind sulfatide. The removal of TSR5 prevents C3b and sulfatide binding. Properdin lacking TSR4 is unable to stabilize the C3b-Bb complex but is able to bind C3b and sulfatide, and shows the presence of monomers and dimers in an electron microscope. Properdin without TSR3 is able to stabilize the C3b-Bb complex, to bind C3b and sulfatide, and forms dimers, trimers, and tetramers. Properdin lacking TSR6 is unable to form oligomers. The N-linked carbohydrate of properdin is not required for oligomerization or stabilization of the C3b-Bb complex. The results implicate TSR5 in both C3b and sulfatide binding, and suggest that TSR4 may also be involved in stabilization of the C3b-Bb complex.

Amino Acid Sequence↗

PubMed1995

The C-terminal portion of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallisable domain that binds calcium and collagen IV.

The extracellular glycoprotein BM-40 consists of three domains, an acidic domain I, a follistatin (FS)-like domain II and a calcium-binding EC domain with an EF-hand related motif. BM-40 and several other related proteins (QR1, SC1/hevin, testican and tsc-36/FRP) are members of a novel modular protein family that share the FS domain followed by an EC domain. We have expressed this pair of FS and EC domains (mutant delta I) and the calcium-binding EC domain alone (mutant delta I, II) of human BM-40 as recombinant proteins in human 293 cells. Circular dichroism demonstrated that both mutants were obtained as folded proteins with a distinct three-dimensional conformation. In addition, mutant delta I, II could be readily crystallized and diffraction patterns with a resolution limit of 2.4 A resolution were obtained. Calcium binding to this fragment was ten times weaker (Kd = 0.8 microM) than for the wild-type protein. Identical reversible increases in alpha-helicity upon calcium binding were observed for the 150-residue long mutant delta I, II and for BM-40 (286 residues). A 26-residue synthetic peptide corresponding to the EF-hand related motif exhibited much weaker calcium binding. The apparent dissociation constant decreased with increasing peptide concentration (from Kd 2.4 mM at 1 microM, to Kd 0.3 mM at 100 microM peptide concentration) and calcium binding was accompanied by dimerization of the peptide. This suggests that for strong calcium binding the EF-hand related motif has to be embedded into a larger protein domain that can form an autonomously folding protein module. The EC domain was also shown by surface plasmon resonance assay to be responsible for calcium-dependent binding to collagen IV with an affinity (Kd = 19 microM) only sixfold lower than that of intact human BM-40.

Amino Acid Sequence↗

PubMed1995

Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminaseC-catalyzed modification.

The extracellular matrix protein BM-40 (osteonectin, SPARC) has recently been shown to be a major target for transglutaminase-catalyzed cross-linking in differentiating cartilage. In the present study we demonstrate that recombinant human BM-40 can be modified with [3H]putrescine in a 1:1 molar ratio by transglutaminaseC (tissue transglutaminase). Residues Gln3 and Gln4 were identified as major amine acceptor sites. This was confirmed with several mutant proteins, including deletions in the N-terminal domain I of BM-40, site-directed mutagenesis of the reactive glutamines, and fusion of the seven-amino acid-long N-terminal sequence (APQQEAL) to an unrelated protein. The results showed that the N-terminal target site is sufficient for modification by transglutaminase but at a low level. For high efficiency amine incorporation an intact domain I is required. The conservation of at least one of the transglutaminase target glutamines in the known vertebrate BM-40 sequences and their absence in an invertebrate homologue point to an important, but yet unknown, role of this modification in vertebrates.

Amino Acid Sequence↗

PubMed1995

Collagen type VI in the human bone marrow microenvironment: a strong cytoadhesive component.

Collagen type VI, which forms characteristic microfibrillar structures, is assembled from three individual alpha(VI) chains that form a short triple helix and two adjacent globular domains. Expression of all three alpha (VI) collagen chains in the human bone marrow (BM) microenvironment could be detected by chain-specific antibodies in tissue sections and in the adherent stromal layer of long-term BM cultures. In functional studies, collagen type VI was shown to be a strong adhesive substrate for various hematopoietic cell lines and light-density BM mononuclear cells. The adhesive site within the molecule seems to be restricted to the triple helical domain of all three alpha (VI) chains, because individual alpha (VI) chains were not active in the attachment assays. Adhesion of the hematopoietic cell lines to collagen VI was dose-dependent and could be inhibited by heparin. Although the triple helix contains several RGD sequences, adhesion of the hematopoietic cell types to collagen VI could be blocked neither by RGD-containing peptides nor by a neutralizing antibody to the beta 1 integrin subunit. In combination with an antiadhesive substrate, the binding properties of collagen VI could be downregulated. These data suggest that this collagen type may play an important role in the adhesion of hematopoietic cells within the BM microenvironment.

