Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.     

Effect of hydrocortisone on skin development in the chick embryo: ultrastructural, immunohistological, and biochemical analysis

The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.     

Bovine bone activin enhances bone morphogenetic protein-induced ectopic bone formation.

A 25-kDa homodimeric protein was purified from demineralized bovine bone extract and identified as activin A. The bovine bone activin enhanced formation of ectopic bone in rat subcutis when implanted in combination with partially purified bovine bone morphogenetic protein (BMP-2, BMP-3) in collagen/ceramic carrier. The implants, removed at 14 days, contained markedly elevated levels of alkaline phosphatase activity. Histological examination revealed an extensive formation of woven bone with very little cartilage. In contrast, a combination of transforming growth factor-beta 2 and BMP promoted formation of bone with an abundance of cartilage. The implants with BMP alone exhibited some osteoinductive activity, while the implants with activin alone showed no activity. These results demonstrate that bone is a rich source of activin and that activin plays an important role in modulating bone formation.     

Purification and characterization of mammalian integrins expressed by a rat neuronal cell line (PC12): evidence that they function as alpha/beta heterodimeric receptors for laminin and type IV collagen

Cells of the rat neuronal line, PC12, adhere well to substrates coated with laminin and type IV collagen, but attach poorly to fibronectin. Adhesion and neurite extension in response to these extracellular matrix proteins are inhibited by Fab fragments of an antiserum (anti-ECMR) that recognizes PC12 cell surface integrin subunits of Mr 120,000, 140,000, and 180,000 (Tomaselli, K. J., C. H. Damsky, and L. F. Reichardt. 1987. J. Cell Biol. 105:2347-2358). Here we extend our study of integrin structure and function in PC12 cells using integrin subunit-specific antibodies prepared against synthetic peptides corresponding to the cytoplasmic domains of the human integrin beta 1 and the fibronectin receptor alpha (alpha FN) subunits. Anti-integrin beta 1 immunoprecipitated a 120-kD beta 1 subunit and two noncovalently associated integrin alpha subunits of 140 and 180 kD from detergent extracts of surface-labeled PC12 cells. Immunodepletion studies using anti-integrin beta 1 demonstrated that these two putative alpha/beta heterodimers are identical to those recognized by the adhesion-perturbing ECMR antiserum. Anti-alpha FN immunoprecipitated fibronectin receptor heterodimers in human and rat fibroblastic cells, but not in PC12 cells. Thus, low levels of expression of the integrin alpha FN subunit can explain the poor attachment of PC12 cells to FN. The PC12 cell integrins were purified using a combination of lectin and ECMR antibody affinity chromatography. The purified integrins: (a) completely neutralize the ability of the anti-ECMR serum to inhibit PC12 cell adhesion to laminin and collagen IV; (b) have hydrodynamic properties that are very similar to those of previously characterized integrin alpha/beta heterodimeric receptors for ECM proteins; and (c) can be incorporated into phosphatidylcholine vesicles that then bind specifically to substrates coated with laminin or collagen IV but not fibronectin. Thus, the ligand-binding specificity of the liposomes containing the purified PC12 integrins closely parallels the substrate-binding preference of intact PC12 cells. These results demonstrate that mammalian integrins purified from a neuronal cell line can, when incorporated into lipid vesicles, function as receptors for laminin and type IV collagen.     

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.     

The cysteine-rich domain protein KCP is a suppressor of transforming growth factor beta/activin signaling in renal epithelia

The transforming growth factor beta (TGF-beta) superfamily, including the bone morphogenetic protein (BMP) and TGF-beta/activin A subfamilies, is regulated by secreted proteins able to sequester or present ligands to receptors. KCP is a secreted, cysteine-rich (CR) protein with similarity to mouse Chordin and Xenopus laevis Kielin. KCP is an enhancer of BMP signaling in vertebrates and interacts with BMPs and the BMP type I receptor to promote receptor-ligand interactions. Mice homozygous for a KCP null allele are hypersensitive to developing renal interstitial fibrosis, a disease stimulated by TGF-beta but inhibited by BMP7. In this report, the effects of KCP on TGF-beta/activin A signaling are examined. In contrast to the enhancing effect on BMPs, KCP inhibits both activin A- and TGF-beta1-mediated signaling through the Smad2/3 pathway. These inhibitory effects of KCP are mediated in a paracrine manner, suggesting that direct binding of KCP to TGF-beta1 or activin A can block the interactions with prospective receptors. Consistent with this inhibitory effect, primary renal epithelial cells from KCP mutant cells are hypersensitive to TGF-beta and exhibit increased apoptosis, dissociation of cadherin-based cell junctions, and expression of smooth muscle actin. Furthermore, KCP null animals show elevated levels of phosphorylated Smad2 after renal injury. The ability to enhance BMP signaling while suppressing TGF-beta activation indicates a critical role for KCP in modulating the responses between these anti- and profibrotic cytokines in the initiation and progression of renal interstitial fibrosis.     

Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and secretion of alpha-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata, and its synthesis by hepatocytes seeded onto a mixed substratum of laminin and fibronectin is down-regulated by fibronectin in a dose-related manner. Similarly, type IV collagen synthesis is less when the cells are seeded on the homologous matrix protein substratum than on heterologous substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as alpha-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.     

Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen

Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.     

Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5-21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.     

Extracellular matrix (ECM) synthesis in muscle cell cultures: quantitative and qualitative studies during myogenesis

When the synthesis of extracellular matrix components was examined in G8-1 murine skeletal muscle cells as a function of differentiation, non-collagen and to an even greater extent collagen synthesis was increased. Specifically, collagen types I, III, IV, laminin and fibronectin were identified by SDS-PAGE. Immunoprecipitation, with specific antibodies revealed that both the cell layer and medium of differentiated multinucleated myotubes contained increased levels of type IV collagen and laminin, decreased levels of type III collagen and fibronectin and equivalent levels of type I collagen compared to mononuclear myoblasts.     

Laminin B1 expression is required for laminin deposition into the extracellular matrix of PC12 cells.

The extracellular matrix of rat pheochromocytoma PC12 cells was shown by indirect immunofluorescence to consist of a network of fibronectin. The matrix did not contain laminin. The cells synthesized messenger RNA for fibronectin, laminin B2, and s-laminin but not for entactin or the B1 and A chains of laminin. Laminin B2 but not laminin B1 was detectable in the culture medium and in cell lysates. A full-length cDNA clone for the B1 chain of laminin was constructed in the plasmid p-444, which contains the neomycin-resistance marker and human beta-actin promoter. PC12 cells were transfected with this recombinant plasmid, and stable neomycin-resistant clones were isolated and characterized. Clones that synthesized laminin B1 messenger RNA were found to deposit a laminin-containing matrix. In many of these clones the deposition of the fibronectin matrix was greatly diminished. The laminin matrix was predominantly localized in the intercellular spaces forming a honeycomb pattern. The morphology of the laminin-synthesizing transfected cells was markedly different from the parental cells. The cells grew in tight clusters that were resistant to dissociating agents. It is concluded that the B1 chain of laminin contains information that is required for the formation of a stable laminin-containing extracellular matrix network either by interaction with cell surface receptors or other extracellular matrix components. Furthermore, expression of the laminin B1 gene may be a central regulatory point in determining extracellular matrix composition during embryogenesis.     

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10(-9) and 10(-7) M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.     

Roles of extracellular matrix components in differentiating teratocarcinoma cells

F9 embryonal carcinoma cells treated with 5 X 10(-8) M retinoic acid and cultured in suspension for 8 days form aggregates consisting of an outer epithelial layer of alpha-fetoprotein-producing visceral endoderm cells. We have previously shown (Grover, A., Oshima, R. G., and Adamson, E. D. (1983) J. Cell Biol. 96, 1690-1696) that the differentiation of F9 cells to visceral endoderm is accompanied by the activation of several genes, and increased laminin synthesis is one of the earliest events. Here we analyze in detail the syntheses and secretion of fibronectin, type IV collagen, and laminin during the 8-day process. Employing immunoprecipitation and enzyme-linked immunosorbent assay, we show that the levels of all three components change with different patterns. Unstimulated F9 cells synthesize and secrete relatively high levels of fibronectin and low levels of type IV collagen. Fibronectin synthesis and secretion decreases to 10% of its original level whereas type IV collagen synthesis rises approximately 3-fold during the differentiation process. Laminin synthesis also rises at least 2-fold, and the proportions of its subunits change as the syntheses of B1 and A accelerate starting on day 2. However, unlike fibronectin and type IV collagen, laminin is largely accumulated in the aggregates. The data suggest that fibronectin has a role in aggregation whereas laminin is important in the differentiation process.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Transforming growth factor-beta1 regulates basement membrane formation by alveolar epithelial cells in vitro

