Ultrastructural colocalization of nidogen-1 and nidogen-2 with laminin-1 in murine kidney basement membranes

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen type IV networks. Recently a new member of the nidogen family, nidogen-2, has been characterized. Preliminary immunohistochemical data indicated that nidogen-1 and nidogen-2 show a similar tissue distribution at the light microscopic level. We have now localized nidogen-1 and nidogen-2, as well as their corresponding mRNAs, at the light and electron microscopic levels in adult mouse kidney, by in situ hybridization and immunogold histochemistry, as well as carrying out double labeling with laminin-1. Both nidogen-1 and nidogen-2 mRNAs are found not only in mesenchymal cells of embryonic tissues, but also in all epithelial and endothelial cells in adult mouse kidney. Both nidogens are ubiquitous basement membrane components in the mouse kidney, being found in glomerular, tubular, and capillary compartments and Bowman’s capsule. Furthermore, a substantial fraction of nidogen-1 and nidogen-2 colocalizes with laminin-1. The results indicate that nidogen-1 and nidogen-2 could well substitute for one another in some of their biological activities in kidney, for example, stabilizing basement membrane networks in vivo. Accepted: 8 December 1999     

Nidogen-1 and nidogen-2 are found in basement membranes during human embryonic development

The recently identified nidogen-2 is a matrix protein showing homology to the well-known basement membrane molecule nidogen-1. Nidogen-1 might well serve as a link between laminin-1 and collagen type IV and thus stabilise certain basement membranes in vivo and play a major role in embryogenesis. However, the exact tissue distribution of nidogen-1 and nidogen-2 during human embryogenesis is still unclear. As a first step towards the elucidation of their possible cell biological functions during human development, we compared the distribution of both nidogens during human organogenesis at the light microscope level. Nidogen-2 and nidogen-1 were found to be ubiquitous components of basement membrane zones underneath developing epithelia of most of the major organ systems. However, in the developing intestine and the pancreas anlage, only nidogen-1 was present in the epithelial basement membrane zones of all developmental stages investigated. Our data suggest that nidogen-2 and nidogen-1, as is known for mouse development, could well participate in cell biological functions during human development. These two proteins might well be able to fulfil identical functions during human organogenesis.     

Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens

Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin gamma1 chain (gamma1III4) is important for kidney development and for nerve guidance. Other proteins may also bind gamma1III4, although human nidogen-2 binds poorly to the mouse laminin gamma1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and gamma1III3-5, which both contain the gamma1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial-mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin gamma1 chain is dependent on epithelial-mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by alpha3beta1 and alpha6beta1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized.     

Expression of nidogens in rat uterus and embryo during decidualization and implantation

The purpose of this study was to demonstrate the expression of nidogen-1 and nidogen-2 and their possible role in decidualization and implantation events during early pregnancy in rats. The tissue samples were examined from pregnant animals between gestational days 1-8 using immunocytochemistry. The uterine luminal epithelium, the glandular epithelium, and the myometrial smooth muscle cells stained strongly from gestational days 1-8 with both nidogen antibodies. At day 4 the decidual reaction areas began to appear in the stromal matrix and immunostaining of both nidogens revealed that the basement membrane of the surface epithelium was discontinuous. The differentiation of stromal cells into decidual cells was seen at gestational day 5 and both nidogens were weakly expressed in the decidualizing cells. At day 6, nidogen-2 immunoreactivity was higher in the primary decidual cells close to the embryo than nidogen-1, and during development of the decidual tissue both nidogens appeared in the endometrial stromal cells. At day 7, while expression of both nidogens declined in the primary decidual cells, their expression was markedly observed in the secondary decidual cells close to the myometrium. At day 8, expression of both nidogens was also observed to increase in the primary decidual cells. While nidogen-2 expression was seen in the parietal endoderm and primary ectoderm of the rat embryos at this developmental stage, nidogen-1 expression was only detected in the parietal endoderm. These results indicate that nidogen-1 and nidogen-2 could play important roles during embryogenesis, decidualization, and implantation in the endometrium of rat uterus.     

Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice

Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.     