Bone Marrow↗

PubMed1995

Structural properties of recombinant domain III-3 of perlecan containing a globular domain inserted into an epidermal-growth-factor-like motif.

A fragment comprising approximately domain III-3 of the basement membrane heparan sulfate proteoglycan perlecan was prepared in recombinant form from kidney cell clones. This fragment was predicted to contain a cysteine-free globular domain inserted within an epidermal-growth-factor(EGF)-like motif (L4 module) and three additional EGF-like motifs (LE module) without large inserts. This prediction was confirmed by electron microscopy, which demonstrated a globule joined to a very short rod-like segment. The globule was selectively destroyed by pepsin, which also demonstrated that its insertion into an EGF-like motif did not prevent the typical disulfide connections known for such motifs. Yet the globule was more stable against neutral proteinases. The fragment showed a distinct content (55-60%) of alpha helical and beta structure and a partially reversible melting of the conformation in 6 M guanidine. Antibodies raised against recombinant domain III-3 demonstrated a complete cross-reaction with tissue-derived perlecan but not with laminin and a distinct basement membrane staining of tissue sections. Most of the epitopes were lost after reduction and alkylation. Together the data demonstrated a proper folding of recombinant domain III-3 similar to its structure in the native protein and provided the first structural evidence for a novel globular protein motif L4 based on an EGF-like scaffold.

Amino Acid Sequence↗

PubMed1995

Integrin-binding and cell-adhesion studies of fibulins reveal a particular affinity for alpha IIb beta 3.

The extracellular matrix proteins fibulin-1 (variants C and D) and fibulin-2 occur in basement membranes and in vessel walls and are thus potential candidates for cellular interactions. Recombinant forms of these proteins were obtained from stably transfected kidney cell clones and examined for cell-adhesion activity and binding to five different purified integrins. The two variants of mouse fibulin-1 were inactive in all these assays. Mouse fibulin-2, however, bound to alpha IIb beta 3 integrin almost as strongly as fibrinogen, while a lower activity was found for alpha V beta 3 and almost none for alpha 5 beta 1 integrin. Synthetic SVPRGDLDG peptide, corresponding to the single RGD site of mouse fibulin-2, was a strong antagonist of alpha IIb beta 3 integrin binding. Its affinity for alpha V beta 3 and alpha 5 beta 1 integrins was, however, 10- to 50-fold lower compared to GRGDS. Mouse fibulin-2 also promoted adhesion of thrombin-stimulated platelets and of some established cell lines which could be inhibited by RGD peptides. Human fibulin-2, in which the RGD sequence is changed to RSS, bound less strongly to alpha IIb beta 3 integrin and showed no cell-adhesion activity. Together these data suggest a potential role in hemostatic control for mouse fibulin-2 and possibly also for human fibulin-2.

Amino Acid Sequence↗

PubMed1995

Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts.

A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.

Animals↗

PubMed1995

Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro.

The purpose of this study was to determine whether nidogen, the linkage protein of the basal lamina, is of epidermal or dermal origin. The development of the basal lamina was studied in an in vitro skin model. Preputial fibroblasts seeded onto a nylon mesh attached, proliferated, and developed a rich extracellular matrix (dermal model). Preputial keratinocytes were added to the dermal model to form a keratinocyte dermal model that ultrastructurally resembled in many respects human skin. Ultrastructural analysis revealed early stages of dermal development, including an incomplete basal lamina, aggregates of dermal filamentous material connecting to the lamina densa, bundles of 10-nm microfibrils, formation of premature hemidesmosomes, anchoring filaments, and anchoring fibrils. The cell origin of nidogen was determined in the dermal model and in the epidermal and dermal components of the keratinocyte dermal model. Specific antibodies and a cDNA probe for nidogen were used for immunofluorescence microscopy, Western and Northern blots, and for in situ hybridization studies. Our data show that fibroblasts are the only source of nidogen during early basal lamina formation. Although fibroblasts can synthesize nidogen and deposit it in the dermal matrix, no basal lamina will form unless they are recombined with keratinocytes. This suggests that the epidermis plays a major regulatory role in the production and assembly of nidogen into the basal lamina.

Basement Membrane↗

PubMed1995

The collagen superfamily.

The collagens represent a large family of molecular proteins which are located in the extracellular matrix. They all share a triple-helical segment of variable length (100-450 nm) but differ considerably in the size and nature of their globular domains. The various collagen types I-XIX known so far in vertebrates are adjusted to different functions including the formation of fibrils, microfibrils and networks. Their biological role is now increasingly studied by genetic approaches.

Animals↗

PubMed1995

Increased expression of type VI collagen in lung fibrosis.

