Altered synthesis of laminin 1 and absence of basement membrane component deposition in (beta)1 integrin-deficient embryoid bodies

2000 ◽  
Vol 113 (2) ◽  
pp. 259-268 ◽  
Author(s):  
M. Aumailley ◽  
M. Pesch ◽  
L. Tunggal ◽  
F. Gaill ◽  
R. Fassler
Keyword(s):  
Basement Membrane ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Mouse Genetics ◽  
Embryoid Bodies ◽  
Membrane Formation ◽  
Beta 1 Integrin ◽  
Laminin 1

Basement membranes are the earliest extracellular matrices produced during embryogenesis. They result from synthesis and assembly into a defined supramolecular architecture of several components, including laminins, collagen IV, nidogen, and proteoglycans. In vitro studies have allowed us to propose an assembly model based on the polymerisation of laminin and collagen IV in two separate networks associated together by nidogen. How nucleation of polymers and insolubilisation of the different components into a basement membrane proceed in vivo is, however, unknown. A most important property of several basement membrane components is to provide signals controling the activity of adjacent cells. The transfer of information is mediated by interactions with cell surface receptors, among them integrins. Mouse genetics has demonstrated that the absence of these interactions is not compatible with development as deletion of either laminin (gamma)1 chain or integrin (beta)1 chain lead to lethality of mouse embryos at the peri-implantation stage. We have used embyoid bodies as a model system recapitulating the early steps of embryogenesis to unravel the respective roles of laminin and (beta)1 integrins in basement membrane formation. Our data show that there is formation of a basal lamina in wild-type, but not in (beta)1-integrin deficient, embryoid bodies. Surprisingly, in the absence of (beta)1 integrins, laminin 1 was not secreted in the extracellular space due to a rapid switch off of laminin (alpha)1 chain synthesis which normally drives the secretion of laminin heterotrimers. These results indicate that (beta)1 integrins are required for the initiation of basement membrane formation, presumably by applying a feed-back regulation on the expression of laminin (alpha)1 chain and other components of basement membranes.

scholarly journals The Absence of Nidogen 1 Does Not Affect Murine Basement Membrane Formation

2000 ◽  
Vol 20 (18) ◽  
pp. 7007-7012 ◽  
Author(s):  
Monzur Murshed ◽  
Neil Smyth ◽  
Nicolai Miosge ◽  
Jörg Karolat ◽  
Thomas Krieg ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Embryonic Stem ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Null Mutation ◽  
Membrane Structures ◽  
Membrane Formation ◽  
Nidogen 1

ABSTRACT Nidogen 1 is a highly conserved protein in mammals,Drosophila melanogaster, Caenorhabditis elegans, and ascidians and is found in all basement membranes. It has been proposed that nidogen 1 connects the laminin and collagen IV networks, so stabilizing the basement membrane, and integrates other proteins, including perlecan, into the basement membrane. To define the role of nidogen 1 in basement membranes in vivo, we produced a null mutation of the NID-1 gene in embryonic stem cells and used these to derive mouse lines. Homozygous animals produce neither nidogen 1 mRNA nor protein. Surprisingly, they show no overt abnormalities and are fertile, their basement membrane structures appearing normal. Nidogen 2 staining is increased in certain basement membranes, where it is normally only found in scant amounts. This occurs by either redistribution from other extracellular matrices or unmasking of nidogen 2 epitopes, as its production does not appear to be upregulated. The results show that nidogen 1 is not required for basement membrane formation or maintenance.

Zebrafish mutants identify an essential role for laminins in notochord formation

Development ◽  
2002 ◽  
Vol 129 (13) ◽  
pp. 3137-3146 ◽  
Author(s):  
Michael J. Parsons ◽  
Steven M. Pollard ◽  
Leonor Saúde ◽  
Benjamin Feldman ◽  
Pedro Coutinho ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Positional Cloning ◽  
Essential Role ◽  
Basement Membranes ◽  
Form Complex ◽  
Organ Formation ◽  
Notochord Cells ◽  
Laminin 1

Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin β1 and laminin γ1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin β1 and laminin γ1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord.

Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo

1995 ◽  
Vol 108 (12) ◽  
pp. 3795-3805 ◽  
Author(s):  
F. Schuler ◽  
L.M. Sorokin
Keyword(s):  
In Situ Hybridization ◽  
Basement Membranes ◽  
Chain Gene ◽  
Muscle Fibres ◽  
Myogenic Cells ◽  
Mouse Tissues ◽  
Laminin 1

The expression of laminin-1 (previously EHS laminin) and laminin-2 (previously merosin) isoforms by myogenic cells was examined in vitro and in vivo. No laminin alpha 2 chainspecific antibodies react with mouse tissues, 50 rat monoclonal antibodies were raised against the mouse laminin alpha 2 chain: their characterization is described here. Myoblasts and myotubes from myogenic cell lines and primary myogenic cultures express laminin beta 1 and gamma 1 chains and form a complex with a 380 kDa alpha chain identified as laminin alpha 2 by immunofluorescence, immunoprecipitation and PCR. PCR from C2C12 myoblasts and myotubes for the laminin alpha 2 chain gene (LamA2) provided cDNA sequences which were used to investigate the in vivo expression of mouse LamA2 mRNA in embryonic tissues by in situ hybridization. Comparisons were made with specific probes for the laminin alpha 1 chain gene (LamA1). LamA2 but not LamA1 mRNA was expressed in myogenic tissues of 14- and 17-day-old mouse embryos, while the laminin alpha 2 polypeptide was localized in adjacent basement membranes in the muscle fibres. In situ hybridization also revealed strong expression of the LamA2 mRNA in the dermis, indicating that laminin alpha 2 is expressed other than by myogenic cells in vivo. Immunofluorescence studies localized laminin alpha 2 in basement membranes of basal keratinocytes and the epithelial cells of hair follicles, providing new insight into basement membrane assembly during embryogenesis. In vitro cell attachment assays revealed that C2C12 and primary myoblasts adhere to laminin-1 and -2 isoforms in a similar manner except that myoblast spreading was significantly faster on laminin-2. Taken together, the data suggest that laminins 1 and 2 play distinct roles in myogenesis.

scholarly journals Gene Structure and Functional Analysis of the Mouse Nidogen-2 Gene: Nidogen-2 Is Not Essential for Basement Membrane Formation in Mice

2002 ◽  
Vol 22 (19) ◽  
pp. 6820-6830 ◽  
Author(s):  
Jürgen Schymeinsky ◽  
Sabine Nedbal ◽  
Nicolai Miosge ◽  
Ernst Pöschl ◽  
Cherie Rao ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Biochemical Characterization ◽  
Gene Knockout ◽  
Basement Membranes ◽  
Membrane Formation ◽  
Gene Knockout Mice ◽  
Almost All ◽  
Nidogen 1 ◽  
Deficient Mice

ABSTRACT 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.

scholarly journals Endothelial Cell-Pericyte Interactions Stimulate Basement Membrane Matrix Assembly: Influence on Vascular Tube Remodeling, Maturation, and Stabilization

2011 ◽  
Vol 18 (1) ◽  
pp. 68-80 ◽  
Author(s):  
Amber N. Stratman ◽  
George E. Davis
Keyword(s):  
Growth Factor ◽  
Basement Membrane ◽  
Basement Membranes ◽  
Heparin Binding ◽  
Matrix Assembly ◽  
Matrix Deposition ◽  
Vascular Basement Membrane ◽  
Membrane Matrix

AbstractExtracellular matrix synthesis and deposition surrounding the developing vasculature are critical for vessel remodeling and maturation events. Although the basement membrane is an integral structure underlying endothelial cells (ECs), few studies, until recently, have been performed to understand its formation in this context. In this review article, we highlight new data demonstrating a corequirement for ECs and pericytes to properly deposit and assemble vascular basement membranes during morphogenic events. In EC only cultures or under conditions whereby pericyte recruitment is blocked, there is a lack of basement membrane assembly, decreased vessel stability (with increased susceptibility to pro-regressive stimuli), and increased EC tube widths (a marker of dysfunctional EC-pericyte interactions). ECs and pericytes both contribute basement membrane components and, furthermore, both cells induce the expression of particular components as well as integrins that recognize them. The EC-derived factors—platelet derived growth factor-BB and heparin binding-epidermal growth factor—are both critical for pericyte recruitment to EC tubes and concomitant vascular basement membrane formationin vitroandin vivo. Thus, heterotypic EC-pericyte interactions play a fundamental role in vascular basement membrane matrix deposition, a critical tube maturation event that is altered in key disease states such as diabetes and cancer.

scholarly journals Renal glomerular proteoglycans. An investigation of their synthesis in vivo using a technique for fixation in situ