The absence of one or both nidogens does not alter basement membrane composition in adult murine kidney

Nidogen-1 and nidogen-2 are major components of all basement membranes and are considered to function as link molecules between laminin and collagen type IV networks. Surprisingly, the knockout of one or both nidogens does not cause defects in all tissues or in all basement membranes. In this study, we have elucidated the appearance of the major basement membrane components in adult murine kidney lacking nidogen-1, nidogen-2, or both nidogens. To this end, we localized laminin-111, perlecan, and collagen type IV in knockout mice, heterozygous (+/-) or homozygous (-/-) for the nidogen-1 gene, the nidogen-2 gene, or both nidogen genes with the help of light microscopic immunostaining. We also performed immunogold histochemistry to determine the occurrence of these molecules in the murine kidney at the ultrastructural level. The renal basement membranes of single knockout mice contained a similar distribution of laminin-111, perlecan, and collagen type IV compared to heterozygous mice. In nidogen double-knockout animals, the basement membrane underlying the tubular epithelium was sometimes altered, giving a diffuse and thickened pattern, or was totally absent. The normal or thickened basement membrane of double-knockout mice also showed a similar distribution of laminin-111, perlecan, and collagen type IV. The results indicate that the lack of nidogen-1, nidogen-2, or both nidogens, plays no crucial role in the occurrence and localization of laminin-111, collagen type IV, and perlecan in murine tubular renal basement membranes.     

Comparative study of clonal growth and differentiation of mesenchymal stromal cells from rat fetal liver at different developmental stages

Fetal liver during period of its hematopoietic activity contains mesenchymal stromal cells (MSC) that are known to play a major role in establishing hematopoietic microenvironment. These cells are capable of clonal growing and multilineage differentiation, but only limited data exist about changes in their properties during prenatal development. We compared cloning efficiency of MSC from liver of 14, 16 and 20 day rat fetuses and evaluated their potentials to in vitro osteo- and adipogenesis and in vivo chondrogenesis after whole organ ectopic transplantation. Content of clonogenic MSC in suspension of liver cells was maximal in 16 day fetuses and to a lesser extent in 20 day ones. MSC derived from 16 day fetuses demonstrated maximal potential to estimated lineages. Osteogenic potential of MSC from 14 day fetuses was comparable to whereas their adipogenic and chondrogenic abilities were inferior to that from 16 day fetuses. Cells from 20 day fetuses had only weak adipogenic potency and failed to differentiate into osteogenic of chondrogenic pathways. The results indicate that both number and differentiation potential of MSC in developing rat liver correlate with dynamics of hematopoiesis in this organ. Detected changes may be ascribed to the decline of hematopoiesis in liver and acquisition its definitive functions.     

Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1.

Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. Here, we report the crystal structure of the mouse nidogen-1 G2 fragment, which contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded beta-barrel with a central helix. The beta-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the beta-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding.     

Loss of nidogen-1 and -2 results in syndactyly and changes in limb development

Nidogens are two ubiquitous basement membrane proteins produced mainly by mesenchymal cells. Nidogen-mediated interactions, in particular with laminin, collagen IV, and perlecan have been considered important in the formation and maintenance of the basement membrane. However, whereas mice lacking both nidogen isoforms or carrying mutations in the high affinity nidogen-binding site upon the laminin gamma1 chain have specific basement membrane defects in certain organs, particularly in the lung, characterization of these mice has also shown that basement membrane formation per se does not need nidogens or the laminin-nidogen interaction. Limb development requires the complex interplay of numerous growth factors whose expression is dependent upon the apical ectodermal ridge. Here, we show that lack of nidogen-1 and -2 results in a specific and time-limited failure in the ectodermal basement membrane of the limb bud. The absence of this basement membrane leads to aberrant apical ectodermal ridge formation. It also causes altered distribution of growth factors, such as fibroblast growth factors and leads to a fully penetrant soft tissue syndactyly caused by the dysregulation of interdigital apoptosis. Further, in certain animals more severe changes in bone formation occur, providing evidence for the interplay between growth factors and the extracellular matrix.     

Sequential morphological characteristics of murine fetal liver hematopoietic microenvironment in Swiss Webster mice

Embryonic hematopoiesis occurs via dynamic development with cells migrating into various organs. Fetal liver is the main hematopoietic organ responsible for hematopoietic cell expansion during embryologic development. We describe the morphological sequential characteristics of murine fetal liver niches that favor the settlement and migration of hematopoietic cells from 12 days post-coitum (dpc) to 0 day post-partum. Liver sections were stained with hematoxylin and eosin, Lennert’s Giemsa, Sirius Red pH 10.2, Gomori’s Reticulin, and Periodic Acid Schiff/Alcian Blue pH 1.0 and pH 2.5 and were analyzed by bright-field microscopy. Indirect imunohistochemistry for fibronectin, matrix metalloproteinase-1 (MMP-1), and MMP-9 and histochemistry for naphthol AS-D chloroacetate esterase (NCAE) were analyzed by confocal microscopy. The results showed that fibronectin was related to the promotion of hepatocyte and trabecular differentiation; reticular fibers did not appear to participate in fetal hematopoiesis but contributed to the physical support of the liver after 18 dpc. During the immature phase, hepatocytes acted as the fundamental stroma for the erythroid lineage. The appearance of myeloid cells in the liver was related to perivascular and subcapsular collagen, and NCAE preceded MMP-1 expression in neutrophils, an occurrence that appeared to contribute to their liver evasion. Thus, the murine fetal liver during ontogenesis shows two different phases: one immature and mainly endodermic (<14 dpc) and the other more developed (endodermic-mesenchymal; >15 dpc) with the maturation of hepatocytes, a better definition of trabecular pattern, and an increase in the connective tissue in the capsule, portal spaces, and liver parenchyma. The decrease of hepatic hematopoiesis (migration) coincides with hepatic maturation.     

Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice.

Previous studies have shown that inhibition of nidogen-laminin binding interferes with basement membrane stabilization in various mouse organ cultures while no overt phenotype has been observed following inactivation of the nidogen-1 gene in mice. We have now used recombinant mouse nidogen-1 and nidogen-2 in order to evaluate a possible compensation between the two isoforms in the knock-out mice. Essentially, a comparable in vitro binding of nidogens-1 and -2 to the same laminin gamma1 chain structure and to several other basement membrane proteins has been revealed. Quantitative radioimmuno-assays have demonstrated high concentrations of nidogen-1 exceeding those of laminin gamma1 and nidogen-2 by factors of 5 and 20-50, respectively, in tissue extracts of wild-type mice. A three- to sevenfold increase in nidogen-2 was observed in heart and muscle of mice with nidogen-1 deficiency and confirmed by a similar increase in the intensity of immunogold staining of these tissues. However, a few of the tissues from mice with the gene knock-out still contained some nidogen-1-like immunoreactivity (1% of wild-type). Furthermore, both nidogen isoforms showed a similar distribution in various organs during embryonic development which, however, as shown previously, changed in some adult tissues. The data support the nidogen-2 compensation hypothesis to explain the limited phenotype observed following elimination of the nidogen-1 gene.     

Laminin gamma3 chain binds to nidogen and is located in murine basement membranes

Recently a novel laminin gamma3 chain was identified in mouse and human and shown to have the same modular structure as the laminin gamma1 chain. We expressed two fragments of the gamma3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3-5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The gamma3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the beta2 VI/V fragment. The gamma3 III3-5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the gamma1 III3-5 fragment. These data suggested that laminins containing the gamma3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against gamma3 VI/V and gamma3 III3-5 showed no cross-reaction with homologous fragments from the gamma1 and gamma2 chains of laminin and allowed the establishment of gamma chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20-100-fold lower content of the gamma3 chain compared with the gamma1 chain in various tissue extracts of adult mice. The expression of gamma3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the gamma3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the gamma3 chain to basement membranes of these tissues.     

Mesenchymal entactin-1 (nidogen-1) is required for adhesion of peritubular cells of the rat testis in vitro

Epithelial-like Sertoli cells isolated from immature rat testis aggregate to form tubule-like structures when cultured on a monolayer of mesenchyme-derived peritubular cells. At the end of this morphogenetic process both cell types are separated by a basement membrane. In this study the gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells was examined using DD-RT-PCR. One of the isolated cDNA clones showed high homology to the cDNA encoding the basement membrane component entactin-1 (nidogen-1). Even though the entactin-1 (nidogen-1) gene is transcribed in peritubular cells, Sertoli cells, and in direct cocultures, the mRNA is translated only by the peritubular cells. No entactin-1 (nidogen-1) was detected in the Sertoli cells by Western blotting. Moreover, peritubular cell monocultures and cocultures showed the presence of one single band at 152 kDa in the supernatant, whereas in cell lysates two bands were detectable at 152 kDa and 150 kDa. Perturbation experiments using monoclonal antibodies directed against entactin-1 (nidogen-1) were performed with peritubular cells and Sertoli cells, respectively, and demonstrated loss of cell adhesion of the peritubular cells, while the Sertoli cells remained adherent. From these data we conclude that entactin-1 is exclusively produced and secreted by mesenchymal peritubular cells, and affects adhesion of peritubular cells in an autocrine manner.     