Pulmonary fibrosis is characterized by disturbances of extracellular matrix protein deposition resulting from fibroblast activation and proliferation. Collagen VI, forming microfibrillar meshworks separate from major fibrillar systems, is thought to serve as an anchoring element between collagen I/III fibrils and basement membranes and as a cell binding substrate. We report on the expression of collagen VI in normal and in fibrotic lungs. Collagen VI is present in vascular and bronchial walls and in the interstitial space of normal lungs. Its turnover is too low to generate a mRNA signal by in situ hybridization. Collagen VI expression is increased in lung fibrosis, and its degree appears independent of the etiology of fibrosis. There was no evidence for differential regulation of gene expression for the alpha 1(VI) and alpha 3(VI) constitutive peptide chains of collagen VI. Collagen VI mRNA is expressed by fibroblasts, mostly with myofibroblast characteristics. Collagen VI was coexpressed with collagen III rather than collagen I in idiopathic bronchiolitis obliterans with organizing pneumonia, acute interstitial pneumonitis of the Hamman-Rich type, and bleomycin-induced fibrosis, but collagen VI overlapped with collagen types I and III in idiopathic pulmonary fibrosis. These coexpression data suggest that collagen VI expression may be an early rather than a late phenomenon in pulmonary fibrosis.

Collagen↗

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↗

PubMed1994

Binding of purified collagen receptors (alpha 1 beta 1, alpha 2 beta 1) and RGD-dependent integrins to laminins and laminin fragments.

Integrins alpha 1 beta 1 and alpha 2 beta 1 when purified by collagen affinity chromatography, showed distinct binding to mouse tumor laminin-1, which has the chain composition alpha 1 beta 1 gamma 1. The binding was, however, about 10-fold lower than to collagen IV. Only little (alpha 1 beta 1) or no binding (alpha 2 beta 1) was observed to two different laminin isoforms (alpha 2 beta 1 gamma 1, alpha 2 beta 2 gamma 1) from human placenta. Binding to laminin-1 was abolished by EDTA and could be specifically inhibited by antibodies to the respective integrin alpha subunit. These antibodies also inhibited cell adhesion to collagens. The binding of soluble integrins was weaker than that of immobilized integrins but could be enhanced by an activating anti(beta 1 integrin). No enhancement was observed for immobilized integrins. Studies with laminin-1 fragments demonstrated lack of binding to the major cell-adhesive fragment E8 from the long arm, fragments E3 and E4, involved in heparin-binding and self-assembly, respectively, and fragment P1, corresponding to the inner segments of the short arms. A larger short-arm fragment (E1XNd), which lacks the N-terminal beta 1 chain domains V and VI, was as active as laminin. Together, these results, suggested the localization of the binding sites for alpha 1 beta 1 and alpha 2 beta 1 to the N-terminal region of the laminin alpha 1 chain. Fragment P1 but not intact laminin-1 bound to alpha V beta 3 integrin in an EDTA-sensitive and RGD-sensitive manner, underscoring previous data on the cryptic nature of the RGD site in laminin-1. Further analyses by surface plasmon resonance assays demonstrated a KD = 50 nM for alpha 2 beta 1/laminin-1 binding and a KD = 450 nM for alpha V beta 3/fragment P1 binding and confirmed the anti-beta 1-mediated increase in affinity for alpha 2 beta 1.

Amino Acid Sequence↗

PubMed1994

Recombinant expression and properties of the Kunitz-type protease-inhibitor module from human type VI collagen alpha 3(VI) chain.

The Kunitz-type inhibitor motif (domain C5) present at the C-terminus of the human collagen alpha 3(VI) chain was prepared in a recombinant form from the culture medium of stably transfected kidney cell clones. The 76-residue protein was disulfide bonded and showed a high stability against protease treatment. The recombinant protein lacked, however, any inhibitory activity for trypsin, thrombin, kallikrein and several other proteases, which could be due to a few unusual substitutions in the region crucial for inhibitor binding. A sensitive radioimmunoassay detected low concentrations of C5 epitopes in normal human serum and fibroblast culture medium and showed a lack of cross-reaction with aprotinin. Antibodies against C5 immunoprecipitated collagen VI obtained from fibroblast medium. The C5 epitopes could not be detected on intact collagen VI purified from guanidine extracts of human placenta. Collagen VI was shown to possess several alpha 3(VI) chain bands (approximately 200 kDa) and reacted strongly with antibodies to an N-terminal recombinant fragment. Immunofluorescence with anti-C5 antibodies failed to stain several human tissues but produced a distinct intracellular staining of cultured fibroblasts. The data indicate the rapid loss of the C5 domain after biosynthesis of collagen VI.

Amino Acid Sequence↗

PubMed1994

Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition.