Immortalized alveolar type II epithelial (SV40-T2) cells formed a continuous, thin lamina densa when they were cultured on collagen fibrils with the supplement of 1.0 ng/ml TGF-beta1. Corresponding to lamina densa formation, immunohistochemical analysis of laminin, type IV collagen, perlecan, and entactin (nidogen) indicated integration of these components in a linear array beneath the SV40-T2 cells. Synthesis of these basement membrane constituents was significantly enhanced by TGF-beta1 in a dose-dependent manner. On the other hand, TGF-beta1 did not affect the synthesis of extracellular matrix-regulatory enzymes and their inhibitors, such as type II transglutaminase, matrix metalloproteinase-2, plasminogen activator inhibitor-1, or tissue inhibitor of matrix metalloproteinase-1. These results indicate that basement membrane formation in the presence of 1.0 ng/ml TGF-beta1 is attributable to enhanced synthesis of basement membrane constituents. However, formation of a continuous basement membrane was inhibited at a TGF-beta1 concentration of 5.0 ng/ml. Synthesis of the basement membrane constituents was further enhanced at this concentration and the extracellular matrix-regulatory enzymes remained unchanged. The deposits of cellular fibronectin and type I collagen beneath SV40-T2 cells were significantly augmented. Thus excessive production of interstitial extracellular matrix components appears to obstruct the integration of basement membrane constituents into a continuous architecture. These results indicate that the basement membrane formation by SV40-T2 cells is achieved at the optimal TGF-beta1 concentration.     

Binding of host extracellular matrix proteins to Mycoplasma fermentans and its effect on adherence to, and invasion of HeLa cells

In the present study, we show that intact Mycoplasma fermentans cells have a wealth of adhesive interactions with components of the extracellular matrix. Mycoplasma fermentans intensively bind plasminogen, and to a lesser extent, fibronectin, heparin, and laminin. The binding of collagen type III, IV, or V was low. The binding of plasminogen, collagen type III, or collagen type V markedly enhanced the adherence of M. fermentans to HeLa cells, whereas the binding of fibronectin, heparin, laminin, or collagen IV induced only a small effect on mycoplasma adherence. Utilizing plasminogen-treated M. fermentans preparations, we detected microorganisms within host HeLa cells by the gentamicin protection assay or by confocal laser scanning microscopy of immunofluorescent preparations. However, no intracellular M. fermentans was detected when M. fermentans preparations treated with fibronectin, heparin, laminin, or collagen type III, IV, or V were utilized.     

Immunomorphological research on the formation of the extracellular matrix in epithelial cell cultures

Formation of extracellular matrix structures in cultures of rat liver epithelial nontransformed cell line IAR2 was studied with antisera to fibronectin, laminin and type IV collagen by immunofluorescence and immunoelectron microscopy of platinum replicas. Fibronectin formed peripheral spots of variable size some of which outlined free cell edges, as well as fibrils located towards the center of single cells or of cellular islands. Similarly distributed structures were seen in isolated matrices. Codistribution of fibronectin and actin was observed only for the peripheral line of fibronectin spots and marginal circular actin bundle. Basement membrane components. laminin and type IV collagen, formed mainly spots of variable size predominantly beneath the cell or each cell in an island. Occasional fibrils were seen also. Essentially the same results were obtained by immunofluorescence and immunogold electron microscopy. Cytochalasin D treated cells displayed spots of both fibronectin and laminin. The relevance of previously postulated receptor-mediated assembly of extracellular matrix structures to the epithelial cells is discussed.     

Activin A/bone morphogenetic protein (BMP) chimeras exhibit BMP-like activity and antagonize activin and myostatin

Activins and bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of growth and differentiation factors that induce signaling in target cells by assembling type II and type I receptors at the cell surface. Ligand residues involved in type II binding are located predominantly in the C-terminal region that forms an extended beta-sheet, whereas residues involved in type I binding are located in the alpha-helical and preceding loop central portion of the molecule. To test whether the central residues are sufficient to determine specificity toward type I receptors, activin A/BMP chimeras were constructed in which the central residues (45-79) of activin A were replaced with corresponding residues of BMP2 and BMP7. The chimeras were assessed for activin type II receptor (Act RII) binding, activin-like bioactivity, and BMP-like activity as well as antagonistic properties toward activin A and myostatin. ActA/BMP7 chimera retained Act RII binding affinity comparable with wild type activin A, whereas ActA/BMP2 chimera showed a slightly reduced affinity toward Act RII. Both the chimeras were devoid of significant activin bioactivity in 293T cells in the A3 Lux reporter assay up to concentrations 10-fold higher than the minimal effective activin A concentration (approximately 4 nM). In contrast, these chimeras showed BMP-like activity in a BRE-Luc assay in HepG2 cells as well as induced osteoblast-like phenotype in C2C12 cells expressing alkaline phosphatase. Furthermore, both the chimeras activated Smad1 but not Smad2 in C2C12 cells. Also, both the chimeras antagonized ligands that signal via activin type II receptor, such as activin A and myostatin. These data indicate that activin residues in the central region determine its specificity toward type I receptors. ActA/BMP chimeras can be useful in the study of receptor specificities and modulation of transforming growth factor-beta members, activins, and BMPs.     

Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.