Nidogen is a prosurvival and promigratory factor for adult Schwann cells

Schwann cells provide a favorable microenvironment for successful regeneration of the injured peripheral nerve. Even though the roles of extracellular matrix proteins in the Schwann cell physiology have long been studied, the precise function of nidogen, a ubiquitous component of the basal lamina, in Schwann cells is unknown. In this study, we show that the protein and mRNA messages for nidogens are up-regulated in the sciatic nerve after sciatic nerve transection. We demonstrate that recombinant nidogen-1 increased the process formation of Schwann cells cultured from adult rat sciatic nerves and that nidogen-1 prevented Schwann cells from serum-deprivation-induced death. In addition, nidogen-1 promoted spontaneous migration of Schwann cells in two-independent migration assays. The Schwann cell responses to the recombinant nidogen-1 were specific because the nidogen-binding ectodomain of tumor endothelial marker 7 inhibited the nidogen responses without affecting Schwann cell response to laminin. Finally, we found that beta1 subunit-containing integrins play a key role in the nidogen-induced process formation, survival, and migration of Schwann cells. Altogether, these results indicate that nidogen has a prosurvival and promigratory activity on Schwann cells in the peripheral nerve.     

Nidogen-1. Expression and ultrastructural localization during the onset of mesoderm formation in the early mouse embryo.

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen Type IV networks and is expressed by mesenchymal cells during embryonic and fetal development. It is not clear which cells produce nidogen-1 in early developmental stages when no mesenchyme is present. We therefore localized nidogen-1 and its corresponding mRNA at the light and electron microscopic level in Day 7 mouse embryos during the onset of mesoderm formation by in situ hybridization, light microscopic immunostaining, and immunogold histochemistry. Nidogen-1 mRNA was found not only in the cells of the ectoderm-derived mesoderm but also in the cytoplasm of the endoderm and ectoderm, indicating that all three germ layers express it. Nidogen-1 was localized only in fully developed basement membranes of the ectoderm and was not seen in the developing endodermal basement membrane or in membranes disrupted during mesoderm formation. In contrast, laminin-1 and collagen Type IV were present in all basement membrane types at this developmental stage. The results indicate that, in the early embryo, nidogen-1 may be expressed by epithelial and mesenchymal cells, that both cell types contribute to embryonic basement membrane formation, and that nidogen-1 might serve to stabilize basement membranes in vivo. (J Histochem Cytochem 48:229-237, 2000)     

Bronchial macrophages in asthmatics reveal decreased CD16 expression and substantial levels of receptors for IL-10, but not IL-4 and IL-7

The role of different subpopulations of bronchial macrophages (BMs) in asthma pathogenesis has not yet been completely elucidated. In addition, little is known about potential in vivo responsiveness of BMs to pro- and anti-inflam-matory cytokines present in the bronchial milieu. We aimed to characterize asthmatic patients' BM subpopulations delineated by common markers of macrophage/monocyte cells, CD16 and CD14, and subsequently to analyze cytokine receptor expression on those subsets. Subjects included eighteen patients with moderate asthma (six steroid-naive and twelve steroid-treated) and ten healthy control subjects. Flow cytometry was used to analyze phenotypical features of BMs including expression of receptors for IL-10, IL-4 and IL-7. Exhaled nitric oxide analysis and induced sputum eosinophil counts were used to assess airway inflammation. BMs from both steroid-naive and steroid-treated asthmatic patients showed significantly decreased expression of CD16, as compared to healthy subjects' BMs. CD16, but not CD14, expression inversely correlated with exhaled nitric oxide levels and sputum eosinophilia. Short-term administration of inhaled cortiocosteroids (ICS) in steroid-naive asthmatic patients led to significant reduction of CD16 expression and enhancement of CD14 expression. Next, we analyzed the expression of receptors for IL-10, IL-4 and IL-7 on the surface of BM subpopulations characterized by different levels of CD14 and CD16 expression. We observed substantial levels of IL-10R on the surface of BMs collected from asthmatic and healthy subjects. Interestingly, IL-10R was found mostly on those macrophages that co-expressed CD14. In contrast, independently on co-expression of CD14, the levels of IL-4R and IL-7R on BMs were low in both asthmatic and healthy subjects. The results suggest that different BM subsets may be differentially involved in regulating the inflammatory response in allergic asthma.     

Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation

Summary Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells.     

Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation.

Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells.     

Cellular composition and regulatory function of fetal liver stroma

Hematopoietic differentiation and formation of hepatic tissue both take place in mammalian liver during its prenatal development. Hematopoietic and hepatic stem cells self-renew, proliferate and differentiate within specific microenvironment that is organized by stromal elements. Stroma of developing liver consists of different cell populations such as mesenchymal stromal cells, Ito cells, portal fibroblasts and myofibroblasts, vascular endothelial and smooth muscle cells, cells undergoing epithelial-to-mesenchymal transition. In this review, their phenotypical and functional properties, possible derivation and role in the regulation of hematopoiesis and hepatogenesis are discussed.     

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.