The inhibitory capacities of six different disintegrins and one related neurotoxin analogue for the binding of RGD-dependent integrins to either fibrinogen, vitronectin or fibronectin were compared in solid phase assays. Echistatin and flavoridin were the most active inhibitors for alpha V beta 3 and alpha 5 beta 1 integrins and moderately exceeded the activity of the natural protein ligands. The same disintegrins together with eristostatin, bitistatin and barbourin were also very potent inhibitors of fibrinogen binding to alpha IIb beta 3 integrin. For all three integrins, albolabrin showed the lowest affinity, but it still clearly exceeded that of synthetic GRGDS. However, assay conditions may determine these relative affinities, as shown for the alpha IIb beta 3 and alpha V beta 3 integrins when used either in immobilized or soluble form. For alpha IIb beta 3, however, a close correlation was found between KD values determined in platelet binding assays and the concentrations required for half maximal inhibition of three disintegrins. The inhibiting capacity of disintegrins in assays with purified integrins also correlated reasonably well with their inhibition of cell attachment to RGD-dependent protein substrates. However, sequence differences in the RGD loops of the various disintegrins may not fully account for the 20-100-fold difference in their binding capacities. This was particularly evident for echistatin and albolabrin, which differ in this region only by two conservative substitutions but have considerably different inhibitory activities. More remote regions of the disintegrins and alignment of disulfide bridges are therefore likely to contribute to their affinity and selectivity.

Amino Acid Sequence↗

PubMed1994

Immunological properties and tissue localization of two different collagen types in annelid and vestimentifera species.

Rabbit antisera against cuticle and interstitial collagens from shallow sea water and hydrothermal vent annelids (Arenicola marina, and the pompeii worm Alvinella pompejana) and the vestimentiferan tube worm Riftia pachyptila showed a clear distinction between the two types of collagens, a broad cross-reactivity among the worm collagens and no reactions with various mammalian collagens. The antibodies reacted with various epitopes found on both triple helical and unfolded collagens. The cuticle collagens were localized by immunofluorescence to the outer surface of the epidermis and in annelids additionally to the anterior part of the digestive tract. The interstitial collagen was detected underneath the epidermis and between distinct muscle layers. Both collagens were also detected in the anterior obturaculum, a tissue unique to vestimentifera. They were located either in the periphery of the tissue (cuticle collagen) or in the central part (interstitial collagen), which appeared to be a large extracellular matrix. Both collagens, however, showed a different supramolecular organization in the obturaculum when compared to the posterior body wall collagens. The identity of the interstitial collagens from the two locations was verified by biochemical analysis. These data demonstrate a very special and rigid matrix structure in the obturaculum, which may adapt it to specific physiological functions.

Animals↗

PubMed1993

Multimeric vitronectin. Identification and characterization of conformation-dependent self-association of the adhesive protein.

The adhesive glycoprotein vitronectin (VN) shows a high degree of conformational flexibility implicating that different molecular forms of the molecular may exist. Conformation-dependent monoclonal antibodies 13H1 or 16A7 that, per se, did not react with plasma VN bound to VN treated with heparin, chaotropes, detergents, pH below 6, or by heating at 56 degrees C. Dependent on the stimulus, recognition of VN by these antibodies varied and preceded heparin binding and self-association of VN resulting in the formation of noncovalently linked multimeric species of the protein. Both monoclonal antibodies also reacted with VN in serum or in platelet releasates as well as with VN in extracellular matrices of endothelial cells and inhibited cell adhesion on immobilized VN. Critical VN levels were needed for concentration-dependent multimerization indicating a nonlinear type of polymerization process. The nature of VN multimers was judged by nondenaturing gel electrophoresis, gel filtration, and sucrose gradient ultracentrifugation and revealed the formation of 3- to 16-mer multimeric species within an M(r) range of 200-1200 kDa representing a mean sedimentation coefficient of 9.6 S. In electron microscopy, multimeric VN occurred as globular specimens with an average diameter of 15-28 nm (monomeric plasma VN, 6-8 nm). In contrast to plasma VN, VN multimers were efficiently stabilized by covalent inter-molecular bonds following chemical or transglutaminase-induced cross-linking. A synthetic peptide comprising the central heparin binding region of VN (residues 348-361) not only bound to plasma VN but induced its multimerization also in plasma. During plasmin proteolysis of VN, fragments were generated that lacked the heparin binding region and that lost the ability to multimerize following urea or detergent treatment, implicating that the highly basic region is essential for multimer formation. These data suggest that non-plasma forms of VN, which are abundant in platelets and subendothelium, represent the prototype conformer of the reactive heparin binding form of VN. Our findings implicate that conformationally altered forms of VN enable the adhesive protein to multimerize in a characteristic fashion and thereby endow extracellular matrix sites with unique multivalent properties.

Amino Acid Sequence↗

PubMed1993

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.

Animals↗