1988 ◽  
Vol 251 (2) ◽  
pp. 411-418 ◽  
Author(s):  
L A Beavan ◽  
M Davies ◽  
R M Mason
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Membrane Fraction ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Basement Membranes ◽  
Chondroitin Sulphate Proteoglycans

Newly synthesized rat glomerular [35S]proteoglycans were labelled in vivo after injecting Na2[35S]SO4 intraperitoneally. At the end of the labelling period (7 h) the kidneys were perfused in situ with 0.01% (w/v) cetylpyridinium chloride. This fixed proteoglycans in the tissue and increased their recovery 2-3-fold during subsequent isolation of glomeruli from the renal cortex. The glomeruli were fractionated by a modified osmotic lysis and detergent extraction procedure [Meezan, Brendel, Hjelle & Carlson (1978) in The Biology and Chemistry of Basement Membranes (Kefalides, N.A., ed.), Academic Press, New York; Kanwar & Farquhar (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4493-4497] to obtain a basement membrane preparation. The proteoglycans released at each stage of the procedure were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC and HNO2 digestion and Sepharose CL-4B gel-permeation chromatography. About 85% of the [35S]proteoglycans synthesized were of the heparan sulphate variety, the remainder being chondroitin sulphate proteoglycans. Three sizes of heparan sulphate proteoglycans were identified. The largest (HS1, Kav. 0.47) accounts for 44% of the total extractable heparan sulphates. About one third of HS1 were extracted from the glomerular basement-membrane fraction with 8 M-urea and 4 M-guanidine hydrochloride but the remainder were released from the glomerulus during preparation of the fraction. The two smaller molecules (HS2, Kav. 0.56 and HS3, Kav. 0.68) accounted for 27% and 28% of the extractable heparan sulphate respectively and were not associated with the basement membrane fraction. HS1, HS2 and HS3 were also isolated from non-fixed glomeruli labelled in vivo but with much lower recovery. In glomeruli labelled in vitro, heparan sulphate accounted for only 35% of the proteoglycans, the remainder being of the chondroitin sulphate type. Proteoglycans similar to HS1, HS2 and HS3 were present in glomeruli labelled in vitro but, in addition, a large, highly charged heparan sulphate (HS1a) was extracted from the glomerular basement-membrane fraction of these glomeruli. It accounted for 6% of the total heparan sulphate.

scholarly journals Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys.

1985 ◽  
Vol 100 (6) ◽  
pp. 1988-2000 ◽  
Author(s):  
D R Abrahamson
Keyword(s):  
Basement Membrane ◽  
Developmental Stages ◽  
Cell Types ◽  
Basement Membranes ◽  
Newborn Rats ◽  
Mature Adult ◽  
Capillary Loop ◽  
Double Label

To examine the origin and assembly of glomerular basement membranes (GBMs), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and intravenously injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S-shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBMs, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickness. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In contrast with these results, in kidneys fixed 4-6 d after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma, and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin-rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least two different processes at separate times in development: (a) fusion of endothelial and epithelial basement membranes followed by (b) addition of new basement membrane from the epithelium into existing GBMs.

scholarly journals Non-muscle alpha-dystroglycan is involved in epithelial development.

1995 ◽  
Vol 130 (1) ◽  
pp. 79-91 ◽  
Author(s):  
M Durbeej ◽  
E Larsson ◽  
O Ibraghimov-Beskrovnaya ◽  
S L Roberds ◽  
K P Campbell ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Kidney Development ◽  
Physiological Role ◽  
Epithelial Differentiation ◽  
Basement Membranes ◽  
Metanephric Mesenchyme ◽  
Mouse Development ◽  
Alpha Dystroglycan ◽  
Laminin 1

The dystroglycan complex is a transmembrane linkage between the cytoskeleton and the basement membrane in muscle. One of the components of the complex, alpha-dystroglycan binds both laminin of muscle (laminin-2) and agrin of muscle basement membranes. Dystroglycan has been detected in nonmuscle tissues as well, but the physiological role in nonmuscle tissues has remained unknown. Here we show that dystroglycan during mouse development in nonmuscle tissues is expressed in epithelium. In situ hybridization revealed strong expression of dystroglycan mRNA in all studied epithelial sheets, but not in endothelium or mesenchyme. Conversion of mesenchyme to epithelium occurs during kidney development, and the embryonic kidney was used to study the role of alpha-dystroglycan for epithelial differentiation. During in vitro culture of the metanephric mesenchyme, the first morphological signs of epithelial differentiation can be seen on day two. Northern blots revealed a clear increase in dystroglycan mRNA on day two of in vitro development. A similar increase of expression on day two was previously shown for laminin alpha 1 chain. Immunofluorescence showed that dystroglycan is strictly located on the basal side of developing kidney epithelial cells. Monoclonal antibodies known to block binding of alpha-dystroglycan to laminin-1 perturbed development of epithelium in kidney organ culture, whereas control antibodies did not do so. We suggest that the dystroglycan complex acts as a receptor for basement membrane components during epithelial morphogenesis. It is likely that this involves binding of alpha-dystroglycan to E3 fragment of laminin-1.

