Primary Structure, Developmental Expression, and Immunolocalization of the Murine Laminin α4 Chain (original) (raw)
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Primary structure, developmental expression, and immunolocalization of the murine laminin ��4 chain
1997
The complete primary structure of the mouse laminin ␣4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature ␣4 chain of 1,792 residues. Northern analysis on whole mouse embryos revealed that the expression was weak at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and cardiac and skeletal muscles. Weak expression was also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin ␣4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistologic staining with affinity-purified IgG localized the laminin ␣4 chain primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium.
Experimental Cell Research, 2002
Protein levels, mRNA expression, and localization of laminin ␣1 and ␣2 chains in development and in adult mice were examined. Recombinant fragments were used to obtain high-titer-specific polyclonal antibodies for establishing quantitative radioimmunoinhibition assays. This often demonstrated an abundance of ␣2 chain, but also distinct amounts of ␣1 chain for adult tissues. The highest amounts of ␣1 were found in placenta, kidney, testis, and liver and exceeded those of ␣2. All other tissue extracts showed a higher content of ␣2, which was particularly high in heart and muscle when compared to ␣1. Content of ␥1 chain, shared by most laminins, was also analyzed. This demonstrated ␥1 chain levels being equal to or moderately exceeding the sum of ␣1 and ␣2 chains, indicating that these isoforms represent the major known laminin isoforms in most adult mouse tissues so far examined. Moreover, we found good correlation between radioimmuno-inhibition data and mRNA levels of adult tissues as measured by quantitative real-time reverse transcriptase-PCR. Embryonic tissues were also analyzed by radioimmuno-inhibition assays. This demonstrated for day 11 embryos comparable amounts of ␣1 and ␥1 and a more than 25-fold lower content of ␣2. This content increased to about 10% of ␣1 in day 13 embryos. The day 18 embryo showed in heart, kidney, and liver, but not yet in brain and lung, ␣1/␣2 chain ratios comparable to those in adult tissues. Immunostaining demonstrated ␣1 in Reichert's membrane (day 7.5), while ␣2 could not be detected before day 11.5. These data were compared with immunohistochemical localization results on several more embryonic and adult tissue sections. Our results regarding localization are consistent with those of earlier work with some notable exceptions. This was in part due to epitope masking for monoclonal antibodies commonly used in previous studies in esophagus, intestine, stomach, liver, kidney, and spleen.
Expression of laminin α1, α5 and β2 chains during embryogenesis of the kidney and vasculature
Matrix Biology, 1996
Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of ix, 13 and y chain subunits. To resolve conflicting data in the literature concerning coexpression of ctl and 132 chains, expression of ixl chain was studied with two different antisera against the E3 fragment of laminin ix1 chain. Expression of the (xl chain was seen in several types of epithelial basement membranes throughout development, but its expression in rat glomerular basement membranes and some other types of epithelial basement membranes occurred only during early stages of development. By contrast, 132 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, 132 chains were detected somewhat earlier, but in situ hybridization revealed that 132 chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of ix1 and 132 chains was mutually exclusive. To explore whether the newly described 0d chain is expressed in locations lacking ix1 chain, expression of ~5 chain was studied by Northern blots and in situ hybridization. The ct5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little ix1 chain. There also appeared to be a developmental trend, with ix1 chain appearing early and ix5 later, in maturing epithelial sheets. The 0d chain could be a major ix chain of the adult glomerular basement membrane.
Developmental Biology, 1997
We have previously shown that mouse and bovine endothelial cells express a novel 400-kDa laminin a chain complexed to b1 and g1 laminin chains. We describe here purification of this laminin isoform from the conditioned medium of a mouse peripheral lymph node endothelial cell line, SVEC. The laminin a chain was isolated from the laminin complex, subjected to Edman digestion, and the amino acid sequences of the resulting peptides were determined. Amino acid sequence revealed 100% identity to the predicted amino acid sequence of the recently reported laminin a5 gene. A monoclonal antibody to the laminin a5 chain was raised (4G6), allowing investigation of its distribution in embryonic, newborn, and mature mouse tissues. The laminin a5 chain was expressed mainly by epithelial, endothelial, and myogenic cells: In both embryonic and mature tissues the laminin a5 chain was strongly expressed by epithelial cells, the bronchi of the lungs and the developing kidney tubules being the sites of strongest expression. However, laminin a5 was not associated with early stages of epithelial cell development, but rather with epithelial cell maturation. Widespread expression of laminin a5 in endothelial cells was apparent only in tissues of mature mice, its appearance correlating approximately with sexual maturity. During embryogenesis and in newborn tissues, laminin a5 occurred in basement membranes of larger blood vessels only, excluding a role in angiogenic processes. Smooth muscle and skeletal muscle cells were the only other cell types which showed considerable laminin a5 expression, with skeletal muscle exhibiting a developmentally regulated pattern of expression: The laminin a5 chain occurred in skeletal muscle fiber basement membranes early in embryogenesis (E13-E15) but decreased with development, remaining strongly expressed only at the neuromuscular junction. The data show that laminin a5 expression is associated with epithelial and endothelial cell maturation, implicating a role for this laminin chain in the maintenance of differentiated epithelial and endothelial cell phenotype.
Tissue distribution of the laminin β1 and β2 chain during embryonic and fetal human development
Journal of Molecular Histology, 2010
Laminins are the major glycoproteins present in all basement membranes. Previously, we showed that perlecan is present during human development. Although an overview of mRNA-expression of the laminin b1 and b2 chains in various developing fetal organs is already available, a systematic localization of the laminin b1 and b2 chains on the protein level during embryonic and fetal human development is missing. Therefore, we studied the immunohistochemical expression and tissue distribution of the laminin b1 and b2 chains in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin b1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin b2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development.
Localization of Laminin 4-Chain in Developing and Adult Human Tissues
Journal of Histochemistry & Cytochemistry, 2002
Recent studies suggest important functions for laminin-8 (Ln-8; α4β1γ1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln α4-chain. Immunoreactivity for the Ln α4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln α4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln α4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln α4- and Ln α2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric gland...
Matrix Biology, 1995
The sequence of the human laminin [32 chain (previously s-laminin) was derived from cloned cDNAs. The complete translation product has 1798 amino acid residues, including a 32-residue signal peptide. The human chain lacks the tripeptide sequence LRE in domain I which is present in the rat polypeptide chain and has been shown to promote motor neuronal cell adhesion. The human gene (LAMB2) was localized to chromosome 3p21 using somatic cell hybrids and fluorescent in situ hybridization analysis. Northern and in situ hybridization analyses from numerous fetal tissues revealed that the [32 chain is generally widely expressed. [32, but not [31, was shown by in situ hybridization to be expressed in fetal brain and renal glomeruli. In fetal skin, [32 was expressed both in epidermal and dermal cells, while [31 was expressed only in the dermis. Expression of [32 in fetal liver was seen in hepatocytes, while no signals were observed for [31. In lung, both [31 and [32 were expressed in alveoli and bronchial smooth muscle cells, whereas only the [32 chain was expressed in bronchial epithelial cells. In striated muscle, however, the [31 chain, but not [32, was expressed. These results indicate different biological roles for the laminin [31 and [32 chains.
Regulation of the beta2 subunit chain of laminin in developing rabbit fetal lung tissue
Differentiation, 1996
Laminins are a family of basement membrane-associated heterotrimeric proteins that are important in mediating the growth, migration, and differentiation of a variety of cell types. The β2 subunit chain is a component of several laminin isoforms, e.g., laminin-3, laminin-4, laminin-7, and possibly other, as yet uncharacterized laminin isoforms. Utilizing monoclonal antibodies directed against the β2 subunit chain of laminin, we detected this protein in fetal, neonatal, and adult lung tissues. The relative amount of laminin β2 subunit chain in fetal lung tissue increased as gestation proceeded, reaching its peak around the time of alveolar type II cell differentiation in the rabbit. The laminin β2 subunit chain was localized in early gestational age rabbit fetal lung tissue primarily in basement membranes of prealveolar ducts, airways, and smooth muscle cells of airways and arterial blood vessels. A rabbit laminin β2 cDNA was generated using RT-PCR and utilized as a probe in northern blot analysis to characterize the levels of laminin β2 mRNA in developing rabbit lung tissue. Similar to the pattern of laminin β2 protein induction observed in fetal lung tissue, laminin β2 mRNA levels were maximal late in gestation. Utilizing a laminin β2 chain cRNA probe and in situ hybridization, we detected laminin β2 mRNA in the epithelial cells of prealveolar ducts, the alveolar wall, and airways, as well as in connective tissue cells, and the smooth muscle cells of airways and blood vessels in fetal and adult lung tissues. In addition, using an in vitro explant model, we determined that alveolar type II cells are capable of synthesizing laminin β2 subunit mRNA and depositing this laminin subunit chain in the basement membrane beneath type II cells. The results of this study are suggestive that the laminin β2 chain may be involved in alveolar epithelial cell differentiation.& b d y :
Characterization of α1, β1, and γ1 laminin subunits during rabbit fetal lung development
Developmental Dynamics, 1995
Laminin-1 is an extracellular matrix protein composed of three polypeptide chains that are designated al, pl, and yl. We investigated the expression of laminin cwl, pl, and y l subunit chains during several stages of rabbit fetal lung development. Utilizing polyclonal antibodies directed against human placental laminin and immunoblot analysis, we found that the highest levels of laminin al, pl, and y l subunit chains in the fetal lung were present on day 26 of gestation (term = 31 days), coincident with the initiation of alveolar epithelial cell differentiation. Levels of the laminin chains were approximately five times higher in fetal lung at day 26 of gestation than in adult lung tissue. Different temporal patterns of laminin al, pl, and y l subunit chain expression were observed, data suggestive that the chains are independently regulated during lung development. Laminin was localized to the basement membranes of bronchi, bronchioles, prealveolar ducts, and blood vessels in fetal lung tissue, as shown by immunostaining with polyclonal laminin antibodies. A similar staining pattern was observed in adult lung tissue, but the alveolar wall was also stained. Laminin was also observed surrounding a few mesenchymal cells in fetal lung on day 19 of gestation; the number of positive mesenchymal cells increased with lung development. Laminin a1 subunit chains, detected using a monoclonal antibody, were present in the basement membranes of bronchi, bronchioles, prealveolar ducts, and blood vessels in fetal lung tissue. No laminin a1 chain staining was observed in the mesenchyme of early fetal lung tissue. Using a monoclonal antibody, laminin P l subunit chains were immunolocalized in the basement membranes of bronchi, bronchioles, in prealveolar ducts, and surrounding some mesenchymal cells in fetal lung tissue. Laminin a1 and p l subunit chains in adult lung tissue were present in basement membranes of airways, blood vessels, and alveoli. Thus, changes in the localization and accumulation of laminin near the time of alveolar type I and type I1 epithelial cell differentiation suggest that laminin may play a role in mediating the differentiation of these cell types during rabbit fetal lung development.