scholarly journals Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes.

1979 ◽  
Vol 81 (1) ◽  
pp. 137-153 ◽  
Author(s):  
Y S Kanwar ◽  
M G Farquhar
Keyword(s):  
Ionic Strength ◽  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Left Kidney ◽  
High Ionic Strength ◽  
Basement Membranes ◽  
Mesangial Matrix ◽  
Anionic Sites

Cationized ferritin (CF) of narrow pI range (7.3-7.5) and the basic dye ruthenium red (RR) have been used as cationic probes to partially characterize anionic sites previously demonstrated in the glomerular basement membrane (GBM). When CF was given i.v. to normal rats and the left kidney was fixed by perfusion 15 min thereafter, clusters of CF molecules were found throughout the lamina rara interna (LRI), lamina rara externa (LRE), and mesangial matrix distributed at regular (approximately 60 nm) intervals. When kidneys were perfused with aldehyde fixative containing RR, small (20 nm) RR-stained particles were seen in the same locations distributed with the same 60 nm repeating pattern, forming a quasiregular, lattice-like arrangement. Fine (approximately 3 nm) filaments connected the sites and extended between them and the membranes of adjoining endothelial and epithelial cells. When CF was given i.v. followed by perfusion with RR in situ, both probes localized to the same sites. CF remained firmly bound after prolonged perfusion with 0.1-0.2 M KCl or NaCl. It was displaced by perfusion with buffers of high ionic strength (0.4-0.5 M KCl) or pH (less than 3.0 or greater than 10.0). CF also bound (clustered at approximately 60 nm intervals) to isolated GBM's, and binding was lost when such isolated GBM's were treated with buffers of high ionic strength or pH. These experiments demonstrate the existence of a quasi-regular, lattice-like network of anionic sites in the LRI and LRE and the mesangial matrix. The sites are demonstrable in vivo (by CF binding), in fixed kidneys (by RR staining), and in isolated GBM's (by CF binding). The results obtained with CF show that the binding of CF (and probably also RR) to the laminae rarae is electrostatic in nature since it is displaced by treatment with buffers of high ionic strength or pH. With RR the sites resemble in morphology and staining properties the proteoglycan particles found in connective tissue matrices and in association with basement membranes in several other locations.

scholarly journals Absence of Basement Membranes after Targeting the LAMC1 Gene Results in Embryonic Lethality Due to Failure of Endoderm Differentiation

1999 ◽  
Vol 144 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Neil Smyth ◽  
H. Seda Vatansever ◽  
Patricia Murray ◽  
Michael Meyer ◽  
Christian Frie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Basement Membrane ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Basement Membranes ◽  
Embryoid Bodies ◽  
Endoderm Differentiation

The LAMC1 gene coding for the laminin γ1 subunit was targeted by homologous recombination in mouse embryonic stem cells. Mice heterozygous for the mutation had a normal phenotype and were fertile, whereas homozygous mutant embryos did not survive beyond day 5.5 post coitum. These embryos lacked basement membranes and although the blastocysts had expanded, primitive endoderm cells remained in the inner cell mass, and the parietal yolk sac did not develop. Cultured embryonic stem cells appeared normal after targeting both LAMC1 genes, but the embryoid bodies derived from them also lacked basement membranes, having disorganized extracellular deposits of the basement membrane proteins collagen IV and perlecan, and the cells failed to differentiate into stable myotubes. Secretion of the linking protein nidogen and a truncated laminin α1 subunit did occur, but these were not deposited in the extracellular matrix. These results show that the laminin γ1 subunit is necessary for laminin assembly and that laminin is in turn essential for the organization of other basement membrane components in vivo and in vitro. Surprisingly, basement membranes are not necessary for the formation of the first epithelium to develop during embryogenesis, but first become required for extra embryonic endoderm differentiation.

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